Untitled - KAFKAS ÜNİVERSİTESİ VETERİNER FAKÜLTESİ DERGİSİ

(MART - NİSAN) (MARCH - APRIL)

ISSN: 1300-6045e-ISSN: 1309-2251

Cilt/Volume: 18 Sayı/Number: 2 Yıl/Year: 2012

KAFKAS ÜNİVERSİTESİVETERİNER FAKÜLTESİ DERGİSİTHE JOURNAL OF THE FACULTY OF VETERINARY MEDICINE

UNIVERSITY OF KAFKAS

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YAZIŞMAADRESİ(AddressforCorrespondence)

Kafkas Üniversitesi Veteriner Fakültesi Dergisi Editörlüğü36040 - Kars / TÜRKİYE

Phone: +90 474 2426807-2426836/5228Fax: +90 474 2426853

E-mail: [email protected]

ELEKTRONİKBASKI(ElectronicEdition)

http://vetdergi.kafkas.edu.tr

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http://vetdergikafkas.org

ISSN:1309-2251

Bu dergi Kafkas Üniversitesi Veteriner Fakültesi tarafından iki ayda bir yayımlanır.This journal is published bi-monthly, by the Faculty of Veterinary Medicine, University of Kafkas.

KafkasÜniversitesiVeterinerFakültesiAdınaSahibi

(Owner)

Prof.Dr. Hidayet Metin ERDOĞANDekan (Dean)

EDİTÖR(Editor-in-Chief)

Prof.Dr. İsa ÖZAYDIN

YABANCIDİLEDİTÖRLERİ(EnglishEditors)

Doç.Dr. Ahmet ÜNVERDoç.Dr. Şükrü Metin PANCARCI

İSTATİSTİKEDİTÖRÜ(StatisticsEditor)

Prof.Dr. Gül ERGÜN

EDİTÖRYARDIMCILARI(AssociateEditors)

Prof.Dr. Mehmet ÇİTİLDoç.Dr. Özgür AKSOYYrd.Doç.Dr. Oktay ÖZKANYrd.Doç.Dr. Duygu KAYA

SEKRETER(Secretary)

Fahri ALTUN

BASKI(Print)

ESER OFSET MATBAACILIKTel: +90 442 2334667

ERZURUM

DANIŞMAKURULU(AdvisoryBoard)

Prof.Dr. Kemal AK İstanbul Üniversitesi Veteriner FakültesiProf.Dr. Belma ALABAY Ankara Üniversitesi Veteriner FakültesiProf.Dr. Mustafa ALİŞARLI Ondokuz Mayıs Üniversitesi Veteriner FakültesiProf.Dr. Feray ALKAN Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Çiğdem ALTINSAAT Ankara Üniversitesi Veteriner Fakültesi Prof.Dr. Kemal ALTUNATMAZ İstanbul Üniversitesi Veteriner FakültesiProf.Dr. Mustafa ARICAN Selçuk Üniversitesi Veteriner FakültesiProf.Dr. Sırrı AVKİ Mehmet Akif Ersoy Üniversitesi Veteriner FakültesiProf.Dr. Metin BAYRAKTAR Fırat Üniversitesi Veteriner FakültesiProf.Dr. Burhan ÇETİNKAYA Fırat Üniversitesi Veteriner Fakültesi Prof.Dr. Nazir DUMANLI Fırat Üniversitesi Veteriner FakültesiProf.Dr. Hüdaverdi ERER Selçuk Üniversitesi Veteriner FakültesiProf.Dr. Ayhan FİLAZİ Ankara Üniversitesi Veteriner FakültesiProf.Dr. Ekrem GÜREL Abant İzzet Baysal Üniversitesi Fen Edebiyat FakültesiProf.Dr. Tolga GÜVENÇ Ondokuz Mayıs Üniversitesi Veteriner FakültesiProf.Dr. Ali İŞMEN Çanakkale Onsekiz Mart Üniversitesi Su Ürünleri FakültesiProf.Dr. Hakkı İZGÜR Ankara Üniversitesi Veteriner FakültesiProf.Dr. Zafer KARAER Ankara Üniversitesi Veteriner FakültesiProf.Dr. Arif KURTDEDE Ankara Üniversitesi Veteriner FakültesiProf.Dr. Erdoğan KÜÇÜKÖNER Süleyman Demirel Üniversitesi Mühendislik Mimarlık FakültesiProf.Dr. Kamil ÖCAL Adnan Menderes Üniversitesi Veteriner FakültesiProf.Dr. Metin PETEK Uludağ Üniversitesi Veteriner FakültesiProf.Dr. Sevim ROLLAS Marmara Üniversitesi Eczacılık FakültesiProf.Dr. Berrin SALMANOĞLU Ankara Üniversitesi Veteriner FakültesiProf.Dr. Nesrin SULU Ankara Üniversitesi Veteriner FakültesiProf.Dr. Ayşe TOPAL Uludağ Üniversitesi Veteriner FakültesiProf.Dr. Ş. Doğan TUNCER Ankara Üniversitesi Veteriner FakültesiProf.Dr. Cevdet UĞUZ Afyon Kocatepe Üniversitesi Veteriner FakültesiProf.Dr. Cengiz YALÇIN Dicle Üniversitesi Veteriner FakültesiProf.Dr. Halis YERLİKAYA Fırat Üniversitesi Veteriner Fakültesi

BuSayınınHakemListesi(alfabetik sıra)TheRefereesListofThisIssue(in alphabetical order)

AKHAN Süleyman Akdeniz Üniversitesi Su Ürünleri Fakültesi Yetiştiricilik BölümüAKTAŞ Abit İstanbul Üniversitesi Veteriner Fakültesi Histoloji ve Embriyoloji Anabilim DalıAKYÜZ Bilal Erciyes Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıALTUNOK Vahdettin Selçuk Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıARSLAN Cavit Kafkas Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıATAKİŞİ Emine TAŞÇI Kafkas Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıATAKİŞİ Onur Kafkas Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıAVCI Mehmet Harran Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıAYAŞAN Tugay Doğu Akdeniz Tarımsal Araştırma Enstitüsü MüdürlüğüAYDENİZÖZ Meral Kırıkkale Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim DalıBAKIREL Tülay İstanbul Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim DalıBALCIOĞLU Murat Soner Akdeniz Üniversitesi Ziraat Fakültesi Zootekni BölümüBALTA Fikri Rize Üniversitesi Su Ürünler Fakültesi Su Ürünleri Yetiştiriciliği BölümüBAŞALAN Mehmet Kırıkkale Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıBAYDAN Emine Ankara Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim DalıBERİK Nermin Çanakkale Onsekiz Mart Üniversitesi Su Ürünleri Fakültesi Avlama ve İşleme Teknolojisi BölümüBİLDİK Ayşegül Adnan Menderes Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıBOLAT Duran Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıBOZKURT Hakan İstanbul Üniversitesi Veteriner Fakültesi Histoloji ve Embriyoloji Anabilim DalıCİRİT Ümüt İstanbul Üniversitesi Veteriner Fakültesi Dölerme ve Suni Tohumlama Anabilim DalıÇELEBİ Selahattin Atatürk Üniversitesi Tıp Fakültesi Mikrobiyoloji Anabilim DalıÇOBAN Özlem EMİR Fırat Üniversitesi Su Ürünleri Fakültesi Su Ürünleri İşleme TeknolojisiDEĞER Yeter Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıDİLER İbrahim Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Yetiştiricilik BölümüDOĞAN Abdullah Kafkas Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim DalıDURAK Yusuf Selçuk Üniversitesi Fen Fakültesi Biyoloji BölümüDÜZLER Ayhan Erciyes Üniversitesi Veteriner Fakültesi Anatomi Anabilim DalıERGÜN Emel Kırıkkale Üniversitesi Veteriner Fakültesi Histoloji ve Embriyoloji Anabilim DalıERGÜNAY Koray Hacettepe Üniversitesi Tıp Fakültesi Tıbbi Mikrobiyoloji Anabilim DalıERTEKİN Ali Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıERYAVUZ Abdullah Afyon Kocatepe Üniversitesi Veteriner Fakültesi Fizyoloji Anabilim DalıEYDURAN Ecevit Iğdır Üniversitesi Ziraat Fakültesi Zootekni BölümüGÜL SATAR Nihal Y. Uludağ Üniversitesi Veteriner Fakültesi Cerrahi Anabilim DalıGÜNER Yusuf Ege Üniversitesi Su Ürünleri Fakültesi Yetiştiricilik BölümüHOŞTÜRK Gülhan Turkay İstanbul Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıİCA Anıl Erciyes Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim DalıİNTAŞ Deniz Seyrek Uludağ Üniversitesi Veteriner Fakültesi Cerrahi Anabilim DalıİŞCAN Muhsin Kaan Erciyes Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıKARABULUT Huriye ARIMAN Rize Üniversitesi Su Ürünler Fakültesi Yetiştiricilik BölümüKARAPEHLİVAN Mahmut Kafkas Üniversitesi Veteriner Fakültesi Biyokimya Anabilim DalıKARATEPE Mustafa Niğde Üniversitesi Bor Meslek YüksekokuluKART Asım Kafkas Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim DalıKEÇECİ Tufan Selçuk Üniversitesi Veteriner Fakültesi Fizyoloji Anabilim DalıKILIÇ Engin Kafkas Üniversitesi Veteriner Fakültesi Cerrahi Anabilim DalıKIRMIZIGÜL Ali Haydar Kafkas Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim DalıKIRPIK Mehmet Ali Kafkas Üniversitesi Fen-Edebiyat Fakültesi Biyoloji BölümüKOCABAĞLI Neşe İstanbul Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıKOÇAK Ömür İstanbul Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıMADEN Mehmet Selçuk Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim DalıNAZLIGÜL Ahmet Adnan Menderes Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıNUR İsmail Hakkı Erciyes Üniversitesi Veteriner Fakültesi Anatomi Anabilim Dalı

ODABAŞIOĞLU Fuat Mustafa Kemal Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıOĞAN Mustafa Uludağ Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıORAL Hasan Kafkas Üniversitesi Veteriner Fakültesi Doğum ve Jinekoloji Anabilim DalıÖZEN Abdullah Fırat Üniversitesi Veteriner Fakültesi Veteriner Hekimliği Tarihi ve Deontoloji Anabilim DalıÖZKAN Muhip Ankara Üniversitesi Ziraat Fakültesi Zootekni BölümüÖZKUL Aykut Ankara Üniversitesi Veteriner Fakültesi Viroloji Anabilim DalıÖZSOY Bülent Mustafa Kemal Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıSAÇAKLI Pınar Ankara Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıSARI Ebru KARADAĞ Kafkas Üniversitesi Veteriner Fakültesi Histoloji ve Embriyoloji Anabilim DalıSARI Mehmet Kafkas Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıSERBEST Ayşe Uludağ Üniversitesi Veteriner Fakültesi Anatomi Anabilim DalıSOLMAZ Hasan Mustafa Kemal Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim DalıŞAHİN Tarkan Kafkas Üniversitesi Veteriner Fakültesi Hayvan Besleme ve Beslenme Hastalıkları Anabilim DalıŞAKİ Cem Ecmel Fırat Üniversitesi Veteriner Fakültesi Parazitoloji Anabilim DalıŞEKER İbrahim Fırat Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıŞİMŞEK Gülcihan Fırat Üniversitesi Veteriner Fakültesi Zootekni Anabilim DalıTEKİN Mehmet Emin Selçuk Üniversitesi Veteriner Fakültesi Zootekni ve Hayvan Besleme Anabilim DalıTÜRKER Hakan Abant İzzet Baysal Üniversitesi Fen Edebiyat Fakültesi Biyoloji BölümüÜNVER Ahmet Kafkas Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim DalıYARDIMCI Hakan Ankara Üniversitesi Veteriner Fakültesi Mikrobiyoloji Anabilim DalıYAŞAR Aşkın Selçuk Üniversitesi Veteriner Fakültesi Veteriner Hekimliği Tarihi ve Deontoloji Anabilim DalıYILDIRIM Murat İstanbul Üniversitesi Veteriner Fakültesi Farmakoloji ve Toksikoloji Anabilim DalıYILMAZ Muhittin Kafkas Üniversitesi Fen-Edebiyat Fakültesi Biyoloji Bölümü

BuSayınınHakemListesi(alfabetik sıra)TheRefereesListofThisIssue(in alphabetical order)

İÇİNDEKİLER(CONTENTS)

ARAŞTIRMA MAKALELERİ(ReseaRch aRticles) Sayfa

(Page)

Kars İlinde Bulunan Mandıraların Etkinliğinin Veri Zarflama Analizi İle ÖlçülmesiDEMIR P, DERBENTLI O, SAKARYA E ..........................................................................................................................................

Effects of Starter Culture Types and Different Temperatures Treatments on Physicochemical, Microbiological and Sensory Characteristics, and Fattty Acid Compositions of Çökelek Cheese Made from Goat Milk SIMSEK B, SAGDIC O .......................................................................................................................................................................

Tıbbi Bitki Özütlerinin Melek Balığı (Pterophyllum scalare) Yumurtasının Açılımı Üzerine EtkisiYILMAZ S, ERGUN S .........................................................................................................................................................................

Mitochondrial DNA D-loop and 12S Regions Analysis of the Long-Crowing Local Breed Denizli Fowl from Turkey KARAMAN M, KIRDAG N ...............................................................................................................................................................

Isolation of Flavobacterium psychrophilum Causing Rainbow Trout Fry Syndrome and Determination of an Effective Antibacterial Treatment in Rainbow Trout (Oncorhynchus mykiss) FryBOYACIOGLU M, AKAR F ...............................................................................................................................................................

Erzurum’da Tüketime Sunulan Kaşar Peynirlerinin Mineral Madde İçeriği ve Ağır Metal KontaminasyonuOZLU H, AYDEMIR ATASEVER M, URCAR S, ATASEVER M ................................................................................................

Karın Kaymağı Peynirinden İzole Edilen Laktobasillerin Tanımlanması TURGUT T, ERDOGAN A, ATASEVER M ....................................................................................................................................

Determination of Pre-parturition and Post-parturition Behaviors of Norduz GoatsYILMAZ A, KARACA S, KOR A, BINGOL M ..............................................................................................................................

Effects of Malathion in Fetal Kidney Tissues in Pregnant Rats: Teratogenic Effects Induced by Different Doses ALP H, SAK ME, EVSEN MS, FIRAT U, EVLIYAOGLU O, PENBEGUL N, SANCAKTUTAR AA, SOYLEMEZ H, TUZCU M .............................................................................................................................................................................................

Kars Yöresinde Atık Yapmış İnek Sürülerinden Alınan Süt ve Vajinal Sıvap Örneklerinden Brusella Etkenlerinin Bakteriyolojik ve Moleküler Tanımlanması ARSLAN T, ERDOGAN F, ERDOGAN M .....................................................................................................................................

eQTL Analysis and Association of MYF6 mRNA Expression with Meat Quality Traits in Pigs CINAR MU, FAN H, NEUHOFF C, GROßE-BRINKHAUS C ..................................................................................................

Investigation on Porcine Aromatase (CYP19) as a Specific Target Gene for Boar Testis CINAR MU, GUNAWAN A .............................................................................................................................................................

Selective Gray and White Matter Staining of the Horse Spinal CordBOLAT D, BAHAR S, SUR E, SELCUK ML, TIPIRDAMAZ S ...................................................................................................

Evaluation for Some Bacterial and Viral Abortions of Dairy Cattle Farms in Burdur District of TurkeyOZTURK D, KALE M, PEHLIVANOGLU F, HASIRCIOGLU S, TURUTOGLU H .................................................................

The Effect of Pepper Gas (OC) on Some Biochemical Parameters in Rats SEYHAN E, MERT N, MERT H .......................................................................................................................................................

Erzurum Yöresinde Satışa Sunulan Kırmızı Etlerde 17 β-östradiol, Dietilstilbestrol ve Zeranol Kalıntılarının Araştırılması SEVER E, OKUMUS B, INCE S .......................................................................................................................................................

Türkiye’de Farklı Bölgelerde Yetiştirilen Holştayn Sığırlarda Bazı Süt ve Döl Verimi Özellikleri GURSES M, BAYRAKTAR M ...........................................................................................................................................................

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Determination of the Metal Contents of Honey Samples from Orumieh in IranSAGHAEI S, EKICI H, DEMIRBAS M, YARSAN E, TUMER I ..................................................................................................

Production of Rhamnolipid (A Biosurfactant) Using Free and Immobilized Cells of Pseudomonas sp.SIDAL U, YILMAZ ES .......................................................................................................................................................................

Etlik Piliç Yemlerine Esansiyel Yağ Karışımı İlavesinin Büyüme Performansı, Karkas Randımanı ve Bazı İç Organ Ağırlıkları Üzerine EtkileriKUCUKYILMAZ K, CATLI AU, ÇINAR M .....................................................................................................................................

Alterations in the Hematological and Biochemical Parameters and Plasma Ion Concentrations of Common Carp, (Cyprinus carpio L., 1758) After Short Term Exposure to Sub-lethal Concentrations of LeadERGONUL MB, ATASAGUN S, KOCATURK K ...........................................................................................................................

Molecular Detection of Anisakis pegreffii in Horse Mackerels (Trachurus trachurus) Sold for Human Consumption in Erzurum Province of TurkeyUTUK AE, PISKIN FC, BALKAYA I ..................................................................................................................................................

Effects of Coenzyme Q10 on the Levels of some Trace Elements in Serum of Rats with Bleomycin Induced Pulmonary Fibrosis CEBI A, CELIKEZEN FC, ERTEKIN A .............................................................................................................................................

Effects of Sepiolite Usage in Broiler Diets on Performance, Carcass Traits and Some Blood ParametersESER H, YALCIN S, YALCIN S, ŞEHU A .......................................................................................................................................

Psychological Symptom Profile of Butchers Working in Slaughterhouse and Retail Meat Packing Business: A Comparative Study EMHAN A, YILDIZ AS, BEZ Y, KINGIR S .....................................................................................................................................

A Comparative Study on Some Quality Properties and Mineral Contents of Yoghurts Produced from Different Type of Milks ERKAYA T, SENGUL M ......................................................................................................................................................................

Potential Nutritive Value of Field Binweed (Convolvulus arvensis L) Hay Harvested at Three Different Maturity StagesCANBOLAT O .....................................................................................................................................................................................

Ophthalmoscopic, Ultrasonographic and Electrophysiologic Findings of Experimentally Induced Intraocular Pressure Increase in RabbitsERGIN I, BESALTI O .........................................................................................................................................................................

The Right Displacement of Abomasum with Ulceration in A CalfALTAN S, ALKAN F, KOC Y .............................................................................................................................................................

Partially Forelimb Amputation and Application of An Artificial Limb (Prosthetics) in A Free-Ranging Red Deer (Cervus elaphus)OLGUN ERDIKMEN D, OZSOY S, AYDIN D, HASIMBEGOVIC H, DONMEZ K .............................................................

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OLGU SUNUMU (CaSE REpORT)

Kafkas Univ Vet Fak Derg18 (2): 169-176, 2012DOI:10.9775/kvfd.2011.3635

SummaryBu çalışmada, Kars ilinde üretimde bulunan 20 adet mandıraya ait etkinlik değerleri veri zarflama analizi yöntemi ile ortaya konulmaya

çalışıldı. Analizler için üç girdi ve bir çıktı kullanılarak çıktı yönelimli model kuruldu. Hesaplamalar sonucunda Kars ilindeki mandıralara ait etkinlik değerleri saptandı. Analiz CCR metodu ve BCC metodu kullanılarak iki farklı şekilde çözüldü ve aralarında farklılık olup olmadığı karşılaştırıldı. CCR modelinin sonuçları uygulanabilirlik açısından uygun bulundu. Analiz sonucunda etkin olmayan işletmelerin etkin olması için tavsiye edilebilecek potansiyel iyileştirmeler elde edildi. İşletmeler yıllık süt işleme kapasiteleri dikkate alınarak ölçek büyüklüğüne göre dört eşit gruba ayrıldı. Gruplar etkinlik skorları bakımından kıyaslandı. Çalışma sonucunda BCC modeline göre yapılan hesaplamada birinci ve ikinci grupta ikişer adet, üçüncü ve dördüncü grupta birer adet olmak üzere toplamda 6 adet karar verme birimi etkin bulundu. CCR modeli üzerinden yapılan hesaplama her gruptan bir adet karar verme birimi etkin bulundu. Etkin olmayan karar verme birimleri için iyileştirmeler hesaplandı. BCC modeli sonuçlarının pratikte uygulanabilir olmadığı görüldü. CCR modeli sonuçlarına göre etkin olmayan işletmeleri etkinlik sınırına çekmek için gerekli olan iyileştirmeler ve ölçeğe göre getirilerin yönü hesaplandı.

Anahtar sözcükler: Etkinlik, Veri zarflama analizi, Mandıra, Kars, Kaşar peyniri

Measurement the Efficiency of Dairies in the Kars Province with Data Envelopment Analysis

SummaryIn this study, 20 dairies in Kars province, values of the efficiency put forward by Data Envelopment Analysis method was attempted.

Three inputs and one output used and output-oriented model is established. Efficiency scores are obtained by the dairies. Analysis is solved using the method in two different ways, CCR and BCC method. Differences between methods were compared. The results of the CCR model are found to be applicable. Potential improvements were obtained for inefficient enterprises. Considering an annual capacity of milk processing enterprises divided into four groups according to the size scale. Efficiency scores of groups were compared. In study 6 decision making unit found efficient, two in the first group, two in second group, one in the third and one in the fourth group. In the calculation one effective decision making unit found from each group. Improvements were calculated for the inactive decision making units. BCC model results were found not suitable in practice. Improvements were calculated to inefficient enterprises take on efficiency frontier and direction of returns to scale according to the results of the CCR model.

Keywords: Efficiency, Data envelopment analysis, Dairy, Kars province, Cheddar cheese

Kars İlinde Bulunan Mandıraların Etkinliğinin Veri Zarflama Analizi İle Ölçülmesi [1]

Pınar DEMİR * Özden DERBENTLİ ** Engin SAKARYA ***

[1]

*

*****

Bu çalışma “AB Uyum Sürecinde Türkiye Hayvancılık Kongresi 2011(Uluslararası Katılımlı), (20-22 Ekim 2011, Ankara - Türkiye)’de poster olarak sunulmuşturKafkas Üniversitesi Veteriner Fakültesi, Hayvancılık Ekonomisi ve İşletmeciliği Anabilim Dalı, TR-36100 Paşaçayırı, TR-36100 Kars - TÜRKİYEİlçe Gıda Tarım ve Hayvancılık Müdürlüğü, TR-17200 Biga, Çanakkale - TÜRKİYEAnkara Üniversitesi Veteriner Fakültesi, Hayvan Sağlığı Ekonomisi ve İşletmeciliği Anabilim Dalı, TR-06110 Dışkapı, Ankara - TÜRKİYE

Makale Kodu (Article Code): KVFD-2011-3635

Hayvancılığa dayalı sanayi, bugün gelişmiş ülkelerde ekonominin ayrılmaz bir parçası haline gelmiştir. Nitekim

Türkiye’de de hayvancılık sektöründe süt ve süt ürünleri sanayi oldukça önemli bir yere sahip olup gıda firmalarının

GİRİŞ

İletişim (Correspondence) +90 474 2426807/5039 [email protected]

RESEARCH ARTICLE

170Kars İlinde Bulunan ...

yaklaşık %16’sını süt ve süt ürünlerine ait sanayi işletmeleri oluşturmaktadır 1.

Kars ilinin, çayır-mera ve hayvan varlığı nedeniyle önem- li bir hayvancılık bölgesi olması, coğrafi şartlarının uygun-luğu, ekonomisinin büyük ölçüde hayvancılığa dayanması ve üretilen kaşar peynirinin Türkiye çapında tanınıyor ol- ması bölgede süt hayvancılığını önemli bir konuma getir- miştir. Kars ilinde bulunan süt sanayi işletmelerini; gele-neksel koşullarda üretim yapan ve genellikle mevsimlik çalışan küçük kapasiteli mandıralar ile modern teknoloji uygulayan büyük kapasiteli mandıra ve süt fabrikaları ola-rak iki grup altında değerlendirmek mümkündür 2. Ancak ilde kaşar peyniri üretiminin büyük bir bölümü, mandıra olarak adlandırılan ve mevsimlik olarak çalışan küçük işlet-melerde gerçekleştirilmektedir 3.

Süt çabuk bozulabilen bir ürün olması nedeniyle mo-dern ve teknoloji düzeyi yüksek tesislerde, kalite ve hijyen koşullarına uygun olarak üretiminin rantabl ve verimli şe- kilde değerlendirilmesi gerekmektedir. Bu nedenle yapılan bu çalışmada Kars ilinde üretimde bulunan 20 adet man-dıraya ait etkinlik değerleri ortaya konulmaya çalışılmıştır. Nitekim, Veri Zarflama Analizi (VZA) parametrik olmayan doğrusal programlama prensiplerine dayanan birimlerarası göreli etkinlik kıyaslaması yapan bir yöntemdir 4. VZA metodu başlangıçta bankacılık ve sigortacılık gibi sektörlere uyarlanmış sonrasında üniversiteler, oteller, telekomüni-kasyon, hastaneler, gıda, eğitim gibi çeşitli sektörlerde uy-gulanmaya başlanmıştır 4-12.

VZA ile tarım ve hayvancılık sektörüne yönelik çalışma- lar da yapılmıştır. Stokes et al.13 Pensilvanya’da süt sığırcı-lığı yapan 34 işletmenin etkinliğini veri zarflama analizi metoduyla incelemişlerdir. Bu çalışmada işletme arazi büyüklüğü, iş gücü ve inek sayısı olmak üzere 3 girdi, üre- tilen süt miktarı ve tereyağı miktarı olmak üzere 2 çıktı ele almışlardır. Theodoridis ve Psychoudakis 14, Yunanistan’da 165 süt sığırı işletmesini ölçek büyüklüklerine göre 4 gruba ayırmış, girdi olarak iş gücü, değişken maliyetler ve sabit maliyetler, çıktı olarak brüt geliri ele almışlardır. Galanopaolus et al.15 Yunanistan’da 100 domuz çiftliğiyle yaptıkları veri zarflama analizinde ölçeğe göre sabit ve değişken etkinlikleri hesaplanmışlardır. Lilienfeld ve Asmild 16

1992-1999 yılları arasında Batı Kansas eyaletinde sulu tarım yapan 43 işletmenin su kullanım etkinliklerini incelemiş-lerdir. Girdi olarak birim başına kullanılan su miktarı, iş gücü, sermaye, tohum, gübre, ilaç, toprağın su tutma kapasitesi alınmıştır. Çıktı olarak üretilen buğday, mısır, sorgum, soya fasulyesi, alfa alfa otu ve silajlık ürünler kabul edilmiştir. Kullanılan sulama tekniği ve teknolojinin su kullanım etkin- liğinde fark oluşturduğu, su kullanımı tasarruflu olan işlet- melerin etkinlik düzeylerinin kullanmayanlara oranla yük-sek olduğu sonucuna varmışlardır.

Özden ve Armağan 17, Aydın ilindeki bitkisel üretim ya-pan işletmelerde veri zarflama analizi metodu ile verimlilik düzeylerini incelemişlerdir. Analize alınan 84 işletmeden

4’ünün etkin diğerlerinin etkinsiz çalıştığını, girdi kullanı-mında azaltmaya gidilerek aynı üretim değerinin elde edi- lebileceği sonucuna varmışlardır. Candemir ve ark.18 İzmir, Manisa, Aydın, Denizli, Bursa illerinde faaliyet gösteren 212 adet tarım kredi kooperatifinin 2001 ve 2008 yılları arasın-daki teknik etkinliklerini ölçeğe göre sabit getiri varsayımı altında veri zarflama analizi yöntemiyle hesaplamışlardır. Bayramoğlu ve ark.19 Tekirdağ ilinde sözleşmeli kanola yetiştiriciliği yapan 130 işletmeye ait ekonomik etkinlik, kaynak kullanım etkinliği, teknik etkinlik, ölçek etkinliği ve saf teknik etkinliği hesaplanmışlardır. Sonuç olarak işletme- lerin yaklaşık %40’ının ekonomik olarak etkinsiz olduğu ve bu işletmelerin maliyetlerinin yüksek olduğu sonucuna varmışlardır. Koyubenbe ve Candemir 20 Küçük Menderes Havzasında Ödemiş, Tire, Bayındır ve Torbalı ilçelerindeki 80 adet süt sığırcılığı işletmesinin ölçeğe göre sabit getiri varsayımı altında etkinliklerini veri zarflama analizi yöntemiyle hem ilçelere göre hem de grup halinde ince-lemiş, sonuçta kaynak kullanımında israfın olduğunu ve optimum ölçekte üretim yapılmadığını tespit etmişlerdir.

Bu çalışma da ise Türkiye’de yöresel özelliği itibariyle tanınan Kars kaşarı peynirinin üretildiği mandıralarda veri zarflama analizi yöntemi ile saf teknik etkinlikleri ve teknik etkinlikleri bulunarak, ölçek etkinlikleri ile ölçeğe göre getirilerinin yönü hesaplanmış, etkin olmayan işletmeler için girdi kullanımında ve çıktı miktarlarındaki iyileştirmeler incelenmiştir.

MATERYAL ve METOT Kars Gıda Tarım ve Hayvancılık Müdürlüğü’nden alınan

verilere göre 2008 yılı itibariyle ilçeler hariç Kars-Merkez köylerinde süt ve süt ürünleri imal eden ve Gıda Tarım ve Hayvancılık İl Müdürlüğü’nden üretim izni almış faal 50 adet süt sanayi işletmesi bulunmakta, bunların 45 adedi süt teşvik primi kapsamına alınmıştır.

Bu çalışmanın materyalini Kars ilinde faaliyette bulunan 20 adet mandıradan elde edilen veriler oluşturmuştur 2. Çalışmada, veri zarflama analizi metodu kullanılarak işlet-melerin verimlilikleri incelenmiş, etkin ve etkin olmayan işletmeler bulunmuştur.

Veri Zarflama Analizi

Veri Zarflama Analizi (VZA) Charnes, Cooper ve Rhodes isimli araştırmacılar tarafından literatüre CCR adıyla geçen (DEA) karar verme birimlerinin etkinliğinin ölçüldüğü non-parametrik bir metottur 21. Orijinal CCR modeli ölçeğe göre sabit getiri varsayımıyla karakterize teknik etkinliğin ölçüldüğü modeldir. Banker, Charnes ve Cooper modeli (BCC) ise ölçeğe göre değişen getiri varsayımına göre geliştirmişlerdir 22.

VZA analize alınan işletmeler arasındaki göreceli verim-liliği ölçen doğrusal programlama yaklaşımıdır. Analiz çok sayıda girdi ve çok sayıda çıktının söz konusu olduğu

171

DEMİR, DERBENTLİSAKARYA

sistemlerde rahatlıkla uygulanabilmektedir. Ancak aynı girdi ve çıktıya sahip işletmelerin karşılaştırmalı ölçümü yapılabilmektedir. Her karar verme birimi (Decision Making Unit = DMU) için model çözülür. Amaç fonksiyonu girdinin minimizasyonu veya çıktının maksimizasyonu şeklinde se- çilir ve model belirlenen amaç fonksiyon üzerinden kurulur. Çözüm sonucunda amaç fonksiyonu 1.00’e eşit olan karar verme birimleri ‘etkin’, 1.00’ e eşit olmayanlar ise etkin olmayan karar verme birimi olarak adlandırılır 21.

Model çözümü sonucunda etkin olmayan karar verme birimleri için referans olan KVB’leri belirlenir ve her KVB’ne ait girdi ve çıktı ağırlık değerleri elde edilir. Elde edilen ağırlık değerleri iyileştirme formülasyonuna yerleştirilerek KVB için hedeflenen girdi ve çıktı miktarları bulunur. Böylece KVB’lerin daha etkin çalışması için önerilebilecek girdi ve çıktı miktarları bulunur 23.

Analizin özelliği doğrusal programlama tekniklerini kullanarak, “en iyi uygulama” gözlemine dayanan bir etkin üretim sınırı oluşturulmasıdır. Etkinlik sınırı etkin karar verme birimlerini kapsayan (zarflayan) bir düzlem oluştur-makta, etkin birimler sınır üzerinde, etkin olmayanlar sını- rın altında yer almaktadır. Böylece hem karar verme birimlerinin etkinliği hesaplanmakta hem de aralarında en iyi performansı gösterenler referans gösterilerek sınır altında kalanların etkinliğe ulaştırılması için yapabilecek iyileştir-meler belirlenebilmektedir 24.

Teknik etkinlik aynı şartlar altında, belli miktardaki gir- diden en yüksek düzeyde çıktı üretilmesi veya aynı çıktının daha az girdi ile elde edilmesi olarak tanımlanabilir 11. BCC yöntemi saf teknik etkinliğin dikkate alındığı bir ölçüm metodudur. CCR yöntemi ise ölçek etkinliğini de dikkate alarak etkinliği ölçmektedir. Bu nedenle BCC analizi ile elde edilen sonuçlar CCR yöntemi ile elde edilen sonuçlardan farklı olabilmektedir. BCC etkinlik sınırı CCR sınırının her zaman altında yer almaktadır bu yüzden etkinlik skoru CCR skorundan büyük ya da ona eşittir. BCC modeli sadece teknik etkinliği ölçmektedir. E etkinliği göstermek üzere ECCR = EÖlçek * EBCC şeklinde yazılabilmektedir 5.

Dual çözümü sonucunda elde edilen -λjk değeri sıfırdan büyük olan tüm karar verme birimleri, etkin olmayan bir karar verme birimi için referans kümesi meydana getirmektedir. Etkin olmak isteyen bir KVB girdi ve çıktı değerlerini referans kümedeki rol model ile eşitlerse etkin duruma geçer. Şu şekilde formülize edilerek iyileştirmeler hesap-lanmaktadır.

Bir KVB referansa göre iyileştirildiğinde iyileştirme önce-sindeki haline kıyasla girdi faktörlerini daha az kullanırken

çıktı faktörlerini en az kendisi kadar üretir 25.

Etkinlik ölçümünde girdilerin ağırlıklı ortalamasının çıktıların ağırlıklı ortalamasına oranı 1’den küçük veya 1’eşit kabul edilmektedir. Böylece karar verme biriminin etkinliği maksimize edilmeye çalışılır. Yukarıda gösterilen kesirli modelin doğrusal hale çevrilmesiyle elde edilen modelin kısıtları şu şekildedir. N tane karar verme birimi için yapılan analizde ‘s’ adet çıktı, ‘m’ adet girdi kullanılmaktadır. ‘x’ girdiyi, ‘y’ çıktıyı göstermektedir 23.

xij = Etkinliği ölçülen j karar birimine ait i. girdi miktarıyrj = Etkinliği ölçülen j karar birimine ait r. çıktı miktarıur = Karar birimi tarafından r. çıktıya verilen ağırlıkvi = Karar birimi tarafından i. girdiye verilen ağırlık

Karar verme birimine ait lamda değerleri toplamı 1’e eşitse ölçeğe göre sabit, 1’den küçükse ölçeğe göre artan, 1’den büyükse ölçeğe göre azalan getiri durumunu gös-termektedir 26.

CCR modeli bir kısıtın eklenmesiyle BCC modeline dö-nüştürülmekte böylece ölçeğe göre değişkenlik varsayımı sağlanmaktadır. Yukarıdaki model primal modeldir. Çıktıya göre etkinlik ölçülmek istendiğinde primal model dual modele çevrilir. Lamdalar toplamının 1’e eşit olduğu kısıtı modele eklenerek BCC etkinliği hesaplanır 22.

Analizde Kullanılan Girdiler ve Çıktı

Çalışmada girdi olarak; mandıraların üreticilerden aldığı çiğ süt miktarı (kg) (Girdi 1), işçi sayısı (adet) (Girdi 2) ve sütün taşınmasında ortaya çıkan nakliye gideri (TL) (Girdi 3) baz alınmıştır. Çıktı ise elde edilen kaşar peynirinin satı-şından elde edilen gelir olarak belirlenmiştir.

Süt kalitesindeki değişim üretilen çıktıyı direkt etkile-mekte, ürün miktarını değiştirmektedir. Kaliteli girdi; işlet-

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me maliyetlerini artırmakta ancak çıktı miktarının artmasını sağlamaktadır. Girdi kalitesindeki olumsuzluk ise işletme maliyetlerini düşürmekte ancak çıktı miktarında azalmaya sebep olmaktadır. Bu nedenle analiz için girdi miktarının kg cinsinden alınması uygun görülmüştür.

İşçi sayısı (Girdi 2) işletmede çalışan işçi sayısını ifade etmektedir. Üretimde bulunan yabancı ve aile işgücü esas alınmıştır. Yabancı işgücüne yapılan aylık ödemeler işletme sahibinin beyanı üzerinden değerlendirilmiştir. Aile işgücü ise yetişkin işgücü birimine çevrildikten sonra asgari ücret üzerinden değerlendirilmiştir.

Nakliye giderleri (Girdi 3) işletme sahibinin beyanı esas alınarak birim süt toplama ve pazarlama-nakliye giderle-rinin toplamı dikkate alınmıştır.

Üretilen kaşardan elde edilen gelir (Çıktı) ise kaşar mik-tarı ile o yıla ait 1 kg kaşarın ortalama fiyatın çarpımından elde edilmiştir. Yapılan çalışmada bölgedeki 1 kg kaşarın ortalama fiyatının 6.5 kg/TL olduğu tespit edilmiştir.

BULGULAR

Elde edilen veriler doğrultusunda 20 mandıraya ait 3 girdi ve 1 çıktıdan oluşan veri seti Tablo 1’de gösterilmiştir.

Analiz için Xpress-Mp Lineer Programming yazılımı kul-lanılmış olup veri seti üzerinde BBC yöntemi kullanılarak veri zarflama analizi uygulanmış, karar verme birimlerine (KVB) ait etkinlik değerleri Tablo 2’de gösterilmiştir.

Tablo incelendiğinde 3, 4, 6, 10, 14, ve 16. KVB’ler olmak üzere toplam 6 KVB teknik etkin olduğu görülmektedir. Etkin olmayan KVB’lerin etkinlik değerleri 0.94 ile 0.78 ara-sında değişmektedir. Etkin olmayan KVB’ler için rol model üzerinden iyileştirmeler hesaplanmıştır (Tablo 2). Sonuca göre KVB’lerin etkin olmak için ölçeklerini artırmak zorunda kaldıkları görülmektedir. Sonuçların gerçeğe uyarlanabilir olması için CCR yöntemi ile analiz tekrarlanmış, sonuçları Tablo 3’te gösterilmiştir.

CCR yöntemi sonucunda 20 işletmeden 4’ü (3, 6, 14 ve 16. KVB’leri) teknik etkin bulunmuştur. Etkin olmayan KVB’- lerin etkinlik skorları 0.96 ile 0.77 arasında değişmektedir.

CCR yöntemi sonuçlarına etkin olmayan karar verme birimleri için girdi miktarlarında yapılması tavsiye edilen iyileştirmeler, girdi miktarlarındaki azalış yüzdeleri Tablo 4’te verilmiştir.

Tablo 4 incelendiğinde etkin olmayan KVB’ler etkin hale geçebilmek için girdi miktarlarında azaltmaya gidildiği görülmektedir. Örneğin 1. KVB işçi sayısında değişiklik yap-madan süt miktarını ve nakliye giderini %8.11 azaltabilirse etkin hale geçmektedir.

Dördüncü ve 10. KVB’lere ait etkinlik değeri BCC yön-temine göre analiz yapıldığında 1.00 değerini verirken CCR yöntemine göre analiz yapıldığında sırasıyla 0.96 ve 0.89 bulunmaktadır. Metot çeşidine göre sonuçlar değiş- mektedir. 13. KVB etkinlik sınırına yaklaşırken süt miktarını %9.53, işçi sayısını %7.14, nakliye giderini %9.49 azalt-maktadır. Etkin olan KVB’leri için girdi ve çıktı düzeyinde değişiklik yapılmasına gerek olmamaktadır.

BBC ve CCR yöntemi sonucuna göre işletmelerin et-kinlik değerleri, referans seti ve ölçek etkinliklerine ilişkin elde edilen değerler Tablo 5’te verilmiştir.

Tabloda her KVB’ye denk gelen referans seti o işletmenin etkin çalışması için taklit edebileceği işletme numaralarını göstermektedir. Örneğin ilk sütunda görülen 1. KVB etkin olmak için isterse BCC referans seti olarak gösterilen 3. sütun hizasında yer alan 3, 4, 6 ya da 16. karar verme birimini taklit etmektedir. 3 numaralı KVB zaten etkin durumdadır, bu yüzden kendini taklit eder, dolayısıyla referans setinde yalnızca kendisi yer alır. Bütün etkin karar verme birimleri referans setinde yalnızca kendisini gösterir.

Tablo 5’te CCR etkinlik değeri için de BCC yöntemine benzer şekilde referans setleri oluşturulmuştur, etkin olan KVB’ler için yine kendiler referans setinde yer almakta, etkin olmayanlar için taklit edilecek KVB’ler refere edilmiştir.

Tablo 1. Veri zarflama analizinde kullanılan veri seti

Table 1. Data used in data envelopment analysis

KVB SütMiktarı (kg)

İşçi Sayısı (Adet)

Nakliye Gideri (TL)

Kaşar Peyniri Geliri (TL) KVB Süt

Miktarı (kg)İşçi

Sayısı (Adet)Nakliye

Gideri (TL)Kaşar Peyniri

Geliri (TL)

1 450.000 4 10.000 280.000 11 1.800.000 10 50.000 975.000

2 450.000 9 15.000 300.000 12 320.000 4 10.000 182.000

3 450.000 2 13.000 287.000 13 1.800.000 15 45.000 1.092.000

4 165.000 2 4.000 112.500 14 3.000.000 11 25.000 1.820.000

5 680.000 5 16.000 420.000 15 1.200.000 8 20.000 620.000

6 1.500.000 9 20.000 975.000 16 300.000 5 5.000 224.000

7 500.000 5 10.000 322.500 17 1.200.000 8 20.000 632.400

8 400.000 5 15.000 227.500 18 1.150.000 10 30.000 600.000

9 1.300.000 7 22.000 770.000 19 1.800.000 10 50.000 1.014.000

10 300.000 2 6.000 175.500 20 1.400.000 13 30.000 780.000

173


Tablo 2. Veri zarflama analizi BCC yöntemi sonuçları, etkin olan ve etkin olmayan karar verme birimleri, iyileştirilmiş girdi ve çıktı miktarları

Table 2. Result of BCC metod, efficient and inefficient decision making units, improved inputs and output

KVB SafTeknik Etkinlik

İyileştirilmiş SütMiktarı

İyileştirilmiş İşçiSayısı

İyileştirilmiş Nakliye Gideri

Mevcut Kaşar Geliri

İyileştirilmiş KaşarGeliri

1 0.91965235 2.415.000 18 42.000 280.000 1.598.500

2 0.93652907 1.800.000 14 25.000 300.000 1.199.000

3 1 450.000 2 13.000 287.000 287.000

4 1 165.000 2 4.000 112.500 112.500

5 0.93185609 2.250.000 16 38.000 420.000 1.486.000

6 1 1.500.000 9 20.000 975.000 975.000

7 0.93928013 2.250.000 16 38.000 322.500 1.486.000

8 0.80296407 915.000 9 22.000 227.500 623.500

9 0.92410995 4.950.000 22 58.000 770.000 3.082.000

10 1 300.000 2 6.000 175.500 175.500

11 0.84336692 4.950.000 22 58.000 975.000 3.082.000

12 0.80958084 915.000 9 22.000 182.000 623.500

13 0.94871795 4.500.000 20 45.000 1.092.000 2.795.000

14 1 3.000.000 11 25.000 1.820.000 1.820.000

15 0.78729106 2.250.000 16 38.000 620.000 1.486.000

16 1 300.000 5 5.000 224.000 224.000

17 0.80306141 2.250.000 16 38.000 632.400 1.486.000

18 0.78330342 1.800.000 14 25.000 600.000 1.199.000

19 0.87742912 4.950.000 22 58.000 1.014.000 3.082.000

20 0.84886818 1.800.000 14 25.000 780.000 1.199.000

Tablo 3. Veri zarflama analizi CCR yöntemi sonuçları, etkin olan ve etkin olmayan karar verme birimleri, iyileştirilmiş girdi ve çıktı miktarları

Table 3. Result of CCR metod, efficient and inefficient decision making units, improved inputs and output

KVB Teknik Etkinlik

İyileştirilmiş SütMiktarı

İyileştirilmiş İşçiSayısı

İyileştirilmiş Nakliye Gideri Mevcut Kaşar Geliri İyileştirilmiş Kaşar

Geliri

1 0.918933 413.520 4 9.189 280.000 280.000

2 0.892857 401.787 7 6.696 300.000 300.001

3 1 450.000 2 13.000 287.000 287.000

4 0.965515 159.310 2 3.379 112.500 112.500

5 0.931028 633.099 5 14.896 420.000 420.000

6 1 1.500.000 9 20.000 975.000 975.000

7 0.939024 469.512 5 9.390 322.500 322.500

8 0.801570 320.628 4 6.680 227.500 227.500

9 0.921303 1.197.692 6 20.269 770.000 769.999

10 0.890687 267.206 2 5.344 175.500 175.500

11 0.836323 1.505.379 8 18.000 975.000 974.999

12 0.801570 256.502 3 5.344 182.000 182.000

13 0.902208 1.628.477 14 40.729 1.092.000 1.094.871

14 1 3.000.000 11 25.000 1.820.000 1.820.000

15 0.787113 944.536 6 15.742 620.000 620.000

16 1 300.000 5 5.000 224.000 224.000

17 0.802856 982.196 7 16.599 632.400 632.400

18 0.772201 888.030 8 21.879 600000 599.999

19 0.869775 1.565.596 9 43.489 1.014.000 1.014.000

20 0.818660 114.6042 11 24.558 780.000 780.000


Tablo incelendiğinde 4. KVB BCC yöntemine göre model kurulduğunda 1, 8 ve 12. KVB’lerine referans olurken CCR yöntemine göre model kurulduğunda hiçbir karar verme birimine referans olmadığı görülmektedir. Yine benzer şekilde 10. KVB BCC yöntemine göre etkin kabul edilerek sadece kendine referans teşkil etmekte, CCR yöntemine göre etkin kabul edilmemektedir.

6. KVB BCC modeline göre 13 defa referans olmaktadır. CCR modeline göre çözüm yapıldığında 16 kez referans olan 16. KVB ön plana çıkmaktadır. Tablo 5’te modele göre

referans olma sıklığının değiştiği görülmektedir. Teknik et-kinlik (CCR) değerinin saf teknik etkinlik (BCC) değerine oranı bize ölçek etkinliğini vermektedir. Yapılan çalışmada 3, 6, 14 ve 16 numaralı KVB’leri ölçeğe göre sabit getiri altında çalıştıkları tespit edilmiştir.

Tablo 5’te ölçeğe göre getirini yönü de gösterilmekte-dir. CCR model çözümü sonucunda elde edilen lamda de- ğerleri toplamı 1.00’den büyük olan KVB’ler ölçeğe göre artan, 1.00’e eşit olan yani etkin olan KVB’ler ölçeğe göre sabit, 1.00’den düşük olan KVB’ler ölçeğe göre artan

Tablo 4. CCR yöntemi sonuçlarına göre etkin olmayan karar verme birimleri için girdi miktarlarında yapılması tavsiye edilen iyileştirmeler, girdi miktarlarındaki azalış (%)

Table 4. Improvements for inefficient decision making units by result of CCR method, decreasing in input (%)

KVB Süt Miktarındaki Değişim

İşçi Sayısındaki Değişim

Nakliye Giderindeki Değişim KVB Süt Miktarındaki

Değişim İşçi Sayısındaki

DeğişimNakliye Giderindeki

Değişim

1 8.11 0.00 8.11 11 16.37 25.00 64.00

2 10.71 22.22 55.36 12 19.84 33.33 46.56

3 0.00 0.00 0.00 13 9.53 7.14 9.49

4 3.45 0.00 15.53 14 0.00 0.00 0.00

5 6.90 0.00 6.90 15 21.29 33.33 21.29

6 0.00 0.00 0.00 16 0.00 0.00 0.00

7 6.10 0.00 6.10 17 18.15 14.29 17.01

8 19.84 25.00 55.47 18 22.78 25.00 27.07

9 7.87 16.67 7.87 19 13.02 11.11 13.02

10 10.93 0.00 10.93 20 18.14 18.18 18.14

Tablo 5. Etkinlik değerleri, referans seti ve ölçek etkinliği

Table 5. Efficieny scores, referans set and scale efficieny

KVB BCC Etkinlik Değeri BCC Refr. Seti CCR Etkinlik Değeri CCR Refr. Seti Ölçeğe Göre Getr. Yönü Ölçek Etkinliği

1 0.91965235 3, 4, 6, 16 0.918933 3, 6, 16 Azalan 0.999217802

2 0.93652907 6,16 0.892857 16 Azalan 0.953368164

3 1 3 1 3 Sabit 1

4 1 4 0.965515 16 Artan 0.965515000

5 0.93185609 3, 6, 16 0.931028 3, 6, 16 Azalan 0.999111354

6 1 6 1 6 Sabit 1

7 0.93928013 3, 6, 16 0.939024 3, 6, 16 Azalan 0.999727312

8 0.80296407 3, 4, 16 0.801570 3, 16 Artan 0.998263845

9 0.92410995 3, 6, 14 0.921303 3, 6, 14 Azalan 0.996962537

10 1 10 0.890687 3, 6, 16 Artan 0.890687000

11 0.84336692 3, 6, 14 0.836323 3, 16 Azalan 0.991647858

12 0.80958084 3, 4, 16 0.801570 3, 16 Artan 0.990104954

13 0.94871795 6, 14 0.902208 3, 16 Azalan 0.950975999

14 1 14 1 14 Sabit 1

15 0.78729106 3, 6, 16 0.787113 3, 6, 16 Azalan 0.999773832

16 1 16 1 16 Sabit 1

17 0.80306141 3, 6, 16 0.802856 3, 6, 16 Azalan 0.999744216

18 0.78330342 6, 16 0.772201 3, 16 Azalan 0.985826157

19 0.87742912 3, 6, 14 0.869775 3, 16 Azalan 0.991276651

20 0.84886818 6, 16 0.818660 3, 6, 16 Azalan 0.964413815

175


getiri durumunu göstermektedir. Örneğin KVB 4 işletme ölçeğini artırabilirse CCR etkinlik sınır çizgisi üzerinde yer alabilmektedir. Aynı şekilde KVB 8, KVB 10 ve KVB 12 öl-çeğe göre artan getiri durumundadır. KVB 1, KVB 2, KVB 5, KVB 7, KVB 9, KVB 11, KVB 13, KVB 15, KVB 17, KVB 18, KVB 19 ve KVB 20 ölçeğe göre azalan getiri durumunu ser- gilemektedir, işletmeler ölçeklerini azaltarak etkinlik sını-rına çekilirler. Bahsedilen ölçeğe göre getiri durumu CCR model sonuçlarından yola çıkarak açıklanmıştır.

Mandıraların günlük işledikleri süt miktarı dikkate alı- narak ölçek büyüklüğüne göre dört sınıfa ayrılmıştır (Tablo 6). Yıllık süt işleme kapasitesi 0-19 ton arası birinci grup, 20-25 ton arası ikinci grup, 26-49 ton arası üçüncü grup, 50 ve üstü ton ise dördüncü grubu oluşturmaktadır. Her grupta 5 işletme yer almıştır. Mandıraların günlük süt iş-leme kapasitesine göre BBC ve CCR etkinlikleri Tablo 6’da verilmiştir.

BCC modeline göre birinci grupta 2, ikinci grupta 2, üçüncü grupta 1, dördüncü grupta 1 işletme etkin bulun-muşken CCR modeline göre her grupta birer adet işletme etkin bulunmuştur. Etkinlik gösteren KVB’lerin her işletme grubunda da yer almaktadır. Belli bir grup için yapılan ve göreli etkinliğin ölçüldüğü bu çalışma, işletme ölçeği küçük olsa bile işletmelerin etkinliği yakalayabildiğini, etkin olma-yan KVB’ler için referans olabileceğini göstermektedir. Ör-neğin 3. KVB 10 ton/yıl süt işleme kapasitesine sahiptir ve CCR (1.00), BCC (1.00) ve ölçek (1.00) etkin bulunmuştur. Oysa 16. KVB 3. KVB’ye göre daha yüksek kapasiteye (50 ton) sahip olmasına rağmen CCR, BCC ve ölçek etkindir.

Bu bağlamda yapılan bu çalışmada işletmelerin gün-lük süt işleme kapasitesi bakımından ölçek değişikliğinin etkinlik üzerinde belirleyici olmayabileceği söylenebilir. Çünkü KVB 16 CCR modeline göre 11, BCC modeline göre 16 kez referans olmaktadır. KVB 16’nın referans sıklığının bu kadar fazla olmasına rağmen onunla aynı grupta yer alan 17, 18, 19 ve 20. KVB’ler etkinlik sınırından uzaklaş-maktadırlar. KVB 18 için etkinlik değeri 0.77’ye kadar düş-mektedir.

TARTIŞMA ve SONUÇ

Kars ili süt hayvancılığı ve süt sanayinin önemli so-runları bulunmaktadır 2. Üretiminin yapıldığı süt sığırcılık işletmeleri genellikle dağınık yapıda, ölçekleri düşük ve üre- timde geleneksel yapıya sahiptirler. Diğer taraftan bölgede süt üretiminin bütün yıla yayılmaması nedeniyle verimliliği düşük olan sütün hijyen ve kalitesinde yaşanan sorunlar önemini korumaktadır. İşletmelerdeki bu irrasyonel yapı, üretim sanayi entegresyonunun gelişimini de olumsuz etki- lemektedir. Ayrıca süt sanayi işletmelerinin kaliteli ham- madde temininde yaşanan sorunlar, süt sanayi işletmele-rinin kapasite kullanım oranının düşük olması ve maliyet-lerin yüksek olması bu işletmelerin karlı ve verimli çalış-malarına olanak vermemektedir 27.

Veri zarflama analizinin en önemli avantajı belli bir modele bağlı kalmadan etkin birimleri göstererek iyileş-tirmeye olanak sağlamasıdır. Dezavantajı ise girdi ve çık-tılardaki en ufak değişikliğin sonuçları değiştirmesidir. Bu nedenle analiz sonuçlarının gerçeği yansıtması için işletme bilgileri mümkün olduğunca sağlıklı alınmalı, girdilerin, çıktıların ve karar verme birimlerinin seçimine dikkat edil-mesi gerekmektedir.

Bu makalede temel olarak BCC ve CCR yöntemlerinden bahsedilmiştir, iki yönteme göre çözüm yapılmış sonuç- lar kıyaslanmıştır. BCC yöntemi sonuçlarına göre işletme-lere iyileştirme oranları verildiğinde bunun pratikte uygu-lanabilir olmadığı görülmüştür. Çünkü etkin olmayan işlet-meler etkinliği yakalamak için girdi ve çıktı miktarlarını büyük oranda değiştirmek zorunda kalmaktadırlar. CCR yöntemi sonuçlarına göre elde edilen iyileştirmeler diğer yönteme göre pratiğe daha uygulanabilir olduğu görün-mektedir.

Bu çalışma ile kaşar peyniri üreten çeşitli ölçek büyük- lüğüne sahip mandıraların veri zarflama yöntemi ile etkinlikleri tespit edilmeye çalışılmıştır. Veri zarflama ana-lizinde mandıralardan elde edilen veriler doğrultusunda

Tablo 6. Süt işleme kapasitesine (ton/gün) göre işletmelerin mandıraların BBC ve CCR etkinlikleri

Table 6. BBC and CCR effficieny of dairies according to milk processing capacity (tonnes/day)

KVB Süt İşleme Kapasitesi

ÖlçekGrubu

BCC Etkinliği

CCR Etkinliği KVB Süt İşleme

KapasitesiÖlçek Grubu

BCC Etkinliği

CCR Etkinliği

1 5 1 0.9196 0.918933 11 30 3 0.8434 0.836323

2 8 1 0.9365 0.892857 12 30 3 0.8096 0.801570

3 10 1 1 1 13 30 3 0.9487 0.902208

4 10 1 1 0.965515 14 40 3 1 1

5 15 1 0.9318 0.931028 15 42 3 0.7873 0.787113

6 20 2 1 1 16 50 4 1 1

7 20 2 0.9393 0.939024 17 60 4 0.8031 0.802856

8 20 2 0.8030 0.801570 18 60 4 0.7833 0.772201

9 25 2 0.9241 0.921303 19 80 4 0.8774 0.869775

10 25 2 1 0.890687 20 100 4 0.8489 0.818660


üreticilerden aldığı çiğ süt miktarı, işçi sayısı ve sütün taşınmasında ortaya çıkan nakliye giderleri girdi bazında, üretilen kaşar peynirinden elde edilen gelir ise çıktı bazında dikkate alınmıştır. Bu kapsamda Demir’in 2 çalışmasın- daki süt sanayi işletmelerine ait en önemli maliyet ka-lemlerinin sırasıyla çiğ süt alım gideri (%71-72), nakliye gideri (%5.9) ve işçilik giderinin (%5.1) olması girdi seçi-minin belirlenmesinde göz önünde bulundurulmuştur.

Galanopaolus et al.15, Yunanistan’da çiftlikler üzerinde veri zarflama analizi yöntemini uygulamış, çıktı olarak hayvan başına düşen işletme brüt karı, girdi olarak ise hayvan başına düşen iş gücü, sermaye, yem ve diğer gider- ler kullanılmıştır. 20-199 arası domuza sahip işletmeleri küçük, 200-399 arasını orta ve 400 ve üstü olanları büyük işletme grubu olarak alınmıştır. Büyük ölçekli işletmelerin küçük ölçekli işletmelerden daha etkin çalıştıkları sonucuna varılmıştır.

Yapılan bu çalışmada ise ölçek büyüklüğüne göre dört grup oluşturulmuş, her grupta en az bir etkin işletme yer almıştır. Elde edilen veriler doğrultusunda sadece işletme ölçeklerinin etkinlik üzerinde belirleyici etkisinin olma-yabileceği, işletmelerin girdi kullanımında yapabilecekleri değişiklikler sayesinde teknik etkinliği yakalayabilecekleri söylenebilir. Nitekim Tablo 4 incelendiğinde etkin olmayan bazı mandıraların etkin hale geçebilmek için üreticilerden aldıkları süt miktarlarında da azaltmaya gitmeleri gerektiği görülmektedir. Bu durum, mandıralarda işlenen süt mik-tarı kadar alınan sütün kalitesinin de ne kadar önemli oldu-ğunu göstermektedir.

Analize konu olan 20 işletmeye ait etkinlik düzeyleri ve varsayılan iyileştirmelerin bilimsel dayanağı VZA ile elde edilen sonuçlardır. İşletmelerin karlı ve verimli çalışabil-mesi kaynakların etkin kullanımıyla mümkündür. İşlet-melerdeki girdilerin azaltılması ve üretilen kaşar miktarının artırılmasıyla etkinliğin sağlanacağı göz önünde bulun-durulmalı, kaynak kullanımındaki etkinliğin işletme geliri ve karlılığı açısından önemli olduğu unutulmamalıdır.

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SummaryÇökelek cheese produced with heated, filtrated and salted yogurt is a traditional cheese belong to Turkey. In this study, Çökelek

cheeses were produced from the coagulums prepared by using cultures including Lactobacillus helveticus, yogurt culture or the mixture of Lb. helveticus and yogurt cultures (1:1). The Çökelek cheeses produced with the cultures were prepared after heating of the coagulums at two different temperatures, 85ºC or 95ºC for 30 min. The produced Çökelek cheeses were stored at 4oC for 45 days. Therefore, in the Çökelek cheese production, using of Lb. helveticus (as a single or mix with yogurt starter cultures) as starter culture and heating at 85ºC may be proposed.

Keywords: Çökelek cheese, Lactobacillus helveticus, Yogurt starter cultures, Goat milk

Keçi Sütünden Üretilen Çökelek Peynirinin Fizikokimyasal, Mikrobiyolojik ve Duyusal Özellikleri ile Yağ Asidi Kompozisyonu

Üzerine, Laktik Starter Kültür Tipi ve Farklı Sıcaklık Uygulamanın Etkisi

ÖzetÇökelek peyniri, ısıl işlem uygulanıp, süzülen ve tuzlanmış yoğurttan yapılan Türkiye’ye özgü geleneksel bir peynirdir. Bu çalışmada,

Çökelek peynirleri, kültür olarak (1) Lactobacillus helveticus, (2) yoğurt kültürü veya (3) Lb. helveticus ve yoğurt kültürü karışımlarının (1:1) eklendiği süt pıhtılarından üretilmiştir. Çökelek peynirleri, belirtilen kültürlerle elde edilen pıhtılarından 85ºC veya 95ºC’lik iki farklı sıcaklıkta ısıtılmasından sonra elde edilmiştir. Üretilen Çökelek peynirleri 4oC’de 45 gün depolanmıştır. Sonuç olarak, çökelek peyniri üretiminde starter kültür olarak Lb. helveticus’un kullanımı (yoğurt bakterileri ile tek veya karışık olarak) ve 85oC’ lik ısıl işlem önerilebilir.

Anahtar sözcükler: Çökelek peyniri, Lactobacillus helveticus, Yoğurt starter kültürleri, Keçi sütü

Effects of Starter Culture Types and Different Temperatures Treatments on Physicochemical, Microbiological and Sensory

Characteristics, and Fattty Acid Compositions of Çökelek Cheese Made from Goat Milk

Bedia ŞİMŞEK * Osman SAĞDIÇ **

***

Suleyman Demirel University , Agricultural Faculty, Department of Food Engineering, TR-32260 Isparta - TURKIYE Yildiz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Food Engineering, TR-34210 Istanbul - TURKIYE


The dairy products produced from by yogurt such as yayik butter, tarhana and Çökelek cheese are very popular products in the Middle East where they are used in the preparation of some popular food dishes. Çökelek cheese is produced by heating of yogurts and then straining in a special cloth bag. It has been made from cow, goat milk and ewe milk for a long time. The goat milk production

ranks third in the world after cattle and buffalo milk production. In the developing countries, dairy goat farming has an increasing important. Goat milks are very important for flavor and quality of Çökelek cheese 1.

Yogurt bacteria and Lactobacillus helveticus are homofermentative thermophilic lactic acid bacteria widely used

INTRODUCTION

İletişim (Correspondence) +90 246 2111541 [email protected]

RESEARCH ARTICLE

178Effects of Starter Culture ...

in the dairy technology. In particular, yogurt bacteria is used in yogurt production 2. Additionally Lb. helveticus being of highly proteolytic activity is used as primary natural whey starter for the manufacture of some Swiss and Italian cheese varieties 3-5. Also Lb. helveticus can be used as starter culture in a variety of fermented dairy products and as a non-starter flavour enhancer, being capable of reducing bitterness and accelerating cheese flavour development 6-9.

The main role of lactic acid bacteria used as starter during cheese production is the production of lactic acid through metabolism of lactose. This action improves the milk coagulation process, makes the curd stronger and protects the final product against contamination. Further- more, glycolysis, proteolysis and lipolysis produced by the enzymes of lactic acid bacteria contribute to the flavour development of the cheeses during the ripening process 10.

Heat has a great effect on proteins. Apart from denaturation, heat-induced chemical reactions involving amino acid residues can significantly alter protein properties. Such heat-induced chemical changes have not been characterized quantitatively so far 11.

Traditionally, in the production of Çökelek cheese does not use starter culture. However, thermophilic yogurt cultures can grow aroma and can be alive during the heating process if Çökelek cheese is produced from yogurt.

The aim of this study was to determine the suitable starter culture and heating temperature combination to produce better Çökelek cheese. For the reason, goat milk and thermophil cultures were selected for Çökelek cheese production. Thermophilic cultures including Lb. helveticus and yogurt bacteria are more heat stabile than other lactic starter cultures 12. Additionally Lb. helveticus has high proteolytic activity and it is a bacterium fast aroma-grown. Çökelek cheeses produced by using different starter cultures and two different temperatures were examined in respect of their physicochemical and microbiological properties, protein fractions, FFAs (free fatty acids), sensory properties during their ripening period. Additionally we added to the milk used in Çökelek cheese yogurt cultures and/or Lb. helveticus as thermophilic starter cultures.

MATERIAL and METHODS

Çökelek Cheese Production

Çökelek cheeses were produced in Ün-Süt Dairy Plant of Suleyman Demirel University. In yogurt production, the method proposed by Tamime and Robinson 13 was followed with the exception that the homogenization and solid standardization stages were ignored. Firstly, goat milk was analyzed for physicochemical, microbiological and sensorial properties and fatty acid compositions. Then, goat milk was standardized to 1.5±0.2% fat and heated up

to 95°C with gentle stirring and kept at this temperature for 5 min. (the first pasteurization), followed by cooling to 42-45oC. The pasteurized milk was divided into three groups. Each group was inoculated with a yogurt culture (Y) (Yo-mix 502, Gemak-Danisco, Ankara, Turkey), Lb. helveticus (H) (LH-B02, Peyma-Chr. Hansen, Istanbul, Turkey) or mixture culture (M) of these (1:1) at a rate of 3% (w/w) ratio. These groups were incubated at 42-45°C until pH 4.6 and then cooled down to 4°C. After overnight refrigeration, the coagulums were manually stirred and then were heated up 85°C and 95°C for 30 min (the second pasteurization). In this respect, six treatment groups including control group were studied in this study: (1) Y95 (Control), (2) Y85, (3) H95, (4) H85, (5) M95, (6) M85 (Traditional Çökelek cheese usually made with yogurt bacteria. Then, yogurt is heated at high temperature. Therefore, the Y95 samples were evaluated as a control group in this study).

All of these treatment coagulums were cooled down to 25°C and then, poured into a layer cheese cloth bag. A drainage procedure was accomplished in a temperature controlled room, set at 25°C. Çökelek cheese treatments were filled into the containers, salted with 1% salt and mixed gently to achieve homogeneity. Obtained Çökelek cheeses treatment groups were packaged as the lots weighing 500 g. and stored at 4°C for 45 days until analysis. Çökelek cheese is a fresh cheese consumed, although the duration of 45 days for sensorial, microbiological and chemical changes that occur in the content of fatty acids stored in order to observe.

Physicochemical Analyses

The titratable acidity (TA, as lactic acid %, acidity of milk - AOAC Official Method 947.05), total solid (TS%, AOAC Official Method 990.20) was determined as outlined by AOAC methods 14,15. Fat (%) of the raw milk was determined using the method of James 12. pH values were measured using a HANNA pH meter (HANNA Instruments, Padova, Italy). Percentage of NaCI and fat of cheese was determined using the method of James 16. NaCl content was expressed as salt concentration. Moisture contents of cheese were determined by AOAC methods (Official Method 926.08) 17. Total nitrogen (TN) and water-soluble nitrogen (WSN) levels were determined according to the method of Grippon et al.18. Protein content was calculated by multiplication of TN content with 6.38. Ripening index of Çökelek cheese samples were calculated using following equation:

Ripening index (%) = (WSN/TN) ×100

Analysis of Fatty Acids

Fatty acid composition was determined using a modified fatty acid methyl ester method as described by Marquard 19. The oil was extracted from 10 g sample by homogenisation with hexane. The reactive was 500 ml of total Na-metaoxid (0.5 g), isooctane (20 ml) and methanol (80 ml) added to the oil (50 µl). Isooctane (500 µl) was added

179ŞİMŞEK, SAĞDIÇ

into the sample following waiting overnight. The fatty acids methyl esters were analyzed in a Hewlett-Packard 6890 series gas chromatograph (Perkin Elmer Auto System XL, San Jose, CA, USA) equipped with a flame ionizing detector (FID), a fused silica capillary column Cp SIL 88, 100 m × 0.25 mm i.d., film thickness 0.2 μm (Chrompack Inc, Bridgewater, NJ,USA). It was operated under the following conditions: oven temperature program, 60°C for 4 min. raised to 175°C at a rate of 13°C·min-1 and than kept at 175°C for 27 min; than at 215°C for 5 min. and raising to 240°C at a rate of 4°C·min-1 and then kept at 240°C for 15 min injector and detector temperatures, respectively. Carrier gas was helium at flow rate of 15 cm·s-1; split ratio, 1/10 ml·min-1. The contents of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids were determined by computing integrator.

Determination of Lactic Acid Bacteria

Streptococcus thermophilus and lactobacilli were enumerated on M17 and MRS agars (Merck, Darmstadt, Germany), respectively, using the pour plate method with incubation at 30°C for 3 days 20,21. Results were expressed as log cfu g-1.

Sensorial Analysis

Çökelek cheeses were tested by a panel of 25 panelists familiar with cheese grading. They scored the cheeses for appearance (9 points), flavour (9 points), odour (9 points) and texture (9 points). The hedonic scale of 9 points was used (1 = dislike extremely; 5 = neither like nor dislike; 9 = like extremely). The panelists were provided with unsalted crackers and mineral water to rinse their palates when necessary. The panelists were asked to evaluate these cheeses and to record any unexpected or unpleasant flavour defects and total score 22.

Statistical Analyses

Analyses of variance for bacterial counts after a log transformation was performed on data obtained at different stages of production. Chemical, microbiological, fatty acids and organoleptic characteristics of the Çökelek samples were subjected to analyses of variance (one-way) using multivariate test using SPSS 15.0 Statistic Package (SPSS, Chicago, Illinois, USA). Statistical significance level was taken as 95%.

RESULTS

The pH, TA (%), TS (%), fat (%), density (g ml-1) and protein (%) of the raw goat milk used in this study were as follows: 6.76±0.46, 0.19±0.01, 13.16±0.25, 3.37±0.49, 1.033±0.00 and 3.18±0.03, respectively.

Yield of cheese is basically important to cheese manufacturers to get large sums of money or profit. The

yield values of Çökelek cheese treatment groups; Y95, Y85, M95, M85, H95 and H85 were found as 27.29%, 27.43%, 28.15%, 25.37%, 28.96% and 25.91%, respectively. Generally, the yield of Çökelek cheeses heated at 95 °C was higher than those of cheese heated at 85°C (except Y95 and Y85 samples), which was thought to be due to the whey proteins, being effective on yield of cheese in heating process at 95°C.

The physicochemical characteristics of Çökelek cheeses during the storage period are presented in Table 1. The pH value of the control sample (4.30±0.48) was higher than those of the other samples and the titratable acidity levels of the Çökelek samples ranged from 2.23 - 2.45% in the 45th day. During the ripening period, titratable acidity increased in each treatment groups. Reason for this increase during storage, the products formed as a result of microbial and enzymatic activity may be due to the acidic character. The total solid, fat, NaCl varied between 25.87% and 28.98%, 4.08% and 5.50%, and 1.64% and 1.83%, respectively. The total solid value of M85 samples was statistically different from others on first days and 15th days. In addition, this value of H95 significantly higher than other samples was identified in 45th day. In general, total solid values of the Çökelek cheese heated at 95oC were higher than those of the others.

Lb. helveticus and secondary pasteurization process applied to the product may have an effect on TS%. The TN% and WSN% increased throughout the ripening period. The ripening indices were determined as 13.59% -20.00% in the all samples. The ripening index of the Çökelek cheese produced with yogurt cultures (85°C) was the highest. In this regard, proteolytic activity of lactic acid bacteria used as starter cultures are thought to have played an effective role. In our study, high temperature led to reduction of bacterial content, this reduction has been effective in reducing the content of the enzymes. TS%, TN%, WSN% or % ripening indexes (RI) were statistically significant among groups for each parameter during storage (P<0.05).

Both lactococci and lactobacilli counts increased in the all samples during the storage period. While the log counts of lactococci were determined between 2.86 and 3.03, those of lactobacilli to ranged from 3.60-4.33 during the storage period (Table 1). The differences between the log counts of lactococci and lactobacilli in the Çökelek cheeses were found significant for each lactic acid bacteria group during storage (P<0.05).

The fatty acid compositions of Çökelek cheeses are presented in Table 2. SFA contents in the treatment groups - (Y95, 85, M95, M85, H95, and H85) were 65.70, 66.88, 68.70, 67.62, 67.48 and 65.92 in the 45th day of storage, respectively. The lowest SFA content was in the control group. The myristic acid level of control cheese (9.56%) was the highest, while palmitic acid level of the sample made with yogurt cultures at 85°C (28.05%) was the highest.


Tabl

e 1.

Che

mic

al a

nd m

icro

biol

ogic

al (l

og c

fu g

-1) v

alue

s det

ecte

d in

Cok

elek

che

eses

Tabl

o 1.

Çök

elek

pey

nirin

de sa

ptan

an k

imya

sal v

e m

ikro

biyo

lojik

(log

kob

g-1

) değ

erle

r

Stor

age

Tim

eN

opH

*TA

* [%

]TS

** [%

]Fa

t* [%

]Fa

t/TS

* [%

]N

aCl*

[%]

NaC

l/TS*

[%

]W

SN**

[%]

TN**

[%]

RI**

[%]

Lact

ic A

cid

Bact

eria

(log

cfu

g-1

)

Stre

ptoc

occu

s th

erm

ophi

lus

**La

ctob

acill

i **

MSD

MSD

MSD

MSD

MSD

MSD

MSD

MSD

MSD

MSD

MSD

MSD

Firs

t day

Y95:

C4.

340.

482.

300.

1027

.15ab

1.51

5.33

0.58

19.6

11.

061.

640.

006.

040.

330.

19fg

0.04

2.19

a0.

048.

52gh

1.73

1.36

gh

0.25

2.53

e-h

0.20

Y85

4.37

0.50

2.32

0.05

26.6

3ab1.

205.

500.

2520

.68

1.33

1.64

0.00

6.16

0.27

0.15

g0.

012.

05bc

0.02

7.17

h0.

521.

66 e

-h0.

202.

56 e

-h0.

25

M95

4.40

0.37

2.16

0.09

26.6

1ab0.

165.

250.

2519

.73

1.00

1.64

0.12

6.16

0.48

0.14

g0.

032.

10ab

c0.

046.

79h

1.41

1.73

e-h

0.11

2.43

fgh

0.35

M85

4.31

0.30

2.13

0.08

25.8

7b0.

625.

330.

5820

.59

1.81

1.68

0.07

7.20

1.11

0.15

g0.

012.

10ab

c0.

056.

99h

0.61

1.43

fgh

0.30

2.20

h0.

26

H95

4.40

0.24

2.21

0.29

27.0

8ab0.

825.

500.

0020

.32

0.62

1.68

0.18

6.18

0.48

0.21

fg0.

022.

09ab

c0.

0210

.02gh

0.77

1.30

h0.

102.

30 g

-h0.

30

H85

4.38

0.17

2.20

0.32

26.3

8ab0.

565.

170.

2919

.57

0.73

1.76

0.12

7.45

1.02

0.23

ef0.

022.

09ab

c0.

0510

.93fg

0.71

1.73

e-h

0.11

2.33

g-h

0.28

15th

day

Y95:

C4.

290.

602.

420.

1627

.15ab

1.51

5.25

0.66

19.2

91.

331.

760.

126.

480.

630.

32cd

0.03

2.17

ab0.

0914

.80cd

e1.

451.

96 c

-e0.

253.

36 b

cd0.

35

Y85

3.93

0.11

2.39

0.15

26.6

3ab1.

205.

170.

3819

.39

0.98

1.68

0.14

6.30

0.45

0.40

ab0.

062.

07ab

c0.

0319

.30ab

2.61

2.33

bcd

0.41

3.23

cde

0.25

M95

4.03

0.04

2.34

0.16

26.6

1ab0.

165.

080.

5219

.10

1.88

1.68

0.07

6.30

0.26

0.25

ef0.

022.

09ab

c0.

0411

.70ef

g0.

582.

46 a

bc0.

053.

16 c

-f0.

40

M85

4.01

0.09

2.33

0.16

25.8

7b0.

625.

170.

7219

.94

2.37

1.79

0.14

6.94

0.56

0.33

cd0.

032.

02c

0.05

16.1

0bcd

1.68

2.10

cde

0.36

2.86

d-h

0.23

H95

4.09

0.19

2.32

0.22

27.0

8ab0.

825.

000.

6618

.52

2.95

1.72

0.07

6.35

0.42

0.29

de0.

022.

03bc

0.05

14.3

0cde

1.39

1.86

d-g

0.05

2.93

d-h

0.32

H85

4.05

0.26

2.31

0.27

26.3

8ab0.

565.

080.

7619

.23

2.51

1.79

0.18

6.79

0.53

0.32

cd0.

022.

08ab

c0.

0415

.25cd

1.36

2.46

abc

0.11

2.96

d-g

0.20

45th

day

Y95:

C4.

300.

482.

400.

0427

.78ab

2.21

4.33

0.95

15.7

84.

341.

830.

075.

970.

710.

34bc

d0.

022.

20a

0.12

15.7

1cd1.

852.

86 a

b0.

254.

33a

0.41

Y85

4.23

0.48

2.45

0.17

27.6

9ab0.

984.

251.

1515

.42

4.43

1.79

0.14

6.63

0.69

0.43

a0.

032.

15ab

c0.

0620

.00a

1.17

3.03

a0.

354.

23a

0.20

M95

4.26

0.39

2.30

0.07

27.3

3ab0.

044.

080.

5214

.94

1.93

1.83

0.07

6.23

0.26

0.29

de0.

032.

11ab

c0.

0613

.60de

f1.

163.

00 a

0.00

4.06

ab

0.46

M85

4.20

0.32

2.23

0.11

27.4

7ab1.

164.

170.

5815

.13

1.52

1.79

0.00

6.71

0.26

0.36

abc

0.03

2.10

abc

0.12

17.3

0abc

1.69

2.86

ab

0.30

3.76

abc

0.15

H95

4.29

0.39

2.31

0.24

28.9

8a0.

854.

750.

4316

.37

1.16

1.83

0.07

6.02

0.14

0.36

abc

0.03

2.08

abc

0.02

17.6

0abc

1.37

2.50

abc

0.10

3.60

a-d

0.26

H85

4.23

0.44

2.40

0.34

27.5

6ab0.

424.

250.

7515

.44

2.86

1.78

0.14

6.32

0.57

0.40

ab0.

022.

09ab

c0.

0719

.10ab

0.66

3.03

a0.

113.

76 a

bc0.

30

M -

arith

met

ical

mea

n, S

D –

stan

dard

dev

iatio

n. M

eans

in th

e sa

me

colu

mn

with

diff

eren

t let

ters

show

stat

istic

ally

sign

ifica

nt d

iffer

ence

s*

stat

istic

ally

not

sig

nific

ant d

iffer

ent P

>0.0

5, *

* st

atis

tical

ly s

igni

fican

t diff

eren

t P<0

.05

C - C

ontr

ol, Y

85 a

nd Y

95 -

inoc

ulat

ed w

ith y

ogur

t cu

lture

, and

hea

ted

at 8

5°C

and

95°C

, res

pect

ivel

y; M

85 a

nd M

95 -

inoc

ulat

ed w

ith y

ogur

t cu

lture

+ L

b. h

elve

ticus

(1:1

), an

d he

ated

at

85°C

and

95°

C,

resp

ectiv

ely;

H85

and

H95

: ino

cula

ted

with

Lb.

hel

vetic

us, a

nd h

eate

d at

85°

C an

d 95

°C, r

espe

ctiv

ely

TA -

titra

tabl

e ac

idity

, TS

- tot

al s

olid

s, TN

- to

tal n

itrog

en, W

SN -

wat

er-s

olub

le n

itrog

en, R

I-Rip

enin

g In

dex


Table 2. Fatty acid values detected in Cokelek cheeses

Tablo 2. Çökelek peynirlerinde saptanan yağ asidi değerleri

Fatty Acids (%)First Day 45th Day

Y95: C Y85 M95 M85 H95 H85 Y95: C Y85 M95 M85 H95 H85

SFA 67.46 68.42 67.49 75.93 70.10 69.87 65.7 66.88 68.70 67.62 67.48 65.92

C4:0 1.00 1.55 1.08 3.12 1.70 1.76 1.75 1.01 1.17 1.48 1.34 0.93

C6:0 0.94 1.46 1.13 2.90 1.50 1.60 1.99 1.15 1.68 1.66 1.78 0.99

C8:0 1.36 1.93 1.50 3.68 1.92 1.94 2.53 1.25 1.80 1.86 1.52 1.07

C10:0 6.62 7.36 6.94 12.76 8.18 7.50 1.98 4.59 5.85 5.50 4.69 3.93

C12:0 3.43 3.18 3.49 4.77 3.86 3.54 3.71 2.37 2.42 2.26 2.14 1.99

C14:0 9.78 8.92 9.57 10.12 9.96 9.62 9.56 8.20 7.40 7.28 7.46 7.23

C16:0 27.09 26.46 26.61 23.59 26.44 26.66 26.62 28.05 26.41 26.24 27.03 27.45

C17:0 0.35 0.31 0.36 0.14 0.32 0.34 0.72 0.75 0.96 1.08 0.74 0.84

C18:0 16.89 17.25 16.81 13.85 16.22 16.91 16.84 19.51 21.01 20.26 20.78 21.43

TUFA 24.25 23.55 23.81 18.63 23.11 22.29 23.47 27.61 26.01 24.67 26.76 27.47

MUFA 21.99 21.28 21.55 16.81 20.83 19.98 21.36 25.09 23.95 22.46 24.63 25.04

C11:1 0.13 0.12 0.14 0.23 0.15 0.14 0.20 0.10 0.12 0.11 0.10 0.10

C16:1 0.97 0.91 0.98 0.28 0.82 0.97 0.36 0.57 0.45 0.40 0.48 0.63

C17:1 0.22 0.10 0.22 0.11 0.11 0.12 0.18 0.25 0.23 0.30 0.11 0.20

C18:1 trans 1.29 1.34 1.16 1.06 1.28 1.25 0.50 1.00 0.60 0.76 1.00 0.85

C18:1 cis 19.38 18.81 19.05 15.13 18.47 17.5 20.12 23.17 22.55 20.89 22.94 23.26

PUFA 2.26 2.27 2.26 1.82 2.28 2.31 2.11 2.52 2.15 2.21 2.13 2.43

C18:2 1.84 1.85 1.84 1.52 1.81 1.82 1.63 1.91 1.48 2.08 1.93 1.84

C18:3 0.42 0.42 0.42 0.30 0.47 0.49 0.48 0.61 0.67 0.13 0.20 0.59

SFA - saturated fatty acids, TUFA - total unsaturated fatty acids, MUFA - mono unsaturated fatty acids, PUFA - poli unsaturated fatty acids C - Control, Y85 and Y95 - inoculated with yogurt culture, and heated at 85°C and 95°C, respectively; M85 and M95 - inoculated with yogurt culture + Lb. helveticus (1:1), and heated at 85°C and 95°C, respectively; H85 and H95: inoculated with Lb. helveticus, and heated at 85°C and 95°C, respectively

Table 3. Sensorial values detected in Cokelek cheeses (points)

Tablo 3.Çökelek peynirlerinde saptanan duyusal değerler (puan)

SampleFirst Day

Appearance** Odour* Texture* Flavour** Total*

Y95:C 6.70±0.44ab 6.17±0.76 6.70±0.85 5.97±0.78ab 25.53±2.75

Y85 6.83±0.91ab 6.36±0.80 6.60±0.95 6.33±0.78ab 26.13±3.43

M95 6.63±0.47ab 6.33±0.93 6.77±0.68 6.40±0.70a 26.13±2.66

M85 6.90±0.70ab 6.33±0.57 6.57±0.80 6.10±0.57ab 25.90±2.55

H95 6.10±0.17b 6.17±0.37 6.33±0.30 5.80±0.17ab 24.40±0.26

H85 6.67±0.47ab 6.27±0.70 6.47±0.61 6.07±0.37ab 25.47±2.07

15th Day

Y95:C 7.07±0.51a 6.60±0.56 6.23±0.75 6.50±0.46a 26.40±2.25

Y85 6.90±0.34ab 6.17±0.40 6.27±0.97 6.27±0.68ab 25.60±2.16

M95 6.73±0.49ab 6.13±0.42 5.87±1.07 5.67±0.57ab 24.40±2.52

M85 6.70±0.60ab 5.90±0.62 5.57±1.20 5.50±0.98ab 23.67±3.30

H95 6.83±0.42ab 6.30±0.26 6.00±0.30 5.37±0.38b 24.50±0.75

H85 6.73±0.06ab 6.30±0.10 6.13±0.37 5.60±0.87ab 24.76±0.71

* Statistically not significant different P>0.05, ** statistically significant different P<0.05C - Control, Y85 and Y95 - inoculated with yogurt culture, and heated at 85°C and 95°C, respectively; M85 and M95 - inoculated with yogurt culture + Lb. helveticus (1:1), and heated at 85°C and 95°C, respectively; H85 and H95: inoculated with Lb. helveticus, and heated at 85°C and 95°C, respectively


During 45 days of ripening period, TUFA contents of the all groups increased.

The sensory evaluation scores give to the treatment groups are shown in Table 3. We did not analysed sensory evaluation of the samples after 15th days, because of bad taste and odour. In control group, the mean values given for appearance, odours, and flavours were the highest at the 15th day. In addition, total sensory score of the samples made with yogurt culture in heating at 95°C was the highest (15th days). Panelists’ preferred traditional Çökelek cheese produced from yogurt cultures. Statistical difference was found to be important for appearance and flavours during storage (P<0.05). Total sensory score of the control group was higher than the others, but the difference is not statistically significant between the samples.

DISCUSSION

The properties of raw goat milk were suitable for yogurt production 1. Cheese yield is of basic importance to cheese manufacturers as small differences in yield translate to large sums of money or profit. Kalantzopoulos 23 stated that the yield capacity of fresh cheese was related to the protein and fat contents of goat milk.

Our study is the first research on the Çökelek cheese produced with starter cultures Therefore, the Çökelek cheeses produced in the study were compared to results of Çökelek cheese produced by different ways in previous very few study.

The pH, TA%, TS%, NaCl%, TN% were relatively higher than the findings reported by some researchers 24-27. Our results compared with the literature, different results were obtained. The reason for this difference, Çökelek cheeses raw material and/or production methods may be different.

Lactococci and lactobacilli counts were lower than those reported by some researchers 24,25. The microbioligal quality of “Çökelek” cheese, which is sold without packaging in Diyarbakır (in Turkey), an average of total microorganism 8.49±0.79 log cfu g-1, heterofermentative lactic acid bacteria 8.58±0.98 log cfu g-1 were determined 28. Discrepancy in the results could be due to the variations in production methods of Çökelek cheeses (especially due to the heating process at high temperature like 95°C in this study).

Lipolysis plays an essential role in the sensory properties of cheese; some free fatty acids (FFAs) have been shown to contribute directly to the aroma characteristics of many types of cheese, or indirectly as precursors of aroma components 29. The fatty acids hexanoic, octanoic and decanoic acids have long been considered responsible for the characteristic aroma of goat cheeses, giving rise to the popular terms caproic, caprilic and capric acids. Additionally, certain branched-

chain FFAs contribute, by themselves, to the goaty flavour of cheese 30-32. Consistent with the results of the previous studies, Myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids were the main fatty acids present in the Çökelek cheeses 33,34. Palmitic and myristic acid are considered hypercholesterolemic, oleic acid and stearic acid are considered hypocholesterolemic 33. The findings of this study show similarity with recorded by Kondyli and Katsiari 35, and Dönmez 36. But the degree of lipolysis undergone by the sample cheeses here was comparatively low, as might be expected, since ripening tends to be relatively short.

The results showed that Lb. helveticus (single or mixture) could be used as starter culture in Çökelek cheese made from goat milk; it did not produce very different results from the other samples. The yield and some physicochemical properties of the Çökelek cheese samples heated at 95°C were better than those of the samples heated at 85°C. However, it was found that the sensorial score, ripening index and lactic acid bacteria counts of the samples heated at 85°C were higher than those of the others. So, the heating of yogurt at 85°C may be preferred to another heating temperature. As a conclusion, both Lb. helveticus and yogurt cultures may be proposed as a starter culture (a single and mix) for Çökelek cheese production.

Acknowledgements

I am grateful to ‘the Chair of Scientific Research Projects’ of Suleyman Demirel University for their financial support (Project number: 966, 2004).

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ÖzetBu çalışmada, bazı kimyasallar ve tıbi bitki özütlerinin melek balığı (Pterophyllum scalare) yumurta açılımı ve sudaki bakteri yükü

üzerine etkisi incelenmiştir. Bu amaçla metilen mavisi 0.5 ppm ve potasyum permanganat 0.2 ve 0.4 ppm uygulanmıştır. Biberiye, civanperçemi ve sumak özütlerinin herbiri ise 1.000, 5.000 ve 10.000 ppm olarak uygulanmıştır. Metilen mavisi 0.5 ppm, potasyum permanganat 0.2 ppm, biberiye 1.000, 5.000, ve 10.000 ppm ve civanperçemi 5.000 ppm uygulamaları sudaki bakteri yükünü azaltmış ve yumurta açılımını önemli oranda arttırmıştır (P<0.05). Biberiye özütünün konsantrasyonu arttıkça bakteri yükü azalmış ve yumurta açılımı artmıştır. Tüm sumak uygulamaları bakteri yükünü azaltmış, ancak yumurta açılımınını olumsuz etkilemiştir. Elde edilen bulgular yumurta açılımı için en uygun tıbbi bitki özütünün biberiye olduğunu göstermiştir. Sonuç olarak bu çalışmada biberiye özütünün sentetitik kimyasallar yerine kullanımının daha uygun ve melek balığı yumurta açılımı için optimum biberiye özüt dozajının 1.000 ppm olduğu bulunmuştur.

Anahtar sözcükler: Pterophyllum scalare, Yumurta açılımı, Metilen mavisi, Potasyum permanganat, Biberiye, Sumak, Civanperçemi

Effects of Medicinal Herb Extracts on Egg Hatching of the Angel Fish (Pterophyllum scalare)

SummaryIn this study, the effects of sythetic chemicals and medicinal herbs on the amount of bacteria in egg water and egg hatching of

the angel fish (Pterophyllum scalare) were investigated. In this goal, methylene blue exposed to 0.5 ppm and potassium permanganate exposed to 0.2 and 0.4 ppm. Each rosemary, yarrow and sumac extracts exposed to 1.000, 5.000 and 10.000 ppm. Methylene blue (0.5 ppm), potassium permanganate (0.2 ppm), rosemary (1.000, 5.000, and 10.000 ppm) and yarrow (5.000 ppm) treatments were significantly decreased bacterial loads and increased egg hatching (P<0.05). With the increasing in the concentration of rosemary extract the bacterial loads decreased and egg hatching increased. All sumac treatments were significantly decreased total bacterial loads, but were negatively affected on egg hatching. The findings were shown that the best group was rosemary for egg hatching. Finally, this study showed that rosemary extract was more suitable to use instead of sythetic chemicals, and optimum rosemary extract dose was 1.000 ppm for the hatching of the angel fish eggs.

Keywords: Pterophyllum scalare, Egg hatching, Methylene blue, Potassium permanganate, Rosemary, Sumac, Yarrow

Tıbbi Bitki Özütlerinin Melek Balığı (Pterophyllum scalare)Yumurtasının Açılımı Üzerine Etkisi

Sevdan YILMAZ * Sebahattin ERGÜN *

* Çanakkale Onsekiz Mart Üniversitesi, Su Ürünleri Fakültesi, Yetiştiricilik Bölümü, TR-17100 Çanakkale, TÜRKİYE


Yetiştiriciliği yapılan balık türlerinde yumurta açılımının yüksek olması hedeflenmektedir. Bu amaçla ekonomik öne- me sahip birçok balık türünde metilen mavisi, potasyum permanganat, hidrojen peroksit, formalin, bakır sülfat, glutaraldehit ve iyot gibi kimyasallar kullanılmıştır 1-3. Melek balıkları da ilk kez 1909 yılında üretilmiş olup ticari ve hobi amaçlı olarak 50 yıldır üretilmektedir 4. Bu balıkların

yetiştiriciliğinde en büyük sorunlardan biri yumurta açılı- mının bakteri ve mantar enfeksiyonları nedeniyle düşük olmasıdır. Melek balıklarının döllenmemiş veya mantarla-şan yumurtaları doğada anaç balıklar tarafından toplan-maktadır 5. Ancak, ticari olarak düşünüldüğünde bu durum mümkün gözükmemektedir. Bunun nedeni 1) anaç balıkların yumurtaları ile aynı ortamda bırakıldıklarında yumurtaları

GİRİŞ


RESEARCH ARTICLE

186Tıbbi Bitki Özütlerinin ...

yemeleri, 2) yumurtalara ve devamında larvalara baktık-ları dönemde daha az beslenmeleri ve bundan dolayı tekrardan yumurta üretimlerinin gecikebilmesi, 3) yumur-taların üreme ortamında bırakıldığında daha düşük açılım göstermeleridir.

Genel olarak melek balıklarının yetiştiriciliğinde yumur- taların bulunduğu zemin üretim ortamından alınıp çeşitli kimyasallar ile yumurta açılım oranı arttırılmaktadır. Bu amaçla melek balığı yumurtalarında metilen mavisi, hidrojen peroksit, acriflavin ve chloramine-T önceki çalışma- larda kullanılmıştır 6,7. Günümüzde ise sentetik kimyasal-ların kullanımı yerine alternatif doğal kaynaklara yönelik çalışmalar hız kazanmıştır. Balık yetiştiriciliğinde de hormon, antibiyotik, vitamin ve diğer birçok kimyasala alter-natif olarak tıbbi bitkilerin kullanılabilirliği araştırılmakta-dır 8. Fakat balık yumurtalarında bitki özütlerinin kullanı- mına yönelik çalışma sayısı oldukça azdır. Daha önce kekik, ateş çiçeği, okaliptüs ve nane bitkilerinin esans yağı karışımları; alabalık 9 ve hint bademi özütü; tilapia 10 balık- larının yumurta açılımının artırılmasında ve mantar enfek-siyonlarının önlenmesinde kullanılmıştır. Ancak biberiye, civanperçemi ve sumak bitkilerinin melek balıklarının yumurta açılımına etkisi üzerine herhangi bir çalışmaya rast-lanılmamıştır.

Bu çalışmada potasyum permanganat, metilen mavisi, biberiye, civanperçemi ve sumak özütlerinin melek balığı yumurtalarının açılım oranına ve bakteri yükü üzerine etki-lerinin tespit edilmesi amaçlanmıştır.

MATERYAL ve METOT

Balık, Yumurta ve Deneme Ortamı

Çalışmada “Tri Colour” varyetesi melek anaçlarından (Pterophyllum scalare) elde edilen yumurtalar kullanılmıştır. Yumurta açılımı ve yumurta kalitesi üzerindeki fiziksel, kimyasal, mikrobiyolojik su koşulları, yem ve anaç balık gibi birçok parametrenin etkisinin en aza indirilmesi ama- cıyla aynı partinin yumurtalarında deneme yürütülmüştür. Kullanılan anaç balıklar ve yumurtalar 26±1°C, 400 μs iletkenlik, 6.7 pH suda ve 12 saat aydınlık, 12 saat karanlık ışık periyodunda tutulmuşlardır. Balıklar günde 2 defa Tetramin Flake Food (Tetra® Germany) marka ticari yemle beslenmişlerdir. Balıkların bulunduğu akvaryum sisteminde günlük %10 su değişimi yapılmıştır. Balıklar 45° açılı yatay bir zemine yumurtladıktan 1 saat sonra yumurtaları na-zikçe sifonlanmıştır. Yumurtaların sifonlanmasından sonra oluşabilecek olası hasarın tespit edilmesi için bir pipet yar- dımıyla ışığa tutulan yumurtaların opaklaşmayan, döllen-miş ve hasarsız olanları seçilmiştir. Sağlıklı yumurtalar içerisinden 390 tanesi tesadüfen 3 tekerrürlü olarak, her biri 0.5 litrelik olan 39 adet silindire konik deneme ortamlarına yerleştirilmişlerdir. Deneme boyunca yumurtaların hava-landırılması hava matoru yardımıyla yapılmış ve hava hor- tumuna takılan cam pisetler kullanılmıştır. Melek yumur-

taları su değişimine hassas olduklarından dolayı bulun-dukları deneme ortamında su değişimi yapılmamıştır.

Bitki Özütlerinin Çıkarılması

Çalışmada kullanılan biberiye (Rosmarinus officinalis); Çanakkale, sumak (Rhus coriaria); Edirne ve civanperçemi (Achillea millefolium); Roiak (Kuzey Doğu Bulgaristan)’dan toplanmıştır. Kurutulup öğütülen bitkilerin özütleri sulu özüt çıkartma metodu modifiye edilerek elde edilmiştir 11. Bitkiler 5 g tartılıp 95 ml saf suda 1 saat süreyle oda sıcak-lığında muhafaza edildikten sonra 10 dakika 50°C de bek-letilip 2 dakika boyunca vortex ile karıştırılmışlardır. Bu ka- rışımlar 0.45 mikronluk filtreden geçirilerek özütler elde edilmiştir.

Sentetik Kimyasalların ve Özütlerin Uygulanması

Yumurtalar deneme ortamına yerleştirildikten sonra sentetik kimyasallar ve doğal özütler bir defa olacak şekilde ilave edilmiştir. Yumurtalar açılıncaya kadar deneme orta-mında yumurtalara uygulanmışlardır. Kontrol grubu (K) suyuna hiçbir uygulama yapılmamıştır. Metilen mavisi daha önce melek balıklarında yapılan bir çalışma referans alı-narak 6 0.5 ppm (M5), potasyum permanganat 0.2 ppm (P2) ve 0.4 ppm (P4), biberiye (B1, B5, B10), sumak (S1, S5 ve S10) ve civanperçemi (C1, C5, C10) özütleri yumurtaların bulunduğu suya sırasıyla 1.000 ppm, 5.000 ppm, 10.000 ppm olacak şekilde uygulanmışlardır.

Yumurta Açılımı

Melek balığı yumurtaları 3. günün sonunda açılmıştır. Zeminde sağlıklı bir şekilde hareket eden besin keseli lar-vaların, açılmamış yumurtaların ve ölü larvaların sayımları yapılarak her bir grup için yüzde açılım oranları formül yardımıyla hesaplanmıştır 12.

Mikrobiyal Yükün Tespit Edilmesi

Yumurtaların açıldığı 3. günün sonunda her bir grubun mikrobiyal yükünü tespit etmek için alınan su örnekleri gerekli seyreltmeler (10-5 ve10-6) yapıldıktan sonra 3 gün Tryptic Soy Agar (TSA) ortamında3 26±1°C derecede inkübe edilmiştir. Bakteri yükünün tespitinde aşağıdaki formül kullanılmıştır.

cfu /ml = Katı besiyerindeki koloni miktarı ÷ 10-X (seyretme miktarı)

İstatistiksel Analizler

İstatistiksel analizler için SPSS 17 paket programı kulla- nılmıştır. Yüzde açılım verilerine arcsin dönüşümü uygulan-dıktan sonra varyans analizi yapılmış ve ortalamalar Tukey testi ve açılımı değiştirme oranları Dunnett t-testi ile %5 önem düzeyinde karşılaştırılmıştır 13.

BULGULAR

Çalışmada farklı deneme ortamlarının kontrol grubuna

187YILMAZ, ERGÜN

göre yumurta açılımı üzerine arttırıcı veya azaltıcı etkileri (Tablo 1) ve melek balığı yumurta açılım oranları (Şekil 1) tespit edilmiştir. Kontrol grubunda açılım 50% bulunurken bu oranı M5: %20, P2: %23.33, B1: %20, B5: %26.67, B10: %30 ve C5: %16.67 arttırmıştır (P<0.05). C1 (-%8.33), C10 (%3.33) ve S1 (-%13.33) grupları kontrol grubuyla benzer bulunmuştur (P>0.05). P4 (-%33.33), S5 (-%30) ve S10 (-%50) grupları ise yumurta açılımını azaltmıştır (P<0.05).

Çalışmada toplam bakteri en fazla kontrol grubunda tespit edilmiştir (Şekil 2). M5, P2 ve P4 grupları bakteri miktarını azaltmıştır (P<0.05). Bitki özütlerinden biberiye, civanperçemi ve sumak uygulamalarının tamamı bakteri yükünü azaltmıştır (P<0.05). B5, B10, C5 ve C10 grupları bakteri miktarı üzerinde birbirleriyle benzer etki göster-miştir (P>0.05).

TARTIŞMA ve SONUÇ

Bu çalışmada melek balığı yumurtalarının bulunduğu ortama ilave edilen bitki özütlerinin ve sentetik kim-yasalların bakteri yükü ve yumurta açılımı üzerinde etkili oldukları tespit edilmiştir.

Sentetik kimyasallardan metilen mavisinin melek balı- ğı yumurtalarında açılım oranını önemli miktarda arttırdığı (Şekil 1) ve bakteri yükünü azalttığı (Şekil 2) bulunmuştur. Benzer olarak melek balıklarında 0, 0.5, 2.5 ve 5 ppm metilen mavisi uygulamaları arasında en yüksek açılımı 0.5 ppm sağlamış (%63.3), ancak gruplar arasında fark tespit edilememiştir 6. Melek balıklarında yapılan başka bir çalışmada ise metilen mavisi, hidrojen peroksit, acriflavin ve chloramine-T uygulamaları yumurta açılımına etki etme-miştir 7. Melek balıklarında yapılan çalışmalarda kimyasal

Table 1. Changing hatching rates of the treatment groups

Tablo 1. Deneme gruplarının açılımı değiştirme oranları

Deneme Grupları Konsantrasyon (ppm) Açılımı Değiştirme Oranı (%) P

Kontrol 0 ---

Metilen Mavisi 0.5 +20.00* 0.038

P. permanganat0.2 +23.33* 0.011

0.4 -33.33* 0.000

Biberiye

1000 +20.00* 0.038

5000 +26.67* 0.003

10.000 +30.00* 0.001

Civanperçemi

1000 -8.33 0.795

5000 +16.67* 0.043

10.000 +3.33 1.000

Sumak

1000 -13.33 0.295

5000 -30.00* 0.001

10.000 -50.00* 0.000

* Dunett t-testi sonuçlarına göre kontrol grubundan istatistiksel açıdan farklı olan grupları göstermektedir

g

f

e

cd

bc

de

aab

ab

de

ab ab

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0102030405060708090

100

K M5 P2 P4 B1 B5 B10 C1 C5 C10 S1 S5 S10

Gruplar

Açı

lım O

ranı

(%))

Her bir deneme grubu ortalama ± standart hatayı temsil etmektedir (n=3)

Farklı harfleri içeren gruplar arasındaki fark istatistiksel olarak önemlidir (P<0.05)

Şekil 1. Deneme gruplarının yumurta açılım-larına etkisi

Fig 1. The effects of treatment groups on egg hatching rates

188Tıbbi Bitki Özütlerinin ...

uygulaması yapılmayan gruplarda dahi açılım oranının %14 ile %84 arasında değişim göstermesinin 7 nedeni, farklı partilerin yumurtalarının kullanılmasıyla açıklanabilir. Bu çalışmadaki melek balıklarında aynı parti yumurtaların kullanılması daha standart veriler elde edilmesini sağlamış olabilir.

Melek balıklarında potasyum permanganatın (P2), me-tilen mavisine (M5) benzer olarak yumurta açılımını arttırdığı (Şekil 1), ancak bakteri yükünü daha düşük oranda azalttığı (Şekil 2) tespit edilmiştir. Melek balığı yumurtalarının açılımı potasyum permanganatın 0.2 ppm uygulamasında %23.33 artmıştır (Tablo 1). Afrika kedibalığı (Clarias gariepinus)’nda yapılan farklı bir çalışmada ise potasyum permanganat 2 ppm dozunda yumurta açılımını %37.8’den %85.6’a çıkar- mıştır 14. Kedibalığında potasyum permanganat daha yük-sek konsantrasyon da kullanılmakla birlikte uygulamalar kısa süreli banyolar şeklinde yapılmıştır. Bu çalışmada ise potasyum permanganat düşük dozda uzun süre (3 gün) uygulanmış ve benzer olarak yumurta açılımının artmasını sağlamıştır. Ancak, artan oranda yumurta açılımını yaklaşık %33 oranında azaltmıştır (Tablo 1). Bunun nedeni yüksek oranda potasyum permanganatın yumurtalarda öldürücü etki yapmasıyla açıklanabilir. Permanganatın farklı balık ve kabuklu türlerinin yumurta ve larvaları üzerinde öldürücü etkisi 15 bu sonucu desteklemektedir.

Bu çalışmada suya uygulanan doğal ve sentetik ürün- lerin antimikrobiyal etkileri sayesinde mikrobiyal yükü azalttığı düşünülmektedir. Ayrıca toplam bakteri miktarı bakımından kimyasal ve bitkisel kaynakların etkilerine bakıldığında tüm grupların bakteri miktarını azalttıkları bulunmuştur (Şekil 2). Ancak sumağın tüm dozajlarında yumurta açılımının daha az olduğu görülmektedir (Şekil 1). Bunun nedeni sumağın potasyum permanganatta oldu-ğu gibi yumurtalar üzerinde toksik etki göstermesiyle açık-lanabilir. Benzer olarak farklı çalışmalarda kullanılan çeşitli sentetik kimyasalların kullanım konsantrasyonu arttıkça bakteri miktarı azalmış, ancak yumurta açılım oranı düşük bulunmuştur 7,16,17. Tıbbi bitkilerin sentetik kimyasallar gibi yüksek miktarda toksik etki göstermeleri içerdikleri

komponentlerden kaynaklanabilir. Bu durumda yüksek miktarlarda uygulandıklarında yumurtaları öldürdükleri söylenebilir.

Bu çalışmada deneme grupları arasında bakteri mikta-rının düşürülmesinde en etkili uygulamanın biberiye ol- duğu tespit edilmiştir. Melek balıklarında elde edilen bul-gulara benzer olarak alabalıklarda enfekte yumurta miktarı farklı bitkilerin esans yağ karışımıyla azaltılmıştır 9. Yapılan çalışmalarda metilen mavisi 18, potasyum permanganat 19, biberiye 20, sumak 11 ve civanperçeminin 21 antimikrobiyal ve antifungal etkileri bildirilmiştir. Bu etkileriyle sudaki bakteri yükünü azalttıkları ve dolayısıyla yumurta açılı-mınıda arttırdıkları düşünülmektedir. Benzer olarak çalış-malarda mikrobiyal yükün azalması yumurta açılımını art- tırmıştır 3,16,22. Ancak bazı araştırmacılar mikrobiyal yük ile yumurta açılımının artması arasında önemli bir ilişki olma- dığını bildirmemişlerdir 12,23,24. Elde edilen farklı sonuçlar yumurtalar üzerinde kullanılan kimyasalların dozajının uygun olmaması, mikrobiyal yük dışında çevresel faktörler, beçler arasındaki farklılıklar, yumurta kalitesindeki deği-şimler, mantar, virus, parazit gibi etkenler ve tümünün birlikte etkileriyle açıklanabilir.

Çalışma sonunda biberiye özütünün 1.000 ppm, 5.000 ppm ve 10.000 ppm uygulamaları, yumurta açılımını benzer oranda arttırmıştır. Bu nedenle 1.000 ppm biberiye özütünün melek balıklarında kontrole göre yeterli yumurta açılımını sağladığı, potasyum permanganat ve metilen mavisi yerine kullanılabileceği sonucuna varılmıştır. İleriki çalışmalarda sumağın daha düşük dozları ve civan-perçeminin ara dozları denenebilir.

Teşekkür

Çalışmada kullanılan kaynakların teminindeki katkıların-dan dolayı Sayın Dr. Özcan ÖZEN’e teşekkürlerimizi sunarız.

KAYNAKLAR

1. Wagner EJ, Arndt RE, Billman EJ, Forest A, Cavender W: Comparison of the efficacy of iodine, formalin, salt, and hydrogen peroxide for control

ab

c cdde

ef

gf

ghghihi

i i0.000.501.001.502.002.503.003.504.00

K M5 P2 P4 B1 B5 B10 C1 C5 C10 S1 S5 S10

Gruplar

Log

N (c

fu/m

l))

Her bir deneme grubu ortalama ± standart hatayı temsil etmektedir (n=3)

Farklı harfleri içeren gruplar arasındaki fark istatistiksel olarak önemlidir (P<0.05)

Şekil 2. Deneme gruplarının yumurta suyun-daki bakteri yüküne etksi

Fig 2. The effects of treatment groups on bacterial loads in egg water

189YILMAZ, ERGÜN

of external bacteria on rainbow trout eggs. N Am J Aquacult, 70 (2): 118-127, 2008.

2. Straus DL, Mitchell AJ, Radomski AA, Carter RR: Laboratory dose confirmation of copper sulfate for treating fungus on channel catfish eggs. N Am J Aquacult, 71 (4): 333-338, 2009.

3. Can E, Saka Ş, Fırat K: Disinfection of gilthead sea bream (Sparus aurata), red porgy (Pagrus pagrus), and common dentex (Dentex dentex) eggs from sparidae with different disinfectants. Kafkas Univ Vet Fak Derg, 16 (2): 299-306, 2010.

4. Yılmaz M, Mutaf BF, İkiz R: Melek Balıklarında (Pterophyllum scalare Lichtenstein, 1823) birinci döl bireylerinde renk-desen açılımının izlenmesi ile ebeveyn genotiplerinin belirlenmesi. Turk J Fish Aquat Sci, 23 (1-2): 173-176, 2006.

5. Swann L: Reproduction of angelfish (Pterophyllum scalare). Illinois-Indiana Sea Grant Program Fact Sheet, Soyink, AS-489: 6 pp, 1993.

6. Perlberg ST, Diamant A, Ofir R, Zilberg D: Characterization of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare (Schultz), and the effect of exposure to methylene blue. J Fish Dis, 31 (3): 215-228, 2008.

7. Sanabria C, Diamant A, Zilberg D: Effects of commonly used disinfectants and temperature on swim bladder non-inflation in fresh-water angelfish, Pterophyllum scalare (Lichtenstein). Aquaculture, 292 (3-4): 158-165, 2009.

8. Citarasu T: Herbal biomedicines: a new opportunity for aquaculture industry. Aquacult Int, 18 (3): 403-414, 2010.

9. Mousavi SM, Mirzargar SS, Mousavi HEZ, Baigi RO, Khosravi A, Bahonar A, Ahmadi MR: Evaluation of antifungal activity of new combined essential oils in comparison with malachite green on hatching rate in rainbow trout (Oncorhynchus mykiss) eggs. Turk J Fish Aquat Sci, 4 (2): 103-110, 2009.

10. Chitmanat C, Tongdonmuan K, Khanom P, Pachontis P, Nunsong W: Antiparasitic, antibacterial, and antifungal activities derived from a Terminalia catappa solution against some tilapia (Oreochromis niloticus) pathogens. Songklanakarin J Sci Technol, 27 (Suppl.1): 359-364, 2005.

11. Nasar-Abbas SM, Halkman AK: Antimicrobial effect of water extract of sumac (Rhus coriaria L.) on the growth of some food borne bacteria including pathogens. Int J Food Microbiol, 97 (1): 63-69, 2004.

12. Komar C, Turnbull JF, Roque A, Fajer E, Duncan NJ: Effect of water treatment and aeration on the percentage hatch of demersal, adhesive

eggs of the bullseye puffer (Sphoeroides annulatus). Aquaculture, 229 (1-4): 147-158, 2004.

13. Logan M: Single factor classification (ANOVA). In, Logan M (Ed): Biostatistical Design and Analysis Using r: A Practical Guide. 1st ed., pp. 254-282, Wiley-Blackwell, London, 2010.

14. Rasowo J, Okoth OE, Ngugi CC: Effects of formaldehyde, sodium chloride, potassium permanganate and hydrogen peroxide on hatch rate of African catfish Clarias gariepinus eggs. Aquaculture, 269 (1-4): 271-277, 2007.

15. Melendre PM, Celada JD, Carral JM, Sáez-Royuela M, Aguilera A: Effectiveness of antifungal treatments during artificial incubation of the signal crayfish eggs (Pacifastacus leniusculus Dana. Astacidae). Aquaculture, 257 (1-4): 257-265, 2006.

16. Treasurera JW, Cochrane E, Grant A: Surface disinfection of cod Gadus morhua and haddock Melanogrammus aeglefinus eggs with bronopol. Aquaculture, 250 (1-2): 27-35, 2005.

17. Mitchell AJ, Radomski AA, Straus DL, Carter R: The effect of hydrogen peroxide on the hatch rate and Saprolegnia spp. infestation of channel catfish eggs. N Am J Aquacult, 71 (3): 276-280, 2009.

18. Peloi LS, Soares RRS, Biondo CEG, Souza VR, Hioka N, Kimura E: Photodynamic effect of light-emitting diode light on cell growth inhibition induced by methylene blue. J Biosciences, 33 (2): 231-237, 2008.

19. Wakabayashi H: Effects of environmental conditions on the infectivity of Flexibacter columnaris to fish. J Fish Dis, 14(3): 279-290, 1991.

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21. Kokoska L, Polesny Z, Rada V, Nepovim A, Vanek T: Screening of some siberian medicinal plants for antimicrobial activity. J Ethnopharmacol, 82 (1): 51-53, 2002.

22. Holcomb M, Cloud JG Ingermann RL: Impact of bacteria on short-term storage of salmonid eggs. Aquac Res, 36 (15): 1555-1561, 2005.

23. Tendencia EA: Bacterial microbiota of eggs from cage-reared and tank-reared grouper, Epinephelus coioides. Bull Eur Ass Fish Pathol, 24 (3): 161-165, 2004.

24. Miguez B, Combarro MP, Guisande C, Vergara AR Riveiro I: Effect of bacterial epiflora on egg hatching of the Atlantic sardine (Sardina pilchardus). FEMS Microbiol Ecol, 50 (2): 111-115, 2004.


SummaryAim of this study is to analyze the genetic structure of long-crowing Denizli chicken, a Turkish local fowl, using nucleotide sequences

of the mitochondrial DNA 12S and D-loop regions. DNA isolation was carried out using feather samples of cocks of this local breed. D-loop and 12S regions were amplified using polymerase chain reaction technique and ca 1230 bp and 950 pb PCR products were obtained respectively. Native genotype Denizli fowl’s D-loop and 12S regions were sequenced and sequencing data were analyzed and compared with the related published data. Nucleotide content of the 12S region showed a 32% A, 19% T, 19% G and 30% C, whilst AT and GC ratios were found as 59.88% and 40.12% respectively for D-loop region. D-loop sequence data analysis clearly identified the Denizli fowl within the clade of long-crowing cocks. The results give the first informative data about the mitochondrial DNA D-loop and 12S regions of this local breed from Turkey.

Keywords: 12S rDNA, D-loop, Denizli fowl, Local breed, mt-DNA

Türkiye Uzun Ötüşlü Yerli Irkı Denizli Tavuğunun Mitokondriyal DNA D-loop ve 12S Bölgeleri Analizi

ÖzetÇalışmanın amacını ülkemiz yerli tavuk ırklarından olan uzun ötüşlü Denizli tavuğunun mitokondriyal DNA 12S ve D-loop

bölgelerinin gen dizi analizine göre genetik yapısının ortaya çıkarılması oluşturmaktadır. Bu amaçla DNA izolasyonu için Denizli horozlarından alınan tüy örnekleri kullanılmıştır. D-loop ve 12S bölgeleri polimeraz Zincir Reaksiyonu (PZR) ile amplifiye edilmiş ve sırasıyla 1230 bç ve 950 bç uzunluğunda PZR ürünleri elde edilmiştir. D-loop ve 12S bölgelerinin gen dizi analizleri çıkarılmış ve daha önce yayınlanmış ilgili gen bölgeleriyle analize tabi tutulmuştur. 12S bölgesinin gen dizisi incelendiğinde %32A, %19T, %19G ve %30C olarak bulunmuştur. D-loop bölgesi için AT ve GC oranları ise sırasıyla %59.88 ve %40.12 olarak gözlemlenmiştir. D-loop bölgesi gen dizi analizi sonuçları Denizli ırkının „uzun ötüşlü ırklar arasında yer aldığını açık olarak ortaya koymuştur. Sonuçlar bu lokal ırkın mitokondriyal DNA’sı hakkında ilk informatif verileri ortaya koymuştur.

Anahtar sözcükler: 12S rDNA, D-loop, Denizli tavuğu, Lokal ırk, mt-DNA

Mitochondrial DNA D-loop and 12S Regions Analysis of the Long-Crowing Local Breed Denizli Fowl from Turkey

Mesut KARAMAN * Nurside KIRDAG *

* Kahramanmaras Sutcu Imam University, Agriculture Faculty, Animal Science Department, TR-46100 Kahramanmaras - TURKEY


Although most animal species were used and domesticated as work animals or as a food source, historical and archaeological findings strongly suggest that chicken was first functioned in religious ceremonials, decorative art or as entertainment. Domesticated fowl species are now widely distributed around the world and are bred in various categories for different purposes such as food source as egg type and meat type, decorative art as ornaments, fighting or game instrument. With regard to

any role, human culture has had a strong influence on the domestication of chickens. Throughout history, however, chickens have been bred for various other purposes. Long-crowing chickens and fighting cocks are typical examples of chickens that have been bred for purposes rather than as human food or decorative ornament or adornment material , and are therefore excellent species for studying the relationship between selective breeding and human culture 1,2. Chickens have played an important role in the

INTRODUCTION

İletişim (Correspondence) +90 344 2191618 GSM: +90 532 6050265 [email protected]

RESEARCH ARTICLE

192Mitochondrial DNA D-loop and ...

development of human culture, particularly in Asian countries such as China, Thailand and Japan.

Four species of Gallus have currently been reported as grey junglefowl (Gallus sonnerati), red junglefowl (Gallus gallus), ceylon junglefowl (Gallus lafayettei) and green junglefowl (Gallus varius), only Gallus gallus is suggested to be the main progenitor of all contemporary domesticated chicken breeds 3,4. This view is consistent with the discussion that the domestication and breeding of chickens was initiated with junglefowls, which then only inhabited Southeast Asian regions such as Indochina and South China 3,4. Exact location of domestication of Gallus gallus, however, is still remain controversial 5 since the findings of these researchers suggest that contemporary fowls have multiple origin in South Asia and Southeast Asia.

Denizli chicken (Gallus gallus domesticus) is a local breed of Turkey and vast majority of them are habituated at Western Anatolia. Because of poor commercial performance they provide an excellent example of seriously threatened populations with immediate risk of extinctions, and therefore, Turkey has undertaken a conservation program operated by the Lalahan Livestock Central Research Institute and supported by Turkish Ministry of Agricultural and Rural Affairs since 1997. The females of this breed are considered as layer type chicken breed although their egg production is about 110-120 eggs for 29 week laying period, whilst their cocks are famous for long-crowing 6. They can crow up to 20 seconds for 1-year-old and up to 24 seconds for 3-year-old mature cocks, and in some cases they could become unconscious following a long crowing session. There is no report (to our best knowledge) that Denizli chicken exists in nature, but semi-selectively bred by local people for their relatively robust body and long-crowing characters. One recent report by Kaya and Yildiz 7 about the estimation of genetic diversity by microsatellite markers seems the only study aiming the exploration of genetic structure of this local breed. Therefore, the origin, domestication story and pyhlogenetic relationship with other fowls in particular with long-crowing cocks of this local chicken remain unknown.

This study aims to examine the mitochondrial DNA regions, D-loop and 12S, sequences of long-crowing Denizli chicken from the viewpoint of molecular phylogeny. The sequence data of the current study was also analyzed after combination with the earlier published data representing game cocks and long crowing cocks located in GenBank web site (http://www.ncbi.nlm.nih.gov/).


Samples

Feather samples of two pure bred cocks were obtained from the Denizli Provisional Directorate of Agriculture in

where this bred is under conservation scheme. The feather samples were washed twice using 70 % EtOH and kept under aseptic conditions at -20oC until required for DNA extraction.

DNA Extraction

Feather samples were cut by a sterile scissors into small pieces and treated using liquid nitrogen to disrupt the cells prior to DNA extraction. Phenol-chloroform based DNA isolation was performed according to Villalta et al.8 (originally devised to extract fish tissue DNA) with the slight modification of the incubation time which was elongated to 48 h in extraction buffer at 55oC. Extracted DNAs were either used freshly or kept in 100 ml of TE buffer [10 mM Tris-HCl (pH=8.0), 1mM EDTA] 9 at -20 for further studies.

Primer Design for D-loop and 12S Regions

Designing of forward and reverse primers for both D-loop and 12S mitochondrial regions were conducted using published mitochondrial DNA sequence data of Gallus gallus derived from the web server of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). For amplification of the D-loop region forward 5’-TTA ACC TAA CTC CCC TAC TAA GTG TA-3’ and reverse 5’-TCT TCC GTA AAA CAC AAA CC-3’primers were used, whilst forward 5’-GGT TTT TGC TAG ACA TAT ACA TGC-3’ and reverse 5’-CAT CAG ATT CAC GTG GAA GGC -3’ primers were designed for the amplification of 12S region. The designed primers for both mitochondrial D-loop and 12S regions were synthesized by a commercial company (Iontek, Istanbul-Turkey).

PCR Amplification

PCR amplification of the extracted DNAs was performed for 50 ml in size. Amplification primers were synthesized by a commercial company (Iontek, Istanbul), dNTPs and Hi-Fi Taq DNA polymerase were supplied by Favorgen (Favorgen Biotech Corp., Taiwan). The optimized denaturation, annealing and extension characteristics of PCR amplification steps for both regions are shown in Table 1. Following the amplification, all PCR products were run in 1% agarose gel and post stained using ethidium bromide solution 0.1% (w/v) for 15min, and finally photographed with the aid of a digital camera (Canon Powershot S3 IS, Japan) under 312nm wavelength ultra violet (UV) trans-limunator light (UVP, UK). The PCR products were purified using PCR clean up kit (Favorgen Biotech Corp., Taiwan) according to manufacture’s instruction prior to sequencing process.

Sequencing Procedure and Data Analysis

Sequencing process was performed by a commercial company (Iontek, Istanbul-Turkey) as direct sequencing from purified PCR products. Sequencing process for D-loop and 12S region were conducted in both forward

193KARAMAN, KIRDAG

and reverse directions and all sequence data were edited using Bioedit 7.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and FinchTV Version 1.4.0 (http://www.geospiza.com/finchtv) following naked eye checking. The sequence data produced were aligned to earlier reported Gallus gallus sequence data using CLUSTAL X program 10 with default parameters. The bootstrap analysis of 1000 replications was performed to assess the statistical confidence of the phylogenetic trees which were constructed by the neighbor joining method. Conversion of the data format and DNA polymophism analysis were carried out with the aid of DNaSP 4.02 11.

RESULTS

PCR Amplification and Nucleotide Ratios of D-loop and 12S Regions

A partial PCR amplification of the D-loop region of the Denizli fowl yielded a ca 1210bp PCR product (Fig. 1) and

1094 bp of that fragment was successfully sequenced and analyzed. The sequence data of D-loop region of Denizli cock was submitted to the GenBank (http://www.ncbi.nlm.nih.gov) and located there with the accession number of EU194446. Nucleotide ratios of the D-loop region were calculated as 26% A, 34% T, 14% G and 26% C. Relatively higher AT ratio compare to GC content for this region was observed, and ratios for these data were found as 59.8% and 40.2% respectively.

For 12S region, newly designed primers amplified a ca 950bp PCR fragment (Fig 1b) and 872bp length of this product was successfully sequenced and analyzed. The sequence data of 12S region of Denizli fowl is deposited on the GenBank web site with the accession number of FJ610338 (http://www.ncbi.nlm.nih.gov). The nucleotide content of the sequenced data was calculated as 32% A, 19% T, 19% G and 30% C. Balanced AT and GC ratios were observed for 12S region since these values were found as 51.8% and 48.2% respectively.

Table 1. The optimized polymerase chain reaction amplification characteristics of mitochondrial D-loop and 12S regions

Tablo 1. Mitokondriyal DNA D-loop ve 12S bölgeleri için Polimeraz Zincir Reaksiyonu optimizasyonu

PCR Steps D-loop 12S

Initial Denaturation 94ºC 4 min

Denaturation 94ºC 1 min

35 cycle

94ºC 1 min

35 cycleAnnealing55ºC 30 sec 55ºC 30 sec

63ºC 30 sec 58ºC 30 sec

Extension 72ºC 2 min 72ºC 2 min

10

1 Fig 1. Polymerase chain reaction amplification of the D-loop (a) and 12S (b) regions yielded 2

a ca1210bp and 950bp PCR products respectively. M: 1kb ladder from Favorgen Biotech. 3

corp. (Taiwan) 4

5

Şekil 1. Polimeraz zincir reaksiyonu (PZR) amplifikasyonu D-loop (a) için ca 1210bç 12S 6

bölgesi (b) için ca950 bç PZR ürünü ortaya koymuştur. M: 1kb merdiven, Favorgen Biotech. 7

corp. (Taiwan) 8

9

10

11

12

13

14

15

16

17

D-loop M M

12S

950bp 1210bp

a b

Fig 1. Polymerase chain reaction amplification of the D-loop (a) and 12S (b) regions yielded a ca1210bp and 950bp PCR products respectively. M: 1kb ladder from Favorgen Biotech. corp. (Taiwan)

Şekil 1. Polimeraz zincir reaksiyonu (PZR) amplifikasyonu D-loop (a) için ca 1210bç 12S bölgesi (b) için ca950 bç PZR ürünü ortaya koymuştur. M: 1kb merdiven, Favorgen Biotech. corp. (Taiwan)


Phylogenetic Trees According to Sequence Data of D-loop and 12S Regions

An unrooted tree was generated using the nucleotide sequence of D-loop region of mitochondrial DNA obtained from 30 samples of fighting, long-crowing and Denizli cocks. The unrooted Upgma tree of 30 samples revealed three divergent clades designated as A, B and C (Fig. 2).

The haplotypes located in clades A and B represent the game-cocks, while clade C composed of long-crowing cocks and Denizli cock. Although Denizli cock shared the same clade with long-crowing cocks, a finer analysis showed that the sequence data of this Turkish local breed was differed from the remaining part of the clade by at least 7 single nucleotide mutations (see FJ610338 - Denizli fowls 12S region accession number).

The sequencing data of mitochondrial 12S region was analyzed for phylogenetic purposes too (Fig. 3). To obtain a better alignment with the earlier reported data in GenBank, first 6 bases (GGAGGA) were excluded from the submitted 12S sequence of the Denizli fowl (Accession no: FJ610338). A rooted tree, for 12S region, was generated using the sequence data of local breed Denizli chicken and 18 published data belong to Gallus gallus derived from GenBank web site.

As seen in Fig. 3, 12S region of the Denizli chicken is quite similar to those various subspecies of Gallus gallus. Less than 1% differences was observed among the studied 12S regions of 18 fowls. Further, genetic structure of 12S region of Denizli fowl was identical to the White Leghorn (Gallus gallus).

11

Fig 2. Unrooted phylogenetic tree of fighting and long-crowing cocks including Denizli local

bred derived from mitochondrial D-loop region. Numbers of the samples and representing

GenBank accession numbers are as follow; 1 (AB098699); 2 (AB098698); 3 (AB098697); 4

(AB098695); 5 (AB098694); 6 (AB098693); 7 (AB098692); 8 (AB098686); 9 (AB098660);

10 (AB098651); 11 (AB098649); 12 (AB098647); 13 (AB098644); 14 (AB098637); 15

(AB098662); 16 (AB098654); 17 (AB098653); 18 (AB098646); 19 (AB098639); 20

(AB098638); 21 (AB114065); 22 (AB114064); 23 (AB114063); 24 (AB114062); 25

(AB114059); 26 (AB114058); 27 (AB114066); 28 (AB114060); Denizli (EU194446)

Şekil 2. Dövüşcü ve Denizli rknnda içerisinde yer aldğ uzun ötüşlü rklarn mitokondriyal

DNA D-loop bölgelerine göre oluşturulmuş köksüz filogenetik yap. İlgili GenBank ulaşm

numaralar ise şu şekildedir; 1 (AB098699); 2 (AB098698); 3 (AB098697); 4 (AB098695); 5

(AB098694); 6 (AB098693); 7 (AB098692); 8 (AB098686); 9 (AB098660); 10 (AB098651);

11 (AB098649); 12 (AB098647); 13 (AB098644); 14 (AB098637); 15 (AB098662); 16

(AB098654); 17 (AB098653); 18 (AB098646); 19 (AB098639); 20 (AB098638); 21

Fighting cocks

Long crowing cocks Clade C

Clade B

Clade A

Fig 2. Unrooted phylogenetic tree of fighting and long-crowing cocks including Denizli local bred derived from mitochondrial D-loop region. Numbers of the samples and representing GenBank accession numbers are as follow; 1 (AB098699); 2 (AB098698); 3 (AB098697); 4 (AB098695); 5 (AB098694); 6 (AB098693); 7 (AB098692); 8 (AB098686); 9 (AB098660); 10 (AB098651); 11 (AB098649); 12 (AB098647); 13 (AB098644); 14 (AB098637); 15 (AB098662); 16 (AB098654); 17 (AB098653); 18 (AB098646); 19 (AB098639); 20 (AB098638); 21 (AB114065); 22 (AB114064); 23 (AB114063); 24 (AB114062); 25 (AB114059); 26 (AB114058); 27 (AB114066); 28 (AB114060); Denizli (EU194446)

Şekil 2. Dövüşcü ve Denizli ırkınında içerisinde yer aldığı uzun ötüşlü ırkların mitokondriyal DNA D-loop bölgelerine göre oluşturulmuş köksüz filogenetik yapı. İlgili GenBank ulaşım numaraları ise şu şekildedir; 1 (AB098699); 2 (AB098698); 3 (AB098697); 4 (AB098695); 5 (AB098694); 6 (AB098693); 7 (AB098692); 8 (AB098686); 9 (AB098660); 10 (AB098651); 11 (AB098649); 12 (AB098647); 13 (AB098644); 14 (AB098637); 15 (AB098662); 16 (AB098654); 17 (AB098653); 18 (AB098646); 19 (AB098639); 20 (AB098638); 21 (AB114065); 22 (AB114064); 23 (AB114063); 24 (AB114062); 25 (AB114059); 26 (AB114058); 27 (AB114066); 28 (AB114060); Denizli (EU194446)

195KARAMAN, KIRDAG

DISCUSSION

Domesticated fowls are widely distributed throughout the world and recent archaeological studies strongly suggest that their domestication has originated in China

nearly 8.000 years ago 12. Domesticated chickens are currently bred for various purposes, not only because of their usefulness for human nutrition but also for their long crowing, outstanding characteristic songs, colorful appearance (as religious symbols), entertainment and

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14

Denizli

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6

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5

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Fig 3. A rooted phylogenetic tree is generated using Denizli fowl and 18 Gallus gallus samples derived from the 12S rDNA sequence data reported in GenBank web server. The sample numbers representing GenBank accession numbers used to obtain the phylogenetic tree were as follow; 1 (DQ648776), 2 (AP003323), 3 (AP003322), 4 (AP003319), 5 (AP003318), 6 (AP003580), 7 (AY235571), 8 (AP003317), 9 (X52392), 10 (AB086102), 11 (AY235570), 12 (AP003321), 13 (EF373905), 14 (EF373873), 15 (DQ885561), 16 (AJ490505), 17 (AJ583547), 18 (AY309497), Denizli (FJ610338)

Şekil 3. GenBank’tan alınmış 18 Gallus gallus örneği ve Denizli ırkına ait 12S rDNA bölgesi gen dizisine gore oluşturulmuş köklü filogenetik yapı. İlgili GenBank ulaşım numaraları ise şu şekildedir; 1 (DQ648776), 2 (AP003323), 3 (AP003322), 4 (AP003319), 5 (AP003318), 6 (AP003580), 7 (AY235571), 8 (AP003317), 9 (X52392), 10 (AB086102), 11 (AY235570), 12 (AP003321), 13 (EF373905), 14 (EF373873), 15 (DQ885561), 16 (AJ490505), 17 (AJ583547), 18 (AY309497), Denizli (FJ610338)


ceremonial purposes 13.

Turkey has two native chickens, Denizli and Gerze fowls, which are seriously threatened with extinction and they are under a governmental conservation program since 1997. The former one, Denizli chicken, is bred mainly because of its long crowing characteristics as hobby pet, rather than egg or meat production. Although several studies were carried out about their egg- and meat production characteristics 14,15, quite few works has focused on their genetic structures 7,16. The current study is the first attempt which points out the mitochondrial DNA D-loop and 12S regions of this local breed of Turkey.

The analysis of mitochondrial DNA D-loop region sequence data has clearly demonstrated that Denizli chicken is more closely related to long crowing fowls rather than fighting cocks. Finer analysis of D-loop sequence data suggested that within the long crowing clade, Denizli cocks could be separated into subclade. The lack of sequence data for mitochondrial DNA 12S region of long crowing cocks on web site of GenBank hampered the opportunity to discus the phlylogenetic position of Denizli chicken with other long crowing cocks using that particular informative region. 12S sequence data located in GenBank (for other Gallus gallus) were compared with the same region of Denizli fowl. The results of this comparison showed that 12S region of Denizli fowl were almost identical to the 12S nucleotide structure of 18 Gallus gallus samples.

Long crowing fowls are in special interest for human all over the world and, in particular, for the people living in Asia. Komiyama et al.17 conjectured that the culture of cockfighting was closely related with that of long crowing and therefore long crowing culture was derived from that of cock fighting. There is no scientific report or cultural knowledge, however, that Denizli fowl is used for cock fighting although they are famed for their long crowing characters and people would like to have them as hobby animals because of their colorful appearance and exclusive songs.

Further studies are required to explore the genetic structure of this local breed which is under severe threaten in extinction. Complete mitochondrial DNA sequence analysis of this local breed will provide us more information and give as an opportunity to conjecture its possible origin and relationship with other chicken breeds in particular with long crowing cocks more accurately.

REFERENCES

1. Komiyama T, Ikeo K, Gojobori T: Where is the origin of the Japanese gamecocks? Gene 317, 195-202, 2003.

2. Liu YP, Zhu Q, Yao YG: Genetic relationship of Chinese and Japanese gamecocks revealed by mtDNA sequence variation. Biochem Genet, 44, 19-29, 2006.

3. Fumihito A, Myiake T, Takada M, Shingu R, Endo T, Gojobori T, Kondo N, Ohno S: Monophyletic origin and unique dispersal patterns of domestic fowls. Proceedings of the National Academy of Sciences, 93, 6792-6795, 1996.

4. Niu D, Fu Y, Luo J, Ruan H, Yu XP, Chen G, Zhang YP: The origin and genetic diversity of Chinese native chicken breeds. Biochem Genet, 41, 163-174, 2002.

5. Liu YP, Wu GS, Yao YG, Miao YW, Luikart G, Baig M, Beja- Pereira A, Ding ZL, Palanichamy MG, Zhang YP: Multiple maternal origins of chickens: Out of the Asian jungles. Mol Phylo Evo, 38, 12-19, 2006.

6. Sarica M: Tavugun evciltilmesi, tavuk ırklari ve hibritler, in Turkoglu, M. and Sarica, M. (Eds): Poultry Sci, pp. 33-71. 2004.

7. Kaya M, Yildiz MA: Genetic diversity among Turkish native chickens, Denizli and Gerze, estimated by microsatellite markers. Biochem Genet 24, 480-491, 2008.

8. Villalta M, Lavado A, Obeso A, Larrayad R, Pavia P, Montoya J, Blasco JM: mtDNA variation in Spanish brown trouts: Evidence of alien genotypes in restocking programmes. Servicio de Investigación Agroalimentaria, Diputación General de Aragón 50080 Zaragoza, Spain, 1997.

9. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual Appendixes 2nd

ed., Cold Spring Harbor Laboratory Press, USA.

1989.

10. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Resc, 25, 4876-4882, 1997.

11. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19, 2496-2497, 2003.

12. West B, Zhou BX: Did chickens go North? New evidence for domestication. J Archaeol Sci, 14, 515-533, 1988.

13. Komiyama T, Ikeo K, Tateno Y, Gojobori T: Japanase domesticated chickens have been derived from Shamo traditional fighting cocks. Mol Phylo Evol 33, 16-21, 2004.

14. Sekeroglu A, Ozen N: Gerze ve Denizli tavuk ırklarının bazı verim özellikleri bakımından karşılaştırılması. Akdeniz Univ J Fac Agric, 10, 41-57, 1997.

15. Atasoy F, Onbasilar EE, Apaydın S: Comparison of egg quality characteristics between Denizli and commercial layer flocks. Lalahan Hay Aras Enst Derg, 41, 89-100, 2001.

16. Aksoy FT, Ertugrul O, Atasoy F. Gurler S, Erdogan M: A study on blood group alles of Denizli fowl. Turk J Vet Anim Sci, 24, 431-434, 2000.

17. Komiyama T, Ikeo K, Gojobori T: The evolutionary origin of long-crowing chicken: Its evolutionary relationship with fighting cocks disclosed by the mtDNA sequence analysis. Gene, 26, 91-99, 2004.


SummaryThe bacterial pathogen Flavobacterium psychrophilum (F. psychrophilum) causes rainbow trout (Oncorhynchus mykiss) fry syndorme

(RTFS) causes economic losses and widespread in many countries. The aims of the present study are isolation of F. psychrophilum, and determination of antibacterial susceptibility and an effective antibacterial treatment in rainbow trout fry. For this purpose, weighted 2-5 g fry were obtained from a private fish farm in west Aegean region of Turkey. It was estimated according to clinical findings that they were naturally infected with F. psychrophilum originate from the rearing unit. Following fenotipical, biochemical and enzyme tests aimed the identification of bacteria. Kirby-Bauer disc diffusion method was used for determination of antibacterial susceptibility. According to antibiotic susceptibility tests, F. psychrophilum was susceptible to oxytetracycline, enrofloxacin, ciprofloxacin and florfenicol. The antibiotic concentrations in the medicated feeds were to give a dose of 75 mg/kg/day oxytetracycline and 10 mg/kg/day enrofloxacin, ciprofloxacin and florfenicol each antibiotics for 10 days. When considered the clinical signs and death rates among the infected rainbow trout fry, oxytetracycline, enrofloxacin and ciprofloxacin could not displayed enough efficacy. However, florfenicol showed much higher efficacy for controlling the infection and at the end of the treatment, the death rate caused by the infection was decreased significantly.

Keywords: Flavobacterium psychrophilum, Antimicrobial Susceptibility, Florfenicol, Treatment

Gökkuşağı Alabalıklarında (Oncorhynchus mykiss) RTFS’ye (Rainbow trout Fry Syndrome) Neden Olan Flavobacterium

psychrophilum Etkeninin Izolasyonu ve Antibakteriyel Sağaltım Seçeneğinin Belirlenmesi

ÖzetGökkuşağı alabalık (Oncorhynchus mykiss) yavrularında Flavobacterium psychrophilum (F. psychrophilum)’un neden olduğu Yavru

Gökkuşağı Alabalığı Sendromu (Rainbow Trout Fry Syndrome, RTFS) birçok ülkede yaygın olarak görülen ve ekonomik kayıplara neden olan bakteriyel bir hastalıktır. Bu çalışmada F. psychrophilum suşunun izolasyonu ve identifikasyonu, antibakteriyel duyarlılığı ve etkili antibakteriyel ilaç/ilaçlarla sağaltım seçeneklerinin belirlenmesi amaçlanmıştır. Bu amaçla Ege Bölgesi’nin batısında bulunan özel bir işletmede, doğal yolla F. psychrophilum ile enfekte olduğu tahmin edilen yavruhane bölümünde meydana gelen enfeksiyon sonucunda, canlı ağırlıkları 2-5 g arasında hastalıklı gökkuşağı alabalık yavrusu kullanıldı. İdentifikasyon amacıyla bakterilerin fenotipik, biyokimyasal ve enzim testleri gerçekleştirildi. Kirby-Bauer disk difüzyon yöntemine göre yapılan antibiyogram test sonuçlarına göre etkenin oksitetrasiklin, enrofloksasin, siprofloksasin ve florfenikole duyarlı olduğu belirlendi. Sağaltım gruplarına oksitetrasiklin 75 mg/kg/gün dozda, enrofloksasin, siprofloksasin ve florfenikol 10 mg/kg/gün dozda ve her biri 10 gün süreyle uygulandı. Gökkuşağı alabalık yavrularında klinik bulgular ve ölüm oranları dikkate alındığında; oksitetrasiklin, enrofloksasin ve siprofloksasinin sağaltım için yeterli etkinlik gösteremedikleri, florfenikolün ise diğer sağaltım gruplarına göre enfeksiyonu kontrol almada önemli derecede etkin olduğu ve sağaltım sonunda enfeksiyondan kaynaklı ölüm oranlarını azaltığı belirlendi.

Anahtar sözcükler: Flavobacterium psychrophilum, Antimikrobiyal duyarlilik, Florfenikol, Sağaltım

Isolation of Flavobacterium psychrophilum Causing Rainbow Trout Fry Syndrome and Determination of an Effective Antibacterial

Treatment in Rainbow Trout (Oncorhynchus mykiss) Fry [1]

Murat BOYACIOGLU * Ferda AKAR *

[1]*

This work was supported by the Adnan Menderes University, Scientific Research Projects Commission VTF-06005 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, TR-09016 Aydin - TURKEY



RESEARCH ARTICLE

198Isolation of Flavobacterium ...

INTRODUCTION

The bacterial pathogen Flavobacterium psychrophilum (formerly Cytopahaga psychrophila or Flexibacter psychrophilus) causes rainbow trout (Oncorhynchus mykiss) fry syndorme (RTFS) and also is the agent of bacterial coldwater disease in larger fish 1-6. RTFS widespread in many countries and causes severe mortalities and economic losses of rainbow trout fry in hatcheries in Turkey as well as in most other European countries. The RTFS is considered to be on of the most serious bacterial diseases of rainbow trout in Turkey, and cumulative mortility rates can be as high as 70% 7-11. Similar mortality rates are seen in aquaculture in other countries 2-6,12-15.

F. psychrophilum has been found in skin mucus, and connective tissue of the fins, gills and operculum of salmonid fish 1,16. Lorenzen 17 demonstrated F. psychrophilum in the lumen and the mucosa/submucosa of the stomach of naturally infected rainbow trout fry, pointing towards the involvement of the gastro-intestinal tract as a portal of entry. On the other hand, it has been isolated from diseased fish and from fish without any sign of disease 1,14. Handling and stress can exacerbate the disease 18. The most manifest sign is anaemia as revealed by pale gills, kidney, intestine and liver. Clinically, the fry appear lethargic, inappetant and hang at the water surface 19. Primarily observed splenomegali, a white, fragile intestine and a hemorrhagic, protruding anus. In some cases, skin erosions have been reported, and these usually are found behind the dorsal fin or on the flank 18. Fish infected with F. psychrophilum as fry may display ataxia, spiral swimming and eventually spinal column deformities 2,13. By comparison to other bacterial fish pathogens, there is no commercial vaccine available to prevent F. psychrophilum 4,20. Antimicrobial therapy is still the most effective (and used) way of combating F. psychrophilum infections 19.

Proper management of fish culture conditions, monitoring and maintenance of high standards for water quality, and adequate sanitation procedures are other essential requisites that may help to avoid and/or limit the severity of RTFS epizootics 20. The risk of antibiotic treatments inadvertently selecting for drug resistance in bacterial fish pathogens that may also cause disease in humans remains a grave concern in fish health control practices 21,22. Antimicrobial agents are released into the surrounding water during medical treatment of bacterial disease 23. Many studies show that F. psychrophilum is resistant to most antibiotics, and antibiotic resistance may be different by geographic region. F. psychrophilum is capable of acquiring resistance quite easily as seen with oxytetracycline, oxolinic acid, and amoxicillin in Danish freshwater fish farms 23,24. Furthermore F. psychrophilum strains were resistante to both nalidixic acid and oxolinic acid in Japan and United States 5. In France, chloramphenicol resistance was more frequent

than florfenicol resistance in F. psychrophilum isolated from cases of RTFS 22. F. psychrophilum spp. were resistante to cephalexin, cephalothin, chloramphenicol, florfenicol, nalidixic acid, gentamicin, kanamycin, erythromycin and oxytetracycline in Australia 25. Of the increasing resistance problem, it is imperative to base the antibiotic to be used upon susceptibility testing in the laboratory.

The aim of the current study was to demonstrate the presence of F. psychrophilum in the west Aegean region of Turkey, and determination of antibacterial susceptibility and an effective antibacterial treatment in rainbow trout fry. Antibiotic susceptibility of strains were determined using disk diffusion method.


Rainbow Trout Samples

A large fish hatchery located in the west Aegean region of Turkey were sampled. Increased mortality occurred in five concrete raceways of rearing unit. From the different raceways a total of 50 (10 of each raceways ) rainbow trout fry (weight = 2-5 g; total length = 3-6 cm) showing signs of infection with F. psychrophilum (dark skin, dorsal fin white spots or lysis, spiral swimming, spinal column deformities, and failure to feed) were brought to the laboratory. In the hatchery, mean (±SD) water temperature was 9.2±1.5ºC, dissolved oxygen was 9.8±0.4 mg/L, and pH was 6.8±0.3. The living animals were carried in containers filled with freshwater, and dead fish were stored on ice in cold boxes. This study was approved by Animal Ethic Committee of University of Adnan Menderes (Decision date 12.04.2007 and number HEK/2007/0004).

Isolation of F. psychrophilum

Samples were taken from the gill, spleen, kidney, liver, intestine and brain of each fish and from observed caudal peduncle lesions. The samples were streaked onto Cytophaga agar (CA) containing 1.5% agar as modified by Bernardet and Kerouault 26 (0.05% tryptone, 0.05% yeast extract, 0.02% beef extract, and 0.02% sodium acetate; pH = 7.2-7.4). All plates were incubated aerobically at 15ºC for 48-96 h. After incubation, the yellow-pigmented colonies were stained using the Gram staining technique, and Gram negative isolates were observed by microscopic examination 27.

Identification of F. psychrophilum

Identification was based on colony morphology, Gram staining, and biochemical tests. Biochemical characteristics of the isolates were determined using catalase, oxidase, indol, urease, methyl red, Voges-Proskauer, hydrogen sulfide (H2S), nitrate reduction, oxidation-fermentation, and motility tests, gelatin, Congo red, Simmons’ citrate, flexirubin-like pigment (20% KOH), growth on CA for 48-

199BOYACIOGLU, AKAR

96 h at 37ºC, and growth on CA and media containing 0.5, 1.5, and 3.0% NaCl (weight/volume). In addition, tests for fermentation (e.g., production of acid from glucose, lactose, and mannitol) were also carried out 27,28. Activities of 19 enzymes were tested using API ZYM strips (bioMe´rieux, Marcy l’Etoile, France). The API ZYM test was performed according to the manufacturer’s instructions, but the strips were incubated at 15ºC for 16-20 h and F. psychrophilum NCIMB (National Collection of Industrial, Marine, and Food Bacteria) 1947 was used as the control.

Determination of Antimicrobial Susceptibility of F. psychrophilum

Antimicrobial susceptibility was assessed by disk diffusion method. This method is used for more-routine screening, when sensitivity is not as important. The Kirby-Bauer disk diffusion method 29 was performed using multidisks (Oxoid, Ltd., Basingstoke, UK) of ampicillin (AMP; 10 µg), amoxicillin-clavulanic acid (AMC; 30 µg), enrofloxacin (ENR; 5 µg), erythromycin (E; 15 µg), florfenicol (FFC; 30 µg), gentamicin (CN; 10 µg), oxytetracycline (OT; 30 µg), penicilin G (P; 10 U), ciprofloxacin (CIP; 5 µg), and sulfamethoxazole-trimethoprim (SXT; 25 µg). Isolates were plated onto Mueller-Hinton agar (Oxoid) that was modified for testing F. psychrophilum, as described by Hawke and Thune 30. The plated isolates were incubated at 15ºC for 48-96 h.

The antimicrobial susceptibility was determined for each isolate by use of the disk diffusion method described by the National Committee for Clinical Laboratory Standards 31. Endpoint determinations were performed after 96 h. Disk diffusion was evaluated as the lowest zone diameter produced by an antimicrobial agent.

Antibiotic Treatment

Oxytetracycline (Terramycin, Pfizer, Turkey), enrofloxacin (Baytril, Bayer Turk, Turkey), ciprofloxacin (Ciprobiotik, Eczacıbaşı, Turkey) and florfenicol (Florocol, Schering-Plough, UK) were selected according to the in vitro study. Commercial rainbow trout fry pellets (1-1.5 mm) (Bayem, Pro-feed, Turkey) were moistened with cornflower oil to ensure attachment of the powdered drugs to the pellets. The drugs were added and carefully mixed with the pellets. The feed for the control fish was moistened with cornflower oil, without a drug 32. The antibiotic concentrations in the medicated feeds were to give a dose of 75 mg/kg/day oxytetracycline and 10 mg/kg/day enrofloxacin, ciprofloxacin and florfenicol each antibiotics for 10 days 20,33,34. Five concrete raceways of rearing unit were used for this purpose. Each group consisted approximately 25 000 naturally infected rainbow trout fry at the start of the experiment. Chi-square tests with Yates’ correction were used to compare the groups.

RESULTS

Twenty isolates that were phenotypically identified as F. psychrophilum were cultured from the gill (14% of 50 fish samples; n = 7 isolates gill), kidney (6%; n=3), liver (6%; n = 3), spleen (4%; n = 2), brain (4%; n = 2), intestine (4%; n = 2) and from observed pathological lesions of the caudal peduncle (2%; n=1). Results obtained from biochemical, cultural, and physiological characteristics and API ZYM profile tests are given in Table 1. The biochemical and morphological characteristics of the isolates closely resembled those of the type srtain F. psychrophilum NCIMB 1947. Mean zone diameter (mm) for each antimicrobial agent are presented in Table 2. The susceptibility of F. psychrophilum isolates to the antimicrobial agents, as indicated by the disk diffusion method, is shown in Table 3.

Disk diffusion results showed that 100% of the isolates were resistant to AMC, 95% were resistant to AMP and SXT, 80% were resistant to P and, 65% were resistant to CN. However, 100% were susceptible to ENR and CIP, 70% were susceptible to FFC, and 55% were susceptible to OT. Also, 50% of the isolates were intermediate susceptible to E (Table 3). In the study, The number of daily death before and after application of antibiotics, depending on the RTFS are presented in Table 4.

The mortality was very higher in the oxytetracycline group compared the other treatment groups. Although oxytetracycline significantly decreased the mortality on the second day (x2

yates = 14.57, Degree of Freedom (DF) = 3, P<0.05). After the third day the mortality were reduced and blocked with florfenicol. However, that was not significantly (P>0.05). After the fourth day of therapy were not significanlty decreased the effects of enrofloxacin and ciprofloxacin (P>0.05). Deaths due to infection in both groups continued after the application of antibiotic. There was no significant difference between groups of enrofloxacin and ciprofloxacin (x2

yates = 0.36, DF = 1, P>0.05) compared the cumulative mortality. Control (x2 = 11200.55, DF = 4, P<0.001), oxytetracycline (x2

yates = 5534.4, DF = 3, P<0.001) and florfenicol (x2

yates = 296.04, DF = 2, P<0.001) groups were significantly different compared the cumulative mortality.

There was no specific findings about RTFS in FFC group after the treatment. Between 5 to 10 fish died every day in this group. Bacteriological examination yielded no F. psychrophilum or other fish pathogens.

Deaths continued to increase in the control group and the other treatment groups (OT, ENR and CIP) after the treatment. For this purpose 10 surviving fish in each raceway were bacteriological examined for 5 days. Eighteen isolates that were phenotypically identified as F. psychrophilum. The isolates were cultured from the control group (4 isolates spleen, one each from brain, liver and


intestine), OT group (2 isolates spleen, 2 isolates brain, one each from kidney and liver), ENR group (one each from spleen, kidney and brain) and CIP group (one each from spleen and kidney).

DISCUSSION

The method of susceptibility testing used most widely in diagnostic laboratories is the agar disk diffusion technique. It is simple to perform, and a single bacterial isolate can easily be tested with several antimicrobial agents 35. The diluted Mueller-Hinton Agar as described by Hawke and Thune 30 has been recommended by Bruun et al. 24 for susceptibility testing of F. psychrophilum.

Data on the antimicrobial susceptibility of F. psychrophilum isolated from rainbow trout fry are limited in Turkey. Kum et al.11 reported that 20 isolates of F.

psychrophilum were resistance to AMC (90%), SXT (75%), CN (70%), E (65%), and sensitive to ENR (10%), OT (20%) and FFC (25%). Didinen et al.10 reported the resistance of 13 F. psychrophilum isolates to (84.6%), E (61.6%), P (53.9%), ENR (23.1%), AMC (15.4%), and CN (7.7%). Ispir et al.37 demonstrated sensitivity to AMC, CN, E, and OT and resistant to P for five isolates of F. psychrophilum. Using the disk diffusion method, Diler et al.8 reported that two F. psychrophilum isolates were sensitive to AMP, AMC, CN, CIP and OT and were resistant to trimethoprim. Similarly, Korun and Timur 38 reported that 20 isolates of F. psychrophilum were sensitive to OT (100% of isolates) but resistant to sulfadimethoxine and trimethoprim (100%), as determined using the disk diffusion method.

In the present study, disk diffusion method indicated that the 20 isolates of F. psychrophilum were susceptible to ENR and CIP (100%), FFC (70%), and OT (55%). Although

Table 1. Biochemical, cultural, physiological characteristics and API ZYM profile results for 20 F. psychrophilum isolates obtained from rainbow trout fry at a fish hatchery in the west Aegean region of Turkey

Tablo 1. Ege Bögesi’nin batısında bulunan Gökkuşağı alabalık işletmesine ait yavruhane bölümünden izole edilen F. psychrophilum (n=20) suşlarının biyokimyasal, kültürel ve fizyolojik karakterleri ile API ZYM profil sonuçları

CharacterReactions

API ZYM ProfileReactions

Isolates (r) Isolates (r)

Color of coloni yellow yellow Alkaline phosphatase 20/20 +

Gram 0/20 - Esterase 0/20 -

Catalase 20/20 + Esterase lipase 20/20 +*

Oxidase (cytochrome oxidase) 20/20 + Lipase 0/20 -

Hydrogen sulfide 0/20 - Leucine arylamidase 20/20 +

Glucose 0/20 - Valine arylamidase 20/20 +*

Lactose 0/20 - Cystine arylamidase 0/20 -

Mannitol 0/20 - Tyripsin 0/20 -

Motility 0/20 - α-chymotrypsin 0/20 -

Nitrate reduction 0/20 - Acid phosphatase 11/20 +

Urease 0/20 - Napthol-AS-BI-phosphohydrolase 17/20 +*

Indol 0/20 - α-galactosidase 0/20 -

Oxidation-Fermentation 5/20 - β-galactosidase 0/20 -

Gelatine 20/20 + β-glucuronidase 0/20 -

Methyl red 0/20 - α-glucosidase 9/20 -

Voges proskauer 0/20 - β-glucosidase 0/20 -

Flexirubin pigment 20/20 + N-acetyl-β-glucosaminidase 0/20 -

Congo red 0/20 - α-mannosidase 0/20 -

Simmon’s citrate 0/20 - α –fucosidase 0/20 -

Growth on CA

5 °C 20/20 +

37 °C 0/20 -

added 0.5 % NaCl 20/20 +

added 1.5 % NaCl 20/20 +

added 3 % NaCl 0/20 -

+ = positive; +* = weak positive; - = negative included in results; r = NCIMB 1947 F. psychrophilum referance strain; CA= Cytophaga agar

201BOYACIOGLU, AKAR

Table 2. Mean zone diameter (mm) from the disk diffusion method used to determine the susceptibility of 20 F. psychrophilum isolates to ten antimicrobial agents

Tablo 2. İzole edilen F. psychrophilum suşlarının (n=20) disk difüzyon metoduyla on antimikrobiyal ajana karşı oluşan zon çapları (mm)

Antimicrobial AgentMean Zone Diameter (mm)

R I S

AMP ≤ 13 14-16 ≥ 17

AMC ≤ 14 15-19 ≥ 20

ENR ≤ 15 16-20 ≥ 21

E ≤ 13 14-22 ≥ 23

FFC ≤ 14 15-18 ≥ 19

CN ≤ 12 13-14 ≥ 15

OT ≤ 14 15-18 ≥ 19

P ≤ 14 - ≥ 15

CIP ≤ 15 16-20 ≥ 21

SXT ≤ 10 11-15 ≥ 16

AMP = ampicillin; AMC = amoxicillin-clavulanic acid; ENR = enrofloxacin; E = erythromycin; FFC = florfenicol; CN = gentamicin; OT = oxytetracycline; P=penicilin G; CIP = ciprofloxacin; SXT = sulfamethoxazole-trimethoprim. Results are presented for isolates demonstrating resistance (R), intermediate response (I), and susceptibility (S) to each agent

Table 3. Number (percentage in parentheses) of F. psychrophilum isolates (n=20) demonstrating resistance, susceptibility, or an intermediate response to antimicrobial agents (codes defined in Table 2), as assessed using the disk diffusion method

Tablo 3. İzole edilen F. psychrophilum suşlarının (n=20) disk difüzyon metoduyla antimikrobiyal ajanlara (kısaltmalar Tablo 2.’de belirtilmiştir) karşı belirlenen duyarlılık ve dirençlilik durumları (parantez içindekiler; hesaplanan yüzdeliklerdir)

Isolate Response

Antimicrobial Agent

AMP AMC ENR E FFC CN OT P CIP SXT

Resistant 19 (95) 20 (100) - 9 (45) 6 (30) 13 (65) 4 (20) 16 (80) - 19 (95)

Intermediate 1 (5) - - 10 (50) - 1 (5) (25) - - 1 (5)

Susceptible - - 20 (100) 1 (5) 14 (70) 6 (30) 11 (55) 4 (20) 20 (100) -

Disk sizes: AMP = 10 µg, AMC = 30 µg, ENR = 5 µg, E= 15 µg, FFC = 30 µg, CN = 10 µg, OT = 30 µg, P = 10 U, CIP = 5 µg, and SXT = 25 µg

Table 4. The number (percentage in parentheses; calculated by the number of live fish of that day) of daily death before and after application of antibiotics, depending on the RTFS

Tablo 4. Antibiyotik uygulama öncesi ve sonrası RTFS’ye bağlı günlük ölüm sayıları (parantez içindekiler; o günkü canlı alabalık sayısı üzerinden hesaplanan yüzdeliklerdir)

Time (Day)Groups

x2

Control OT ENR CIP FFC

Before application of antibiotics 397 (1.59) 372 (1.49) 373 (1.49) 434 (1.74) 365 (1.46) 8.38 NS

After application of antibiotics

1 112 (0.46) 114 (0.46) 106 (0.43) 130 (0.53) 106 (0.43) 3.42 NS

2 168 (0.69)* 86 (0.35)** 132 (0.54) 129 (0.53) 142 (0.58) 11.58

3 312 (1.28)* 135 (0.55) 96 (0.39) 106 (0.44) 121 (0.50) 209.61

4 286 (1.19)* 243 (1.00)* 126 (0.52) 126 (0.52) 96 (0.40) 160. 68

5 615 (2.59)* 367 (1.53)* 130 (0.54) 137 (0.57) 81 (0.34)** 765.46

6 887 (3.84)* 335 (1.41)* 140 (0.58) 136 (0.57) 63 (0.26)* 1471. 66

7 911 (4.10)* 496 (2.12)* 156 (0.65) 146 (0.61) 41 (0.17)* 1706.23

8 1167 (5.48)* 963 (4.21)* 133 (0.56) 137 (0.58) 22 (0.09)* 2427.27

9 1346 (6.68)* 1114 (5.09)* 187 (0.79) 148 (0.63) 8 (0.03)* 2806.93

10 1217 (6.47)* 988 (4.76)* 195 (0.83) 174 (0.74) 6 (0.03)* 2363.28

Cumulative mortality 7143 (28.57)* 4972 (19.89)* 1524 (6.10) 1494 (5.98) 804 (3.22)* 11200.55

OT = oxytetracycline; ENR = enrofloxacin; CIP = ciprofloxacin; FFC = florfenicol, * P<0.01; ** P<0.05, NS = Not significant


the deaths originate from the rearing unit of hatchery, it is noteworthy that the isolates exhibited quite a range in susceptibilities to the different antibiotics. This may suggest that the fish were infected with multiple clones of F. psychrophilum, each exhibiting different susceptibilities to the antibiotics. Nevertheless, antibiotics used in treatment are not effective except FFC. This reveals a negative correlation between in vitro and in vivo effect. In Europe, RTFS has been successfully controlled using OT at 75-300 mg/kg/d for 10-14 day 20. However, isolates of F. psychrophilum with resistance to OT are increasingly being described in England 39 and Denmark 14,23,24,36. Previous investigations of antimicrobial resistance in F. psychrophilum have only dealt with the in vitro aspect. For example, MIC values can be useful indicators of the probable clinical efficacy, but these results have not been verified by in vivo testing 2,39,40. Branson 18 reported from the antibiotics tested, enrofloxacin, sarafloxacin and florfenicol showed potential for in in vivo trials but no therapeutic efffect has been found in subsequent trials with enrofloxacin.

Therapeutic activity remained at low levels, although on the second day of OT treatment to reduce mortality. After the third day of this activity was decreased, while providing a significant therapeutic efficacy initially of CIP and ENR. The reason for this might be chelation or resistance. Burka et al.41 reported OT and quinolones may be inactivated by exposure to Calcium2+ and Magnesium2+ in the water and the bowel of the fish. Resistance mechanisms have been examined in recent studies as potential causes of antimicrobial aquaculture treatment failures. Resistance to tetracyclines and/or oxytetracyclines has been commonly reported. Resistance to oxytetracycline in F. psychrophilum is caused by uptake of transposons carrying tetracycline resistance determinants or the resistance is caused by other unspecific changes in membrane permeability 36. Alvarez et al.4 described the resistance genes tetQ conferred tetracycline resistance. DNA gyrase (GyrA) is an important target for quinolones in F. psychrophilum 5 and can, as such, only be transferred vertically in bacterial populations 24.

Most of the F. psychrophilum isolates in this study were sensitive to FFC (70%). FFC was found to be highly effective in controlling RTFS. After the second day was reduce mortality. Perhaps FFC resistance has not yet occurred. Resistance of F. psychrophilum to FFC has rarely been observed in England 39, and Nematollahi et al.19 indicated that F. psychrophilum in salmonids exhibited no resistance to ENR or FFC. Michel et al.22 and Akinbowale et al.22 noted that F. psychrophilum resistance to FFC has not become widespread in France or Australia, despite the aquacultural use of FFC in those countries.

However, because of the increasing resistance problem, it is imperative to base the antibiotic to be used upon

susceptibility testing in the laboratory. The emergence of resistant bacteria may have resulted from improper use of antimicrobial agents. As a result of increasing incidence of resistant bacteria and recurrent outbreaks of disease shortly after a treatment, investigations on alternative treatments and prophylactic schemes should be given high priority. These results were considered indicative of FFC potential in controlling outbreaks of RTFS. Future studies of effective antibiotic application for the treatment of RTFS, this study can be used as a reference.

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203BOYACIOGLU, AKAR

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ÖzetAraştırma, Erzurum’da tüketime sunulan taze ve olgunlaşmış kaşar peynirlerinde mineral madde ve ağır metal içeriklerinin

belirlenmesi amacıyla yapıldı. Perakende satış yerlerinden toplanan 50 adet (37 taze ve 13 adet olgunlaşmış kaşar peyniri numunesi) peynir numunesinde mineral madde ve ağır metal kontaminasyon düzeyleri indüktif eşleşmiş plazma optik emisyon spektroskopisi (ICP-OES) ile belirlendi. Peynir örneklerindeki ortalama mineral madde ve ağır metal içerikleri; kalsiyum 2265.57±614.14 mg/kg, potasyum 1555.46±552.31 mg/kg, sodyum 4693.31±1886.12 mg/kg, magnezyum 129.34±32.05 mg/kg, demir 1.65±0.80 mg/kg, çinko 15.66±3.41 mg/kg, bakır 0.39±0.35 mg/kg, mangan 0.15±0.09 mg/kg, nikel 0.27±0.24 mg/kg, kurşun 1.77±1.72 mg/kg ve arsenik 0.0001±0.001 mg/kg olarak tespit edildi. Taze ve olgunlaşmış kaşar peynirlerinin mineral madde ve ağır metal içerikleri arasında önemli fark (P>0.05) saptanmadı.

Anahtar sözcükler: Kaşar peyniri, Mineral madde, Ağır metal, ICP-OES

Mineral Contents and Heavy Metal Contamination in Kashar Cheeses Consumed in Erzurum Province, Turkey

SummaryThis study was carried out to determine the mineral substances and heavy metal contents of fresh and ripened kashar cheeses

consumed in Erzurum province, Turkey. A total 50 samples (37 fresh and 13 ripened kashar samples) were obtained randomly from retail outlets. Mineral substances and heavy metals contamination levels were detected with inductively coupled plasma-optical emission spectrometry (ICP-OES). The average levels of the mineral substances and heavy metals in the kashar cheeses were determined as; calcium 2265.57±614.14 mg/kg, potassium 1555.46±552.31 mg/kg, sodium 4693.31±1886.12 mg/kg, magnesium 129.34±32.05 mg/kg, iron 1.65±0.80 mg/kg, zinc 15.66±3.41 mg/kg, copper 0.39±0.35 mg/kg, manganese 0.15±0.09 mg/kg, nickel 0.27±0.24 mg/kg, lead 1.77±1.72 mg/kg and arsenic 0.0001±0.001 mg/kg. It was not determined any statistical differences in mineral substances and heavy metal levels between fresh and ripened kashar cheese samples.

Keywords: Kashar cheese, Mineral substance, Heavy metal, ICP-OES

Erzurum’da Tüketime Sunulan Kaşar Peynirlerinin Mineral Madde İçeriği ve Ağır Metal Kontaminasyonu

Hayrunnisa ÖZLÜ * Meryem AYDEMİR ATASEVER * Sevda URÇAR * Mustafa ATASEVER *

* Atatürk Üniversitesi Veteriner Fakültesi Gıda Hijyeni ve Teknolojisi Bölümü, TR-25240 Erzurum - TÜRKİYE


Süt ve ürünleri, yapısında bulunan makro ve mikro ele- mentlerden özellikle kalsiyum içeriğinden dolayı beslen-mede oldukça önemlidir 1. Sütün mineral içeriği; hayvanın genetik özelliklerine, laktasyon dönemine, çevresel koşul- lara, mera tipine ve toprağın kirliliğine bağlı olarak deği- şebilir 2. Birçok gıda sektöründe olduğu gibi süt sektöründe de ağır metal kontaminasyonu süt üretim ve ürüne işleme aşamasında gerçekleşebilmektedir. Süt üreten işletmelerde kontaminasyon, genellikle çevresel kaynaklardan (örn., toprak ve su gibi) ve yemlerden bulaşabilir. Ayrıca sağım,

depolama ve işleme sırasında kullanılan makine ve ekip-manlardan kaynaklanabilir 3. Çünkü süt ve peynir gibi asidik nitelikli besinlerin sağımında veya üretiminde kullanılan alet ve ekipmanın bileşimindeki metallerin çözünerek ürü-ne geçme riski diğer gıdalara oranla daha kolay olabilir 4,5. Teknolojik işlemler sırasında süt ve süt ürünlerinin muha-fazasında kullanılan metal kaplardan ve işletme suyundan kaynaklanan metalik kontaminasyondaki başlıca element-ler bakır, çinko, demir, kalay, kurşun, kadmiyum ve arse-niktir 6.

GİRİŞ


RESEARCH ARTICLE

206Erzurum’da Tüketime Sunulan ...

Gelişen endüstrilerin ve daha modern bir yaşam sağlama amacıyla sürdürülen çabaların olumsuz sonuçlarından olan besinlerin yüksek düzeyde ağır metallerle kontaminasyonu günümüzde toplum sağlığını tehdit eden en önemli prob- lemlerden biridir. Ağır metallerin bir kısmı (örn.; demir, çinko ve bakır gibi) yaşam için esansiyel olup önemli enzim sistemlerinin fonksiyonları için gereklidir. Bir kısmının ise insan fizyolojisinde ki fonksiyonları henüz belirlene-memiştir. Bununla birlikte bazıları (örn.; kurşun, ve civa gibi) iz seviyelerde bile potansiyel olarak toksik etki gös-terebilir. Metaller insan vücudunda özellikle belirli doku ve organlarda (örn.; karaciğer, kemik ve böbrek gibi) birikme eğilimindedir. Metallerin toksisitesi en çok beyin ve böb-rekte olurken farklı şekillerde de zararlı etkileri olabilir (örn.; arsenik kansere neden olabilir). Besinlerle alınan ağır me-tallerin vücutta birikme oranı; metalin kimyasal formuna, kişinin yaşı ve beslenme durumuna bağlı olarak değişkenlik gösterebilir 7.

Kaşar peynirinde mineral madde içeriği ve ağır metal kontaminasyonlarının belirlendiği çalışmaların 8-12 yanı sıra, üretim aşamalarında meydana gelen ağır metal kontami-nasyonlarının incelendiği çalışmalara3 da literatürde rast-lanmıştır.

Bu araştırmayla, Erzurum piyasasında tüketime sunu-lan taze ve olgunlaşmış kaşar peynirlerinin kalsiyum, potasyum, sodyum, magnezyum, mangan, demir, çinko, bakır, nikel, kurşun ve arsenik düzeylerinin belirlenmesi amaçlandı.

MATERYAL ve METOT

Örneklerin Alımı

Erzurum piyasasında tüketime sunulan yerel ve ulusal firmalara ait orijinal vakum ambalajlı 37 adet taze ve 13 adet olgunlaşmış kaşar peyniri örneği satış yerlerinden temin edildi. Örnekler soğuk zincir altında laboratuvara getirildi ve analizleri yapılıncaya kadar 4±1°C’de muhafaza edildi.

Örneklerin Çözümlenmesi

Analizi yapılacak kaşar peyniri örneklerinde organik bi- leşikleri yok etmek ve inorganik bileşikleri de çözünür faza geçirmek amacıyla yapılan çözümleme işlemleri kapalı sistem mikrodalga yakma metodu kullanıldı. Bu amaçla Ethos Touch Control (Milestone) mikrodalga çözümleme sistemi ve aksesuarları (TFM kaplar) ile Microwave Digestion Rotor (1000/10 yüksek basınç) ve basınç-sıcaklık kontrol sensörleri kullanıldı. Örneklerin çözümlenmesinde %65’lik nitrik asit (Merck, 1.00452.2500) ve %30’luk hidrojen peroksit (Merck, 1.08597.1000) kombinasyonundan yararlanıldı. Çözünür-leştirme işlemi sonrası oda sıcaklığında soğutulan kapların kapakları açıldıktan sonra çözünen numuneler ultra distile su ile 50 ml’ye seyreltilerek viallere aktarıldı.

Metal kontaminasyonunu önlemek amacıyla örneklerin çözündürülmesi esnasında kullanılan tüm malzemeler, 1+9

(v/v) HNO3/ultra distile su ile birkaç kez çalkalandı. Daha sonra ultra distile su ile çok sayıda yıkanan ve durulanan malzemeler etüvde kurutuldu.

Örneklerin Mineral ve Ağır Metal DüzeylerininICP-OES ile Belirlenmesi

Kaşar peyniri örneklerinde bulunan mineraller ve ağır metaller her biri için ICP-OES’in software’inde yer alan ve cihazın analizler için uygun gördüğü dalga boylarında standart kalibrasyon çözeltiler ile kalibrasyon eğrileri çi- zildi. Bu eğriler, cihaza verilen standart çözeltinin 5 farklı konsantrasyonuna karşılık cihazın ölçümleri sonucu hesap-ladığı konsantrasyonlara göre oluşturuldu.

Çözünen örneklerin, ICP-OES (Optima 2000 DV) ciha-zında her metal için belirtilen dalga boyunda köre karşı okuması yapıldı. Çözelti halinde cihaza verilen örnekler argon gazı eşliğinde gaz fazına geçirildi ve bir süre sonrada yaydıkları rezonans ışınları tespit edilerek ölçümleri ger-çekleştirildi.

İstatistiksel Değerlendirmeler

Elde edilen sonuçların değerlendirilmesinde SPSS paket programı kullanılarak örneklerin minimum, maksimum, ortalama değer ve standart hataları belirlendi. Taze ve ol- gunlaşmış kaşar peynir örnekleri arasındaki mineral madde içeriğindeki farklılıklar t- testi analiziyle incelendi.

BULGULAR

Erzurum ilinde çeşitli perakende satış yerlerinden alınan taze ve olgunlaşmış kaşar peynirlerinde mineral madde ve ağır metal içerikleri; kalsiyum 2265.57±614.14 mg/kg, potasyum 1555.46±552.31 mg/kg, sodyum 4693.31±1886.12 mg/kg, magnezyum 129.34±32.05 mg/kg, demir 1.65±0.80 mg/kg, çinko 15.66±3.41 mg/kg, bakır 0.39±0.35 mg/kg, mangan 0.15±0.09 mg/kg, nikel 0.27±0.24 mg/kg, kurşun 1.77±1.72 mg/kg ve arsenik 0.0001±0.001 mg/kg olarak saptandı. Olgun kaşar peynirlerinde tespit edilen ortalama kalsiyum, sodyum, çinko, bakır, nikel ve kurşun miktarları taze kaşar peynirlerinde nispi olarak yüksek olmasına rağmen istatistiksel olarak önemli (P>0.05) bulunmadı (Tablo 1).

TARTIŞMA ve SONUÇ

Kaşar peyniri numunelerinde kalsiyum ve sodyum içerikleri Mendil’in 8 (3869±385 µg/g ve 4994±489 µg/g), Kılıç ve ark.’nın 9 İzmir’de (888.42±165.72 mg/100g ve 557.10±74.89 mg/100 g) ve Yüzbaşı ve Demirözü’nün 10 Ankara’da (6026.00±908 mg/kg ve 5082.00±1972 mg/kg) yaptıkları çalışmalarda belirledikleri değerlerden daha dü- şük bulunmuştur. Potasyum içeriği ise Mendil’in 8 322±32 µg/g, Kılıç ve ark.’nın 9 129.63±22.22 mg/100 g, Yüzbaşı ve Demirözü’nün 10 723.00±147 mg/kg olarak elde ettiği

207

ÖZLÜ, AYDEMİR ATASEVERURÇAR, ATASEVER

bulgularından oldukça yüksek kaydedilmiştir. Kaşar pey-nirlerinin ortalama magnezyum içeriği Mendil 8 tarafından 36.8±2.9 µg/g olarak belirlenen değerden oldukça yüksek olarak tespit edilirken Yüzbaşı ve Demirözü’nün 10 320.00 107 mg/kg olarak belirlediği değerden daha düşük olduğu saptanmıştır.

Olgunlaşmış kaşar peynir numunelerinin içerdikleri kalsiyum ve sodyum miktarları taze kaşar peynir numu-nelerine oranla biraz yüksek bulunurken; potasyum ve magnezyum içerikleri daha düşük bulunmuştur (Tablo 1). Olgunlaşmayla birlikte peynirdeki nem kaybı sonucu özel- likle sodyum miktarında oransal olarak bir artışın olması muhtemeldir. Kaşar peynirlerindeki mineral madde düzey- lerindeki bu farklılıklar hayvanın yetiştirildiği toprağın yapısından, mevsimsel değişimlerden, kullanılan sütün içe- riğinden ve farklı peynir üretim tekniklerinden kaynak-lanmış olabilir. Nitekim birçok araştırmacı 3-5,13 da peynir üretim tekniklerinin ve çevresel şartların üretilen pey-nirlerin mineral madde içeriklerinde farklılığa yol açabile-ceğini vurgulamıştır.

Kaşar peynirlerindeki demir içeriği Mendil’in 8 7.5±0.6 µg/g, Yüzbaşı ve Demirözü’nün 10 4.11±1.92 mg/kg ve Acar’ın 12 8.36±0.7 mg/kg olarak tespit ettiği değerlerden, çinko içeriği ise Yüzbaşı ve Demirözü’nün 10 Ankara’da yap-tığı çalışmada 38.00±5.17 mg/kg, Yalçın ve Tekinşen’in 11 Konya’da yaptığı çalışmada 27.15±0.71 mg/kg olarak elde

ettikleri değerlerden düşük bulunmuştur. Bununla birlikte, kaşar peynirlerinin ortalama çinko içeriği Acar’ın 12 Ankara’da yaptığı çalışmada 15.3±1.8 mg/kg olarak belirlediği çinko düzeyi ile paralellik gösterirken, Mendil’in 8 10.6±1.05 µg/g olarak tespit ettiği değerden daha yüksek bulunmuştur. Farklı araştırmalarda peynirlerin çinko içeriğinde saptanan farklılıkların, peynir yapımında kullanılan sütten ve üretim aşamasındaki alet ve gereçlerden kaynaklanmış olabileceği düşünülmektedir. Bu durum Yalçın ve Tekinşen11 tarafından da ifade edilmektedir. Ayrıca sütteki çinkonun %85’inin kazein misellerine bağlı olduğu ve asidik pH değerlerinde serbest hale geçerek pıhtıdan ayrıldığı bildirilmiştir14.

Araştırmada kaşar peyniri numunelerindeki ortalama bakır değerinin Mendil’in 8 0.27±0.015 µg/g, Yüzbaşı ve Demirözü’nün 10 0.64±0.24 mg/kg ve Acar’ın 12 0.26±0.04 mg/kg olarak elde ettiği değerlerden daha düşük olduğu tespit edilmiştir. Peynirlerde belirlenen bakır düzeyi sütün muhafaza edildiği metal kapların bileşiminde bulunan ba- kırın sütle etkileşime girerek çözünmesinden veya hayvan-ların beslenmesinde kullanılan yemlerin bakır içeriği yük-sek olan tarım ilaçları ile kontaminasyonundan dolayı arta- bileceği, buna karşın üretim esnasında sütteki bakırın çözünerek peynir altı suyuna geçmesiyle azalabileceği düşünülmüştür. Bu durum Metin 5 ve Temurci ve Güner 15 tarafından da desteklenmiştir.

Kaşar peynirlerinde ortalama kurşun düzeyi 1.77±1.72

Tablo 2. Kaşar peynirindeki ağır metal içerikleri ile ilgili literatürler

Table 2. Heavy metal contents in kashar cheeses concerned with literatures

Fe Zn Cu Pb Mn Ni Literatür

4.11±1.92 (mg/kg) 38.00±5.17 (mg/kg) 0.64±0.24 (mg/kg) - - - Yüzbaşı ve Demirözü 10

7.5±0.65 (µg/g) 10.6±1.05 (µg/g) 0.27±0.015 (µg/g) 0.53±0.055 (µg/g) 1.0±0.1 (µg/g) 0.18±0.01 (µg/g) Mendil 8

8.9±0.3 (mg/kg) 86.9±3.1 (mg/kg) 1.2±0.1 (mg/kg) 42.9±2.3 (µg/kg) - - Yüzbaşı ve ark.3

5.7±1.1 (mg/kg) 58.3±1.7 (mg/kg) 1.5±0.1 (mg/kg) 374.1±89.0 (µg/kg) - - Yüzbaşı ve ark.3

8.36 ± 0.7 (mg/kg) 15.3 ±1.8 (mg/kg) 0.26±0.04 (mg/kg) 0.29±0.02 (mg/kg) Acar 12

15.42±0.40 (mg/kg) 27.15±0.71 (mg/kg) 1.35±0.04 (mg/kg) 0.12±0.02 (mg/kg) 0.43±0.03 (mg/kg) Yalçın ve Tekinşen 11

Tablo 1. Kaşar Peyniri Örneklerinin Mineral Madde ve Ağır Metal İçerikleri (mg/kg)

Table 1. Mineral Substance and Heavy Metal Contents in Kashar Cheeses (mg/kg)

Mineral ve Ağır Metaller

Taze Kaşar Olgunlaşmış Kaşart-değeri

Taze + OlgunlaşmışKaşar

n Ort±Std. hata Min. Mak. n Ort±Std. hata Min. Mak. Ort±Std. hata

Ca 37 2254.03±101.38 965.15 3215.00 13 2298.39±174.90 1113.50 3199.80 -0,481 2265.57±614.14

K 37 1593.76±90.89 450.70 2855.10 13 1446.46±154.73 789.00 2757.50 -0,552 1555.46±552.31

Na 37 4640.36±318.20 1164 9122.00 13 4844.04±500.30 2374.00 8674.00 -2,624 4693.31±1886.12

Mg 37 130.22±5.51 65.83 210.09 13 126.84±7.91 94.75 182.05 -0,860 129.34±32.05

Fe 37 1.71±0.15 0.17 3.75 13 1.49±0.14 0.62 2.23 0,376 1.65±0.80

Zn 37 15.29±0.53 8.23 21.05 13 16.75±1.06 9.83 21.34 -0,020 15.66±3.41

Cu 37 0.34±0.06 - 1.20 13 0.52±0.11 - 0.91 1,671 0.39±0.35

Mn 37 0.16±0.02 0.04 0.39 13 0.14±0.02 0.07 0.24 -0,113 0.15±0.09

Ni 37 0.26±0.04 - 0.72 13 0.30±0.07 - 0.67 1,685 0.27±0.24

Pb 37 1.60±0.29 - 5.16 13 2.25±1.62 - 4.65 -0,481 1.77±1.72

As 37 0.00±0.00 - 0.00 13 - - - -0,552 0.00±0.00

208Erzurum’da Tüketime Sunulan ...

mg/kg olarak saptandı. Peynir örneklerinin 14 tanesinde kurşun tespit edilmezken en yüksek değer 5.16 mg/kg olarak ölçülmüştür. Mendil’in 8 0.53±0.055 µg/g, Acar’ın 12

0.29±0.02 mg/kg ve Ayar ve ark.’nın 16 1.10±0.01 mg/kg olarak belirlediği kurşun düzeyleri bu araştırmada elde edilen değerlerden düşük bulunmuştur. Farklılıkların süt üretimi yapılan çiftlikler, peynir işletmeleri ile satış yerlerinin sanayi kuruluşlarına ve otoyollara olan mesafesiyle ilişkili olabileceği sonucuna varılmıştır.

Araştırmada mangan kontaminasyonu tüm örneklerde, nikel ise kırk yedi numunede tespit edilmiştir. Elde edilen ortalama nikel (0.27±0.24 mg/kg) ve mangan (0.15±0.09 mg/kg) içerikleri Mendil’in 8 yaptığı çalışmada kaşar peynirlerinde 0.18±0.01µg/g ve 1.0±0.1µg/g olarak belirlediği nikel ve mangan düzeylerinden daha düşük olduğu kay- dedilmiştir. Peynir üretiminin ısıtma ve haşlama aşamala-rında kullanılan çelik kapların bileşiminde bulunan nikel ve mangan gibi metallerin ürüne geçebileceği belirtilmiştir 17.

İncelenen örneklerde ortalama arsenik düzeyi 0.0001±0.001 mg/kg olup sadece 4 adet taze kaşar pey-nirinde tespit edilmiştir. Ayar ve ark.16, kaşar peynirlerinde ortalama arsenik miktarını 0.021±0.307 mg/kg olarak sap-tamışlardır. Demirözü ve Saldamlı 18 ise taze ve olgunlaşmış beyaz peynirlerde arsenik düzeylerini sırasıyla 10.85±0.34 ng/g ve11.35±0.29 ng/g olarak belirlemişlerdir. Son yıllara kadar gıdaların arsenik ile kontaminasyonu tarımda kulla- nılan ilaçlar sebebiyle daha yüksek düzeylerde seyreder-ken, günümüzde bu tür insektisit, herbisit ve pestisitlerin kullanılması yasaklandığından kontaminasyon riski azal-mıştır 17.

Sütün bileşimindeki mineral madde ve ağır metallerin ortalama içerikleri kalsiyum 1221-1259 mg/L, potasyum 1424-1550 mg/L, sodyum 310-523 mg/L, magnezyum 89-228 mg/L, demir 0.70-2.40 mg/L, çinko 0.96-4.84 mg/L, bakır 0.19-0.29 mg/L, mangan 0.02-0.038 mg/L, nikel 0-0.730 µg/L, kurşun 4-100 µg/L ve arsenik 30-100 µg/L olduğu belirtilmektedir 6,19. Kaşar peyniri üretiminde ran-dıman ortalama %10 civarındadır 20. Mineral maddeler gibi ağır metallerde belirli oranda süte geçebilmektedir. Bu nedenle kaşar peynirindeki ağır metal düzeyinin peynir yapımında kullanılan sütün ağır metal kontaminasyonuna göre değişebilir. Yüzbaşı ve ark.’nin 3 Ankara’da yaptığı çalış- mada da ağır metal içeriği daha yüksek olan sütten yapı- lan kaşar peynirinin de ağır metal içeriğinin yüksek oldu- ğunu tespit etmişlerdir (Tablo 2). Ancak ağır metal konta-minasyonunun özellikle sütün pıhtılaştırılması aşamasında arttığını, daha sonraki işlem basamaklarında ise azaldığını gözlemlemişlerdir.

Türk Gıda Kodeksi’nin 21 Gıda Maddelerinde Bulaşanların Maksimum Limitlerinin Belirlenmesi Hakkındaki Tebliğ’de kaşar peynirine ait maksimum limitler olmamasına karşın ağır metallerin bazı gıdalarda kabul edilebilir değerleri belirtilmiştir. Araştırmada elde edilen ortalama değerlerden sadece nikelin Kodekste belirtilen değerlerin (0.1-0.2 mg/kg)

üzerinde olduğu tespit edilmiştir. Ayrıca bazı kaşar peyniri numunelerinin kurşun içeriği de belirtilen limitlerden (0.02-2 mg/kg) yüksek bulunmuştur. Peynir üretim tekniklerinin standart hale getirilmesi, üretim ve muhafaza aşamasında kullanılan alet ve ekipmanların inert yapıda olması, sanayi bölgelerinde veya trafiğin yoğun olduğu bölgelere yakın yerlerde hayvan yetiştiriciliğinin yapılmaması ve süt işletme- lerinin bu tür yerlerden uzaklara kurulması önerilmektedir.

KAYNAKLAR

1. Aydemir Atasever M, Çanakçı Adıgüzel G, Atasever M, Özturan K: Determination of aflatoxin M1 levels in some cheese types. Kafkas Univ Vet Fak Derg, 16, 87-91, 2010.

2. Gonzalez-Martin I, Hernandez-Hierro JM, Revilla I, Vivar-Quintana A, Lobos Ortega I: The mineral composition (Ca, P, Mg, K, Na) in cheeses (cow’s, ewe’s and goat’s) with different ripening times using near infrared spectro-scopy with a fibre-optic probe. Food Chem, 127, 147-152, 2011.

3. Yüzbaşı N, Sezgin E, Yıldırım Z, Yıldırım M: Changes in Pb, Cd, Fe, Cu and Zn levels during the production of kasar cheese. J Food Quality, 32, 73-83, 2009.

4. Bakırcıoğlu D, Bakırcıoğlu Kurtuluş Y, Uçar G: Determination of some trace metal levels in cheese samples packaged in plastic and tin containers by ICP-OES after dry, wet and microwave digestion. Food Chem Toxicol, 49, 202-207, 2011.

5. Belgaied JE: Release of heavy metals from Tunisian traditional earthenware. Food Chem Toxicol, 41, 95-98, 2003.

6. Metin M: Süt Teknolojisi. Sütün Bileşimi ve İşlenmesi. 4. Baskı, s. 801-806, Ege Üniversitesi Mühendislik Fakültesi, No: 33, Bornova, İzmir, 2001.

7. Hu H: Human Health and Toxic Metals. In, McCally M (Ed): Life Support: The Environment and Human Health. MIT Press, 2002.

8. Mendil D: Mineral and trace metal levels in some cheese collected from Turkey. Food Chem, 96, 532-537, 2006.

9. Kılıç S, Karagözlü C, Uysal H, Akbulut N: İzmir piyasasında satılan bazı peynir çeşitlerinin kalsiyum, fosfor, sodyum ve potasyum düzeyleri üzerine bir değerlendirme. Gıda, 27 (3): 229-234, 2002.

10. Yüzbaşı N, Demirözü B: Determination of some essential minerals in cheese and milk. Gıda, 27 (6): 449-504, 2002.

11. Yalçın Ö, Tekinşen KK: Konya’da tüketime sunulan beyaz salamura, tulum ve kaşar peynirlerinin ağır metal içeriklerinin araştırılması. Etlik Vet Mikrobiyol Derg, 22, 5-10, 2010.

12. Acar O: Determination and evaluation of copper, lead, iron and zinc contamination levels in cheese and tahini halva by atomic absorption spectro-metry. Int J Food Safety, 13, 45-53, 2011.

13. Cichoscki AJ, Valduga E, Valduga AT, Tornadijo ME, Fresno JM: Characterization of Prato cheese, a Brazilian semi-hard cow variety: Evolution of physico-chemical parameters and mineral composition during ripening. Food Control, 13, 329-336, 2002.

14. Fresno JM, Prieto B, Urdiales R, Sarmiento RM, Carballo J: Mineral content of some Spanish cheese varietes. Differentiation by source of milk and by variety from their content of main and trace elements. J Sci Food Agr, 69, 339-345, 1995.

15. Temurci (Usta) H, Güner A: Ankara’da tüketime sunulan süt ve beyaz peynirlerde ağır metal kontaminasyonu. Atatürk Üniv Vet Bil Derg, 1 (1-2): 20-28, 2006.

16. Ayar A, Sert D, Akın N: The trace metal levels in milk and dairy products consumed in middle Anatolia-Turkey. Environ Monit Assess, 152, 1-12, 2009.

17. Vural H: Ağır metal iyonlarının gıdalarda oluşturduğu kirlilikler. Ekoloji, 8, 3-8, 1993.

18. Demirözü-Erdinç B, Saldamlı I: Variation in some heavy metals during the production of white cheese. Int J Dairy Technol, 53 (3): 96-99, 2000.

19. Demirci M: Sütün mineral maddeleri ve insan beslenmesindeki önemi. Atatürk Üniv Zir Fak Derg, 12 (1): 195-207, 1981.

20. Tekinşen OC, Tekinşen KK: Süt ve Süt Ürünleri. s. 226-230, Selçuk Üniversitesi Basımevi, Konya, 2005.

21. Türk Gıda Kodeksi: Gıda Maddelerindeki Bulaşanların Maksimum Limitleri Hakkında Tebliğ. Tarım ve Köyişleri Bakanlığı Tebliğ No: 2008/26, Ankara, 2008.


ÖzetBu çalışmanın amacı, geleneksel olarak üretilen Karın Kaymağı Peynirinden izole edilen laktobasilleri tanımlamak ve bu peynirin

bazı fiziksel, kimyasal ve mikrobiyolojik özelliklerini belirlemektir. Araştırmada, Karın Kaymağı Peynirinden izole edilen 107 adet izolatın API 50 CHL kiti ile tanımlanması yapılmıştır. Tanımlanan Lactobacillus suşlarının (82 adet); %47.56’sının Lactobacillus plantarum tip 1 ve tip 2, %21.9’unun L. brevis tip 1 ve tip 3, %10.97’nin L. delbrueckii ssp. delbrueckii, %9.75’nin L. acidophilus tip 3 ve %2.43’ünün ise L. fermentum olduğu bulunmuştur. Tanımlanan izolatlardan Leuconostoc mesenteroides ssp. mesenteroides/dextranicum tip 2’nin ise (25 adet) floranın %24.4’ünü oluşturduğu bulunmuştur. Karın Kaymağı Peynir örneklerinde yapılan analizler sonucu; ortalama kurumadde, yağ, protein miktarlarının sırasıyla %74.66, %43.36 ve %25.51 olduğu bulunmuştur. Toplam aerobik mezofilik bakteri (TAMB) sayısının 6.477-8.17 log kob/g, laktik asit bakteri (LAB) sayısının 6.649-8.041 log kob/g ve maya ve küf sayısının ise 3.556- 5.308 log kob/g arasında değiştiği saptanmıştır. Peynir örneklerindeki koliform grubu bakteri, Enterobacteriaceae ve S. aureus sayılarının ise tespit edilebilir seviyenin altında olduğu bulunmuştur (<10 log kob/g).

Anahtar sözcükler: Karın Kaymağı Peyniri, API 50 CHL, Laktik asit bakterileri, Tanımlama

Identification of Lactobacilli Isolated from Cheese Karın Kaymak

SummaryThis study was carried out to identify lactobacilli isolated from the Cheese Karin Kaymagi produced traditionally techniques and

to determine some physical, chemical and microbiological properties. In this study, 107 isolates obtained from Cheese Karin Kaymagi were identified with the API 50 CHL system. The Lactobacillus strains were identified (82 pieces) as follows; 47.56% were Lactobacillus plantarum type 1 and type 2, 21.9% were L. brevis type 1 and type 3, 1.97% were L. delbrueckii ssp. delbrueckii, 9.75% were L. acidophilus type 3 and 2.43% were L. fermentum. Leuconostoc mesenteroides ssp. mesenteroides/dextranicum type 2 isolates as identified constituted 24.4% of bacterial flora. In the Cheese Karin Kaymagi samples analysed, the average dry matter, fat, protein amounts were found as 74.66%, 43.36% and 25.51%, respectively. The count of total aerobic mesophilic bacteria were found to vary between 6.477-8.17 log cfu/g, lactic acid bacteria counts (LAB) 6.649- 8.041 log cfu/g, and yeast and mold counts 3.556- 5.308 log cfu/g. In the cheese samples, coliform group bacteria, Enterobacteriaceae and S. aureus counts were found under the acceptable limit (<10 log kob/g).

Keywords: Cheeese Karin Kaymagi, API 50 CHL, Lactic acid bacteria, Identification

Karın Kaymağı Peynirinden İzole Edilen Laktobasillerin Tanımlanması

Tamer TURGUT * Ahmet ERDOĞAN * Mustafa ATASEVER **

***

Atatürk Üniversitesi, Erzurum Meslek Yüksekokulu, TR-25240 Erzurum - TÜRKİYEAtatürk Üniversitesi, Veteriner Fakültesi, TR-25240 Erzurum - TÜRKİYE


Peynir, sütün pıhtılaştırılıp peynir altı suyunun ayrıl-masından sonra pıhtının değişik şekillerde işlenmesiyle elde edilen, besin değeri ve dayanıklılığı yanında çok sa-yıda çeşidiyle toplumun gelişen zevk ve isteklerine cevap verebilen önemli bir süt ürünüdür 1. Ülkemizde 50’den fazla peynir çeşidinin üretildiği bilinmektedir. Bu peynirlerin birçoğu yalnızca belirli bir coğrafi bölgede üretilmekte ve

üretildiği yerde tüketilmektedir 2. Ekonomik değere sahip Beyaz, Kaşar ve Tulum peynirlerimizden başka yöresel olarak aile işletmelerinde üretilen peynir çeşitlerinden birisi de Karın Kaymağı Peyniridir. Karın Kaymağı Peynirinin ismi, üretimde kullanılan ambalaj materyali olan işkembeden gelmektedir. Karın Kaymağı Peyniri, iç tüketime yönelik olarak üretilen, olgun, yağ oranı yüksek, besin değeri oldukça

GİRİŞ


RESEARCH ARTICLE

210Karın Kaymağı Peynirinden ...

yüksek sarımsı renkte, düzgün tekstürde ve yapılışı yöre-ye özgü bir peynir çeşididir. Doğu Anadolu Bölgesinde Erzurum ve Kars İlleri ile özellikle Kars’ın Sarıkamış ilçesi ve köylerinde üretilmektedir 3. Karın Kaymağı Peynirinin üre-timine yönelik detaylı bilgi veren çalışmalar mevcuttur 3,4.

Laktik asit bakterilerinden Lactobacillus genusu türleri Gram (+), katalaz (-), düzgün çubuk, kokobasil veya uzun zincir oluşturan çubuk şeklindeki bakterilerdir. Lactobacillus genusu türleri doğada oldukça yaygındır. Silajlarda, hayvan ve insanların sindirim sisteminde ve normal süt flo-rasında bulunurlar. Protein parçalama özellikleri tür ve suşa göre değişmekle beraber peptitleri aminoasitlere kadar parçalarlar. Bu sebeple yoğurt ve benzeri ürünlerin yapımında ve sert peynirlerin olgunlaştırılmasında starter kültür olarak kullanılırlar 5. Lactobacillus türleri aynı za-manda gıdalarda en yaygın olarak kullanılan probiyotik bakterilerdir 6. Günümüzde probiyotik bakteri içeren bir-çok gıda ve özellikle süt ürünleri geliştirilip marketlerdeki yerini almıştır 7,8.

Karın Kaymağı Peyniri sahip olduğu kendine has özel-likleri ile önemli bir peynir çeşidimizdir. Karın Kaymağı Peyniri üzerinde yapılan araştırmalar üretim tekniği ile fiziksel, kimyasal ve mikrobiyolojik kalitesinin belirlenmesi üzerinedir. Yapılan çalışmalarda bulunan fiziksel kimyasal ve mikrobiyolojik değerlerin birbirinden farklı olduğu ve laktik asit bakteri ve maya ve küf sayısının oldukça yüksek olduğu dikkat çekmektedir 3,9-11. Karın Kaymağı Peynirinin mikroflarasını oluşturan laktobasillerin belirlenmesi, üreti- minin endüstriyel düzeyde gerçekleştirilmesi için zorun-ludur. Böylelikle bu peynir çeşidinin daha geniş kitlelere ulaştırılması ve ülkemiz peynir çeşitliliğinin artırılması mümkün olacaktır. Bu araştırmanın amacı, geleneksel ola-rak üretilen Karın Kaymağı Peynirinden laktobasilleri izole edip, API 50 CHL kitiyle tanımlanması ve örneklerin çeşitli kimyasal değerleri ile bazı mikrobiyolojik özelliklerini belirlemektir.

MATERYAL ve METOT

Peynir Örnekleri

Araştırmada, 2009-2010 yılları arasında Kars ili Sarıkamış İlçesi Isısu köyünden toplanan 11 adet Karın Kaymağı Peyniri örneği kullanılmıştır. Kendi orjinal ambalajında (işkembe) alınan örnekler soğukta muhafaza edilerek mümkün olan en kısa süre içinde laboratuara getirilmiştir. Analiz süresince örnekler buzdolabı koşullarında (4°C’de) muhafaza edilmiştir. Alınan örneklerde önce mikrobiyolo-jik analizler için numune alınmış, ardından kimyasal ana-lizler yapılmıştır.

Mikrobiyolojik Analizler

Mikrobiyolojik analizler için, 10 g örnek steril plastik poşete tartılmış, üzerine 90 ml steril fizyolojik su (%0.85 NaCl (Merck 106404) ilave edilerek Stomacherde (IUL

Masticator, Spain) homojenize edilmiştir. Daha sonra bu homojenizattan serum fizyolojik (%0. 85 NaCl) kullanılarak uygun dilüsyonlar hazırlanmıştır.

Toplam aerobik mezofilik bakteri (TAMB) sayımı, Plate Count Agar (PCA) (Merck 1.05463) besiyerinde aerobik şartlarda 30°C’de 72 saat süre ile Harrigan’a göre 12, Laktik asit bakteri (LAB) sayımı De Man Rogosa Sharpe Agar (MRS) (Merck 1.10660) besiyerinde anaerobik ortamda (Anaerocult A sistem, Merck 1.13829) 37°C’de 72 saat süre ile Hagen and Narvhus’a göre 13, Koliform grubu bakteri ve Enterobacteriaceae sayısı sırasıyla Violet Red Bile Agar (VRB) (Merck 1.14606) ve Violet Red Bile Dekstrose (VRBD) agar (Merck 1.10275) besiyerinde 30°C’de 24 saat süre ile S. aureus Egg-yolk Tellurite ilaveli Baird-Parker Agar (BPA) (Fluka 11705) besiyerinde 37°C'de 24 saat süre ile Harrigan’a göre 12 ve Maya ve küf sayısı Potato Dextrose Agar (PDA) (Merck 1.10130) besiyerinde 22-25°C’de 4-5 gün süre ile Özkaya ve Kuleaşan’a 14 göre yapılmıştır.

Laktobasillerin İzolasyonu ve Tanımlanması

Laktobasillerin tür düzeyinde tanımlanabilmesi için; standardize edilmiş tanımlama sistemi API (Analytical Profile Index) 50CHL test kitlerinden (Biomerieux, Marcy l’Etoile, France) yararlanılmıştır. API kiti; 49 farklı karbon-hidrat kaynağını, dehidre substart olarak içeren 50 adet kuyucuktan oluşmaktadır. Tanımlanması yapılacak olan izolatlar anaerobik ortamda MRS Agar üzerinde 37°C’de 72 saat sureyle tek koloni düşecek şekilde aktifleştirilmiş ve bu süre sonunda her bir petri kutusunda gelişen 2-3 mm çapındaki beyaz - krem renkli, yuvarlak - eliptik koloniler seçilmiş ve 2 kez MRS agarda (Merck 1.10660) saflaştırılmıştır. Saflaştırılan bu izolatlardan Gram pozitif, katalaz negatif ve çubuk şeklindeki bakteriler tanımlama testleri için seçilmiştir. Seçilen izolatlardan her örmek için 10 koloni seçilip tekrar MRS Agara çizilerek ekilmiştir. Anaerobik ortamda gelişen 24 saatlik koloniler steril eküvyon çubuğuyla toplanıp 2 ml’lik süspansiyon med- yum (ref. 70700) içerisinde 2 Mc Farland (ref. 70900) yo-ğunluk elde edilinceye kadar inoküle edilmiştir. Bu işlem- ler her bir izolat için ayrı ayrı yapılmıştır. Karbonhidrat- ların fermente edilmesiyle asidik ortam oluşmakta ve buna bağlı olarak pH düşmektedir. pH düşmesine bağlı olarak indikatörler sayesinde renk değişiklikleri oluşmaktadır. 24 saatlik inkübasyon sonunda tüplerdeki mavi-morumsu renk negatif, sarı, sarı-yeşil renk oluşumu ise pozitif olarak değerlendirilmiş ve suşun biyokimyasal profili elde edilmiştir. Sonuçlar pozitif ve negatif şeklinde sonuç kağı-dına işaretlenmiş ve tanımlama, bilgisayar programı (API Lab Plus Program, Biomerieux) yardımıyla elde edilmiştir.

Kimyasal Analizler

Örneklerde toplam kurumadde miktarı (%) gravimetrik metotla, yağ miktarı (%) Gerber metodu ile yağsız kurumadde miktarı, toplam kurumadde miktarından yağ miktarının çıkarılmasıyla, asitlik derecesi % laktik asit cin-

211

TURGUT, ERDOĞAN ATASEVER

sinden titrasyon metodu ile belirlenmiştir 15. Örneklerdeki % azot miktarı mikrokjeldahl yöntemiyle belirlenmiş ve bu miktarın 6,38 faktörü ile çarpılmasıyla protein miktarı (%), bulunmuştur. Tuz miktarı Mohr yöntemi ve pH değeri pH metre (WTW 3510İ) kullanılarak belirlenmiştir 16.

BULGULAR

Karın Kaymağı Peynirine ait bazı fiziksel ve kimyasal analiz sonuçları Tablo 1’de verilmiştir. Tablo 1’de görüleceği gibi kurumadde oranı en düşük %59.73, en yüksek %85.43 ve ortalama olarak %74.66 bulunmuştur. Karın Kaymağı Peyniri örneklerinin yağ oranları %35-52 gibi geniş bir ara- lıkta değişmiş ve ortalaması %43.36 bulunmuştur. İncele- nen örneklerde protein miktarı oranı ise ortalama %25.51 olmuştur Bu sonuçlar Karın Kaymağı Peynirinin yüksek besin değerine sahip olduğunu göstermektedir.

Peynir örneklerin % asitlik değerleri 1.262-2.088 ve pH değerleri 4.78-5.08 arasında değişmiş ortalamaları ise sıra- sıyla %1.665 ve 4.93 olmuştur. Araştırmada Karın Kaymağı Peyniri örneklerindeki % tuz miktarları 3.14-5.21 arasında bulunmuş ve ortalaması %4.1 olmuştur. Kül miktarı ortala-masının ise %4.68 olduğu saptanmıştır (Tablo 1).

Karın Kaymağı Peynir örneklerine ait bazı mikrobiyolo-jik analiz sonuçları (log kob/g), Tablo 2’de verilmiştir.

Tablo 2’den görüleceği gibi, Karın Kaymağı Peyniri örneklerin toplam aerobik mezofilik bakteri sayıları (TAMB) 6.477-8.17 log kob/g, laktik asit bakteri (LAB) sayıları 6.649-8.041 log kob/g, maya ve küf sayıları ise 3.556-5.308 log kob/g arasında değişmektedir. İncelenen tüm peynir ör- neklerde koliform grubu bakteri, Enterobacteriaceae sayısı ve S. aureus sayısı tespit edilebilir seviyenin altında (<10 log kob/g) bulunmuştur. Bu sonuçlar Karın Kaymağı Pey- nirinin mikrobiyolojik kalitesinin yüksek olduğunu göster-mektedir. Karın Kaymağı Peynirinin yapımında kullanılan işkembenin daha önceden temizlenmiş, 8-10 dak. haşlan-mış ve kurutulmuş olduğu bildirilmektedir 4.

Karın Kaymağı Peynirinden izole edilip API 50 CHL ile tanımlanan izolatların dağılımı Tablo 3’te verilmiştir.

Tablodan izlenebileceği gibi izole edilip tanımlanan 107 adet suşun 82 tanesinin (%76.6) Lactobacillus türü olduğu tespit edilmiştir. Tanımlanan izolatlardan Leu. mesenteroides ssp mesenteroides/ dextranicum tip 2 ise (25 adet) floranın %24.4’nü oluşturmaktadır.

Tablo 1. Karın Kaymağı Peyniri örneklerinin bazı kimyasal analiz sonuçları

Table 1. Some chemical analysis results of Cheese Karın Kaymak samples

Örnek No KM, % Yağ, % Yağsız KM, % Protein, % Asitlik, % pH Kül, % Tuz, %

1234567891011

80.8785.4377.1081.7976.6977.4060.7074.5659.7770.3376.70

4850475248383544383542

32.8735.4330.1029.7928.6939.4025.7030.5621.7725.3334.70

29.9230.1127.8627.4521.8922.3219.1131.1924.8522.6823.26

2.0882.0522.0141.9081.9451.3321.4301.2961.5521.2621.440

5.044.985.024.914.924.854.864.785.064.884.96

4.256.414.185.664.144.534.494.654.124.504.62

3.985.213.974.293.744.213.854.033.144.474.25

Ort. 74.66±2.454 43.36±1.845 30.39±1.527 25.51±1.205 1.665±0.1007 4.93±0.026 4.68±0.215 4.10±0.153

KM: Kuru madde

Tablo 2. Karın Kaymağı Peynirine ait bazı mikrobiyolojik analiz sonuçları (log kob/g)

Table 2. Some microbiological analysis results of Karın Kaymagı Cheese (log cfu/g)

Örnek No

TAMBSayısı

LABSayısı

Koliform Grubu Sayısı

EnterobacteriaceaeSayısı

S. aureusSayısı

Maya - KüfSayısı

1234567891011

6.8388.1706.7786.9498.0907.0256.7786.4776.9828.1046.914

7.0937.6026.6496.8868.0376.9737.1467.0787.1038.0417.113

<10<10<10<10<10<10<10<10<10<10<10

<10<10<10<10<10<10<10<10<10<10<10

<10<10<10<10<10<10<10<10<10<10<10

3.6334.4153.5564.3235.1634.2794.2243.9755.3084.8694.477

Ort. 7.1914±0.184 7.2474±0.136 <10 <10 <10 4.3838±0.1696

212Karın Kaymağı Peynirinden ...

TARTIŞMA ve SONUÇ

Araştırmada, 11 adet Karın Kaymağı Peynir örneğinden izole edilen 107 adet suşun tanımlanması yapılmış ve peynirin bazı fiziksel kimyasal ve mikrobiyolojik nitelikleri belirlenmiştir.

Bu çalışma sonucunda elde edilen bulgular daha önce Karın Kaymağı Peyniri üzerine yapılmış araştırmalarla kar- şılaştırıldığında; çalışmamızda bulduğumuz %74.66 kurumadde, %43.36 yağ, %25.51 protein ve %1.665 asitlik değerleri ortalamalarının Çakmakçı ve ark.9 ile Yangılar ve Dağdemir 17 tarafından bulunan değerlerden daha yüksek olduğu görülmektedir. Çakmakçı ve ark.9 ortalama kurumadde oranını %69.1, yağ oranını %39.0 ve protein oranını %19.10 olarak bildirmişlerdir. Yangılar ve Dağdemir17 tarafından verilen %44.98 kuru madde, %8.95 yağ ve %28.47 protein değerleri ise hem bizim hemde Çakmakçı ve ark.9 tarafından bulunan değerlerden daha düşüktür. Bunun durum Karın Kaymağı Peynirinin yapımında belirli bir standardın olmadığını göstermektedir. Çalışmada, % kül ve tuz miktarı için bulduğumuz değerler her iki araştır-macı tarafından verilen değerlerden daha düşük olmuştur.

Karın Kaymağı Peynirinin mikrobiyolojik özellikleri üze- rine yapılan çalışmalarda, Özdemir ve ark.11 ortalama TAMB sayısının 7.08 log kob/g, LAB sayısının 6.11 log kob/g ve maya küf sayısının 4.96 log kob/g olduğunu, Çakmakçı ve ark.10 ise TAMB sayısının 7.69-9.89 log kob/g, LAB sayı- sının 7.09-9.89 log kob/g ve maya küf sayısının 3.30-6.61 log kob/g arasında değiştiğini belirtmişlerdir. Bizim bul- duğumuz sonuçlar ise TAMB ve LAB sayısı için Çakmakçı ve ark.10 tarafından bulunan sonuçlardan düşük, Özdemir ve ark.11 tarafından bulunan sonuçlardan yüksek olmuş-tur. Araştırmada saptadığımız maya küf sayısı ise diğer iki araştırıcı tarafından verilen değerlerden daha düşük bulunmuştur.

Karın Kaymağı Peynir örneklerde koliform grubu bakteri, sayısı tespit edilebilir seviyenin altında (<10 log kob/g) bulunmuştur. Özdemir ve ark.11 tarafından yapılan çalış- mada da koliform grubu bakteri sayısının <1-1.69 log kob/g

arasında değiştiği saptanmıştır. Bu bakımdan sonuçlar birbirleriyle uyumludur.

Tulum Peyniri ve Van Otlu Peyniri gibi diğer yöresel peynirlerdeki LAB’nin belirlenmesi üzerine çalışmalar ya-pılmasına karşın Karın Kaymağı Peynirinde bu amaçla yapılmış çalışma bulunmamaktadır. Yaptığımız çalışmada tanımlanan suşların sayı ve oranları şu şekildedir: L. plantarum tip 1 (33 adet) %40.24, L. brevis tip 1 (11 adet) %13.4, L. delbrueckii ssp. delbrueckii (9 adet) %10.97, L. acidophilus tip 3 (8 adet) %9.75, L. brevis tip 3 (7 adet) %8.53, L. plantarum tip 1 ve L. delbrueckii ssp lactis tip 1 (6 şar adet) %7.31 ve L. fermentum ise tip 1-2 (2 adet) %2.43. Çalışmamızda, laktobasil türleri Karın Kaymağı Peynirinin dominant florasını teşkil etmişlerdir. Sağdıç ve ark.18, Van yöresine özgü otlu peynirden izole ettikleri suşların L. plantarum, L. casei ssp. casei, L. brevis, Pediococcus pentosaceus, P. acidilactici, Enterococcus faecalis ve E. faecium olduğunu, dominant florayı ise L. plantarum, L. casei ve P. pentosaceus’un oluşturduğunu bildirilmişlerdir. Bizim çalışmamızda da L. plantarum ve L. brevis dominant florayı oluşturmaktadır.

Öner ve ark.19, Tulum peynirinde üzerine yaptıkları çalışmada izole edip tanımladıkları 202 adet suştan 98 tanesinin Lactobacillus ssp. (%48.5) olduğunu ve en fazla sayıda tanımlanan suşun ise L. paracasei ssp. paracasei (%12.4) olduğunu tespit etmişlerdir. Öksüztepe ve ark.20 tarafından Tulum peynirlerinin olgunlaşması sırasında mik-rofloranın değişimi üzerine yapılan başka bir çalışmada da, olgunlaşmanın ilerleyen safhalarında laktobasillerin tulum peynirinde baskın florayı oluşturduğunu, L. casei ssp. casei ve L. plantarum’un olgunlaşmada önemli rol oynadığını belirtmişlerdir.

Bu çalışma ile Karın Kaymağı peynirine özgü laktobasillerin tanımlanması yapılmış ve sonuç olarak laktobasil türlerinin Karın Kaymağı peynirinin dominant florasını teş- kil ettiği saptanmıştır. Geleneksel usullerle üretilen Karın Kaymağı peynirinden izole edilip tanımlanan suşların gelecekte bu peynirin modern usullerle üretilmesi sıra-sında en uygun starter kültür tiplerinin belirlenmesi ve kullanılmasına katkı sağlayacağı ve bu peynire has tat,

Tablo 3. Karın Kaymağı Peynirinden izole edilen ve tanımlan suşların dağılımı

Table 3. Distribution of the strains isolated and identified from the Karın Kaymagı Cheese

Tanımlanan Suş 1 2 3 4 5 6 7 8 9 10 11 Toplam

L. brevis tip1L. brevis tip3L. plantarum tip1L. plantarum tip 2Leu. mesenteroides ssp. mesenteroides/dextranicum 2L. delbrueckii ssp lactis tip1L. delbrueckii ssp delbrueckii L. fermentum tip 1 L. fermentum tip 2 L. acidophilus tip 3

--4-3

-2--1

2-3-2

11--1

--5-3

----1

---43

21---

223-2

-1---

12-2-

2-11-

3-4-2

-----

-13-3

-2--1

2-3-1

1---2

1-4-2

-2---

-24-4

----2

117

336

25

69118

213

TURGUT, ERDOĞAN ATASEVER

koku ve yapının korunmasına yardımcı olacağı düşünül-mektedir. Bu sayede yöresel peynir çeşitlerimize özgü mikrobiyal zenginliğin korunması ve gelecek kuşaklara aktarılması yolunda bir adım daha atılmış olacaktır.

KAYNAKLAR

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2. Hayaloğlu AA: Türkiye’nin peynirleri - Genel bir perspektif. Türkiye 10. Gıda Kongresi, s. 729-732, 21-23 Mayıs, Erzurum, 2008.

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4. Çetinkaya A, Yaman H, Elmalı M, Karadağoğlu G: Geleneksel lezzetler - Kars Gravyer Peyniri. Gıda Müh Derg, 10 (22): 39-40, 2006.

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7. Helland MH, Wicklund T, Narvhus JA: Growth and metabolism of selected strains of probiotic bacteria in milk- and water- based cereal puddings. Int Dairy J, 12, 579-589, 2004.

8. Saarela M, Rantala M, Hallamaa K, Nohynek L, Virkajärvi I, Mättö J: Stationary-phase acid and heat treatment for improvement of the viability of probiotic lactobacilli and bifidobacteria. J Appl Microbiol, 96, 1205-1214, 2004.

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tekniği ve bazı fiziksel ve kimyasal özellikleri, Gıda, 20 (4): 199-203, 1995.

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12. Harrigan WF: Laboratory Methods in Food Microbiology, 3rd ed., 532 p., Academic Press, London, 1998.

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14. Özkaya DF, Kuleaşan H: Maya ve Küf. Gıda Mikrobiyolojisi ve Uygulamaları. II. Baskı. Sim Matbaacılık, Ankara, 2000.

15. Kurt A, Çakmakçı S, Çağlar A: Süt ve Mamulleri Muayene ve Analiz Metotları Rehberi. 7. Baskı. Atatürk Üniversitesi Ziraat Fakültesi Yayınları, No: 18, Erzurum, 1996.

16. Metin M, Öztürk GF: Süt ve Mamulleri Analiz Yöntemleri. I. Baskı. Ege Meslek Yüksekokulu Basımevi, Bornova - İzmir, 2002.

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18. Sağdıç O, Şimşek B, Küçüköner E: Microbiological and physico-chemical characteristics of Van herby cheese, a traditional Turkish dairy product. Milchwissenschaft, 58 (7-8): 382-385, 2003.

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20. Öksüztepe G, Patır B, Çalıcıoğlu M: Identification and distribution of lactic acid bacteria during the ripening of Şavak Tulum Cheese. Turk J Vet Anim Sci, 29, 873-879, 2005.


SummaryThe objective of this study was to determine of pre-parturition and post-parturition behaviors of Norduz Goats. Animal subjects

consisted of 18 primiparous single-birth does aged 2-3 years. During the kidding time, the goats were recorded with digital video cameras for one hour pre parturition and 24 h post-parturition in order to register parturition traits. Twelve does (67%) gave birth while being recumbent and six (33%) while standing (P<0.01). The majority of kidding (n=12, 67 %) occurred between 12.00 -18.00 h, followed by 18.00-24.00 h (n=4, 22%) and 00.00-06.00 h (n=2, 11%). The majority of does (n= 83 %) accepted and nursed their kids after parturition; however, 3 does (17%) rejected their kids after parturition. Of those does who accepted their kids, 14 (93%) refrained from feeding throughout the observation period, whereas only 1 (7%) left her kid to feed during this period (1/15). The duration of parturition, the duration of placenta expulsion, the latency to first sniffing, the latency to first licking, the latency to first suckling, the duration of first suckling, the latency to first standing, and the duration of standing at the birth site were 21.99±2.49 min., 120.74±6.98 min., 0.64±0.39 min., 0.82±0.22 min., 22.65±2.37 min., 0.62±0.13 min., 17.50±2.42 min. and 4> hrs., respectively. These results clearly suggest that in Norduz goats the parturition behavior occurs within four hours after the parturition, and also Norduz goats are observed to be having a normal maternal behavior regarding with investigated behavioral characteristics

Keywords: Norduz goat, Licking, Rejection, Behavior

Norduz Keçilerinde Doğum Öncesi ve Doğrum Sonrası Davranışlarının Belirlenmesi

ÖzetBu çalışmanın amacı doğum öncesi ve doğum sonrası Norduz keçilerinde doğum davranışını belirlemek amacıyla yapılmıştır.

Hayvan materyalini 18 tekiz doğuran Norduz keçisi (2-3 yaşlı) oluşturmuştur. Keçilerin doğum davranışları barınakta kurulan dört kamera ile kaydedilmiştir. Deneme materyali keçilerin %67’si (n= 12) yerde yatarak doğum yaparken %33’ü (n= 6) ayakta durur pozisyonda doğum yapmıştır (P<0.01). Norduz keçilerinde doğumların %67’si (n= 12) 12.00-18.00 arasında, %22’si (n= 24) 18.00-24.00 saatleri arasında %11’i (n=2) ise 00.00-06.00 saatleri arasında gerçekleşmiştir. Doğum sonrası 15 keçi (%83) yavrusuna ilgi göstermiş ancak 3 baş (%17) yavrusunu reddetmiştir. Yavrusunu kabul eden 15 baş keçiden 14’ü yem yeme davranışında bulunmazken sadece bir baş keçi yeme davranışı göstermiştir. Keçilerde doğum süresi, plasentanın atılma süresi, ilk koklamaya kadar geçen süre, ilk yalamaya kadar geçen süre, ilk emiştirmeye kadar geçen süre, ilk emiştirme süresi, oğlakların ayakta kalmasına kadar geçen sure ve doğum yerinde kalma süresi ortalamaları sırasıyla 21.99±2.49, 120.74±6.98, 0.64±0.39, 0.82±0.22, 22.65±2.37, 0.62±0.13, 17.50±2.42 dakika ve 4> saat olarak saptanmıştır. Bu sonuçlar tekiz doğuran Norduz keçilerinde doğum davranışının doğumdan sonra ilk dört saat içerisinde gerçekleştiğini ve aynı zamanda incelenen davranışsal özellikler bakımında normal bir analık davranışa sahip oldukları gözlemlendi.

Anahtar sözcükler: Norduz keçi, Yalama, Reddetme, Davranış

Determination of Pre-parturition and Post-parturition Behaviors of Norduz Goats

Ayhan YILMAZ * Serhat KARACA ** Aşkın KOR ** Mehmet BİNGÖL **

***

Department of Animal Sciences, Hizan Vocational School, Bitlis Eren University, TR-13000 Bitlis - TURKEYDepartment of Animal Sciences, Agricultural Faculty, Yüzüncü Yıl University, TR-65080 Van - TURKEY


The present production systems have limited the opportunity to exhibit natural maternal care behavior of

livestock species making some manipulative changes, especially in semi-intensive or intensive commercial farms.

INTRODUCTION


RESEARCH ARTICLE

216Determination of Pre-parturition ...

In a plan of intensive livestock production system, however, it has been a vital issue to consider high standards of animal welfare and thus to develop the efficient management techniques which permit to exhibit the natural behavior requirements 1,2.

Goat is different from sheep an in terms of the organization of maternal care. The goat, called as “hider” species, leaves her offspring behind while grazing during the first few days after parturition. In pre-parturient period the goats tended to isolate themselves from its flocks for proper maternal care and bonding. For goats, the organization of maternal care is established within 4 h after parturition 3-6.

For mammals, although fertilization is an essential process, the ability to rear young is also a critical factor in this process. In fact, the presence of well-adapted maternal behavior at parturition is necessary for the survival of the newborn. Recently, pre-weaning mortality has been a growing problem in animal breeding worldwide, with the majority of deaths occurring during the neonatal or postnatal period. Abnormal parturition behavior in sheep and goats plays an important role in the post-parturition mortality of kids. Besides poor management systems, mis-mothering is the main cause of pre-weaning mortality. Improvements in management systems can reduce the likelihood of mismothering and improve survival rates among kids. In order to accurately assess the economic aspects of animal improvement programs, behavioral characteristics must be evaluated along with the other factors; however, little is known about the behavioral characteristics related to parturition behavior in goats. Knowledge of the material and topographic features of paddocks and the time periods during which births may be expected to occur are also important 7-9.

The aim of the present study was to determine behavioral characteristics of primiparous goats of Norduz (single-birth) one hour pre-parturition and four hour post-parturition pattern of goats.


The study was carried out on goat flock in the Yüzüncü Yıl University Faculty of Agricultural in Van, Turkey (38°29’ 39”N, 43°22’48”W). Eighteen primiparous Norduz goats (18 single-birth) were selected for the study. The pregnant does were housed as a single flock in a barn two weeks before the parturition and throughout the parturition period (about 25 does; minimum area: 1.5 m2 per doe). Does were fed twice a day with alfalfa and barley mixture, and given to access the water (at 08.00 and 14.00). All does were permitted to choose their own parturition sites within the barn in which they were normally housed and to remain there throughout the experiment. At the same time, does and their kids were permitted to remain together

throughout the observation period. Digital video cameras (Sony Super Had CCD, 3.5-8 mm 1:1/4 lens) were placed in all four corners of the barn in order to provide a full, continuous view of all animals throughout the experiment. Images were transferred to computers and recorded on DVD. The video cameras were recorded continuously for 24 h, but we only evaluated during one h pre-parturition and at 4 h post-parturition for behavior parameters examined. The parturition behavior of Norduz goats was evaluated according to Ramίrez et al.10, Romano and Piaggoi 11 and Martinez et al.9. These parturition behaviors were the birth shape (%), the time of birth (%), the rejection (%), the eating (%), the licking (%), the duration of parturition (min), the duration of placenta expulsion (min), the latency to first sniffing (min), the latency to first licking (min), the latency to first suckling (min), the duration of first suckling (min), the latency to first standing (min) and the duration of stay at birth site (h), respectively.

Data was analyzed using the SAS 12 statistical software program, with the two-proportion Z test used to make comparisons between the groups.

RESULTS

The behavioral characteristics of Norduz primiparous does in pre-parturition are shown in Table 1. Twelve single-birth does (67%) gave birth while recumbent, the remaining six (33%) gave birth standing. There is significant differences regarding the shape of birth (P<0.01). Also, we found that in Norduz primiparous does a large proportion of kidding occurred between 12.00 and 18.00 h, with the lowest percentage occurring between 00.00 and 06.00 h.

Table 1. Behavioral characteristics of Norduz goats during parturition

Tablo 1. Doğum sırasında Norduz keçilerinde davranışsal özellikler

Behavioral Characteristics %

Birth shape

Recumbent % 67 (12)a

Standing % 33 (6)b

Time of birth

12.00-18.00 h 67 (12/18)a

18.00-24.00 h 22 (4/18)b

00.00-06.00 h 11 (2/18)b

Rejection (%) 17 (3/18)

Eating (%) 6 (1/15)

Licking

Head (%) 57 (8/15)a

Genital area (%) 36 (5/15)a

Rest of body (%) 7 (2/15)b

a,b The differences among the values for every behavior characteristic are statistically important (P<0.01)

217

YILMAZ, KARACAKOR, BİNGÖL

The 22% of Norduz primiparous does were kidded between 18.00 and 24.00 h (P<0.01).

At the same time, fifteen single-births does accepted their kids after parturition, and nursed near to their kids throughout observation while three single-births does (3/18) rejected their kids after the parturition. Fourteen single -births does accepting their kids did not eat throughout the observation, and stayed near their kids at postpartum, whereas only one doe ate (1/15).

The behavioral characteristics of Norduz primiparous does pre parturition are shown in Table 1. The areas of the newborns’ bodies were divided to evaluate the place of licking, where does first directed their actions, like the head, the genital area, the rest of body. The licking of head, the genital area, the rest of body in Norduz primiparous does were 57%, 36%, and 7%, respectively (P<0.01). All Does mainly directed to their kids’ head immediately after birth, except for the rejections.

The behavioral characteristics of Norduz primiparous does pre parturition are shown in Table 2. The duration of parturition, the duration of placenta expulsion, the latency to first sniffing, the latency to first licking, the latency to first suckling, the duration of first suckling, the latency to first standing, and the duration of standing at the birth site were 21.99±2.49 min, 120.74±6.98 min, 0.64±0.39 min, 0.82±0.22 min, 22.65±2.37 min, 0.62±0.13 min, 17.50±2.42 min and 4> h, respectively.

DISCUSSION

In this study, the majority of does (n=12, 67%) gave birth while recumbent, whereas the remaining does (n=6, 33%) gave birth standing. The difference in birth posture was statistically significant (P<0.01). These findings were in line with those of Ramίrez et al.10, who reported most goats birthed while lying down (73%). The majority of kidding in primiparous goats (n=12, 67%) occurred between 12.00-18.00 h, followed by 18.00-24.00 h (n=4, 22%) and 00.00-06.00 h (n=2, 11%). Differences in the time of kidding were statistically significant (P<0.01). Lickliter 13 found 72%of births

occurred between 11.00-16.00 hr; Boch et al.14 reported that goat parturition to have an unmodal distribution, with 90.6% of deliveries occurring between 06.00-20.00 h, the majority at midday and the fewest at around midnight; Das and Tomer 15 found that 80% of births occurred between 06.00-1800 h, with a major peak at about 16.00 h; Yamin et al.16 reported that Angora goats give birth more frequently during daylight hours; and Romano and Piaggio 10 found that 78% of births occurred during daylight hours, with 65.7% occurring between 09.00-17.00 h (P<0.01). In contrast to these findings, Stevens 17 and George 18 found that 28% and 20%, respectively, of births occurred during daylight hours, and Lindahl 19 found no significant differences in the percentage of births occurring during the day (06.00-18.00 h) and night (18.00-06.00 h) (45.6% and 54.4%, respectively; P>0.05).

The majority of does (n=15, 83%) accepted and nursed their kids after the parturition, 3 does (17%) rejected their kids after the parturition. Of those does who accepted their kids, 14 (93%) refrained from feeding throughout the observation period, whereas 1 doe (7%) left her kid to feed during this period (1/15). First licking was directed primarily at the head (57%), followed by the genitals (36%) and the remainder of the body (7%), with the differences between areas of first licking statistically significant (P<0.01). With the exception of those does that rejected their kids, licking was directed mainly at the head of the kid immediately after birth. Licking is known to stimulate the respiratory system,

thereby facilitating breathing; to help removing the fetal membrane; and to stimulate peripheral blood circulation of the kid following birth. The phenomenon of licking in goats has been outlined in numerous studies 10,13,20. In contrast to small ruminant animals, Owens et al.21 found that the majority of cows (21/23) start licking their calves near the abdomen and sides, paying particular attention to breaking the umbilical cord. In sum, Norduz goats exhibited highly developed maternal behavior, in general caring exclusively for their offspring in the period immediately following birth, and most births occurred during day time and the birth shape was lying down.

Table 2. Parturition-related characteristics of primiparous Norduz goats

Tablo 2. Tekiz doğuran Norduz keçilerinde doğumla ilgili özellikleri

Behavioral Characteristics n X±Sx Minimum Maximum

Duration of parturition, min 18 21.99±2.49 8.10 43.00

Duration of placenta expulsion, min 18 120.74±6.98 33.50 158.0

Latency to first sniffing, min 16 0.64±0.39 0.05 6.40

Latency to first licking, min 15 0.82±0.22 0.05 6.40

Latency to first suckling, min 15 22.65±2.37 11.12 43.15

Duration of first suckling, min 15 0.62±0.13 0.08 1.62

Latency to first standing, min 18 17.50±2.42 7.12 50.27

Duration of stay at birth site, h 15 4> - -

218Determination of Pre-parturition ...

The present study found the mean duration of parturition in Norduz primiparous does to be 21.99±2.49 min. This value was similar to the value given in Das and Tomer 15 for Beetal does (10-30 min), but lower than that given by Martinez et al.9 for Murciano Granadina goats (60.48 mean). The differences between study findings may be due to differences in contraction times. In the present study, the mean duration of placenta expulsion was 120.74±6.98 min (minimum: 33.50 min; maximum: 158.0 min) (Table 2). Although Das and Tomer 15 reported a lower mean value (49 min) for complete expulsion of the placenta, their finding is within the range of minimum and maximum values found in the present study.

Immediately following birth, does in this study typically stood up, turned around and began vigorously sniffing and licking their kids. First sniffing was observed to occur immediately after birth (0.64±0.39 min). This finding is similar to that of Martinez et al.9, who found a value of 47.86 s for first sniffing, and to Lickliter 12, Sambraus and Wittmann 20 and Malfatti et al.22, who indicated that sniffing and licking commenced immediately following parturition in small ruminants. However, this finding differed from that of Ramίrez et al.10, who found a latency of 8.7±0.8 min for first sniffing among single-birth does. In fact, in our study, the range of latency to first licking varied widely from 0.05 min to 6.40 min. In the present study, latency to first licking was 0.82±0.22 min and occurred immediately after first sniffing. Latency to first licking was shorter than in Ramίrez et al.10, who reported a time of 13.3±1.0 s. for single-birth kids, but longer than Martinez et al.9, who reported latency to first licking to be 98.61 s. In general, the findings of this study regarding suckling are in line with those of other authors 12,19,21. Most kids in this study (15 of 18) were able to suckle during the first hour post-partum; however, three kids were rejected by their mothers and were unable to suckle.

Mean latency to first suckling was 22.65±2.37 min. This finding is in contrast to Martinez et al.9, who reported 37.32 min to mean suckling for single-birth kids. Duration of first suckling in the present study was 0.62±0.13 min, which is similar to that of Sambrauss and Wittmann 20, who reported the duration of first suckling to be 0.63 min, but it differs from Ramίrez et al.10 and Martinez et al.9, who reported shorter durations for first suckling. In line with Ramίrez et al.10and Martinez et al.9, all kids in the present study succeeded in standing during the first hour of life. Mean latency to first standing of kids in the present study was 17.50±2.42 min, which is similar to the findings for single-birth kids reported by Martinez et al.9, Sambrauss and Wittmann 20 and Ramίrez et al.10, who observed latency to standing to be 16.87 min, 18.7 min and 19.37 min, respectively. In addition, with the exception of the 3 does who rejected their kids, the does in this study did not leave their kids during the 4-hour post-partum observation period. This finding is important in terms of the bond developed between mother and kid.

In conclusion, in pre-parturient period Norduz goats isolated from themselves their flocks and most births occurred during day time, and the birth shape was lying down. Consistent with findings of other studies, the maternal care was established immediately following the birth. Immediately following the birth, does in this study typically stood up, turned around and began vigorously sniffing and licking their kids, and thus within 4 h after the parturition all postnatal behaviors evaluated except for the duration of standing at the birth was developed. Especially, in intensive selected populations, ability of parturition behavior shows an increasing decrease and causes seriously the lost of economic in developed countries using modern production systems. Therefore, in intensive production system planning based on natural behavioral characteristics of domestic animals, producers should be considered the selection related to maternal behavior because domestication decrease the ability of maternal behavior.

Acknowledgements

We would like to thank Dr. Abdullah Yeşilova for his statistical support and the Yüzüncü Yıl University, Faculty of Agriculture in Van, Turkey for providing the animals used.

REFERENCES

1. Miranda-dela L, Mattiello GC: The importance of social behavior for goat welfare in livestock farming. Small Rumin Res, 90, 1-10, 2010.

2. Arslan C: İneklerde beslenme davranışları. Kafkas Univ Vet Fak Derg, 15 (4): 641-648, 2009.

3. Addae PC, Awotwi EK, Oppong-Anane K, Oddoye EOK: Behavioural interactions between West African dwarf nanny goats and their single-born kids during the first 48 hours post-partum. Appl Anim Behav Sci, 67, 77-88, 2000

4. Poindoron P: Mechanisms of activation of maternal behavior in mammals. Reprod Nut Dev, 45, 341-351, 2005.

5. Poindoron P, Terrazas A, Navarro Montes de Oca ML, Serafin N, Hernández H: Sensory and physiological determinants of maternal behavior in the goat (Capra hircus). Hormones and Behavior, 52, 99-105, 2007.

6. Poindoron P, Levy F, Keller M: Maternal responsiveness and maternal selectivity in domestic sheep and goats: The two facets of maternal attachments. Developmental Psychobiology, 49, 54-70, 2007.

7. Awemu EM, Nwakalor LN, Abubakar BY: Environmental influence on preweaning mortality and reproductive performance of Red Sokoto does. Small Rumin Res, 34, 161-165, 1999.

8. Mellor DJ, Stafford KJ: Animal welfare implications of neonatal mortality and morbidity in farm animals. Vet J, 168,118-133, 2004.

9. Martinez M, Otal J, Ramίrez A, Hevia ML, Quiles A: Variability in the behavior of kids born of primiparous goats during the first hour after parturition: Effect of the type of parturition, sex, duration of birth, and maternal behavior. J Anim Sci, 87, 1772-1777, 2009.

10. Ramίrez A, Quiles A, Hevia M, Sotillo F: Behavior of Murciano-Granadino goat in the hour before parturition. Appl Anim Behav Sci, 44, 29-35, 1998.

11. Romano JE, Piaggio J: Time of parturition in Nubian goats. Small Rumin Res, 33, 285-288, 1999.

12. S.A.S: User’s Guide: Statistics. SAS Inst. Inc., Cary, NC, 2010.

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13. Lickliter RE: Bahviour associated with parturition in the domestic goat. Appl Anim Behav Sci, 13, 335-345, 1985.

14. Bosc M, Guillimin P, Bourgy G, Pignon: Hourly distribution of time of parturition in the domestic goat. Theriogenology, 30, 23-33, 1988.

15. Das N, Tomer OS: Time pattern on parturition sequences in Beetal goats and crosses: Comparison between primiparous and multiparous does. Small Rumin Res, 26, 157-161, 1997.

16. Yamin M, Payne G, Blackshaw JK: The Time of birth and the choice of birth sites by Booroola Merino ewes and Angora goats. Appl Anim Behav Sci, 45, 89-96, 1995.

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shelters at lambing? Appl Anim Etiology, 17, 149-155, 1981.

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19. Lindahl IL: Time of parturition in ewes. Anim Behav, 12, 231-234, 1964.

20. Sambrauss HH, Wittmann M: Beoachtungen zu geburtsabrauf and saugverhalten von ziegen. Tierarztl Prax, 7, 359-365, 1989.

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SummaryThe aim of this study was to investigate the teratogenic effects of Malathion (ML) induced by different doses on fetal kidney tissues

in pregnant rats. A total of 28 Sprague-Dawley pregnant rats were randomly divided into 4 groups of 7 rats each. Depending on ML dose, four groups were formed, including (I) control, (II) ML 2.5 (ML 2.5 mg/kg/day, orally), (III) ML 5 (5 mg/kg/day, orally), and (IV) ML 10 (10 mg/kg/day, orally). ML application started when the male and female were put together (when mating started). Daily ML application was continued until birth. It was determined that in parallel with dose of ML, ML resulted in toxic effects on serum enzymes (acetyl-cholinesterase (AChE), amylase and lipase) and kidney tissues of pregnant rats, and also -regardless of ML dose in fetal kidneys- it led to teratogenic effects in all the doses. Biochemical data wasconfirmed by histopathologic data. We concluded that ML leads to kidney damage in both pregnant and fetal rats as a result of its teratogenic and toxic effects.

Keywords: Fetus, Pregnant rat, Organophosphate, Prenatal exposure, Teratogenic effect

Gebe Ratlara Farklı Dozlarda Uygulanan MalathionunFetüs Böbrek Dokusu Üzerine Teratojenik Etkileri

ÖzetBu çalışmanın amacı gebe ratlara düşük dozlarda subakut uygulanan Malathion (ML)’un fetüs böbrek dokusu üzerine teratojenik

etkisini araştırmaktır. Toplam 28 Sprague-Dawley gebe rat randomize olarak her grupta 7 adet gebe rat olacak şekilde 4 gruba ayrıldı. ML’un dozuna bağlı olarak, gruplar; kontrol, ML 2.5 (2.5 mg/kg/gün dozunda oral yoldan (p.o) ML uygulandı), ML 5 (5 mg/kg/gün, p.o) ve ML 10 (10 mg/kg/gün, p.o) olmak üzere 4 gruba ayrıldı. ML uygulamsı erkekler ve dişilerin aynı ortama konulmasından itibaren başladı (çiftleşmeden itibaren). Günlük ML uygulamasına doğuma kadar devam edildi. ML’un, dozuna paralel bir şekilde gebe ratlarda böbrek dokusu ve serum enzimleri (asetilkolinesteraz (AChE), lipaz, amilaz) üzerine toksik etkiler oluşturduğu, ayrıca yavru rat böbreklerinde ise ML’un dozuna bağlı olmayarak, tüm dozlarda teratojenik etkiye neden olduğu belirlendi. Histopatolojik veriler biyokimyasal verileri doğruladı. ML’un düşük dozlarının bile hem anne hem de yavru böbrekleri üzerine toksik ve teratojenik böbrek hasarına neden olduğu sonucuna varıldı.

Anahtar sözcükler: Fetüs, Gebe rat, Organofosfat, Prenatal maruziyet, Teratojenik etki

Effects of Malathion in Fetal Kidney Tissues in Pregnant Rats: Teratogenic Effects Induced by Different Doses

Harun ALP * Muhammet Erdal SAK ** Mehmet Sıddık EVSEN ** Ugur FIRAT *** Osman EVLIYAOGLU **** Necmettin PENBEGUL ***** Ahmet Ali SANCAKTUTAR ***** Haluk SOYLEMEZ ***** Mehmet TUZCU ******

***

*******

***********

Mustafa Kemal University, Medical Faculty, Department of Pharmacology, TR-31040 Hatay - TURKEYDicle University, Medical Faculty, Department of obstetrics and gynecology, TR-21280 Diyarbakir - TURKEYDicle University, Medical Faculty, Department of Pathology, TR-21280 Diyarbakir - TURKEYDicle University, Medical Faculty, Department of Biochemistry, Diyarbakir- TURKEYDicle University, Medical Faculty, Department of Urology, TR-21280 Diyarbakir - TURKEYCumhuriyet University, Veterinary Faculty, Department of Pathology, TR-58140 Sivas - TURKEY


Toxicologic evidences indicate that low-level exposure to organophosphate pesticides (OP) may affect neuro-

development and growth in developing animals and children. Several studies have revealed that fetotoxicity

INTRODUCTION


RESEARCH ARTICLE

222Effects of Malathion in ...

and marked neurochemical changes may occur due to repeated exposures to OP during gestation 1-9. Recent biological studies on females and children have reported that there is a widespread OP exposure 10-15. There are concerns about the potential health effects of pre- and post-natal exposure of children to pesticides, therefore the Food Quality Protection Act (FQPA) established a stringent health-based standard for pesticide residues in food to assure protection from pesticide exposure and to strengthen health protection from pesticide risks for sensitive populations 1,12.

Children are particularly sensitive to the health risks of pesticide exposure since their internal organs are not fully developed. For instance, their immune systems may not be able to protect them against pesticides, and their excretory systems may not be able to excrete these toxic chemicals. Indeed, pes ticide exposure may permanently affect development negatively by blocking the absorption of nutrients. Especially children of farmworkers are at risk for pesticide exposure. Their parents may bring pesticide residues from the agricultural fields into the house and thus the pesti cides may drift from fields into areas where children play 16.

Long-term exposure of the pregnant women to OP can cause toxic effects both on their bodies and their fetuses. However, the number of studies on subacute toxicity of OP on maternal and fetal organs is scarce. Also, there are no studies which compare maternal and fetal tissues in this sense. Malathion (ML) (O,O-dimethyl-S-1.2-bis ethoxy- carbonyl ethyl phosphorodithioate), which is a common OP in the world, was selected as the toxic material of the present study since it usually has lower toxicity than other pesticides. Malaoxon is the bioactivation metabolite of ML, causing higher toxicity. Malaoxon inhibits acetyl-cholin-esterase (AChE), causing the accumulation of acetylcholine within synapses and consequently leading to the overstimulation of postsynaptic receptors and producing rapid twitching of voluntary muscles, incoordination, convulsions, paralysis and ultimately death 17-23. The aim of this study was to investigate the teratogenic effects of ML induced by subacute low doses on fetus kidney tissues in pregnant rats.


The study was approved by Dicle University Animal Ethical Committee and was carried out in accordance with the ‘‘Animal Welfare Act and the Guide for the Care and Use of Laboratory animals prepared by Dicle University Animal Ethical Committee’’. Female Sprague-Dawley rats (250±50 g) were obtained from the Animal laboratory at Dicle University. The rats were housed in clean polypropylene cages (having six rats per cage) and maintained under controlled room temperature (23±2°C) with a photoperiod of 12 h light and 12 h dark cycle. The rats were given standard pellet diet and water ad libitum throughout the

experimental period.

In the study, at first, a total of 40 Sprague-Dawley female rats were divided into 4 groups of 10 rats each. ML was applied in various low doses (2.5-5 and 10 mg/kg/day, orally). The rats were caged for 3 days with one male and one female in each cage. Once ML was applied, the rats were mated. After 3 days, male rats were removed from the cages and vaginal smear was applied to the female rats to determine pregnancy. Only the pregnant females were studied. A total of 28 Sprague-Dawley pregnant rats were randomly divided into 4 groups of 7 rats each. Depending on dose, the rats were divided into 4 groups including (I) control, (II) ML 2.5 (2.5 mg/kg/day, orally), (III) ML 5 (5 mg/kg/day, orally), and (IV) ML 10 (10 mg/kg/day, orally). ML application started when the male and female were put together (when mating started). Daily ML application was continued until birth.

After the birth and anesthesia with ketamine, (85 mg/kg, intraperitoneal, Ketalar, Pfizer), blood samples were obtained from mother rats using intracardiac puncture in sterile tubes without anticoagulant. After a one-hour clotting in the room temperature and centrifugation (1500 g, 10 minutes, 4°C), the sera were carefully harvested and stored at -20°C till biochemical analysis.

Biochemical Analysis

The enzyme amylase, lipase and AChE activities in serum were determined with Roche Cobas Integra 800 autoanalyser via enzymatic colorimetric method using Roche brand commercial kits in 409/659 nm. The enzyme activities were expressed as U / L. Measurement range of the tests were 3-2000 U/L (0,05-33 µkat/L) for amylase, 200-14000 U/L (3,34-234 µkat/L) for AChE and 3-1200 U/L (0,05-5,01 µkat/L) for lipase. All analyses were performed in Biochemistry Department of Dicle University Medical Faculty.

Histopathologic Analysis

Immediately after the death of the rats, kidney tissues were processed in 10% formaldehyde solution via cassette autotechnic tissue processing equipment (Leica ASP 300). Once the processing was completed, the tissues were embedded in paraffin blocks and the sections (5 μm in thickness) were taken by microtome instrument onto lysine laminin. The preparations stained with haematoxylin and eosin (H&E) were evaluated under a light microscope at x100 magnification (Olympus BX51) by a pathologist blinded to the study groups.

Histopathologic evaluation consisted of tissue damages including the intensity of cellular hydropic degeneration along with neutrophil and mononuclear cell infiltration, degenerative changes, nuclear loss, necrosis and fibrosis. Each organ was graded 23 on a scale (Fig. 1). For the kidney, for instance, the occurrence of tubular epithelium damage

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and proteinous accumulation in the tubular lumen due to the filtration failure were examined and graded. Each parameter was scored between 0 and 3 (0: normal, 1: mild, 2: moderate, and 3: severe) depending on the situation of lesions (Table 1).

Statistical Analysis

Activities of enzymes were analyzed using One-way ANOVA and Tukey test. A value of P<0.05 was considered statistically significant.

RESULTS

In the present study, depending on ML dose, histo- pathologically vacuolar degeneration, necrosis and spills were observed in kidney tubular epithelium of the ML groups. In the F1 and P1 groups, swelling of the tubular cells with brush border loss were present. In the P2 and F2 groups, in addition to swelling and brush border loss of the tubular cells, nuclear losses in the tubular cells were seen. Apart from these complications, the P3 and F3 groups presented with degenerations and numerous nuclear losses in the tubular cells (Fig. 1). Moreover, regardless of ML dose, widespread degenerations and necrosis (nuclear loss) were observed in all the fetal groups. The lesions were scored depending on the scale (Fig. 1). The results conclusively indicated that Malathion results in toxic effects on kidney tissues in pregnant rats depending on the dose, and it causes teratogenic effects in fetal kidneys at all doses. In the biochemical evaluation of pregnant rats, a significant difference was observed between the control

group and the ML group in terms of enzyme results. It was determined that ML leads to a decrease in amylase and cholinesterase activities and an increase in lipase activities. Also, the in-group comparisons among ML groups revealed that, depending on dose, the use of ML inhibits AChE and amylase activities while increasing lipase activity (Table 2).

DISCUSSION

Widely used in agriculture, OP has several important features such as environmental safety, limited persistence, and selective toxicity to insects with respect to mammals 24. Therefore, ML was selected as the toxic material for the present study. Previous studies argued that ML inhibits AChE, causing the accumulation of acetylcholine within synapses, and consequently leading to overstimulation of postsynaptic receptors. Among these, Asini et al.25 demonstrated that repeated ML administrations cause a decrease in the circulating AChE activity in rats. Akhgar et al.26 and Rezg et al.27 also observed that the plasma AChE activity in rats was seriously decreased in the case of subchronic exposure to ML.

It has been accepted that a 20% AChE inhibition causes the deleterious effects of OP 28, and when the ratio is greater than 50%, there is a life-threatening situation 29. Thus, a decrease in the AChE activity is considered as a significant marker for OP poisoning. In the present study, depending on dose, the AChE inhibition ratio reached 10.4% in ML 2.5 group, 36.1% in ML 5, and 44% in ML 10. These results indicated that the doses of 5 mg/kg/day and 10 mg/kg/day may cause deleterious effects while the doses greater

Table 2. Comparison of serum enzymes among groups (mean ± Standard deviation)

Tablo 2. Çalışma gruplarında serum enzimlerinin karşılaştırılması

Group Amylase (U/L) Lipase (U/L) Cholinesterase (U/L)

Control 2752±361 4.24±0.58 1066±52

Malathion 2.5 2019±273a 5.22±0.35 956±98

Malathion 5 1801±102b 5.70±0.62b 681±91b,d

Malathion 10 1688±99c 7.74±1.30c,e,f 596±29c,e

a, b, c; difference between control with Malathion 2.5, 5, 10 respectivelyd, e; difference between Malathion 2.5 with Malathion 5, 10 respectivelyf; difference between Malathion 5 with Malathion 10 for a,c,d,e,f values: P<0.001, for b value: P = 0.011

Table 1. Grades for kidney lesions

Tablo 1. Böbrek lezyonlarının dereceleri

Grades Evaluation

Grade 0 No Lesion

Grade 1 Swelling of tubular cells coupled to the loss of brush border, Alteration of the tubule up to a third with nuclear loss without nuclear thickening and epithelial cells into the lumen

Grade 2 Swelling of tubular cells coupled to the loss of brush border Alteration of the tubule up to 2 thirds with nuclear loss and proteinous mass into the lumen

Grade 3 Cell degeneration Tubular alterations in more than 2 thirds with nuclear loss, proteinous mass into the lumen and neutrophil/mononuclear cell infiltrates in the interstitium


Fig 1. Fetal (F) and Pregnant (P) rat kidney tissues (H&E stain, x100). Normal histomorphological appearance of the pregnant and fetal kidney tissues of control groups (“P control” and “F control” respectively). Swelling of the tubular cells (down and left arrows) with brush border loss (right arrows) [P1 and F1 respectively]. Swelling and brush border loss of the tubular cells (right arrows) with some nuclear loss (up and down arrows) [P2 and F2 respectively]. In addition to swelling and brush border loss (right and left arrows), degeneration and many nuclear loss of the tubular cells (up and down arrows) are seen [P3 and F3]

F: Fetal rats; F1: Fetal rats treated with Malathion (2.5 mg/kg/day, p.o), F2: Fetal rats treated with Malathion (5 mg/kg/day, p.o), F3: Fetal rats treated with Malathion (10 mg/kg/day, p.o), P: Pregnant rats; P1: Pregnant rats treated with Malathion (2.5 mg/kg/day, p.o), P2: Pregnant rats treated with Malathion (5 mg/kg/day, p.o), P3: Pregnant rats treated with Malathion (10 mg/kg/day, p.o)

Şekil 1. Fetus (F) ve gebe (P) sıçan böbrek dokuları (H&E boyama, x100). Kontrol gruplarına ait fetal ve gebe sıçan böbrek dokularında normal histomorfolojik görünüm (sırasıyla “P control” ve “F control”). Fırçamsı kenar kayıpları (aşağı ve sol oklar) ile birlikte tubuler hücrelerde şişme (sağ oklar) (Sırasıyla P1 ve F1)). Tubuler hücrelerde bazı nükleer kayıpların ( yukarı ve aşağı oklar) eşlik ettiği fırçamsı kenar kaybı ve şişme (sağ oklar) (Sırasıyla P2 ve F2). Tubuler hücrelerde fırçamsı kenar kaybı ve şişme (sağ ve sol oklar) yanı sıra dejenerasyon ve birçok hücrede nükleer kayıp (yukarı ve aşağı oklar) görülmektedir (sırasıyla P3 ve F3)

F: Fetal sıçan; F1: Malathion uygulanmış fetal sıçan (2.5 mg/kg/gün, oral), F2: Malathion uygulanmış fetal sıçan (5 mg/kg/gün, oral), F3: Malathion uygulanmış fetal sıçan (10 mg/kg/gün, oral), P: Gebe sıçan; P1: Malathion uygulanmış gebe sıçan (2.5 mg/kg/gün, oral), P2: Malathion uygulanmış gebe sıçan (5 mg/kg/gün, oral), P3: Malathion uygulanmış gebe sıçan (10 mg/kg/gün, oral)

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than 10 mg/kg/day may be life-threatening. However, the toxic indicators of ML are not only limited to inhibition of AChE. OP may cause several effects on other parameters such as lipase and amylase activities. Precise measurement of these enzymes is very important in that they indicate organ damages. In previous studies, it was revealed that OP may change lipase and amylase activities depending on pancreatitis and other organ damages. Alp et al. found that ML (200 mg/kg, single dose, orally) markedly depletes the AChE activity while significantly increasing the GGT (g-glutamyltransferase) and amylase activities 23. Gokalp et al.30 reported that high doses of diazinon, used as an OP, significantly inhibits and decreases serum amylase activities in rats due to pancreatitis. In the present study, in parallel with dose of ML, it was found that ML inhibits AChE activity while increasing lipase activity as suggested by Gokalp et al.30, and decreasing amylase activity as contrary to the result by Alp et al.23.

According to the histopathologic analyses in previous studies, OP is likely to cause kidney damage. Alp et al.23

stated that degenerative changes associated with necrosis and inflammatory cell infiltration were evident in the kidneys of the rats intoxicated with ML. In a study by Kalender et al.31, vascular dilation and glomerular atrophy were observed in kidney tissues 4 weeks after the administration of methyl parathion as OP to rats. In another study, it was observed that diazinon leads to tubular swelling, hyperplasia and cell infiltration in rabbit kidneys 32. Sulak et al.33 also administered the OP methidathion to male rats over a 4-week period, concluding that methidathion causes a reduction in AChE activity and kidney damage, together with tubular epithelial cell degeneration, focal tubular necrosis, fibrosis and infiltration. In the present study, similar to the studies aforementioned, it was revealed that ML, in parallel with the dose, causes vacuolar degeneration, necrosis and spills in kidney tubular epithelium. In addition, it leads to mono-nuclear cell infiltrations and intertubular bleeding. Regardless of ML dose, wide spread degenerations and necrosis was observed in all the fetal kidneys of the ML group. In the present study, similar to histopathologic results, it was found that ML, in parallel with dose of ML, inhibits AChE activity while increasing lipase activity and decreasing amylase activity. The biochemical results were verified by histopathologic results.

According to these results, we conclude that even low doses of ML have strong toxic effects on both pregnant and fetal kidney tissues, causing teratogenic kidney damage.

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23. Alp H, Aytekin I, Atakisi O, Hatipoglu NK, Basaralı K, Ogun M, Buyukbas S, Altintas L, Ekici H, Alp A: The effects of caffeic acid phenethyl ester and ellagic acid on the levels of malondialdehyde, reduced glutathione and nitric oxide in the lung, liver and kidney tissues in acute diazinon toxicity in rats. J Anim Vet Adv, 10 (11): 1488-1494, 2011.

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25. Akhgari M, Abdollahi M, Kebryaeezadeh A, Hosseini R, Sebzevari O: Biochemical evidence for free radical induced lipid peroxidation as a mechanisim for subchronic toxicity of MAL in blood and liver of rats. Hum Exp Toxicol, 22, 205-211, 2003.

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Biolog, 330, 143-147, 2007.

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29. Gokalp O, Buyukvanli B, Cicek E, Ozer ME, Koyu A, Altuntas I, Koylu H: The effects of diazinon on pancreatic damage and ameliorating role of vitamin E and vitamin C. Pest Biochem Physiol, 81, 123-128, 2005.

30. Kalender S, Kalender Y, Durak D, Ogutcu A, Uzunhisarcikli M, Cevrimli BS, Yildirim M: Methyl parathion induced nephrotoxicity in male rats and protective role of vitamins C and E. Pest Biochem Physiol, 88, 213-218, 2007.

31. Yehia MAH, El-Banna SG, Okab AB: Diazinon toxicity affects histo-physiological and biochemical parameters in rabbits. ExpToxicol Pathol, 59, 215-225, 2007.

32. Sulak O, Altuntas I, Karahan N, Yildirim B, aAkturk O, Yilmaz HY, Delibas N: Nephrotoxicity in rats induced by organophosphate insecticide methidathion and ameliorating effects of vitamins E and C. Pest Biochem Physiol, 83, 21-28, 2005.


ÖzetBu çalışmada, sperm kanalları olan, abdominal masaj yoluyla gamet toplanabilen, fonksiyonel olarak erkekleştirilmiş gökkuşağı

alabalığı (Oncorhynchus mykiss) anacı üretimi için uygun hormonal cinsiyet dönüşüm prosedürü araştırılmıştır. Bu amaçla 4 grup deneme yapılmıştır. Oral uygulama grubunda, serbest yüzmeye başlayan post larvalar ilk yem alma aşamasından itibaren 600 derece-gün süresince kilogramında 1.0-3.0 mg 17α-metiltestosteron içeren yemlerle beslenilmiştir. Banyo uygulama gruplarında ise, 0.5-1.0 mg l-1 17α-metiltestosteron, 11β-hidroksiandrostenedion ya da 17α-metildihidrotestosteron keseli larval dönem boyunca (döllenmeden itibaren 370-560 derece-gün) kısa süreli (2 saatlik) tek, 48 saat aralı 2-3 ya da 1 hafta aralı çift banyo şeklinde verilmiştir. Çalışma sonuçları, gökkuşağı alabalığında fonksiyonel erkekleştirilmiş dişi anaç üretimi için en uygun hormon prosedürünün döllenmeden sonraki 470-800 derece-gün’leri kapsayan, periyodik banyolar veya periyodik banyolar ve düşük dozda oral uygulamalar şeklinde bir prosedür olabileceğini, androjen banyolarının fonksiyonel erkekleştirilmiş dişi anaçlar üretmekte daha etkili olduğunu, düşük konsantrasyonlarda (≤1 mg kg-1 yem) verildiğinde oral 17α-metiltestosteron uygulamalarının da yüksek oranda fonksiyonel erkek birey üretebileceğini göstermiştir.

Anahtar sözcükler: Oncorhynchus mykiss, 17α-metiltestosteron, 17α-metildihidrotestosteron, 11β-hidroksiandrostenedion, fonksiyonel hormonal cinsiyet değişimi

The Production of Functional Sex-Reversed Male Rainbow Trout (Oncorhynchus mykiss)

SummaryIn this study, appropriate hormonal sex reversal procedure for the production of functional sex-reversed male rainbow trout

(Oncorhynchus mykiss) brooders with intact sperm ducts was investigated. For this purpose, 4 groups of experiment were conducted. In oral administration group, free swimming larvae starting from the first feeding were fed 1.0-3.0 mg 17α-methyltestosterone kg-1 diets for 600 degree-days. In the remaining experimental groups, 0.5-1.0 mg l-1 17α-methyltestosterone, 11β-hidroksiandrostenedione or 17α-methyldihydrotestosterone were administered throughout the sac fry stage (370-560 degree-days post fertilization) as short (2 h) single, 48 h apart 2-3 or 1 week apart double baths. Results of the study demonstrated that appropriate androgen procedure for the production of functionally masculinized rainbow trout females should be covering the 470-800 degree-days post fertilization, but it could be periodic baths or periodic baths and oral administration of low concentrations, at low concentrations (≤1 mg kg-1 feed) oral administration of 17α-methyltestosterone could yield high ratios of functional sex reversed males.

Keywords: Oncorhynchus mykiss, 17α-methyltestosterone, 17α-methyldihydrotestosterone, 11β-hidroksiandrostenedione, functional hormonal sex reversal

Fonksiyonel Erkekleştirilmiş Gökkuşağı Alabalığı (Oncorhynchus mykiss) Üretimi

Tülin ARSLAN * Fatime ERDOĞAN ** Mete ERDOĞAN **

***

Mugla University, Faculty of Fisheries, Department of Aquaculture, TR-48000 Mugla - TURKEY Mugla University, Ortaca Vocational School, Department of Fisheries, TR-48600 Mugla - TURKEY


Ülkemizde entegre kültürü yapılan tek tatlı su balığı olan gökkuşağı alabalığı, Oncorhynchus mykiss Walbaum 1792, yetiştiriciliğinde çoğu ülke verimi artırmak amacıyla

üretimde tamamı dişi populasyonlar kullanmaktadır 1-3. Çünkü gökkuşağı alabalığı erkekleri birinci yaşları içeri- sinde seksüel olgunluğa erişmeye başlarlar. Erginleşmeyle

GİRİŞ

İletişim (Correspondence) +90 252 2111893 [email protected], [email protected]

RESEARCH ARTICLE

228Fonksiyonel Erkekleştirilmiş ...

birlikte yemle alınan enerjinin büyük bir kısmı gonad gelişimine yönlendirildiğinden, erkeklerde büyüme yavaş- lamakta, kaslarda depolanan protein ve lipit miktarların- daki düşüşlere ve kas pigmentasyonunda değişime bağlı olarak et kalitesi de düşmektedir 1,3.

Tek cinsiyetli balık populasyonlarının üretiminde ekonomik olması, yüksek teknoloji ve uzmanlık gerektirme- mesi ve homozigotluk oranının artması gibi istenmeyen genetik yan etkilerinin olmaması dolayısıyla en çok tercih edilen üretim tekniği, hormonal cinsiyet dönüşüm metodudur 2,4-6. Pazara sunulan son ürün üzerinde hormon kullanılmasını gerektirmediğinden sofralık balık üreti- minde bu metot dolaylı olarak uygulanır 2,4,7. Dolaylı hor-monal cinsiyet dönüşüm uygulamasında, hormonlarla cinsiyeti değiştirilmiş bireyler (genotipik ve fenotipik cinsiyeti farklı bireyler) anaç olarak kullanılarak çaprazlama yoluyla tek cinsiyetli populasyonlar üretilir 2,4,7. Gökkuşağı alabalığında çaprazlama yoluyla tamamı dişi populasyonlar üretmekte kullanılan erkekleştirilmiş dişi anaçlar yavruların ilk yemleme aşamasından itibaren 60-90 gün süreyle kilogramında 0.5-3 mg 17α-metiltestosteron içe- ren yemlerle beslenmesiyle üretilmektedir 1,5,8,9. Bu pro-sedür gonadal cinsiyeti başarılı bir şekilde değiştirmekle birlikte, üretilen erkeklerde sperm kanalları oluşmamak- tadır 1,10,11. Sperm kanalları olmayan erkekleştirilmiş dişiler-den gamet elde etmek ancak gonadların ameliyatla vücut dışına çıkarılmasıyla mümkündür. Bu yüzdende geç ergin- leşen ve 2-3 yılda büyütülen bu anaçlar ancak bir defa kullanılabilmektedir. Ayrıca normal (genotipik) erkekler gibi abdomene uygulanan yumuşak bir basınçla sperm bırakamadıklarından gonadal olgunluklarının önceden kontrol edilmesi de mümkün değildir. Vücut renginin ko- yulaşması, alt çenenin uzaması gibi karakterler genotipik erkeklerde olduğu gibi gonad olgunluğu hakkında bil- gilendirici olmakla birlikte, ikincil cinsiyet karakterleri iyi gelişmiş erkekleştirilmiş dişilerin sperm kalitesi her zaman aynı derecede iyi olmayabilmektedir 12. Bunun sebebi olarak sperm kanalı olmayan bu erkeklerde, spermin son olgunlaşma aşamasını kanaldaki seminal plazma orta- mında tamamlayamaması gösterilmektedir 12. Bu nedenle, erkekleştirilmiş dişi anaçlardan elde edilen sperm kullanıl- madan önce bir süre olgunlaştırma solüsyonlarında bek- letilir. Aksi halde olgunlaşmasını tamamlayamamış spermin yumurtaları dölleme kapasitesi çok düşük olabilmekte ve bu durum önemli ekonomik kayıplar yaşanmasına yol açmaktadır 12.

Gökkuşağı alabalığında ilk yemleme sırasında gonad- ların somatik gelişimi zaten başlamıştır 13-15. Sitolojik deği- şim (primordial üreme hücrelerinin oogonia veya sper- matogonia’a dönüşümü) veya ilk mayoz (primer oosit-lerin oluşumu) ise ilk yemlemeden sonra olmaktadır 13-15. Dolayısıyla, ilk yemlemeyle başlatılan hormon uygulama- larından elde edilen erkekleştirilmiş dişilerde sperm kanal- larının oluşmaması uygulamanın zamanlaması ile ilgili bir sorun olabilir. Fonksiyonel, sperm kanalları olan erkek-

leştirilmiş dişi anaçlar üretmek için hormon uygulamala- rının gonadların somatik değişimi sırasında, keseli larval dönemde başlatılması gerekebilir. İşte bu öngörü doğrul-tusunda gerçekleştirilen araştırmamızın amacı fonksiyonel, sperm kanalları olan ve abdominal masaj yoluyla sperm bırakabilen erkekleştirilmiş dişi gökkuşağı alabalığı anacı üretimi için uygun hormonal cinsiyet dönüşüm pro-sedürünü araştırmaktır.

MATERYAL ve METOT

Bu araştırmanın saha çalışmaları Muğla ili, Fethiye ilçe- sinde yıl boyu sabit sıcaklıkta (8.7±0.1ºC) kaynak suyu ile üretim yapan ticari bir gökkuşağı alabalığı kuluçkahane işletmesinde gerçekleştirilmiştir. Saha çalışmalarına 2005 yılı Aralık ayı sonlarında, gökkuşağı alabalığının bölgedeki doğal üreme sezonu (Kasım-Ocak) içerisinde başlanılmış-tır. Çalışmada, işletmede üretilen değişik yaşlardaki besin keseli ve besin kesesini tüketmiş larvalara doğal veya sentetik androjenler (11β-hidroksiandrostenedion (OHA, Sigma A3009), 17α-metiltestosteron (MT, Sigma M7252), 17α-metildihidrotestosteron (MDHT, Sigma M5626)) oral ya da banyo yolu ile verilmiştir.

Oral veya banyo yolu ile hormon uygulamaları için gerekli miktardaki androjen hassas terazide tartıldıktan sonra %96’lık etanol içerisinde çözdürülmüş ve 1 mg androjen ml-1 konsantrasyonunda stok solüsyonlar hazır- lanmıştır. Hazırlanan bu stok solüsyonlar denemelerde kullanılan konsantrasyonlara seyreltilerek hormonlu yemlerin hazırlanmasında ve banyo uygulamalarında kullanıl- mıştır. Hormonlu yemler Arslan ve ark.’nın çalışmasında 4

tarif edildiği şekilde hazırlanmıştır. Hormon banyoları larvalara 40 L hacimli plastik küvetlerde verilmiştir. Küvetler banyo uygulamalarından hemen önce taze su ile dol-durulmuş, planlanan konsantrasyonda androjen içeren stok solüsyonlar banyo suyuna eklenerek kuvvetli bir ha-valandırmayla tüm suya homojen bir şekilde karışmaları sağlanmıştır. Hormon banyoları sırasında, keseli larvalar işletmedeki dikey inkubatörlerden gözenekli tepsilerle birlikte alınarak, banyo küvetlerine yerleştirilmiştir. İki saatlik banyo süresi boyunca, küvetlerdeki su oksijen tüpüne bağlı hava taşı vasıtasıyla, büyük hava kabarcıkları oluşmasına müsaade etmeden hafifçe havalandırılmıştır. Banyo süresinin sonunda, larvalar yine tepsiler içersinde hormon banyosu küvetlerinden çıkarılarak, temiz su ile birkaç sefer yıkanmış ve dikey inkubatörlerine geri yerleş-tirilmiştir. Dikey inkübatörlerde besin kesesini tüketen larvaların bakımında ve oral yolla hormon uygulamaları sırasında 80x240 cm2 boyutlarında kapalı alan, fiberglas yapay yeme alıştırma tankları kullanılmıştır. Bu tanklarda 1-2 g’a ulaşan deneme balıkları gonad gelişimlerinin değerlendirildiği büyüklüklere ulaşıncaya kadar değişik boyutlarda, açık alan beton havuzlarda bakılmışlardır. Beton havuzlarda büyütme sürecinde deneme balıklarına hiç boylama yapılmamış, stoklama yoğunlukları düşük (<5 kg/m3) tutulmuştur. Çalışma süresince deneme balıkları

229

ARSLAN, ERDOĞANERDOĞAN

büyüklüklerine bağlı olarak doyana kadar günde 1-8 defa, ağız açıklıklarına uygun büyüklükte %45-57 oranlarında ham protein içeren alabalık yemleriyle (Trouw Nutrition TR) beslenilmiştir.

Saha çalışmalarının ilk yılında, sonraki yıllarda anaç olarak kullanmak ve denemeleri tamamı dişi populasyonlar üzerinde yapabilmek amacıyla, mevcut hormonal cinsiyet değişim prosedürü kullanılarak erkekleştirilmiş dişi populasyonlar üretilmiştir. Bu amaçla, besin kesesini tüketip, serbest yüzmeye başlayan on biner adet post larva iki tekrarlı olarak ilk yemlemeden itibaren kilogramında 3 mg MT içeren yemle 600 derece-gün süresince beslenilmiştir.

Saha çalışmalarının ilk yılında ve takip eden 2 yılın üreme sezonlarında, birbiriyle bağlantılı 3 grup banyo yoluyla hormon uygulama denemesi yapılmıştır. Bu denemelerde, eksternal hormonlara en duyarlı gelişim dönemi olarak rapor edilen 8,16 yumurtaların açılmasından-dış beslenmeye geçiş dönemi içerisindeki dört biner keseli larvaya yumurta açılmasının tamamlandığı 43. günden (döllenmeden itibaren 370 derece-gün) başlanarak haftalık periyotlarda 0.5 mg l-1 OHA, MT ya da 0.5-1 mg l-1 MDHT içeren tek veya iki farklı sıklıkta 2-3 hormon banyosu verilmiştir (Tablo 1). Kontrol grupları hormon banyoları ile eş zamanlı olacak şekilde ve hormon banyolarıyla aynı miktarda etanol içeren alkol banyolarına tabi tutulmuştur. Çalışma boyunca yapılan tüm denemeler 2 tekrarlı olarak gerçekleştiril-miştir. Sadece son yıldaki banyo denemelerinde 4 tekrarlı bir kontrol grubu kullanılmıştır.

Çalışmanın oral hormon uygulaması üzerine yapılan

son grup denemesinde, mevcut hormonal cinsiyet deği- şim prosedürüne göre yeme katılan MT dozunun gök- kuşağı alabalığının gonad gelişimi üzerine etkileri araştı-rılmıştır. Bu denemede, besin kesesini tüketip serbest yüzmeye başlayan on biner adet post larva iki tekrarlı olarak ilk yemlemeden itibaren kilogramında 1, 2 ya da 3 mg MT bulunduran yemler ile 600 derece-gün süresince beslenilmiştir.

Hormon uygulamalarından bir sonraki üreme sezonunda, gonad gelişiminin tamamlandığı bilinen 7-8. ay sonrasında, 100 g ve üzerine ulaşan deneme balıkları hormon uygulamalarının gonad gelişimi üzerindeki etkisini değerlendirmek üzere örneklenmiştir. Örneklemelerde, uygulamaların her bir tekrarından rastgele olarak 100-110 adet balık alınmıştır. Örneklenen balıklar aşırı dozda anestetik (2-fenoksi ethanol) ile öldürüldükten sonra bir bistüri yardımıyla abdomenleri açılmış ve gonad mor- folojileri önce makroskobik olarak incelenmiştir. Makro- skobik incelemeler sonrası, bütün halde ve hava kesesine yapışık olarak çıkarılan gonadlar %10’luk nötralize-formalin içerisinde fikse edilmiştir. Fikse edilen gonad örnek-lerinden fast green boyası ve gonadal-ezme metodu kul- lanılarak yaş preparatlar hazırlanmıştır 17. Ayrıca gonadal dokunun detaylı incelenebilmesi için her bir hormon uygulamasından 5-10 balığın gonadları kullanılarak Harris-hematoksilen ve eosin ile boyanmış kalıcı preparatlar ha-zırlanmıştır. Yaş ve kalıcı preparatların mikroskobik ince- lemesiyle gonadlar gözlemlenen hücre tiplerine ve iç morfolojik özelliklerine göre dişi, erkek veya interseks ola-rak sınıflandırılmıştır.

Tablo 1. Gökkuşağı alabalığında fenotipik cinsiyeti değiştirmede kullanılan 11β-hidroksiandrostenedion (OHA), 17α-metiltestosteron (MT) ve 17α-metildihidrotestosteron (MDHT) banyolarının zamanları, uygulama yoğunlukları ve dozajları

Table 1. Timing, intensities and dosages of 11β-hydroxyandrostenedione (OHA), 17α-methyltestosterone (MT) and 17α-methyldihydrotestosterone (MDHT) immersions used for changing the direction of phenotypic sex in rainbow trout

Uygulama Başındaki Larval Gelişim Aşaması Uygulama Yoğunluğu Androjen (Doz)

%100 yumurta açılması(Döllenmeden itibaren 370 derece-gün)

48 saat aralı 2 banyoKontrolOHA (0.5 mg l-1)MT (0.5 mg l-1)

48 saat aralı 3 banyo MT (0.5 mg l-1)

%100 yumurta açılmasından 1 hafta sonra(Döllenmeden itibaren 420-450 derece-gün)

Tek banyoKontrolMDHT (0.5 mg l-1)MDHT (1 mg l-1)

48 saat aralı 2 banyo OHA (0.5 mg l-1)MT (0.5 mg l-1)


1 hafta aralı 2 banyo MT (0.5 mg l-1)MDHT (0.5 mg l-1)

%100 yumurta açılmasından 2 hafta sonra(Döllenmeden itibaren 470-490 derece-gün)

Tek banyo KontrolMDHT (0.5 mg l-1)


1 hafta aralı 2 banyo MDHT (0.5 mg l-1)

%100 yumurta açılmasından 3 hafta sonra(Döllenmeden itibaren 560 derece-gün) Tek banyo MDHT (0.5 mg l-1)


Başarılı sayılabilecek hormon uygulamalarında, örnek- lemelerden arta kalan balıklar sperm kanalı gelişimi ve cinsiyet dönüşümünün işlevselselliğinin tam olarak değer- lendirilebilmesi için seksüel olgunluğa eriştikleri 2. yaşla-rına kadar açık alan beton havuzlarda büyütülmüştür. Bu balıklarda sperm kanalı gelişimi ikinci yaşlarındaki üreme sezonunda kontrol edilmiş ve abdominal masaj yoluyla sperm veren kanallı (fonksiyonel) erkeklerin oranı tespit edilmiştir. Abdominal masajla yoluyla gamet toplanama- yan bireyler aşırı dozda anestetik ile öldürüldükten sonra bir bisturi yardımıyla abdomenleri açılmış ve gonad mor- folojileri makroskobik olarak incelenmiştir. Makroskobik incelemeler sonrası, bütün halde, hava kesesine yapışık olarak çıkarılan gonadlar %10’luk nötralize-formalin içeri- sinde fikse edildikten sonra gonadal-ezme metodu kulla- nılarak mikroskobik olarak değerlendirilmiştir. Bu değer- lendirilmelerde grimsi veya süt beyaz renkte gonadları olgun ve/veya olgunlaşmakta olan sperm hücreleri içeren bireyler erkek, ipliksi pembe renkte gonadları yoğun bağ doku ve az sayıda değişime uğramamış hücre içeren bireyler steril (kısır) olarak sınıflandırılmıştır. Sınıflandırma gruplarındaki balık sayıları yüz ile çarpıldıktan sonra incelenen deneme tekrarındaki toplam balık sayısına bö-lünerek yüzde erkek, dişi, interseks ve steril birey oranları tespit edilmiştir.

Veriler SAS (Statistical Analysis System, SAS Instute Inc., Carry, NC, ABD) yazılımının 8.2 sürümü kullanılarak analiz edilmiştir. Yüzde erkek ve interseks oranları analizler öncesi arcsin karekök transformasyonuna tabi tutul-muştur. Deneme gruplarının cinsiyet oranları arasında fark olup olmadığı tek yönlü varyans analizi (one way-ANOVA) ile saptanmıştır. ANOVA testinin α = 0.05 önem derecesinde istatistiki belirgin fark işaret ettiği durumlarda, hormon uygulamalarının ortalamaları önce Dunnett t-testi kullanılarak ilgili kontrol grubu ortalamaları ile karşılaştırılmıştır. Daha sonra uygun durumlarda, Duncan çoğul aralık testi kullanılarak, ortalamalar kendi deneme grupları içersinde etki derecelerine göre sınıflandırılmıştır.

BULGULAR

Oral ya da banyo yoluyla hormon uygulamaları, kul-lanılan dozlarda gökkuşağı alabalığında toksisiteye sebep olmamıştır. Hormon uygulamaları boyunca ve sonunda, bütün deneme gruplarının yaşam oranlarının benzer ve çalışılan kuluçkahanenin başarı standartları içerisinde (%70-80) olduğu gözlenmiştir.

Banyo uygulamalarının tümü erkek oranlarında artışa sebep olmuştur. Fakat OHA banyoları ve %100 yumurta açılmasında ya da 1 hafta sonra verilen 48 saat aralı 3 MT banyosunun erkek oranlarında oluşturduğu artış istatistiki olarak önemli (P>0.05) bulunmamıştır (Tablo 2). Farklı larval yaşlarda verilen tek ya da çift MT veya MDHT banyolarının tümü ise erkek oranlarında istatistiki belirgin (P<0.05) artışa sebep olmuştur (Tablo 2, 3). Erkekleşme oranları ile MT banyo uygulamalarının başlatıldığı larval yaşlar ara-sında belirgin bir bağlantı tespit edilemezken, MDHT banyo uygulamalarının başlatılması için en uygun larval yaşın %100 yumurta açılmasından 2 hafta (döllenmeden itibaren 470 derece-gün) sonra olduğu tespit edilmiştir (Tablo 3). Bu dönemde verilen 0.5 mg l-1 çift MDHT banyosu erkek oranlarında istatistiki olarak en yüksek artışa (ortalama %57.4) sebep olmuştur. Bunlara ilaveten, banyo denemeleri gökkuşağı alabalığını erkekleştirmek için 0.5 mg l-1 MDHT dozunun yeterli olduğunu, uygun larval geli- şim aşamasında verildiğinde birden fazla MDHT banyo-sunun erkekleşme oranları üzerinde olumlu etki ettiğini ve MDHT banyolarının gökkuşağı alabalığında cinsiyet değişi- mini yönlendirmede MT banyolarından daha etkili oldu- ğunu ortaya koymuştur (Tablo 3). Ayrıca periyodik androjen banyo uygulamalarında 48 saat yerine, haftalık aralıklar kullanılmasının daha uygun olduğunu göstermiştir (Tablo 3).

Oral uygulamalar, ilk yemleme aşamasından itibaren 600 derece-gün süresince kilogramında 1-3 mg MT içeren yemlerle besleme, genotipik dişilerin tamamına yakınında gonadal gelişimi etkilemiş ve gonadal değişimin yönünü

Tablo 2. Farklı yaşlardaki keseli larvalara verilen 48 saat aralı 2-3 periyodik 11β-hidroksiandrostenedion (OHA) ve 17α-metiltestosteron (MT) banyolarının gökkuşağı alabalığının cinsiyet oranları üzerine etkileri. Aynı sütunda farklı küçük harflerle işaretlenmiş ortalamalar istatistiki belirgin farklar göstermektedir (P<0.05)

Table 2. Effects of administering 48 hours apart 2-3 periodic 11β-hydroxyandrostenedione (OHA) and 17α-methyltestosterone (MT) immersions at different pre-larval stages on sex ratios of rainbow trout. Means in the same column followed by different lower case letters are significantly different (P<0.05)

Uygulama Başındaki Larval Gelişim Aşaması Uygulama Yoğunluğu Androjen (Doz) N (±SS) % Ortalama

Erkek (±SS)% Ortalama

İnterseks (±SS)% Toplam Etkilenen

%100 yumurta açılması48 saat aralı 2 banyo

KontrolOHA (0.5 mg l-1)MT (0.5 mg l-1)

91 (7)103 (7)87 (4)

58.0 (2.1) b66.5 (3.5) b76.0 (2.8) a

--

2.5 (1.3) a

-8.5

20.5

48 saat aralı 3 banyo MT (0.5 mg l-1) 102 (5) 66.8 (2.7) b 1.0 (1.3) a 9.8

%100 yumurta açılmasından 1 hafta sonra

48 saat aralı 2 banyo OHA (0.5 mg l-1)MT (0.5 mg l-1)

103 (10)89 (3)

67.0 (1.4) b75.5 (0.7) a

--

9.017.5

48 saat aralı 3 banyo MT (0.5 mg l-1) 104 (8) 56.9 (2.6) b 1.4 (0.6) a 10.3

%100 yumurta açılmasından 2 hafta sonra 48 saat aralı 2 banyo MT (0.5 mg l-1) 93 (4) 79.0 (1.5) a 2.1 (1.4) a 23.1

231


değiştirmekte etkili olmuştur (Tablo 4). Yüzde ortalama erkek oranları %97.2-99.5 arasında değişmiş ve yemdeki MT miktarına bağlı herhangi bir farklılık göstermemiştir (P>0.05). Bununla birlikte, spermatogenezin başladığı 2. yaşta (266-699 g) yapılan tetkiklerde önemli oranlarda steril bireyin tespit edilmesi (Tablo 5), spermatogenez öncesi 7-8. aylarda yapılan değerlendirmelerde steril bireylerin erkek olarak sınıflandırıldığını işaret etmiştir. Steril birey-lerin oranları ise yemdeki MT miktarıyla doğru orantılı bir artış göstermiştir (Tablo 5).

Spermatogenezin başladığı 2. yaşta yapılan tetkikler, oral uygulamaların %22’lere varan kısır birey oluşumuna sebep olduğunu gösterirken, hormon uygulamasına tabi tutulmayan kontrol populasyonlarının birinin de %2 ora-nında kısır birey içerdiğini ortaya koymuştur (Tablo 5). Ayrıca 48 saat aralıkla verilen kısa süreli MT banyolarının düşük oranlarda da olsa kısırlaşmaya sebep olabileceğini işaret etmiştir. Diğer yandan, oral uygulamalarda sperm kanalı gelişiminin yeme eklenen MT miktarına bağlı ol-

duğu ve kullanılan MT miktarı düştükçe kanallı erkek ora- nın arttığı görülmüştür. Bununla birlikte, fonksiyonel, sperm kanalına sahip ve abdominal masajla sperm toplanabilen erkekleştirilmiş dişi anaç üretmekte androjen banyolarının oral MT uygulamalarından daha etkili olduğu tespit edil- miştir (Tablo 5).

TARTIŞMA ve SONUÇ

Hormonlarla cinsiyetin başarılı bir şekilde yönlendi-rilmesi ve fonksiyonel olarak fenotipik cinsiyeti değiştirilmiş bireylerin elde edilmesi hormon uygulamalarının geli-şime bağlı zamanlamasına, uygulamanın süresine, kulla- nılan hormonun türüne ve dozuna bağlıdır 2,5,6. Gonokorist balıkların sitolojik değişime uğramamış gamet hücreleri uzun süre cinsiyet hormonlarının etkisine duyarlı kala- bilmektedir. Fakat hormon uygulamalarının gonadların sitolojik değişimden hemen önce başlatılması her zaman fonksiyonel cinsiyet değişimi ile sonuçlanmayabilir. Sito-

Tablo 3. Farklı yaşlardaki keseli larvalara verilen tek ya da 1 hafta aralı çift 17α-metildihidrotestosteron (MDHT) ve 48 saat ya da 1 hafta aralı çift 17α-metiltestosteron (MT) banyolarının gökkuşağı alabalığının cinsiyet oranları üzerine etkileri. Aynı sütunda farklı küçük harflerle işaretlenmiş ortalamalar istatistiki belirgin farklar göstermektedir (P<0.05)

Table 3. Effects of administering single or 1 week apart double 17α-methyldihydrotestosterone (MDHT) and 48 hours or 1 week apart double 17α-methyltestosterone (MT) immersions at different pre-larval stages on sex ratios of rainbow trout. Means in the same column followed by different lower case letters are significantly different (P<0.05)

Uygulama Başındaki Larval Gelişim Aşaması Uygulama Yoğunluğu Androjen (Doz) N (±SS) % Ortalama

Erkek (±SS)% Ortalama

İnterseks (±SS)% Toplam Etkilenen


Tek banyoKontrolMDHT (0.5 mg l-1)MDHT (1 mg l-1)

96 (4)95 (0)96 (5)

1.2 (0.4) f11.6 (2.5) e13.5 (1.5) e

-11.6 (3.0) a5.4 (1.7) ab

-22.017.7

1 hafta aralı 2 banyo MT (0.5 mg l-1)MDHT (0.5 mg l-1)

98 (4)100 (2)

28.1 (0.2) c33.0 (1.0) b

3.4 (1.5) b6.4 (2.7) ab

30.338.2

48 saat aralı 2 banyo MT (0.5 mg l-1) 107 (9) 20.9 (0.1) d 4.7 (0.9) ab 25.1


Tek banyo KontrolMDHT (0.5 mg l-1)

100 (8)98 (3)

0.5 (0.4) f37.0 (3.6) b

-2.8 (3.6) b

-39.3

1 hafta aralı 2 banyo MDHT (0.5 mg l-1) 96 (3) 57.9 (2.3) a 2.6 (2.3) b 60.0

%100 yumurta açılmasından 3 hafta sonra Tek banyo MDHT (0.5 mg l-1) 98 (2) 29.7 (3.3) bc 2.5 (1.7) b 31.7

Tablo 4. Farklı dozlarda 17α-metiltestosteron (MT) içeren yemlerle beslemenin gökkuşağı alabalığının cinsiyet oranları üzerine etkileri. Aynı sütunda farklı küçük harflerle işaretlenmiş ortalamalar istatistiki belirgin farklar göstermektedir (P<0.05)

Table 4. Effects of feeding with different dosages of 17α-methyltestosterone (MT) containing diets on sex ratios of rainbow trout. Means in the same column followed by different lower case letters are significantly different (P<0.05)

Deneme Grubu N (±SS) % Ortalama Erkek (±SS) % Ortalama İnterseks (±SS)

Kontrol 96 (6) 45.0 (2.1) b -

3 mg MT/kg yem 93 (8) 99.5 (0.7) a -

Kontrol 96 (4) 1.2 (0.4) c -

3 mg MT/kg yem 92 (2) 98.0 (1.0) a -

2 mg MT/kg yem 90 (3) 97.2 (2.6) a 2.8 (2.6) a

1 mg MT/kg yem 85 (2) 98.1 (2.8) a 1.0 (1.4) a

* Post larvalar ilk yemlemeden itibaren 600 derece-gün sürecince kilogramında 1-3 mg MT içeren yemlerle beslenmiştir (Starting from first feeding post larvae were feed 1-3 mg/kg MT containing diets for a duration of 600 degree-days)


lojik değişimden hemen önce başlatılan hormon uygula- malarının farklı balık türlerinde gonadların yalnızca game-tik elementlerini etkilediği, ama somatik elementlerini değiştirmediği tespit edilmiştir 13,18-20. Çünkü gonadların somatik değişimi özellikle de gonadal kanal sisteminin oluşumu ve gonadal hücrelerin fizyojik değişimi arasında zamanlama türlere bağlı farklılıklar gösterebilmektedir 14. Yani gökkuşağı alabalığında olduğu gibi sperm kanalları oluşmamış, morfolojik olarak testisten çok ovaryuma ben- zeyen ama yalnızca erkek üreme hücresi içeren gonadlar gelişebilir 1,9,11. Bu türlerde cinsiyetin fonksiyonel olarak değiştirilmesi ve üreme özellikleri ile fenotipik eşdeğer- lerine benzer bireylerin oluşturulması, hormon uygula-malarının gonadların somatik değişimi sırasında başlatıl-masını gerektirebilir.

Nitekim gökkuşağı alabalığının yakın akrabası olan ve gonadlarının sitolojik değişimi larvaların serbest yüzme aşamasına denk gelen Pasifik salmonlarında, O. kisutch ve O. tshawytscha 21,22, ilk yemlemeden itibaren oral yolla veri-len steroidler çok çeşitli sonuçlar üretmiş ve genelde fenotipik cinsiyeti yönlendirmede başarısız olmuştur 5. Gonad-ların somatik büyüme aşamasına denk gelen yumurtaların medyan açılması sırasında verilen 2 saatlik tek androjen veya östrojen banyoları ise fonksiyonel olarak tamamı erkek veya tamamı dişi populasyonlar üretmekte başarılı olmuştur 21-23. Bu çok kısa steroid uygulamalarının yüksek başarısı uygulamaların gonadların fizyolojik olarak hor- monların etkisine en açık olduğu dönemde verilmiş ol-

masına 22 ve verilen steroidlerin salmon larvalarının nispe-ten büyük besin kesesi tarafından absorbe edilerek, banyo uygulamalarından çok sonra bile larvalara aktarılabilme- sine bağlanmıştır 24.

Buna karşılık gonadların sitolojik değişimi larvaların serbest yüzmeye başladığı ay içersinde gerçekleşen gökku- şağı alabalığı 14-16 ve Atlantik salmonunda, Salmo salar 2,5,14, ilk yemlemeden itibaren yemle verilen androjen veya östrojenler, fenotipik cinsiyeti yönlendirmede başarılı so- nuçlar üretmekle birlikte, bu uygulamayla erkekleştirilen bireylerde sperm kanalı oluşmadığı rapor edilmiştir 1,10,11. Bu durum uygulamaların zamanlamasıyla ilişkilendirildi- ğinden, gonadal değişimin hem somatik hem de sitolojik elementlerini etkileyebilmek için her iki türde de keseli larval dönemde banyo yoluyla steroid uygulamaları yapıl- mıştır. Atlantik salmonunda yapılan uygulamalar, yumur- taların medyan açılmasından sonra, 2-4. haftalarda (döl- lenmeden 570-690 derece-gün sonra) verilen 3 saatlik 2 adet 0.4 mg l-1 MDHT banyosunun %100 erkek populasyonlar üretmekte başarılı olduğunu göstermiştir 25. Ayrıca bu yolla üretilen erkekleştirilmiş dişilerin %84-92 oranında fonksiyonel, kanallı bireylerden oluştuğu rapor edilmiş- tir 25. Feist ve ark.’nın ön çalışmasında 8 ise, gökkuşağı ala- balığı yumurtalarına (açılmadan 1 hafta önce) ve larva- larına (medyan açılmada, açılmadan 1-2 hafta sonra) verilen 1-4 adet, 2 saatlik 0.4 mg l-1 MT ya da OHA banyosunun %0-76 oranında erkekleşmeye sebep olduğu ve bu yolla erkekleştirilen dişilerin %60-100 oranında kanallı fonksi-

Tablo 5. Banyo ya da oral yolla verilen 11β-hidroksiandrostenedion (OHA), 17α-metiltestosteron (MT) ve 17α-metildihidrotestosteron (MDHT) uygulamalarının gökkuşağı alabalığının gonad gelişimine etkileri

Table 5. Effects of immersion or oral 11β-hydroxyandrostenedione (OHA), 17α-methyltestosterone (MT) and 17α-methyldihydrotestosterone (MDHT) administrations on gonadal development of rainbow trout

Deneme Grubu

Uygulama Başındaki Larval Gelişim Aşaması

Uygulama Yoğunluğu

% Ortalama Erkek (±SS) N % Ortalama

Kanallı Erkek % Ortalama Steril Birey

Kontrol %100 yumurta açılması 48 saat aralı 2 banyo 45.0 (2.1) 100 98.0 2.0

OHA (0.5 mg l-1)

%100 yumurta açılması48 saat aralı

2 banyo

66.5 (3.5)

50 82.0 2.0%100 yumurta açılmasından 1 hafta sonra 67.0 (1.4)

MT (0.5 mg l-1)

%100 yumurta açılması

48 saat aralı 2 banyo

76.0 (2.8)

75 80.0 8.0%100 yumurta açılmasından 1 hafta sonra 75.5 (0.7)

%100 yumurta açılmasından 2 hafta sonra 79.0 (1.5)

MDHT (0.5 mg l-1)


1 hafta aralı 2 banyo 33.0 (1.0) 50 90.0 -


1 hafta aralı 2 banyo 57.9 (2.3) 75 96.0 -

3 mg MT/kg yem

Yeme yeni kalkan -

99.5 (0.7) 75 44.0 20.0

3 mg MT/kg yem 98.0 (1.0) 100 18.0 22.0

2 mg MT/kg yem 97.2 (2.6) 100 35.0 13.0

1 mg MT/kg yem 98.1 (2.8) 100 53.0 3.0

233


yonel bireylerden oluştuğu rapor edilmiştir. Ayrıca banyo uygulamaları için en uygun zamanın medyan açılmadan 1 hafta (döllenmeden yaklaşık 425 derece-gün) sonra olduğu gözlenmiştir 8. Buna karşılık bu çalışmada, en yüksek ba- şarı döllenmeden sonraki 470-560 derece-gün’lerde verilen banyo uygulamalarında elde edilmiş ve MDHT ban- yolarının MT banyolarından daha etkili olduğu tespit edil- miştir. Ama benzer şekilde, banyo yoluyla üretilen erkek-leştirilmiş dişilerin yem uygulamalarına göre daha yüksek oranda (%80-96) fonksiyonel, kanallı bireylerden oluştuğu görülmüştür.

Banyo uygulamaları daha yüksek oranda fonksiyonel, kanallı erkek üretmekle birlikte, keseli larval dönemin deği- şik zamanlarında verilen banyo uygulamalarının hiçbiri gökkuşağı alabalığında tamamı veya tamamına yakını erkek populasyonlar oluşturmakta başarılı olamamıştır. Pasifik veya Atlantik salmonlarındaki gibi populasyondaki tüm bireylerin etkilenebildiği, eksternal androjenlerin etkisine duyarlılığın maksimum olduğu kısa kritik bir gelişim dönemine rastlanmamıştır. Diğer yandan, yumurta açılma- sından-keseli larval dönemin son haftasına kadar (döllen-meden itibaren 370-560 derece-gün arası) verilen tüm tek veya çift MT ve MDHT banyoları erkek oranlarında önemli artışa sebep olmuştur. Fakat yumurta açılmasından sonraki 2. ve 3. haftalarda (döllenmeden sonraki 470-560 derece-gün’lerde) verilen çift MDHT banyosu aynı dönemde verilen tek banyo uygulamalarından daha etkili olmuştur. Keseli larval dönem sonrasında, uzun bir süreçte (600 derece-gün boyunca) oral yolla verilen MT ise genotipik dişilerin tamamına yakınında gonadal gelişimi etkilemiş ve gonadal değişimin yönünü değiştirmekte daha etkili olmuştur. Ayrıca yeme eklenen MT miktarı düştükçe, erkekleştirilen dişilerde sperm kanallı, fonksiyonel birey oranının arttığı (%18’den %53’e yükseldiği) gözlenmiştir. Benzer bir çalışmada da, yemdeki MT oranının 3 mg’dan 1 mg/kg’a düşürmenin fonksiyonel erkek oranını %26.4’ten %54.7’ye yükselttiği görülmüştür 4. Bu sonuçlar, kanal olu- şumu ve fonksiyonel cinsiyet değişiminin, uygulamanın zamanlaması yanında uygulamada kullanılan hormonun dozajı ile de ilgili olduğunu ifade etmektedir. Ayrıca gök- kuşağı alabalığında eksternal androjenlere en duyarlı peri-yodun döllenmeden sonraki 470 derece-gün civarında baş- lamakla birlikte, keseli larval dönem sonrasında da devam ettiğini işaret etmektedir. Dişi ovaryumlarında mayozun keseli larval dönemden sonraki haftalar içersinde (800 derece-gün civarında) başladığı da 15 göz önünde bulun- durulursa, bu sonuçlar gökkuşağı alabalığında fonksiyonel monoseks erkek populasyonlar üretmek için uygun andro-jen prosedürünün en azından döllenmeden sonraki 470-800 derece-gün’leri kapsayan, periyodik banyolar veya periyodik banyolar ve düşük dozlu oral uygulamalar şek-linde bir prosedür olması gerektiğini ortaya koymaktadır.

Yumurtaların açılmasından bir hafta (döllenmeden iti-baren 420-450 derece-gün) sonra birer hafta yerine daha yakın (48 saat) aralıkla verilen MT banyolarında, banyo sayısı

artıkça erkekleşme oranlarının düştüğü görülmüştür. Banyo yoluyla verilen steroidler salmonid larvalarının nispeten büyük besin kesesi tarafından absorbe edilmektedir 24. Dolayısıyla sık periyotlarda androjen banyosu verilen larvalar, besin kesesinde birikim sonucu daha seyrek periyotlarda androjen banyosu verilen keseli larvalara göre daha yüksek dozajlarda androjene maruz kalmış olabilirler. Diğer yandan, birer hafta arayla verilen benzer konsantrasyonda MT banyolarında banyo sayısı arttıkça erkekleşme oran- larında azalma olduğu gözlemlenmiştir ve banyo uygula- malarının etkinsinin artan banyo sayısı ile azaldığı rapor edilmiştir 8. Ama o çalışmada 8, keseli larvalar daha yük-sek su sıcaklığında (11.5ºC) bakılmışlardır. Yüksek su sıcak- lıklarında bakılan larvaların metabolik hızı ve besin kesesini tüketme süresi, dolayısıyla maruz kaldıkları gerçek andro-jen dozu, larvaların daha düşük bir sıcaklıkta (8.7±0.1ºC) bakıldığı bu çalışmaya göre daha yüksek olmuş olabilir. MT östrojene aromatize olabilen bir androjen olduğun- dan 26, yüksek dozda MT verilen balıklarda aromatizasyon sonucu erkekleşme oranlarında düşüş ve hatta dişi oran- larında artışlar (paradoksal dişileşme) görülmüştür 23,27,28. Bu sebeple, periyodik MT banyo uygulamalarında gözlem-lenen erkekleşme oranlarında azalma, banyo sayısından çok larvaların maruz kaldığı gerçek androjen dozajı ile bağlantılı olabilir.

Öte yandan, östrojene aromatize olabilen androjenlerin yüksek dozları kısa süreli uygulamalarda paradoksal dişi- leşmeye sebep olurken, tüm androjenlerin yüksek dozları uzun süreli uygulamalarda sterilizasyona (kısırlaşmaya) sebep olabilmektedirler 5,6,27,28. Çalışmamızda da uzun süreli yem uygulamalarında, yemdeki MT konsantrasyonu art- tıkça kısır birey oranlarının arttığı gözlenmiştir. Daha ilginci, kısa süreli uygulama olarak düşünülebilecek MT banyo uygulamalarında kısır birey oranın, kontrol populasyonun üzerinde olduğunun gözlenmesidir. Bu durum banyo uy- gulamalarında kullanılan 0.5 mg l-1 MT dozunun çalışma- mızdaki gibi sık periyotlarda (48 saat arayla) verildiğinde, besin kesesinde birikim sonucu kısırlaşmaya sebep ola-bilecek kadar yüksek konsantrasyonlara ulaşabileceğini ve yüksek dozda kısa süreli uygulamalarında sterilizasyona sebep olabileceğini işaret etmektedir.

Salmonidlerde cinsiyet temelde ikili kromozomal ve dişi homogametik (XX/XY), genetik bir mekanizma tara-fından belirlenmektedir 29. Bu tür bir mekanizmanın katı kontrolü altında, erkekleştirilmiş dişi yavrularının tamamı- nın dişi olması beklenir. Fakat çalışmamızda, erkekleştiril- miş dişi gökkuşağı alabalığı yavruları arasında %0-2 ora-nında erkeklerin olduğu tespit edilmiştir. Benzer şekilde salmonidlerde yapılan diğer çalışmalarda da erkekleştirilmiş dişi yavruları arasında az sayıda erkeğe rastlanmıştır 8,25,30. Bu durum, balıklarda cinsiyete etki eden genlerin spesifik kromozomlar üzerinde toplanarak cinsiyet kromozomlarını oluşturmasını, yani cinsiyet kromozomu evrimini tamam- lamamış olmasına ve otozomal kromozomlar üzerindeki gen veya genlerinde cinsiyeti belirlemede etkili olabilme-


sine bağlanmaktadır 29-31. Bu koşullar altında, tamamı dişi populasyonlar üretebilmek ancak üretimde kullanılacak anaçların cinsiyeti etkileyen bu otozomal gen veya genlere karşı seçilimi ile mümkün olabilecektir. Monoseks dişi populasyonlar üretmek isteyen üreticiler anaç olarak kul- landıkları dişi ya da erkekleştirilmiş dişilerin %100 dişi yavrular ürettiğini takip etmeli ve yavruları %100 dişi olmayan bireyleri anaç populasyonundan çıkarmalıdır.

Teşekkür

Bu araştırma için ana finansal destek Türkiye Bilimsel ve Teknolojik Araştırma Kurumu tarafından 104V134 nolu proje kapsamında sağlanmıştır. Projenin destekleyici kuru- luşu olan Önder Alabalık Şirketi saha çalışmaları için fiziki alan sağlamak yanında, yavru, yem ve işçilik anlamında projeye finansal katkıda bulunmuştur. Yazar ticari bir işletmenin yoğun üretim baskısı altında, böylesine uzun soluklu bir araştırmada desteklerini esirgemeyen tüm Önder Alabalık ailesine ve özellikle Su Ürünleri Müh. Aytaç Levent IŞIK’a teşekkürü bir borç bilir.

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SummaryThe aim of this research was to measure mRNA expression of porcine MYF6 (myogenic factor 6, also known in the medical literature

as herculin and myogenic regulatory factor 4, MRF4) and to perform association study with meat quality traits as well as to unravel the transcriptional regulation of this gene by expression QTL (eQTL) study. For this purpose, Duroc × Pietrain F2 resource population (DuPi; n = 313) were used for association and eQTL study. The mRNA levels in Longissimus dorsi muscle tissue of MYF6 gene were evaluated by using qRT-PCR to identify association between gene expression and meat quality traits as well as to analyse eQTL. The mRNA expression of MYF6 associated with conductivity24L (P<0.01) and pH24L (P<0.1). Expression of MYF6 gene was higher in animals with high pH and conductivity of muscle. Linkage analysis using GridQTL revealed 4 trans-regulated eQTL on four porcine autosomes. Significant eQTL [P<0.01, CW (chromosome-wide)] were found for MYF6 on SSC2. A suggestive eQTL (P<0.05, CW) was identified on SSC8. These results revealed that gene expression of MYF6 associated with the meat quality traits and this gene could be potential functional candidate gene for meat quality traits in pigs. However, the analysis of eQTL also suggested that additional genes encoding for transcription factors (TF) could be considered, via fine-mapping underlying the eQTL peaks, in order to understand interaction among these genes.

Keywords: eQTL, Pig, MYF6, mRNA expression, Meat quality traits

MYF6 mRNA İfadesinin Domuzlarda Et Kalitesi ile İlişkisi ve eQTL Analizi

ÖzetBu çalışmanın amacı literatürde herculin ve myogenic factor 4 (MRF4) olarak ta bilinen domuz MYF6 (myogenic factor 6) geninin et

kalite özellikleri ile ilişkisi belirlenmesi ve transkripsiyon seviyesinde gen ifadesinin kontrölünün QTL (eQTL) yöntemiyle incelenmesidir. Bu amaçla, Duroc × Pietrain F2 deneysel populasyonu (DuPi; n = 313) asosiyasyon ve eQTL analizleri icin kullanılmıştır. MYF6 geninin mRNA düzeyleri Longissimus dorsi kas dokusu hücrelerinde asosiyasyon ve eQTL analizi icin qRT-PCR kullanılarak ölçülmüştür. MYF6 genin mRNA düzeyi iletkenlik24L (P<0.01) ve pH24L (P<0.1) ile ilişkili bulunmuştur. MYF6 ifade düzeyi iletkenliğin ve pH’nın yüksek olduğu kaslarda daha yüksek olarak bulunmuştur. GridQTL kullanılarak yapılan bağlantı analizinde dört domuz otozomal kromozomu üzerinde 4 trans-kontrollü eQTL tespit edilmiştir. SSC2 üzerinde MYF6 ifadesi için istatistiki olarak önemli eQTL [P<0.01, KÇ (kromozomal çapta)] bulunmuştur. SSC8 üzerinde önerim düzeyinde bir eQTL tespit edilmiştir (P<0.05, KÇ). Bu sonuçlar MYF6 ifadesinin et kalite özellikleri ile ilişkili olduğunu ve bu genin domuzlarda et kalitesinden sorumlu fonksiyonel bir aday gen olabileceğini göstermiştir. Ancak eQTL analizleri sonucunda, genler arasındaki etkileşimin anlaşılabilmesi için, detaylı haritalama çalışmaları ile eQTL bölgesindeki transkripsiyon faktörlerini (TF) kodlayan genlerin incelenmesi gerekmektedir.

Anahtar sözcükler: eQTL, Domuz, MYF6, mRNA ifadesi, Et kalite kriterleri

eQTL Analysis and Association of MYF6 mRNA Expression with Meat Quality Traits in Pigs

Mehmet Ulaş ÇINAR * Huitao FAN * Christiane NEUHOFF * Christine GROßE-BRINKHAUS *

* Institute of Animal Sciences, Animal Breeding and Husbandry Group, University of Bonn, 53115 Bonn - GERMANY


A significant number of genes 1-4 and quantitative trait loci (QTL) (www.animalgenome.org/cgi-bin/QTLdb/SS) have been reported for meat and carcass quality traits in pig. The myogenic factor 6 (MYF6/myogenic regulatory

factor 4, MRF4) gene codes for the basic helix-loop-helix (bHLH) transcription factor (TF) belonging to MyoD family 5. Its expression accompanies the processes of differentiation and maturation of myotubes during embryogenesis and

INTRODUCTION


RESEARCH ARTICLE

236eQTL Analysis and Association ...

continues on a relatively high level after birth, affecting the muscle phenotype 6. The expressed MYF6 is involved in the processes of differentiation and maturation of myotubes during embryogenesis and dominates quantitatively over the other myogenic regulatory factors (MRFs) in adult muscle 6-9. Increases in MYF6 mRNA and protein may play a role in the differentiation of adult fibers 10. This gene has 10-fold higher postnatal expression than the other genes of the MRF family 7. The lack of active MYF6 protein causes an anomaly in the growth of muscle, known as myopathy. On the other hand, it was demonstrated that MYF6 also acts as a determining factor in the absence of myogenic factor 5 (MYF5) and myogenic differentiation 1 (MYOD1) during the onset of myogenesis in mice 11. This is the main reason why MYF6 is regarded as the principal factor influencing skeletal muscle phenotype 12. Transcriptional regulation complexity of the genes encoding the myogenic regulatory factors has been reviewed in vertebrates and concluded as skeletal myogenesis is coordinated by the activation of the MRFs in response to signals that are interpreted by their associated regulatory elements in different precursor cells during development 13. This explains the existence of numerous regulatory elements such as cis-, trans- or miRNAs at large distances to MYF6 points make very complex pattern of the gene regulation with significant differences between species.

Growth rate and muscularity are economically important in pig breeding. The development of skeletal muscle depends on the number and size of muscle fibers. Because of its functions and supported by results of linkage studies, MYF6 is considered as candidate gene for growth related traits in meat-producing animal species 14. Wyszynska-Koko et al.6

has shown the association between SNP in coding and promoter region of MYF6 and production traits in pigs. Porcine MYF6 was mapped to porcine chromosome 5 (SSC5) 15 where different QTLs for meat and carcass quality traits were identified in pigs (http://www.animalgenome.org/cgi-bin/QTLdb/SS/index, August 2011). mRNA expression of MYF6 was shown to be differentially regulated between different pig breeds due to genetic 16 or epigenetic factors 8. Association between mRNA expression and meat quality traits were shown in pigs 17,18. Moreover, expression variation of meat quality related genes enables us to identify the effects of QTL for gene expression called expression QTL (eQTL) 19. However, to best of our know-ledge, no study has been devoted to association between mRNA expression of MYF6 and meat quality in pigs in a large animal population. Therefore, the aim of this work was to study MYF6 through association and expression as well as eQTL study to prove its candidacy for meat quality traits.


Populations and Phenotypes

Genomic DNA and phenotypic data were obtained

from a reciprocal cross of Duroc and Pietrain (DuPi, n = 313) F2. The structure and breeding of the DuPi population was described in a previous study 20. All pigs were kept at the experimental research farm “Frankenforst”, Institute of Animal Science, University of Bonn (Germany). All pigs were slaughtered in a commercial slaughter house. Meat quality traits analysed in this study cover indicators of water holding capacity including drip loss, thawing loss, cooking loss, pH at 45 min post-mortem (p.m.) in loin (pH1L), pH at 24 h p.m. in loin (pH24L), conductivity at 45 min p.m. in loin (Conductivity1L) and conductivity at 24 h p.m. in loin (Conductivity24L). Conductivity and pH-values were measured using Star-series equipment (Rudolf Matthaeus Company, Germany) in both the longissimus dorsi muscle between 13th/14th ribs. Drip loss was scored based on a bag-method with a size-standardized sample from longissimus dorsi collected at 24 h p.m. that was weighed, suspended in a plastic bag, held at 4°C for 48 h, and thereafter re-weighed. To determine cooking loss, a loin cube was taken from the longissimus dorsi, weighed, placed in a polyethylene bag and incubated in water at 75°C for 50 min. Shear force was recorded in Instron testing machine. The bag was then immersed in flowing water at room temperature for 30 min and the solid portion was re-weighed. Thawing loss was determined similarly after at least 24 h freezing at -20°C. Drip loss, cooking loss, and thawing loss were calculated as a percentage of weight loss based on the start weight of a sample. Carcass quality traits were collected according to guidelines of German performance test 21. The numbers of records, mean values and standard errors are shown in Table 1.

Expression Study Using Quantitative Real-Time PCR (qRT-PCR)

Total RNA from the skeletal muscle samples of the 313 DuPi animals were isolated using TriZol® reagent (Sigma-Aldrich). cDNA was synthesised by reverse transcription PCR using 2 μg of total RNA, SuperScript II reverse trans- criptase (Invitrogen) and oligo(dT)12 primer (Invitrogen). Gene specific primers for the qPCR were designed using the Primer Express software (Applied Biosystem); primers are listed in Table 2.

The qPCR was conducted using the following program: 95°C for 3 min, 40 cycles 95°C for 15 sec/60°C for 45 sec on the ABI Prism 7000 Sequence Detection System (Applied Biosystem). Each PCR reaction contained 10 μl iTaqTM SYBR® Green Supermix with ROX PCR core reagents (Bio -Rad), 2 μl of cDNA and an optimized amount of primer were mixed with double-distilled water to a final reaction volume of 20 μl. All samples were analysed twice (technical replication) and the arithmetic mean of the Ct values were further used for association analysis using SAS v9.2 (SAS Institute Inc., Cary, NC). The geometric mean of three housekeeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase), RPL32 (ribosomal protein 32) and TBP (TATA box binding protein) was used for normalisation of

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CINAR, FAN, NEUHOFFGROßE-BRINKHAUS

the target genes. The delta Ct (ΔCt) values were calculated as the difference between target gene and geometric mean of the reference genes: ΔCt = Cttarget gene – Cthousekeeping gene. Ct values were Logarithm (Log) transformed and used for further analysis. The higher Ct value indicated lower expression and lower Ct value indicated higher gene expression.

Association of Expression Profiles with Meat Quality Traits

Association of gene expression levels with meat quality traits was analysed using generalized linear model (PROC GLM) in SAS. In this model, season of slaughter, family (Dam × Sire) and sex of the animal were assumed as fixed effects; slaughter weight and gene expressions were included as a linear covariate for meat quality traits. The following GLM model was used:

yijk = µ + sexi + ysj + familyk + βSW + βGE + eijk

where yijk is the observation of the trait (meat quality); µ is the population mean; Sexk is the effect of k-th sex (k = 1 for male and 2 for female); ysj is the effect of j-th season of slaughter (j = 1 through 12; four seasons in 3 years); Familyk is the fixed effect of k-th family (k = 1 through 19); βGE is the linear effect of normalized gene expression as covariate (βGE = MYF6) and βSW is the linear effect of slaughter weight as covariate and eijk is the random residual error.

Marker Analysis and eQTL Study

A linkage map with the total length of 2159.3 cM and an average marker interval of 17.7 cM was constructed. F0, F1 and F2 animals of the DuPi population were genotyped at 122 markers loci covering all porcine autosomes. Marker positions and details of genotyping procedures were described by Liu et al.20 and for SSC1 by Große-Brinkhaus et al.22. F2 QTL interval mapping was performed using the web-based program GridQTL 23 based on a least square method. Single line-cross QTL analysis was carried out. Relevant fixed effects for gene expression values including class effects and covariates were tested the using GLM procedure of SAS. Residuals of the GLM model were used for the QTL analysis. Therefore, the basic QTL regression model used for eQTL analysis was:

yijk = µ + sexi + ysj + familyk + βSW + eijk

where yijk is the observation of the trait (gene expression); µ is the population mean; sexk is the effect of k-th sex (k = 1 for male and 2 for female); ysj is the effect of j-th season of slaughter (j = 1 through 12; four seasons in 3 years); familyk is the fixed effect of k-th family (k = 1 through 19); and βSW is the linear effect of slaughter weight as covariate and eijk is the random residual error.

Chromosome-wide (CW) 5% significance thresholds were determined using 1000 permutations 24. The confidence

Table 1. Data collection in the DuPi population and traits measured with mean and standard errors

Tablo 1. DuPi populasyonundaki veri seti ve ölçülen özelliklerin ortalaması ve standart hatası

Trait N Mean Standard Error Minimum Maximum

pH1L 313 6.56 0.21 5.91 7.02

pH24L 313 5.49 0.08 5.30 5.84

Con1L (ms) 313 4.35 0.63 2.80 6.00

Con24L (ms) 313 2.79 0.81 1.60 9.20

Drip loss (%) 313 2.07 0.96 0.50 5.60

Thawing loss (%) 313 8.20 1.86 3.30 13.60

Cooking loss (%) 313 24.68 1.97 17.20 29.40

Shear force (N) 313 35.18 6.62 21.96 61.21

pH1L, pH24L: pH in longissimus dorsi muscle at 13th/14th rib at 45 minutes and 24 h p.m., respectively; Con1L, Con24L: conductivity in longissimus dorsi muscle at 13th/14th rib at 45 min and 24 h p.m., respectively, N: Newton, ms: millisecond

Table 2. Primer sequences used for qRT-PCR analysis

Tablo 2. qRT-PCR analizi için kullanılan primer dizileri

Gene Name Primers Sequence Tm Product Size GenBank ID

MYF6 F: TGGATCAGCAGGACAAAATGR: TGTTTGTCCCTCCTTCCTTG 55°C 171 bp AY188502

GAPDH F: ACCCAGAAGACTGTGGATGGR: ACGCCTGCTTCACCACCTTC 60°C 247 bp DQ845173

RPL32 F: AGCCCAAGATCGTCAAAAAGR: TGTTGCTCCCATAACCAATG 54°C 164 bp AY550039

TBP F: AGCAGCACAGTACGAGCAAR: GATGGACGTTCGGTTTAGG 60°C 124 bp DQ178129


interval (CI) was calculated by bootstrapping in QTL Express. We considered only the QTL which cross LOD score ≥1.7 in this study. Percentage of F2 variance explained by the model was calculated as

25

Where MSR is the residual MS from the reduced model, omitting QTL but including all fixed effects, and MSF is the residual MS from the full model, including QTL and all fixed effects.

RESULTS

Association of Expression with Meat and Carcass Quality Traits

The gene expression as well as association study and

eQTL analysis were conducted in 313 DuPi animals. Association between gene expression and meat quality traits were estimated with a GLM model. The main fixed effects influencing meat quality characteristics were season of slaughter followed by sex (except Con24L, drip loss and cooking loss) and family (except pH24L, con1L and drip loss). Slaughter weight has no effect on meat quality traits except for cooking loss and shear force. Significant (P values) values of the class effects were given in Table 3.

According to the result of the linear model, estimated R-square value explained 58% of pH24L and 58% of Con24L

variation by MYF6 expression (Table 3). Suggestive association was detected between MYF6 expression with pH24L (P<0.1). A significant association was detected between MYF6 mRNA expression and Con24L (P<0.01). Moreover, in the DuPi population, one of the important meat quality parameter drip loss was found to be negatively correlated with pH24L and positively correlated with Con24L (Table 4).

Table 4. Correlation among meat quality traits in the DuPi population

Tablo 4. DuPi populasyonunda et kalite özellikleri arasındaki korrelasyon

Trait r, (p) pH1L pH24L Con1L Con24L Drip Loss Thawing Loss Cooking Loss Shear Force

pH1L 1 0.14(0.007)

-0.28(<.0001)

-0.50(<.0001)

-0.43(<.0001)

0.03(0.57)

-0.14(<.0001)

-0.16(0.0041)

pH24L 1 0.01(0.72)

0.00073(0.98)

-0.20(0.0002)

-0.15(0.0051)

-0.16(0.0022)

0.02(0.71)

Con1L 1 0.20(0.0002)

0.23(<.0001)

-0.01(0.82)

0.04(0.37)

0.13(0.01)

Con24L 1 0.38(<.0001)

0.01(0.81)

0.10(0.06)

0.25(<.0001)

Drip loss 1 0.14(0.007)

0.15(0.003)

0.15(0.0067)

Thawing loss 1 -0.03(0.50)

0.04(0.44)

Cooking loss 1 0.23(<.0001)

Shear force 1

Table 3. Association between MYF6 mRNA expression and meat quality traits in the DuPi population

Tablo 3. DuPi populasyonunda MYF6 mRNA ekspresyonu ve et kalite kriterleri arasindaki asosiyasyon

TraitsClass Effects

R2 Gene Expression Season of Slaughter Sex Slaughter Weight Family

pH1L 0.60 ns *** * ns ***

pH24L 0.58 * *** *** ns ns

Con1L 0.48 ns ** *** ns ns

Con24L 0.58 *** *** ns ns ***

Drip loss 0.47 ns *** ns ns ns

Cooking loss 0.65 ns *** ns *** ***

Thawing loss 0.54 ns *** * ns ***

Shear force 0.63 ns *** ** ** ***

ns = not significantThree significance levels were used: *** (P<0.01); ** (P<0.05); * (P<0.1)P-values showed by the star, derived from total variation explained by the class effects (R2)

%Var =R

FR

MSMSMS x 100

239


Differential Expression of MYF6

In the animals with extreme phenotype of pH1L, pH24L and Con24L (n = 10) selected for differential expression,

the gene expression was significantly higher in animals with low pH1L compared to animals with high pH1L (data not shown) (Fig. 1). Animals with higher pH24L had higher MYF6 gene expression compared to animals with low pH24L (Fig. 1). The MYF6 mRNA expression was higher in animals with high conductivity at 24 h p.m. value compared to animals with low conductivity at 24 h p.m. (Fig. 1).

Expression QTL (eQTL)

A total of four eQTL were detected for MYF6 expression on four porcine autosomes. On SSC2, a 1% chromosome-wide significant eQTL at 26 cM close to the marker S0141 explained 5.12% of the phenotypic variation (Table 5). A suggestive eQTL was identified on SSC5 at 116 cM close to marker IGF1. Moreover, a 5% chromosome-wide significant eQTL was detected on SSC8 between the markers SW2611 and S0086 which explains 3.14% of total variation. Another suggestive eQTL for MYF6 was detected on SSC10 at 133 cM close to marker SW951 (Table 5). Experiment-wide thresholds (8.36 and 9.80, P<0.05 and P<0.01, respectively) were also calculated; however no eQTL in this study reached the experiment-wide thresholds.

DISCUSSION

Expression of MYF6

In recent few decades, pig breeding focused on improving growth rate and muscularity. This causes gradual decrease in meat quality and deterioration of carcass traits such as intramuscular fat content, colour, hardness or water content in meat 16. The commercial Duroc and Pietrain breeds differ extremely for their muscle phenotypes (i.e., myofiber numbers and myofiber types) 26. In vertebrates skeletal myogenesis is coordinated by the activation of the myogenic regulatory factors (MRFs) in response to signals that are interpreted by their associated regulatory elements in different precursor cells during

Table 5. Expression QTL (eQTL) mapping results for the mRNA expression of MYF6 gene on swine autosomes identified in the DuPi population

Tablo 5. Domuz otozomlarında tanımlanan DuPi populasyonunda MYF6 geninin mRNA ifadesi için ekspresyon QTL (eQTL) haritalama sonuçları

SSCa Traitb F-ratioc Position (CI95)d Closest Markerse Additive (SE)f Dominance (SE)g Variation (%)h

2 MYF6 8.1** 26 (0 – 202) SW263 – S0141 0.14 (0.21) 2.43 (0.60) 5.12

5 MYF6 3.28 116 ( 0 – 113) IGF1 -0.05 (0.17) 0.82 (0.32) 1.71

8 MYF6 5.26* 41 (16.5 – 113.5) SW2611 – S0086 -0.24 (0.19) 0.71 (0.27) 3.14

10 MYF6 3.98 133 (0 – 129) SW951 – SWR67 -0.55 (0.21) 0.55 (0.44) 2.22a Sus scrofa chromosomeb Gene abbreviations according to NCBI databasec Significance level was used: 5% chromosome-wide significance level, as suggestive level. The eQTL those can reach the level marked by stard Positions of detected eQTL in Kosambi cM with the 95% confidence interval (CI95) given in parentheses according to the bootstrapping approache The closest markers were those markers around the peak, as near as possiblef Additive effects, expressed as the deviation of the Duroc-Pietrain alleles. SE = standard errorg Dominance effects, expressed as the deviation of the Duroc-Pietrain alleles. SE =standard errorh Fraction of phenotypic variance explained by each eQTL as a percentage of the residual variance in the F2 population* Significant on a chromosome-wide level with P≤0.05** Significant on a chromosome-wide level with P≤0.01

Fig 1. Differential expression of MYF6 mRNA (a) animals with high and low pH24L, (b) animals with high and low Con24L. Lower Ct values represent higher gene expression and vice versa

Şekil 1. MYF6 mRNA’sının ifade farklılığı (a) yüksek ve düşük pH24L özelliğe sahip hayvanlar, (b) yüksek ve düşük Con24L özelliğine sahip olan hayvanlar. Düşük Ct değerleri yüksek gen ifadesini göstermektedir


development 27. MYF6 is the most abundantly expressed myogenic factor in postnatal muscle where it quantitatively predominates over the other MyoD family transcripts 28. MYF6 is considered as one promising candidate gene for growth- and meat quality-related traits in adult pigs 5. Recently, MYF6 mRNA and protein expression was shown to be significantly up-regulated in purebred Pietrain pigs compared to purebred Duroc pigs in the founder family of DuPi F2 animals 8. Polymorphism in MYF6 in the promotor region and exon 1 are signicantly correlated with weight of loin and ham, right half-carcass weight and daily gain 29. The same researchers did not observe any mRNA expression differences among genotype classes. However, in our study, we found that MYF6 expression was associated with meat pH at 24 h post-mortem (Table 3) and showed that expression was higher in animal with high muscle pH and conductivity indicating that this gene was expressed higher in muscle with lower drip loss because in the DuPi population, muscle pH was negatively correlated with drip loss (r = -0.20, P = 0.0002). The same negative correlation between muscle pH and drip loss has also been reported 30. Similar results were also obtained by Liu et al.31; they also identified association between MYF5 SNP and longissimus dorsi pH in pigs. Te Pas et al.32 stated that genetic variation at the porcine MYF5 gene locus could also be used as a marker to study association of the MYF6 locus with muscle development-related meat traits. SNP in bovine MYF5 were found to be associated with meat traits in cattle 33. In chicken, although MYF5 found to be associated with several carcass traits, no polymorphism was detected in MYF6 gene 34. In the study of Ropka-Molik et al.16 expression for the MYF6 gene did not show statistically significant differences between ages of development in analyzed Poland Landrace and Pietrain breeds. The highest mRNA level of MYF6 gene was observed in gracilis muscle in this study among two breeds, but a statistically significant (P<0.01) difference was only found in Pietrain pigs.

Expression QTL Analysis

Analysis of expression quantitative trait loci (eQTL) provides a means for detecting transcriptional regulatory relationships at a genome-wide scale 35. Many investigations have reported the successful mapping of eQTL in rats, mice, humans or pigs with different sample sizes 36-39. But experimental data from genetic investigations of gene expression in farm animals are still limited 37. Mapping of eQTL to the specific gene indicates that cis- changes are responsible for the different expression levels, whereas mapping positions of eQTL that are different from the positions of the corresponding genes indicate trans- regulation. In this study, four eQTLs on four different porcine autosomes were detected. Different Lod scores were reported as threshold for a QTL 25,40,41. Here in our study a Lod score value of 1.7 was chosen based on the median of presented thresholds in literature 40,41 to indicate an eQTL as suggestive. Among them, an eQTL

on SSC5 might be a putative cis-regulated eQTL since the confidence interval of this eQTL did incorporate the chromosomal location of the MYF6 gene. Moreover, the highest peak of this eQTL (116 cM) was closed to the marker IGF1, very close to the chromosomal location of MYF6. The power of this putative eQTL on SSC5 is low which might be due to low number of animals in the population. However, the DNA variations of this gene in promoter and exon 1 were not affecting the expression of the MYF6 gene in a previous study 16 and the confidence interval of this eQTL is very wide indicating that the eQTL on SSC5 is better to be considered as a trans-eQTL. The other three eQTLs showed trans-regulation of this gene which were consistent with the previous reports for pigs 42 and other species 35,43.

Most genes are regulated through complex networks, and function and expressions are influenced by itself as well as different other genes, transcription factors and miRNAs 34. Moreover, distant eQTL may affect their target genes in many possible ways such as protein-coding regions, cis-regulatory DNA motifs, transcription factor that is polymorphic in its DNA binding region, or other functional nucleotide sequences 35. The MRFs trigger a cascade of transcription factors and downstream structural genes. Due to the close vicinity of MYF6 and MYF5 genes on SSC5, these two genes have a common regulatory region and the expression of both genes is in part activated together 44. Analyses of the mechanisms behind promoter-enhancer specificity in the MYF6/MYF5 locus have unveiled a new class of element, termed Transcription balancing sequences which explains the cis-acting effects observed in the various MYF6 and MYF5 alleles 13. On the other hand, Black et al.45 showed that MYF6 promoter is trans-activated by myogenin, MyoD, MYF5 and by the myocyte enhancer factor-2 (MEF2) factors in mice. In our study a significant eQTL (Table 5) was detected close to marker S0141. An important gene called insulin-like growth factor 2 (IGF2) locates distal part of the p arm on SSC2. Regulatory mutation of IGF2 and relation to muscle growth in pigs gene was shown previously 46. Alzhanov et al.47 identified distal transcriptional enhancer directly stimulates the transcriptional activity of an IGF2 promoter-reporter gene in differentiating myoblasts.

Ponsuksili et al.37 stated that the expression of genes associated with traits that have low heritability, like drip loss or meat pH is under the control of several trans-regulated genes. The detected eQTL in this study explain a low proportion of phenotypic variation. Trans-eQTL usually reflects genetic regulation that is dispersed across many loci with small effects and each explains small phenotypic variance 48. Moreover, trans-regulated genes appear more in complex traits (i.e., under polygenic control) than the cis-regulated genes and are likely to reflect the additive outcome of genetic, epigenetic, and environmental regulation 49. In our study, all detected

241


eQTLs were found to have higher dominance effects (Table 5). Duroc and Pietrain pig breeds were divergent for meat quality and growth traits, recently their mRNA expression for MYF6 gene was found to be differentially regulated 8. Heterosis might be one of the possible reasons for this over dominancy. However, it is not possible to proof this with the available data in our hand. To link an eQTL to the genetic background of a classical phenotypic trait of interest, it is necessary to establish a relationship between the variation of that classical phenotypic trait and its corresponding ‘pQTL’ (pheneQTL) position on one hand and the expression level of that particular transcript or the mapping position of the eQTL on the other hand 37. Among these QTL, phenotypic QTL for pH and conductivity reported on SSC2 20,50 are close to our detected eQTL for MYF6 in the DuPi population.

As a conclusion, significant and suggestive associations were found between gene expression and meat quality traits in the DuPi animals and higher expression of the MYF6 might contribute to lower drip loss and higher muscle pH. According to eQTL analyses it is difficult to say that our candidate gene is cis-regulated. Moreover, DNA variations in promoter of MYF6 including MYF5 were requested as further study to test whether SNPs affecting its transcription abundance. Therefore, MYF6 could be a trans-regulated gene which might be due to the presence of the widely distributed genetic regulators such as transcription factors and non-coding RNAs in the whole genome as well as non-genetic mechanisms (epigenetics). Finally, MYF6 could be suggested as a potential candidate gene for meat quality traits in pigs. However, further studies are necessary to validate these results with other breeds especially in larger outbreed populations and further analysis of detected overlapping eQTLs and QTL regions can dissect genes and regulators that are responsible for meat quality traits in pigs.

Acknowledgements

This project was supported by the German Research Foundation (DFG), “Functional Genetic Principles of Water Binding Capacity in Pork “DRIP FOR 753” Germany. Authors are indebted to Ms. Nadine Leyer for technical assistance during the experiments.

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SummaryCytochrome P450 aromatase is the key enzyme in estrogen biosynthesis, encoded by CYP19 gene. Impairment of spermatogenesis

associated with a decrease in sperm motility and inability to fertilize oocytes in mice due to the lacking of CYP19 was observed. However, it is little known about the CYP19 roles in boar spermatogenesis and fertility. Therefore, the aim of this research was to investigate the mRNA and protein expression of CYP19 in boar reproductive tissues from boars with different sperm quality. For mRNA and protein expression study, a total of six boars were divided into two groups with Group 1 (G-I) and Group 2 (G-II) where G-I was characterized for relatively a better sperm quality. The result showed that the CYP19 transcript was not expressed throughout the male reproductive system. mRNA expression of CYP19 was higher only in testis. CYP19 expression was similar in testis collected from G-I and G-II boars. The CYP19 protein expression results from western blot were different with the results of qRT-PCR. The CYP19 protein was higher in testis collected from G-II than G-I boars. The CYP19 protein localization in testis showed a strong staining only in the cytoplasm Leydig cell. These results shed new light on the roles of porcine CYP19 in spermatogenesis as a specific target gene for testis.

Keywords: mRNA, Protein, Immunofluorescence, Testis, Boar spermatozoa

Erkek Domuzlarda Testisler İçin Hedef Bir Gen Olan Aromataz (CYP19) Üzerine Araştırma

ÖzetSitokrom P450 aromataz östrojen üretiminde önemli bir enzim olup, CYP19 geni tarafından ifade edilir. CYP19 geninin yokluğunda

farelerde sperm üretiminin zarar gördüğü ve ilişkili olarak spermlerin yüzme ve yumurtaları dölleme kabiliyetinin azaldığı gözlenmiştir. Buna karşılık CYP19’un domuz sperm üretiminde ve yumurtaların döllenmesinde nasıl bir rol oynadığı bilinmemektedir. Bu nedenle, bu calışmanın amacı CYP19 mRNA ve protein ifadesini farklı sperm kalite özelliklerine sahip olan erkek domuzların üreme organı dokularında araştırmaktır. mRNA ve protein ifadesi çalısması için altı erkek domuz, grup 1 (G-I) ve grup 2 (G-II) olarak iki gruba ayrılmıştır. G-I hayvanlar göreceli olarak G-II hayvanlara göre daha iyi sperm özelliklerine sahiptir. Sonuçlar CYP19 mRNA ifadesinin tüm erkek üreme organlarında ifade edilmediğini göstermiştir. CYP19’un mRNA ifadesi en çok testis dokularında gözlenmiştir ve ayrıca G-I ve G-II domuzlardan alınan testis örneklerinde aynı düzeyde tespit edilmiştir. CYP19’un Western-Blot protein ifadesi sonuçları qRT-PCR sonuçlarından farklı olarak gözlenmiştir. CYP19 protein ifadesi G-II hayvanlarda G-I hayvanlara göre daha yüksek düzeyde gözlenmiştir. Testiste CYP19 protein lokalizasyonu sitoplazma Leydig hücrelerinde güçlü bir sinyal göstermiştir. Sonuçlar, CYP19 geninin domuzlarda sperm üretimi için testislerde spesifik bir hedef gen olduğuna dair yeni bulgular ortaya koymuştur.

Anahtar sözcükler: mRNA, Protein, İmmunfloresans, Testis, Domuz spermatozoa

Investigation on Porcine Aromatase (CYP19) as a Specific Target Gene for Boar Testis

Mehmet Ulaş ÇINAR * Asep GUNAWAN *

* Institute of Animal Sciences, Animal Breeding and Husbandry Group, University of Bonn, 53115 Bonn, GERMANY


Aromatase is the only enzyme responsible for the irreversible bioconversion of androgens into estrogens. This enzyme is a complex composed of an ubiquitous NADPH cytochrome P450 reductase and a specific cytochrome P450 aromatase encoded by the CYP19 gene 1.

Estrogens have been for a long time considered as a specific female hormone; however, the presence of estrogens in the male gonad is now well documented 2. Indeed the androgen/estrogen balance is essential for normal sexual development and reproduction in mammals. In

INTRODUCTION


RESEARCH ARTICLE

244Investigation on Porcine ...

the mammalian testis, maintenance of this balance is under a fine tuning via endocrine and paracrine factors, but is also related to the aromatase activity 2. Although in humans and some higher primates, there is a more extensive distribution of estrogen biosynthesis including placenta, adipose tissue, liver and skin 3, according to the steroid levels assayed in the testicular artery and vein, testes are a major source of estrogens 4 and aromatase has been immunolocalized in Leydig cells 5,6. In the testis of mammals, gonadotropins and testosterone together with numerous locally-produced factors are responsible for the induction and/or the maintenance of spermatogenesis 7.

Deficieny of CYP19 and effects on spermatogenesis were shown in different mammalian species. Impairment of spermatogenesis associated with a decrease sperm motility and inability to fertilize oocytes was reported due to lacking of CYP19 gene in mice 8. In buffalo, higher expression of CYP19 was found in spermatozoa obtained from good quality of semen as compared to spermatozoa from poor quality of semen 9. Similarly, the higher expression CYP19 was found in motile spermatozoa as compared to non-motile 10. CYP19 mRNA and protein expressed higher in adult stallions compared to colts 1. In the adult stallions, the testis, among the tissues analyzed, found to be the major source of aromatase that shows gene expression is specifically enhanced at this level, and is responsible for the high estrogen synthesis 1.

Testis is responsible for the induction and/or the maintenance of spermatogenesis and is the major source for CYP19 enzyme 11. However, there is no information regarding the expression of CYP19 in reproductive tissue from different quality of boar sperm and very few known about the role of CYP19 in boar spermatogenesis. Therefore, this research was aimed to investigate the mRNA and protein expression of CYP19 in boar reproductive tissues from boars with different sperm quality.

MATERIALS and METHODS

Samples for mRNA and Protein Expression Analysis

Boars from the artificial insemination station SuisAG (Sempach, Switzerland) were selected based on extreme phenotypes [high/low sperm concentration (SCON), sperm motility (SMOT), and sperm volume (SVOL)]. The SCON

(average sperm concentration) was highly negative (r = -0.8) correlated with SVOL (average semen volume), whereas SCON was highly positive (r = 0.7) correlated with SMOT (average sperm motility). Moreover, SVOL was highly negative (r = -0.8) correlated with SMOT. Therefore, grouping was done on the basis of SCON, SVOL and SMOT (Table 1). A total of six animals were selected and equally divided into group I (G-I) with high SCON (>262.32 ×106 ml), high SMOT (>76.59%) and low SVOL (<215.24 ml/ejaculation) and group II (G-II) with low sperm concentration and motility, and high sperm volume (Table 1). The difference between the two groups was calculated using proc t-test in SAS. There were differences for SCON (P<0.05) and for SVOL (P<0.01) between G-I and G-II, whereas for the SMOT the difference was not significant (P = 0.12). Reproductive tissues (testis, head of epididymis, body of epididymis, tail of epididymis, vas deferens, bulbourethral gland, vesicular glands and prostate gland), non reproductive tissues (brain, liver and skeletal muscle tissue) and semen samples (spermatozoa) of six boars (Duroc, Large White and Landrace) were collected for the mRNA and protein study 12.

Semi-Quantitative PCR

Total RNA was isolated using TRI Reagent (Sigma-Aldrich) from different reproductive and non reproductive tissues of breeding boars mentioned in previous chapter. RNA was purified using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA was treated using on-column RNase-Free DNase set (Promega) and quantified sphectrophotometrically (Nano Drop, ND8000 Thermo Scientific). Furthermore, RNA integrity was checked by 2% agarose gel electrophoresis. First-strand cDNA were synthesized from individual RNA using Superscript II enzyme (Invitrogen). cDNA amplification was performed by using specific forward and reverse primers (forward: 5´-ttag caagtcctcaagtgtg -3´ and reverse: 5´- ccaggaagaggttgtt agag-3´) derived from porcine CYP19 sequence (GenBank accession: U37311). Amplification was performed with an initial heating at 95°C for 5 min followed by 35 cycles of 95°C for 30 sec, annealing temperature at 52°C for 30 sec and 72°C for 30 sec, on the PCR Thermal Cycler (Bio-Rad). PCR product were electrophoresed on a 1.5% agarose gel and visualized upon staining with ethidium bromide. Amplification of GAPDH (forward: 5´-acccagaagactgtgga tgg-3´ and reverse: 5´-acgcctgcttcaccaccttc-3´) (GenBank accession No. AF017079) served as housekeeping gene.

Table 1. Means, standard errors (S.E.), number of boars and ranges of traits selected for mRNA and protein expression study

Tablo1. mRNA ve protein ifadesi için seçilen erkek domuzlara ait özelliklerin ortalamaları, standart hataları, erkek domuz sayıları ve örneklem genişliği

TraitsSelected Animals (n = 6) G-I (n = 3) G-II (n = 3)

Mean S.E. Mean S.E. Mean S.E.

SCON (106/ml)SVOL (ml)SMOT (%)

262.32215.2476.59

87.9734.933.71

335.94185.0779.03

50.7816.331.89

188.70245.4074.14

22.547.423.60

245ÇINAR, GUNAWAN

Quantitative Real-Time PCR (qRT-PCR)

For qRT-PCR, total RNA and cDNA synthesis from different reproductive tissues of two divergent groups of animals (G-I and G-II) were done as described above. The same primers pair used in semi-quantitative PCR were also used in qRT-PCR. Nine-fold serial dilution of plasmids DNA were prepared and used as template for the generation of the standard curve. In each run, the 96-well microtiter plate contained each cDNA sample, plasmid standards for the standard curves and no-template control. To ensure repeatability of the experiments, each plate was run in three replications. Quantitative real-time RT-PCR (qRT-PCR) was set up using 2 μl first-strand cDNA template, 7.6 μl deionized H2O, 0.2 μM of upstream and downstream primers and 10 μl 1× Power SYBR Green I master mix with ROX as reference dye (Bio-Rad). The thermal cycling conditions were 3 min at 94°C followed by 40 cycles of 20 sec at 94°C and 1 min at 60°C. Experiments were performed using the ABI prism®7000 (Applied Biosystems) qRT-PCR system. An amplification-based threshold and adaptive baseline were selected as algorithms. The housekeeping gene GAPDH (forward: 5´-acccagaagactgtggatgg-3´ and reverse: 5´-acgcctgcttcaccaccttc-3´) derived from porcine sequence (GenBank accession No. AF017079) was used for the data normalization. Final results were reported as the relative abundance level after normalizing with mRNA expression level of the housekeeping gene. Differences in CYP19 mRNA expression were analyzed with the simple t-test in SAS software (SAS Institute Inc., ver. 9.2). Values of P<0.05 were considered to indicate statistically significant differences.

Western Blotting

Total protein was isolated from the tissues of the six boars which were also used for mRNA isolation. Total protein was isolated by using TRI Reagent (Sigma-Aldrich), before protein separation in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (gradient 4-18%) and transferring onto a nitrocellulose membrane (Amersham Biosciences). The membranes were further kept in blocking buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween-20 and 1% Polyvinylpyrolidone) for 1 h at room temperature; the membrane was incubated overnight at 4°C with the anti-CYP19 antibody purified from goat polyclonal antibody (Cat.nr.14245; Santa Cruz) in the blocking medium (diluted 1:500). Non-specific binding of antibody was washed off with six changes of 0.1% PBST. The horseradish peroxidase conjugated donkey anti-goat IgG secondary antibody (Cat.No. Sc2020; Santa Cruz) was used as the secondary antibody (diluted 1:5000). The membrane was incubated for 1 hour at room temperature with secondary antibody, followed by washing with six changes of 0.1% PBST. The chemiluminesce was detected by using the ECL plus western blotting detection system (Amersham Biosciences) and visualized by using Kodak BioMax XAR film (Kodak). GAPDH was used as a loading

control and for normalization. The membrane was stripped by incubation in 2% SDS, 100 mM Tris-HCl, 0.1% beta-mercaptoethanol for 30 min at 60°C and re-probed with GAPDH antibody (Cat.No. Sc20357; Santa Cruz).

Protein Localization by Immunofluorescence

Due to the limitations of fresh samples from G-I and G-II boars, we collected fresh testis sample from a healthy breeding boar after slaughtering for protein localization. Immunofluorescence staining was performed on 8 μm cryostat sections of snap frozen tissues. All sections were kept in -80ºC for further analysis. To block unspecific staining, sections were incubated for 30 minutes at room temperature with 5% bovine serum albumin in PBS (50 nM sodium phosphate, pH 7.4; 0.9% NaCl). Sections were incubated overnight at 4ºC with the CYP19 goat polyclonal primary antibody (Cat.nr.14245; Santa Cruz) diluted at 1:50 in PBST followed by six times (10 min to time) washing with PBS. Then, the sections were incubated 1 hour at room temperature with the biotinylated donkey anti-rabbit IgG-B conjugated with fluorescein isothiocynate (FITC) reactive water-soluble fluorescent dye (Cat nr. Sc2090; Santa Cruz) (dilution 1:200) which was used as a secondary antibody for CYP19. Then sections were washed six times (10 min to time) with PBS. Finally, the samples were counterstained with vectashield mounting medium (Vector Laboratories) containing 40,6-diamidino-2-phenyl indole (DAPI) and covered with a cover glass slip. The staining was observed by confocal laser scanning microscope (Carl Zeiss). In case of negative controls, PBS was used instead of the primary antibody.

RESULTS

mRNA Expression by Semi-Quantitative PCR

Among reproductive and non-reproductive tissues CYP19 gene expression was detected only in testis (Fig. 1). When semi-quantitative PCR was applied in reproductive tissues from G-I and G-II boars, CYP19 mRNA expression was observed in testis among all different reproductive tissues (Fig. 2a). The semi-quantitative reverse transcription PCR result of GAPDH showed no remarkable differences among tissues (Fig. 1 & Fig. 2a).

mRNA and Protein Expression Study in Testis from G-I and G-II Boars

Semi-quantitative PCR results showed that the CYP19 mRNA was highly expressed in testis samples from G-I and G-II boars (Fig. 2a). Results of semi-quantitative PCR overlapped with the results of the qRT-PCR (Fig. 2b). qRT-PCR showed that no significant mRNA expression difference in testis between G-I and G-II boars (Fig. 2b). CYP19 protein with 58 kDa molecular weight was detected in testis of G-I and G-II boars (Fig. 3a). Protein expression result of western blot appeared to be inconsistent with


the results of the qRT-PCR (Fig 3b & Fig. 2b). The western blot result showed that the CYP19 protein expression was higher in the testis of G-II boars compared to G-I boars (Fig. 3b).

Localization of CYP19 Protein in Boar Reproductive Tissues by Immunofluorescence

Sections of testis were stained through the same

optical panel for the cell surface CYP19 protein expression (Fig. 4). Immunoreactive CYP19 protein was observed as strong staining only in the cytoplasm of the Leydig cells in testis. No immunostaining was detected in Sertoli cells (Fig. 4a).

DISCUSSION

CYP19 mRNA and Protein Expression in Boar Reproductive Tissues

In this study, we measured the CYP19 mRNA gene expression in various boar tissues for the first time. More- over, CYP19 gene was measured in various reproductive organs from boars with divergent sperm quality traits.

Fig 1. mRNA expression of CYP19 in reproductive and non-reproductive tissues by semi-quantitative PCR

Şekil 1. CYP19 geninin üreme ve üreme dışı dokularda yarı-kantitatif PCR mRNA ifadesi

Fig 2. mRNA expression of CYP19 in reproductive tissues (testis, head, body and tail of the epididymis), (2a) CYP19 mRNA expression in different reproductive tissues from G-I and G-II boars by semi-quantitative PCR, (2b) CYP19 mRNA expression in different reproductive tissues from G-I and G-II boars by qRT-PCR

Şekil 2. CYP19 geninin üreme dokularında (testis, epididimisin baş, vücut, ve kuyruk kısmı) mRNA ifadesi, (2a) CYP19 mRNA ifadesinin üreme dokularında yarı-kantitatif PCR ile G-I ve G-II erkek domuzlarda gösterimi, (2b) CYP19 mRNA ifadesinin üreme dokularında qRT-PCR ile G-I ve G-II erkek domuzlarda gösterimi

Fig 3. CYP19 protein expression in testis from G-I and G-II boars by Western Blotting, (3a) Western-Blot results of CYP19 and GAPDH proteins in testis from G-I and G-II boars, (3b) Quantification of Western-Blot results between two group boars

Şekil 3. G-I ve G-II erkek domuzlarda CYP19 protein ifadesinin Western-Blot ile gösterilmesi, (3a) G-I ve G-II erkek domuzlarda CYP19 ve GAPDH proteinlerinin Western-Blot ile gösterilmesi, (3b) Western-Blot sonuçlarının iki grup erkek domuz arasında sayısallaştırılmış görüntüsü

247ÇINAR, GUNAWAN

mRNA and protein expression of CYP19 was measured quantitatively by using qRT-PCR and western-blot respectively. CYP19 protein was also localized in testis section to proof the presence of this protein in Leydig cells. Results demonstrated that the porcine CYP19 mRNA expression was observed only in testis (Fig. 2 & Fig. 3) and CYP19 transcript was not widespread throughout the male reproductive tract. Importantly, CYP19 expressed highly in testis of boars collected from G-I and G-II.

In our study CYP19 transcript was not detected throughout the male reproductive system except in testis which is in accordance with the previous reports describing that CYP19 expression is found only in testis in human 13 and stallion 1. The porcine CYP19 gene is expressed in a tissue-specific fashion in three principal sites, the gonads, the placenta, and the preimplantation blastocyst 14. Tissue-specific expression of the CYP19 promoted survival of the CYP19 genes 15. Although promoter that drives ovarian CYP19 expression is well conserved in mammalian species, expression in the pig testis is driven by a different promoter than that utilized in the ovary 16. Testis is major source for CYP19 enzyme and corresponds to daily sperm production 11

is supporting our findings. CYP19 enzyme catalyses the synthesis of estrogens from androgens and play roles in the sexual development, reproduction and in behaviour 14. In the mammalian testis, gonadotropins and testosterone together with numerous locally-produced factors are responsible for the induction and/or the maintenance of spermatogenesis 7. Levallet et al.17 and Janulis et al.18 showed that the highest amount of CYP19 mRNA in testis is related to the estrogen production. Gist et al.19 detected CYP19 in the testis and suggested that testicular estrogens might have a regulatory influence on the spermatogenesis in the testis. Investigation on spermatogenesis in knockout mice (ArKO) revealed that lack in functional aromatase (CYP19) enzyme is unable to convert C19 steroids (androgens) to C18 steroids (estrogens) 20. CYP19 deficient mice indicated that spermatogenesis required the presence estrodiol-17 beta (E2). E2 is necessary to stimulate glucose uptake, oxidative metabolism and motility. CYP19 deficient mice (ArKO) are reported to have disrupted spermatogenesis associated

with a decrease in sperm motility and inability to fertilize oocytes 8,20,21. The presence of CYP19 transcripts could be a marker of male gamete quality since existence of it reported to be influence the motility and the acrosome reaction 22. However, our results showed that CYP19 mRNA and protein expression are tended to be higher in G-II boars but the mRNA and protein expression differences between G-I and G-II were not statistically significant. Moreover, it is important to note that all boars used in this study were used for breeding purpose by the breeding company which means all boars were good enough. The differences for SCON, SVOL and SMOT between two groups of boars were not extreme. The G-II boars had comparatively poor quality semen when compared to G-I.

Protein Localization of CYP19

Immunoreactive CYP19 protein was observed strong staining only in cytoplasm of Leydig cells in testis. These results are in good agreement with the previous study in boar 23, horses 6,24, ram 25 and human 13,26. However, some studies detected immunoreactive CYP19 in both the Leydig cells and seminiferous tubules in rat 17,27, mouse 28

and rooster 29. Importantly, this study confirmed that immunoreactive CYP19 is restricted only to the Leydig cells in the testis in mammalian species like horses 5,30, pig 16,23 and rams 25. It has been reported that the major function of Leydig cells is to produce estrogen and being the source of estrogen biosynthesis in rat 30 and human 31. Moreover, Hess et al.30 showed in male horse that there is an age-dependent shift in the localization of immunoreactive CYP19 from the Leydig cell to Sertoli cells. In adult animals, highest CYP19 activity are found in the Leydig cells 1 but in immature rat before puberty it is found more in Sertoli cell 30. The boar used for localization in this study was an adult breeding boar which supports our finding for localization of CYP19 only in the Leydig cells. The mRNA and protein expression study of the CYP19 imply that it may have a role in spermatogenesis and specific target gene in testis in pigs. Therefore, the results of this study could be valuable to shed light on the roles of CYP19 in spermatogenesis in boars.

Fig 4. Localization of CYP19 protein in testis of boar, (4a) Immunofluorescence detection of CYP19 in Leydig cells. Leydig cells were stained with CYP19 (arrows), (4b) The cell nuclei were counterstained with DAPI, (4c) Merged images, (4d) Negative control

Şekil 4. CYP19 proteinin erkek domuz testisinde lokalizasyonu, (4a) CYP19’un Leydig hücrelerinde immunoflorasan tesbiti, (4b) Hücre cekirdeklerinin DAPI ile işaretlenmiş görüntüsü. (4c) Birleştirilmiş görüntü, (4d) Negatif kontrol


Acknowledgments

This study was financially supported by FBF (Förderverein Biotechnologieforschung e. V.), Bonn, Germany.

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in the developing and adult rat brain. J Steroid Biochem, 61 (3-6): 359-364, 1997.

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29. Kwon S, Hess RA, Bunick D, Nitta H, Janulis L, Osawa Y, Bahr JM: Rooster testicular germ-cells and epididymal sperm contain P450 aromatase. Biol Reprod, 53 (6): 1259-1264, 1995.

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SummaryThe ratio of gray and white matter is an important clinical parameter in the diagnosis of diffuse and compressive diseases of

the spinal cord. Although histological methods are used to determine this parameter, there are some difficulties encountered in histological studies related to tissue size. The aim of this study was to evaluate possible modifications to overcome these difficulties. In the study, nine tissue samples taken from the C6 segment of a female Shetland pony and selected by systematic random sampling were used. The dehydration process of the spinal cord of the horse was supported by applying a vacuum. Paraffin blocks were prepared and cut into 10 µm sections to be stained separately with the different staining methods. Six different staining methods, including Modified May - Grunwald - Giemsa (MMGG), were compared and used to image entire slides. The stains, Hematoxylin & eosin (H&E), May-Grunwald-Giemsa (MGG), Masson’s trichrome (MT), AgNORs, Kluver Barrera (KB) and MMGG, were evaluated macroscopically and microscopically by participants who were unaware of which staining methods had been used. The staining methods were scored from worst (1) to best (5) using a Likert scale. Vacuum application was found to reduce the difficulties related to inadequate tissue dehydration. MMGG was selected as the best staining method in differentiating gray and white matter in the spinal cord of the horse.

Keywords: Gray matter ratio, Horse, Imaging, Spinal cord, Stain comparison

At Omuriliğinin Gri ve Ak Maddesinin Seçici Boyanması

ÖzetMedulla spinalis’te yer alan gri ve ak madde oranları diffuz ve kompresif omurilik hastalıklarının teşhisinde önemli klinik para-

metrelerdendir. Bu parametrelerin tespitinde histolojik metotlar kullanılmasına rağmen, histolojik çalışmalarda doku büyüklüğüne bağlı bazı güçlüklüklerle karşılaşılmaktadır. Bu çalışmanın amacı, bahsedilen zorlukları aşmaya yönelik olası modifikasyonları değerlendirmektir. Çalışmada, Shetland pony ırkına ait C6 segmentinden sistematik rastgele örnekleme kuralına sadık kalınarak elde edilen 9 doku kesiti kullanıldı. Medulla spinalis’in dehidrasyon işlemi sırasında vakum uygulaması yapıldı. Dokuların parafin blokları hazırlandı, dokular 10 µm kalınlığında kesilerek farklı histolojik boyalar ile boyandı. Modifiye May - Grunwald - Giemsa (MMGG)’nın yer aldığı 6 farklı boyama metodunun boyama performansları karşılaştırıldı. Hematoxylin & eosin (H&E), May-Grunwald-Giemsa (MGG), Masson’s trichrome (MT), AgNORs, Kluver Barrera (KB) ve MMGG ile boyanan kesitler tek kör grup tarafından makroskopik ve mikroskopik olarak değerlendirildi. Boyaların performansları Likert skalası kullanılarak en kötü (1) en iyi (5) olmak üzere değerlendirildi. Vakum uygulamasının yetersiz doku dehidrasyonundan kaynaklanan problemleri ortadan kaldırdığı görüldü. MMGG, at medulla spinalis’inde gri ve ak madde ayrımında en başarılı boya olarak tespit edildi.

Anahtar sözcükler: Gri madde oranı, At, Görüntüleme, Medulla spinalis, Boya karşılaştırması

Selective Gray and White Matter Staining of the Horse Spinal Cord

Durmus BOLAT * Sadullah BAHAR ** Emrah SUR *** Muhammet L SELCUK ** Sadettin TIPIRDAMAZ **

***

***

Kirikkale University, Faculty of Veterinary Medicine, Department of Anatomy, Campus, TR-71451Yahsihan, Kirikkale - TURKEY Selcuk University, Faculty of Veterinary Medicine, Department of Anatomy, Campus, TR-42000 Selcuklu, Konya - TURKEY Selcuk University, Faculty of Veterinary Medicine, Department of Histology and Embryology, Campus, TR-42000 Selcuklu, Konya - TURKEY


The part of the central nervous system situated within the vertebral canal, namely, the spinal cord, is composed of 42-43 segments in horses, 8 of which are cervical, 18 thoracic, 6 lumbar, 5 sacral and 5-6 caudal. The spinal cord, enveloped by the meninges, extends from the foramen

magnum to the level in-between the first and second sacral vertebrae. In general, the spinal cord is dorso-ventrally compressed and elliptic with two enlargements at the cervical intumescence and the lumbar intumescence, and terminates forming the medullary cone. In histological

INTRODUCTION


RESEARCH ARTICLE

250Selective Gray and White ...

sections, the gray matter, resembling the letter H in shape and composed of nerve and glia cells and blood vessels, is observed to be situated centripetally, whilst the white matter, composed mostly of myelinated axons and blood vessels, is located peripherally. The central canal is located in the center of histological sections. Of all domestic animals, horses have the largest spinal cord, which measures 180-200 cm in length and 250-300 g in weight 1.

The segmental anatomical features of the spinal cord have been demonstrated in the cat 2, monkey 3, dog 4, sheep 5, goat 6, impala 7, horse and cattle 8. Furthermore, histological and some histomorphometric features of the spinal cord have been ascertained in research conducted in humans 9-11, rats 12, sheep 13, donkeys 14 and horses 15. However, during the conduct of research on sections pertaining to spinal cord segments of humans, goats, sheep, donkeys and horses, the necessity for full images arose in order to obtain histomorphometric data. For this purpose, several methods were applied, including direct drawings using millimetric paper and a calliper 6,13,14, indirect drawings 15, photograph combining 9, projection 10

and digital tablets 11. Literature review revealed that different fixation, processing and staining methods were applied for histological tissue processing in the researches referred to above, and yet limited information was provided by the researchers on the methods applied.

The gray and white matter in the central nervous system have different anatomical and cellular properties. Investigation of chronic diseases that affect the central nervous system, such as multiple sclerosis and schizophrenia, using the ratio of gray and white matter is of great importance 16-18. Imaging techniques, for instance Magnetic Resonance Imaging (MRI) and Diffusion Tensor Imaging (DTI) 19-21, and histological methods are used routinely to determine this parameter 12,22. Although obtaining data using imaging methods is very rapid and practical, some difficulties (in tissue processing, staining and imaging) are encountered during application of the histological process, owing to the size of tissue samples.

A search of the literature found that a limited number of morphometric and histological studies have been conducted on the spinal cord of the horse. The aim of this study was to overcome difficulties related to tissue processing, to decide which stain is best for the clear differentiation of gray and white matter of the spinal cord of horse, to acquire tissue images as a whole, and to compare the staining performance of various stains macroscopically and microscopically.


Material

A 15-year-old female Shetland pony (230 kg) that was

suffering from various orthopedic disorders was sent to the Department of Anatomy from the Equestrian Facility of the Faculty of Veterinary Medicine, Selcuk University for use in this study. The research was approved by the ethical committee of Faculty of Veterinary Medicine, Selcuk University (2011/16). The animal was anesthetized by administration of 10% chloral hydrate (80 mg/kg, IV) 23 and killed under general anesthesia by draining blood from the common carotid artery. Ten percent neutral formaldehyde was administered to the animal through a cannula in the common carotid artery after death. The spinal cord was dissected out from the vertebral column by laminectomy after the cadaver had been kept in a container of 10% neutral formaldehyde for 10 days. Segmentation was performed over the spinal cord following removal of the dura mater and pia mater. The C6 segment was used in this study; it was 68.8 mm in length and weighed 12.8 g.

Methods

Sampling and Tissue Processing

The C6 segment was divided into 18 pieces using a tissue slicer prior to histological processing. Each tissue sample was 3.8 mm in length. Systematic random sampling was performed on the separated pieces 24. A sampling ratio of 1/2 was used, and nine tissue samples were obtained for routine histological processing. The tissue samples were placed on trays, taking into account their cranial-caudal orientation, and dehydrated in an ethanol series. A vacuum was applied during the second application of 96% ethanol (200 mmHg, 30 min), the third application of 100% ethanol (200 mmHg, 1 h), the third application of xylene (200 mmHg, 30 min), and in paraffin (56-58ºC, 300 mmHg, the last 2 h), and the samples were blocked in paraffin wax. Samples in paraffin blocks were sectioned consecutively on a rotary microtome at a thickness of 10 μm, and six sections were obtained from each block. The sections were kept in an incubator (37ºC) for 24 h, stained according to the order given in Table 1, and mounted with Entellan under a glass coverslip.

Table 1. Section series and stains

Tablo 1. Kesit sayısı ve boyalar

Section Number Stain Reference

1 H&E (11)

2 MMGG Table 2

3 MGG (12, 13)

4 MT (14)

5 AgNORs (15)

6 KB (16)

H&E (Hematoxylin & eosin), MGG (May-Grunwald-Giemsa), MT (Masson’s trichrome), KB (Kluver Barrera), MMGG (modified May-Grunwald-Giemsa)

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BOLAT, BAHAR, SURSELCUK, TIPIRDAMAZ

Preparation of Giemsa Solution

Giemsa stock solution (Merck KGaA, Darmstadt, Germany) was added at 1 drop per ml to 250 mL distilled water using a Pasteur pipette (The solution prepared in this way is not recommended to be used more than two times).

Acquiring Images from Slides Using an Office Scanner

Images of gross biological structures (brain or cerebellum) that are difficult to view under the micro-

scope can be obtained with the help of a standard office scanner 31. In this study, all slides were scanned as positive image using a standard office flatbed scanner (Hp Scanjet G4010) at 300 dpi for macroscopic evaluation.

Area Calculation and Evaluation of Variations among Stains with Point Counting Frame

The point counting method has been used frequently to assess morphological parameters such as the area, volume, and area or volume ratio 32-34. This method was used to predict possible variations among stains that could affect measurement of the areas of gray matter and gross section. For this purpose, the grid function of the image analysis software ImageJ was applied to the scanned images. The area per point value was set at 0.4 mm2 (0.2 × 0.2 mm) and 4 mm2 (2 × 2 mm) to acquire an optimum CE value 24 for gray matter and the gross section respectively (Fig. 1). The areas of gray and gross section

on the scanned images were calculated three times by each of six different operators using the random function of the software. The results were analyzed using ANOVA (Table 3).

Evaluation of the Ability of White and Gray MatterStaining with A Survey

This part of the study was designed as a single blinded experiment. The survey group was composed of 20 healthy and non-colorblind students who were selected randomly

Table 3. The mean cross-sectional areas of gray matter and gross section and responses of survey participants (median)

Tablo 3. Transversal kesitlerde ortalama gri ve ak madde oranları (mean±SE) ve anket sonuçları (median)

Stain Gray Matter (mm2) Spinal Cord (mm2) Median Score

H&E 11.97±0.641 138.20±0.577 1d

MMGG 11.91±0.255 139.01±0.376 5a

MGG 11.72±0.513 138.52±0.565 3.5b

MT 12.00±0.391 138.05±0.425 2c

AgNORs 11.64±0.444 138.15±0.465 2cd

KB 11.99±0.320 138.52±0.565 4a

There was no statistical difference among the areas of the structures with different staining methods (P>0.05, ANOVA) a,b,c,d Different letters in the same column are significantly different (P<0.05, Mann-Whitney U test)

Table 2. Modified May-Grunwald-Giemsa staining procedure

Tablo 2. Modifiye edilmiş May-Grunwald-Giemsa boya prosedürü

Staining Stages

1 Xylene, 5 min 8 Rinse in distilled water 15 96% alcohol dip once

2 Xylene, 5 min 9 Tap water for 5 min. 16 96% alcohol dip once

3 100% alcohol, 3 min 10 Rinse in distilled water 17 100% alcohol dip once

4 100% alcohol, 3 min 11 Place in 250 ml May-Grunwald stock solution at room temperature for 10 min 18 100% alcohol dip once

5 96% alcohol, 3 min 12 Rinse in distilled water 19 Xylene 2 min

6 80% alcohol, 3 min 13 Place in Giemsa solution (250 ml) in 56°C incubator for 45 min 20 Xylene 2 min

7 70% alcohol, 3 min 14 Cool at room temperature 21 Xylene 2 min

Fig 1. Superimposed point counting frame on cross-section of spinal cord using ImageJ (area per point = 4 mm2, bar=5 mm)

Şekil 1. Medulla spinalis kesitleri üzerine ImageJ kullanılarak uygulanan noktalı alan ölçüm cetveli (bir noktanın alanı = 4 mm2, bar=5 mm)


from the senior students of the Faculty of Veterinary Medicine. The students were given a figure (Fig. 2) that showed six different stains and were asked to evaluate these stains in terms of the differentiation of gray and white matter according to a Likert scale (1: worst, 5: best). The results were analyzed using the Mann-Whitney U test (Table 3).

Light Microscopic Evaluation of Sections

The ability of the six dyes to stain neurons, glia, ependymal cells, endothelium, axon and dendrites were investigated using a Likert scale and the results are given in Table 4.

RESULTS

Collapsed areas were detected particularly in the gray matter region in paraffin blocks of tissues to which vacuum had not been applied during dehydration. Ruptures occurred during the cutting of these blocks with the rotary microtome, and low quality staining was observed especially in that area. This problem was solved by the application of vacuum. As a result of calculations made using the point counting frame superimposed on each slide stained with the different staining methods, the mean cross-sectional surface area of C6 was found to be 138±0.197 mm2 and the mean area of gray matter

Table 4. Microscopic assessment using Likert scale

Tablo 4. Likert skalası kullanılarak mikroskopik değerlendirme

Stains

White Matter Gray Matter

Epen

dym

al c

ells

Diff

eran

tiat

ıon

of g

ray

and

whi

te m

atte

r

Diff

eran

tiat

ıon

of a

xon

and

dend

rite

App

eara

nce

of N

issl

bod

ies

Axo

n

Glia

Endo

thel

Neu

ron

Glia

Endo

thel

N C N C N C N C N C N C

H&E 3 4 - 4 - 4 4 4 - 4 - 4 4 1 3 4

MMGG 4 3 - 2 - 2 2 3 - 2 - 1 1 5 2 2

MGG 2 3 - 3 - 3 3 3 - 3 - 2 2 2 3 3

MT 3 4 - 5 - 4 4 4 - 5 - 5 5 2 4 4

AgNORs 3 4 - 5 - 3 2 4 - 5 - 5 3 3 1 1

KB 4 4 - 3 - 5 5 4 - 3 - 4 4 5 5 5

N: Nucleus, C: Cytoplasm

Fig 2. The image used by the survey group to evaluate the performance of stains using a Likert scale (bar= 5mm) (A: H&E, B: MMGG, C: MGG, D: MT, E: AgNORs, F: KB)

Şekil 2. Anket grubu tarafından Likert skalası kullanarak boya performanslarının değerlendirilmesinde kullanılan resim dosyası (bar= 5mm) (A: H&E, B: MMGG, C: MGG, D: MT, E: AgNORs, F: KB)

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BOLAT, BAHAR, SURSELCUK, TIPIRDAMAZ

was estimated to be 11.87±0.170 mm2. The ratio of gray matter to gross section was determined to be 8.58%. A statistical difference was not detected (P>0.05, Table 3) among the areas of gray and white matter calculated from sections made using the six different stains.

The calculated coefficient of error value (CE) for the gray matter was 0.053 and for the spinal cord was 0.048. Statistical analysis of the responses given by the survey group showed that MMGG and KB were thought to be the best staining methods for differentiation of gray and white matter (Table 3, Fig. 2).

DISCUSSION

The application of a vacuum during the dehydration process is not recommended because of the fragile character of central nervous system tissue 25. In the present study, collapse caused by inadequate dehydration of the surface of paraffin blocks were seen, especially in the gray matter region, as a result of the routinely applied dehydration process without vacuum application. These problems were solved with vacuum application, as described in the section describing sampling and tissue processing. Different staining techniques have been utilized to differentiate gray and white matter macro-scopically 35,36 and microscopically 9,37 because of the poor contrast between these structures. Although KB is the staining method used most commonly for the central nervous system, MMGG (Table 2) is a preferable staining method to differentiate gray and white matter in the spinal cord of the horse because of its selective quality for this area and rapid reliable results (Fig. 3, Table 4).

The ratio of gray and white matter is used as an important parameter in the diagnosis of several diseases (schizophrenia, multiple sclerosis) in the central nervous system. Although imaging methods such as MRI are used most commonly to obtain this information 16,17,21, its use is limited in large domestic animals. Histological methods are frequently preferred when investigating the spinal cord of these animals 13,15. However, the entire structure must be displayed to estimate the ratio from histological sections. For this reason, a photographic camera 9,10, Edingerschen Apparatus 15, slide scanner 38 and office scanner 31 have been used to view the entire sections taken from large biological structures. In the current study, stained slides were scanned as JPEG files using a standard flatbed office scanner with a positive image scan option at 300 dpi. Measurement of the scanned images with a point counting frame estimated the ratio of gray matter to be 8.58% in the C6 segment (Table 3). The ratios of gray matter in C6 segments were reported as 11.86% of donkey 14, 18.3% of human 10, and 35-40% of rat 12 respectively. Braun 15 reported that the ratio of gray matter was 12.7% in the C6 segment of horse. It is thought that the dissimilarity between the two results could have resulted from differences in methodology. However, use of an office scanner to obtain images of large structures such as the brain and cerebellum was found to be a rapid method to view the spinal cord of the horse. Under microscopic examination, H&E, AgNORs, MT and KB for glia, MT and AgNORs for endothelial cells, KB for differentiation of axons and dendrites and also Nissl bodies, MT and AgNORs, especially for ependymal cells, were found to be good staining options (Fig. 3, Table 4).

Fig. 3. Cross-sectional images obtained by 4× and 100× objectives under a light microscope (A: H&E, B: MMGG, C: MGG, D: MT, E: AgNORs, F: KB; Upper-case letter: 4×, Lower-case letter: 100×)

Şekil 3. Işık mikroskopu ile 4× ve 100× objektif kullanılarak elde edilen kesit görüntüleri (A: H&E, B: MMGG, C: MGG, D: MT, E: AgNORs, F: KB; Büyük harf: 4×, Küçük harf: 100×)


The current study showed that a vacuum can be applied during dehydration of large structures such as the spinal cord of the horse in order to acquire acceptable results. MMGG is a useful staining method for the differentiation of gray and white matter, and it is advised to be used when the spinal cord is examined both macroscopically and microscopically. Use of an office scanner is a cheap and practical method to view and scan large biological tissues. These methods are suggested as tools for use in morphometric studies related to the central nervous system.

Acknowledgements

The Authors are grateful to senior students of Faculty of Veterinary Medicine, Selcuk University for their kindly assistance.

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SummaryIn this study, the presence of antibodies to Brucella abortus, Chlamydophila abortus, Coxiella burnetii, Bovine herpesvirus-1 (BHV-1),

Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-4 (BHV-4) were investigated in dairy cattle herds with abortion history in Burdur province, Southwest region of Turkey. The blood samples were collected from 932 dairy cattle ≥2 years of age from 10 herds not vaccinated against for mentioned infections and all of the serum samples were tested for B. abortus by ELISA. Seronegative herds for B. abortus were investigated for C. burnetii and C. abortus. While seropositivity for B. abortus and C. burnetii were detected 25.3% (236/932) and 10.2% (19/186), antibodies against C. abortus was not detected. Seronegative herds for B. abortus, C. burnetii and C. abortus were analysed for antibodies to BHV-1, BVDV and BHV-4 and seropositivity were found as 43.5% (40/92), 81.5% (75/92), 42.4% (39/92), respectively. BVDV antigen was determined in 2.2% (2/92) of the samples. As a result, we determined that C. abortus is not an important agent causing abortion in dairy herds in Burdur, but the seropositivity of B. abortus, C. burnetii, BHV-1, BVDV and BHV-4 are high. Thus, the herds should be tested for these infections regularly and control measures must be taken.

Keywords: Abortion, ELISA, Dairy cattle, Brucella abortus, Chlamydophila abortus, Coxiella burnetii, Bovine herpesvirus-1, Bovine viral diarrhea virus, Bovine herpesvirus-4

Burdur Yöresinde Süt Sığırı İşletmelerinin Bazı Bakteriyel ve Viral Abortlar Yönünden Değerlendirilmesi

ÖzetBu çalışmada, Türkiye’nin güneybatı bölgesinde yer alan Burdur ili’nde yavru atma problemli süt sığırlarında Brucella abortus,

Chlamydophila abortus, Coxiella burnetii, Bovine herpesvirus-1 (BHV-1), Bovine viral diarrhea virus (BVDV) ve Bovine herpesvirus-4 (BHV-4)’e karşı antikorların varlığı araştırıldı. Kan örnekleri araştırılan enfeksiyonlara karşı aşılanmamış 10 sürüden 2 yaş ve üzeri 932 hayvandan toplandı. Serumlar önce B. abortus yönünden ELISA ile test edildi. B. abortus negatif bulunan serum örnekleri C. burnetii ve C. abortus’a karşı antikorların varlığı yönünden araştırıldı. B. abortus ve C. burnetii için pozitiflik sırasıyla %25.3 (236/932) ve %10.2 (19/186) belirlenirken, C. abortus antikorları tespit edilemedi. B. abortus, C. burnetii ve C. abortus negatif bulunan sürülerde BHV-1, BVDV ve BHV-4’e karşı antikorların varlığı araştırıldı. Seropozitiflik bu hastalıklar için sırasıyla %43.5 (40/92), %81.5 (75/92), %42.4 (39/92)olarak belirlendi. BVDV antijen ise örneklerin %2.2’sinde tespit edildi. Sonuç olarak, C. abortus’un Burdur’da süt sığırı işletmelerinde aborta neden olan önemli bir ajan olmadığı, B. abortus, C. burnetii, BHV-1, BVDV ve BHV-4’ün seropozitifliğinin yüksek olduğu belirlendi. Bu nedenle, sürülerin bu enfeksiyonlar yönünden düzenli olarak test edilmesi ve gerekli kontrol önlemlerinin alınması gerektiği sonucuna varıldı.

Anahtar sözcükler: Abort, ELISA, Süt sığırı, Brucella abortus, Chlamydophila abortus, Coxiella burnetii, Bovine herpesvirus-1, Bovine viral diarrhea virus, Bovine herpesvirus-4

Evaluation for Some Bacterial and Viral Abortions of Dairy Cattle Farms in Burdur District of Turkey

Dilek OZTURK * Mehmet KALE ** Faruk PEHLIVANOGLU * Sibel HASIRCIOGLU ** Hulya TURUTOGLU *

***

Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Department of Microbiology, TR-15100 Burdur - TURKEY Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Department of Virology, TR-15100 Burdur - TURKEY


Bovine abortion is a significant cause of economic losses in dairy cattle herds 1. Although the cause of abortion depends on several factors including idiopathic, metabolic

or hormonal abnormalities, nutritional deficiencies, trauma and infections, the infectious agents including bacterial, viral, fungal and protozoan agents are the most important

INTRODUCTION


RESEARCH ARTICLE

256Evaluation for Some Bacterial ...

factors associated with abortions among dairy herds worldwide 2,3. The infections causing abortion may vary, presumably due to climate, production types, management practices and geographic factors in different regions 4. Effective preventive measures for abortions require not only prompt and accurate diagnosis, but also understanding of the multicausal factors involved 5.

The aim of this study was to investigate Brucella abortus, Chlamydophila abortus, Coxiella burnetii, Bovine herpes-virus-1, Bovine viral diarrhea virus and Bovine herpesvirus-4 infections of dairy cattle herds with abortion history.


Samples

This study was conducted in 10 dairy cattle herds with abortion history in Burdur province, Southwest region of Turkey. Blood samples were collected from 932 dairy cattle ≥2 years of age. Blood samples were taken into tubes without anticoagulant. After clotting, the tubes were centrifuged, and the serum samples were separated and kept at -20°C until used. All of the serum samples were tested for B. abortus by ELISA. The seronegative samples for B. abortus were investigated for C. abortus and C. burnetii by ELISA. After, seronegative herds for B. abortus, C. burnetii and C. abortus were analysed for antibodies to BHV-1, BVDV and BHV-4. Leucocyte were obtained from blood samples, taken into tubes with EDTA, by centrifugation at 2.500 rpm/25 min. and stored in deep freezer at -20°C till used. All of animals were not vaccinated against BHV-1, BVDV, BHV-4 and B. abortus before.

Antibody Detections in Sera by ELISA

Presence of antibodies against B. abortus, C. burnetii and C. abortus (Institute Pourquier, France), BHV-1, BVDV and BHV-4 (Bio-X Diagnostics, Belgium) were investigated by ELISA. The tests were carried out according to the kit procedure.

BVDV Antigen - ELISA

The kit of BVD/MD Antigen Mix Screening ELISA (Institute Pourquier, France) was used to determine the existence of antigen in the leucocyte samples. The test was carried out according to the kit procedure.

RESULTS

The seropositivity for B. abortus was found in 25.3% (236/932) of the sera. While seropositivity for C. burnetii was found in 10.2% (19/186) of the samples, C. abortus was not detected in any of the sera (0/225). In seronegative herds for B. abortus, C. burnetii and C. abortus, seropositivity for BHV-1, BVDV and BHV-4 were found as 43.5% (40/92), 81.5% (75/92), and 42.4% (39/92), respectively (Table 1).

Antibodies rates of BHV-1 + BVDV + BHV-4, BHV-1 + BHV-4, BHV-1 + BVDV and BVDV + BHV-4 were 21.7% (20/92), 21.7% (20/92), 40.2% (37/92), 37% (34/92), respectively. Also, the rates at which cattle were detected for both BHV-1 + BVDV (antigen) + BHV-4 and BHV-1 + BVDV (antigen) were 1.1% (1/92) (Fig. 1).

BVDV antigen was only detected as 2.2% (2/92) in the leucocyte samples in aborted cattle.

Table 1. The rates of seropositivity for B. abortus, C. burnetii, C. abortus, BHV-1, BVDV, BHV-4 in dairy cattle herds with abortion history

Tablo 1. Abort hikayesi bulunan süt sığırı işletmelerinde B. abortus, C. burnetii, C. abortus, BHV-1, BVDV, BHV-4’ün seropozitiflik oranları

Bacterial and Viral Agents Number of Serum Samples Positive %

B. abortus 932 236 25.3

C. burnetii 186 19 10.2

C. abortus 225 0 0

BHV-1 92 40 43.5

BVDV 92 75 81.5

BHV-4 92 39 42.4

Fig 1. Multiple infection rates of BHV-1, BVDV and BHV-4 in dairy cattle herds with abortion history

(Ab: Antibody, Ag: Antigen a: BHV-1 (Ab) + BVDV (Ab) + BHV-4 (Ab), b: BHV-1 (Ab) + BHV-4 (Ab), c: BHV-1 (Ab) + BVDV (Ab), d: BVDV (Ab) + BHV-4 (Ab), e: BHV-1 (Ab) + BVDV (Ab-/Ag+) + BHV-4 (Ab), f: BHV-1 (Ab) + BVDV (Ab-/Ag+)).

Şekil 1. Abort hikayesi bulunan süt sığırı sürülerinde BHV-1, BVDV and BHV-4 için çoklu enfeksiyon oranları

257

OZTURK, KALE, PEHLIVANOGLUHASIRCIOGLU, TURUTOGLU

DISCUSSION

Abortion has caused serious economic losses world-wide. Regional differences in the prevalence of abortion-causing diseases can be a result of a variety of reasons such as methods of herd health management, climate, types of cattle operations, feed, vaccination programmes, geographic factors, number of pregnancies and sanitation 1,4. Brucellosis is also very common and an economically important zoonotic disease in Turkey 6,7 and worldwide 8,9. In this study, the seropositivity for B. abortus was found to be 25.3% in dairy cattle herds with abortion history in Burdur province. The prevalence studies for B. abortus have been conducted in different parts of Turkey by researchers 6,7,10. In these studies 6-9, prevalence have changed from 1.4% to 18% and quite low than this study. There is one study related with the seropositivity of brucellosis in dairy cattle herds with abortions problems in Burdur which reported the rate of brucellosis as 10.1% by Rose Bengal plate test (RBPT), and 6.8% by serum agglutination test (SAT) during 2007-2008 11. This study results was rather lower than present study. The reason of this may be due to higher sensitivity and specificity of ELISA compared to RBPT and SAT. Seroprevalance of brucellosis was reported in between 3.3-22.2% in other countries 8,9. We thought that these differences may originate from factors such as use of different detection methods, climate, vaccination programmes, sampling method of animals, intensification of animal movements due to the high potential of the region and lack of herd screening for B. abortus. This study supports researchers who state that prevalence of infections causing abortion may be affected by the mentioned factors 1,4,12.

Abortions caused by Chlamydophila spp. in cattle are rather widespread worldwide and are generally associated with C. abortus infections. Because Chlamydophila infections are not considered as a major threat to the cattle industry in Turkey, there are less studies of cattle than sheep or goats 13,14. In this study, the antibodies for C. abortus can not be detected in blood serum samples of cattle. In the South East part of Turkey, seroprevalance of C. abortus was determined to be 8.3% in aborted dairy cattle 15. The seroprevalance for Chlamydophila infection was also reported in between 0.4-57% in other countries by ELISA 16-18. Chlamydophila spp. was not responsible from abortion cases of cattle in this region, because positive serum samples were not detected.

In this study, the prevalence of C. burnetii was 10.2%, while studies conducted in different parts of Turkey have results ranging from 4.3% to 44.5% in cattle herds with abortion problems 19-21. Özyer et al. 19 reported that the seropositivity of C. burnetii was 44.5% in cattle with abortion history by complement fixation test (CFT). The seropositivity of C. burnetii was 22.6% in Erzurum 20 and 16.3% in 16 locations of Eastern Turkey 21 by ELISA. In

Aydin, Kirkan et al.22 were found that C. burnetii was 4.3% in cattle by polymerase chain reaction (PCR). The prevalance of C. burnetii infection was found between 5% and 44.9% in other countries 23-25. The seropositivity of C. burnetii was found in 39% of cattle by indirect fluorescence in Zimbabwe 23. Serbezov et al.24 reported that the occurence of C. burnetii was 5% to 31% in cattle by CFT in Bulgaria. The seropositivity was determined 44.9% in dairy cattle of Northern Italy by ELISA 25. These differences may be associated with the differences of sensitivity and specificity of diagnostic methods used for diagnosis of C. burnetii infections.

In the present study, the seropositivity of BHV-1, BVDV and BHV-4 was found to be 43.5%, 81.5% and 42.4%, respectively. The presence of BHV-1 antibodies detected in 25-60% 4,26,27 and for BVDV were detected 75-80% 28,29, for BHV-4 were found 36-88% 30-32, in aborted cattle. However, there are also contradictory studies 33-35. But, Kale et al.36 investigated presence of BHV-1, BVDV and BHV-4 infections in 204 dairy cows 3 to 5 years of age that aborted at 5-7 months of gestation in same area 36. They found lower rates of BHV-1 (21.6%), BVDV (25.5%) and BHV-4 (29.4%) seropositivity than the current study. As a result, there has been an increase in the infections of BHV-1, BVDV and BHV-4 in the region. In Kars, Yildirim et al.37 reports that the seropositivity of BHV-1 and BHV-4 were found 61.4%, 29.3% by ELISA and the presence of BVDV specific antibody was found 52.9% by virus neutralization test in aborted cattle. The seropositivity of BHV-1 and BVDV are higher than this study. Often, no obvious clinical signs are seen in herds with fetal losses due to BVD virus and abortions occuring days or several weeks following maternal infection. In the study, seroprevalance of BVDV antigen was detected as 2.2% in aborted cattle. Research has shown <10% of abortion is related to BVDV 38,39. The seroprevalence of BHV-1, BVDV and BHV-4 had higher levels than that of other studies which viral and bacterial mix in the study 40. However, there are also higher seroprevalence results 41 than seen in the study. Results of this study indicate that BHV-1, BVDV and BHV-4 are a significant viral cause of abortion in the area. Bovine abortion can be caused by several pathogens and evidence of multiple agent involvement has been indicated 42. Furthermore, in the research, while seroprevalance of BHV-1 + BVDV have the highest and 40.2%, BHV-1 + BVDV (negative antibody/positive antigen) + BHV-4 and BHV-1 + BVDV (negative antibody/positive antigen) was the lowest and 1.1%. In context, it is important to point out that in mixed cases it is extremely difficult to establish the actual agent responsible for the abortion 27.

In conclusion, we determined that seroprevalence of B. abortus, C. burnetii, BHV-1, BVDV and BHV-4 in dairy cattle farms in Burdur were higher than those in other parts of Turkey, but C. abortus is not a major agent causing abortion in cattle population in the region. Thus, the herd surveys should be performed regularly for such infections in the region.

258Evaluation for Some Bacterial ...

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SummaryIn this dissertation, the effect of the widely used pepper gas (oleoresin capsicum, OC) derived from the cayenne pepper was studied in

the blood gases, electrolytes and some biochemical parameters on the rats with different dosages and periods. In the pre-dosage phase, 12 of 47 Wistar albino rats were used as the live material. Commercially available 90 g OC sprays were provided. It was experimentally measured that 6 g of OC was sprayed per second. 6 rats were used as control group. Experimental Group 1 was 9 rats which were kept 5 min in an OC sprayed closed enviroment for 4 sec (24 g); Experimental Group 2 was 10 rats which were kept 8 min in an OC sprayed closed enviroment for 8 sec (48 g); Experimental Group 3 was 10 rats which were kept 15 min in an OC sprayed closed enviroment for 12 sec (72 g). The rats were kept as a group of 5 of each in 50x50x50 cm experiment cube and OC was applied for the abovementioned periods and dosages. The blood samples were taken from the heart after the 1-2 min sedation application. The potential hydrogen (pH), carbon dioxide partial pressure (pCO2), concentration of total carbon dioxide (ct CO2), O2% saturation, HCO3, base excess (BE), base excess of extracellular fluid (B(ecf )), Na, K, Ca, Cl and glucose levels were measured by Rapidlab 865 System and auto analyser. When the control group values were compared to the other three groups, pH, ct CO2 and HCO3 (except the Group 3) were decreased, pCO2, pO2, O2% saturation, K, Cl, glucose, BE and B(ecf ) were increased and no significant change was observed in Na and Ca levels. The pH change (P≤0.05), pO2 (P≤0.01), HCO3 (P≤0.01); %O2 saturation (P≤0.001), K (P≤0.05), Cl (P≤0.05); B(ecf ) (P≤0.05) levels were evaulated the changes according to inter-group and control group. As a result, no fatality were encountered in rats which were exposed different dosages and amounts of OC in a closed enviroment except difficulty in breathing and conjuctivity. Despite that supporting biochemical changes for respiratoric asidose were encountered, it was understood that pepper gas could especially be used as a safe riot control agent in open air.

Keywords: Bicarbonate, Blood gases, Electrolyte, OC, Pepper Gas, Rat

Ratlarda Biber Gazının (OC) Bazı Biyokimyasal Parametreler Üzerine Etkisi

ÖzetBu çalışmada Cayenne bitkisinden elde edilen ve bu amaçla çok kullanılan biber gazının (oleoresin capsicum, OC), ratlar üzerinde farklı

süre ve dozda uygulanması sırasında kan gazları, elektrolitler ve bazı biyokimyasal parametreler üzerine etkisi incelendi. Araştırmada 12 tanesi ön doz çalışmasında kullanılmak üzere toplam 47 adet Wistar albino rat canlı materyal olarak kullanıldı. Ticari olarak satılan 90 g’lık spreyler halinde OC temin edildi. Saniyede 6 g OC püskürtüldüğü denemelerle hesaplandı. 6 adet rat kontrol grubu, 9 adet rat 4 saniye OC sıkılmış (24 g) ortamda 5 dak. bırakılan deneme 1; 10 adet rat 8 saniye (48 g) OC sıkılmış ortamda 10 dakika bırakılan deneme 2; 10 adet rat ise 12 saniye (72 g) OC sıkılmış ortamda 15 dak. bırakılan deneme 3 grubunu oluşturdu. Ratlar 50x50x50 cm’lik bir deney küpüne beşerli gruplar halinde konularak, yukarıda belirtilen doz ve sürede OC uygulandı. Süre sonunda hayvanlar eter ile 1-2 dak. sedasyon işlemine tabii tutulup kalpten kan örnekleri alındı ve hemen pH, parsiyel karbondioksit basıncı (pCO2), toplam karbondioksit (ct CO2), %O2 saturasyonu, bikarbonat (HCO3), baz fazlalığı (BE), hücre dışı sıvı baz fazlalığı (B(ecf )), Na, K, Ca, Cl ve glukoz düzeyleri Rapidlab 865 cihazı ve otoanalizörle ölçüldü. Kontrollerin değerleri diğer üç grup ile karşılaştırıldığında pH, ct CO3, üçüncü grup hariç HCO3 düzeylerinde azalma, pCO2, pO2, %O2 saturasyonu, K, Cl, glukoz, BE ve B(ecf ) düzeylerinde artma, Na, Ca düzeylerinde ise dikkati çeken değişim saptanmadı. pH değişimi (P≤0.05), pO2 (P≤0.01), HCO3 (P≤0.01), %O2 saturasyonunda (P≤0.001), K (P≤0.05), Cl (P≤0.05), B(ecf ) (P≤ 0.05) düzeylerinde önemle deneme grupları arasında veya kontrollere göre değişim hesaplandı. Sonuç olarak kapalı ortamda farklı dozda OC miktarına, farklı sürelerde maruz kalan ratlarda solunum güçlüğü ve konjuktivit görülmesine rağmen hiçbir ölüm olayına rastlanılmadı. Kan gazları değerleri incelendiğinde respiratorik asidoz oluşumuna destek veren biyokimyasal değişimlere rastlanmış olmasına rağmen ve biber gazının özellikle açık alanlarda toplumsal olaylarda güvenli olarak kullanılabilecek gaz olabileceği kanısına varıldı.

Anahtar sözcükler: Biber gazı, Bikarbonat, Elektrolit, Kan gazları, Rat, OC

The Effect of Pepper Gas (OC) on Some Biochemical Parameters in Rats [1]

Ercan SEYHAN * Nihat MERT ** Handan MERT **

[1]

***

This paper has been prepared from doctorate dissertation of “The Effect of Pepper Gas (OC) on Some Biochemical Parameters in Rats”, Yuzuncu Yil University, Institute of Health Sciences, PhD Thesis in Department of Biochemistry, Van, 2010

Jandarma Kriminal Daire Baskanligi, TR-06580 Beytepe, Ankara - TURKEYYüzüncüyıl University, Veterinary Faculty, Biochemistry Department, TR-65080 Van - TURKEY



RESEARCH ARTICLE

260The Effect of Pepper Gas (OC) ...

INTRODUCTION

For as long as human existed on earth, there has been an ongoing need for law enforcement and justice. To maintain the continuity of this balance, law enforcement agencies has got the responsibility of force continium. Law enforcement agencies sometimes perform their duties during life treatening situations. In such cases, they only have moments to decide the level of force to be applied. Law enforcement agencies had limited numbers of tools to stop the aggresive behaviors of substance addicted or mentally ill persons for many years. Even the police force or the suspects got injured due to the inadequate means of force continuum means.

For many years, law enforcement agencies have used chloracetophenone (CN) and cholorobenzylidene malono-nitrile (CS) against the suspects to stop the assaults and control the riot 1. But these agents could not be used effectively because of their impact dependancy on exposure distance and ambient temperature. Particularly, serious problems were encoutered related with the decontamination procedures.

The police force started to use oleoresin capsicum, commonly known as pepper gas in public. Oleoresin capsicum is an organic substance derived from cayenne pepper which is imflammatory and adhesive because of its oily resin surface 2,3.

The short and long term effects as well as the possible negative effects on the suspects under arrest must be finely evaluated by the law enforcement agencies. The main reason behind is that every technique enables the police force to arrest and cuff the aggressive suspects is highly significant.

OC is commonly accepted as an effective and safe means of tool by law enforcement agencies to control the riot 4,5. The effectiveness of OC mostly depends on the inflammatory effect rather than the distance sprayed or ambient temperature. This makes OC more efficient for controling the drug addicteds and mentally ills than CN and CS 6. It is also known that OC provides more effectiveness on aggressive animals while carrying out duties.

OC is basically different from the traditional riot control agents of CS and CN. The tradional chemical riot control agents mostly cause pain and tears but OC generally has inflammatuar effect. Inhaling OC causes an intense inflammatuar effect on trachea and breathing is temporarily restricted to short and shallow breaths. The other side effects include involuntarily closing the eyes, coughing, chocking, lack of upper body strenght and nausea 7.

OC is a substance having the same inflammatuar effect of many commonly known plants. The direct touch to

the substance which has capsaicin content causes local irritation, burning and lacrimation. Long or repeated OC exposure dosages can cause dermatitis, adverse nasal and gastrointestinial effects in the lungs 8.

Medical researchers still have difficulty to define the pathological symptoms of OC gas. Despite that OC is stated as a very safe riot control agent in some studies, it is also stated that there is not enough physical data if it does not have long term side effects or cause fatality. This situation can lead to that the long term side effects of OC had not been systematically put forward enough. Today, there is insufficient data about the OC’s long term side effects. Except some studies performed on animals, there is not enough data about the mutagenic and carcinogenic long term side effects of OC 9,10. Even some researchers state that single dose does not have significant long term cancer risk, there is not enough study about it 9. Besides, not enough studies were found about the biochemical parameter changes in blood of materials exposed in a closed enviroment or in the open air.

In this study, It was aimed to determine the changes occure in the biochemical blood parameters of OC use in a closed enviroment.


As live material, 47 male Wistar albino rats, each bet-ween 190-220 g of weight provided from Fırat University Experimental Research Center (FUDAM), were used. Six rats in every cage were kept in an air conditioned room with 23ºC controlled room temperature. Ad libitum feeding and care were done to the end of trial period. Special rat food and ad libitum water were given in the 7 days adaptation period. The cages were cleaned daily.

SR 10 ST Rubber Gas Mask (MKEK, Code No: 214) which is protective for biological and chemical agents was used 11.

A special glass cube with 50x50x50 cm dimentions and 5 mm glass thickness was built. The upper lid was designed as an easy open lid with a rubber isolation stopping the gas leak. A circle with 3 cm diameter was cut on the side window of the experiment cube to spray OC in easily. The gas leak was prohibited by a sticky plastic material closing the circle.

The pepper gas was commercially purchased 90 grams of 5% pepper gas with March 2011 expiration date were used in the experiment. It was experimentally evaluated that OC spray canister releases 6 g of OC per second to enviroment because it emties fully in 15 sec.

Twelve rats were used as preliminary study to get an overwiev of the OC effect on rats. The practicability of experiment cube was tried by different spraying durations and dosages. Then, 35 rats were grouped for different applications.

261SEYHAN, MERT, MERT

Group I, 6 rats as Control Group (Control)

Group II, 9 rats kept 5 minin cube with 4 sec OC spraying (24 g) (Experiment 1)

Group III, 10 rats kept 10 min in cube with 8 sec OC Spraying (48 g) (Experiment 2)

Group IV, 10 rats kept 15 min in cube with 12 sec (72 g). (Experiment 3)

Five rats of each group except control group were taken to the experiment cube at every turn. OC was sprayed into the cube as much as determined for each group. Then the upper lid and the secured 3 cm diameter spraying hole were closed firmly. Rats were kept in the cube due to the predetermined durations of 5, 10 and 15 min. The special hygienic clothes, gloves and protective mask was worn.

The rats were sedated by a short preanesthetic application with ether and the blood samples were taken and sent to laborotory for analysis. The blood gases were analysed by Bayer Health Care Lab 865 and K, Cl, Ca and glucose levels were analysed by otoanalyser in biochemistry lab of Yuzuncuyil University Research Hospital 12.

In this study, ethical approval was taken from YYU Ethical Council.

RESULTS

In this study, the effect of pepper gas on blood gases, different electrolites and bicarbonate ions in rats were studied and the results were shown at three different tables (Table 1-3). The statistical importance between control and the other groups means was shown by (*) and the statistical importance of the comparisons made between experiment groups was done by Duncan Test and shown by capital letters. The blood gas and pH values of all the rats were presented at Table 1.

Considering the pH values, the 7.208 mean value in the control group was decreased to 7.099-7.084-7.093 at OC exposed rats for 4, 8 and 12 sec. The change with respect to controls showed P≤0.05, P≤0.07 and P≤0.05 statistical importance.

pCO2 levels with respect to controls showed increase in all three OC exposed groups. The significant physiological

Table 1. The pH and Blood Gas Changes in the rats of different duration and quantity OC Exposure

Tablo 1. Farklı süre ve miktarda OC uygulanan ratlardaki pH ve kan gazlarının değişimi

Group n pHX ± SEM

pCO2 (mmHg)X ± SEM

pO2 (mmHg)X ± SEM

ctCO2 (mmol/L)X ± SEM

O2SAT (mmol/L)X ± SEM

Control 6 7.208±0.015 49.167±3.027 78.500±9.511 21.733±0.837 87.083±5.643

Experiment 1 9 7.099±0.038 * 56.000±3.520 133.556±15.779 b 18.733±1.132 a 94.267±2.760

Experiment 2 10 7.084±0.027 50.900±2.742 146.400±6.809 b 16.300±0.447 b 97.930±0.293**

Experiment 3 10 7.093±0.036* 57.200±5.164 187.400±12.151 a 14.510±0.796 b 98.060±0.833**

* P≤0.05, ** P≤0.01 , a,b: Different letters in the same column show P≤0.05 importance

Table 2. The bicarbonate, BE and Base (ecf) levels change in healthy and OC exposed rats

Tablo 2. Bikarbonat, BE ve Baz (ecf) düzeylerinin sağlıklı ve farklı OC uygulanmış ratlardaki değişimi

Group n HCO3 (mmol/L)X ± SEM

BE (mmol/L)X ± SEM

B(ecf) (mmol/L)X ± SEM

Control 6 19.600±0.694 -9.217±0.894 -8.567±0.695

Experiment 1 9 17.022±1.097 -13.100±1.602 -12.667±1.584*b

Experiment 2 10 14.910±0.446 -13.930±0.959 -14.190±0.835ab

Experiment 3 10 25.260±12.213** -16.200±1.009 -16.590±0.914 a

* P≤0.05, ** P≤0.01 , a,b: Different letters in the same column show P≤0.05 importance

Table 3. The changes in serum electrolyte and glucose levels during different OC exposure doses and duration

Tablo 3. Farklı doz ve sürede OC uygulanması sırasında serum elektrolit ve glukoz düzeylerindeki değişimler

Gorup n Na (mmol/L)X ± SEM

K (mmol/L)X ± SEM

Ca (mg/dL)X ± SEM

Cl (mmol/L)X ± SEM

Glucose (mg/dL)X ± SEM

Control 6 135.500±1.147 4.570±0.097 10.605±0.323 96.167±0.477 142.167±13.489

Experiment 1 9 136.300±0.573 4.898±0.139 10.084±0.164 99.889±0.857 235.889±15.697

Experiment 2 10 136.500±0.500 5.270±0.233* 10.425±0.192 100.500±0.582 244.100±12.885

Experiment 3 10 135.200±1.133 5.688±0.414* 10.107±0.227 99.300±1.713* 248.600±11.211

* P≤0.05


increase (56-50.9-57.2 mmHg) was not considered as significant. Only the increase at the third group was accepted as P≤0.05 significant.

pO2 value at control group was 78.5 mmHg while it increased to 133.556-146.4-187.4 mmHg at all other three groups. This increase was not considered as statistically significant with respect to control groups but the comparisons between them made by Duncan test was determined statistically significant as P≤0.05.

The comparison of controls with other three groups mean values showed no statistical importance. But the comparison between other groups showed P≤0.05 meaningful statistical chance.

O2% saturation was determined as 87.083-98.060%. The comparison between control and the other three groups, this difference in saturation showed P≤0.001 statistical significant change in 2nd and 3rd groups but no significant change was determined in the 1st group (P≥0.05). In the Duncan Test observation, the difference between all three groups except controls was not found meaningful (P>0.05).

When total bicarbonate levels of control group was compared with the other three groups, it was only observed in the 3rd group a statistical importance of P≤0.001. The control group mean value of 19.6 mmol/L increased to 25.260 mmol/L in the 3rd group. At the interpretation between the experiment groups made by Duncan Test, the difference was not found meaningful (P≥0.05) (Table 2).

The BE levels at control and other groups were changed between -9.217-16.200 mmol/L. These observed increases was not found statistically significant at the comparisons made by Duncan Test neither between controls and other groups nor between experiment groups (P≥0.05) (Table 2).

As the Base (ecf ) level at the control group was -8.567 mmol/L, increase in other groups were determined as increased -12.667, -14.190 and -16.590 mmol/L and the statistical importance of P≤0.05 between control and the 1st group was found. The comparison made by Duncan Test between groups showed P≤0.05 statistical importance (Table 2).

No statistical importance was determined in Na and Ca value changes (Table 3). The Na level was 135.5 mmol/L in the control group while it was between 135.2-136.5 mmol/L levels in the other groups.

The Ca level at the controls was 10.605 mg/dL and between 10.084-10.425 mg/dL values in other three groups. No importance was determined neither in Na nor Ca levels between controls and the other three groups comparisons. The comparison made in mean values between experiment groups showed no importance (P≥0.05).

The serum K level of controls was lower than the experiment groups means. A significant increase of P≤0.05 was determined in the comparisons of 2nd and 3rd group means but the comparison with Duncan Test between experiment groups showed no importance (P≥0.05).

The Serum Cl level in OC exposed rats was found as changed between 96.167 mmol/L and 100.5 mmol/L. A significant change as P≤0.05 level was found between controls and the 3rd group but the comparison between experiment groups showed no statisitcal importance (P≥0.05).

The glucose level, a significant energy metabolism in organism, was found as 142.167 mg/dL in the control group but it showed increase due to amount of OC exposure and time period. The values of experiment groups were respectively 235.889, 244.1,248.6 mg/dL. The comparison of these values with control groups and the comparison of the three experiment groups by Duncan Test was not considered significant (P≥0.05) (Table 3).

The initial clinical symptoms observed in the OC exposed rats were tendency of dispersing and reunion, sensitivity in the eyes, conjunctivitis and respiratory distress. Due to the increase in exposure quantitiy from 24 g to 72 g, rats were dispersed and tried to put their heads under cage bedplace by the burning and pain feelings (Fig. 1-4).

In the histopathological examination, the organs at the control group were observed as normal histologic appearance (Fig. 5). The most meaningful change was observed in the lungs at the OC exposed groups. The changes in the lungs were more distinctive and broad depending on the OC exposure duration and dose. It was observed in the lungs that there were capillar hyperemia in interalveolar septum, mononuclear cell infiltration and obvious thickenning due to oedema, mostly lymphoid cell as perivascular and peribronchioler, less pneumonia charecterized by cellular exudation formed by leucocyte (Fig. 6).

Fig 1. The 190-220 g weight Wistar albino rats used in experiments

Şekil 1. Araştırmada kullanılan 190-220 g ağırlığına sahip Wistar albino rat


DISCUSSION

During riot control operations, the chemical agents are widely used to incapasitate the crowd without causing any illness or permanent injury. Incapacitating effects are based on unpleasant symptoms involving eyes, skin, mouth, nose and respiratory tract 13.

OC or pepper gas was considered as the main consideration of chemical agents to be used. They contain capsaicin which leads to irritation of the trigeminal cell pain receptors located in the mouth, nose, stomach and the mucous membrana 14.

Many riot control gases were developed with OC using different solvents. But no link with OC has been observed

Fig 2. The conjunctivitis and eye sensitivity case at the rats with 8 seconds (48 g) OC exposure

Şekil 2. 8 saniye süre ile 48 g OC uygulanan ratlarda gözde meydana gelen hassasiyet ve konjuktivit olgusu

Fig 3. The case of general condition disorder and effort to put their heads under cage bedplace at the rats with 8 seconds (48 g) OC exposure

Şekil 3. OC spreyi ile 8 saniye (48 g) biber gazı uygulanan ratlarda genel durum bozukluğu ve hayvanın altlık içine kafasını sokma girişimi

Fig 4. The case of putting heads under cage bedplace to ease the pain and disorder at the rats with 12 seconds (72 g) OC exposure

Şekil 4. 12 saniye süre ile biber gazı uygulanan (72 g) ratlarda duyulan rahatsızlık ve acı hafifletmek için altlık içine kafasını sokma işlemi

Fig 5. Normal hystologic appearence of lung parenchyma. H.E.X 300

Şekil 5. Akciğer parankiminin normal histolojik görünümü. H.E.X 300

Fig 6. Group VII: Ccapillar hyperemia in interalveolar septum, mono-nuclear cell infiltration and obvious thickenning due to oedema (star marking), leucocyte as leucocyte perivascular (gren arrow) and lymphoid cell infiltration (black arrow) H.E.X 300

Şekil 6.VII. Grup: İnteralveoler septumda kapilar hiperemi ve mono-nükleer hücre infilitrasyonu sonucu belirgin kalınlaşma (yıldız işareti), perivasküler olarak eozinofil, lökosit (yeşil ok) ve lenfoid hücre (siyah ok) infilitrasyonu, H.E.X 300


to result in severe health problems and even death. As a result of public diffusion for personal protection, the use of riot control chemicals were brought to agenda. Various biological screening tests have been experimented. Among the exisitng test involving animals, plethysmography is the most common one to evaluate the effects of active substances on the respiratory tract. Using a nose-only exposure integrated system, minute ventilation, frequency and tidal volume in awake rats were studied.

In a study, the effects of the solutions usually found in tear gas sprays on the respiratory tract of awake rats exposed to aerosols generated with a Collision Nebuliser were studied. The results indicate that the respiratory effects observed during acute exposure to CS (5%) are more significant than those observed during exposure to OC (7%). In addition, for both tear gases the respiratory effects observed during exposure to their solvents alone, are in the same range as those observed during exposure to the product as a whole 15.

The necessity to stop dangerrous and higly agitated persons leads enforcement forces to search for strong incapacitating properties of chemicals. These incapacitating properties are the result of the chemical’s direct action on nevre endings 16. The effects and signs observed concern eyes, skin and respiratory tract 17.

Effects involving the repiratory tract are potentially the most capable of having incapacitating consequences and consist in a burning sensation of the throat, coughing and chest constricting. Persons exposed can involuntarily gasp, come into apnoea shallow 17. The effects produced by those potent gases strongly dependant on the concetration of the compound and the exposure duration. It was reported in literature that these effects are generally transient and no long term sequelae 13. However cases of asthma aggravation have been reported and questions on the safety of tear gases used against persons with pre- existing lung disease have been raised 18. In addition, several fatalities, probably due to respiratory effects, have been reported after exposure to tear gas in confined space 19. The use of these chemical agents for self defence if their use are no longer well controlled, leads to search for safer agents. The screening of riot control agents for safety includes conventional acute, subacuteand chronic toxicity in order to identify any specificially vulnerable target organs esepially the respiratory organs 20.

In addition main studies are made with solvents which are not clasically used in tear gas sprays. It must be noted that OC was used with several solvents such as dichloromethane, trichlorethylene, tetrachlorethylene, which potentially are highly irritant to the respiratory system. Fatalities have been reported after tear gas bombs containing OC dissolved in one of these solvents. Nevertheless, involvement of OC could not be established 14.

Steffee et al.21 stated that intense pain in the conjunctiva, systematic physiological changes and intense disorder feeling were observed when OC was sprayed to mucose membrans. The behaviours of putting head under cage bedplace, gathering and dispersing to ease the pain were observed at the rats when they have been exposed to OC with different dosages because of redness in conjunctiva, diffuculty in breathing and burning feeling.

Dicpinigaitis et al.22 stated in a compiling on capsaicin that they evaluated a total of 4388 subjects undergoing capsaicin cough challenge testing. No serious adverse reaction to capsaicin inhalation challenge was reported. In the small fraction of studies that mentioned any adverse reactions to capsaicin inhalation, these were limited to minor complaints, most commonly transient throat irritation but not any long term side effects.

Any death or extreme abnormality in breathing were not obseved even spraying 72 g of OC into 0.5 m3 experiment cube. No death were seen in this consentration of OC in 15 min. No serious symptoms were observed even it was a closed enviroment. When subjects with sensory hyper-reactivity undergo capsaicin cough challenge testing, the same syptoms develop as happening from exposure to numerous environmental triggers, such as nasal and ocular irritation, rhinorrhea, hoarseness, phlegm production and dyspnea 22,23. One study specifically evaluating broncho-constriction after capsaicin inhalation demonstrated a dose-dependant fall that lasted less than 60 seconds 24.

In the present study, a relief was observed at the first few minutes in the rats exposed to OC with high dosage when we take them out to get their blood. In short, it was seen that possible side effects on trachea and lung were no longer existing.

Moreover, 109 of 122 studies, all of whom confirmed that there in fact had been no serious adverse effects associated with capsaicin inhalation 27.

Some previous animal and cell culture studies 4,28 have suggested that capsaicin may be mutagenic an/or tumorigenic. However, subsequent studies 27,28 have failed to demonstrate such capsaicin-induced effects have even demonstrated a chemoprotective effect of capsaicin against vinylcarbamate and N-nitrosodimethylamine induced mutagenesis and tumorigenesis 29.

A study 30 of 81 individuals presenting to an emergeny department after olearesin capsicum aerosol exposure found that none of the patients required hospitalization due to toxicity and pepper spray did not result in abnormal spirometry findings, hypoxemia, or hypoventilation 31.

In a study carried out to examine the potential harmful effects on corneal structure, innervation and sensitivty of a spray containing the capsaicin (OC), it was stated that OC causes immediate changes in mechanical and chemical


sensitivity that may persist for a week, a single exposure of OC appears harmless to corneal tissues 32. The same study also states that the changes are possibly associated with damage of corneal nevre terminals of mainly unmyelinated polymodal nociceptor fibers and shows that the structural and functional effects of OC spray on conjunctiva and cornea are mild and temporary. This interpretation does not apply to the solvents such as 92% trichloroethylene which can cause severe corneal damage 33. It was already known that isoprophyl alcohol used in OC does not cause any damage. The damage caused in eye appications of OC is mostly because of alcohol rather than the OC itself 34.

OC is an inflammatory agent that causes pain, erythema and edema. It is safer and more effective than other riot control agents and its effects on the eyes and skin are transient. When sprayed to eyes, it causes a burning sensation and blurring vision. Gasping and couhing are the main symptoms when inhaled. In another study, it was stated that there was no significant change in O2 and CO2 levels in vital capacity of lungs 35.

The pH and some other parameters must be in a limited change for the regular survival functions of life. In this study, pH value was 7.21 at the control group. But it was decreased between 7.10-70.84 in the OC exposed rats and showed decreasing tendency.

The pCO2 levels increased respectively to 56.00, 52.10, 59.00 mmHg in the 1st, 2nd and 3rd experiment group from 49.17 Increase in pCO2 takes place in hypoventilation and insufficient blood stream to lungs and insufficient clearence of CO2 from lungs. In this way, CO2 retention increases respectively pCO2 level and amount of carbonic acid. At the end pH in blood will decrease. pCO2 increase and pH decrease will stimulate ventilation and try to stop CO2 retention.

The increasing ventilation due to high pCO2 is not enough to normalize pCO2. If the pCO2 increases too much, respiration center cannot be stimulated and hypoxia starts to form. If it is not treated, hypoxia can form because acidosis and O2 will be diffused slowly. When the blood pCO2 increase is not too high, the respiration center will be sensitive to slightly pCO2 increase. The respiratoric asidosis answers this with a counter-ventilation. In the present study, respiratoric asidosis in the rats can be decided by looking at the pH and pCO2 changes. These two parameters differed paralel to controls and compatible with the respiratory acidosis classic literature 36-38.

Increase in bicarbonate level shows alcalosis and decrease shows acidosis. The normal blood bicarbonate levels are stated as 22-26 mEq/L. The actual bicarbonate is the real bicarbonate value in the blood. But the standart bicarbonate is normal pCO2 value and pO2 is the bicarbonate existing in blood. In the present study, the actHCO3 value of control group rats was 19.60 mmol/L. But it was between 17.02-14.91-13.11 mmol/L after exposed to OC. The pCO2

increase and decrease both in pH and HCO3 reinforces the acidoses symptoms.

K+ which is essential for celullar excitability is an important intracelullar cation. Its value in the extracelullar liquid is 3.5-5 mEq/L and 150 mEq/L in intracellular liquid. Majority of potassium dispersed mostly in muscle cells as well as erythrocyte and liver. K goes in cell in alcalosis but goes out of cell in acidosis. In the present study, serum K level was 4.57 mmol/L in control group but it increased in the experiment groups and reached 4.90-5.27 and 5.65 mmol/L levels. Serum K level is followed to monitor the K metabolism. In acidosis, collected H is taken inside the cell and K changes its place toward extracelullar liquid. K accumulates extracellularly parallel to this undesired change. In alcalosis, H ions go out of the cell and extracelullar K goes to intracellular compartment. K level decreases. Increasing extracellular K and decreasing intracelullar K levels incite the hyperkalemia. In hyper-kalemia, weak muscle, deep tendon reflex loses and seldomly skeleton muscle paralysis are seen 36.

The blood glucose level controlled by insulin, glucagon and adrenaline can be affected from hunger, stres, fear, excitement and diabetes 39. In the present study, the blood glucose level in control group was 142 mg/dL but it was 235-244 and 248 mg/dL in the experiment groups due to the OC exposure. However, statistical importance of this could not be determined. It is obvious that the reason of sharp change in glucose was the stimulation of glucose producing mechanism of blood in the course of adrenaline produced by fear and excitement in the rats under OC exposure. The other reason was also sharp increase of the blood glucose level because of the dispersion of muscle glycogen. This shows that pepper gas adversely affected the rats’ behaviors and metabolism.

As a result, no fatality was observed in the rats when they were kept inside the 0.5 m3 glass cube in fives and sprayed OC for 4, 8 and 12 sec durations in which the rats were respectively exposed to OC of 24, 48 and 72 g. All the rats in control group was observed with intensive redness in the eyes, conjunctiva and difficuly in breathing. In the analyses of blood gases, decrease in pH, increase in pCO2, decrease in HCO3 and increase in K support the respiratory acidosis formation but no fatality was obseved in any OC exposure group.

The pepper gas used often by police force to control the riot can be a safe agent due to the results obtained if the the person was not subject to asthma or chronic lung disease. It can be also stated that OC does not cause any fatality but sensitivity and burning in the eyes, conjunctivitis and coughing symptoms.

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22. Dicpinigaitis PV, Rakesh VA, Alva MD: Safety of capsaicin cough challenge testing. Chest, 128, 196-202, 2005.

23. Ternesten-Hasseus E, Farbrot A, Lowhagen O: Sensitivity to metha-choline and capsaicin in patients with unclear respiratory symptoms. Allergy, 57, 501-507, 2002.

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27. Muralidhara K, Narasimhamurthy K: Non-mutagenicity of capsaicin in albino mice. Food Chem Toxicol, 26, 955-958, 1998.

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29. Surh YJ, Lee RC, Park KK: Chemoprotective effects of capsaicin and diallylsulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-Nitrosodimethylamine. Carcinogenesis, 16, 2467-2471, 1995.

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31. Chan TC, Vilke GM, Clausen J, Schmith P, Snowden T, Neuman T: The effect of oleoresin capsicum “pepper spray” inhalation on respiratory function. JOFS, 47, 299-304, 2002.

32. Vesaluoma MH, Sankila EM, Juana G, Müller LJ, Petroll W, Moilanen AO, Forsius H, Tervo MT: Autosomal recessive cornea plana: In vivo corneal morphology and corneal sensitivity. IOVS, 41, 2120-2126, 2000.

33. Tervo T, Joo F, Huikuri K, Toth I, Palkama A: Fine structure of sensory nerves in the rat cornea: an experimental nevre degeneration study. Pain, 6, 57-70, 1979.

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38. Müsellim B: Arter kan gazları. In, Yıldırım N (Ed): Akciğer Fonksiyon Testleri. Fizyolojiden Klinik Uygulamaya. s. 209-221, Turgut Yayıncılık, Ankara, 2004.

39. Mert N: Veteriner Klinik Biyokimya. s. 1-50, Uludağ Üniv. Güç. Vak. Yayınları, No: 12, Ceylan Matbaacılık, 1996.


ÖzetBu çalışmada sığır etlerinde anabolik amaçlı kullanılan 17 β-östradiol, dietilstilbestrol ve zeranol hormon kalıntılarının belirlenmesi

amaçlandı. Bu çalışmada test materyali olarak kullanılan toplam 204 örnek (51 boyun, göğüs, but ve idrar örnekleri) Erzurum’daki mezbahalardan temin edildi. Ekstrakte edilen örneklerdeki hormon kalıntıları Enzyme-Linked Immunosorbent Assay (ELISA) ile belirlendi. Çalışmada, 16 et örneğinde 17 β-östradiol (4), dietilstilbestrol (4) ve zeranol (8) ≥0.5 ng/kg, diğer taraftan 6 idrar örneğinde dietilstilbestrol (4) ve zeranol (2) ≥0.5 ng/L düzeyinde bulundu. Bununla birlikte, örneklerdeki hormonların belirlenmesi ve konsantrasyonları bağımsız akredite labotaruvarda (TUBİTAK-MAM) LC-MS-MS ile doğrulandı. Sonuç olarak et ve et ürünlerinde hormon bulunması tüketiciler için zararlı olduğundan dolayı, Türkiye’de anabolik maddelerin kullanımı sıklıkla kontrol edilmelidir.

Anahtar sözcükler: 17 β-östradiol, Dietilstilbestrol, Zeranol, ELISA, Et

Investigation of Residual 17 β-estradiol, Diethylstilbestrol, and Zeranol in Red Meat Sold in Erzurum Province, Turkey

SummaryThe aim of this study was to detect hormone residues such as 17 β-estradiol, diethylstilbestrol, and zeranol used anabolic agents in

bovine meat. In the study, a total of 204 samples used as test materials (51 neck, thoracic, leg meat and urine samples) were obtained from local abattoirs in Erzurum. The hormone residues in the sample extracts were detected by Enzyme-Linked Immunosorbent Assay (ELISA). 17 β-estradiol (4), diethylstilbestrol (4), and zeranol (8) were detected in 16 meat samples at ≥0.5 ng/kg, on the other hand, diethylstilbestrol (4) and zeranol (2) were detected in 6 urine samples at ≥0.5 ng/L in this study. However, concentrations and identities of the hormones in the samples were confirmed by independent accredited laboratory (TUBITAK-MAM) via LC–MS–MS. In conclusion, since presence of hormones in meat and meat products is harmful for consumers, utilization of anabolic agents must be strictly controlled in Turkey.

Keywords: 17 β-estradiol, Diethylstilbestrol, Zeranol, ELISA, Meat

Erzurum Yöresinde Satışa Sunulan Kırmızı Etlerde 17 β-östradiol, Dietilstilbestrol ve Zeranol Kalıntılarının Araştırılması [1]

Emine SEVER * Biray OKUMUŞ * Sinan İNCE **[1]

***

Bu çalışma Tarımsal Araştırmalar Genel Müdürlüğü tarafından TAGEM/GY/03/01/175 proje numarasıyla desteklenmiştir T.C. Gıda, Tarım ve Hayvancılık Bakanlığı, Veteriner Kontrol Enstitüsü Müdürlüğü, TR-25070 Erzurum - TÜRKİYEAfyon Kocatepe Üniversitesi, Veteriner Fakültesi, Farmakoloji ve Toksikoloji Anabilim Dalı, TR-03200 Afyonkarahisar - TÜRKİYE


Ülkemiz de dahil olmak üzere bir çok ülkede hayvansal protein açığı vardır ve gün geçtikçe hızlı nüfus artışına bağlı olarak bu açık büyümektedir. Dünya nüfusundaki artışa paralel olarak, hayvancılık sektöründe hormonlar gibi geliş- meyi hızlandırıcı çok sayıda madde kullanılmaktadır. Böyle hayvanlardan elde edilen et ve diğer ürünlerin insan sağ-lığına zarar verebilecek miktarda kalıntı içermesi ciddi bir sorundur. Halen tüm dünyada, hayvanlarda kullanılan hor-

monlar ve sonrasında hayvanlardan elde edilen etlerdeki kalıntı düzeyleri tartışma konusu olmaktadır 1.

Androjenik-anabolik maddeler, vücutta azotun tutulma-sını sağlayarak protein sentezini artırmakta ve kemiklerde özellikle uzunlamasına büyümeyi hızlandırmaktadır. Bu etkileri ile anabolik maddeler hayvanlarda canlı ağırlık ka- zancını %10-25, yemden yaralanmayı %5-10 arasında art-

GİRİŞ

İletişim (Correspondence) +90 272 2281312/142 [email protected], [email protected]

RESEARCH ARTICLE

268Erzurum Yöresinde Satışa Sunulan ...

tırmaktadır 1. Günümüzde, hayvansal ürünlerde ilaç ve kimyasal madde kalıntılarının önüne geçmek amacıyla gerekli yasal düzenlemeler yapılmıştır. Fakat bu duruma ne kadar sağlıklı bir şekilde uyulduğu kuşkuludur. Yapılan deneysel araştırmalarda, hormon uygulamalarıyla beraber bazı has- talık olguları ile kanser, mutajetinite ve teratojenite gibi özel toksik etkilerin oranındaki artışların belirtilmesi bu var-sayımı güçlendirmektedir. Bu durum, giderek bilinçlenen, kaliteli ve sağlıklı ürün satın almak isteyen tüketicilerde kaygı uyandırmaktadır 2,3.

Bu türden kalıntı içeren besinleri tüketen insanların cinsiyet özelliklerinde değişiklikler yanında çeşitli doku ve organlarda tümör sıklığında artış olabileceği öngörülmek-tedir. Özellikle gebelik önleyici amaçla olmak üzere, uzun süreli kullanılma durumunda östrojenik maddeler, başta meme bezi, uterus, testis, böbrek, kemik ve diğer doku ve organlarda tümörlerin gelişmesine sebep olmaktadırlar. Ayrıca, anabolizan maddeleri ihtiva eden gıdaların uzun süreli alınmasının hormonal sistemi de olumsuz yönde etkilediği belirtilmektedir 4-6.

Ülkemizde kırmızı et ve sakatatlarda anabolizan kalın- tılarının araştırıldığı çalışmalar az sayıdadır. Bu çalışma ile Erzurum’da faaliyet gösteren ruhsatlandırılmış mezbahalardan kesim sonrası tüketime sunulan kırmızı et örnekle- rinde 17 β-östradiol, dietilstilbestrol ve zeranol kalıntıla-rının araştırılması ve “güvenli gıda sağlama” başlığı altında halk sağlığı açısından değerlendirmesi amaçlanmıştır.

MATERYAL ve METOT

Çalışmada kullanılan 17 β-östradiol, dietilstilbestrol ve zeranol standartları Sigma’dan, analizlerin yapılacağı Enzyme-Linked Immunosorbent Assay (ELISA) test kitleri de Ridascreen marka olarak temin edildi. Analizlerde kullanılan diğer kimyasal madde ve malzemeler Erzurum Veteriner Kontrol ve Araştırma Enstitü laboratuvarından sağlandı. Örneklerin okutulması ve kalıntı miktarlarının belirlenmesi amacıyla BIOTEK (ELx800) marka ELISA cihazı kullanıldı.

Çalışma materyali olarak kullanılan kırmızı et örnekleri,

2010 yılı Mayıs-Temmuz ayları arasında Erzurum’da faaliyet gösteren mezbahalara değişik zamanlarda gidilerek, 51 adet erkek büyükbaş hayvandan temin edildi. Kesim son-rasını takiben karkasların boyun, göğüs ve but kısımların- dan et örnekleri, aynı hayvanın idrar keselerinden idrar örnekleri steril numune kaplarına alındı. Alınan örnekler soğuk zincir altında en kısa süre içerisinde Erzurum Vete-riner Kontrol ve Araştırma Enstitüsü’ndeki laboratuvara ge-tirildi ve derin dondurucuda -20ºC’de işleneceği zamana kadar muhafaza edildi. Et ve idrar örneklerinin ekstraksiyon işlemleri Ridascreen test kitlerinde (R-biopharm; R3301) belirtilen yönteme göre yapıldı.

Çalışmada örneklerden elde edilen verilerin değerlen-dirilmesinde; örneklerin sayıları, bu sayılara karşılık gelen ortalama miktarları ve standart hataları ile minimum ve maksimum değerleri verildi.

BULGULAR

Kalıntı varlığının belirlenmesi amacıyla toplamda 204 adet olan; 51 adet idrar örneği dietilstilbestrol ve zeranol, her biri 51 adet olan boyun, göğüs ve but kırmızı et örnekleri ise 17 β-östradiol, dietilstilbestrol ve zeranol yönünden ELISA ile analiz edildi. 17 β-östradiol, dietilstilbestrol ve zeranolün ELISA ile elde edilen standart eğrileri sırasıyla Şekil 1A, Şekil 1B ve Şekil 1C’de verildi.

Kırmızı et örneklerine ait ELISA ile belirlenen 17 β- östradiol kalıntı miktarları Tablo 1’de verildi. Numunelerin analizinde bulunan 17 β-östradiol miktarlarının bütün et örneklerinde tespit edildiği ve numunelerin içerdiği 17 β-östradiol düzeylerinin en çok 100 ile 300 ng/kg arasında dağılım gösterdiği belirlendi.

Kırmızı etlerde tespit edilen dietilstilbestrol kalıntı miktarları incelendiğinde 4 hayvanın boyun ve 5 hay- vanın göğüs ve budunda 100 ile 200 ng/kg arasında, ayrıca 1 hayvanın boyun ve göğüs ile 2 hayvanın budunda 500 ng/kg üzerinde olduğu ELISA ile tespit edildi (Tablo 2). Zeranol miktarlarının kırmızı etlerde tespit edilen kalıntı miktarları ise Tablo 3’te verildi. ELISA ile yapılan analiz neticesinde boyun, göğüs ve but için sırasıyla 13, 16

Şekil 1. 17 β-östradiol (A); dietilstilbestrol (B) ve zeranol (C) standart eğri grafikleri

Fig 1. Standard curve graphics of 17 β-estradiol (A), diethylstilbestrol (B), and zeranol (C)

269SEVER, OKUMUŞ, İNCE

Tablo 1. Boyun, göğüs ve but örneklerinde ELISA ile bulunan 17 β-östradiol için numune sayısı, ortalama miktarları, minimum ve maksimum değerleri

Table 1. Numbers of sample, average, maximum and minimum values of 17 β-östradiol determinated by ELISA in neck, thoracic, and leg meats

Miktar (ng/kg)

Boyun Göğüs But

Numune Sayısı Ortalama ± SD Min-Max

DeğerlerNumune

Sayısı Ortalama ± SD Min-Max Değerler


Değerler

0-100 9 42.09±37.94 0-83.5 9 40.41±37.29 0-88.2 10 60.87±31.10 0-84

100-200 17 151.74±25.20 102.7-195 21 168.18±24.29 124.8-198.6 16 146.80±26.58 100.4-199.8

200-300 16 244.88±26.62 208.7-297.8 11 251.28±30.77 207.3-296.1 14 240.45±20.16 210-280.9

300-400 3 337.05±39.76 304.6-393 4 339.80±24.75 315.3-379.9 7 340.25±28.30 306.5-397.6

400-500 4 408.25±5.10 402-413.6 4 439.77±20.47 409-466.7 4 459.22±25.29 434.9-494.4

500- > 2 530.23 500.6-559.9 2 512.55 503.2-521.9 - - -

Tablo 2. Boyun, göğüs ve but örneklerinde ELISA ile bulunan dietilstilbestrol için numune sayısı, ortalama miktarları, minimum ve maksimum değerleri

Table 2. Numbers of sample, average, maximum and minimum values of diethylstilbestrol determinated by ELISA in neck, thoracic, and leg meats

Miktar (ng/kg)

Boyun Göğüs But


DeğerlerNumune



Değerler

0 46 - - 45 - - 44 - -

0-100 - - - - - - - - -

100-200 4 124.92±19.98 103.1-148.8 5 125.01±16.69 108.8-151.7 5 138.47±23.06 116.4-181.5

200-300 - - - - - - - - -

300-400 - - - - - - - - -

400-500 - - - - - - - - -

500- > 1 795.35 795.35 1 877.93 877.93 2 888.60 718.3-1058.8

Tablo 3. Boyun, göğüs ve but örneklerinde ELISA ile bulunan zeranol için numune sayısı, ortalama miktarları, minimum ve maksimum değerleri

Table 3. Numbers of sample, average, maximum and minimum values of zeranol determinated by ELISA in neck, thoracic, and leg meats

Miktar (ng/kg)

Boyun Göğüs But


DeğerlerNumune



Değerler

0 38 - - 35 - - 36 - -

0-100 - - - - - - - - -

100-200 - - - - - - - - -

200-300 6 261.20±38.32 203.1-296.8 8 239.65±21.12 209.9-276.3 6 235.13±29.66 201.4-293.3

300-400 4 342.81±23.15 325.4-382.7 3 336.72±17.54 316.2-359 4 332.93±14.07 312.7-347.5

400-500 1 403.62 403.62 2 430.52 417.7-443.3 2 426.67 422.7-430.6

500- > 2 551.46±14.67 536.8-566.1 3 554.21±35.12 505.2-585.7 3 581.82±80.07 519-694.8

Tablo 4. İdrar örneklerinde ELISA ile bulunan dietilstilbestrol ve zeranol için numune sayıları, ortalama miktarları, minimum ve maksimum değerleri

Table 4. Numbers of sample, average, maximum and minimum values of diethylstilbestrol and zeranol determinated by ELISA in urine

Miktar (ng/L)Dietilstilbestrol Zeranol

Numune Sayısı Ortalama ± SD Min-Max Değerler Numune Sayısı Ortalama ± SD Min-Max Değerler

0 - - - 26 - -

0-100 - - - - - -

100-200 - - - - - -

200-300 1 283.19 283.19 11 249.91±19.36 211.2-271.5

300-400 7 346.17±18.85 319.5-376 8 325.02±9.02 308.4-340.6

400-500 12 441.64±30.03 403.9-497.5 4 427.59±33.14 402.2-484.1

500- > 31 600.75±88.90 502.4-859.5 2 826.25±116.18 710-942.3


ve 15 et örneğinde tespit edilen düzeyin 200 ng/kg ve üzerinde olduğu gözlendi. Yine aynı yöntemle idrar örnek-lerinin yarıdan fazlasında dietilstilbestrol kalıntı miktarı- nın 500 ng/L üzerinde olduğu belirlendi. Bununla birlikte 25 idrar örneğinde zeranol kalıntısının belirlendiği, bu örneklerdeki kalıntı miktarının ise en çok 200 ile 400 ng/kg arasında dağılım gösterdiği saptandı (Tablo 4).

Bu elde edilen verilere ek olarak, TUBITAK-MAM’ın laboratuvarlarında kırmızı et ve idrar örnekleri 17 β- östradiol, dietilstilbestrol ve zeranol yönünden LC-MS- MS (Thermo Electron, USA) ile analiz edildi. Analizler neticesinde et ve idrar örneklerinde 17 β-östradiol, dietilstilbestrol ve zeranol düzeylerinin < 1 µg/kg olduğu rapor edildi. İdrar örneklerinin analizi neticesinde 17 β-östradiol, dietilstilbestrol ve zeranol kromotogram analiz grafikleri sırasıyla, Şekil 2A, Şekil 2B ve Şekil 2C’de verildi.

TARTIŞMA ve SONUÇ

Hayvan yetiştiriciliğinde anabolizan maddelerin bilinç- sizce kullanımı sonucunda et ve ürünlerinde kalıntıların bulunması tüketicilerin sağlığı açısından birçok sakınca- lar oluşturmaktadır 7-9. Avrupa Birliği, anabolik hormon-ların kasaplık hayvan yetiştiriciliğinde büyüme hızlandırıcı olarak kullanımını yasaklamış, Amerika Birleşik Devlet-leri Gıda ve Tarım İdaresi (FDA) ise doğal kökenli bazı hormonların (östradiol ve testosteron) hayvan yetiştiricili-ğinde kullanımına izin vermiştir 10. Ülkemizde, Türk Gıda Kodeksinde anabolizan amaçla kullanıma uygun maddelerin (zeranol gibi) gıda değeri olan hayvanlara uygulanması ilgili tebliğle yasaklanmıştır 11.

17 β-östradiol hayvanların tedavisinde (östrus senkro-nizasyonu) kullanılan östrojenik aktivite gösteren doğal steroid yapıda bir hormondur. Buna ilaveten yetiştiricilikte anabolik amaçlı büyümeyi ve karkas kalitesini artırmak içinde kullanılmaktadır 12,13. Bu çalışmada 150 adet et ör- neklerinde bulunan 17 β-östradiol miktarlarının sadece 4 ünün 0.5 µg/kg düzeyi aştığı, ayrıca örneklerin LC-

MS-MS ile analizinde de 17 β-östradiol düzeyinin < 1 µg/kg olduğu belirlendi. Etlerde GC-IT-MS ve GC-MS ile anabolik madde varlığının araştırıldığı çalışmalarda, 17 β- östradiolünde içinde bulunduğu diğer anabolik maddelerin etlerdeki oranlarının 0.1-0.4 µg/kg düzeyde olduğu 14,15, bu miktarlarında incelenen etlerde tespit edilen 17 β- östradiol miktarları ile aynı aralıkta olduğu gözlendi. Ayrı- ca, tespit edilen miktarların numune sayılarında farklılık göstermesi anabolizan maddelerin uygulama yerlerinin farklı olması, hayvanların yaşı ve metabolik aktiviteleri ile deneylerin analizleri sırasında oluşabilen hatalardan kaynak- lanmış olabileceğini akla getirmektedir. 17 β-östradiolün organizmada sentezlenen steroid yapıda bir hormon ol- ması, ayrıca ulusal ve uluslararası belirlenmiş bir maksimum kalıntı düzeyinin bulunmaması nedeniyle, bu çalışmada düşük düzeylerde bulunan bu miktarların tüketiciler için sağlık riski yaratmayacağı kanaatine varılmıştır.

Dietilstilbestrol gelişmeyi hızlandırıcı etkisi kuvvetli olan sentetik östrojenik bir bileşiktir. Karsinojenik etkiye sahip olmasından ve organizma tarafından metabolize ol- madığından anabolik amaçlı kullanımı yasaklanmış maddedir 16-18. Xu ve ark 18 anabolizan maddeler için yaptıkları yöntem belirleme ve örnek analizi sonuçlarında ELISA ve LC-MS-MS yöntemleriyle 6 tavuk eti örneğine 1 ve 2 µg/kg enjekte ettikleri dietilstilbestrolü sırasıyla ELISA örneğinde 0.67-1.45, LC-MS-MS ile 0.67-1.53 olarak bildirmektedirler. Çalışma sonunda her iki yönteminde yenilebilir dokularda dietilstilbestrol için kullanılabileceğini, immunoassay yön- teminin tarama ya da durum analizi yönünden değerlen-dirilebileceğini vurgulamışlardır. Bu çalışmada araştırılan boyun, göğüs ve but örneklerinden 4 örnekte 500 ng/kg (%2.7) üzerinde ve sadece but örneğindeki bir numunede düzeyin 1 µg/kg’ı aştığı, 14 örnekte 100-200 ng/kg (%8) arasında ve 135 örnekte (%89.3) ise dietilstilbestrol varlığı tespit edilmediği belirlendi. Ayrıca örneklerin LC-MS-MS ile analizinde de dietilstilbestrol düzeyinin < 1 µg/kg olduğu belirlendi. Nazlı ve ark.16 yaptıkları araştırmada, İstanbul’da satışa sunulan 60 adet et örneğinden 21 ade- dinde (%35) ≥10 µg/kg düzeyinde dietilstilbestrol tespit

Şekil 2. İdrar örneklerinde 17 β-östradiol (A), dietilstilbestrol (B) ve α-zearalenol (C) kromotogram analiz grafikleri

Fig 2. Chromatogram analysis graphics of 17 β-estradiol (A), diethylstilbestrol (B), and α-zearalenol (C) in urine samples

271SEVER, OKUMUŞ, İNCE

ettiklerini belirtmişler ve satışa sunulan taze etlerin insan sağlığı açısından riskli olduğunu vurgulamışlardır. Nazlı ve ark.’nın 16 elde ettikleri verilerle kıyaslandığında bu çalış-mada tespit edilen dietilstilbestrol varlığının daha az olduğu fakat bulunan düzeyler tüketime sunulan etlerin insan sağlığı açısından riskli konumda olduğunu da gös-termektedir.

Zeranol β-rezorsilik asit-lakton-steroid yapıda olmayan anabolik bir maddedir. Uygulama yeri ve zamanına bağlı olarak zeranol kas ve yağ dokusunda en düşük, safra, idrar ve dışkıda ise en yüksek miktarlarda bulunmaktadır 19. Dünya Sağlık ve Tarım Örgütünün ortak komitesi zeranol için hayvanların yenilebilir dokularında tolerans düzeyini 2 µg/kg olarak belirlemişlerdir. Nazlı ve ark.16 yaptıkları çalışmada, İstanbul piyasasında satılan 60 adet et örneğinin tamamında zeranol kalıntısı tespit etmişler ve 23 örneğin insan sağlığına risk oluşturacak miktarda yüksek olduğunu belirlemişlerdir. Bu çalışmada 44 adet et örneğinde (%29.3) zeranol miktarının 200-694 ng/kg arasında ve bulunan miktarların bildirilen tolerans düzeyinin de aşağısında olduğu belirlendi. Buna ilaveten, örneklerin LC-MS-MS ile analizinde zeranol düzeyinin < 1 µg/kg olduğu belirlendi.

Bu çalışmada idrar örneklerinin LC-MS-MS ile analizinde dietilstilbestrol, 17 β-östradiol ve zaranol düzeyinin < 1 µg/L olduğu belirlendi. ELISA ile incelenen idrar örnek-lerinin 25’inde (%49) zeranol ve hepsinde dietilstilbestrol kalıntısının belirlenmesi kasaplık için yetiştirilen hayvanlara illegal olarak hormon veya eşdeğeri madde verildiğini, bu düzeylerin 1 µg/L’nin altında olması ise yetiştiricilerin hay- vanlarını kesim öncesi süreye bağlı olarak kesime getir-diklerini göstermektedir. Bu sonuca benzer olarak, Türk ve Liman 20 yaptıkları çalışmada Kayseri Bölgesinde erkek besi sığırlarında incelenen 45 adet sığır idrarından 8’inde ELISA yöntemiyle 250-500 ng/L düzeyinde zeranol kalıntısı tespit etmişler ve çalışma sonunda parenteral yoldan bu hayvanlara zeranolun kullanıldığını fakat halk sağlığını tehdit edecek düzeyde olmadığını vurgulamışlardır.

Çalışmada ELISA yöntemiyle ng/L düzeyinde analiz ya- pıldığı, LC-MS-MS ile bunun µg/kg ile sınırlı olduğu göz-lenmiştir. ELISA yönteminin bu maddeler için iyi sonuç ve hızlı tarama imkanı verdiği fakat çapraz reaksiyon sonucu birçok madde ile de sonuç alındığı düşünüldüğünde yapı- lacak analizlerde ikinci bir yönteminde kullanılması gerekmektedir. Birçok araştırıcıda yaptıkları çalışmalar sonu-cunda etler için ELISA yöntemiyle alınan hormon ve ilaç kalıntı değerlerinin aynı zamanda ileri cihaz ve tekniklerle de doğrulanması gerekliliğini vurgulamışlardır 14,15,21,22.

Ülkemizde bu ve benzeri maddelerin tespiti ve kont- rolü amacıyla kalıntı izleme planı oluşturulmuştur. Ulusal Kalıntı Kontrol Planı, hayvansal orijinli gıdaların güvenli-ğinden emin olmak üzere yıllık bazda uygulanan program-lardır. Bu programların uygulamasında Avrupa Birliği mev-zuatı ile uyumlaştırması tamamlanmış Türk mevzuatı esas alınmaktadır. Ulusal kalıntı kontrolünde “Canlı Hayvanlar

ve Hayvansal Ürünlerde Belirli Maddeler İle Bunların Kalıntılarının İzlenmesi İçin Alınacak Önlemlere Dair Yönetmelik” (RG: 19.01.2005, No.25705) esas alınmaktadır. Kalıntı kontrol çalışmaları, bu yönetmelik ve yönetmeliğe bağlı “2006/05 sayılı Su ürünleri, Kanatlı Hayvan ve Etleri, Bal ve Çiğ Sütte Kalıntı İzleme Genelgesi”nde belirtilen kurallara göre yürütülmektedir 23.

Sonuç olarak; yapılan bu çalışma hayvanlarda hormon (dietilstilbestrol ve zeranol) veya benzeri maddelerin kul-lanıldığını, Erzurum ve bölgesi için etlerde tespit edilen düzeylerin, halk sağlığı açısından, kesim öncesi bekletme sürelerine uyulmadığı takdirde risk oluşturabileceğini gös-termektedir. Ayrıca, oluşabilecek tehlikeli bir durumun giderilebilmesi için bölgedeki yetiştiricilerin ve hekimlerin bilinçlendirilmesi ve düzenli bir şekilde kesim öncesi ve sonrasında hayvanlardan örnekler alınarak ilgili kurumlar tarafından hızlı ve kontrollü bir tarama ile tespit işlemlerinin titizlikle yapılması gerekliliği sonucuna varılmıştır.

KAYNAKLAR

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21. Sun MM, Zhao YW, Liang Y, Qian JR, Li LH, Wang SH: Determination of residual diethylstilbestrol in chicken by fluoroimmunoassay. Phys Test Chem Anal Chem Anal B, 5, 1-4, 2010.

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ÖzetBu çalışmanın amacı, Türkiye’nin farklı bölgelerinde yetiştirilen Holştayn sığırların süt ve döl verimi özelliklerinin belirlenmesi, bu

özellikler üzerine bazı çevresel faktörlerin etkilerinin araştırılmasıdır. Bu amaçla, Türkiye’nin farklı coğrafi bölgelerinde yetiştirilen Holştayn sığırların verim kayıtları, işletmelerin sağım sistemlerine bağlı sürü yönetimi programlarından dijital olarak alınmıştır. Ceylanpınar, Dalaman, Koçaş ve Tahirova Tarım İşletmelerinde 2001-2009 yılları arasında yetiştirilen toplam 3550 baş sığırın 7623 laktasyonuna ait toplam 18470 süt verimi kaydı ile 8091 döl verimi kaydı kullanılmıştır. Süt ve döl verimi özellikleri üzerine çevre faktörlerinin etkileri en küçük kareler metodu ile değerlendirilmiştir. En küçük kareler ortalamaları 100 günlük süt verimi için 2719.66±13.52, 200 günlük süt verimi için 5246.94±25.69, 305 günlük süt verimi için 7395.35±45.75 kg, ilk buzağılama yaşı için 809.32±2.07 gün, servis periyodu için 127.43±5.41 gün ve buzağılama aralığı için 395.86±2.58 gün olarak bulunmuştur. İncelenen tüm faktörlerin (işletme, buzağılama yılı, buzağılama mevsimi, buzağılama yaşı ve laktasyon sayısı) 100, 200 ve 305 günlük süt verimi üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). İşletme, buzağılama yılı ve buzağılama mevsiminin ilk buzağılama yaşı üzerine etkisi önemli bulunmuştur (P<0.01). Buzağılama yılının servis periyodu üzerine etkisi önemsiz bulunurken, işletme, buzağılama yaşı, buzağılama mevsimi ve laktasyon sayısının servis periyodu üzerine etkisi önemli bulunmuştur (P<0.05). Buzağılama aralığı üzerine incelenen tüm faktörlerin etkisi önemli bulunmuştur (P<0.001). Türkiye’nin farklı bölgelerinde yetiştirilen Holştayn sığırlar arasında süt ve döl verim özellikleri yönünden önemli farklar tespit edilmiştir. Bu farkların genetik faktörlerin yanında işletmeler arasındaki iklimsel ve yönetimsel farklılıklardan kaynaklandığı söylenebilir.

Anahtar sözcükler: Holştayn, Süt verimi, Döl verimi, Çevresel faktörler, Sağım sistemi, Sürü yönetimi

Some Milk Production and Reproductive Traits of Holstein Cattle Raised in Different Regions of Turkey

SummaryThe objective of this study was to detect milk production and reproductive traits of Holstein breed raised in different regions

of Turkey, to investigate effects of some environmental factors on these traits. For this purpose, production records were collected digitally from herd management programme of milking management systems of agricultural enterprises from Holstein cows raised in different geographical regions of Turkey. Total 8091 reproduction and 18470 milk yield records from 7623 lactation of 3550 cows raising in Ceylanpınar, Dalaman, Koçaş ve Tahirova agricultural enterprises were used. The Effects of environmental factors on milk and reproductive traits were determined by the least square methods. Least square means for 100 days milk yield 2719.66±13.52, 200 days milk yield 5246.94±25.69, 305 days milk yield 7395.35±45.75 kg, first calving age 809.32±2.07 days, open days 127.43±5.41 days, calving interval 395.86±2.58 days were found. The effects of all factors (enterprise, calving year, calving season, calving age and lactation number) were found significant on 100, 200, 305 days milk yield (P<0.001). The effects of enterprise, calving year and calving season were found significant on first calving age (P<0.01). While the effect of calving year was not found significant, the effects of enterprise, calving season, calving age and lactation number were found significant on open days (P<0.05). The effects of all factors were found significant on calving interval (P<0.001). Significant differences were found for the milk production and reproductive traits between Holstein cows raised in different geographical regions of Turkey. These differences were based on climatic and administrative differences between enterprises as well as genetic factors.

Keywords: Holstein, Milk yield, Reproductive traits, Environmental factors, Milking system, Herd management

Türkiye’de Farklı Bölgelerde Yetiştirilen Holştayn Sığırlarda Bazı Süt ve Döl Verimi Özellikleri

Murad GÜRSES * Metin BAYRAKTAR **

***

Fırat Üniversitesi Veteriner Fakültesi Genetik Anabilim Dalı, TR-23119 Elazığ - TÜRKİYEFırat Üniversitesi Veteriner Fakültesi Zootekni Anabilim Dalı, TR-23119 Elazığ - TÜRKİYE



RESEARCH ARTICLE

274Türkiye’de Farklı Bölgelerde ...

GİRİŞ

Türkiye, sığır varlığı bakımından dünyanın önde gelen ülkeleri arasında yer almasına rağmen, hayvan başına düşen verimler yönünden yeterli düzeyde değildir. Türkiye 2009 yılı verilerine göre 10.859.942 baş sığır varlığı ile dünyanın 21. ülkesi olmasına karşın, sağmal hayvan başına düşen yıllık 2802 kg süt verimi ile 8.000 kg’ın üzerinde süt verimine sahip İsrail, A.B.D., Güney Kore, Suudi Arabistan, Danimarka, İsveç, Kanada ve Finlandiya gibi ülkelerin oldukça gerisinde 58. sırada yer almaktadır 1.

Dünyada yıllık üretilen toplam 696.554.346 ton sütün %83.34’ü (580.481.508) sığırlardan elde edilirken Türkiye’de yıllık üretilen toplam 12.542.186 ton sütün %92.35’i (11.583.313) sığırlardan ve bu miktarın %82.11’i (10.298.863) de kültür ırk ve melezlerinden elde edilmektedir 1,2. Bu veriler ışığında ve Holştayn ırkının, Türkiye’de en yaygın yetiştirilen sütçü sığır ırkı olduğu göz önüne alındığında,

Holştayn ırkı sığırların süt verimi üzerine etkili genetik ve çevre faktörlerinin belirlenmesinin ekonomik önemi daha iyi anlaşılmaktadır.

Türkiye’de yetiştirilen Holştayn ırkı sığırların süt ve döl verim özelliklerini ortaya koymaya yönelik birçok araş- tırma yapılmıştır. Süt verim özellikleri üzerine yapılan araştırmalar Tablo 1’de, döl verim özellikleri üzerine yapılan araştırmalar Tablo 2’de özetlenmiştir. Süt verim özellikleri üzerine yapılan araştırmaların neredeyse tamamı ayda bir yapılan kontrol sağımlarından yararlanılarak, 305 gün- lük ve laktasyon süt veriminin tahmin edilmesi esasına dayanarak gerçekleştirilmiştir. Döl verim özelliklerinin belirlenmesi amacıyla yapılan çalışmalar da işletmelerin doğum, tohumlama, verim ve diğer kontrol defterleri esas alınarak gerçekleştirilmiştir.

Türkiye’de son 10 yıllık dönemde 100 baş ve üzeri modern süt sığırcılığı işletmelerinin sayısının giderek artması, yüksek kapasiteli ve bilgisayar destekli sağım sistemlerine

Tablo 1. Holştayn ırkı sığırların süt verim özellikleri üzerine yapılan araştırma sonuçları

Table 1. The results of studies on milk yield traits of holstein cattle breed

Bölge - İl İşletme Dönem 305 GSV LSV Kaynak

1 Marmara-Ege-Akdeniz ANAFI Projesi 1990-1993 - 6404.77 3

2 Marmara - İstanbul Özel 1987-1995 4455.25±55.2241 4556.64±60.767* 4

3 Marmara - İstanbul Özel 1987-1995 4530.17±57.8162 4625.04±62.705** 4

4 Marmara - İstanbul Özel 1987-1995 4275.90±36.439 4296.74±39.985 5

5 İç Anadolu-Aksaray Koçaş TİM 1988-1995 4784±27.35 4966±32.96 6

6 İç Anadolu-Ankara Polatlı TİM 1992-1995 4597.3±64.1 4859.4±61.8 7

8 Akdeniz-Muğla Dalaman TİM 1989-1996 7337.08±53.66 - 8

9 Kahramanmaraş Kahramanmaraş TİM <1997 4398 9

10 Karadeniz-Samsun Gelemen TİM 1982-1997 4564.8±42.04 4925.8±39.71 10

11 Karadeniz-Samsun Gelemen TİM 1982-1997 4171±31.3 - 11

12 Ege-Marmara (17 il) 1207 İşletme 1985-1999 5592±9.7 - 12

13 Akdeniz-Hatay Reyhanlı TİM 1987-1999 5085.5±1010.2 - 13

14 Akdeniz-Hatay Reyhanlı TİM 1990-1999 6208.42±69.39 6427.90±75.03 14

15 Marmara-Balıkesir Tahirova TİM 1990-1999 6170.85±67.06 6311.68±74.91 15

16 İç Anadolu-Aksaray Koçaş TİM 1994-2000 - 6400.3±85.15 16

17 Karadeniz-Amasya Gökhöyük TİM 1996-2002 6467.0±80.9 6273.0±100.4 17

18 Türkiye Geneli TDSYMB 1984-2004 6729.2±33.3 7160.6±33.0 18

19 Ege-Afyon Özel İşletme 2004 - 7057.705±170.743 19

20 İç Anadolu-Aksaray Koçaş TİM 1995-2005 - 6937.63±109.46 20

21 İç Anadolu-Ankara Bala TİM 1998-2005 - 7704.25±111.90 21

22 Akdeniz-Burdur Burdur DSYB 2000-2005 5735.67±70.49 - 22

23 İç Anadolu -Ankara Lalahan HMAE 1990-2006 - 5969.75±255.24 23

24 İç Anadolu -Ankara Polatlı TİM 1993-2006 5606.92±75.49 - 24

25 İç Anadolu -Ankara Polatlı TİM 1997-2006 6976.1±48.8 7473.4±59.6 25

26 Ege-Afyon Afyon DSYB 2005-2006 6884.111±162.880 - 26

27 Marmara-Balıkesir Tahirova TİM 2000-2007 6810.14±56.38 7574.39±55.22 27

28 Karadeniz -Tokat Kazova TİM 2003-2007 7460.5±142.99 7518.9±140.12 28

29 Ege-Aydın Aydın DSYB 1998-2008 6379.41 7142.85 29

305 GSV: 305 Günlük Süt Verimi, LSV: Laktasyon Süt Verimi, TİM: Tarım İşletmesi Müdürlüğü, DSYB: Damızlık Sığır Yetiştiricileri Birliği, HMAE: Hayvancılık Merkez Araştırma Enstitüsü, * Almanya Orjinli, ** Türkiye Orjinli

275GÜRSES, BAYRAKTAR

geçişi de beraberinde getirmiştir. Modern sağım sistemleri ve sürü yönetimi programlarının kullanılmasıyla birlikte hayvanların bireysel olarak takip edilmesi ve dijital olarak kayıt altına alınması mümkün hale gelmiştir. Hayvanların pedigri, tohumlama, doğum ve veteriner işlemleri kayıtla- rıyla birlikte süt verim kayıtları da gerçek zamanlı olarak kayıt altına alınmaya başlanmıştır. Kuşkusuz bu sistemler- den elde edilecek gerçek veriler, aylık kontrol sağımlarının tahminlenmesiyle elde edilen verilere göre damızlık değe-rinin daha doğru tespit edilmesine olanak sağlayacaktır.

Bu çalışma ile Türkiye’nin farklı bölgelerinde yetiştirilen Holştayn ırkı sığırlara ait bazı süt ve döl verimi özelliklerinin belirlenmesi, bu özellikler üzerine çevre faktörlerinin etkilerinin ortaya çıkartılması ve elde edilecek sonuçlar doğrul-tusunda genetik ve çevresel faktörler yönünden uygun sürü yönetimi stratejisinin tespit edilmesi amaçlanmıştır.

MATERYAL ve METOT

Materyal

Araştırma materyalini, TİGEM’e bağlı Ceylanpınar, Dalaman, Koçaş ve Tahirova Tarım İşletmelerinde yetiştiri-

len toplam 3550 baş Holştayn inek ile bu hayvanların 2001-2009 yılları arasında 7623 laktasyonuna ait toplam 18470 süt verimi kaydı ile 8091 döl verimi kaydı oluş-turmuştur. Verilerin işletmelere göre dağılımı Tablo 3’te sunulmuştur.

Metot

Çalışma kapsamında, Türkiye’nin farklı bölgelerinde yer alan Holştayn ırkı sığırların yetiştirildiği işletmeler seçil- miştir. İşletmeler arasındaki çevresel faktörlerin eliminas-yonu açısından aynı merkez tarafından yönetilen ve aynı sağım sistemi ve sürü yönetimi programını kullanan işletmeler tercih edilmiştir. Bu amaçla, TİGEM’e bağlı Güney Doğu Anadolu Bölgesi’nden Ceylanpınar, Akdeniz Bölgesi’nden Dalaman, İç Anadolu Bölgesi’nden Koçaş ve Marmara Bölgesi’nden Tahirova Tarım İşletmesi çalışma kapsamına alınmıştır.

Çalışma kapsamında toplanan tüm veriler, direk işlet- melerin bilgisayar destekli sağım sistemlerine (Westfalia Surge) bağlı sürü yönetimi programlarından (Dairy Plan C21) özel bir bilgisayar yazılımı yardımıyla dijital olarak alınmıştır. Süt verimi özelliklerinin belirlenmesinde, sürü

Tablo 2. Holştayn sığırların döl verim özellikleri üzerine yapılan araştırma sonuçları

Table 2. The results of studies on reproductive traits of holstein cattle breed

Bölge - İl İşletme Dönem İBY SP BA Kaynak

1 Marmara-Ege-Akdeniz ANAFI Projesi 1990-1993 - - 418.86 3

2 Marmara - İstanbul Özel 1987-1995 30.10±0.257* 87.86±2.601 363.96±2.763 5

3 İç Anadolu - Aksaray Koçaş TİM 1988-1995 27.70±0.11* 93.33±1.57 12.30±0.05* 30

4 Karadeniz-Samsun Gelemen TİM 1982-1997 30.6±0.15* 110.2±3.40 388.5±3.39 10

5 Karadeniz-Samsun Gelemen TİM 1982-1997 - - 398±2.16 11

6 Akdeniz- Kahramanmaraş Kahramanmaraş TİM <1997 860 - 390 9

8 Akdeniz - Hatay Reyhanlı TİM 1993-1998 892.12±116.98 103.39±13.82 394.01±72.24 14

9 Ege-Marmara (17 il) 1207 işletme 1985-1999 28.4±0.04* 121±4.56 401±0.59 12

10 Akdeniz - Hatay Reyhanlı TİM 1987-1999 - 112.0±61.36 381.5±64.29 13

11 Marmara - Balıkesir Tahirova TİM 1990-1999 782.24±56.59 120.31±38.19 397.39±38.17 31

12 İç Anadolu - Aksaray Koçaş TİM 1994-2000 830.6±4.72 109.7±2.55 389.3±2.92 16

13 Doğu Anadolu-Erzurum DATAE 1995-2001 936.7±33.2 119.9±6.6 402.4±7.1 32

14 Karadeniz - Amasya Gökhöyük TİM 1996-2002 827.4±4.5 122.4±6.0 393.4±5.1 33

15 Marmara-Kırklareli Sarımsaklı TİM 1983-2003 28.15±2.50* - 407.07±78.59 34

16 Ege-Aydın Özel 1994-2003 - 114.5±1.7 394.9±1.9 35

17 Türkiye Geneli TDSYMB 1984-2004 845.8±6.6 125.6±3.3 - 18

18 İç Anadolu -Aksaray Koçaş TİM 1997-2005 855.43±2.66 110.57±6.28 382.30±6.42 20

19 İç Anadolu -Ankara Bala TİM 1998-2005 826.21±4.91 100.68±4.31 401.86±4.49 21

20 Akdeniz - Burdur Burdur DSYB 2000-2005 842.79±8.54 124.37±3.32 398.47±2.94 22

21 İç Anadolu -Ankara Lalahan HMAE 1990-2006 869.01±13.53 - 437.58±10.81 23

22 İç Anadolu -Ankara Polatlı TİM 1997-2006 - 149.60±4.355 427.88±4.36 36

23 İç Anadolu-Ankara Polatlı TİM 1997-2006 823.9±6.07 135.8±3.96 411.2±2.23 25

24 Ege-Afyon Bolvadin DSYB 2005-2006 26.17±0.22* 146.519±10.030 424.804±9.898 26

25 Ege-Aydın Aydın DSYB 1998-2008 861.99±113.20 102.25±54.47 413.78±74.29 29

İBY: İlk Buzağılama Yaşı, SP: Servis Periyodu, BA: Buzağılama Aralığı, TİM: Tarım İşletmesi Müdürlüğü, DSYB: Damızlık Sığır Yetiştiricileri Birliği, HMAE: Hayvancılık Merkez Araştırma Enstitüsü, * Buzağılama Aralığı Ay


yönetim programında en az 100 günlük süt verim kaydı mevcut tüm sağlıklı hayvanlar değerlendirmeye alınmıştır.

Süt ve döl verimi özellikleri üzerine çevre faktörlerinin (işletme, buzağılama yılı, buzağılama mevsimi, buzağıla- ma yaşı, laktasyon sayısı) etkileri En Küçük Kareler Metodu kullanılarak belirlenmiştir. Kullanılan matematiksel model; Yijklm = µ+ai+bj+ck+dl+fm+eijklm şeklinde olup, bu modelde yer alan terimlerden Yijklm = herhangi bir hayvanın incelenen verim özelliği değerini, µ: popülasyonun beklenen ortalamasını, ai: işletmenin etkisini (i: 1-4; Ceylanpınar, Dalaman, Koçaş, Tahirova), bj: buzağılama yılının etkisini ( j: 1-6; 2001/02/03, 2004, 2005, 2006, 2007, 2008/09), ck: buzağılama mevsiminin etkisini (k: 1-4; kış, ilkbahar, yaz, sonbahar), dl: buzağılama yaşının etkisini (l: 1-6; 2, 3, 4, 5, 6, 7+), fm: laktasyon sırasının etkisini (m: 1-6; 1, 2, 3, 4, 5, 6+), eijklm = incelenen faktörler dışındaki faktörlerin etki miktarını (hata terimi) temsil etmektedir. İstatistiksel analizler Minitab® 16.1.1 paket programı kullanılarak ger- çekleştirilmiştir. İstatistiksel olarak önemli bulunan para-metrelerde Tukey çoklu karşılaştırma testi kullanılarak alt gruplar karşılaştırılmıştır 37.

BULGULAR

Ceylanpınar, Dalaman, Koçaş ve Tahirova Tarım İşlet- melerinde yetiştirilen Holştayn sığırların 100, 200 ve 305 günlük süt verimine ait en küçük kareler ortalamaları Tablo 4’te, ilk buzağılama yaşı, servis periyodu ve buzağılama aralığı özelliklerine ait en küçük kareler ortalamaları Tablo 5’te verilmiştir.

TARTIŞMA ve SONUÇ

Süt Verimi Özellikleri

Türkiye’nin farklı bölgelerinde yetiştirilen Holştayn sığır-ların 100 günlük süt verimi ortalamaları 2719.66±13.52, 200 günlük süt verimi ortalamaları 5246.94±25.69, 305

günlük süt verimi ortalamaları 7395.35±45.75 kg olarak bulunmuştur. Bu çalışma ile Türkiye’de sığırların 100 ve 200 günlük süt verimleri ilk kez belirlenirken, 305 günlük süt verimi 6.000 kg’ın altında 4-7,10-13,22,24 ve 6.000-7.000 kg arasında 14,15,17,18,25-27,29 bildirilen araştırmalardan yüksek, 7460.5±142.99 olarak bildirilen araştırmaya yakın bulun-muştur 28.

İşletmenin 100, 200 ve 305 günlük süt verimi üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde Parlak 26 işletmenin 305 günlük süt verimine etkisini önemli bulmuştur. Toksoy 19 ise işletmenin lak-tasyon süt verimi üzerine etkisini önemsiz bulmuştur. İşletmelerin 100, 200 ve 305 günlük süt verimi yönünden Koçaş>Tahirova>Dalaman>Ceylanpınar şeklinde sıralan-dığı tespit edilmiştir. İşletmeler arasındaki farklar, genetik ve çevresel faktörlerden kaynaklanmaktadır. İşletmelerin farklı boğa sperması ve damızlık materyal kullanması genetik farklılıkların temelini oluşturmaktadır. Çalışma sonucunda 100, 200 ve 305 günlük süt verimi yönünden Koçaş ve Tahirova İşletmelerinin Dalaman ve Ceylanpınar işletmelerine göre daha yüksek süt verimi ortalamalarına sahip olduğu görülmüştür. Bu durumun, Akdeniz ve Güneydoğu Anadolu Bölgelerinde yer alan Dalaman ve Ceylanpınar işletmelerinin mevsim koşullarının İç Anadolu ve Marmara Bölgelerinde yer alan Koçaş ve Tahirova işletmelerine göre daha sıcak geçmesinden kaynaklandığı düşünülmektedir. 305 günlük süt verimi yönünden işlet- meler arasındaki fark göz önüne alındığında, en yüksek verim ortalamasına (8078 kg) sahip Koçaş Tarım işlet-mesinde genetik yapının ve uygulanan sürü yönetiminin daha iyi olduğu da söylenebilir.

Buzağılama yılının 100, 200 ve 305 günlük süt verimi üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde birçok araştırmacı buzağılama yılının 305 günlük süt verimi 8,9,13,24, laktasyon süt verimi 16,23 ve hem 305 hem de laktasyon süt verimi 4-7,10,14,15,17,18,25,27,28

üzerine etkisinin önemli olduğunu bildirirken, bunların aksine bazı araştırmacılar 305 günlük süt verimi 22,26

Tablo 3. Verilerin işletmelere göre dağılımı

Table 3. Distribution of data for enterprises

İşletme Ceylanpınar Dalaman Koçaş Tahirova Toplam

Hayvan Sayısı 1506 641 849 554 3550

Laktasyon Kaydı 2593 1548 2078 1404 7623

Süt Verim Kaydı 6029 3866 5060 3515 18470

100 Gün 2593 1548 2078 1404 7623

200 Gün 2111 1384 1880 1290 6665

305 Gün 1325 934 1102 821 4182

Döl Verim Kaydı 2536 1774 2350 1431 8091

İBY 848 613 792 421 2674

SP 625 298 399 200 1522

BA 1063 863 1159 810 3895

İBY: İlk Buzağılama Yaşı, SP: Servis Periyodu, BA: Buzağılama Aralığı


ve laktasyon süt verimi 21 üzerine etkisini önemsiz bul-muşlardır.

Buzağılama mevsiminin 100, 200 ve 305 günlük süt ve-rimi üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde birçok araştırmacı 305 günlük süt verimi 8,9,13,15,22,24, laktasyon süt verimi 19-21, hem 305 hem de laktasyon süt verimi 4,17,18,25,28,38 üzerine buzağılama mevsiminin etkisini önemli bulmuşlardır. Ayrıca bazı araştır- macılar buzağılama ayının 305 günlük süt verimine 3, bazı- ları da hem 305 hem de laktasyon süt verimine 10 etkisinin önemli olduğunu bildirmişlerdir. Bunların aksine bazı araş- tırmacılar 305 günlük süt verimi 26, laktasyon süt verimi 23 ve hem 305 hem de laktasyon süt verimine 5-7,14,27 buza- ğılama mevsiminin etkisin önemsiz olduğunu belirt- mişlerdir. Buzağılama mevsiminin 100 günlük süt verimi

yönünden kış>ilkbahar>sonbahar>yaz, 200 ve 305 günlük süt verimi yönünden kış>sonbahar>ilkbahar>yaz şeklinde sıralandığı tespit edilmiştir.

Buzağılama yaşının 100, 200 ve 305 günlük süt verimi üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde buzağılama yaşının 305 günlük süt verimi 24,26, laktasyon süt verimi 27, hem 305 hem de laktasyon süt verimi 4,5,28 üzerine etkisini önemli bulan araştırmalar mevcuttur. Bunların aksine bazı araştırmacılar buzağılama yaşının 305 günlük süt verimi 22, laktasyon süt verimi 16,19 ve hem 305 hem de laktasyon süt verimi 14,15

üzerine etkisinin önemsiz olduğunu bildirmişlerdir.

Laktasyon sayısının 100, 200 ve 305 günlük süt verimi üzerine etkisi farklı düzeylerde (P<0.01 ve P<0.001) önemli

Tablo 4. Süt verimi özelliklerine ait en küçük kareler ortalamaları (X± Sx)

Table 4. Least square means for milk yield traits (X± Sx)

İşletme n100 Gün

n200 Gün

n305 Gün

*** *** ***

Ceylanpınar 2593 2492±18.88c 2111 4854±36.69d 1325 6853±64.77c

Dalaman 1548 2610±20.32b 1384 4966±38.52c 934 6894±66.15c

Koçaş 2078 2896±18.76a 1880 5661±35.50a 1102 8078±63.42a

Tahirova 1404 2880±20.10a 1290 5506±37.90b 821 7757±66.48b

Buzağılama Yılı *** *** ***

2001/02/03 301 2717±40.24cd 298 5232±73.00bc 192 7382±125.93ab

2004 356 2910±37.10a 355 5535±67.17a 229 7718±115.43a

2005 789 2531±25.54e 776 5029±46.72c 522 7134±78.50b

2006 1787 2632±18.36d 1667 5078±34.81c 1176 7167±59.09b

2007 2092 2729±17.16bc 2024 5271±32.10b 1436 7519±54.21a

2008/09 2298 2799±16.73ab 1545 5337±36.42ab 627 7452±78.32a

Buzağılama Mevsimi *** *** ***

Kış 2105 2849±18.20a 1869 5516±34.70a 1304 7578±58.50a

İlkbahar 1794 2783±19.90b 1718 5207±37.09b 1161 7320±63.85b

Yaz 1767 2566±19.69d 1661 4985±36.90c 838 7158±69.59b

Sonbahar 1957 2682±18.71c 1417 5279±38.08b 879 7526±68.43a

Buzağılama Yaşı *** *** **

2 2459 2516±50.55b 2321 4809±97.42c 1586 6914±170.16b

3 1696 2693±37.65a 1505 5107±73.44b 922 7246±128.77b

4 1327 2817±32.87a 1114 5360±64.38ab 659 7641±113.00a

5 901 2822±32.25a 739 5430±63.42a 442 7541±112.01ab

6 540 2779±38.63a 433 5397±75.99ab 271 7620±132.75ab

7 + 700 2692±49.43ab 553 5379±99.14ab 302 7410±172.85ab

Laktasyon No *** *** ***

1 2677 2623±46.53bc 2506 5316±89.33bc 1707 7691±155.03ab

2 2004 2889±31.77a 1741 5640±61.77a 1061 7931±107.81a

3 1277 2812±28.15a 1080 5454±55.04ab 630 7590±97.30ab

4 784 2755±33.36ab 630 5243±65.64bc 384 7274±115.48bc

5 447 2697±49.09ab 369 5049±97.12cd 222 7114±169.89bc

6 + 434 2542±61.25c 339 4780±123.41c 178 6772±219.39c

Genel Ortalama 7623 2719.66±13.52 6665 5246.94±25.69 4182 7395.35±45.75

* P<0.05, ** P<0.01, *** P<0.001, a-d: aynı sütunda farklı harflerle gösterilen grup ortalamaları arasındaki farklar önemlidir (P<0.05)


bulunmuştur. Bu sonuçların aksine bazı araştırmalarda laktasyon sayısının 305 günlük süt verimi 26, laktasyon süt verimi 16,19,21, hem 305 hem de laktasyon süt verimi 7,14 üze- rine etkisini önemsiz bulunurken, bu çalışma sonuçları ile benzer birçok araştırmada laktasyon sayısının 305 günlük süt verimi 8,9,13,22,24, laktasyon süt verimi 20,23,39,40 ve hem 305 hem de laktasyon süt verimi 6,10,15,17,18,25,27,28 üzerine etkisini önemli bulunmuştur. Laktasyon sayısının 100 günlük süt verimi yönünden 2>3>4>5>1>6+, 200 günlük süt verimi yönünden 2>3>1>4>5>6+, 305 günlük süt verimi yönün-den 2>1>3>4>5>6+ şeklinde sıralandığı tespit edilmiştir.

Döl Verimi Özellikleri

Çalışma sonucunda Holştayn sığırların döl verimi özel- liklerinden ilk buzağılama yaşı 809.32±2.07 gün, servis peri-

yodu 127.43±5.41 gün ve buzağılama aralığı 395.86±2.58 gün olarak bulunmuştur.

Çalışmayla 809.32±2.07 gün veya başka bir ifade ile yaklaşık 27 ay olarak bulunan ilk buzağılama yaşı, 840 gün veya 28 aydan daha yüksek bulunan araştırmalar- dan 5,9,10,12,14,18,20,22,23,29,32,34 daha düşük, 782.24±56.59 gün olarak bulunan araştırmadan 31 daha yüksek, 820-830 gün ve 26-27 ay olarak bulunan araştırmalara 16,21,25,26,30,33 yakın bulunmuştur. Çalışma sonucunda ilk buzağılama yaşının birçok araştırmadan daha düşük bulunmasının güney bölgelerde yer alan Ceylanpınar ve Dalaman işlet- melerinden kaynaklandığı, bu bölgelerdeki ortalama hava sıcaklığının Türkiye’nin birçok bölgesinden daha yüksek olması nedeniyle hayvanların daha erken pubertaya ulaş-tıkları düşünülmektedir.

Tablo 5. Döl verimi özelliklerine ait en küçük kareler ortalamaları (X± Sx)

Table 5. Least square means for reproductive traits (X± Sx)

İşletme nİBY

nSP

nBA

*** *** ***

Ceylanpınar 848 794.2±3.40b 625 129.6±5.49a 1063 404.1±3.69a

Dalaman 613 786.0±3.37b 298 131.8±6.04a 863 397.2±3.38ab

Koçaş 792 833.8±3.04a 399 116.9±5.91b 1159 390.1±3.23b

Tahirova 421 823.3±4.04a 200 131.5±5.88a 810 392.1±3.49b

Buzağılama Yılı *** ÖD ***

2001/02/03 122 811.2±7.24ab 58 370.1±10.45c

2004 125 810.0±7.13ab 161 393.0±6.40bc

2005 315 799.3±4.50b 5 147.1±19.74 335 395.5±4.52bc

2006 664 801.3±3.20b 57 117.6±6.01 606 398.6±3.47bc

2007 766 811.8±2.93ab 83 127.7±5.11 1209 403.6±2.58b

2008/09 682 822.3±3.08a 1377 117.3±1.80 1526 414.4±2.47a

Buzağılama Mevsimi ** *** ***

Kış 746 805.7±3.16b 236 113.1±6.02c 1035 386.4±3.31b

İlkbahar 776 802.5±3.27b 224 143.4±6.05a 793 394.6±3.58ab

Yaz 556 812.0±3.67ab 465 132.4±5.66b 967 402.4±3.44a

Sonbahar 596 817.2±3.48a 597 120.9±5.56c 1100 400.1±3.26a

Buzağılama Yaşı * ***

2 326 111.1±8.93b

3 321 117.4±7.21b 1099 329.3±6.01e

4 304 125.7±6.69ab 1074 348.4±5.03d

5 231 139.1±6.61a 731 411.2±4.53c

6 141 135.1±7.19ab 429 471.9±4.98b

7 + 199 136.1±8.45ab 562 518.5±6.07a

Laktasyon No * ***

1 384 147.8±8.51a

2 416 139.2±6.88ab 1545 547.4±5.19a

3 291 122.2±6.49bc 1018 439.2±4.12b

4 200 119.0±6.88bc 638 371.7±4.24c

5 98 118.3±8.20c 356 324.8±6.14d

6 + 133 118.2±9.24c 338 296.1±7.70e

Genel Ortalama 2674 809.32±2.07 1522 127.43±5.41 3895 395.86±2.58

* P<0.05, ** P<0.01, *** P<0.001, ÖD: Önemli değil (P>0.05), a-e: aynı sütunda farklı harflerle gösterilen grup ortalamaları arasındaki farklar önemlidir (P<0.05)


Çalışmayla 127.43±5.41 gün olarak bulunan servis periyodu 115 günün altında bulunan araştırmalardan 5,10,13,14,

16,20,21,29,30,35 yüksek, 140 günün üzerinde bulunan araştırma- lardan 26,36 düşük, 115-135 gün arasında bulunan araş- tırmalara 12,18,22,25,31-33 yakın bulunmuştur. Servis periyo-dunun uterusun involüsyonu için gerekli 60-90 günlük süreden daha yüksek bulunması, işletmelerin kızgınlık ve tohumlama zamanının takibi konusunda problem yaşadıklarını düşündürmektedir.

Çalışmayla 395.86±2.58 gün olarak bulunan buzağıla- ma aralığı 405 günün üzerinde bulunan araştırmalardan 3,23,25,26,29,34,36 daha düşük, 380 günün altında bulunan araştır-madan 5 daha yüksek, 380-405 gün arasında bulunan araş-tırmalara 9-14,16,20-22,31-33,35 ise yakın bulunmuştur.

İşletmenin ilk buzağılama yaşı, servis periyodu ve buzağılama aralığı üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). İşletmenin ilk buzağılama yaşı üze- rine etkisi ilk kez bu çalışma ile belirlenmiştir. Parlak 26

bu çalışma sonuçlarıyla benzer şekilde işletmenin servis periyodu üzerine etkisini önemli bulurken, çalışma sonuç- larından farklı olarak buzağılama aralığı üzerine etkisini önemsiz bulmuştur. İlk buzağılama yaşı yönünden işlet- melerin Koçaş>Tahirova>Ceylanpınar>Dalaman, servis periyodu yönünden Tahirova>Dalaman>Ceylanpınar>Koçaş, buzağılama aralığı yönünden Ceylanpınar>Dalaman> Koçaş>Tahirova şeklinde sıralandığı tespit edilmiştir. İlk buzağılama yaşının güney bölgelerde yer alan işletmelerde (Dalaman ve Ceylanpınar) daha düşük bulunmuştur. Bu durumun sıcak iklim şartları nedeniyle hayvanların daha erken pubertaya ulaşması ve dolayısıyla daha erken to- humlanmasından kaynakladığı düşünülmektedir. Koçaş işletmesinin servis periyodu ve buzağılama aralığı süresinin diğer işletmelere göre daha düşük bulunması, bu işletmede daha iyi bir sürü yönetimi olduğunu göstermektedir.

Buzağılama yılının, servis periyodu üzerine etkisi önemsiz bulunurken, ilk buzağılama yaşı ve buzağılama aralığı üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde birçok araştırmacı buzağılama yılının ilk buzağılama yaşı 18,30, buzağılama aralığı 9,10,13,32,33,36

ve hem ilk buzağılama yaşı hem de buzağılama aralığı 25,34

üzerine etkisini önemli bulmuşlardır. Bunlardan faklı olarak bazı araştırmacılar buzağılama yılının ilk buzağılama yaşı 32,33 ve buzağılama aralığı 22,26,30,31,35 üzerine etkisini önemsiz bulmuşlardır. Çalışma bulguları ile benzer şekilde buzağılama yılının servis periyodu üzerine etkisi önemsiz bulan araştırmaların 16,22,26,30,31,33,35 yanı sıra önemli bulan araştırmalar 5,9,10,13,18,25,32,36 da mevcuttur.

Buzağılama mevsiminin, ilk buzağılama yaşı (P<0.01), servis periyodu ve buzağılama aralığı üzerine etkisi yüksek düzeyde önemli bulunmuştur (P<0.001). Benzer şekilde buzağılama mevsiminin ilk buzağılama yaşı 5,18,33, servis periyodu 5,10,13,16,18,21,25,32,36 ve buzağılama aralığı 3,5,10,25,26,32,34,36

üzerine etkisini önemli bulan araştırmaların yanında buza- ğılama mevsiminin, ilk buzağılama yaşı 16,21,23,25,30,32,34, servis

periyodu 9,20,22,26,30,31,33,35 ve buzağılama aralığı 9,13,16,20-23,30,31,33,35 üzerine etkisini önemsiz bulan araştırmalar da mevcuttur.

Buzağılama yaşının, servis periyodu (P<0.05) ve buza- ğılama aralığı (P<0.001) üzerine etkisi önemli bulunmuştur. Bu çalışma ile benzer şekilde buzağılama yaşının, servis periyodu ve buzağılama aralığı üzerine etkisini önemli bulan araştırmaların 31,36 yanında buzağılama yaşının, servis periyodu ve buzağılama aralığı üzerine etkisi önemsiz bulan araştırmalar da mevcuttur 16,22,26. Buzağılama yaşının servis periyodu yönünden 5>(7+)>6>4>3 ve buzağılama aralığı yönünden (7+)>6>5>4>3 şeklinde sıralandığı tespit edilmiştir. Genel olarak, buzağılama yaşı arttıkça servis periyodu ve buzağılama aralığının da arttığı söylenebilir.

Laktasyon sayısının servis periyodu (P<0.01) ve buza-ğılama aralığı (P<0.001) üzerine etkisi önemli bulunmuştur. Benzer şekilde laktasyon sayısının, servis periyodu 16,18,25,31 ve buzağılama aralığı 25,31 üzerine etkisini önemli bulan çalışmalardan farklı olarak laktasyon sayısının, servis peri-yodu 9,10,13,20-22,26,30,32,33,35 ve buzağılama aralığı 3,9,10,13,16,20-

23,26,30,32-35 üzerine etkisi önemsiz bulan araştırmalar da mevcuttur. laktasyon sayısının, servis periyodu yönün- den 1>2>3>4>5>(6+) ve buzağılama aralığı yönünden 2>3>4>5>(6+) şeklinde sıralandığı tespit edilmiştir. Genel olarak, laktasyon sayısı arttıkça servis periyodu ve buzağı-lama aralığının azaldığı da söylenebilir.

Sonuç olarak, Türkiye’nin farklı bölgelerinde yetiştirilen Holştayn sığırlar arasında süt ve döl verim özellikleri yönünden önemli farklar tespit edilmiştir. Bu farkların, genetik faktörlerin yanında işletmeler arasındaki iklimsel ve yönetimsel farklılıklardan kaynaklandığı söylenebilir. Ayrıca, bilgisayar destekli sağım sistemleri ve buna bağlı sürü yönetimi programları, tahminleme yolu ile elde edilen verilere göre, hayvanların damızlık değerinin belirlenmesi ve uygulanacak seleksiyon ve ıslah programının tespiti için daha doğru veriler sağlayacaktır.

Araştırma sonucunda 7395.35±45.75 kg olarak bulu-nan 305 günlük süt verimi Türkiye ortalamasının üzerinde olmasına rağmen gelişmiş ülkelerin gerisinde kalmaktadır. Koçaş, Tahirova, Dalaman, Ceylanpınar Tarım İşletmelerinin Türkiye’nin elit sürülerine sahip olduğu ve yetiştiricilere damızlık temini gibi önemli bir görev üstlendikleri göz önüne alındığında çevresel faktörlerde yapılacak düzen-lemelerin yanında genetik kapasitesi yüksek boğa veya spermalarının ithalatı, embriyo transferi ve genotipik se-leksiyon uygulamaları ile hayvanların genetik kapasiteleri yükseltilerek, verimlerde kalıcı artışlar sağlanmalıdır.

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SummaryIn this study the concentrations of metals in 89 different honey samples were collected, from four different areas of Orumieh City,

in Iran from September of 2009 were investigated. Most of the samples were obtained from beekeepers located at a distance of 10 km from the roadside, to their accommodation. Pb, Co, Cr, Fe, Mn, Zn, As and Ni were detected in all of the honey samples. The contents of metals in honey samples were found to be in the range of 0.04±0.1 ppm, 0.001±0.002 ppm, 7.09±9.4 ppm, 0.6±0.9 ppm 0.06±0.1 ppm, 9.99±26.5 ppm, 0.0008±0.0011 ppm, 0.003±0.005 ppm for Pb, Co, Cr, Fe, Mn, Zn, As and Ni respectively. At the end of the study the Pb levels were found to be lower than the maximum residue limits of the European Union. Other metal levels were within acceptable levels. It was detected that the honey which was collected was of good quality from the point of metal contents.

Keywords: Atomic absorption spectrometry, Honey, Iran, Metal, Orumieh

İran’ın Orumieh Şehri’nden Toplanan Ballarda Metal Düzeylerinin Belirlenmesi

ÖzetBu çalışmada İran’ın Orumieh Şehri’nin 4 farklı bölgesinden (Tergever, Mergever, Sero ve Merkez) 2009 Eylül ayında toplanan 89

farklı bal örneklerinde metal kalıntı (Pb, Co, Cu, Fe, Mn, Zn, As, Ni) düzeyleri araştırıldı. Örneklerin çoğunluğu yol kenarlarına en az 10 km mesafede konaklayan arıcılardan sağlandı. Pb, Co, As, Ni analizleri Grafit Fırınlı Atomik Absorpsiyon Spektrometre cihazı ile, Fe, Cu, Mn ve Zn analizleri ise Alevli Atomik Absorpsiyon Spektrometre cihazı ile yapıldı. Analizler sonucunda bütün bal örneklerinde Pb, Co, Cu, Fe, Mn, Zn, As ve Ni tespit edildi. Analiz edilen bal örneklerinde Pb 0.04±0.1 ppm, Co 0.001±0.002 ppm, Cu 7.09±9.4 ppm, Fe 0.6±0.9 ppm, Mn 0.06±0.1 ppm, Zn 9.99±26.5 ppm, As 0.0008±0.0011 ve Ni 0.003±0.005 düzeyinde tespit edildi. Bu çalışma sonucunda belirlenen Pb düzeylerinin Avrupa Birliği tarafından kabul edilen maksimum kalıntı limitlerini aşmadığı, diğer metal düzeylerinin ise kabul edilebilir düzeylerde olduğu görüldü. Metal kalıntı yönünden balların iyi kalitede olduğu tespit edildi.

Anahtar sözcükler: Atomik absorpsiyon spektrometresi, Bal, İran, Metal, Orumieh

Determination of the Metal Contents of Honey Samples from Orumieh in Iran

Shahram SAGHAEI * Husamettin EKICI ** Mevlut DEMIRBAS *** Ender YARSAN * Ilyas TUMER ***

*

**

***

University of Ankara, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, TR-06110 Ankara - TURKEYUniversity of Kirikkale, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, TR-71451 Kirikkale - TURKEYPendik Veterinary Control Institute, TR-34890 Istanbul - TURKEY


Honey has been used for different aims such as a whole food in all parts of the world, treatment for various ailments and diseases, in medicine and also as a biological monitor for the determination of heavy metals, radioactivity in the region and to determine environmental quality in polluted

environments 1-4. There are various reviews about honey which used as a method for determining the amount of heavy metals of the region. Beekeeping is considered to be one of the most important agricultural activities in the world. Nowadays it is estimated that are 56 million bees

INTRODUCTION


RESEARCH ARTICLE

282Determination of the Metal ...

worldwide and that 1.2 million tons honey is produced by them in one year 5-7.

Iran is the one of the most important honey producers country in the world. According to official statistics in 2007, 2% of world’s total honey production came from Iran with 36.000 tons. Approximately 10.000 tons of honey is produced in Orumieh City yearly 8,9. The aim of this study is to determine the residue of some heavy metals (Pb, Co, Cr, Fe, Mn, Zn, As and Ni) in honey samples collected from four different regions of Orumieh City and to determine potential health risks to humans.


In total eighty-nine samples were collected, made up from the following regions Tergever (26 samples), Mergever (22 samples), Serow (19 samples) and Center region (22 samples) of Orimieh in 2009 September (Fig. 1). Natural honey, natural + sweet honey, wax honey were collected from Tergever and Mergever regions. Natural honey and natural + sweet honey were collected from Serow and Center regions. The honey samples were stored in plastic molds and kept at room temperature (25°C) until analyzed.

One gram of honey samples from each region were mixed with magnesium acetate (1 mg/mL). The mixture was placed in a porcelain crucible. After drying at 100°C for 2 h, the samples were ashed at 600°C. Care was taken during heating so that no excess foaming took place.

The ash was extracted with nitric acid (HNO3) 2N and was diluted to 30 mL. The contents of lead (Pb), cobalt (Co), chrome (Cr), iron (Fe), manganese (Mn), zinc (Zn), arsenic (As) and nickel (Ni) were determined directly in the ash solution using atomic absorption spectroscopy (GF 3000 model AAS, Graphite Furnace GF 3000, Auto Sampler GBC PAL 3000, GBC Scientific Equipment Pty Ltd, Australia). The accuracy of the instrument was periodically checked with a known standard. Calibration curves were prepared using dilutions of stock solutions. The results were read three times and the mean values and the relative standard deviations were computed.

Data was analyzed statistically by one-way analysis of variance (ANOVA). When significant treatment effects were detected, DUNCAN’S multiple range test was used to identify specific differences between treatment means at a probability level of P<0.05.

RESULTS

The means and standard deviation of the ash contents of 89 honey samples for Pb, Co, Cr, Fe, Mn, Zn, As and Ni were founds as 0.04±0.1 ppm, 0.001±0.002 ppm, 7.09±9.4 ppm, 0.6±0.9 ppm 0.06±0.1 ppm, 9.99±26.5 ppm, 0.0008±0.0011 ppm, 0.003±0.005 ppm, respectively.

Statistical comparisons were calculated for regional differences and varieties of honey (Table 1 and Table 2). Pb, Mn and Fe levels were statistically similar in terms of natural + sweet honey with natural honey while wax

Table 1. Metal concentration according to different honey samples*

Tablo 1. Farklı bal örneklerine göre metal konsantrasyonları *

Honey Samples Pb Co Mn Fe Cr Zn As Ni

natural + sweet (n:33)

0.07±0.04 a

(0.394-0.190)0.001±0.0008(0.0006-0.003)

0.08±0.02a

(0.026-0.116)0.7±0.3a

(0.344-2.542)8.4±3.5

(3.642-17.314)24.3±19.2cb

(0.953-42.490)0.0006±0.00063

(0.0000028-0.0028)0.003±0.001

(0.0011-0.0086)

natural honey (n:40)

0.07±0.04a

(0.383-0.253)0.002±0.001

(0.0003-0.005)0.1±0.05a

(0.018-0.305)0.7±0.2a

(0.378-1.935)8.1±5.3

(3.369-33.765)19.9±15.07b

(0.702-68.49)0.0011±0.00105

(0.0000264-0.0038)0.006±0.01

(0.0015-0.068)

waxy honey (n:16)

0.1±0.08b

(0.048-0.389)0.002±0.001

(0.0007-0.007)0.06±0.05b

(0.031-0.222)1.1±1.04b

(0.341-4.349)7.6±3.4

(2.968-13.635)11.4±17.9ab

(0.896-70.55)0.0009±0.00065

(0.000131-0.0025)0.005±0.004

(0.0019-0.017)*as ppm: mean±Standard deviation and rangea.b Mean within in the same columns with different letters are statistically significant (P<0.05)

Fig 1. Locations of study areas: Tergever, Mergever, Serow and Center

Şekil 1. Çalışmanın yapıldığı bölgeler: Tergever, Mergever, Serow ve Merkez

283

SAGHAEI, EKICIDEMIRBAS, YARSAN, TUMER

honey showed statistically difference from the others. Furthermore significant differences were found between natural+sweet honey and wax honey samples for Zn levels (Table 1). On the other hand regional comparisons were examined in this study. Significant differences were also found for Pb, Co, Mn and Zn levels in different regions. Tergever and Central regions showed similarities for these four metals (Table 2).

DISCUSSION

Heavy metals are natural components of the Earth’s crust. They cannot be degraded or destroyed. To a small extent they enter our bodies via food, drinking water and air as trace elements. Some heavy metals (e.g., copper, selenium, zinc) are essential to maintain the metabolism of the human body. In this study, heavy metals were analyzed and measured by atomic absorption spectrophotometer. The results indicated that Zn had highest concentration followed by Cr, Fe, Mn, Pb, Ni, Co and As.

Demirezen and Aksoy 3 reported that the ranges of the metal contents of honey samples from Kayseri as 0.11-0.18 ppm for cadmium, 0.15-0.66 ppm for copper, 2.2-11 ppm for zinc, 0.2-0.8 ppm for nickel and 0.1-0.85 ppm for lead. Fredes and Montenegro 10 determined that the contents of Pb, Cd, Cu, Fe and Mn in honey as 0.02±0.03 ppm, 0.01±0.01 ppm, 0.91±0.66 ppm, 3.13±1.44 ppm, 1.26±1.29 ppm, respectively. Frazzoli et al.11 reported that Cd and Pb contents ranged from 0.2 to 1.37 ng g-1 and from 4.6 to 30.5 ng g-1 in flower honey, while the highest concentrations were presented by honeydew honey.

Pb, Cd, Cu, Fe and Zn contents in honey were determined as 0.10005 ppm, 0.25005 ppm, 5.00005 ppm and 3.105 ppm, respectively 12. Erbilir and Erdogrul 4 found that the means of Cd, Mn, Fe and Mg in honey as 0.32 ppm, 0.03 ppm, 0.36 ppm and 10.45 ppm, respectively. On the other hand, Ni was not detected in the honey samples in the same study.

Devillers et al.13 reported that the means (ranges) of Pb, Cd, Cu, Fe, Mn and Zn contents in honey from France as 0.793 ppm (0.28-1.08 ppm), 0.152 ppm (0.08-0.25 ppm), 0.305 ppm (0.06-1.71 ppm), 11.03 ppm (0.56-86.76), 3.685 ppm (0.11-42.81 ppm) and 1.343 ppm (0.17-6.42 ppm) respectively. Also, Adebiyi et al.14 determined the means (ranges) of Cu, Fe, Mn and Zn contents in honey from Nigeria as 21 ppm (10-35 ppm), 220,6 ppm (136-407 ppm), 3 ppm (0-5 ppm) and 63.4 ppm (31-106 ppm).

The results of this study revealed that the Pb levels were higher than the results of Frazzoli et al.11, but lower than those of Demirezen and Aksoy 3 and Devillers et al.13, and similar to Antonescu and Mataescu 12 and Fredes and Montenegro 10. Moreover, Fe levels that were found in this study were lower than the results of Antonescu and Mataescu 12, Devillers et al.13, Adebiyi et al.14, Fredes and Montenegro 10 and Erbilir and Erdogrul 4. The levels of Mn were higher than Erbilir and Erdogrul’s 4 results, but lower than results of Fredes and Montenegro 10, Devillers et al.13 and Adebiyi et al.14. Results from this study for Zn were higher than the results of Demirezen and Aksoy 3, Devillers et al.13 and Antonescu and Mataescu 12, however, lower than those of Adebiyi et al.14. Results from this study for Ni were lower than the results of Demirezen and Aksoy 3. European Union (EU) maximum residue limit for Pb in honey is 1 mg/kg. The maximum residue limit for other metals in honey are not defined by EU, therefore it is difficult to evaluate the metal residues in honey from a toxicological aspect 15.

It was concluded that a little metal residue in honey samples collected from Orimieh city and the level of the residues were below the permitted limit. These results showed that the honey samples collected from Orimieh city were far from the polluted sources (i.e. highways with intense traffic, mining, industry, different botanical origins). Therefore, it is recommended that the beekeeping activities must be far from pollution. With regards to other studies, our results are in accordance with the acceptable limits of EU standards for lead. Thus the present study showed that analyzed honey samples were good quality.

Table 2. Metal concentration according to different regions*

Tablo 2. Farklı bölgelere göre metal konsantrasyonları*

Regions Pb Co Mn Fe Cr Zn As Ni

Tergever(n:26)

0.08±0.03a

(0.048-0.20)0.002±0.001a

(0.0009-0.073)0.09±0.04a

(0.032-0.222)0.8±0.4

(0.485-2.721)7.6±4.07

(2.968-18.597)14.6±9.8c

(0.896-38.14)0.0009±0.00086

(0.000071-0.0032)0.005±0.003(0.002-0.017)

Mergever(n:22)

0.1±0.07b

(0.038-0.38)0.001±0.001a

(0.0005-0.0037)0.06±0.06b

(0.026-0.30)0.6±0.3

(0.34-1.62)7.09±2.4

(3.69-12.98)9.9±12.9 ac

(0.70-52.95)0.0008±0.00046

(0.0000448-0.00195)0.004±0.002

(0.0022-0.098)

Serow(n:19)

0.04±00.01c

(0.039-0.389)0.001±0.0007 b

(0.0003-0.0035)0.09±0.02a

(0.03-0.16)0.9±0.5

(0.48-2.54)8.8±3.5

(4.27-19.33)23.7±10.9c

(8.02-60.58)0.0011±0.0012

(0.0000264-0.0034)0.004±0.005

(0.0018-0.068)

Center(n:22)

0.07±0.04a

(0.039-0.253)0.002±0.001a

(0.0008-0.0044)0.1±0.02a

(0.018-0.17)0.8±0.1

(0.49-1.14)9.4±6.3

(3.369-33.765)26.5±12.2bc

(0.90-68.49)0.0008±0.0009

(0.0000028-0.0038)0.003±0002

(0.0011-0.013)

*as ppm: mean±Standard deviation and rangea.b Mean within in the same columns with different letters are statistically significant (P<0.05)

284Determination of the Metal ...

REFERENCES

1. Conti ME: Lazio region (central Italy) honeys: A survey of mineral content and typical quality parameters. Food Control, 11 (6): 459-463, 2000.

2. Fernández-Torres R, Pérez-Bernal JL, Bello-López MA, Callejón-Mochón M, Jiménez-Sánchez JC, Guiraúm-Pérez A: Mineral content and botanical origin of Spanish honeys. Talanta, 65 (3): 686-691, 2005.

3. Demirezen D, Aksoy A: Determination of heavy metals in bee honey using by inductively coupled plasma optical emission spectrometry (ICP-OES). GU J Sci, 18 (4): 569-575, 2005.

4. Erbilir F, Erdogrul O: Determination of heavy metals in honey in Kahramanmaras, City, Turkey. Environmental Monitoring and Assessment, 109, 181-187, 2005.

5. Downey G, Hussey K, Jelly JD, Walshe TF, Martin PG: Preliminary contribution to the characterization of artisanal honey produced on the island of Ireland by palynological and physicochemical data. Food Chemistry, 91 (2): 347-354, 2005.

6. Yarsan E, Karacal F, Ibrahım IG, Dikmen B, Koksal A, Das YK: Contents of some metals in honeys from different regions in Turkey. Bull Environ Contam Toxicol, 79, 255-258, 2007.

7. Pohl P: Determination of metal content in honey by atomic absorption and emission spectrometries. TrAC Trends in Analytical Chemistry, 28 (1): 117-128, 2009.

8. Anonymous: http://www.farmna.ir/Pages/News-7496.html. Accessed: 24 May 2011.

9. Anonymous: http://www.farmna.ir/Pages/News-7498.html. Accessed: 24 May 2011.

10. Fredes C, Montenegro G: Heavy metals and other trace elements contents in Chilean honey. Cien Inv Agr, 33 (1): 50-58, 2006.

11. Frazzoli C, D’Ilio S, Bocca B: Determination of Cd and Pb in honey by SF-ICP-MS: Validation figures and uncertainty of results. Analytical Letters, 40, 1992-2004, 2007.

12. Antonescu C, Mateescu C: Environmental pollution and its effects on honey quality. Roum Biotechnol Lett, 6 (5): 371-379, 2001.

13. Devillers J, Dore JC, Marenco M, Poirier-Duchene F, Galand N, Viel C: Chemometrical analysis of 18 metallic and nonmetallic elements found in honeys sold in France. J Agric Food Chem, 50, 5998-6007, 2002.

14. Adebiyi FM, Akpan I, Obiajunwa EI, Olaniyi HB: Chemical/physical characterization of Nigerian honey. Pakistan J Nutr, 3 (5): 278-281, 2004.

15. Bogdanov S: Contaminants of bee products. Apidologie, 37, 1-18, 2006.


SummaryThis study presents a method for the production of rhamnolipid, a biosurfactant, by Pseudomonas sp. Pseudomonas sp. cells that

were grown in nutrient agar were inoculated into sterile liquid medium. Following an incubation period of 24 h, 2 ml of cells were inoculated into a different liquid medium and the results were obtained at the end of 26 hours incubation time. In our study, the effects of temperature, pH, and glucose concentration on rhamnolipid production were also investigated. Later, the same procedure was applied to immobilized cells that were kept away from the free microorganisms. The production of rhamnolipid by free cells was found to be much higher than that of immobilized cells. Free cells could be used for rhamnolipid production effectively.

Keywords: Biosurfactant, Rhamnolipid, Pseudomonas sp., Immobilized cells, Free cells

Serbest ve Tutuklanmış Pseudomonas sp. Hücrelerinden Ramnolipid (Biyosürfektan) Eldesi

ÖzetBu çalışmada, bir büyosürfektan olan ramnolipidin Pseudomonas sp.’den üretimi araştırılmıştır. Nutrient agarda üretilen

Pseudomonas sp. örnekleri sıvı besiyerine aktarılmıştır. 24 saatlik inkübasyon sonrası, 2 ml’lik kültür örnekleri ramnolipid üretimi için, farklı bir sıvı besiyerine aktarılarak 26. saatte sonuçlar gözlenmiştir. Çalışmada, ayrıca ramnolipid üretimine sıcaklığın, pH’ın ve glukoz konsantrasyonunun etkisi de incelenmiştir. İmmobilize (tutuklanmış) hücreler içinde aynı metotlar uygulanmıştır. Ramnolipid üretimi, serbest hücrelerde tutuklanmış hücrelerden daha yüksek bulunmuştur. Serbest hücreler, etkin ramnolipid üretimi için kullanılabilirler.

Anahtar sözcükler: Biyosürfektan, Ramnolipid, Pseudomonas sp., Tutuklanmış hücre, Serbest hücre

Production of Rhamnolipid (A Biosurfactant) Using Free and Immobilized Cells of Pseudomonas sp.

Uğur SIDAL * Ebru Şebnem YILMAZ ** *

**Celal Bayar University, Faculty of Art and Science, Department of Biology, TR-45030 Manisa - TURKEYMustafa Kemal University, Faculty of Art and Science, Department of Biology, TR- 31040 Hatay - TURKEY


Many microorganisms are able to synthesize different types of biosurfactants when grown on various carbon sources. These biosurfactants include low molecular weight glycolipids, lipopeptides, and high molecular weight lipid -containing polymers such as lipoproteins, lipopoly- saccharide-protein complexes, and polysaccharide-protein -fatty acid complexes 1,2. It is well known that several strains of Pseudomonas can accumulate surface-active compounds characterized as rhamnolipids when grown on different carbon sources 3. The rhamnolipids produced by Pseudomonas aeruginosa have been widely studied and are reported to be a mixture of the homologous species RL1 (Rha C10C10), RL2 (Rha C10), RL3 (Rha2C10C10) and RL4 (Rha2C10) 4.

In recent years, the number of investigators studying biosurfactant-producing microorganisms has dramatically increased. Biosurfactants have several advantages over synthetic surfactants for potential industrial applications. The most important advantage of biosurfactants when compared to synthetic surfactants is their ecological acceptance, owing to their low toxicity and biodegradable nature 3,5. Some of these biosurfactants also have been investigated for their potential to act as biologically active compounds for pharmaceuticals 6.

Microorganisms used for industrial applications are selected to provide the best possible combination of characteristics for the specific process and equipment

INTRODUCTION


RESEARCH ARTICLE

286Production of Rhamnolipid ...

being used. The selected strains should be tolerant of high concentrations of carbohydrates and biosurfactants. P. aeruginosa is capable of using different carbon sources to produce rhamnolipids: glycerol, vegetable oils, hydrocarbons and others 3,5. Biosurfactant production is possible using either free or immobilized cells in culture. Immobilization involves the restriction of cell mobility within a defined space, which provides high cell concentrations and permits the reuse of the cells 7,10. Both techniques have some advantages and disadvantages, e.g., economical and technical. The use of immobilized whole cells in industrial processes has attracted considerable attention during the past few years due to its advantages over traditional processes, and the development of cell immobilization techniques has gained momentum in recent years due to its potential application in different areas of biotechnology, including biosurfactant production. The aim of this study was to compare the productivity of rhamnolipid production in batch systems in which either free or immobilized Pseudomonas sp. cells were grown using glucose as a substrate.


Microorganism

Pseudomonas sp. BKK041 was obtained from the culture collection of the Department of Biology, Faculty of Science and Arts, Mustafa Kemal University (Hatay, Turkey). The stock culture was maintained on a nutrient agar slant at 4°C.

Culture Medium

The stock culture (24 h in nutrient agar) was tranferred into a 250-ml Erlenmayer flask containing 50 ml of liquid medium (1.28 g NaNO3, 0.87 g K2HPO4, 0.1 g MgSO4

.7H2O, 0.1 g NaCl, 0.2 g KCl, 6.5 g Tris (hydroxymethyl amino- methane), 20.0 g glucose, 5 ml Mineral Salt Solution [0.116g FeSO4 (NH4)2SO4.6H2O, 0.232 g H3BO3, 0.41 g CoCl2.6H2O, 0.008 g CuSO4.5H2O, 0.008 g MnSO4.H2O, 0.022 g (NH4)6Mo7O24.4H2O, 0.174 g ZnSO4.7H2O in 1 L] in 1 L), then incubated for 24h at 30°C 11. Culture media were sterilized in an autoclave.

Rhamnolipid Determination

Two ml of the seed culture was inoculated into a 250-ml flask containing 50 ml of liquid culture medium. All samples were incubated at 150 rpm for 26 h. The culture broth was then centrifuged at 5.000 rpm for 12 min. Rhamnolipid production was determined by a colorimetric assay at 480 nm using a Shimadzu UV-VIS spectrophotometer. For this purpose, 2 ml of the cell-free broth (supernantant) was used. Rhamnolipid concentration was estimated by the phenolsulphuric acid method 12.

Glucose Determination

The glucose level was determined according to Miller 13

by the dinitro salicylic acid method at 550 nm using a Shimadzu UV-VIS spectrophotometer.

Cell Immobilization

Immobilization of cells was performed according to Cheetham et al.8 with a minor modification. For this purpose, 0.6g of sodium alginate was dissolved in 20 ml of distilled water, then mixed with previously centrifuged cells. This mixture was poured into a beherglass that contained 0.5 M CaCl2. All tests were performed in triplicate.

RESULTS

In the present study, we have investigated batch culture system effects on rhamnolipid production in free and immobilized Pseudomonas sp. BKK041 cells. The results shown in Table 1 indicate that there is a substantial influence of temperature on biosurfactant production. The highest biosurfactant concentration (600 mg/L) was obtained at 30°C. Higher and lower temperature levels inhibited rhamnolipid production 14,17. The glucose utilization curve showed a linear pattern throughout 26 hours of fermentation.

Table 2 shows the pattern of biosurfactant production by the free and immobilized cells of Pseudomonas sp. BKK041 in different pH conditions. These results indicate that pH variation had an appreciable effect on rhamnolipid production, as its concentration varied over a wide range

Table 1. Values of rhamnolipid and final glucose concentration at different temperatures

Tablo 1. Farklı sıcaklıklardaki son glukoz konsantrasyonu ve ramnolipid miktarları

Temperature(°C)

Free Cells Immobilized Cells

Rhamnolipid(mg/L)

Final GlucoseConcentration (g/L)

Rhamnolipid(mg/L)


20 50 0.025 0.2 0.250

25 470 0.375 4 0.300

30 600 3.450 300 3.200

35 10 3.175 3 2.000

40 2 0.250 0.2 0.250

287SIDAL, YILMAZ

of pHs. An initial pH value of 7 was found to be the most suitable for Pseudomonas sp. to produce rhamnolipid 14-18.

The effect of glucose concentration on rhamnolipid production is shown in Table 3. The maximum bio-surfactant concentration (387.5 mg/L) was obtained at an initial glucose concentration of 2% (w/v). Higher and lower glucose concentrations inhibited rhamnolipid production 17-20.

DISCUSSION

The results shown in Tables 1-3 represent the average of triplicate trials. When the different batch processes

are compared, it can be seen that free cells gave better results than immobilized cells (Fig. 1-3). The reasons for this difference may be explained as a loss of activity during immobilization or the diffusion limitations between alginate gel surfaces and microorganisms. Similar results have been reported by Jamuna et al.21, Kanwar et al. 22, and Goksungur and Guvenç 23. However, their studies were on different substrates and used different microorganisms. If the limitations with the rhamnolipid production techniques can be solved, the advantages of free cells utilization would increase, in both batch and continuous culture systems.

It is well known that biosurfactants are surface-active agents produced by bacteria, yeasts, and fungi, and include

Table 2. Values of rhamnolipid and final glucose concentration at different pH values

Tablo 2. Farklı pH değerlerindeki son glukoz konsantrasyonu ve ramnolipid miktarları

pH


Rhamnolipid(mg/L)


Rhamnolipid(mg/L)


5.0 2.50 0.250 0.25 0.450

6.0 12.5 0.500 0.10 0.900

7.0 410 4.000 320 3.400

8.0 25 0.400 20 2.100

Table 3. Values of rhamnolipid and final glucose concentration at different glucose concentrations

Tablo 3. Farklı glukoz konsantrasyonlarındaki son glukoz ve ramnolipid miktarları

Glucose Concentration (%)


Rhamnolipid(mg/L)


Rhamnolipid(mg/L)


0.5 2.5 0.050 0.25 1.250

1.0 5 0.125 1 2.000

1.5 300 1.375 10 1.500

2.0 387.5 3.200 210 2.200

2.5 200 1.725 100 1.100

0

100

200

300

400

500

600

Rhamnolipidamount (mg/L)

20 25 30 35 40Temperature (°C)

Free cellsImmobilized cells

Fig 1. The effect of temperature on rhamnolipid production

Şekil 1. Ramnolipid üretimine sıcaklığın etkisi

288Production of Rhamnolipid ...

products such as fatty acids, glycerides, phospholipids, lipopeptides, antibiotics, and glycolipids. Thus, in recent years there has been much interest in the production of biosurfactants from microorganisms for use in different applications in many industries. For example, demand for biosurfactants has increased in the oil and cosmetics industries, among others, because biosurfactants are easily degradable by microorganisms, compared with synthetic surfactants, which are highly resistant to biodegradation or are only partially biodegradable 24. The rhamnolipids from Pseudomonas aeruginosa were first described by Jarvis and Johnson 25, and studies on the biosynthesis of these compounds in optimal in vitro conditions were carried out by others 26-28, who reported that these glycolipids were secreted into the medium during the stationary phase of growth 29.

For these reasons, many studies have been conducted to improve the production of biosurfactants. Biosurfactant production optimization appears to be one of the main fields of research, owing to the low product yields 30. However, fermentation technology for biosurfactant production using immobilized cells has not been applied widely yet. In conclusion, this study showed that, the yield of the biosurfactant rhamnolipid was low using the

immobilized cell technique. We suggested that it may be possible to improve this method in further studies. In addition, Pseudomonas sp. BKK041 free cells presented good potential for the production of a rhamnolipid-type biosurfactant.

Acknowledgments

This work was supported by Mustafa Kemal University’s Scientific Research Project Fund (Hatay, Turkey).

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050

100150200250300350400450

Rhamnolipid amount (mg/L)

5 6 7 8

pH


Fig 2. The effect of pH on rhamnolipid production

Şekil 2. Ramnolipid üretimine pH’ın etkisi

050

100150200250300350400

Rhamnolipid amount (mg/L)

0,5 1 1,5 2 2,5

Initial glucose concentration (% w/v)


Fig 3. The effect of initial glucose concentration on rhamnolipid production

Şekil 3. Ramnolipid üretimine başlangıç glukoz konsantrasyonunun etkisi

289SIDAL, YILMAZ

growth on Brazilian native oils. Process Biochem, 41, 483-488, 2006.

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14. Mercade ME, Manresa MA: The use of agroindustrial by products for biosurfactant production. J Amr Oil Chem Society, 71, 61-64, 1994.

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23. Goksungur Y, Güvenç U: Batch and continuous production of lactic acid from beet molasses by Lactobacillus delbrueckii IFO 3202. J Chem Tech Biotechnol, 69, 399-404, 1997.

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30. Abouseoud M, Yataghene A, Amrane A, Maachi R: Biosurfactant production by free and alginate entrapped cells of Pseudomonas fluorescens. J Ind Microbiol Biotechnol, 35, 1303-1308, 2008.


ÖzetYeme esansiyel yağ karışımı (EYK) ilavesinin etlik piliçlerin büyüme performansı, karkas randımanı ve bazı iç organ ağırlıkları üzerine

etkilerini belirlemek amacıyla 2 deneme yürütülmüştür. Her bir denemenin hayvan materyalini günlük yaşta ve karışık cinsiyette 400 adet etlik civciv oluşturmuştur. Her iki denemede de piliçlerin yarısına temel yem karması verilirken, diğer yarısına ise altı farklı esansiyel yağın karışımından oluşmuş EYK ilave edilmiş yem verilmiştir. Yeme EYK ilavesinin birinci denemede etlik piliçlerin büyüme performansı üzerine istatistiki olarak herhangi bir etkisi olmazken (P>0.05), ikinci denemede hem üç hem de altıncı hafta canlı ağırlıklarını artırmış ve yemden yararlanmayı iyileştirmiştir (P<0.01). Yeme EYK ilavesinin etlik piliçlerin yem tüketimi ve ölüm oranı üzerine denemelerin her ikisinde de etkisi olmamıştır (P>0.05). Yem EYK ilavesi birinci denemede karkas randımanını artırmıştır (P<0.05). Ayrıca yeme EYK ilavesi ikinci denemede etlik piliçlerin pankreas ağırlığını artırmıştır (P<0.05). Bu çalışmadan elde edilen sonuçlar etlik piliç yemlerinde EYK’nın performans artırıcı yem katkı maddesi olarak kullanılabileceğini göstermektedir.

Anahtar sözcükler: Etlik piliç, Esansiyel yağ karışımı, Performans, Karkas randımanı, Organ ağırlığı

The Effect of Dietary Essential Oil Mixture Supplementation on the Broiler Performance, Carcass Yield and Some Internal

Organ Weight

SummaryTwo experiments were conducted to evaluate the effects of dietary essential oil mixture supplementation (EOM) on the

performance, carcass yield and some organ weights of broilers. A total of 400 one-day-old broiler chicks (Ross-308) per one experiment were randomly assigned to 2 groups. Half of the broilers were given untreated basal diets while the remaining birds were fed on the basal diet supplemented with EOM containing a blend of six essential oils. Dietary EOM supplementation did not promote growth in the experiment 1 (P>0.05), while EOM increased body weight and improved feed conversion ratio between 0-3 and 0-6 weeks in the experiment 2 (P<0.01). Feed intake and mortality was not affected by EOM supplementation in both trials (P>0.05). Supplementation diet with EOM increased carcass yield in the experiment 1 (P<0.05). In addition, pancreas weight was increased in response to EOM in the second trial. Overall results showed that an EOM can be used as performance enhancer in broiler diets.

Keywords: Broiler, Essential oil mixture, Performance, Carcass yield, Organ weight

Etlik Piliç Yemlerine Esansiyel Yağ Karışımı İlavesinin Büyüme Performansı, Karkas Randımanı ve Bazı İç Organ Ağırlıkları

Üzerine EtkileriKamil KÜÇÜKYILMAZ * Abdullah Uğur ÇATLI * Mustafa ÇINAR *

* Erbeyli İncir Araştırma Enstitüsü Müdürlüğü, TR-09600 İncirliova, Aydın - TÜRKİYE


Antibiyotiklerin uzun yıllardan beri hayvan yemlerinde kullanımına bağlı olarak ortaya çıkan bakteriyel direnç ve hayvansal organizmadaki kalıntı riski nedeniyle 2006 yılı başından itibaren AB ülkelerinde ve ülkemizde antibiyotiklerin yeme katılması yasaklanmıştır 1,2. Bu durum anti-

biyotiklere alternatif diğer yem katkı maddelerinin araştı- rılması konusundaki çalışmaları hızlandırmıştır. Probiyotik, prebiyotik ve organik asitlerin yanı sıra bitkisel kökenli doğal yem katkı maddeleri (bitki ekstratları, tüm bitki kısımları ve esansiyel yağlar) önem kazanmaya başlamıştır 3.

GİRİŞ


RESEARCH ARTICLE

292Etlik Piliç Yemlerine ...

Geniş spektruma sahip antibakteriyel aktivitelerinin yanı sıra antioksidan, antifungal, enzimatik ve antikoksidiyal aktiviteleri çok sayıda bilimsel çalışmalarla ortaya konan esansiyel yağların kanatlı hayvanların yemlerine büyütme faktörü olarak katılmalarıyla ilgili çalışmalar son 10 yıl içinde ağırlık kazanmıştır. Esansiyel yağların tek başına ve karışım halinde yeme katılmasının etlik piliçlerin besi performanslarını artırdığını bildiren çalışmaların 4-6 yanısıra, endojen enzimatik aktiviteyi destekleyerek organik besin maddelerinin sindirilebilirliğini artırdığı gösterilmiştir 7.

Yapılan çalışmalar sonucunda eterik yağların spesifik karışımlarının hayvanın sağlığı ve verimi üzerindeki olumlu etkilerinin yıllardır kullanıla gelmekte olan geleneksel bü- yütme faktörleri ile mukayese edilebilir avantajlara sahip olduğu bildirilmiştir. Esansiyel yağ karışımlarının hayvan sağlığı ve verimler üzerindeki etkisinin uygulanan dozu ile ilişkili olduğu da ayrıca belirtilmiştir 8.

Esansiyel yağların iyi bilinen antimikrobiyal etki meka- nizmalarından beklenilen faydanın sağlanabilmesi kümes-lerdeki manejman koşullarıyla doğrudan ilişkilidir 6. Manej- man koşullarındaki olumsuzlukların (aşırı yerleşim sıklığı, kümes sıcaklığında istenmeyen değişimler, altlık kalitesinde kötüleşme, civciv sağlığı ve kümes hijyenindeki aksaklıklar, vb.) kanatlı hayvan yemlerine katılan tüm performans artırıcı yem katkı maddelerinin çalışma yöntemi ve etkinliği üzerine büyük önemi vardır. Zira bazı araştırıcılar uygun çevre koşullarında bu tür katkıların beklenilen başarıyı gösteremediklerini ileri sürmüşlerdir 9-11.

Esansiyel yağlarla yapılan çalışmaların sonuçları arasın-daki tutarsızlıklar çalışmalardaki manejman koşularının değişkenliğine atfedilmiştir. Bu çalışmada, esansiyel yağ karışımı (EYK)’nın etlik piliçlerin besi performansı, karkas randımanı ve bazı iç organ ağırlıkları üzerine etkileri manejman koşularından kaynaklanan eşitsizliği minimize etmek amacıyla iki deneme kapsamında incelenmiştir.

MATERYAL ve METOT

Bu çalışma aynı metot uygulanmak suretiyle 2 deneme halinde yürütülmüştür. Çalışmanın birinci denemesi Ekim-Kasım, ikinci denemesi Nisan-Mayıs ayları arasında yapılmıştır. Araştırmanın hayvan materyalini her bir deneme için 400 adet olmak üzere günlük yaşta ve karışık cinsiyette toplam 800 adet etlik civciv (Ross 308) oluş-turmuştur. Civciv çıkış ağırlığı ile performans arasındaki olası interaksiyonların önlenmesi amacıyla ticari kuluçka- haneden alınan civcivlerin benzer ağırlıkta olmasına dikkat edilmiştir. Piliçlerin yarısına temel yem karması verilirken, diğer yarısına ise ülkemiz florasında yetişen aromatik bit- kilerden elde edilen bir EYK ilave edilmiş temel yem kar-masından verilmiştir. EYK yerli bir ürün olup (Herbromix®

- Herba A.Ş. İzmir), altı farklı esansiyel yağın karışımından oluşmuştur (kekik, defne, rezene, ada çayı, mersin yaprağı ve portakal kabuğu yağı). Denemede kullanılan EYK’nın

aktif madde bileşenlerinin içeriği Tablo 1’de verilmiştir. Toplam 48 g EYK mikroenjeksiyonlu püskürtücü vasıtasıyla 952 g zeolite emdirilerek hazırlanan preparat bir ton yeme karıştırılmak suretiyle yem karmalarına 48 mg/kg düzeyinde EYK ilave edilmiştir.

Her iki denemede 2 grup ile yürütülmüştür. Her bir grup 4 tekerrüre ayrılmış ve her tekerrür de 50 adet (25 erkek-25 dişi) etlik civcivden oluşturulmuştur. Civcivler yarı açık perdeli tip etlik piliç kümesinin talaş altlıklı yer bölmelerine m2’ye 14 adet civciv düşecek şekilde yerleş- tirilmiştir. Her bölmede 2 adet yemlik ve 1 adet suluk bulundurulmuştur. Kümes içi sıcaklığı ilk 3 gün için 32°C’ye ayarlanmış, daha sonra 21. günlük yaşa kadar kademeli olarak 22°C’ye düşürülmüş ve deneme sonuna kadar 22°C’ye sabitlenmiştir. Yem ve su ad libitum olarak verilmiş, günde 23 saat aydınlatma yapılmıştır.

Deneysel yem karmaları mısır, buğday, soya ve ayçiçeği tohumu küspesi esaslı olarak hazırlanmıştır. Her iki denemenin yemleri hazırlanırken aynı hammaddeler ve formü-lasyon kullanılmıştır. Birinci denemede kullanılan yem hammaddeleri ikinci denemenin başlangıcına kadar 18°C sıcaklık ve %70 nisbi rutubeti geçmeyen depo koşulla- rında muhafaza edilmiştir. Yem karmalarının analizinde AOAC’dan 12 yararlanılmış, metabolik enerjinin hesaplan-masında TSE’de 13 bildirilen eşitlik kullanılmıştır. Etlik piliç-lere 0-21 günler arasında etlik civciv, 22-42 günler arasında etlik piliç yemi verilmiştir (Tablo 2).

Denemenin 21. ve 42. günlerinde tüm hayvanlar bireysel olarak tartılarak canlı ağırlıkları belirlenmiş, yem tüke- timi ve yemden yararlanma değerinin hesaplanmasında her bir bölmede tüketilen karma yem miktarı dikkate alın-mıştır. Yemden yararlanma değeri birim canlı ağırlık artışı için tüketilen yem miktarı olarak ifade edilmiştir. Deneme sü- resince ölen piliçler günlük olarak kaydedilerek yemden

Tablo 1. Esansiyel yağ karışımının aktif madde bileşenleri

Table 1. The active substances of essential oil mixture

Bileşik Elde Edildiği Bitki

Karvakrol Kekik

1,8-sineol Defne, Adaçayı

Linalool Kekik

γ- terpinen Tümünde

p- simen Tümünde

Limonen Portakalda

α- pinene Tümünde

α- terpineol/borneol Tümünde ve ağırlıklı olarak kekikte

β- karyofillen Tümünde ve ağırlıklı olarak kekikte

Timol Kekik

(E)- anetol Rezene

α- terpinen Tümünde

293

KÜÇÜKYILMAZÇATLI, ÇINAR

yararlanma değerinin hesaplanmasında dikkate alınmıştır.

Etlik piliçler 42 günlük yaşta kesilmiştir. Karkas randı- manı ve iç organ ağırlıklarını belirlemek amacıyla her iki denemede de grup ortalamalarına benzer ağırlıkta her gruptan 24 adet (12 erkek ve 12 dişi) olmak üzere toplam 48 adet etlik piliç kesilmiştir. Kesim için ayrılan piliçler kesimden önce 10 saat aç bırakılarak sindirim kanalının bo- şalması sağlanmıştır. Kesilen piliçler 24 saat +4°C’de bekle-tilip soğuk karkas ağırlıkları belirlenmiştir. Karkas ağırlığı canlı ağırlığa oranlanarak karkas randımanı bulunmuştur. Kesim esnasında piliçlerden çıkarılan iç organlar (Karaciğer, dalak, ince bağırsak, kalın bağırsak, pankreas, kör bağırsak) tartılarak canlı ağırlığa bölünmüş ve oransal ağırlıkları be-lirlenmiştir.

Denemeden elde edilen verilerin istatistiki analizi SAS paket programından 14 yararlanarak yapılmıştır. Yüzde (%)

ile tanımlanan veriler, değerlendirilmeden önce arc- sine transformasyonuna tabi tutulmuştur. Gruplar arasındaki farklılıkların önemlilik kontrolü t testi ile yapılmıştır.

BULGULAR

Çalışmanın birinci ve ikinci denemesine ait canlı ağırlık, yem tüketimi, yemden yararlanma değeri ve ölüm oranları sırasıyla Tablo 3 ve Tablo 4’te verilmiştir.

Yeme EYK ilavesi birinci denemede etlik civciv ve piliçlerin 3. ve 6. canlı ağırlıkları üzerine istatistiki olarak herhangi bir etkide bulunmazken (P>0.05), ikinci denemede hem üç hem de altıncı hafta canlı ağırlıklarını artırmıştır (P<0.01). İkinci denemede EYK ilave edilmiş yem verilen grubun canlı ağırlığı kontrol grubuna kıyasla 3. haftada 30 g, 6. haftada ise 62 g daha yüksek bulunmuştur.

Tablo 2. Denemede kullanılan karma yemlerin yapısı ve kimyasal analiz sonuçları

Table 2. Ingredients and chemical composition of the experimental diets

Yemler (%)

Etlik Civciv Yemi (g/kg)

Etlik Piliç Yemi (g/kg)

Kimyasal Analiz Sonuçları

Etlik Civciv Yemi (%)

Etlik Piliç Yemi (%)

Mısır 313.98 351.95 Kuru madde 90.04 89.42

Buğday 220.00 220.00 Ham Protein 21.12 19.02

Soya küspesi (%48) 222.10 177.01 M.E. (kcal/kg) 3011 3100

Tam yağlı soya 60.00 60.00 Ham Yağ 6.55 7.68

Ayçiçeği toh.küspesi 110.00 110.00 Ham Sellüloz 4.87 4.80

Bitkisel yağ 36.38 46.17 Ham Kül 6.51 6.28

Kireç taşı 12.17 11.73 Kalsiyum 0.90 0.85

D.C.P. 14.15 13.09 Toplam Fosfor 0.66 0.62

Tuz 2.50 2.50 Lizin 2 1.25 1.05

Vit.Premix* 2.50 2.50 Metiyonin + sistin 2 0.88 0.78

Min.Premix** 1.00 1.00 Metiyonin 2 0.52 0.45

Koksidiyostat 0.50 0.50

Metiyonin 1.88 1.40

Lizin 1.84 1.15

Esansiyel yağ karışımı 1.00 1.00* 2.5 kg vitamin karışımı 12.000.000 IU Vit. A, 1.500.000 IU Vit. D3, 30.000 mg Vit. E, 5.000 mg Vit. K3, 3.000mg Vit. B1, 6.000 mg Vit. B2, 5.000 mg Vit. B6, 30 mg Vit. B12, 40.000 mg Nicotinamid, 10.000 mg Calcium-D-pentothenate, 750 mg Folik asit, 75 mg D-Biotin, 375.000 mg Choline Chloride içerir** 1 kg mineral karışımı 80.000 mg mangan, 40.000 mg demir, 60.000 mg çinko, 5.000 mg bakır, 400 mg iyot, 100 mg kobalt, 150 mg selenyum, 10.000 mg antioksidan içerir2 Hesaplanmış içerik

Tablo 3. Yeme EYK ilavesinin etlik piliçlerin büyüme performansı üzerine etkileri (1. Deneme)

Table 3. Growth performance of broilers fed on diets supplemented with EOM (Experiment 1)

Gruplar Civciv ÇıkışAğırlığı (g)

0-21 gün 0-42 gün

21. Gün Canlı Ağırlığı (g)

Yem Tüketimi (g) YYO Ölüm Oranı

(%)42. Gün Canlı

Ağırlığı(g)Yem

Tüketimi (g) YYO Ölüm Oranı(%)

Kontrol 43.90 613 958.1 1.685 1.00 2240 4262 1.945 3.66

EYK 43.93 624 983.1 1.693 1.00 2259 4248 1.917 2.00

Ort. Std. hata 0.07 5.45 12.05 0.03 0.68 21.27 47.40 0.02 0.73

P değeri 0.8698 0.1555 0.1593 0.8587 1.000 0.4085 0.8369 0.3487 0.1957


Yeme EYK ilavesinin etlik piliçlerin 1-21 ve 1-42 günler arasındaki yem tüketimi üzerine denemelerin her ikisinde de etkisi olmamıştır (P>0.05). Etlik piliçlerin 1-21 ve 1-42 günler arasındaki yemden yararlanma oranı üzerine yeme EYK ilavesinin etkisi birinci denemede önemsiz bulunurken (P>0.05), ikinci denemede önemli bulunmuştur. İkinci de-nemede EYK ilave edilmiş yem verilen etlik piliçlerin 1-21 ve 1-42 günler arasındaki yemden yararlanma oranları kont-rol grubuna kıyasla sırasıyla %2.78 ve %3.82 düzeyinde iyi-leşmiştir (P<0.01).

Yeme EYK ilavesi etlik piliçlerin 1-21 ve 1-42 günler ara- sındaki ölüm oranı üzerine iki denemede de etkide bulun-mamıştır (P>0.05).

EYK ilavesinin etlik piliçlerin karkas randımanı ve çalış-mada incelenen iç organ ağırlıkları üzerine etkilerine ait bulgular Tablo 5 ve Tablo 6’da verilmiştir. Karkas randımanı üzerine EYK ilavesinin etkisi birinci denemede önemli (P<0.05), ikinci denemede önemsiz (P>0.05) bulunmuştur. Yeme performans artırıcı yem katkı maddesi olarak ilave edilen EYK birinci denemede kesim randımanını kontrol

grubuna kıyasla %1.42 düzeyinde artırmıştır.

Yeme EYK ilavesinin incelenen tüm iç organ ağırlıkları üzerine olan etkisi birinci deneme önemsiz (P>0.05) bulun-makla birlikte, ikinci denemede EYK pankreas ağırlığını artırıcı etkidei bulunmuştur (P<0.05).

TARTIŞMA ve SONUÇ

Bu çalışmanın ikinci denemesinin sonuçlarına benzer şekilde aynı EYK ile önceki yapılan çalışmalarda 4,5,15 benzer dozda (48 mg/kg) yeme EYK karışımı ilavesinin canlı ağırlığı artırdığı belirlenmiştir. EYK’nın canlı ağırlık kazancı üzerinde sağladığı iyileşmenin sayısal düzeyde olduğunu bildiren Bozkurt ve ark.16 ile bu çalışmanın birinci denemesine ait sonuçlar benzerdir. Farklı esansiyel yağların kullanıldığı benzer çalışmalarda da etlik piliç yemlerine esansiyel yağ katılmasının kontrol grubuna kıyasla canlı ağırlık kazancını arttırdığı belirlenmiştir 9,17-19. Buna karşılık diğer kimi araş- tırmalarda ise yeme esansiyel yağların tek başına veya karı-şımlarının ilave edilmesinin canlı ağırlık kazancı üzerinde

Tablo 4. Yeme EYK ilavesinin etlik piliçlerin büyüme performansı üzerine etkileri (2. Deneme)

Table 4. Growth performance of broilers fed on diets supplemented with EOM (Experiment 2)

Gruplar Civciv ÇıkışAğırlığı (g)

0-21 gün 0-42 gün

21. Gün Canlı Ağırlığı (g)

Yem Tüketimi (g) YYO Ölüm Oranı

(%)42. Gün Canlı

Ağırlığı (g)Yem

Tüketimi (g) YYO Ölüm Oranı(%)

Kontrol 43.25 693b 1120 1.723a 0.50 2289b 4639 2.065a 1.00

EYK 43.12 723a 1139 1.675b 0.50 2351a 4585 1.986b 0.50

Ort. Std. hata 0.07 4.65 13.01 0.015 0.32 15.48 38.27 0.017 0.32

P değeri 0,7965 0.0001 0.3141 0.0474 1.000 0.0050 0.3343 0.0003 0.3684

a, b: Aynı sütunda farklı harf taşıyan ortalamalar arasındaki farklılıklar önemlidir (P<0.05)

Tablo 5. Etlik piliç yemlerine esansiyel yağ karışımı ilavesinin karkas randımanı ve bazı iç organ ağırlıkları (%) üzerine etkileri (1. deneme)

Table 5. Carcass yield and some internal organ weight of broilers (%) fed on diets supplemented with essential oil mixture (Experiment 1)

Gruplar Karkas Randımanı Karaciğer Dalak İnce

BağırsakKalın

Bağırsak Pankreas Kör Bağırsak

Kontrol 77.18b 2.41 0.10 3.21 0.18 0.29 0.47

EYK 78.28a 2.26 0.10 3.07 0.19 0.25 0.47

Ort. Std. hata 0.33 0.06 0.006 0.14 0.03 0.014 0.03

P değeri 0.0323 0.1193 0.5566 0.4658 0.6956 0.0809 0.8261


Tablo 6. Etlik piliç yemlerine esansiyel yağ karışımı ilavesinin karkas randımanı ve bazı iç organ ağırlıkları (%) üzerine etkileri (2. deneme)

Table 6. Carcass yield and some internal organ weight of broilers (%) fed on diets supplemented with essential oil mixture (Experiment 2)

Gruplar Karkas Randımanı Karaciğer Dalak İnce Bağırsak Kalın Bağırsak Pankreas Kör Bağırsak

Kontrol 77.62 2.05 0.11 2.76 0.17 0.25b 0.37

EYK 77.62 2.08 0.12 2.89 0.18 0.30a 0.40

Ort. Std. hata 0.48 0.06 0.007 0.11 0.01 0.01 0.02

P değeri 0.9952 0.7333 0.1901 0.3851 0.3300 0.0112 0.2500


295

KÜÇÜKYILMAZÇATLI, ÇINAR

belirgin bir iyileşme sağlamadığı bildirilmiştir 20-24.

Benzer EYK ile yapılan önceki çalışmalarda 5,6,16 EYK‘nın yem tüketimi üzerine olumsuz bir etkisi belirlenmemiştir. Esansiyel yağlarla yürütülen önceki çalışmalarda da aromatik etkilerinden kaynaklanan iştahı arttırıcı veya azaltıcı belirgin etkileri görülmemiş ve etlik piliçlerin toplam yem tüketimi üzerine önemli bir etkileri bulunmamıştır 21-23. Esansiyel yağların bileşimi ve aktif madde içeriğindeki de-ğişimler, uygulanan manejman koşullardaki farklılıklar, deneysel yemlerin bileşimi ve besin madde içeriğindeki değişikliklere rağmen yürütülen çalışmalarda yem tüketi- mi açısından önemli düzeyde farklılık bulunmaması dikkat çekicidir.

Yemden yararlanma oranı üzerine EYK’nın etkisi bakı- mından önceki çalışmalarda farklı sonuçlar alınmıştır. Etlik piliç yemine antibiyotik (avilamycin) yerine 3 farklı düzeyde (24, 48 ve 72 ppm) EYK ilave edilmesi durumunda, 21. ve 42. gün itibariyle en iyi yemden yararlanma değerinin yeme 48 ve 72 ppm düzeyinde EYK ilave edilen gruplardan elde edildiği; kontrol grubu ile antibiyotik ve 24 ppm EYK verilen etlik piliçlerin ise bunlardan daha kötü yemden yararlanma kabiliyetine sahip oldukları belirlenmiştir 4. Aynı araştırıcıların 5 yaptıkları diğer bir çalışmada ise yeme 36 ppm ve 48 ppm EYK ilavesinin 0-3 ve 0-6 hafta yemden yararlanma değerleri üzerine etkisinin olmadığı bildiril-miştir. Bir diğer çalışmada ise EYK ilave edilmiş yemle bes-lenen piliçlerin yemden yararlanma değerinin başlangıç ve büyütme dönemlerinde de kontrol grubundan daha iyi olduğu bildirilmiştir 16.

Yeme EYK ilavesi etlik piliçlerin ölüm oranı üzerine iki denemede de önemli etkide bulunmamış olup, daha önce aynı EYK karışımı ile yapılan çalışmalarla 5-7,15,16 benzer so- nuçlara ulaşılmıştır.

Etlik piliç yemine 48 ppm ve 72 ppm EYK ilave edil-mesinin karkas randımanını önemli düzeyde artırdığı yö-nündeki bildirişler 5,6, bu çalışmanın birinci denemesinin sonuçlarıyla benzerdir. EYK’nın birinci denemede kontrol grubuna kıyasla karkas randımanını artırmış olması benzer miktarda yem tüketimi karşılığında vücutta daha fazla kas kitlesi birikimi sağladığını göstermektedir. Etlik piliçlerin ekonomik değere sahip olan kısmının vücut kasları olduğu düşünüldüğünde, EYK’nın karkas randımanını artırmak suretiyle etlik piliç yetiştiriciliğindeki verimliliği iyileştirdiği görülmektedir. Buna karşılık diğer bazı çalışmalarda 6,18,21,24 esansiyel yağların karkas randımanını etkilemediği bildi-rilmiştir.

Yeme esansiyal karışımı ilavesi ikinci denemede etlik piliçlerin pankreas ağırlığını artırmış olup, bunun esansiyel yağların safra asidi salgısını arttırmak suretiyle bağırsak mukozası ve pankreasın enzim salgısını teşvik etmelerin- den 25 kaynaklandığı düşünülmektedir. Ayrıca esansiyel yağların antimikrobiyal etkilerinin bağırsak mikroflorasın- daki patojen yükünü azaltarak bağırsak ağırlığını düşür-

düğü tespit edilmiştir 5. Daha önceki yapılan çalışmalarda da bu denemelerde kullanılan EYK’nın ve farklı eterik yağ-ların iç organ ağırlıklarını etkilemediği bildirilmiştir 6,18,23,26,27.

Bu çalışmada yeme katılan EYK’nın etlik piliçlerin canlı ağırlık kazancı ve yemden yararlanma değeri ile karkas randımanı üzerinde sağladığı önemli iyileşmeyi EYK’nın sahip olduğu antimikrobiyal etkinin yanı sıra, sahip olduğu enzimatik aktiviteyle besin madde sindirilebilirliğini arttı- rarak gerçekleştirdiği düşünülmektedir. Nitekim, esansiyel yağların ileal sindirilebilirliği önemli düzeyde yükselterek yemin sindirilebilirliğini artırdığı ve dolayısıyla etlik piliç- lerin yemden yararlanma kabiliyetini iyileştirdiği belirlen-miştir 28.

Benzer metodoloji ile yürütülen iki ayrı denemede EYK’nın kimi özellikler üzerine farklı etkiler göstermesi ma-nejman faktörlerinden dikkate alınmayan bazılarının (ör- neğin etçi damızlıkların maternal bağışıklık düzeylerindeki farklılıklar) esansiyal yağların performans artırıcı etkinli- ğini farklı biçimde yönlendirebileceğini gösterir nitelik-tedir. Nitekim ebeveynlere verilen yemlerin besin madde bileşimlerinin civcivlerin performanslarını etkileyebilece- ğini gösteren bilimsel kanıtlar mevcuttur 29-31. Ayrıca da- mızlık yaşı, yumurta depolama süresi ve kuluçka şartlarının etlik civcivlerin büyüme performanslarını etkiyebileceği bil- dirilmiştir 32.

Sonuç olarak, çalışmada etlik piliç yemlerine EYK ila-vesinin birinci denemede yemden yararlanma oranını iyi-leştirmesi, ikinci denemede vücuttaki yenilebilir kısımları artırmış olması, aromatik bitkilerin esansiyel yağlarının performans artırıcı yem katkı maddesi olarak kullanım ola- naklarının bulunduğunu göstermektedir.

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SummaryIn this study the 96 h acute toxicity (LC50) of lead (Pb) for common carp (Cyprinus carpio L., 1758) was investigated and the fish

was exposed to sub-lethal concentration of lead for 96 h. The hematological and biochemical parameters (red blood cell count, hemoglobin, hematocrit, white blood cell count and leucocrit) and plasma glucose, lactate and total protein levels and plasma ion concentrations of fish were investigated and compared to control fish. It was found that lead had significant effects on red blood cell count, hemoglobin levels, plasma glucose and lactate levels and plasma ions, while hematocrit, white blood cell count, leuocrit and plasma total protein levels remained unchanged.

Keywords: Lead, Lactate, Acute toxicity, LC50, Plasma ions, Probit

Kurşunun Sub-lethal Dozlarına Maruz Bırakılan Sazan Balıklarında (Cyprinus carpio L., 1758) Hematolojik

ve Biyokimyasal Parametreler ve Plazma İyon Konsantrasyonlarındaki Değişimler

ÖzetBu çalışmada kurşunun (Pb) sazan balıkları (Cyprinus carpio L., 1758) için için 96 saatlik LC50 değeri belirlenmiş ve balıklar 96 saat

süreyle kurşunun sub-lethal konsantrasyonuna maruz bırakılmıştır. Çalışmada balıkların alyuvar sayısı, hemoglobin ve hematokrit seviyesi, akyuvar sayısı ve lökokrit gibi hematolojik parametrelerinin yanısıra plazma glukoz, laktat ve total protein seviyesi gibi biyokimyasal parametreleri ile birlikte plazma iyon konsantrasyonları da incelenmiş ve kontrol grubu balıklar ile karşılaştırılmıştır. Kurşunun alyuvar sayısı, hemoglobin seviyesi, plazma glukoz ve laktat seviyesi ve plazma iyonları üzerinde önemli bir etkisi olduğu, akyuvar sayısı, lökokrit ve plazma total protein seviyesinin ise değişmediği bulunmuştur.

Anahtar sözcükler: Kurşun, Laktat, Akut toksisite, LC50, Plazma iyonları, Probit

Alterations in the Hematological and Biochemical Parameters and Plasma Ion Concentrations of Common Carp, (Cyprinus

carpio L., 1758) After Short Term Exposure to Sub-lethal Concentrations of Lead

Mehmet Borga ERGÖNÜL * Sibel ATASAĞUN * Kübra KOCATÜRK *

* Ankara University, Faculty of Science, Department of Biology, TR-06100 Tandoğan, Ankara - TURKEY


The contamination of aquatic environments with a wide range of pollutants has become a matter of great concern over the last few decades. Among these pollutants, heavy metals are natural trace components of the aquatic environment, but their levels have increased due to domestic, industrial, mining and agricultural activities 1,2, thus, constitute a considerable part of the

aquatic pollution. Discharge of heavy metals into aquatic environment can change both aquatic species diversity and ecosystem, due to their toxicity and accumulation. In addition, although, physiologic roles of heavy metals such as iron, zinc, copper and to a lesser extent chromium are known, metals such as lead and cadmium are not believed to be essential for health even in trace amounts 3. Chronic

INTRODUCTION


RESEARCH ARTICLE

298Alterations in the Hematological ...

low level exposure to heavy metals has adverse effects not only on aquatic animals but also on human beings via food chain, due to the fact that there is no effective mechanism for their elimination from the body 4. Thus, accurate information on heavy metal toxicity on aquatic organisms is needed.

Among heavy metals, lead (Pb) is non-essential, non-beneficial and toxic to many organisms even at very low concentrations 5-8. Lead toxicity may occur from chronic waterborne exposure at concentrations as low as 4 µg l-1 8

which is in the range of concentrations reported from freshwater environments 1, thus creating a demand for further studies. Although, a number of studies focus on the toxicity of lead on various fish species including eel (Anguilla anguilla L., 1758) 5, Nile Tilapia (Oreochromis niloticus L., 1758) 9, rainbow trout (Oncorhynchus mykiss Walbaum, 1792) 7,10, streaked prochilod (Prochilodus lineatus Valenciennes, 1837) 11, tench (Tinca tinca L., 1758) 12, and carp (Cyprinus carpio L., 1758) 6,13, little is known on the physiological effects of lead on common carp.

Investigations on the toxic effects of heavy metals on fish may be accompanied by the analysis of changes in certain hematological and biochemical blood parameters. Hematological and biochemical profile in fish is proved to be a sensitive index for the evaluation of fish metabolism under metallic stress and can be used as an indicator for pollution. The hematological parameters of fish such as red blood cell count, hematocrit, hemoglobin, white blood cell count and leucocrit may be used to assess the functional status of the oxygen carrying capacity of the blood stream and as an indicator of metal pollution in aquatic environment 12,14. Changes in carbohydrate metabolism measured as plasma glucose and lactate can also be used as general stress indicators in fish. Ions of body fluids have various functions such as contributing a majority of the osmotically active particles, to provide buffer systems and the mechanisms for the regulation acid-base balance 15.

The present study was performed on common carp

(C. carpio) which has a widespread distribution in many Asian and European countries, since it is one of the most commercially important and widely cultivated freshwater fish. Thus, available data on carp biology provide a comprehensive database for further studies. Although, it has been the subject of many toxicological studies, there are no studies demonstrating the changes in the hematological and biochemical parameters of common carp exposed to Pb. Hence the aim of this study was to determine the acute toxicity (96 h LC50) of Pb for carp with a static test system and to investigate the possible effects of sub-lethal lead exposure on carp by determining certain hematological and biochemical parameters such as red blood cell count, hemoglobin, hematocrit, white blood cell count and leucocrit, plasma glucose, lactate and total protein levels and plasma concentrations of ions such as sodium (Na+), potassium (K+) and chloride (Cl-).


Test fish, common carp (C. carpio), were captured with gill nets from Lake Mogan (Ankara, Turkey). Fish were brought to laboratory and placed in 800 l tanks and allowed for at least 2 weeks for acclimatization to the laboratory conditions. Mean weight and length of fish was 210.2±8.9 g and 20.2±3.3 cm, respectively. Fish were maintained in aerated dechlorinated tap water at 17.7±1.47°C and fed with commercial feed (45% crude protein) at a rate of 2% body weight. Fish were not fed for 48 h prior to and during the experiments. The physico-chemical properties of the water were as follows; pH 7.71±0.49, electrical conductivity (EC) 217.47±16.95 µS cm-1, dissolved oxygen 6.39±0.45 mg l-1, total hardness and total alkalinity 77.5 mg l-1 and 80 mg l-1 as CaCO3, respectively.

After acclimatization period, randomly selected 10 fish were transferred to 100 l aquaria for the determination of 96 h LC50 of lead. Lead was added to the water as lead nitrate Pb(NO3)2 (Sigma-Aldrich). Ten concentrations were tested to estimate lethal concentration of lead (Table 1).

Table 1. Tested lead concentrations and mortality rates of fish

Tablo 1. Test edilen kurşun konsantrasyonları ve balıklardaki mortalite oranı

Exposure Concentration (mg Pb2+ l-1)

Number Exposed

Number Responded

Mortality Rate

Expected Mortality Rate

Calculated Mortality Rate

1 10 0 0.00 0.00 0

5 10 0 0.00 0.00 0.0005

10 10 1 0.10 0.10 0.0448

15 10 2 0.20 0.20 0.2212

20 10 4 0.40 0.40 0.4568

25 10 6 0.60 0.60 0.6566

30 10 7 0.70 0.70 0.6890

35 10 8 0.80 0.80 0.8799

40 10 10 1.00 1.00 0.9307

45 10 10 1.00 1.00 0.9600

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ERGÖNÜL, ATASAĞUNKOCATÜRK

Half of the water in the aquaria was replaced once (at 48 h intervals) during bioassays and lead nitrate solution was added to water to keep the required concentration constant. Dead fish were counted at 12 h intervals and removed from aquaria. After bioassay, probit analysis was performed with EPA 16 probit analysis program (v 1.5).

After calculation of the 96 h LC50 of lead, fish were exposed to sub-lethal concentration (10% of lethal concentration = 2 mg Pb2+ l-1) of lead for 96 h. After exposure, blood samples were taken from the caudal vein from fish from both exposure (N=10) and control (N=10) groups with heparinized syringes. Control fish were handled in the same way. Hemoglobin (Hb) levels were determined with a commercial kit (Roche) according to the instructions supplied with. Hematocrit (Hct) and leucocrit (Lct) measurements were made according to Blaxhall and Daisley 17 and McLeay and Gordon 18, respectively. Red blood cell (RBC) and white blood cell (WBC) counts were made with Improved Neubauer hemocytometer, using Natt-Herrick as the diluent and stain 19. Blood plasma was obtained by centrifugation at 14.000 rpm for 10 min and stored in eppendorf tubes at -35°C until analysis. Plasma sodium (Na+), potassium (K+), chloride (Cl-), glucose, total protein levels (Teco Diagnostics) and plasma lactate levels (Randox) were determined by using biochemical kits according to the instructions supplied with.

Data are presented as mean±standart deviation (SD) and were compared with One-Way ANOVA using the statistical packet program (SPPS v 9.0) at a significance level of P<0.05.

RESULTS

The data obtained from the toxicity tests of lead nitrate on C. carpio were evaluated by using EPA probit Analysis Program 14 and the 96 h LC50 value was found as 20.97 mg Pb2+ l-1 (95% confidence limits; 17.43-24.29) (Table 2). No

mortality was observed during 96 h at lead concentration of 1.00 and 5.00 mg l-1 and mortality rate was 100% at 40 and 45 mg l-1. No mortality was observed for control fish.

Parameters related to red blood cell system showed marked changes. Red blood cell count and hemoglobin levels were elevated by 47.47% and 25.42% respectively (Fig. 1a, 1b). Hematocrit levels did not show a marked difference compared to control (Fig. 1c) (P>0.05). No significant differences were observed between exposure and control groups in respect to white blood cell counts and leucocrit levels (Fig. 2a, 2b).

The changes in plasma ion concentrations for carp exposed to lead at a concentration of 2 mg l-1 (10% of the 96 h lethal concentration) for 96 h are shown in Fig. 3a-3c. Plasma Na+ and Cl- levels decreased by 23.18% and 17.12%, respectively, compared to control fish. Plasma K+ levels increased by 53.43% compared to control fish. Plasma glucose levels of fish exposed to lead increased by 60.39% (Fig. 4a). Plasma lactate levels also showed an increase (47.42%) (Fig. 4b). Total protein levels of fish exposed to lead showed no significant alterations (Fig. 4c).

Table 2. Estimated LC values for carp exposed to lead

Tablo 2. Kurşuna maruz bırakılan sazan balıkları için hesaplanan LC değerleri

Point Exposure Concentration (mg l-1)

95% Confidence Limits

LC1.00 7.60 3.75 - 10.61

LC5.00 10.23 5.99 - 13.27

LC10.00 11.99 7.67 - 14.99

LC15.00 13.34 9.05 - 16.31

LC50.00 20.97 17.43 - 24.29

LC85.00 32.95 28.04 - 43.30

LC90.00 36.67 30.69 - 50.76

LC95.00 42.96 34.86 - 64.65

LC99.00 57.84 43.84 - 102.78

a b c

Fig 1. Red blood cell (RBC) counts (a), hemoglobin (b) and hematocrit levels (c) of fish. Dark bars represent exposure group, light bars represent control group, * P<0.05

Şekil 1. Balıklarda alyuvar sayısı (a), hemoglobin (b) ve hematokrit seviyesi (c). Koyu sütunlar deneme grubunu, açık sütunlar kontrol grubunu temsil etmektedir, * P<0.05


DISCUSSION

In the present study, the 96 h LC50 value of lead for carp was found as 20.97 mg Pb2+ l-1. Martinez et al.11 found that 96 h LC50 for P. lineatus was 95 mg Pb2+ l-1. Al-Akel and Shamsi 9 reported the 96 h LC50 as 12.45 and 22.65 mg Pb2+ l-1 for O. niloticus and Clarias gariepinus (Burchell, 1822), respectively. Hodson et al.10 stated that 21 day LC50 was 2.4 mg Pb2+ l-1 for O. mykiss. Rogers et al.7 showed that for same fish species 96 h LC50 was 1.0 mg Pb2+ l-1. Shah 12

found that 96 h LC50 for T. tinca was 300 mg Pb2+ l-1. The 96 h LC50 for C. carpio was reported as 0.44-0.80 mg Pb2+ l-1 and 8.2-1291 mg Pb2+ l-1 by Alam and Maughan 13 and Datta and Das 6, respectively. The values obtained for lead by toxicity tests by different authors show a high degree of variation, even for the same species. It is known that such variation might be due to several factors influencing toxicity, such as temperature, pH, dissolved oxygen as well as the fish species itself 6,7. Furthermore, it is also known that in static bioassays, lead content may vary depending on the absorption, adsorption and precipitation 11.

a b

Fig 2. White blood cell (WBC) counts (a) and leucocrit levels (b) of fish. Dark bars represent exposure group, light bars represent control group

Şekil 2. Balıklarda akyuvar sayısı (a) ve lökokrit seviyesi (b). Koyu sütunlar deneme grubunu, açık sütunlar kontrol grubunu temsil etmek-tedir

Fig 3. Plasma Na+ (a), Cl- (b) and K+ (c) concentrations of fish. Dark bars represent exposure group, light bars represent control group, * P<0.05

Şekil 3. Balıklarda plazma Na+ (a), Cl- (b) ve K+ (c) konsantrasyonları. Koyu sütunlar deneme grubunu, açık sütunlar kontrol grubunu temsil etmektedir, * P<0.05

a b c

Fig 4. Plasma glucose (a), lactate (b) and total protein (c) levels of fish. Dark bars represent exposure group, light bars represent control group. * P<0.05

Şekil 4. Balıklarda plazma glukoz (a), laktat (b) ve total protein (c) seviyesi. Koyu sütunlar deneme grubunu, açık sütunlar kontrol grubunu temsil etmektedir, * P<0.05

a b c

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Since blood is a pathophysiological reflector of the whole body, hematological parameters may provide a reliable data for the health status of fish 14 and may be used as indicator of pollution 12,20. It was observed that sub-lethal lead exposure for 96 h caused a marked increase in red blood cell count and hemoglobin levels of carp. However, it was found that hematocrit levels remained unchanged. Elevated numbers of red blood cells and hemoglobin levels without a marked increase in hematocrit levels, may indicate the increase of newly formed immature red blood cell population and shortening the life span of mature red blood cells, which may give rise to the pattern observed in this study; a shift in red blood cell population from mature red cells to newly formed immature cells. Such a pattern may also give acceleration to release of new red blood cells from the erythropoetic organs and synthesis of hemoglobin to compensate for the loss in oxygen carrying capacity and compensating for red blood cell loss of fish exposed to lead 9-11. Besides, the increase in hemoglobin levels may be a response to replace abnormal hemoglobin which might have been oxidized or denatured by the metal that enter the red blood cells, which in turn stimulate erythropoietic tissues 21.

It was found that lead exposure had no effect on the white blood cell count and leucorit levels of carp in this study. However, Shah and Altındağ 20 found that lead exposure had significant effects on white blood cell counts and leucocrit levels; with both decreases and increases depending on the exposure time and concentration. Al-Akel and Shamsi 9 reported that lead exposure had no effect on white blood cell count of O.niloticus. Santos and Hall 5 reported that thrombocyte counts remained unchanged. They also reported that lymphocyte counts increased while neutrophil counts decreased. According to our results it is reasonable to conclude that sub- lethal lead exposure did not cause an immunological impairment in carp.

Ions of body fluids in fish have various functions such as contributing a majority of the osmotically active particles and providing buffer systems for the regulation acid-base balance. In freshwater fishes ions such as Na+, Cl- and K+ have a significant role in keeping the body fluids hyperosmotic 15 and it is suggested that plasma ion levels may be employed for quantifying toxic effects of metals 22. In the present study it was found that plasma Na+ and Cl- levels were decreased and plasma K+ levels were increased. The pattern observed for plasma ions in the present study was also reported for common carp exposed to diazinon 23, cypermethrin 14 and acidic pH 24. Rogers et al.7 found that plasma K+ levels did not show any significant differences while a decrease was observed for plasma Na+ and Cl- in rainbow trout exposed to lead. Santos and Hall 5 found no differences in the plasma Na+ and K+ levels of eel exposed to lead (Cl- levels were not mentioned). In freshwater fish, it is known that

Na+ and Cl- ions are taken up by active transport across osmoregulatory surfaces, especially by gills. The decrease in both plasma Na+ and Cl- ions during lead exposure in this study may be attributed to the increased permeability of gill epithelium which lead to elevation in the efflux of Na+ and Cl- from branchial epithelium and increased water uptake by the gills 15,25, or a decrease and/or inhibition of Na+/K+-ATPase activity 7. It is possible that the decrease in plasma Cl- levels might be due to a shift of Cl- into red blood cells due to the effect of lowered plasma pH and/or a penetration of Cl- into the intracellular compartment to balance the efflux of lactate to intercellular compartment that leads to decrease blood pH. The increase in the plasma K+ levels may be the result of an efflux from intracellular compartment. Potassium is the dominant intra-cellular cation and plasma ionic dilution would favor efflux of K+ into extracellular fluid. On the other hand, it is also possible that the alterations observed on the plasma ions may simply result from a non-specific stress response of fish.

Plasma or serum glucose level has been used a sensitive indicator of stress in fish. In the present study plasma glucose levels showed an increase when compared to control fish. Lactate is a closely related parameter to glucose metabolism and has been used as an indicator of anaerobic metabolism. In the present study, lactate levels also significantly increased. Although there are a few studies that show lead has no effect on plasma lactate levels in fish 7, several authors reported an increase both in glucose and lactate levels in fish 5,26,27. It is known that plasma lactate levels increase typically in stressed fish, particularly if any aspect of the stressor results in a decrease in oxygen availability 28. Thus it is acceptable to conclude that lead exposure may lead to a shift from aerobic to anaerobic metabolism in carp. There were no significant differences between the total plasma protein levels of exposure and control groups. A similar pattern was observed for eel exposed to lead 5.

In conclusion, in the present study it is found that lead at sub-lethal concentrations had marked effects on red blood cell system, plasma ion concentrations and plasma glucose and lactate levels. However, we did not find any marked alterations in the white blood cell count and leucocrit levels. Taking the whole picture into account, it seems that sub-lethal lead exposure effects mainly the respiratory system of carp by altering the function of gills via ion balance alteration and/or enzyme activity in gills 7 and/or simply by physical blockage of gill surface by excess mucus 12 caused by lead exposure which in turn stimulated erythropoietic tissues to compensate for the decrease in oxygen levels which is also proved by the increase in lactate levels. It is also reasonable to speculate that lead also had deleterious effects on mature red blood cell life span which might also has given rise to the release of immature red blood cells.


REFERENCES

1. WHO (World Health Organization): Environmental Health Criteria 165. International Programme on Chemical Safety. Geneva, 1995.

2. USEPA (United States Environmental Protection Agency): National Recommended Water Quality Criteria. Office of Water, Washington, DC, 2006.

3. Canlı M, Atlı G: The relationship between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species. Environ Pollut, 121, 129-136, 2003

4. Bahemuka TE, Mubofu EB: Heavy metals in edible green vegetables grown along the sites of the Sinza and Msimbazi rivers in Dares Salaam, Tanzania. Food Chem, 66, 63-66, 1999.

5. Santos MA, Hall A: Influence of organic lead on the biochemical blood composition of the eel (Anguilla anguilla). Ecotoxicol Environ Saf, 20, 7-9, 1990.

6. Datta S, Das RC: Influence of some abiotic environmental factors on acute toxicity of inorganic lead to Cyprinus carpio var communis and Catla catla in simulated toxic aquatic environment. Toxicol Environ Chem, 85, 203-219, 2003.

7. Rogers JT, Richards JG, Wood CM: Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout. Aquat Toxicol, 64, 215-234, 2003.

8. Grosell M, Gerdes RM, Brix KV: Chronic toxicity of lead to three freshwater invertebrates - Brachinous calyciflorus, Chironomus tentans and Lymnea stagnalis. Environ Toxicol, 25, 97-104, 2006.

9. Al-Akel AS, Shamsi MJK: A comparative study of the toxicity of lead and its impact on the carbohydrate metabolism and some hematological parameters of cichlid fish Oreochromis niloticus and catfish Clarius gariepinus from Saudi Arabia. Toxicol Environ Chem, 74, 19-28, 2000.

10. Hodson PV, Blunt BR, Spry DJ: Chronic toxicity of water borne and dietary lead to rainbow trout in lake Ontario water. Water Res, 12, 869-878, 1978.

11. Martinez CBR, Nagae MY, Zaia CTB, Zaia DAM: Acute morphological and physiological effects of lead in the neotropical fish Prochilodus lineatus. Braz J Biol, 64, 797-807, 2004.

12. Shah SL: Hematological parameters in tench Tinca tinca after short term exposure to lead. J Appl Toxicol, 26, 223-228, 2006.

13. Alam MK, Maughan OE: Acute toxicity of heavy metals to common carp (Cyprinus carpio). J Environ Sci Health, 30, 1807-1816, 1995.

14. Suvetha L, Ramesh M, Saravanan M: Influence of cypermethrin toxicity on ionic regulation and gill Na/k-ATPase activity of a freshwater

teleost Cyprinus carpio. Environ Toxicol Pharm, 29, 44-49, 2010.

15. Wendelaar Bonga, SE, Lock RAC: Toxicants and Osmoregulation in fish. Neth J Zool, 42, 478-493, 1992.

16. EPA (Environmental Protection Agency): LC50 software programme, version 1.5. Center for Exposure Assessment Modelling (CEAM). Distribution Center, 1999.

17. Blaxhall PC, Daisley KW: Routine haematological methods for use with fish blood. J Fish Biol, 5, 771-781, 1973.

18. McLeay DJ, Gordon MR: Leucocrit: A simple hematological technique for measuring acute stress in salmonid fish, including stressful concentrations of pulp mill effluent. J Fish Res Board Can, 34, 2164-2175, 1977.

19. Stoskopf MK: Fish Medicine. Saunders, Philadelphia, 1993.

20. Shah SL, Altındağ A: Alterations in the immunological parameters of tench (Tinca tinca) after acute and chronic exposure to lethal and sublethal treatments with mercury, cadmium and lead. Turk J Vet Anim Sci, 29, 1163-1168, 2005.

21. Cyriac PJ, Antony A, Nambisan PNK: Hemoglobin and hematocrit levels in the fish Oreochromis mossambicus after short term exposure to copper and mercury. Bull Environ Contam Toxicol, 43, 315-320, 1989.

22. Mayer FL, Versteeg DJ, Mckee MJ, Formoar LC, Graney RL, Mccume DC, Rattner BA: Physilogical and non-specific biomarkers. In, Huggest RJ, Kimerle RA, Mehrle-Jr PM, Bergman HL (Eds): Biomarkers: Biochemical, Physiological and Histological Markers of Anthropogenic Stress. pp. 5-86, Lewis Press, Boca Raton, 1992.

23. Luskova V, Svoboda M, Kolarova J: The effect of dianizon on blood plasma biochemistry in carp (Cyprinus carpio). Acta Vet Brno, 71, 117-123, 2002.

24. Mathan R, Kurunthachalam S, Priya M: Alterations in plasma electrolyte levels of s freshwater fish Cyprinus carpio exposed to acidic pH. Toxicol Environ Chem, 92, 149-157, 2010.

25. Singh NN, Das VK, Srivastava AK: Insecticides And Ionic Regulation In Teleosts: A Review. Zoologica Poloniae, 47, 21-36, 2002.

26. Kennedy CJ, Sweeting RM, Johansen JA, Farrell AP, McKeown BA: Acute effects of chlorinated resin exposure on juvenile rainbow trout. Environ Toxicol Chem, 14, 977-982, 1995.

27. Santos MA, Pacheco M: Anguilla anguilla stress biomarkers recovery in clean water and secondary treated pulp mill effluent. Ecotoxicol Environ Saf, 35, 96-100,1996.

28. Thomas PM, Pankhurst NW, Bremner HA: The effect of stress and exercise on post-mortem biochemistry of Atlantic salmon and rainbow trout. J Fish Biol, 54, 1177-1196, 1999.


SummaryThis study was planned to identify six parasites detected in horse mackerels (Trachurus trachurus) sold for human consumption

in Erzurum province. DNA extraction was performed on six parasites diagnosed as nematode. Species identification was done by PCR and PCR-RFLP analysis of ITS (ITS-1, 5.8S subunit rRNA gene and ITS-2) region of ribosomal DNA (rDNA) and partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) and mt-CO2 genes. According to the PCR and PCR-RFLP results, all parasites were identified as Anisakis pegreffii. Partial sequence of mt-CO1 gene of one randomly selected parasite was corresponding with A. simplex and mt-CO2 genes of two parasites were corresponding with A. pegreffii. With this study, A. pegreffii was molecularly detected for the first time in Turkey.

Keywords: Anisakis pegreffii, Fish, PCR, PCR-RFLP, Sequencing

Türkiye’nin Erzurum İlinde İnsan Tüketimi İçin Satılan İstavrit Balıklarında (Trachurus trachurus) Anisakis pegreffii’nin

Moleküler Tanısı

ÖzetBu çalışma, Erzurum ilinde insan tüketimi için satılan istavrit balıklarında (Trachurus trachurus) tespit edilen altı parazitin tür tanısını

yapmak amacıyla planlanmıştır. Nematod olarak belirlenen altı parazitten DNA ekstraksiyonu yapılmıştır. Tür tanısı, rDNA’nın ITS bölgelerinin (ITS1, 5.8 alt ünite rRNA ve ITS2) PCR ve PCR-RFLP analizleri ve mitokondrial sitokrom c oksidaz alt ünite 1 (mt-CO1) ve mt-CO2 genlerinin kısmi olarak sekanslanması ile yapılmıştır. PCR ve PCR-RFLP sonuçlarına göre tüm parazitlerin Anisakis pegreffii olduğu belirlenmiştir. Rastgele seçilen bir parazitin mt-CO1 geninin kısmi sekans sonucu A. simplex, iki parazitin mt-CO2 geninin kısmi sekans sonuçları ise A. pegreffii ile uyuşmuştur. Bu çalışma ile A. pegreffii Türkiye’de ilk kez moleküler olarak tespit edilmiştir.

Anahtar sözcükler: Anisakis pegreffii, Balık, PCR, PCR-RFLP, Sekanslama

Molecular Detection of Anisakis pegreffii in Horse Mackerels (Trachurus trachurus) Sold for Human Consumption in Erzurum

Province of TurkeyArmagan Erdem UTUK * Fatma Cigdem PISKIN * Ibrahim BALKAYA**

***

Etlik Veterinary Control Central Research Institute, Parasitology and Bee Diseases Laboratory, TR-06020 Ankara - TURKEYUniversity of Ataturk, Faculty of Veterinary Medicine, Department of Parasitology, TR-25240 Erzurum - TURKEY


The species of the genus Anisakis are the parasites of the stomach and intestine of pinnipeds (seal, sea-lion, walrus) and cetaceans (dolphin, narwhal, porpoise, whale). Larvae of Anisakis spp. have been reported in the body cavities or tissues of a great variety of marine and anadromous teleosts as well as squids and prawns 1-3.

Anisakiasis is an important fish-borne zoonosis.

Humans become accidental hosts by eating raw or poorly cooked marine fish or squid harboring larvae. Human anisakiasis occurs worldwide; the majority of cases are reported in Asian countries where consumption of sea- food is common. Difficulty in swallowing, stomach pain, abdominal pain, nausea, vomiting, diarrhea, isolated swellings to urticaria and life-threatening anaphylactic shocks are the symptoms of the disease. These symptoms

INTRODUCTION


RESEARCH ARTICLE

304Molecular Detection of ...

can be slight to severe 1,2.

Recent molecular studies on Anisakis revealed the existence of two main clades. One encompassing the species showing the larval stage is indicated as Anisakis Type I, and a second sharing the larval morphology as Anisakis Type II. The first clade includes the species of A. simplex complex (A. pegreffii, A. simplex sensu stricto and A. simplex C), A. typica, A. ziphidarum and Anisakis sp. The second includes the species A. physeteris, A. brevispiculata and A. paggie 4. These species are genetically characterized by molecular techniques, such as PCR (polymerase chain reaction), PCR-RFLP (restriction fragment length poly-morphism), RAPD-PCR (Random Amplified Polymorphic DNA) and DNA sequencing 5-11.

The aim of this study was to identify nematode species isolated from horse mackerels (Trachurus trachurus) sold for human consumption in Erzurum province of Turkey by use of PCR, PCR-RFLP and DNA sequencing techniques.


Parasite Materials

Six parasites were detected in horse mackerels (T. trachurus) sold for human consumption in Erzurum province of Turkey. The origin of the fishes was determined as Black Sea. Samples were put into 70% ethanol until use.

DNA Extraction

DNA was isolated from ethanol preserved parasites. Each parasite was cut into small pieces. Prior to DNA extraction, broken material was washed with 5X PBS and then digested for 6 h at 56°C with 250 μl TEN-SDS (50 mM Tris-HCl, pH 8, 5 mM EDTA, 100 mM NaCl, 10% SDS) containing 2 mg/ml Proteinase K. After digestion, 125 μl of 6M NaCl was added and the samples were vortexed for 15 seconds. Then, the samples were centrifuged for 5 minutes at 13.000 g in eppendorf tubes. Finally, the supernatants were transferred to new clean eppendorf tubes, and DNA was extracted using the classical phenol-chloroform and precipitation method. After drying, the DNA was suspended in 50 μl Tris-EDTA buffer (pH 7.6) 12, 13.

PCR-RFLP Analysis

Universal primers A (5′-gtcgaattcgtaggtgaacctgcggaa ggatca-3′) and B (5′-gccggatccgaatcctggttagtttcttttcct-3′) were used for the amplification of rDNA (ITS1, 5.8 subunit rRNA and ITS2) 5,7,14-16. PCR was carried out in a final volume of 50 μl, containing 23.75 μl DNase, RNase free steril distilled water (Biobasic, Inc), 5 μl 10X PCR buffer, 6 μl 25 mM MgCl2, 5 μl 1 mM dNTP mix, 2.5 μl of each primer (50 pmol), 5 μl of template DNA, and 0.25 μl of TaqDNA polymerase (1.25 IU) (MBI, Fermentas). PCR conditions were: 5 min at 95°C (initial denaturation), 35 cycles of 1 min at 95°C, 1 min at 48°C and 1 min at 72°C and finally 5 min at 72°C (final extension).

PCR products were digested with the restriction enzyme Hinf1 (MBI, Fermentas) by following the manufacturer’s instructions. Digested products were separated on agarose gels (3%), stained with ethidium bromide, visualised and photographed on an UV transilluminator (UVP).

Species-Specific PCR

APE1 (5′-gagcagcagcttaaggcagaggc-3′) and B (5′-gccg gatccgaatcctggttagtttcttttcct-3′) primer pair was specific to A. pegreffii and used for the partial amplification of 18S-28S rRNA gene 5. PCR mix and PCR conditions were same as above. PCR products were separated on agarose gels (1.5%), stained with ethidium bromide, visualised and photographed on an UV transilluminator (UVP).

Sequence Analysis

JB3 (5′-ttttttgggcatcctgaggtttat-3′) and JB4.5 (5′-taaa gaaagaacataatgaaaatg-3′) primer pair was used to amplify partial mt-CO1, and 211F (5′-ttttctagttatatagattgrttyat-3′) and 210R (5′-caccaactcttaaaattatc-3′) primer pair was used to amplify partial mt-CO2 genes of the nematodes 17,18. PCR was carried out in a final volume of 50 μl, containing 25.75 μl DNase, RNase free steril distilled water (Biobasic, Inc), 5 μl 10X PCR buffer, 5 μl 25 mM MgCl2, 4 μl 1 mM dNTP mix, 2.5 μl of each primer (50 pmol), 5 μl of template DNA (100-200 ng), and 0.25 μl of TaqDNA polymerase (1.25 IU ) (MBI, Fermentas). PCR conditions were: 5 min at 95°C (initial denaturation), 35 cycles of 1 min at 95°C, 1 min at 50°C and 1 min at 72°C and finally 5 min at 72°C (final extension). PCR products were separated on agarose gels (1.5%), stained with ethidium bromide, visualised and photographed on an UV transilluminator (UVP).

Amplicons of mt-CO1 gene of one parasite and mt-CO2 gene of two parasites were sequenced by a commercial company (Refgen, Ankara, Turkey). Obtained sequences were edited and aligned with CLC main workbench soft-ware 19; then, sequence analysis was undertaken by BLAST algorithms and databases from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov).

RESULTS

Digestion of approximately 1000 bp region of rDNA (ITS1, 5.8 subunit rRNA and ITS2) (Fig. 1) with Hinf1 produced the same banding pattern in all six parasites analyzed. All parasites showed three bands of 250, 300, 370 bp which were specific to A. pegreffii (Fig. 2). PCR products of 672 bp were amplified in all of six parasites with primer pair APE1 and B, which is specific for A. pegreffii (Fig. 1). Mt-CO1-PCR yielded 446 bp and mt-CO2-PCR yielded 629 bp of amplification products (Fig. 1). When compared to GenBank results, mt-CO1 sequence (GenBank accession no JN102304) was corresponding with A. simplex (94-98%), and mt-CO2 results (GenBank accession no JN827308 and JN827309) were corresponding with A. pegreffii (97-99%).

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DISCUSSION

Molecular markers have been successfully used for the species level identification of Anisakis species. Mattucci et al.10 reported A. pegreffii for the first time in a paraffin embedded granuloma taken from a man with intestinal anisakiasis in Italy. Santoro et al.20 reported A. pegreffii in loggerhead sea turtles (Caretta caretta) from Central Mediterranean of southern Italy. Valentini et al.21 determined the genetic relationship among nine species of Anisakis sp. and reported A. pegreffii in common dolphin (Delphinus delphis) from Spanish coasts of Northeast Atlantic Ocean. In these studies, mt-CO2 gene was used for the detection and phylogenetic analyses of A. pegreffii.

Umehara et al.14 studied on 85 patients with anisakiasis in Hokkaido and Kyushu in Japan. According to this study, 84 out of 85 patients were infected with A. simplex s.s., and one was infected with A. pegreffii. Same researchers reported these two parasites and a hybrid genotype from fish and cetacean in Japanese waters 7. Lee et al.16 studied on 60 larvae obtained from 90 sea fishes and 15 squids. Among 60 isolates, 47 were identified as A. pegreffii, 10 as A. typica, one as A. simplex s.s. and two as hybrid genotypes. Species level identification of parasites was achieved by PCR-RFLP analysis of ITS region.

Quiazon et al.22 detected A. simplex s.s. and A. pegreffii in marine fishes in Japanese waters by PCR-RFLP analysis of ITS region and sequencing of mt-CO2 gene. Abe et al.23 reported A. simplex s.s from a 65-year-old Japanese woman with epigastralgia. Umehara et al.8 reported A. typica in addition to A. simplex s.s. and A. pegreffii in hairtail fish caught in coasts of Taiwan and Japan. Pontes et al.15 studied on 150 anisakid larvae in three different species of fish, and they detected A. simplex s.s., A. pegreffii, A. ziphidarum, A. physeteris, A. typica and Anisakis sp. in Madeiran waters (Atlantic Ocean, Portugal). Parasites were identified by sequencing and PCR-RFLP analysis of ITS region 15,23.

In PCR-RFLP analysis, primer pairs A-B and NC2-NC5 were used for the amplification of ITS region, AniF-Ani1R for 5.8 rRNA and JB3-JB4.5 for mt-CO1 genes 7,8,14-16,22-25. The most used restriction enzymes were HinfI, HhaI and TagI for the analysis of both ribosomal and mitochondrial genes 7,8,14-16,22-25. In the studies in which A and B primer pair was used, RFLP patterns of HinfI provided the discrimination of A. pegreffii, A. simplex s.s. A. physeteris, A. typica, A. ziphidarum, Anisakis sp. A and hybrid genotypes of Anisakis sp., while the digestion profiles of HhaI were same in A. pegreffii, A. simplex s.s. A. physeteris and A. ziphidarum and different only between A. typica and Anisakis sp. A. Likewise, digestion patterns of TagI were not sufficient to discriminate Anisakis species 7,14-16,22,25. In this study, we used universal primers A and B for the amplification of ITS region, and HinfI for digestion of ITS products. All six parasites showed three bands of 250,

Fig 1. RFLP analysis of six nematodes using Hinf1 enzyme M: 100 bp marker, 1-6: Banding patterns of nematode samples obtained from horse mackerels

Şekil 1. Altı nematodun Hinf1 enzimi kullanarak yapılan RFLP analizi M: 100 bç marker, 1-6: İstavrit balıklarından elde edilen nematodların RFLP kalıpları

Fig 2. PCR results of six nematodes obtained from horse mackerels, M: 100 bp marker, 1: 446 bp amplicon of mt-CO1 gene, 3: 629 bp amplicon of mt-CO2 gene, 5: 672 bp amplicon of 18S-28S rRNA gene, 7: 1000 bp amplicon of rDNA (ITS1, 5.8 subunit rRNA and ITS2), 2,4,6,8: Negative controls (sterile distilled water)

Şekil 2. İstavrit balıklarından elde edilen altı nemotodun PCR sonuçları, M: 100 bç’lik marker, 1: Mt-CO1 geninin 446 bç’lik ürünü, 3: Mt-CO2 geninin 629 bç’lik ürünü, 5: 18S-28S rRNA geninin 672 bç’lik ürünü, 7: rDNA (ITS1, 5.8 altünite rRNA ve ITS2)’nın 1000 bç’lik ürünü, 2,4,6,8: Negatif kontroller (steril distile su)

306Molecular Detection of ...

300, 370 bp which were specific to A. pegreffii. Researchers obtained the same banding pattern for A. pegreffii in different studies 5,7,14,16,22,25.

Umehara et al.5 and Fang et al.6 developed species-specific primers from the ITS region of rDNA and used in multiplex PCR for the rapid identification of anisakid nematodes. Umehara et al.5 achieved to differentiate A. simplex s.s., A. pegreffii and A. physeteris, and Fang et al.6, A. simplex s.s., A. pegreffii from other anisakid nematodes. In these two studies, primer pairs APE1-B and APEF-NC2 were used for the detection of A. pegreffii. We used APE1-B primer pair for the detection of A. pegreffii and we obtained 672 bp amplification products as obtained by Umehara et al.5.

Kim et al.11 determined the complete mitochondrial genome of A. simplex and defined the phylogenetic relationship of A. simplex with 13 different nematode species. Noguera et al.26 studied on 42 Atlantic salmons (Salmo salar) in Scotland. They detected Anisakis type I larvae in the morphologic examination. For species level identification, larvae were analyzed by PCR-RFLP of ITS region and sequencing of ITS and mt-CO1 genes. At the end of the study, all larvae were identified as A. simplex s.s. Cross et al.27 detected A. simplex s.s. in 37 Atlantic herrings (Clupea harengus) caught off the north-west coast of Scotland. They made species level detection by sequencing of large subunit mt-CO1 gene of 161 nematodes.

In sequencing reactions, primer pairs A-B and NC2-NC5 were used for the amplification of ITS region, AnCO1F-AnCO1R for mt-CO1 and 211F-210R for mt-CO2 genes 8,10,15,20-25,27. In this study, we used primer pairs JB3-JB4.5 and 211F-210R for the amplification and sequencing of mt-CO1 and mt-CO2 genes, respectively. Mt-CO1 result was corresponding with A. simplex (94-98%), and mt-CO2 results were corresponding with A. pegreffii (97-99%) when compared to GenBank results.

In Turkey, there is limited data on A. simplex. This parasite was reported in red mullet (Mullus surmuletus) caught near Gökceada, anchovies (Engraulis encrasicolus) from Marmara sea, and mackerel (T. trachurus), scad (Trachurus mediterranus), whiting (Merlangius euxinus) as well as sardines (Sardina pilchardinus) caught on Canakkale coast and throughout the Dardanelles Strait of Turkey 28-30. In these studies, species identification was based on morphological features of parasites. Even though morphology is useful for the diagnosis of Anisakis spp., the species of A. simplex complex are so far morphologically indistinguishable at both adult and larval stage; consequently, only genetic and molecular methods can be used reliably to identify them at all the developmental stages. Although A. typica and A. ziphidarum are distinguishable at their male adult stages, this is not possible at their larval stage 4. In this study, we determined A. pegreffii from horse mackerels (T. trachurus) by PCR and PCR-RFLP analysis of ITS region

of rDNA and partial sequencing of mt-CO1 and mt-CO2 genes for the first time in Turkey. This study suggests that Anisakis species can easily be diagnosed by molecular techniques at species level. We consider that further studies should be carried out in Turkey to detect different Anisakis species and understand clearly the transmission ways, epidemiology and control of the disease.

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9. Sánchez JM, Artacho-Reinoso ME, D´ıaz-Gavilán M, Valero-López A: Structure of Anisakis simplex s.l. populations in a region sympatric for A. pegreffii and A. simplex s.s. Absence of reproductive isolation between both species. Mol Biochem Parasitol, 141, 155-162, 2005.

10. Mattiucci S, Paoletti M, Borrini F, Palumbo M, Palmieri RM, Gomes V, Casati A, Nascetti G: First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Ascaridida) in paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy. BMC Infect Dis, 11, 1-6, 2011.

11. Kim KH, Eom KS, Park JK: The complete mitochondrial genome of Anisakis simplex (Ascaridida: Nematoda) and phylogenetic implications. Int J Parasitol, 36, 319-328, 2006.

12. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY, 1989.

13. Utuk AE, Simsek S, Koroglu E, McManus DP: Molecular genetic characterization of different isolates of Echinococcus granulosus in East and Southeast Regions of Turkey. Acta Tropica, 107, 192-194, 2008.

14. Umerha A, Kawakami Y, Araki J, Uchida A: Molecular identification of the etiological agent of the human anisakiasis in Japan. Parasitol Int, 56, 211-215, 2007.

15. Pontes T, Amelio SD, Costa G, Paggi L: Molecular characterization of larval anisakid nematodes from marine fishes of Madeira by a PCR-based approach, with evidence for a new species. J Parasitol, 9, 1430-1434, 2005.

16. Lee MH, Cheon DS, Choi C: Molecular genotyping of Anisakis species from Korean sea fish by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Food Cont, 20, 623-626, 2009.

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17. Bowles J, Blair D, McManus DP: Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol, 54, 165-174, 1992.

18. Nadler SA, Hudspeth DSS: Phylogeny of the Ascaridoidea (Nematoda: Ascaridida) based on three genes and morphology: Hypotheses of structural and sequence evolution. J Parasitol, 86, 380-393, 2000.

19. Knudsen B, Knudsen T, Flensborg M, Sandmann H, Heltzen M, Andersen A, Dickenson M, Bardram J, Steffensen PJ, Mønsted S, Lauritzen T, Forsberg R, Thanbichler A, Bendtsen JD, Görlitz L, Rasmussen J, Tordrup D, Værum M, Ravn MN, Hachenberg C, Fisker E, Dekker P, Schultz J, Hein AMK, Sinding JB: CLC Main Workbench, Version 5.5, CLC bio, Finlandsgade 10-12, Katrinebjerg, 8200 Aarhus N, Denmark, 2007.

20. Santora M, Mattiucci S, Paoletti M, Liotta A, Uberti BD, Galiero G, Nascetti G: Molecular identification and pathology of Anisakis pegreffii (Nematode: Anisakidae) infection in the Mediterranean loggerhead sea turtle (Caretta caretta). Vet Parasitol, 174, 65-71, 2010.

21. Valentini A, Mattiucci S, Bondanelli P, Webb SC, Mignucci-Giannone AA, Colom-Llavina MM, Nascetti G: Genetic relationships among Anisakis species (Nematoda: Anisakidae) Inferred from mitochondrial cox2 sequences, and comparison with allozyme data. J Parasitol, 92, 156-166, 2006.

22. Quiazon KMA, Yoshinaga T, Ogawa K: Distribution of Anisakis species larvae from fishes of the Japanese waters. Parasitol Int, 60, 223-226, 2011.

23. Abe N, Tominaga K, Kimata I: Usefulness of PCR-restriction fragment length polymorphism analysis of the internal transcribed spacer region of rDNA for identification of Anisakis simplex complex. Jpn J Infect Dis, 59,

60-62, 2006.

24. Suzuki J, Murata R, Hosaka M, Araki J: Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes. Int J Food Microbiol, 137, 88-93, 2010.

25. Quiazon KMA, Yoshinaga T, Ogawa K: Morphological differences between larvae and in vitro-cultured adults of Anisakis simplex (sensu stricto) and Anisakis pegreffii (Nematoda: Anisakidae). Parasitol Int, 57, 483-489, 2008.

26. Noguera P, Collins C, Bruno D, Pert C, Turnbull A, McIntosh A, Lester K, Bricknell I, Wallace S, Cook P: Red vent syndrome in wild Atlantic salmon Salmo salar in Scotland is associated with Anisakis simplex sensu stricto (Nematoda: Anisakidae). Dis Aquat Org, 87, 199-215, 2009.

27. Cross MA, Collins C, Campbell N, Watts PC, Chubb JC, Cunningham CO, Hatfield EMC, MacKenzie K: Levels of intra-host and temporal sequence variation in a large COI sub-units from Anisakis simplex sensu stricto (Rudolphi 1809) (Nematoda: Anisakisdae): implications for fisheries management. Mar Biol, 151, 695-702, 2007.

28. Akmirza A: Gökçeada civarında avlanan Tekir (Mullus surmuletus L) balığının metazoon parazitleri. İstanbul Üniv Vet Fak Derg, 26, 129-140, 2000.

29. Tuncel VA, Akmirza A: Karadeniz ve Marmara’da avlanan hamsi (Engraulis encrasicolus (Linnaeus, 1758), balığının endoparazitlerinin karşılaştırılması. İst Üniv Su Ürünleri Derg, 20, 17-26, 2006.

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SummaryCoenzyme Q10 (CoQ10) is an antioxidant and an electron carrier in the mitochondrial matrix. The aim of this study was to study the

protective effect of CoQ10 on the levels of Fe, Cu, Zn and Mn in serum with bleomycin induced pulmonary fibrosis in rats. Thirty Wistar albino rats were randomly divided into three groups: bleomycin alone, bleomycin + CoQ10, and physiological saline alone (control group). The bleomycin group was given 7.5 mg/kg body weight (single dose) bleomycin hydrochloride intratracheally. The bleomycin + CoQ10 group was also instilled with bleomycin hydrochloride but received administrations of CoQ10 twice a week. The control group was treated with physiological saline alone. Animals were euthanized 14 d after intratracheal instillation of bleomycin. Fe, Cu, Zn and Mn were measured in the serum of rats. Atomic absorption spectroscopy was used to measure the Fe, Cu, Zn and Mn levels. Our data may suggest that bleomycin impairs the equilibrium of Fe, Cu, Zn and Mn which are important in the antioxidant system and CoQ10 tends to rechange the imbalance of some trace elements in pulmonary fibrosis.

Keywords: Coenzyme Q10, Bleomycin, Pulmonary fibrosis, Trace element, Rat

Koenzim Q10’ nin Bleomisinin Teşvik Ettiği Akciğer Fibrozisli Ratların Serumlarındaki Bazı İz Elementlerin Seviyelerine Etkisi

ÖzetKoenzim Q10 (CoQ10) mitokondrial matrikste bulunan antioksidan bir elektron taşıyıcısıdır.Bu çalışmanın amacı bleomisin

uygulanarak oluşturulmuş akciğer fibrozisinde CoQ10’ nin Fe, Cu, Zn ve Mn düzeyleri üzerine koruyucu etkisini araştırmaktı. Otuz Wistar albino ırkı rat yalnız bleomisin uygulanan grup, bleomisin + CoQ10 uygulanan grup, ve yalnız serum fizyolojik uygulanan grup (kontrol grubu) olmak üzere 3 gruba ayrıldı. Bleomisin grubuna bleomisin hidroklorid 7.5 mg/kg canlı ağırlık oranında intratrakeal olarak tek doz şeklinde uygulandı. Bleomisin + CoQ10 grubuna da bleomisine ilave olarak haftada iki kez CoQ10 uygulandı. Kontrol grubuna ise yalnız serum fizyolojik uygulandı. Bleomisin uygulamasından 14 gün sonra ratlara ötenazi uygulandı. Serumlarında Fe, Cu, Zn ve Mn düzeyleri saptandı. Fe, Cu, Zn ve Mn düzeylerinin belirlenmesinde atomik absorbsiyon spektrofotometresi kullanıldı. Elde ettiğimiz veriler, bleomisinin antioksidan sistemde önemi olan Fe, Cu, Zn ve Mn dengesini bozduğunu ve CoQ10’ in akciğer fibrozisinde ağır metal ve iz element dengesizliğini yeniden değiştirdiğini düşündürmektedir.

Anahtar sözcükler: Koenzim Q10, Bleomisin, Akciğer fibrozisi, İz element, Rat

Effects of Coenzyme Q10 on the Levels of some Trace Elements in Serum of Rats with Bleomycin Induced Pulmonary Fibrosis

Ayşegül ÇEBİ * Fatih Çağlar ÇELİKEZEN ** Ali ERTEKİN ***

***

***

Faculty of Health Sciences, Giresun University, TR-28340 Piraziz/ Giresun - TURKEYFaculty of Science and Art, Bitlis Eren University, TR-13000 Bitlis - TURKEYFaculty of Veterinary, Ondokuz Mayıs University, TR-55139 Samsun - TURKEY


Pulmonary fibrosis is a chronic interstitial lung disease. The lung parenchyma is damaged by varying patterns of inflammation and fibrosis with a high mortality rate and poor response to available medical therapy 1,2. The etiology of this disease is unknown. However, lung inflammation

is a major underlying component of a wide variety of pulmonary fibroproliferative disorders. Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, peroxynitrite, and hydroxyl radical are the major mediators of the lung inflammatory processes 3,4.

INTRODUCTION


RESEARCH ARTICLE

310Effects of Coenzyme Q10 on ...

Bleomycin generates reactive oxygen metabolites, including superoxide and hydroxyl radicals. Generation of the reactive species in the lung tissue results in DNA injury, lipid peroxidation, alteration in lung prostaglandin synthesis and degradation, and an increase in lung collagen synthesis 5. Bleomycin-induced fibrosis is the most commonly used animal model and appears as a significant drug-induced lung disease in the clinical setting 3.

The complex bleomycin-Fe has been the most studied because bleomycin joins the DNA and Fe at the same time, and release of free radicals happens in the presence of molecular oxygen 6. Fe is typically in the Fe (III) form and will need to be reduced prior to any Fenton activity. Lung lining fluid contains antioxidants, such as glutathione (GSH) and ascorbic acid, which can reduce Fe(III) to Fe(II) 7.

Coenzyme Q10 (CoQ10), also known as ubiquinone, is a vitamin like substance present in all human cells in the membranes of endoplasmic reticulum, peroxisomes, lysosomes, and the inner membrane of mitochondria. It is an important component of the electron transport chain in mitochondria responsible for generation of energy. Furthermore, its reduced form serves as an important antioxidant in both mitochondria and lipid membranes where it protects the body against the deleterious effects of reactive oxygen species (ROS). Therefore, the enzyme has been therapeutically employed in many disorders for its cytoprotective and antioxidant properties 8.

Huang et al.9 reported that many trace elements play an important role in a number of biological processes by activating or inhibiting enzymes, by competing with other elements and metalloproteins for binding sites, by affecting the permeability of cell membranes or by other mechanisms. Trace elements function as activators of enzyme systems or as constituents of organic compounds. Trace elements are necessary for humans and animals 10.

The purpose of this study was to evaluate the effects of CoQ10 on the serum levels of some of the trace elements (Fe, Cu, Zn and Mn) in rats with bleomycin induced pulmonary fibrosis.


Thirty male Wistar albino rats, six months old, weighing 180-200 g, were obtained from the animal laboratory of Yuzuncu Yil University. All procedures involving animals were approved by the institutional ethics committee (Document No: 28.02.2007/04). Rats were housed in specific cages. A12-h light/dark cycle was maintained and the rats had free access to water and food ad libitum. The animals were divided randomly in the following three groups: Control group (n=10): Sterile physiological saline solution was given intraperitoneally. Bleomycin treated group (n=10): Bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) was dissolved in 250 μL of phosphate- buffered saline (PBS) solution and instilled into the animals at a dose of 7.5 mg/kg body weight (single dose) intratracheally under chloroform anesthesia. The animals were shaken to facilitate distribution of the bleomycin and physiological saline 11. Bleomycin+CoQ10 treated group (n=10): CoQ10 was given as CoQ10 (Sigma, St. Louis, MO, USA) at a dose of 10 mg/kg body weight (twice a week) during the experiment 12. To evaluate the serum trace elements of bleomycin induced pulmonary fibrosis, animals were euthanized 14 d after its instillation. Blood samples were collected for analysis. They were centrifuged at 1500 rpm for 15 min and serum was obtained. The levels of trace elements (Fe, Cu, Zn and Mn) were analyzed by atomic absorption spectrophotometry (Unicam 929 atomic absorption spectrophometer).

All analyses were made using the SPSS statistical software package. Data are expressed as means±standard deviation. Statistical analyses were made using the independent sample t test.

RESULTS

The levels of Fe, Cu, Zn and Mn in the groups are indicated in Table 1. The concentration of Fe was lower in bleomycin + CoQ10 administrated groups as compared to the other groups (P<0.05). The levels of Zn were significantly decreased in all groups as compared to the

Table 1. Concentrations of Fe, Zn, Mn and Cu in serum of the control, bleomycin administered and bleomycin + CoenzymeQ10-administered groups

Tablo 1. Bleomisin, bleomisin + Coenzyme Q10 uygulanmış ve kontrol gruplarının serumlarında Fe, Cu, Zn ve Mn konsantrasyonları

Parameters n BleomycinM±SD

Bleomycin + CoenzymeQ10M±SD

ControlM±SD

Fe (µg/dl) 10 0.446±0.14 0.211±0.20 c 0.597±0.38

Zn (µg/dl ) 10 0.188±0.06 b 0.101±0.09 a 0.250±0.03

Mn (µg/dl ) 10 0.006±0.00 a 0.005±0.00 c 0.003±0.00

Cu (µg/dl ) 10 0.225±0.08 b 0.143±0.14 b 0.315±0.04

a P<0.001, b P<0.01, c P<0.05

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control. It was found that the levels of Mn were significantly increased in all groups than that of the control. The level of Mn was lower in CoQ10 + bleomycin administrated group as compared to the only bleomycin administered group. The concentration of Cu was significantly decreased in all groups as compared to the control. The level of Cu was higher in bleomycin administered group than that of the CoQ10 + bleomycin treated groups.

DISCUSSION

Bleomycin has been used as cytostatic treatment of many malignant tumors, such as germ cell tumors, lymphomas, head and neck tumors, and Kaposi’s sarcomas 13,14. Its mechanism of action is breaking the DNA double helix by the production of free radicals, which is oxygen and iron dependent 14,15. Pulmonary fibrosis has more common side effect of bleomycin in cancer treatment. In a previous study, Mert et al.16 showed that α-tocopherol and vitamin D3 levels in the lung tissue of the bleomycin treated rats decreased appreciably more than that of the control group. Hence, the histological changes observed in the bleomycin administered rats. Large fibrous areas in the interalveolar septa tissue, peribronchial and perivascular regions were observed with apparent infiltration of lymphocytes. In another study, Ertekin et al.3 have investigated the preventive effect of vitamin E on bleomycin induced rats. In their study, the amounts of Cu, Fe, and carbonic anhydrase found in the lung were increased in both bleomycin and bleomycin+vitamin E administered rats. Zn and Mn levels were decreased, except for the Mn level in the bleomycin group. The levels of Zn, Mn, and Cu were decreased in both experimental groups compared to the control group, whereas Fe and carbonic anhydrase activity increased in comparison to the control group. Dede et al. 17 had also showed that the lungs of rats induced with bleomycin were damaged and α-tocopherol prevented it from certain damage as indicated by the difference of the concentrations in major elements of serum. The levels of Fe and Mg in the group of bleomycin were lower than that of the control group and the levels of Cu, Ca and K were not changed among the groups.

In this study, we have investigated the protective effect of CoQ10 on some trace elements (Fe, Cu, Zn, Mn) in the serum of rats with lung fibrosis induced by bleomycin. Cu was decreased significantly in bleomycin and bleomycin + CoQ10 administered groups (P<0.01). The level of Zn was significantly decreased bleomycin treated group (P<0.01). Zn is a part of superoxide dismutase which is an important antioxidant enzyme in the cell. The change of the level of Zn means an imbalance of the antioxidant system. The Mn concentrations were higher in all of the groups than that of the control. Mn is also part of superoxide dismutase enzyme. The less increase of the level of Mn in bleomycin + CoQ10 treated group than that of the bleomycin

administered group may imply an amelioration in the antioxidant system. Because, CoQ10 is an antioxidant and found in the mitochondria. Manganese superoxide dismutase (MnSOD) is the major ROS detoxifying enzyme of cells because of its localization to mitochondria. Altered function or expression of MnSOD can have remarkable consequences on mitochondrial function and the overall health of cells due to oxidative damage to various mitochondria-localized metabolic processes, leading to the development of different diseases 18. CoQ10 may show an effect in the mitochondrial matrix tending to regulate Mn balance. However it is difficult to say that the protective effect of CoQ10 barely by measuring trace elements. It could be more helpful that to measure the antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase. In another study, the effects of CoQ10 on the levels of serum biochemistry and malondialdehyde (MDA) in rats were investigated. The decreased levels of alkaline phosphatase (ALP) and increased levels of phosphorus (P) in CoQ10 treated rats were found compared to the control rats 19.

In a research, serum CoQ10 levels were measured at rest and during incremental exercise in 21 patients with chronic obstructive pulmonary disease (COPD) and 9 patients with idiopathic pulmonary fibrosis (IPF). The mean of serum CoQ10 levels were found lower in both experimental groups than that of the healthy subjects. Hence, they investigated the effects of the oral administration of CoQ10 on pulmonary function and exercise performance in patients with COPD. Serum CoQ10 levels were found significantly higher in association with an improvement in hypoxemia at rest, whereas pulmonary function was unchanged 20.

Reju et al.21 indicated that in the cancerous tissue of testis, the concentrations of K, Cr and Cu were higher while the concentrations of Fe, Co and Zn were lower when compared to those in normal tissue of testis. The concentrations of Cl, Ca, Ti and Mn were in agreement in both cancerous and normal tissues of testis. Cunzhi et al.22 investigated the relationship between trace elements and the incidence of cervical cancer. They measured tissue and serum levels of six elements Cu, Zn, Fe, Mn, Ca and Se and Cu/Zn ratio in patients belonging to cervical cancer and uterine myoma. Their results show that the tissue contents of Zn, Se and Ca were significantly lower and the Cu and Fe levels and Cu/Zn ratio were significantly higher in cervical cancer tissue. The same trend was also observed in serum levels also.

Free radical species affect all important components of cells such as lipids, proteins, carbohydrates and nucleic acids. It is known that CoQ10 is an important free radical scavenger. In the present study, we may conclude that bleomycin impairs the equilibrium of Fe, Cu, Zn and Mn which are important in the antioxidant system. We

312Effects of Coenzyme Q10 on ...

can not suggest that CoQ10 has protective effect on bleomycin induced pulmonary fibrosis as measuring trace elements. However, we can say that CoQ10 rechanges to imbalance of trace elements in pulmonary fibrosis. Further investigations are needed to find how the mechanism works related the trace elements and CoQ10 in pulmonary fibrosis.

REFERENCES

1. Atzori L, Chua F, Dunsmore SE, Willis D, Barbarisi M, McAnulty RJ, Laurent GJ: Attenuation of bleomycin induced pulmonary fibrosis in mice using the heme oxygenase inhibitor Zn-deuteroporphyrin IX-2,4-bisethylene glycol. Thorax 59 (3): 217-223, 2004.

2. MacNee W, Rahman I: Oxidants/antioxidants in idiopathic pulmonary fibrosis. Thorax 50, 53-58, 1995.

3. Ertekin A, Deger Y, Mert H, Mert N, Yur F, Dede S, Demir H: Investigation of the effects of-tocopherol on the levels of Fe, Cu, Zn, Mn, and carbonic anhydrase in rats with gleomycin-induced pulmonary fibrosis. Biol Trace Elem Res, 116 (3): 289-300, 2007.

4. Oury TD, Thakker K, Menache M, Chang LY, Crapo JD, Day BJ: Attenuation of bleomycin-induced pulmonary fibrosis by a catalytic antioxidant metalloporphyrin. Am J Respir Cell Mol Biol, 25 (2): 164-169, 2001.

5. Sleijfer S: Bleomycin-induced pneumonitis. Chest 120, 617-624, 2001.

6. Hay J, Shahzeidi S, Laurent G: Mechanisms of bleomycin-induced lung damage. Arch Toxicol, 65, 81-94, 1991.

7. Ruda TA, Dutta PK: Fenton chemistry of Fe(III)-exchanged zeolitic minerals treated with antioxidant. Environ Sci Technol, 39 (16): 6147-6152, 2005.

8. Ruiz-Jimenez J, Priego-Capote F, Mata JM, Quesada JM, Luque de Castro MD: Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme-Q10) in human serum by lipid chromatography tandem mass spectrometry to evaluate the oxidative stres. J Chromatography A, 1175, 242-248, 2007.

9. Huang YL, Sheu JY, Lin TH: Association between oxidative stress and changes of trace elements in patients with breast cancer. Clin Biochem, 32 (2): 131-136. 1999.

10. Shena SG, Lia H, Zhaoa YY, Zhangb QY, Suna HW: The distribution

patterns of trace elements in the blood and organs in a rabbit experimental model of Cu pollution and study of haematology and biochemistry parameters. Environ Toxicol Pharmacol, 19, 379-384, 2005.

11. Ozyurt H, Sogut S, Yıldırım Z: Inhibitory effect of caffeic acid phenethyl ester on bleomycine-induced lung fibrosis in rats. Clin Chim Acta, 339, 65-75, 2004.

12. Kilinc C, Ozcan O, Karaoz E, Sunguroglu K, Kutluay T, Karaca L: Vitamin E reduces bleomycin-induced lung fibrosis in mice: Biochemical and morphological studies. Basic Clin Physiol Pharmacol, 4 (3): 249-269, 1993.

13. Chen XL, Li WB, Zhou AM: Role of endogenous peroxynitrite in pulmonary injury and fibrosis induced by bleomycin A5 in rats. Acta Pharmacol Sin, 24, 697-702, 2003.

14. Cheson BD: Pharmacology of cancer chemotherapy: miscellaneous chemotherapeutic agents. In, De Vita Jr VT, Hellmann S, Rosenberg AS (Eds): Cancer Principles and Practice of Oncology. pp. 452-459, Lippincott Willians & Wilkins, Philadelphia, 2001.

15. Hay J, Shahzeidi S, Laurent G: Mechanisms of bleomycin-induced lung damage Arch. Toxicol, 65, 81-94, 1991.

16. Mert H, Yoruk I, Ertekin A, Dede S, Deger Y, Yur F, Mert N: Vitamin levels in lung tissue of rats with bleomycin induced pulmonary fibrosis. J Nutr Sci Vitaminol, 55 (2): 186-190, 2009.

17. Dede S, Mert H, Mert N, Yur F, Ertekin A, Deger Y: Effects of alpha-tocopherol on serum trace and major elements in rats with bleomycin-induced pulmonary fibrosis. Biol Trace Elem Res Winter, 114 (1-3): 175-184, 2006.

18. Miao L, St. Clair DK: Regulation of superoxide dismutase genes: Implications in disease. Free Radic Bio Med, 47, 344-356, 2009.

19. Kısmalı G: Effects of coenzyme Q10 on blood biochemistry in rats. Kafkas Univ Vet Fak Derg, 15 (2): 191-194, 2009.

20. Fujimoto S, Kurihara N, Hirata K, Takeda T: Effects of coenzyme Q10 administration on pulmonary function and exercise performance in patients with chronic lung diseases. Clin Investig, 71 (8): 162-166, 1993.

21. Naga Raju GJ, John Charles M, Bhuloka Reddy S, Sarita P, Seetharami Reddy B, Rama Lakshmi PVB, Vijayan V: Trace elemental analysis in cancer-afflicted tissues of penis and testis by PIXE technique. Nuc Inst and Meth in Phys Res, B 229, 457-464, 2005.

22. Cunzhi H, Jiexian J, Xianwen Z, Jingang G, Shumin Z, Lili D: Serum and tissue levels of six trace elements and copper/zinc ratio in patients with cervical cancer and uterine myoma. Biol Trace Elem Res, 94, 113-122, 2003.


SummaryThis study was carried out to determine the effects of dietary sepiolite on performance, carcass traits and some blood parameters

of broilers. A total of 204 daily Ross 308 female broiler chicks were allocated into one control group and two treatment groups each containing 68 chicks. Sepiolite was used at the level of 0%, 0.5% and 1% for the diets of control group and the first and second treatment groups, respectively. The experimental period lasted 6 weeks. Supplemental sepiolite improved body weight (P<0.01) and overall body weight gain (P<0.05). No differences were observed in feed intake, feed efficiency, carcass yield and the relative weights of gizzard, liver, heart, spleen and Bursa Fabricus among groups. The relative weight of abdominal fat and the levels of serum cholesterol and serum triglyceride were reduced with 1% sepiolite inclusion in the diet. Blood serum levels of total protein were not affected by sepiolite. It is concluded that 1% sepiolite in the diets of broiler increase body weight gain and reduce the relative weight of abdominal fat and the levels of serum cholesterol and triglyceride.

Keywords: Blood parameters, Broiler, Performance, Carcass traits, Sepiolite

Broyler Karma Yemlerinde Sepiyolit Kullanımının Performans, Karkas Özellikleri ve Bazı Kan Parametreleri Üzerine Etkileri

ÖzetBu araştırma broyler karma yemlerinde sepiyolit kullanımının performans, karkas özellikleri ve bazı kan parametreleri üzerine

etkilerini belirlemek amacıyla yapılmıştır. Toplam 204 adet günlük Ross 308 dişi broyler civciv her biri 68 adet içeren bir kontrol ve iki deneme grubuna ayrılmıştır. Sepiyolit, kontrol grubu, birinci ve ikinci deneme grupları karma yemlerinde sırasıyla %0, %0.5 ve %1 düzeylerinde kullanılmıştır. Deneme 6 hafta sürdürülmüştür. Sepiyolit ilavesi canlı ağırlık (P<0.01) ve toplam canlı ağırlık kazancını (P<0.05) artırmıştır. Yem tüketimi, yemden yararlanma, karkas randımanı ile taşlık, karaciğer, kalp, dalak ve Bursa Fabricus relatif ağırlıkları bakımından gruplar arasında farklılık gözlenmemiştir. Karma yemde %1 düzeyinde sepiyolit bulunması relatif abdominal yağ ağırlığı, serum kolesterol ve serum trigliserit düzeylerini azaltmıştır. Serum toplam protein düzeyi sepiyolitten etkilenmemiştir. Sonuç olarak, broyler karma yemlerinde %1 düzeyinde sepiyolit bulunması canlı ağırlık kazancını artırmış, relatif abdominal yağ ağırlığı ile serum kolesterol ve trigliserit düzeylerini azaltmıştır.

Anahtar sözcükler: Broyler, Kan parametreleri, Karkas özellikleri, Performans, Sepiyolit

Effects of Sepiolite Usage in Broiler Diets on Performance, Carcass Traits and Some Blood Parameters

Handan ESER * Sakine YALÇIN ** Suzan YALÇIN *** Adnan ŞEHU **

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Abant İzzet Baysal University, Mudurnu Süreyya Astarcı Vocational School of Higher Education, TR-14800 Bolu - TURKEYAnkara University, Faculty of Veterinary Medicine, Department of Animal Nutrition and Nutritional Diseases, TR-06110 Dışkapı, Ankara - TURKEYSelçuk University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, TR-42075 Konya - TURKEY


Sepiolite is a natural ingredient, clay family known as sepiolite-palygorskite. It is a hydrated magnesium silicate, Si12Mg8O30(OH2)4(OH)4.8H2O. Sepiolite has high porosity and surface area, strong absorptive power, high structural

stability, chemically inert and strong capacity to form stable suspensions at low concentrations. It reduces dust losses, improves the durability and hardness of pellets. Sepiolite is shown to be useful in following applications such as

INTRODUCTION


RESEARCH ARTICLE

314Effects of Sepiolite Usage in ...

absorbents, environmental deodorants, catalyst carriers, polyesters, asphalt coatings, paints, pharmaceutical uses, decolorizing agents, filter aids, anticaking agents, phytosanitary carriers, cigarette filters, plastisols, rubber, animal nutrition, detergents, cosmetics, agriculture (soil conditioning, fluid carriers for pregerminated seeds, seed coating, fertilizer suspensions), grease thickeners, and drilling fluids. Sepiolite is used as an adsorbent for toxins, bacteria and viruses in the intestine, as lubricants of ground diets and as a pelleting agent during feed processing procedures 1-3.

Sepiolite may replace growth factors, antibiotics and anticoccidials in the diets of monogastric animals. Ayed et al.4 reported that dietary sepiolite supplementation at the levels of 0.5%, 1% and 2% improved growth performances and feed efficiency. Chickens fed a diet containing 1.5% sepiolite showed a reduction in the intestinal transit time 5

which might correspond to a better nutrient utilisation. In Turkey, the use of sepiolite in animal nutrition is still limited. Therefore the objective of this trial was to assess the effects of sepiolite supplementation in broiler diets on growth performances, carcass traits and some blood parameters.


Animals, Diets and Experimental Design

The animals were treated according to the Animal Care and Use Regulation “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purpose, 1996”.

A total of 204 Ross 308 female broiler chicks aged one day were used. They were divided into one control group and two treatment groups each containing 68 chicks. Each group was divided into four replicates as subgroups, each comprising 17 chicks. Feed and water were provided for ad libitum consumption and the diets were presented in mash form. The experimental period lasted 6 weeks. Broilers were fed on starter diets during 1-21 days and fed on grower diets during 22-42 days. Sepiolite was used at the level of 0%, 0.5% and 1% for the diets of control group and the first and second treatment groups, respectively. Diets were formulated to be isonitrogenous and isocaloric. The ingredients and chemical composition of the diets are presented in Table 1.

Table 1. The ingredient and chemical composition of diets

Tablo 1. Karma yemlerin yapısı ve kimyasal bileşimi

Ingredients (%)

Starter Diets (1-21 Days) Grower Diets (22-42 Days)

Control S1 S2 Control S1 S2

Corn 53.95 52.85 51.80 51.90 50.90 49.90

Soybean meal 29.50 29.50 29.50 29.15 28.75 28.40

Full fat soya 9.00 9.30 9.55 9.50 10.2 10.85

Fish meal 2.00 2.00 2.00 2.00 2.00 2.00

Vegetable oil 2.50 2.80 3.10 4.20 4.40 4.60

Sepiolite 0 0.50 1.00 0 0.50 1.00

Limestone 1.20 1.20 1.20 1.30 1.30 1.30

Dicalcium phosphate 1.10 1.10 1.10 1.20 1.20 1.20

Salt 0.25 0.25 0.25 0.25 0.25 0.25

DL-Methionine 0.20 0.20 0.20 0.20 0.20 0.20

Lysine 0.05 0.05 0.05 0.05 0.05 0.05

Vitamin-mineral premix 1 0.25 0.25 0.25 0.25 0.25 0.25

Chemical Composition (Analyzed)

ME 2 (kcal/kg) 3122 3105 3105 3200 3210 3212

Crude protein (%) 22.05 22.00 22.06 21.89 21.94 21.85

Calcium (%) 0.95 1.00 0.95 0.98 1.02 1.03

Total phosphorus (%) 0.63 0.66 0.66 0.64 0.66 0.69

S1: Diet containing 0.5% sepiolite, S2: Diet containing 1% sepiolite1 Supplied the following per kilogram of diet: 12.000 IU vitamin A, 2.400 IU vitamin D3, 30 mg vitamin E, 2.5 mg vitamin K3, 2.5 mg vitamin B1, 6 mg vitamin B2, 4 mg vitamin B6, 20 mg vitamin B12, 25 mg niacin, 8 mg calcium-D-panthotenate, 1 mg folic acid, 50 mg vitamin C, 50 mg D-biotin, 80 mg Mn, 60 mg Zn, 60 mg Fe, 5 mg Cu, 1 mg I, 0.5 mg Co, 0.15 mg Se2 Metabolizable energy content of diets was calculated using the nutrient contents 9

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ESER, YALÇINYALÇIN, ŞEHU

Measurements, Sample Collection and Laboratory Analysis

The nutrient composition of basal diets were determined according to the AOAC 6. The samples were ashed in a muffle furnace prior to the analysis of calcium 7

and total phosphorus 8. Metabolizable energy levels of samples were estimated using the following equation of Carpenter and Clegg indicated by Leeson and Summers 9 :

ME, kcal/kg = 53 + 38 [(crude protein,%) + (2.25 x ether extract, %) + (1.1 x starch, %) + (sugar, %)]

Chicks were weighed individually at the beginning of the experimental period and weekly to determine body weight and body weight gain. The birds were daily observed for evaluating mortality. Feed consumption was recorded weekly and expressed as g per bird per week and the feed conversion ratio was calculated as g feed per g body weight gain.

At day 42, 16 broilers from each group (4 from each replicate) were weighed and slaughtered by severing the jugular vein. Hot carcasses were weighed to determine the carcass yield. Weights of abdominal fat, gizzard, liver, heart, spleen and Bursa Fabricius were also determined to calculate relative weights.

At day 41, 16 broilers from each group (4 from each replicate) were randomly selected and bled from the brachial vein. Blood samples were taken in the tubes containing no anticoagulant and centrifuged at 3220 g for 8 min. Serum was collected and stored at -20oC for determination of total protein, cholesterol and triglyceride by Vitros 350 autoanalyser (New York, USA; Product code 680-2153) using their accompanying commercial kits (Vitros Chemistry Products, Ortho-Clinical Diagnostics, Johnson-Johnson Company, New York, USA).


Statistical analysis were done using SPSS programme (SPSS Inc., Chicago, IL, USA). The normality of data distribution was checked using the Kolmogorov-Smirnov test. One-way ANOVA was performed to examine differences among groups. The significance of mean differences between groups were tested by Tukey. Values were given as mean and standard error of mean 10. Level of significance was taken as P<0.05.

RESULTS

The effects of dietary supplementation of sepiolite on body weight in broilers were given in Table 2. Dietary sepiolite supplementation increased body weight significantly (P<0.01). The group fed 1% sepiolite had the highest body weight at day 7. No differences were observed between the two doses of sepiolite for the mean of body weight after 7 days. Body weight gain during 1-21 days (P<0.01) and during 1-42 days (P<0.05) was improved by 1% sepiolite supplementation (Table 3). Dietary treatments did not affect feed intake and feed conversion ratio of broilers (Table 3). During experimental period one bird from control group and one bird from group fed diets supplemented 1% sepiolite dead. The effects of dietary supplementation of sepiolite on carcass yield and relative organ weights in broilers were shown in Table 4. Sepiolite had no significant effect on carcass yield and the relative weights of gizzard, liver, heart, spleen and Bursa Fabricus, but the relative weight of abdominal fat was decreased with 1% sepiolite supplementation (P<0.05).

The inclusion of sepiolite in the diet of broilers reduced the levels of serum cholesterol (P<0.001) and triglyceride (P<0.05). Serum total protein was not affected by the usage of sepiolite (Table 5).

Table 2. Effects of dietary supplementation of sepiolite on body weight in broilers

Tablo 2. Karma yemlere sepiyolit ilavesinin broylerlerde canlı ağırlık üzerine etkileri

Age (Day)

Groups

SEM P ValueControl S1 S2

n Body Weight (g) n Body Weight (g) n Body Weight (g)

1 68 38.7 68 38.4 68 38.6 0.1 0.671

7 67 125c 68 138b 67 147a 1 <0.001

14 67 350b 68 375a 67 386a 3 <0.001

21 67 654b 68 694a 67 717a 5 <0.001

28 67 1077b 68 1119a 67 1133a 7 <0.001

35 67 1562b 68 1609a 67 1625a 8 0.004

42 67 2037b 68 2079a 67 2090a 6 <0.001

S1: Group fed starter and grower diets containing 0.5% sepiolite, S2: Group fed starter and grower diets containing 1% sepioliteDifferent superscripts a,b,c in the same row indicate significant differences between groups (P<0.05 or more)


Table 3. Effects of dietary supplementation of sepiolite on body weight gain, feed intake and feed conversion ratio in broilers

Tablo 3. Karma yemlere sepiyolit ilavesinin broylerlerde canlı ağırlık artışı, yem tüketimi ve yemden yararlanma üzerine etkileri

Parameters

Groups


Body weight gain (g/bird)

1-21 days 616b 656ab 678a 9 0.006

22-42 days 1383 1385 1374 6 0.757

1-42 days 1999b 2041ab 2052a 9 0.015

Feed intake (g/bird)

1-21 days 865 870 872 6 0.916

22-42 days 2829 2833 2832 10 0.988

1-42 days 3695 3704 3704 10 0.925

Feed conversion ratio (g feed intake/g body weight gain)

1-21 days 1.41 1.33 1.29 0.02 0.077

22-42 days 2.05 2.05 2.06 0.01 0.656

1-42 days 1.85 1.81 1.80 0.01 0.118

n=4S1: Group fed starter and grower diets containing 0.5% sepiolite, S2: Group fed starter and grower diets containing 1% sepioliteDifferent superscripts a,b in the same row indicate significant differences between groups (P<0.05 or more)

Table 4. Effects of dietary supplementation of sepiolite on carcass yield and relative organ weights in broilers

Tablo 4. Karma yemlere sepiyolit ilavesinin broylerlerde karkas randımanı ve relatif organ ağırlıkları üzerine etkileri

Parameters(%)

GroupsSEM P Value

Control S1 S2

Carcass yield 68.7 69.1 69.1 0.2 0.766

Gizzard 1.66 1.53 1.53 0.04 0.209

Liver 2.46 2.43 2.62 0.04 0.104

Heart 0.64 0.63 0.59 0.01 0.244

Spleen 0.14 0.15 0.14 0.01 0.908

Bursa Fabricus 0.11 0.12 0.12 0.01 0.792

Abdominal fat 1.73a 1.39ab 1.36b 0.06 0.024


Table 5. Effects of dietary supplementation of sepiolite on blood serum parameters in broilers

Tablo 5. Karma yemlere sepiyolit ilavesinin broylerlerde kan parametreleri üzerine etkileri

Parameters

Groups


Cholesterol (mg/dl) 116.6a 109.8a 94.6b 1.9 <0.001

Triglyceride (mg/dl) 57.2a 50.6ab 43.5b 2.2 0.041

Protein (g/dl) 3.93 3.79 3.89 0.05 0.458


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ESER, YALÇINYALÇIN, ŞEHU

DISCUSSION

In the present study body weight was enhanced by incorporating sepiolite into broiler diets. At the end of the experiment (on day 42) the body weight of broilers receiving sepiolite was significantly higher (P<0.001) than that of control group. The effect of sepiolite on body weight gain was more important (P<0.01) between 1 and 21 days of age than in the grower period. Dietary sepiolite supplementation improved overall body weight gain (P<0.05) however had no effect on feed intake and feed efficiency. In agreement with the present study improving effects of sepiolite on growth performances in poultry were reported by Ayed et al.4. However Ouhida et al.11 showed that sepiolite supplementation at 1% and 2% had no significant effect on body weight, feed intake and feed efficiency. Palygorskite (a clay with similar physical properties to sepiolite) supplementation at 1% in broiler diets had no significant effect on feed efficiency 12. Alzueta et al.13 reported that addition of sepiolite to the diets of broiler chickens had no influence on the nutrient utilization and intestinal digesta viscosity.

Improvement in growth performance with the usage of sepiolite might be explained that sepiolite may increase the digesta retention time and this increment may allow the endogenous enzyme activity to be more effective in the digestion of fat, protein and carbohydrates and improve their absorption 5,14. Ouhida et al.14 also reported that sepiolite decreased the viscosity of jejunum digesta and may cause a reduction in the antinutritive effects of high viscosity.

In the present study, one bird from control group and one bird from group fed diets supplemented 1% sepiolite dead during 42 days. Mortality was not related with sepiolite. Similarly some researchers observed that sepiolite 4 usage up to 2% and palygorskite 12 at 1% in the broiler diets had no effect on mortality rate.

As shown in Table 4 dietary sepiolite supplementation had no significant effect on carcass yield and the relative weights of gizzard, liver, heart, spleen and Bursa Fabricus but the relative weight of abdominal fat decreased with 1% sepiolite addition to the diets. In agreement with the present study, some researchers showed that hot carcass yield4 and the relative weights of gizzard, liver, spleen and heart5 were not affected from sepiolite supplementation. Safaei Katouli et al.15 reported that the relative weights of liver, spleen, heart and Bursa Fabricus were not affected by the usage of clay minerals (kaolin, bentonite and zeolite) in broilers. Miazzo et al.16 also showed that the relative weights of liver, kidney, heart, gizzard and spleen were not changed by 0.3% sodium bentonite supplementation in broiler diets.

In this study dietary inclusion of sepiolite did

not significantly affect serum total protein. Sepiolite supplementation at the level of 1% reduced serum levels of cholesterol and triglyceride. Similarly Pappas et al.12 reported that serum total protein of broilers aged 42 days was not affected by the usage of palygorskite. Safaei Katouli et al.17 observed that kaolin and zeolite at the level of 1.5% as clay minerals decreased serum triglyceride levels significantly however serum total protein in groups fed diets containing 1.5, 3.0% kaolin and 3% zeolite was significantly higher in compared to control group. The increase in serum protein could be due to the action of kaolin and zeolite on the enhanced digestibility of certain nutrients 17. Clay based hydrated sodium calcium aluminosilicate did not result in any significant changes in serum chemistry in trials with chicks 18-22.

Sepiolite usage in barley-wheat based diets may be more useful to counteract negative effects of soluble non starch pollysaccharides in the diet by reducing the viscosity of jejunum digesta 14.

The results of this study indicate that 1% sepiolite in the diets improve body weight gain and reduce the relative weight of abdominal fat and the levels of serum cholesterol and triglyceride without any adverse effects on feed efficiency and carcass traits. The improvement in performance is the highest with the diet having 1% sepiolite in the starter period. Further studies are necessary to investigate the effects of dietary sepiolite under stressed conditions such as health, environmental or nutritional challenges.

REFERENCES

1. Galan E: Properties and applications of palygorskite-sepiolite clays. Clay Miner, 31, 443-453, 1996.

2. Murray HH, Pozo M, Galan E: An Introduction to palygorskite and sepiolite deposits-location, geology and uses. In, Galan E, Singer A (Eds): Developments in Palygorskite-Sepiolite Research: A New Outlook on These Nanomaterials, 1st ed., pp. 85-91, Elsevier B.V., Amsterdam, The Netherlands, 2011.

3. Viseras C, Lopez-Galindo A: Pharmaceutical application of some Spanish clays (sepiolite, palygorskite, bentonite): some preformulation studies. Appl Clay Sci, 14, 69-82, 1999.

4. Ayed MH, Zghal I, Rekik B: Effect of sepiolite supplementation on broiler growth performances and carcass yield. Proceedings, Western Section, American Society of Animal Science, 59, 169-172, 2008.

5. Tortuero F, Fernandez Gonzalez E, Martin ML: Effects of dietary sepiolite on the growth, visceral measurements and food passage in chickens. Arch Zootec, 41, 209-217, 1992.

6. AOAC: Official Methods of Analysis. 17th ed., Chapter 4, pp. 1-41, AOAC International, Maryland, 2000.

7. Farese G, Schmidt JL, Mager M: An automated method for the determination of serum calcium with glyoxal bis (2-hydroxyanil). Clinical Chem, 13, 515-520, 1967.

8. ADAS: The analysis of agricultural materials. Ministry of Agriculture, Fisheries and Food, Agricultural Development and Advisory Service, 2nd ed,, Her Majesty’s Stationery Office, London, 1981.

9. Leeson S, Summers JD: Commercial Poultry Nutrition. 3rded, pp. 80, University Books. Guelph, Canada. 2005.


10. Dawson B, Trap RG: Basic and Clinical Biostatistics. 3rd ed., pp. 161-182, Lange Medical Books/McGraw-Hill Medical Publishing Division, New York, 2001.

11. Ouhida I, Perez JF, Piedrafita J, Gasa J: The effect of sepiolite in broiler chicken diets of high, medium and low viscosity. Productive performance and nutritive value. Anim Feed Sci Technol, 85, 183-194, 2000.

12. Pappas AC, Zoidis E, Theophilou N, Zervas G, Fegeros K: Effects of palygorskite on broiler performance, feed technological characteristics and litter quality. Appl Clay Sci, 49: 276-280, 2010.

13. Alzueta C, Ortiz LT, Rebole A, Rodriguez ML, Centeno C, Trevino J: Effects of removal of mucilage and enzyme or sepiolite supplement on the nutrient digestibility and metabolyzable energy of a diet containing linseed in broiler chickens. Anim Feed Sci Technol, 97, 169-181, 2002.

14. Ouhida I, Perez JF, Gasa J, Puchal F: Enzymes (b-glucanase and arabinoxylanase) and/or sepiolite supplementation and the nutritive value of maize-barley-wheat based diets for broiler chickens. Brit Poultry Sci, 41,617-624, 2000.

15. Safaei Katouli M, Boldaji F, Dastar B, Hassani S: Effect of different levels of kaolin, bentonite and zeolite on broiler performance. J Biological Sci, 10, 58-62, 2010.

16. Miazzo R, Peralta MF, Magnoli C, Salvano M, Ferrero S, Chiacchiera SM, Carvalho ECQ, Rosa CAR, Dalcero A: Efficacy of sodium bentonite

as a detoxifier of broiler feed contaminated with aflatoxin and fumonisin. Poultry Sci, 84, 1-8, 2005.

17. Safaei Katouli M, Jafariahangari Y, Baharlouei A: An evaluation on the effects of dietary kaolin and zeolite on broilers blood parameters, T4, TSH and growth hormones. Pakistan J Nutr, 10, 233-237, 2011.

18. Bailey CA, Latimer GW, Barr AC, Wigle WL, Haq AU, Balthrop JE, Kubena LF: Efficacy of montmorillonite clay (NovaSil PLUS) for protecting full-term broilers from aflatoxicosis. J Appl Poult Res, 15, 198-206, 2006.

19. Dwyer MR, Kubena LR, Harvey RB, Mayura K, Sarr AB, Buckley S, Bailey RH, Phillips TD: Inorganic absorbents and cyclopiazonic acid in broiler chickens. Poultry Sci, 76, 1141-1149, 1997.

20. Kubena LF, Harvey RB, Huff WE, Elissalde MH, Yersin AG, Phillips TD, Rottinghaus GE: Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and diacetoxyscirpenol. Poultry Sci, 72, 51-59, 1993.

21. Miles RD, Henry PR: Safety of improved Milbond-TX when fed in broiler diets at greater than recommended levels. Anim Feed Sci Technol, 138, 309-317, 2007.

22. Rosa CA, Miazzo RR, Magnoli C, Salvano M, Chiacchiera SM, Ferrero S, Saenz M, Carvalho ECQ, Dalcero A: Evaluation of the efficacy of bentonite from the South of Argentina to ameliorate the toxic effects of aflatoxin in broilers. Poultry Sci, 80, 139-144, 2001.


SummaryIn this study we aimed to test the hypothesis that butchers have more psychological problems than any other occupation. For

this purpose, we applied SCL-90-R (Symptom Check List-90 Revised) to 43 butchers working in a slaughterhouse and 39 butchers working in meat processing or packing in supermarkets, and as a control group 82 office workers. In the research, a statistical significance between butchers and the control group in that the Global Severity Index (GSI) scores (P<0.001) was found. In terms of the GSI scores, there was no significant relationship between the slaughter house workers and the supermarket workers at P<0.05. However, there were significant relationships among the somatization, anxiety, anger-hostility, and psychotism (P<0.01). As a result, when comparing the two groups, butchers, especially those who work in slaughterhouses, have higher levels of psychological disorders than the office workers.

Keywords: Butchers, Slaughterhouse, Behavioral disorder, Psychological distress

Mezbaha ve Et Perakende İşletmelerinde Çalışan Kasapların Psikolojik Durumlarının Karşılaştırmalı Olarak Araştırılması

ÖzetBu çalışmada kasapların diğer meslek gruplarına göre daha fazla psikolojik problemler yaşadığı hipotezinin kantitatif olarak test

edilmesi amaçlanmıştır. Bu amaçla, 43’ü mezbaha, 39’u perakende dükkan ve süpermarkette çalışan toplam 82 kasap ve kontrol grubu olarak 82 büro çalışanı ile görüşülerek SCL-90-R (Symptom Check List-90 Revised) testi uygulanmıştır. Araştırmada kasaplar ile kontrol grubu arasındaki Global Severety Index (GSI) skorları arasındaki fark anlamlı bulunmuştur (P<0.001). Mezbaha ve entegre tesiste çalışan kasaplar ile perakende dükkan ve süpermarketlerde çalışan kasapların GSI skorları arasında P<0.05 düzeyinde anlamlı bir fark bulunmazken, bu iki grup arasında da somatizasyon, anksiyete, öfke-düşmanlık, psikotizm skorlarının farklı olduğu tespit edilmiştir. Sonuç olarak kasaplar, özellikle de mezbahada çalışan kasaplar, büro çalışanlarına kıyaslandığında daha yüksek düzeyde psikopatoloji yaşamaktadır.

Anahtar sözcükler: Kasaplar, Kesimhane, Davranış bozukluğu, Psikolojik bozukluk

Psychological Symptom Profile of Butchers Working in Slaughterhouse and Retail Meat Packing Business:

A Comparative Study

Abdurrahim EMHAN * Ahmet Şener YILDIZ ** Yasin BEZ *** Said KINGIR ****

***

***

****

Dicle Üniversitesi, İktisadi ve İdari Bilimler Fakültesi, İşletme Bölümü, TR-21280 Diyarbakır - TÜRKİYE Dicle Üniversitesi, Veteriner Fakültesi, Hayvan Sağlığı Ekonomisi ve İşletmeciliği Anabilim Dalı, TR-21280 Diyarbakır - TÜRKİYE Dicle Üniversitesi Tıp Fakültesi, Ruh Sağlığı ve Hastalığı Anabilim Dalı, TR-21280 Diyarbakır - TÜRKİYESiirt Üniversitesi, Sağlık Meslek Yüksekokulu, İktisadi İdari Bilimler Programı, TR-56100 Siirt -TÜRKİYE


According to the type of occupation, because of their work under physical or psychological negative effects, there are a lot of studies in the literature. For example, workers

working in the coal mines suffer from various respiratory tract diseases 1,2. Similarly workers, working under high noise have much more common hearing problems 3 that

INTRODUCTION

İletişim (Correspondence) +90 412 2488020/8662 GSM: +90 505 2960921 [email protected]

RESEARCH ARTICLE

320Psychological Symptom Profile...

have been reported by several researchers.

In terms of emotional context for instance: The kinds of works affect workers’ behavioral levels. Most of the employee who work for the institutions that serve to the problematic areas such as mental health hospitals, intensive care units, nursing homes, and orphanages may develop psychosomatic disorders 4-7

Butchers work both physically and emotionally in a negative environment. Therefore, there have been reported that butchers have a higher rate of physical disorders 8-10, work accident or injuries 11,12, alcohol and drug use, higher absenteeism, and staff turnover 13,14.

Because slaughterhouse workers constantly kill animals, it has been reported that they suffer from anxiety, panic, depression, increased paranoia, a sense of disintegration, high levels of drugs, and alcohol use 15,16. In addition, over time, some of its employees enjoy the killing, and this situation become psychotic antisocial and statements prepared by the proposed development of the ground 17) Crime rate among butchers has been found to be higher than other occupational groups 18,19.

While it has been reported that employees work in abattoirs and butchers in the meat packing industry suffer from physical diseases in a large number of scientific research in the literature, investigating the effects of psychological studies is very limited. In the studies which are related to the psychological aspects of the subject, almost all used the qualitative methods 13,14,18,19. Due to adverse working conditions, the purpose of this study is to test the hypothesis that assumes that butchers have more psychological problems than the other job groups by using quantitative method. Unlike, similar works which used qualitative method, this study used quantitative methods for testing the hypothesis. It is believed that this study can provide useable knowledge for the behavioral scientists, the administrators or the investors of this industry.


Study Design

To test the study hypothesis, three groups were formed; the first group was butchers working in slaughterhouses, the second group was butchers working in the meat processing or packing in supermarkets, and the third group was office workers as a control group. The SCL-90-R scale was applied to the three groups.

In Diyarbakir province, where 3 slaughterhouses have been actively running, we reached 51 butchers working in slaughterhouses, only 45 butchers to fill out the survey. For the agreed second group, we accessed 51 butchers working in meat processing or packing at supermarkets, only 42 butchers agreed to fill out the survey. For the third group,

as a control group office workers were accessed in the same city and we obtained 85 surveys. Finally we got 172 surveys and demographic variables. Eight surveys were excluded from the analysis due to significant lack of information thus, 164 forms were evaluated for further analysis.

Data Collection Materials

Demographic Form: This was prepared by the authors to collect demographic variables including age, marital status, and education.

Symptom Check List-90 Revised (SCL-90-R): It is a psycho- metric scale named for screening any psychopathologies. This is a 90 item multidimensional questionnaire to screen for a broad range of psychological problems revised by Derogatis in 1994. Each of the 90 items is rated on five- point Likert scale of distress, ranging from “not at all” (0) to “extremely” (4). Subsequently the answers are combined in the 10 primary symptom dimensions: Somatization, obsessive - compulsiveness, interpersonal sensitivity, depression, anxiety, anger-hostility, phobic anxiety, paranoid ideation, psychotism, and others. The scale provides also a global severity index (GSI) score which is an indicator of general psychological distress.


The obtained data were analyzed by using SPSS version 17.0. Parametric variables were compared between groups with independent t test. Statistically significance level was set as P<0.05.

RESULTS

A total of 82 butchers (43 working in slaughterhouses, 39 working in markets) and most of the office workers have been included in the study. All participants were male in gender. In terms of age, education level and marital status butchers and office workers as control subjects were similar. Their demographical variables and psychological scale results are given in Table 1. They were similar in terms of age distribution. More participants were married and graduated from primary school in the butchers group than office workers group. As seen from Table 1, butchers scored higher in all sub-scales scores and in GSI score than the control subjects.

When SCL-90-R subscale scores of butchers working in slaughterhouses were compared with butchers working in markets; the former group scored significantly higher in somatization, anxiety, anger-hostility, and psychotism sub-scales than the latter one (Table 2).

DISCUSSION

Butchers and office workers participating in the study were similar. When the butchers and office workers

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EMHAN, YILDIZBEZ, KINGIR

compared with, marital status and education levels, the marriage rate among butchers is higher than the office workers and the butchers’ educational level was lower than the other group. Butchers have lower education levels as it was expected to be.

All the butchers’ GSI scores and all the other sub-scale scores are higher than the control group. Therefore, these results supported the hypothesis that the butchers have more psychopathology. In this case, a two-way relationship may be taken into consideration. Namely, some butchers

Table 1. Comparison of demographical variables and SCL-90-R scores between butchers and office workers.

Tablo 1. Kasaplar ile ofis çalışanlarının SCL-90 ve demografik değişkenlerin karşılaştırılması

Variables Butchers (N=82)(%)

Controls (N=82) (%)

Statistics(χ2or t) P

Age

20-30 37 (45%) 41 (50%)

1.37* 0.5131-40 32 (39%) 25 (30%)

41-50 13 (16%) 16 (20%)

Marital status

Married 62 (76%) 40 (49%)12.55* 0.001

Unmarried 20 (%24) 42 (51%)

Education

Primary school or literate 59 (72%) 7 (9%)

80.57* 0.001High school 17 (20%) 20 (24%)

University 6 (8%) 55 (67%)

SCL-90-R (mean±SD)

Somatization 1.50±0.91 0.83±0.61 5.51 0.001

Obsessive-compulsiveness 1.34±0.76 0.99±0.70 3.13 0.002

Interpersonal sensitivity 1.29±0.75 0.85±0.71 3.93 0.001

Depression 1.26±0.84 0.76±0.71 4.13 0.001

Anxiety 1.09±0.80 0.59±0.57 4.69 0.001

Anger-hostility 1.47±0.97 0.80±0.83 4.74 0.001

Phobic anxiety 0.82±0.67 0.40±0.57 2.28 0.001

Paranoid ideation 1.40±0.86 0.97±0.79 3.30 0.001

Psychotism 0.99±0.70 0.64±0.76 3.03 0.003

Other 1.62±0.89 0.92±0.75 5.44 0.001

GSI 1.28±0.70 0.77±0.60 4.99 0.001

* shows χ2 value, SCL-90-R: Symptom check-list-90 revised (* χ2 değerini gösterir, SCL-90-R: Yeniden gözden geçirilmiş SCL-90)

Table 2. Comparison of SCL-90-R subscale scores between butchers working in slaughterhouse and working in markets

Tablo 2. Marketlerde ve mezbahalarda çalışan kasaplar arasında SCL-90-R karşılaştırması

SCL-90-R Subscales Butchers Working in Slaughterhouse(N = 43)

Butchers Working in Markets(N = 39)

Statistics( t ) p

Somatization 1.77±0.90 1.21±0.83 2.93 0.004

Obsessive-compulsiveness 1.43±0.84 1.26±0.65 1.01 0.312

Interpersonal sensitivity 1.33±0.79 1.26±0.72 0.43 0.662

Depression 1.27±0.86 1.25±0.81 0.12 0.897

Anxiety 1.32±0.89 0.84±0.59 2.85 0.005

Anger-hostility 1.67±1.06 1.24±0.81 2.02 0.046

Phobic anxiety 0.94±0.77 0.67±0.49 1.95 0.055

Paranoid ideation 1.33±0.86 1.46±0.86 0.69 0.491

Psychotism 1.13±0.80 0.82±0.53 2.05 0.043

Other 1.73±0.88 1.49±0.89 1.18 0.239

GSI 1.40±0.73 1.14±0.63 1.74 0.085

SCL-90-R: Symptom check-list-90 revised (SCL-90-R: Yeniden gözden geçirilmiş SCL-90)

322Psychological Symptom Profile...

have more psychopathology due to their job, some people with psychopathology may also have chosen the profession of butchering which is the effect and which is the cause, is unclear. In the situation it is not possible to reach conclusion.

However, it can be said there is a relationship between butchers and an increased risk of psychopathology. On the other hand, in terms of somatization, anxiety, anger hostility, phobic anxiety and psychotism, slaughterhouse workers’ scores are higher than meat packing business workers. The results are consistent with the results of previous studies 15,16.

The results of slaughterhouse workers having higher GSI scores showed that this group needs medical help. GSI scores on the course of 1 is associated with clinically significant psychopathology. The average scores of 1.40±0.73 for slaughterhouse workers, for the meat packing business workers 1.14±0.63, respectively. In addition, in somatization, anxiety, anger hostility, and psychotism scores, the slaughterhouse workers have higher scores than meat packing business. When taking into consideration the health spending and loss of labor, it can be said that the actual cost of the meat may be higher than the current cost.

Throughout history, the profession of butchery has been considered to be an unwanted job 20,21. During the period of Ottoman Empire, Ahi Organizations, which were a kind of job trade association, and tradesmen guild, foundations considered the job of butchery among the problematic jobs. So this organization did not want to include it in their members 22. According to this organization, it was considered the profession of butchery was a bloody job and it was assumed that butchers’ compassion and kindness declined over time due to their job.

In this study, there have been the highest levels of somatization and anger-hostility observed among butchers. The two psychopathologies in the above mentioned assumptions match up with the previous studies. Another indirect support for this assumption is that there have been reported high crime rates among butchers 18,19. On the other hand it is also known that butchers working in the slaughterhouses behave ruthlessly or they have remorse feelings from time to time 14. Butchers with high somatization, depression and anxiety could be associated with remorse feelings and the brutal behavior may be associated with high levels of anger and hostility. It can be thought that these psychological problems may cause diminished workforce and productivity which eventually affect adversely the profitability of enterprises.

As a result, when it was comparing butchers, especially the ones working in a slaughterhouse, and office workers, butchers have had a high level of psychopathology. Considering butchers’ working conditions, this situation can provide much usable knowledge to the behavioral scientists, the administrators or the investors in this industry.

In further studies, slaughterhouse workers occupational accidents, illness, absenteeism and turnover rates will be examined by comparing them with the other sectors.

REFERENCES

1. Cimrin AH, Demiral Y, Ergor A, Uz Basaran S, Komus N, Ozbirsel C: Dust exposure levels and pneumoconiosis prevalence in a lignite coal miners. Tüberküloz ve Toraks Dergisi, 53, 268-274, 2005.

2. Parihar YS, Patnaik JP, Nema BK, Sahoo GB, Misra IB, Adhikary S: Coal workers’ Pneumoconiosis: A study of prevalence in coal mines of Eastern Madhya Pradesh and Orissa States of India. Ind Health, 35, 467-473, 1997.

3. Guvercin Ö, Aybek A: Noise problem in stone pulverizing and sieving plants. KSÜ Fen ve Mühendislik Dergisi, 6, 101-107, 2003.

4. Stansfeld SA, Fuhrer R, Head J, Ferrie J, Shipley M: Work and psychiatric disorder in the whitehall li study. J Psychosom Res, 43, 73-81, 1997.

5. Acker GM: The Impact of clients’ mental illness on social workers’ job satisfaction and burnout. Health Soc Work, 24, 112-119, 1999.

6. Egan M, Kadushin G: Job satisfaction of home health social workers in the environment of cost containment. Health Soc Work, 29, 287-296, 2004.

7. Barth RP, Lloyd EC, Christ SL, Chapman MV, Dickinson NS: Child Welfare Worker Characteristics and Job Satisfaction: A National Study. Soc Work, 53, 199-209, 2008.

8. Viikari JE: Neck and Upper Limb Disorders among Slaughterhouse Workers. An Epidemiologic and Clinical Study. Scand J Work Env Hea, 9, 283-290, 1983.

9. Roto P, Kivi P: Prevalence of epicondylitis and tenosynovitis among meatcutters. Scand J Work Env Hea, 10, 203-205, 1984.

10. Örtenger R, Andersson GBJ, Petersen I, Sabel B: An ergonomic study of work methods and physical disorders among professional butchers. Appl Ergon, 18, 43-50, 1987.

11. Winders B, Nibert D: Consuming the surplus: expanding “meat” consumption and animal oppression. Int J Sociol Soc Pol, 24, 76-96, 2004.

12. Stull DD, Broadway MJ: Slaughterhouse blues: The meat and poultry industry in North America. Wadsworth/Thomson Learning Inc., Canada, 2004.

13. Kristensen TS: Sickness absence and work strain among danish slaughterhouse workers: An analysis of absence from work regarded as coping behaviour. Soc Sci Med, 32, 15-27, 1991.

14. Eisnitz GA: Slaughterhouse: The shocking story of greed, neglect, and inhumane treatment inside the U.S. meat industry Newyork. Prometheus Books, 1997.

15. Broadway MJ: Meatpacking and Its Social and Economic Consequences for Garden City, Kansas in the 1980s. Urban Anthropology, 19, 321-344, 1990.

16. MacNair RM: Perpetration-induced traumatic stress: The psychological consequences of killing. greenwood publishing, USA, 2002.

17. Dillard J: A Slaughterhouse nightmare: Psychological harm suffered by slaughterhouse employees and the possibility of redress through legal reform. Georgetown Journal on Poverty Law Policy, 1-18, 2007. http://ssrn.com/abstract=101640. Accessed: 05 Jan. 2011,

18. Broadway MJ: Planning for change in small towns or trying to avoid the slaughterhouse blues. J Rural Stud, 16, 37-46, 2000.

19. FitzGerald AJ, Kalof L, Dietz T: Slaughterhouses and increased crime rates an empirical analysis of the spillover from “the jungle” into the surrounding community. Organ Environ, 22, 158-184, 2009.

20. Faroqhi S: Osmanlıda Kentler ve Kentliler (Towns and Townsmen of Ottoman). Çeviri (Translation): N. Kalaycıoglu. Türkiye Araştırmaları Tarih ve Yurt Yayınları, İstanbul, 1994.

21. Greenwood A: Istanbul’s meat provisioning. a study of the celepkeşan system. Doctoral Thesis, University of Chicago. Chicago, 1988.

22. Tarim CH: Tarihte Kırşehri-Gülşehri ve Babailer-Ahiler-Bektaşiler. Üçüncü Baskı. Yeniçağ Matbaası, İstanbul, 1948.


SummaryThe objective of this study was to determine and compare the some quality properties and mineral contents of yoghurts made

from different types of milks. For this purpose; physical, chemical, microbiological and sensory properties of yoghurts produced from cows’, buffaloes’, ewes’ and goats’ milks were examined during 28 day storage at 4°C. Some major and minor mineral contents of yoghurts were also determined. Total solids, protein, ash, Ca and P contents of ewes’ yoghurt were significantly higher compared with the other yoghurts. An increase in viscosity was observed with increasing total solids and protein contents of yoghurts. Zn and Fe contents of cows’ yoghurt were higher than the other yoghurts. L. bulgaricus and S. thermophilus counts of all yoghurts showed an increasing until day 7 of storage then a decreasing until the end of storage. Although cows’ and ewes’ yoghurts were the most acceptable during storage, goats’ yoghurt was the lowest scored by panelists.

Keywords: Yoghurt, Milk type, Quality properties, Mineral content

Farklı Tür Sütlerden Üretilen Yoğurtların Bazı Kalite Özellikleri ve Mineral İçerikleri Üzerine Karşılaştırmalı Bir Araştırma

ÖzetBu çalışmanın amacı; farklı tür sütlerden üretilen yoğurtların bazı kalite özellikleri ile mineral içeriklerini tespit etmek ve

karşılaştırmaktır. Bu amaçla, inek, manda, koyun ve keçi sütlerinden üretilen yoğurtların 4°C’de 28 günlük muhafaza süresince fiziksel, kimyasal, mikrobiyolojik ve duyusal özellikleri incelenmiştir. Ayrıca, yoğurtların bazı major ve minör mineralleri de araştırılmıştır. Diğer yoğurtlarla karşılaştırıldığında koyun yoğurdunun toplam kuru madde, protein, kül, Ca ve P içeriklerinin daha yüksek olduğu görülmüştür. Yoğurtların toplam kuru madde ve protein içeriklerinin artışına bağlı olarak viskozite değerleri de artmıştır. İnek yoğurdunun Zn ve Fe içerikleri diğer yoğurtlara nazaran daha yüksek bulunmuştur. Yoğurtların L. bulgaricus ve S. thermophilus sayıları muhafazanın 7. gününe kadar artmış, daha sonra muhafaza süresinin sonuna kadar azalmıştır. İnek ve koyun yoğurtları duyusal açıdan muhafaza süresince en kabul edilebilir olmalarına rağmen, keçi yoğurdu panelistler tarafından en düşük puanları almıştır.

Anahtar sözcükler: Yoğurt, Süt çeşidi, Kalite özellikleri, Mineral içeriği

A Comparative Study on Some Quality Properties and Mineral Contents of Yoghurts Produced From Different Type of Milks [1]

Tuba ERKAYA * Mustafa ŞENGÜL *[1]

*This study was supported by the Atatürk University Research Center (Project No: 2007⁄106)Department of Food Engineering, College of Agriculture, Atatürk University, TR-25240 Erzurum - TURKEY


An increase interest in the production and consumption of fermented dairy products has been observed due to their flavourful and healthful properties. Yoghurt, obtained by the lactic acid fermentation of milk by addition of homofermentative yoghurt starter bacteria, is the most well known fermented dairy product around the world. Although the origin of yoghurt is not known precisely it is thought that it might be as the Middle East according to the historical records 1. Yoghurt is rich in calcium,

phosphorus, magnesium, vitamin A and riboflavin 2. It also contains high levels of viable yoghurt bacteria and their metabolites which are beneficial for health; so that many undesired microorganisms couldn’t grow up in yoghurt. Thus, it is accepted to be a safety product 3.

The milk used for production of yoghurt is important in terms of quality parameters of yoghurt including flavour textural and compositional characteristics. Although milks

INTRODUCTION


RESEARCH ARTICLE

324A Comparative Study on ...

from other animal species, such as ewes, goats and buffaloes are utilized to the human diet in various parts of the world, a great number of studies have focused on cows’ milk. Yoghurt is mostly made from cows’ milk and a very limited extent from ewes’, goats’ or buffaloes’ milks in Turkey. However, for home-made yoghurt, cows’, buffaloes’, goats’ and ewes’ milks or their mixture are frequently used in different regions of Turkey. On the other hand, these milks are very popular in countries around the world, especially Mediterranean region 1. Buffaloes’ milk has an important nutritional value because of its high level of fat content particularly, which is responsible for its high energetic and nutritive properties 4. Furthermore, buffaloes’ milk is the second most produced milk in the world with 82 billion liters produced each year (12.5% of milk produced in the world), after cows’ milk (84% with 551 billion liters) 5, although it is the least produced milk in Turkey. Ewes’ milk, the other milk type, contains higher amount of proteins than cows’ milk (58 g/kg and 33 g/kg, respectively) and does not require fortification of the milk in the production of yoghurt 6. Besides, ewes’ yoghurt has a pleasant creamy-sour flavour, considered by many to be better than cows’ yoghurt 7. On the other hand, goats’ milk and its products, such as yoghurt and cheese, are becoming increasingly popular in the world for its high nutritional value, easy assimilation of components, therapeutic, antioxidative and antiallergenic properties, but it is a possible problem of musky, rancid and goaty flavour and aroma in goats’ milk products 8.

Yoghurt can be a good dietary source of essential minerals. It could contribute importantly to the recommended daily intake of calcium and magnesium to maintain the physiological processes in the body 9. Moreover, bioavailability of calcium from yoghurt is higher than from milk. The acidic pH of yoghurt ionizes calcium and hence facilitates intestinal calcium absorption 10. Phosphorus, magnesium and zinc are also in an available form in yoghurt for absorption and utilization by the body 11. In addition, yoghurt contains noticeable quantities of sodium and potassium 1. The levels of essential minerals present in dairy products depend on the technological treatments, type of milk used and accuracy of analysis 12.

The traditional fermented milk products made from the milks of cows, ewes and goats are still competitive with newer products. There have been a number of separate studies on physiochemical, rheological, microbiological, sensory properties and mineral contents of yoghurts made from these milks. However, only a few comparative research data have been documented on some quality properties 13,14 and mineral contents of yoghurts made from different types of milks 15. Furthermore, information about the chemical composition and physical characteristics of buffaloes’ yoghurt is scarce, compared to cows’, ewes’ and goats’ yoghurts. The comparison of physiochemical, rheological, microbiological, sensory properties and mineral

contents in yoghurts made from these four milks has not been reported in the literature. Therefore, the aims of this study were to determine the some quality properties and mineral contents of the yoghurts made from cows’, ewes’, goats’ and buffaloes’ milks and to contribute to the literatures comparing the differences among these yoghurts.


Milks and Starter Cultures

Cows’ milk was supplied by dairy farm of Atatürk University in Erzurum province of Turkey. Ewes’, goats’ and buffaloes’ milks were obtained from different villages in Erzurum province. Direct-to-vat system yoghurt starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus) coded YC350 was used in yoghurt-manufacturing supplied from Chr.Hansen-Peyma, Istanbul, Turkey.

Manufacture of Yoghurts

The yoghurts were manufactured according to the protocol proposed by Tamime and Robinson 1 in duplicate. The raw cows’, ewes’, goats’ and buffaloes’ milks were separately strained using a cloth filter. Each milk was held to 85°C for 25 min using batch pasteurization and cooled down to 44±1°C. The heat treated milk was inoculated with thermophilic starter culture (S. thermophilus and L. bulgaricus). The inoculation rate was 20 g/100 L milk for all samples. Then, each inoculated milk was distributed into 200-mL sterile glass cups and incubated at 44±1°C until it reached pH 4.6. After fermentation, yoghurt samples removed and stored at 4±1°C for 28 days and analyzed 1, 7, 14, 21 and 28 d of cold storage.

Chemical Analyses

Total solids, fat, ash, titratable acidity 16 and protein contents 17 of milks and yoghurt samples were determined. The pH was measured with a pH meter (model WTW pH-340-A, Weilheim, Germany) fitted with a combined glass electrode.

Syneresis

The yoghurt samples were analyzed for syneresis throughout storage according to the method described by Atamer and Sezgin 18 Twenty-five grams of yoghurt samples were weighed and filtered. After 120 min of drainage at 4±1°C, the amount of collected whey (mL) in a flask was recorded and expressed as an index of syneresis.

Apparent Viscosity

The apparent viscosities of yoghurt samples were measured during storage using Visco Star-L Fungilab visco- meter equipped with a number 6 spindle and operated

325ERKAYA, ŞENGÜL

at a speed of 20 rpm. All of the measurements were performed in duplicate and sample temperature was 4±1°C. The yoghurt samples were stirred gently for 10 s before the viscosity measurement. The readings were taken from instrument directly at the point of 30th s and were recorded in centipoise 19.

Mineral Analysis

Mineral contents (Ca, Mg, Na, P, K, Fe, Mn, Ni, S and Zn) of yoghurt samples were determined using an Inductively Coupled Plasma Optical Emission Spectrometry (ICP/OES (Perkin-Elmer, Optima 2100 DV, Shelton, CT, USA)) and following the method described by Güler 20. Decomposition of samples was performed in a microwave oven (Berghof speed wave, Germany). For this purpose, about 0.5 g yoghurt sample was weighed into the digestion vessels. Concentrated nitric acid (10 mL) was added and after that, digestion was carried out to each sample at 210°C and 176 psi pressure for 10 min. After cooling, the carousels were removed from the oven, 30% hydrogen peroxide (2 mL) was added to samples and then second digestion was applied at 195°C and 95 psi pressure for 5 min. The vessels were immediately closed after the addition of oxidants. At the end of the digestion process, the samples were diluted with distilled water to a suitable concentration, and were filtered through Whatman no. 42 filter paper. All diluted digests were eventually analyzed by an ICP-OES.

Microbiological Analysis

For each yoghurt sample, 11 g were weighted and diluted aseptically in 99 mL of sterile peptone water (0.1% w⁄v). Serial dilutions were made in 0.1% sterile peptone water and appropriate dilutions were poured plated in duplicate. The counts of S. thermophilus were enumerated on M17 agar (Oxoid Ltd, Basingstoke, Hampshire, UK) by incubating the plates aerobically at 37°C for 48 h 21. For enumeration of L. bulgaricus MRS agar (Oxoid Ltd) was used and the plates were incubated anaerobically at 37°C for 72 h. Anaerobic conditions were provided using Anaerocult A sachets (Merck). Plates containing 20-200 colonies were enumerated and the results are expressed as colony-forming units per gram (cfu/g) of yoghurt sample.

Sensory Analysis

Sensory analysis was carried out in yoghurt samples by a group of six panelists from the academic staff working in the Dairy Department. Taste and aroma, odor, mouth-feel, texture, acidity and general acceptability of samples were scored on a scale of 1-9 on days 1, 7, 14, 21 and 28 of storage. Samples were left at the room temperature for 10-15 min and they served with a glass of water a slice of bread 22.

Statistical Analysis of Data

The data were analyzed statistically using SPSS statistical

software programme version 13 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Duncan’s Multiple Range Test was used to determine significant differences among results.

RESULTS

Milk Composition

The compositions of raw cows’, ewes’, goats’ and buffaloes’ milks were total solids contents 12.06%, 17.40%, 14.15%, 16.56%; fat contents 3.80%, 5.90%, 4.70%, 7.40% and ash contents 0.70%, 0.91%, 0.78%, 0.77%, respectively. The respective pH and titratable acidity of cows’ milk were 6.49 and 0.20%; those of ewes’ milk were 6.64 and 0.24%, those of goats’ milk were 6.55, 0.19% and those of buffaloes’ milk were 6.78 and 0.15%. Mineral contents of milks used in yoghurt production are shown in Table 1.

Physical and Chemical Characteristics of Yoghurts

The results of the chemical and physical analyses of the yoghurt samples during storage are presented in Table 2. Ewes’ yoghurt had the highest total solids (18.59%), protein (7.02%) and ash (0.97%) contents whereas buffaloes’ yoghurt had the highest fat (8.26%) content (P<0.05).

The mean pH values of ewes’ and goats’ yoghurts were higher than cows’ and buffaloes’ yoghurts (Table 2). Milk type and storage time affected significantly (P<0.05) titratable acidity of yoghurt samples. As shown in Table 2, ewes’ yoghurt had the highest mean titratable acidity value but cows’ yoghurt had the lowest mean value of titratable acidity. The lowest mean value of titratable acidity was found on day 1st of storage, but the highest value was found on day 28th of storage, and these differences were

Table 1. Mineral contents (mg/kg) of milks used in yoghurt production

Tablo 1. Yoğurt üretiminde kullanılan sütlerin mineral içerikleri (mg/kg)

MineralsMilk Types

Cow Buffalo Ewe Goat

Major Elements

Ca 1137 1509 1512 1312

K 1359 1065 1077 1496

Mg 97 145 148 152

Na 335 334 457 469

S 497 684 948 817

P 869 1146 1209 1051

Minor Elements

Fe 35.03 13.59 35.29 33.57

Mn 0.15 0.18 0.19 0.12

Ni 0.62 0.99 1.11 0.92

Zn 43 49 81 113


statistically (P<0.05) significant.

The syneresis values of yoghurt samples were affected significantly (P<0.05) by milk type and storage time and the changes were shown in Table 2. The mean syneresis values of yoghurts decreased (P<0.05) on day 7 but showed an increase (P< 0.05) at day 14 followed by a decrease (P<0.05) until day 28 of storage. Concerning the rheological properties of yoghurts, there were significant (P<0.05) differences between yoghurt samples for apparent viscosities. The results showed that ewes’ yoghurt had higher viscosity than the other yoghurts.

Mineral Composition

Table 3 shows the mean values of major and minor

elements in yoghurts and their changes during storage. Most of the major and minor mineral contents were in accordance with the ash contents of each yoghurt with a few exceptions (Tables 2 and 3). There were significant differences among the yoghurt samples in terms of major element contents such as Ca, K, Mg, Na, S and P (Table 3). The mean values of Ca in all the yoghurt samples were found to be higher than those of P values. However, the mean Ca and P contents were 1879 ppm and 1441 ppm in ewes’ yoghurt as the maximum content, respectively. When examined the data presented in Table 3, it was observed that the mean values of major elements in yoghurts generally increased (P<0.05) on day 7 but showed a decrease (P<0.05) at day 14 followed by an increase (P<0.05) until day 28 of storage. The highest mean value

Table 3. Mineral contents (mg/kg) of yoghurts made from different milks and their statistical evaluations in terms of milk source and storage time

Tablo 3. Farklı sütlerden üretilen yoğurtların mineral içerikleri(mg/kg) ve süt çeşidi ile depolama süresi bakımından istatistiksel değerlendirmesi

Yoghurt Samples

Mineral Substances

Major Elements Minor Elements

Ca K Mg Na S P Fe Mn Ni Zn

Cow 1268±232a 1571±295b 114±16.80a 390±58.30a 683±110a 921±181a 18.84±5.75 0.16±0.11 0.97±0.49 60.68±24

Buffalo 1697±152b 1164±101a 156±15.14b 344±27.08a 866±153b 1175±69b 13.57±8.34 0.15±0.06 1.11±0.47 63.95±22

Ewe 1879±378b 1133±206a 186±32.63b 567±95.70b 1229±286c 1441±300c 12.79±10.44 0.19±0.07 1.16±0.43 72.99±21

Goat 1643±438b 1800±524b 187±42.38b 568±133.8b 1014±274b 1188±281b 16.79±12.38 0.14±0.07 0.97±0.49 75.03±38

Storage Time (days)

1 1403±252a 1211±295a 135±29.84a 399±81.09a 737±188a 108±166 27.67±12.86b 0.23±0.07b 1.07±0.42 71.46±37

7 1701±397bc 1752±685b 177±54.22b 530±199.9b 923±284b 1249±313 15.98±11.43a 0.17±0.08ab 0.89±0.55 111.13±36

14 1490±325ab 1363±303a 153±29.35ab 445±101.1ab 900±249ab 1093±242 11.54±9.53a 0.12±0.06a 0.92±0.27 60.48±22

21 1678±493abc 1407±223a 167±43.49b 473±129.5ab 1029±331bc 1193±387 8.58±3.69a 0.13±0.07a 1.00±0.42 73.26±28

28 1836±320c 1353±318a 169±39.88b 488±120.8b 1152±283c 1283±297 13.72±9.73a 0.17±0.08ab 1.38±0.54 69.57±291 Means are average of two trials. Different letters indicate (P<0.05) between yoghurt samples and days of storage

Table 2. The mean values of some physical and chemical characteristics of yoghurts made from different milks and their statistical evaluations in terms of milk source and storage time

Tablo 2. Farklı sütlerden üretilen yoğurtların fiziksel ve kimyasal özelliklerine ait ortalama değerler ve süt çeşidi ile depolama süresi bakımından istatistiksel değerlendirmesi

Yoghurt Samples

Physical and Chemical Characteristics

Total Solids (%)

Ash (%)

Fat (%)

Protein (%)

Titratable Acidity (%) pH Whey Seperation

(ml/25g)Viscosity

(cP)

Cow 12.12±0.14a 0.72±0.02a 3.91±0.13a 3.56±0.14a 1.01±0.08a 4.05±0.11a 8.32±1.54d 3254±476a

Buffalo 17.87±0.35c 0.86±0.02c 8.26±0.28d 4.72±0.06b 1.17±0.12b 4.09±0.08a 3.39±0.83b 13302±1558c

Ewe 18.59±0.20d 0.97±0.03d 6.66±0.21c 7.02±0.19d 1.52±0.15c 4.16±0.11b 1.51±0.51a 21380±1077d

Goat 15.06±0.11b 0.84±0.02b 5.35±0.22b 4.81±0.12c 1.14±0.08b 4.17±0.11b 6.48±0.52c 6412±934b

Storage Time (days)

1 15.91±2.76a 0.840±0.08a 5.95±1.74a 4.94±1.25a 1.09±0.18a 4.26±0.07c 4.77±2.45b 10033±7221a

7 16.12±2.78b 0.853±0.08b 6.26±1.88c 5.03±1.31ab 1.15±0.20b 4.16±0.08b 4.31±2.47a 10817±7169b

14 15.84±2.76a 0.846±0.09ab 6.05±1.69b 4.96±1.33a 1.18±0.19b 4.12±0.07b 5.68±3.41c 10948±7651bc

21 15.83±2.70a 0.854±0.12b 6.07±1.61b 5.11±1.47b 1.27±0.21c 4.03±0.08a 5.02±3.35b 12533±7905d

28 15.84±2.69a 0.850±0.11b 5.88±1.70a 5.09±1.33b 1.36±0.24d 4.02±0.03a 4.85±2.93b 11104±7409c

1 Means are average of two trials. Different letters indicate (P<0.05) between yoghurt samples and days of storage

327ERKAYA, ŞENGÜL

of K (1800 ppm) was found in goats’ yoghurt whereas the highest Na contents were determined in ewes’ (567 ppm) and goats’ (568 ppm) yoghurts. Of the minor elements, Mn and Ni were found at higher in ewes’ yoghurt. No significant differences (P>0.05) were observed among Fe, Mn, Ni and Zn contents in all yoghurts.

Counts of Yoghurt Bacteria

The mean viable counts of S. thermophilus and L. delbrueckii spp. bulgaricus in the yoghurt samples and the changes during storage are shown in Table 4. The effect of milk type on S. thermophilus counts was found to be significant (P<0.05).

Sensory Results

Results of the sensory evaluation of cows’, ewes’, goats’ and buffaloes’ yoghurts on a scale from 1 (very bad) to 9 (excellent) are shown in a radar plot in Figs. 1a, b, c and d, respectively. In general, cows’ and ewes’ yoghurts received similar scores by panelists for odor, taste and aroma and general acceptability characteristics. Goats’ yoghurt was the least acceptable for its non-typical yoghurt taste and liquid texture. Ewes’ yoghurt received the highest texture scores and it was parallel to its viscosity values.

a b

c

d

Fig 1. (a) Sensory profile of yoghurt made from cows’ milk during storage (b) Sensory profile of yoghurt made from ewes’ milk during storage (c) Sensory profile of yoghurt made from goats’ milk during storage (d) Sensory profile of yoghurt made from buffaloes’ milk during storage

Şekil 1. (a) İnek sütünden üretilen yoğurdun depolama süresince duyusal değerlendirmesi (b) Koyun sütünden üretilen yoğurdun depolama süresince duyusal değerlendirmesi (c) Keçi sütünden üretilen yoğurdun depolama süresince duyusal değerlendirmesi (d) Manda sütünden üretilen yoğurdun depolama süresince duyusal değerlendirmesi

Table 4. The mean counts (log cfu/g) of yoghurt bacteria counts of yoghurts made from different milks and their statistical evaluations in terms of milk source and storage time

Tablo 4. Farklı sütlerden üretilen yoğurtların ortalama yoğurt bakterisi sayıları (log kob/g) ve süt çeşidi ile depolama süresi bakımından istatistiksel değerlendirmesi

Yoghurt Samples

Yoghurt Bacteria

L. bulgaricus S. thermophilus

Cow 8.10±0.52a 8.18±0.36c

Buffalo 8.17±0.30a 8.49±0.32d

Ewe 7.99±0.36a 7.20±0.50a

Goat 8.19±0.30a 7.66±0.43b

Storage Time (days)

1 8.29±0.22bc 7.77±0.28a

7 8.44±0.35c 8.15±0.61b

14 8.06±0.23b 8.11±0.62b

21 8.04±0.32ab 7.84±0.65ab

28 7.73±0.35a 7.54±0.82a

1 Means are average of two trials. Different letters indicate (P<0.05) between yoghurt samples and days of storage


DISCUSSION

Gross Chemical Composition of Yoghurt Samples

There were significant differences (P<0.05) in the gross chemical composition of yoghurts due to source of milk used. The chemical composition of cows’ yoghurt was lower than the others. The gross chemical values of yoghurts were similar to that found by Güler and Sanal 15 in cows’, ewes’ and goats yoghurts.

Titratable Acidity and pH

The pH values of all yoghurts significantly decreased from 4.26 to 4.02 throughout storage (P<0.05). This could be attributed to further metabolic activity of starter cultures during storage 23. Although ewes’ yoghurt had the highest mean pH value, it also had the highest titratable acidity. This can be due to higher buffering capacity from increasing protein content in the milk 24. So, in this study a decrease in pH of yoghurts with increasing protein contents and an increase in acidity of yoghurts with increasing total solids were observed. The changes of pH and titratable acidity values for yoghurts during storage were similar to those indicated in the literatures 24,25.

Syneresis and Viscosity

Syneresis, which defined as shrinkage of a gel occurs with expulsion of liquid, is a common defect in fermented milk products 26. The highest mean value of syneresis (8.32 mL/25g) was in cows’ yoghurt and the lowest mean value (1.51 mL/25g) was in ewes’ yoghurt. This could be related to total solids of the samples. It was reported that common reasons for the occurrence of syneresis include the use of a high incubation temperature, excessive whey protein to casein ratio, low solids content and physical mishandling of the product during storage and retail distribution 27. Vargas et al.28 found that similar result in yoghurts made from mixtures cows’ and goats’ milks, suggesting that the addition of goats’ milk led to lower syneresis.

The mean viscosity values of yoghurts increased up to 21st day but decreased at 28th day of storage (Table 2). This increase could be also depended on protein contents of yoghurts as well as total solids. On the contrary, Tarakçı 29 found that the viscosity values of yogurt samples decreased during storage period. The effect of different types of milks on viscosity and incubation time was studied by Jumah et al.30. They found that sheep milk reached the highest viscosity value, followed by caprine and bovine milk. The differences in viscosity among types of milk appeared to be due to the differences in total solids contents of the milks. Because, the viscosity value of yoghurt rise with the increase of its total solid content.

Mineral Composition

Mineral analyses are essential for determining the

quality and safety of milk and milk products 20. Ewes’ yoghurt showed higher mineral contents, mainly Ca, S and P than cows’, goats’ and buffaloes’ yoghurt. As known, milk and milk products, especially ewes’ milk and its products, are excellent dietary sources of Ca, P and Mg and they can provide a significant amount of calcium in a bioavailable form 2,10,15. Mg, is related to Ca and P function, may bind to the non-phosphorylated binding sites in the caseins 11. It has been reported that Mg absorption was faster with diets based on caseins, mainly β-casein, compared to those based on lactoserum proteins 31. In this study, Mg contents in ewes’ (186 ppm), goats’ (187 ppm) and buffaloes’ yoghurts (156 ppm) were determined similar, with cows’ yoghurt (114 ppm) being the lowest. According to this, it can be recommended that commercial yoghurts manufactured from mostly cows’ milk should be mixed with ewes’, goats’ and buffaloes’ milks for providing enhancement of Mg absorption. The highest mean value of K was found in goats’ yoghurt. These findings were similar to that reported by Güler and Şanal 15. Raynal-Ljtovac et al.32 also reported that goats’ milk was distinguished by its high K content. The levels of major elements obtained in this study were higher than values found by Stelios and Emmanuel 33 for goats’ and ewes’ yoghurts. In general, the results obtained in this study were accordance with the changes of ash contents of the yoghurts during storage.

The essential minor elements have four major functions as stabilizers, as essential elements for hormonal function, as elements of structure and as cofactors in enzymes 34. Of the minor elements, Fe and Zn were found at higher levels in cows’ yoghurt (Table 3). Guler and Sanal 15 also reported 0.72 ppm as the maximum content of Fe in cows’ yoghurt. The mean minor element contents of yoghurt samples determined in this study were higher than reported by some researchers 15,20. This may be due to possibility of contamination from metal containers 35.

Counts of Yoghurt Bacteria

The viable counts of S. thermophilus were higher in buffaloes’ yoghurt (8.49 log cfu/g) than in the other samples. This could be due to its high fat and fat soluble vitamins stimulate growth of microorganisms. The number of S. thermophilus and L. delbrueckii spp. bulgaricus significantly increased up to day 7 and decreased up to end of storage (P<0.05). Similar results were reported by and Güler-Akın and Akın 36. There were no significant (P>0.05) differences between yoghurt samples in terms of the viable counts of L. delbrueckii spp. bulgaricus.

Sensory Evaluation

From Figs. 1 a, b, c and d it can be seen that the total scores of yoghurts decreased at 28th day of storage because of development of acidity and loss of structure. Although at the beginning of storage (1st and 7th days of storage) cows’ and buffaloes’ yoghurts received high scores for odor, taste

329ERKAYA, ŞENGÜL

and aroma, mouth feel and general acceptability, ewes’ and goats’ yoghurts received lower scores. This could be related to more intensive favorable or unfavorable flavour of yoghurts at the beginning of storage. The yoghurt samples received similar scores for acidity characteristic and the scores decreased during storage. This result was accordance with the instrumental measurements.

In conclusion, the highest mean values of total solid, protein, and ash contents were found in ewes’ yoghurt. However, the highest fat content was determined in buffaloes’ yoghurt. The viscosity values of yoghurt samples were affected during storage by total solids, protein, pH and syneresis values. The highest Ca and P contents were determined in ewes’ yoghurt. Therefore, ewes’ yoghurt may be considered as an important source in respect to these elements. While cows’ and ewes’ yoghurts were received the highest scores, goats’ yoghurt was the lowest scored by panelists due to its musky, rancid and goaty flavour.

REFERENCES

1. Tamime AY, Robinson RK: Yoghurt Science and Technology. 2nd ed., 619 p, Woodhead Publishing: Cambridge, 1999.

2. Wu H, Hulbert GJ, Mount JR: Effects of ultrasound on milk homogenization and fermentation with yogurt starter. Innovative Food Sci Emerg, 1, 211-218, 2000.

3. Rasic JL, Kurmann JA: Yoghurt. p. 427, Technical Dairy Publishing House, Copenhagen, 1978.

4. Ménard O, Ahmad S, Rousseau F, Briard-Bion V, Gaucheron F, Lopez C: Buffalo vs. cow milk fat globules: Size distribution, zeta-potential, compositions in total fatty acids and in polar lipids from the milk fat globule membrane. Food Chem, 120, 544-551, 2010.

5. IDF-International Dairy Federation: The World Dairy Situation 2007. Bulletin No. 423/2007, 2007.

6. El-Zahar K, Chobert JM, Sitohy M, Dalgalarrondo M, Haertle T: Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage. Nahrung, 47 (3): 199-206, 2003.

7. Kurmann JA: Yogurt made from ewe’s and goat’s milk. Bulletin Int Dairy Fed, 202, 153-166, 1986.

8. Pandya AJ, Ghodke KM: Goat and sheep milk products other than cheeses and yoghurt. Small Ruminant Res, 68, 193-206, 2007.

9. De La Fuente MA, Montes F, Guerrero G, Juarez M: Total and soluble contents of calcium, magnesium, phosphorus and zinc in yoghurts. Food Chem, 80, 573-578, 2003.

10. Unal G, El SN, Kilic S: In vitro determination of calcium bioavailability of milk, dairy products and infant formulas. Int J Food Sci Nutr, 56, 13-22, 2005.

11. Mckinley MC: The nutrition and health benefits of yoghurt. Int J Dairy Technol, 58, 1-12, 2005.

12. Miller GD, Jarvis JK, McBean LD: Handbook of Dairy Foods and Nutrition. 2nd ed., Washington, DC: CRC Press, 1999.

13. Yaygın H: Studies of the content of acetaldehyde and other volatile aromatic compounds of yoghurt from cow, sheep, goat and buffalo milk. XXI. International Dairy Congress Brief Communications. Vol. I, pp. 294. Moscow, 1982.

14. Yaygın H, Mehanna NMA: A comparative study on some volatile flavour components of yoghurt made from milk of different species. Ind J Dairy Sci, 41 (4): 432-436, 1988.

15. Güler Z, Şanal H: The essential mineral concentration of Torba yoghurts and their wheys compared with yoghurt made with cows’, ewes’ and goats’ milks. Int J Food Sci Nutr, 60 (2): 153-164, 2009.

16. Metin M: Süt ve Mamülleri Analiz Yöntemleri. No: 24, s. 439, Ege Üniversitesi Basımevi, Bornova-İzmir, 2008.

17. IDF-International Dairy Federation: Standard Method 20B: Milk. Determination of Nitrogen Content. IDF, Brussels, Belgium, 1993.

18. Atamer M, Sezgin E: Yoğurtlarda kurumadde artırımının pıhtının fiziksel özellikleri üzerine etkisi. Gıda, 11 (6): 327-331, 1986.

19. Abrahamsen RK, Holman TB: Yoghurt from hyperfiltrated, unfiltrated and evaporated milk and from milk with added milk powder. Milchwissenshaft, 35 (7): 398-402, 1980.

20. Güler Z: Levels of 24 mineral elements in local goat milk, strained yoghurt and salted yoghurt (Tuzlu yoğurt). Small Ruminant Res, 71, 130-137, 2007.

21. Torriani S, Gardini F, Guerzoni ME, Dellaglio F: Use of response surface methodology to evaluate some variables affecting the growth and acidification characterisitics of yoghurt cultures. Int Dairy J, 6, 625-636, 1996.

22. Bodyfelt FW, Tobias J, Trout GM: The Sensory Evaluation of Dairy Products. In, Bodyfelt FW, Tobias J, Trout GM (Eds): The Sensory Evaluation of Dairy Products. pp. 166-226, Van Nostrand Reinhold, New York, 1988.

23. Bonczar G, Wszolek M, Siuta A: The effects of certain factors on the properties of yoghurt made from ewe’s milk. Food Chem, 79, 85-91, 2002.

24. Lı J, Guo M: Effects of polymerized whey proteins on consistency and water-holding properties of goat’s milk yogurt. Food Chem Toxicol, 71 (1): 34-38, 2006.

25. Mumtaz S, Ur-Rehman S, Huma N, Jamil A, Nawaz H: Xylooligo-saccharide enriched yoghurt: physicochemical and sensory evaluation. Pakistan J Nutr, 7 (4): 566-569, 2008.

26. Lucey JA, Singh H: Formation and physical properties of acid milk gels: A review. Food Res Int, 30 (7): 529-542, 1998.

27. Lucey JA: Cultured dairy products: An overview of their gelation and texture properties. Int J Dairy Technol, 57, 77-84, 2004.

28. Vargas M, Chafer M, Albors A, Chiralt A, Gonzalez-Martinez C: Physicochemical and sensory characteristics of yoghurt produced from mixtures of cows’ and goats’ milk. Int Dairy J, 18, 1146-1152, 2008.

29. Tarakçı Z: Influence of kiwi marmalade on the rheology characteristics, color values and sensorial acceptability of fruit yogurt. Kafkas Univ Vet Fak Derg, 16 (2): 173-178, 2010.

30. Jumah RY, Shaker RR, Abu-Jdayıl B: Effect of milk source on the rheological properties of yoghurt during the gelation process. Int J Dairy Technol, 54 (3): 89-93, 2001

31. Pantako TO, Passos M, Desrosiers T, Amiot J: Effect of dietary milk proteins on time-dependent variations in plasma Fe, Mg and Zn levels in aorta and portal vein in rats. Le Lait, 72, 553-573, 1992.

32. Raynal-Ljutovac K, Lagriffoul G, Paccard P, Guillet I, Chilliard Y: Composition of goat and sheep milk products: An update. Small Ruminant Res, 79, 57-72, 2008.

33. Stelios K, Emmanuel A: Characteristics of set type yoghurt made from caprine or ovine milk and mixtures of the two. Int J Food Sci Technol, 39, 319-324, 2004.

34. Feinendegen LE, Kasperek K: Medical Aspects of Trace Element Research. In, Bratter P, Schramel P (Eds): Trace Element Analytical Chemistry Medicine and Biology. pp. 1-36, Berlin, Walter de Gruyter & Co., 1980.

35. Park YW: Nutrient profiles of commercial goat milk cheeses manufactured in the United States. J Dairy Sci, 73, 3059-3067, 1990.

36. Güler-Akın MB, Akın MS: Effects of cysteine and different incubation temperatures on the microflora, chemical composition and sensory characteristics of bio-yogurt made from goat’s milk. Food Chem, 100, 788-793, 2007.


SummaryThe aim of this study was to determine the potential nutritive value of Convolvulus arvensis L. using chemical composition and

in vitro gas production technique. Gas productions of Convolvulus arvensis hay were determined at 0, 3, 6, 12, 24, 48, 72 and 96 h incubation times and their gas production kinetics were described using the equation y = A (1-exp-ct). As a result of this study, maturity had a significant effect on the chemical composition, in vitro gas production, metabolisable energy (ME) and organic matter digestibility (OMD) of Convolvulus arvensis (P<0.05). Dry matter (DM), cell wall contents neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) of Convolvulus arvensis hay increased whereas crude protein (CP), ether extract (EE) and ash content decreased with maturity. The CP contents of Convolvulus arvensis hay ranged from 16.63 to 23.83%. The NDF, ADF and ADL contents of Convolvulus arvensis hay ranged from 34.00 to 54.04, 28.76 to 40.34 and 5.26 to 12.18% respectively. The potential gas production and estimated parameters decreased with increasing maturity of Convolvulus arvensis hay (P<0.05). The potential gas production (A) ranged from 61.59 to 71.77 ml. The ME and OMD ranged from 9.31 and 11.71 MJ/kg DM and 63.19 to 79.17% respectively. In conclusion, although the nutritive value of Convolvulus arvensis plant hugely decreased with increasing maturity it appears that Convolvulus arvensis plant could be grazed or harvested at these more advanced stages and still provides forage with an adequate quality for ruminant animals to meet their nutrient requirements.

Keywords: Chemical composition, Convolvulus arvensis hay, Digestibility, In vitro gas production, Nutritive value

Üç Farklı Dönemde Hasat Edilen Tarla Sarmaşığı (Convolvulus arvensis L) Otunun Potansiyel Besleme Değeri

ÖzetBu çalışmanın amacı, farklı zamanda hasat edilen tarla sarmaşığının potansiyel besleme değerinin in vitro gaz üretim tekniği ile

saptanmaktır. Tarla sarmaşığının in vitro gaz ölçümleri fermentasyonun başlamasından itibaren 3, 6, 12, 24, 48, 72 ve 96 saat aralıklarla yapılmış ve gaz üretimine ait parametreler ise y = A (1-exp-ct) fonksiyonu kullanılarak saptanmıştır. Hasat zamanı tarla sarmaşığının kimyasal bileşimi, in vitro gaz üretimi, metabolik enerji (ME) ve organik madde sindirimine (OMSD) önemli düzeyde etki etmiştir (P<0.05). Tarla sarmaşığı bitkisi olgunlaştıkça kuru madde (KM), hücre duvarı bileşenlerinden nötral deterjan lif (NDF), asit deterjan lif (ADF) ve asit deterjan lignin (ADL) içerikleri yükselmiş, diğer taraftan ham protein (HP), ham yağ (HY) ve kül içeriği ise azalmıştır. Tarla sarmaşığı otunun ham protein içeriği %16.63 ile 23.83 arasında değişmiş, NDF, ADF ve ADL içerikleri sırasıyla %34.00 ile 54.04, 28.76 ile 40.34 ve 5.26 ile 12.18 arasında bulunmuştur. Potansiyel gaz üretimi ve tahmin edilen parametreler de tarla sarmaşığının olgunlaşmasıyla birlikte azalmıştır (P<0.05). Potansiyel gaz üretimi 61.59 ile 71.77 ml arasında, ME düzeyi 9.31 ile 11.71 MJ/kg KM arasında, OMSD ise %63.19 ile 79.17 arasında değişmiştir. Sonuç olarak, tarla sarmaşığı otunun besleme değeri bitkinin olgunlaşmasına bağlı olarak önemli miktarda azalmasına rağmen, tarla sarmaşığı otu hasat dönemi ilerlemiş dönemlerde bile, ruminant hayvanların ihtiyaçlarını karşılayabilecek miktarda yeterli kalitede ot sağlayabileceği söylenebilir.

Anahtar sözcükler: Besleme değeri, İn vitro gaz üretimi, Kimyasal kompozisyon, Sindirim derecesi, Tarla sarmaşığı otu

Potential Nutritive Value of Field Binweed (Convolvulus arvensis L) Hay Harvested at Three Different Maturity Stages

Önder CANBOLAT *

* Uludag University, Faculty of Agriculture, Department of Animal Nutrition, TR-16059 Bursa - TURKEY


It is well established that forages play an important role for ruminant animals since forages provide energy,

protein and minerals. Forages also provide fiber to ruminants for chewing and rumination. Field bindweed

INTRODUCTION


RESEARCH ARTICLE

332Potential Nutritive Value of ...

(Convolvulus arvensis L) is one of the most problematic weeds in agricultural fields in the most parts of world. Field bindweed causes important economic losses through reduction of germination and yield of wheat as 14 and 80% 1. On the other hand field bindweed was hand pulled by some farmers and given to their ruminant animals as forage to meet nutrient requirements. Therefore it is possible that this practice would be encouraged if field bindweed could be promoted as forage for ruminant animals. However the potential nutritive value of some forages obtained at different harvest maturity is well determined 2-5 there is limited information about the nutritive value of field bindweed hay obtained at different maturity stages. Kazemi et al.6 reported that field bindweed contained 17.41% crude protein, 27.50% NDF, 27.33% ADF and 17.50% ash. Therefore more information on variation in concentration of nutrients, presence of tannin and digestibility of field bindweed is required for an accurate evaluation of potential use of field bindweed as forage in ruminant ration. Recently some researches suggested that chemical composition and in vitro gas production technique can be used to determine the potential nutritive value of previously uninvestigated plants 2-5,7. Therefore the aim of this study was to determine the potential nutritive value of field bindweed (Convolvulus arvensis L) harvested at three maturity stages using chemical composition and in vitro gas production technique.


Field bindweed (Convolvulus arvensis L) plants were harvested at three maturity stages [pre-flowering (15.07.2011), flowering (26.07.2011) and seeding stages (08.08.2011)]. Field bindweed plants were hand harvested from three replicate plots of 5x2 m established in the experimental field in July-August, 2011 in Kahramanmaras, Turkey. The experimental field was not irrigated and cultivated. The soil at the experimental plots is a clay loam. The pH of soil was 7.25. The area is located at 630 m above sea level. The mean annual rainfall and temperature were 713 mm and 11.95oC.

Samples were shade dried and representative dry samples from each plot was taken to laboratory and milled in a hammer mill through a 1 mm sieve for subsequent analysis.

Dry matter (DM) was determined by drying the samples at 105oC overnight and ash by igniting the samples in muffle furnace at 525oC for 8 h. Nitrogen (N) content was measured by the Kjeldahl method 8. Crude protein was calculated as N x 6.25.

Neutral detergent fiber (NDF), ADF and ADL contents were determined by the method Van Soest et al.9 Condensed tannin was determined by butanol-HCl method as described by Makkar et al.10.

Field bindweed hay samples (0.200 g DM) milled through a 1 mm sieve were incubated in vitro with diluted rumen fluid (10 ml rumen fluid + 20 ml culture medium) in triplicate calibrated glass syringes of 100 ml following the procedures of Menke et al.11. Rumen fluid was obtained from cows fed a daily ration containing maize silage and concentrates. The cows (4 years old and 600 kg live weight) had free access to water throughout the experiment. Rumen fluid was obtained using stomach tube from two lactating and pregnant cows fed a daily ration containing 20 kg maize silage and 8 kg concentrates (18% CP and 2750 Kcal ME kg-1). Rumen samples was collected before the morning meal in the thermos flaks and taken immediately to the laboratory where it was strained through 4 layers of cheesecloth and kept at 39oC. The rumen fluid was flushed with CO2. The rumen fluid was added to buffered mineral solution in the ratio of 1:2 respectively. The syringes were prewarmed at 39oC before the injection of 30 mL rumen fluid-buffer mixture into each syringe followed by incubation in a water bath at 39oC. Gas production was recorded at 3, 6, 12, 24, 48, 72 and 96 h after incubation and corrected for blank incubation. Cumulative gas production data were fitted to non-linear exponential model as:

Y = A (1 − exp−ct)

Where Y is gas production at time‘t’, A is the potential gas production (ml/200 mg DM), c is the gas production rate constant (h−1) and t is the incubation time (h).

Time (h) to produce 50 and 95% of potential gas production using the equation suggested by Şahin et al.12.

t50=0.693/c

t95=2.996/c

ME (MJ/kg DM) content of field bindweed was calculated using equation of Menke et al.11 as follows:

ME (MJ/kg DM) = 2.20 + 0.136 GP + 0.057 CP

where GP = 24 h net gas production (ml/200 mg DM); CP = Crude protein.

Organic matter digestibility (%) of field bindweed was calculated using equation of Menke et al.11 as follows:

OMD (%) = 14.88 + 0.889GP + 0.45CP +0.0651 XA

where XA: ash content (%).

One-way analysis of variance (ANOVA) was used to determine the effect of maturity stage on the chemical composition, gas production kinetics, and some estimated parameters such as ME and OMD of field bind-weed hay using SPSS 13. Significance between individual means was identified using the Tukey’s multiple range tests. Mean differences were considered significant at (P<0.05) 14.

333CANBOLAT

RESULTS

The effect of maturity stage on the chemical composition of field bindweed hay is presented in Table 1. The maturity stage has significant effect on the chemical composition of field bindweed hay. Dry matter (DM), NDF, ADF and ADL of field bindweed hay increased whereas CP, ash and EE content decreased with maturity. Dry matter (DM), CP, ash, EE, NDF, ADF, ADL, CT of field bindweed hay ranged from 21.34 to 30.40, 16.63 to 23.83, 3.47 to 7.97, 2.41 to 4.92, 34.00 to 54.04, 28.76 to 40.34, 5.26 to 12.18 and 0.57 to 0.84% respectively.

The effect of maturity stages on gas production at different time intervals is presented in Fig. 1. At all incubation times, gas production at pre-flowering stage was significantly higher than those of flowering and seeding stages.

The effect of maturity stage on gas production kinetics, ME, OMD of field bindweed hay is represented in Table 2.

The maturity had a significant effect on the gas production kinetics (c, A, t50 and t95) and estimated parameters such as ME and OMD. The gas production kinetics and estimated parameters such as ME and OMD of

Table 1. The effect of maturity stage on the chemical composition of field bindweed hay (n=3)

Tablo 1. Hasat zamanının tarla sarmaşığı otunun kompozisyonuna etkisi (n=3)

Nutrients (%)

Maturity StagesSEM Sig.

Pre-flowering Flowering Seeding

DM 21.34c 23.39b 30.40a 0.579 ***

CP 23.83a 20.71b 16.63c 0.330 ***

Ash 7.97a 5.13b 3.47c 0.113 ***

EE 4.92a 3.70b 2.41c 0.110 ***

NDF 34.00c 40.35b 54.04a 0.723 ***

ADF 28.76c 34.23b 40.34a 0.535 ***

ADL 5.26c 7.39b 12.18a 0.266 ***

CT 0.84 0.72 0.57 0.113 NS

a b c Row means with common superscripts do not differ (P>0.05); s.e.m.: standard error mean; Sig.: significance level; DM: Dry matter %, CP: Crude protein, EE: Ether extract, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, ADL: Acid detergent lignin, CT: Condensed tannin, NS: Non-significant; *** (P<0.001)

Fig 1. The effect of maturity stage on gas production of field bindweed hay

Şekil 1. Hasat zamanının tarla sarmaşığı otunun gaz üretimine etkisi

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100

Incubation time (h)

Gas

pro

duct

ion

(mL)

Pre-flowering

Flowering

Seeding

334Potential Nutritive Value of ...

field bindweed hay decreased with increasing maturity.

The gas production rate (c), potential gas production (A), time to produce 50 and 95% of potential gas production ranged from 0.08 to 0.099, 61.59 to 71.77 ml, 6.96 to 8.68 h and 30.13 to 37.54 h. On the other hand, ME and OMD of field bindweed hay ranged from 9.31 to 11.71 MJ /kg DM and 63.19 to 79.17% respectively.

DISCUSSION

There was marked changes in the chemical composition of field bindweed with increasing maturity. The crude protein content of field bindweed hay is in excess of that proposed as the minimum requirements for lactation (12% of DM) and growth (11.3% of DM) in ruminants 15. High crude protein levels suggests field bindweed hay with potential as N supplements to ruminants fed low quality forages during dry season. The concentration of field bindweed hay harvested at seeding stage was comparable with that reported by Kazemi et al.6 who found that field bindweed contained 17.41% crude protein. It was well established that CP content of plants decreases with increasing maturity. This decrease in CP contents results from decrease in CP in leaves, while stems, which had a lower protein concentration, represented a larger pro-portion of the available herbage in more mature forages 16. Several researchers estimated the daily reduction in CP contents of plant using the difference between CP of hay obtained at pre-flowering and seeding stages, divided by the time (days) required to reach from flowering to seeding stage 5,7,17. In the current study, the reduction in CP content of field bindweed hay was 3.01 g/kg/day. This reduction was considerably higher than those reported by Minson 17, Kamalak and Canbolat5 and Kamalak et al. 7

who reported that the average decline in crude protein concentration with advancing maturity averaged 1, 0.82 and 2.34 g/kg/day respectively. It is well known that high

level of CT in forages may adversely affect of the microbial and enzyme ctivities 18-21. However, in this experiment, the condensed tannin levels of field bindweed hay harvested at three maturity stages were lower than those considered detri mental to ruminant animals.

There was also marked changes in the gas production and estimated parameters of field bindweed with increasing maturity. The decline in gas production and estimated parameters is possibly associated with increase in cell wall contents (NDF, ADF and ADL) of field bindweed with maturity. It is well known that cell wall contents are more indigestible fractions of plant. Blummel and Orskov 22

suggested that gas production is associated with volatile fatty acid (VFA) production following fermentation of substrate so the more fermentation of a substrate the greater the gas production, although the fermentation end products do influence more closely with gas production. As a result, there was reduced gas production from the indigestible fractions with increasing maturity. The reduction in CP concentra tion and gas production resulted in OMD and ME concentration of field bindweed also decreased with increasing maturity. These results obtained in the current study are consistent with findings of Kamalak 4, Kamalak and Canbolat 5 and Kamalak et al.7. Even if there is a reduction in ME content of field bindweed with maturity, the ME content of field bindweed obtained at pre-flowering was similar to that of maize silage which is commonly regarded as a very high energy forage with a typical ME concentration of 11.7 MJ kg-1 DM 23. Thus it is suggested that field bindweed could be used not only as a basic forage in the ruminant ration, but also as a high energy feed.

In conclusion, although there was a marked decline in nutritive value of the forage of field bindweed hay with advancing maturity, even at the seeding stage, the forage had high CP content and was quite digestible. Field bindweed hay showed promising potential for

Table 2. The effect of maturity stage on the gas production kinetics, metabolisable energy and organic matter digestibility of field bindweed hay

Tablo 2. Hasat zamanın tarla sarmaşığı otunun gaz üretim parametrelerine, metabolik enerji ve organik madde sindirim derecesine etkisi

EstimatedParameters

Maturity StagesSEM Sig.

Pre-flowering Flowering Seeding

c 0.099a 0.079b 0.080b 0.0035 **

A 71.77a 68.51b 61.59c 0.877 ***

t50 6.96b 8.67a 8.68a 0.365 **

t95 30.13b 37.51a 37.54a 1.581 **

ME 11.71a 10.50b 9.31c 0.147 ***

OMD 79.17a 71.11b 63.19c 0.976 ***

a b c Row means with common superscripts do not differ (P>0.05); s.e.m.: standard error mean; Sig.: significance level; NS: Non-significant, c: gas production rate (%); A:: potential gas production (mL); ME: Metabolisable energy (MJ /Kg DM); OMD: Organic matter digestibility %; ** P < 0.01; *** P<0.001

335CANBOLAT

ruminant animals. However, further studies especially on animal responses, is required to confirm the nutritional characteristics indicated in the current study.

REFERENCES

1. Bogatek R, Gniazdowka A, Stepien J, Kupidlowska E: Convolvulus arvensis L. allelochemicals mode of action in germinating wheat seeds. Proceedings of the 4th World Congress on Allelopathy, 11-14 August, Wagga Wagga, Australia, pp. 263-266, 2005.

2. Kamalak A, Canbolat O, Gurbuz Y, Erol A, Ozay O: Effect of maturity stage on chemical composition, in vitro and in situ dry matter degradation of tumbleweed hay (Gundelia tournetortii L.), Small Rum Res, 58 (2): 149-156, 2005.

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implication in gas production and true digestibility in vitro techniques. Brit J Nutr, 73 (6): 897-913, 1995.

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SummaryThe aim of this study was to determine ophthalmoscopic, ultrasonographic and electrodiagnostic response of the experimentally

induced increasing intraocular pressure (IOP) in 6 New Zealand rabbits. IOP was induced by cauterizing 3 vortex and 3 episcleral veins. The cases were evaluated by ophthalmoscopy, ultrasonography (USG), electroretinography (ERG) and visual evoked potentials (VEP). Significant elevation in intraocular pressure (IOP) was seen in all rabbits on the postoperative 1st day. Although IOP values did not significantly differ between treated and control eyes from the 2nd to the 4th week, exchange of cornea-anterior lens capsule and posterior lens capsule-retina measurements were persistent. In ERG, implicit time of a and b wave of the treated eyes increased significantly during 4 weeks, however, the amplitude of both waves were markedly lower than control eyes until the end of 2nd week. In VEP, the decrease in N1 wave amplitude was marked in 4th week. These results indicated that even though the increasing IOP came back to the normal limits, ERG and USG parameters did not return to baseline. As a result, in clinical cases, even if IOP return to normal limits after treatment, intraocular structures and the retina should be evaluated and treatment should be advanced in this direction.

Keywords: Rabbit, Intraocular pressure, Tonometry, Ultrasonography, Electroretinography

Tavşanlarda Deneysel Göz İçi Basınç Artışının Oftalmoskopik, Ultrasonografik ve Elektrofizyolojik Bulguları

ÖzetBu çalışmanın amacı, altı tavşanda oluşturulan deneysel göz içi basınç (GİB) artışının oftalmoskopik, ultrasonografik ve

elektrofizyolojik bulgulara etkisinin araştırılmasıdır. Çalışmada her bir Yeni Zelanda tavşanının sol gözündeki 3 vorteks ve 3 episkleral ven koterize edilerek GİB artışı sağlanmıştır. Olgular oftalmoskopi, ultrasonografi (USG), elektroretinografi (ERG) ve görsel uyandırılmış potansiyeller (GUP) yönünden değerlendirilmiştir. Tüm tavşanlarda postoperatif 1. haftanın sonuna kadar göz içi basıncında belirgin bir artış ortaya çıkmıştır. İkinci ve 4. haftalarda GİB’deki değişim belirgin olmamış, ancak kornea-lens ön kapsülü aralığındaki artış ve lens arka kapsülü-retina aralığındaki azalmanın kalıcı olduğu görülmüştür. ERG’de a ve b dalga amplitüdlerinde 2. haftanın sonuna kadar belirgin bir düşme görülse de, dalgaların implisit zamanlarındaki artış 4 hafta boyunca sürmüştür. VEP’de ise N1 dalga amplitüdü çalışmanın 4. haftasına kadar belirgin şekilde azalmıştır. Bu bulgular artan GİB normale dönse bile ERG ve USG parametrelerindeki değişimin normale dönmediğini göstermektedir. Sonuç olarak klinik GİB artışı olgularında sağaltım sonrası basınç normale dönse dahi göz içi yapıların ve retinanın klinik olarak ERG ve USG yönünden değerlendirilmeye devam edilmesi ve sağaltımın elde edilen veriler doğrultusunda ilerletilmesi sağlanmalıdır.

Anahtar sözcükler: Tavşan, Göz içi basıncı, Tonometri, Ultrasonografi, Elektroretinografi

Ophthalmoscopic, Ultrasonographic and Electrophysiologic Findings of Experimentally Induced Intraocular Pressure

Increase in Rabbits [1]

İrem ERGİN * Ömer BEŞALTI *

[1]*

This study was summarized from the first author’s PhD Thesis Ankara University, Faculty of Veterinary Medicine, Department of Surgery, TR-06110 Ankara - TÜRKIYE


Resultant retina and optic nerve damage due to increased intraocular pressure (IOP) account for the most common reason of retinal ganglion cell death and loss of

vision 1. The exact mechanism for the ganglion cell death is unknown, but different hypotheses have been introduced. According to the mechanical pressure hypothesis, the

INTRODUCTION


RESEARCH ARTICLE

338Ophthalmoscopic, Ultrasonographic...

pressure on retinal layer causes deformations in optic nerve axons. To the vascular hypothesis, elevated IOP affects the retinal vascularization. This pressure causes slow blood flow and retinal ischemia 2. Early determination of pressure elevation is essential for irreversible damage in conjunction with elevated pressure on the neurologic structures of the eye. As in human medicine, loss of vision due to elevated IOP is very common in veterinary medicine. In order to determine the damage in the eye caused by elevated IOP, several experimental models are developed 1,3-5. In these models, the target is to block humour aquous drainage. The most commonly employed method to block humour aquous drainage is the episcleral vein cauterization model 5, which are shown on rats and mice 4. There are many papers refer to model of acute and chronic IOP induced hyperpressure in rabbits with the use of laser induced, betamethasone, alpha-chymotrypsine, etc. 6,7. However, no studies have been done about episcleral and vortex veins cauterization model in rabbits. Several ultrasonographic experiments on rats and mice have been done, in order to understand about changes in anterior chamber caused by the increased in IOP 8,9.

The aim of this study was to perform time dependant, tonometric, ophthalmoscopic, ultrasonographic and electro- physiological changes of the IOP elevation due to episcleral and vortex vein cauterization model in rabbits.

MATERIALS and METHODS

Animals

This study was approved by the Institutional Animal Ethics Committee (AU-HADYEK 2006∕35). Six adult (two years of age) male, New Zealand rabbits (Oryctolagus cuniculus) weighing 2-2.5 kg were maintained in an environmentally controlled room with 12 h light/12 h dark daily cycles (the room light intensity is 160 lux), a temperature of 21oC, food and water provided ad libitum. This study adhered to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and was approved by Ankara University Faculty of Veterinary Medicine Ethic Committee on Animal Care.

Experimental Intraocular Pressure Model

Rabbits were anesthetized by intramuscular injection of xylazine (5 mg/kg, Alfazyne 2%, Ege Vet, Turkey) and ketamine (35 mg/kg, Ketasol 10%, Richter, Austria). The eyes were anesthetized topically with 0.5% proparacaine HCl (Alcaine®, Alcon, Belgium) eye drops. The general anesthesia lasted approximately 40 min for the complete surgical procedure. IOP was induced in the left eye of each animal and right eye was served as a control. Two millimeter incisions into the conjunctiva and Tenon’s capsule were made on dorsal, ventral and lateral quadrants of the limbus of left eye. One dorsal episcleral vein, located near the

superior rectus muscle, one temporal episcleral vein, near the lateral rectus muscle and one ventral episcleral vein, between the ventral rectus muscle and oblique muscle were isolated from the surrounding tissues. A bipolar cautery was applied to the episcleral veins in the left eye. After episcleral veins cauterization, three vortex veins, which were under the dorsal, ventral and lateral rectus muscle, were isolated from the tissues, and cauterized. Antibiotic ointment containing oxytetracycline-polymyxin B-sulfate (Terramycine®, Pfizer, Germany) was applied topically after each procedure for 5 days.

Intraocular Pressure Monitoring

The right and left eye IOP’s of awake animals were measured using a digital tonometer (Tonopen Vet, Reichert Opthalmic Instruments, USA) every day for 28 days after the surgery. For IOP measurements, the eyes were anesthetized topically with 0.5% proparacaine HCl (Alcaine®, Alcon, Belgium) eye drop. All measurements were taken four times, at the same time of day in order to avoid circadian IOP changes.

Ultrasonography

B mode ultrasonography (Esaote Biomedica AU5, Genova, Italy) was performed in control and treated rabbit eye after instillation of a topical anesthetic and application of acoustic transmission gel on the 7.5-MHz linear transducer tip postoperative first day and every week after surgery. Measurements were performed by measuring the distance between the cornea (C) and the anterior lens capsule (ALC); the posterior lens capsule (PLC) and the retina (R) and overall globe size horizontally.

Ophthalmoscopy

Rabbits were anesthetized and their pupils were dilated by local application of 1% tropicamide (Tropikamid®, Roche, Germany) eye drop in order to examine the retinal and choroidal vasculature. In this way, the vasculature of control and hypertensive eyes was compared. Ophthalmoscopic examinations were performed at pretest (before the treatment), on the 1st day, in the 1st, 2nd and 4th weeks post-operatively. Flexible endoscope (VetVu, Swiss Precision Products, USA) was used for obtain eye fundus images.

Electroretinography (ERG)

To quantify potential damage to the retina due to the elevation of the IOP, electroretinography was performed at postoperative 1st day, in the 1st, 2nd and 4th weeks post-operatively in both eyes, under general anesthesia (10). The rabbits were placed on ventral recumbency, their heads were on dorsal position. An eye speculum was inserted between the lids to keep them open and to retract the nictitating membranes. 0.9% saline solution was used intermittently to keep the corneas moist. A five channels EMG∕EP device’s evoked potentials measuring

339ERGİN, BEŞALTI

system (Medelec/Synergy Oxford, USA) was used for ERG recordings. Three stainless-steel electrodes were used for recordings. The active electrode was placed approximately 1cm below the center of the lower eyelid subcutaneously. The reference was placed ipsilateral to the active electrode and about 2 cm lateral to the outer cantus of the eye. The ground electrode was placed on the rabbit’s scapular region subcutaneously. The animals were dark adapted for minimum of 15 min. ERG was performed with flashes (1 cd sec-1∕m-2). At each stimulus intensity, the recordings were repeated (until two successive identical curves were obtained). A flash ERG routine was delivered at a 2 Hz frequency. Amplitudes and implicit times for a wave and b wave were measured. The a wave amplitude was measured from the baseline to the trough of the a wave, while the b wave measured from the trough of the a wave to the peak of the b wave. The a wave implicit time was measured from the flash onset to the a wave trough and the b wave implicit time was measured from the flash onset to the b wave peak.

Visual Evoked Potentials (VEP)

The same device was used for VEP recordings. Active electrode was placed in midline, subcutaneously, 2 cm rostral to external occipital protuberance; reference electrode was placed in midline, approximately 3 cm behind the eyes and ground electrode was placed in midline, frontal region, 2 cm in front of the eyes. VEP’s were recorded using a LED goggle stimulator, to deliver 8 µsec flash duration with an intensity of 2 cd sec-1∕m-2. For VEP recordings, responses were filtered between 1-100 Hz. Usually 200 responses were averaged in order to sufficiently improve the signal to noise ratio. More than one negative and positive deflection was noted on VEP recordings on control eyes. In order to evaluate primary cortex, only first negative (N1) and positive (P1) deflections were taken. The N1 wave amplitudes were measured from the baseline to the peak of the negative N1 wave, whereas the P1 wave amplitudes were measured from the trough of the N1 wave to the peak of the positive P1 wave. Then, the implicit times of both N1 and P1 waves were measured.


Friedman’s test was performed to the effect of time on the IOP, ERG, VEP and USG parameters which were expressed as mean ±SE. Mean data from experimental group was compared with the data from control group on each experimental time zone by using Wilcoxon test. P<0.05 was considered significant.

RESULTS

Tonometry

Cauterization of vortex and episcleral veins caused temporary elevation of the IOP in rabbits’ treated eye.

The largest increase in IOP following cauterization was observed at the next day after the surgery. At this time, all treated eyes developed significant elevation of the IOP (26.33±1.03 mmHg) compared to controls (12.00±0.63 mmHg). This significant elevation of the pressure was continued until the first week. However, a progressive and slow reduction was observed from the first week till the end of the experiment. IOP values were not significantly differing between control and treated eyes at 4 weeks postoperatively (Fig. 1). Thus, the mean IOP in treated eyes at this period was close (16.65±2.4 mmHg) to the mean IOP of control eyes (12.00±0.63 mmHg).

Ultrasonography

In B-mode ultrasonography, three major echoes; cornea, posterior capsule and retina were seen. The cornea was represented as a curved hyper echoic interface. Anterior chamber of the eye, lens cortex and nucleus, and the aqueous and vitreous humors were anechoic. The optic disc appeared as a hypoechoic structure in the retina. No remarkable difference was found between the eye structures taken from the left and right eyes of the rabbits.

On the postoperative first day, the distance between C-ALC in treated eyes was significantly higher than control eyes. This substantial difference lasted until the end of the postoperative 1st week. By the end of the postoperative 2nd week, measurements in treated eyes were lower than the first week. However, the measurements were still higher than those of control group. By the end of the postoperative 4th week, although the distance between C-ALC values were much lower than the values obtained on the 1st day, a significant increase was noted compared to control group (P<0.05). When the distance between PLC-R was evaluated, the values for treated group were markedly lower than those of control group until the end of the postoperative 4th week (Fig. 2). When the horizontal length was evaluated, the value for experiment group was higher than control group (P<0.05). This increase lasted until the end of the postoperative 1st week. By the postoperative 2nd week, measurements in treated eyes decreased and this lasted until the end of the postoperative 4th week (Fig. 3).

Fig 1. Mean intraocular pressure (IOP) values (±SE, n=6 rabbits)

Şekil 1. Ortalama göz içi basınç (GİB) değerleri (±SE, n=6 rabbits)


Ophthalmoscopy

In the ophthalmoscopic examination of the control group, we observed healthy eye merangiotic fundus structures (Fig. 4a). However in the treated eyes, non-vascularized areas were noted. Vascular congestions were seen in cauterized areas (Fig. 4b). In the 2nd week fundus examination collateral vein formations in different parts of nonvascularized areas were noted. By the end of the postoperative 4th week, treated eye’s fundus had the same structures as the control group.

Electroretinography (ERG)

The amplitude of a and b wave of the treated eyes was

low compared to control eyes until the end of the study, but the difference between the groups was marked by the end of 2nd week. In treated eyes, a and b wave implicit time was significantly higher than control eyes from the postoperative 1st day to the 4th week (Table 1 and 2).

Visual Evoked Potentials (VEP)

The decrease in N1 wave amplitude in treated group compared to control group was marked on the post-operative 1st day and the 1st week, until the end of the postoperative 4th week. The implicit time of N1 wave was elongated in treated group compared to control group until the end of 4th week, however, the difference between time zones and control eyes was not significant (Table 3). The changes for amplitude and implicit time of P1 wave was not significant between treated and control eyes (Table 4).

a

b

Fig 2. Mean values of control and treated eye distance between cornea-anterior lens (a) and between posterior lens-retina (b)

Şekil 2. Kontrol ve deney gözlerde kornea-lensin ön yüzü (a), lensin arka yüzü-retina arası mesafe ölçümlerinin ortalama değerleri (b)

Fig 3. Mean values of horizontal length of control and treated eye

Şekil 3. Kontrol ve deney gözlerin horizontal çap ölçüm değerleri

a

b

Fig 4. a. Merangiotic fundus structures in control eyes, b. In treated eye fundus, nonvascularized area and vascular congestions on postoperative 1st day after cauterization

Şekil 4. a. Kontrol gözlerde merangiotik fundus yapısı, b. Koterizasyon işleminden sonraki 1. gün göz fundusundaki damarsız bölgenin ve damar konjesyonlarının görüntüsü

341ERGİN, BEŞALTI

DISCUSSION

In this experimental study, episcleral vein and vortex vein cauterization was employed in combination to form a chronic pressure elevation in six adult (2 years of age) male, New Zealand rabbit eyes. Prominent elevation in IOP values in treated eyes nearly continued 2 fold of control eyes during postoperative 1st week. Changes in IOP in this study were similar with the previous study employed with rat model 10,11. The gradual decrement in IOP values was reached to what’s noted in control eyes at the end of 4th week. This decrement should be compensated by the changes in C-ALC, PLC-R and horizontal length.

Retinal circulation is weak in rabbits, and choroidal vessels are very important for the metabolic requirement of the retina 12. Vein cauterization model can cause some serious damage in choroidal circulation, and the function of retinal cells, especially photoreceptors will be affected. In one study suggests that changes in a and b waves of ERG are resultant of loss of function in retinal cells due to damage in choroidal circulation 11. However, even

if vein cauterization model causes damage in choroidal circulation, this will not affect the ERG traces completely 10. In the present study, the changes in ERG parameters were in the same time changes with IOP. Because of the disruption of choroidal circulation and resultant increasing IOP, retinal function should be affected. The values obtained from ERG recordings revealed that the retinal function has not come back to the normal.

Serious damage to choroidal veins in cauterized area was seen in postoperative 1st day to the 1st week. Starting from 1st week of onset of decrease in pressure, collateral veins started forming in nonvascularized areas. At the end of the study, all treated eyes mean IOP measurements were the close to the control eye measurements, and the collateral vein network occupied the whole non-vascularized area. In contrast, the retinal veins look normal at the first day when the IOP was at the highest level. Alterations in the fundus of some animals and changes in IOP levels dependant on time being parallel with supported previous studies.

Elevated IOP, besides its effects on retina and optic

Table 1. Mean first negative peak (a) amplitudes and implicit time values. a,b: groups in the same row with different letters are different (- : P>0.05)

Tablo 1. İlk negatif dalga (a) amplitüd ve implisit zaman ortalama değerleri. a,b: aynı sırada farklı harfleri taşıyan gruplar farklıdır (- : P>0.05)

a WavePostoperative 1st Day Postoperative 1st Week Postoperative 2nd Week Postoperative 4th Week

Control Treated Control Treated Control Treated Control Treated

Amplitude, µV 1.39±0.55a 0.41±0.17bA 1.45±0.44a 0.69±0.31bB 1.22±0.44a 0.9±0.39bC 0.98±0.51- 0.86±0.57-C

Implicit time, ms 15.5±3.38a 19.3±3.56bA 13.96±2.91a 16.8±2.45bB 15.20±3.72a 18.15±2.9bC 12.24±4.49a 19.7±2.15bA

Table 2. Mean first positive peak (b) amplitudes and implicit time values. a,b: groups in the same row with different letters are different. - : P>0.05

Tablo 2. Ilk pozitif dalga (b) amplitüd ve implisit zaman ortalama değerleri. a,b: aynı sırada farklı harfleri taşıyan gruplar farklıdır. - : P>0.05

b WavePostoperative 1st Day Postoperative 1st Week Postoperative 2nd Week Postoperative 4th Week

Control Treated Control Treated Control Treated Control Treated

Amplitude, µV 23.31±7.36a 15.86±6.18bA 23.1±5.60a 18.7±9.24bB 22.3±10.43a 19.1±9.18bC 22.96±5.85- 22.08±6.4-D

Implicit time, ms 23.05±4.34a 29.03±7.0bA 23.15±5.2a 28.0±2.39bA 23.55±2.06a 30.15±8.0bA 22.86±2.05a 25.9±1.06bB

Table 3. Mean first negative peak (N1) amplitudes and implicit times values. a,b: groups in the same row with different letters are different. - : P>0.05

Tablo 3. İlk negatif dalga (N1) amplitüd ve implisit zaman ortalama değerleri. a,b: aynı sırada farklı harfleri taşıyan gruplar farklıdır. - : P>0.05

N1 WavePostoperative 1st Day Postoperative 1st Week Postoperative 4th Week

Control Treated Control Treated Control Treated

Amplitude, µV 3.12±2.71a 1.92±0.61bA 2.74±2.56a 0.72±0.21bB 1.63±0.83a 1.50±0.0bC

Implicit time, ms 24.10±8.74- 25.00±9.49- 24.20±6.94- 26.01±3.20- 23.26±8.72- 24.00±6.27-

Table 4. Mean first positive peak (P1) amplitudes and implicit times values. a,b: groups in the same row with different letters are different. - : P>0.05

Tablo 4. Ilk pozitif dalga (P1) amplitüd ve implisit zaman ortalama değerleri. a,b: aynı sırada farklı harfleri taşıyan gruplar farklıdır. - : P>0.05

P1 WavePostoperative 1st Day Postoperative 1st Week Postoperative 4th Week

Control Treated Control Treated Control Treated

Amplitude, µV 8.02±3.51- 6.16±3.78- 6.78±1.62- 5.63±3.24- 6.76±2.04- 4.5±2.82-

Implicit time, ms 13.00±2.78- 14.94±7.92- 12.41±4.14- 14.82±4.12- 13.46±6.38- 15.3±6.36-


nerve, causes numerous structural changes in the eye. In an ultrasonographic study which is the same model we used for rabbits in rats 13, during the time zone with marked elevation in IOP levels, it was observed that no changes took place in depth of anterior camera. In the present study, differences of the distance between C-ALC, and PLC-R were persistent for 4 week. In addition Horizontal length of the eye had increased until first week and decreased gradually as IOP. During the observation period, treated eye’s IOP measurements being higher than control group just at the first week, not at the 2nd and 4th weeks.

Increasing the distance of C-ALC and decreasing the distance of PLC-R were persistent, and the values were significantly different than control eyes. However, the increasing IOP was stayed at the significantly highest level at 1st week, and get at the level of control eyes for the later periods. These findings can be explained as; increasing IOP has been compensated by the moving of the lens or attached structures (zonular fibers, corpus ciliare, iris) backwards.

When the photoreceptors of the retina are stimulated by light, phototransduction, which is the conversion of the energy of light into a neuroelectrical response, occurred in the cells. The information from the photoreceptors is then processed through the retina, and finally, retinal ganglion cells are stimulated. This processing of visual information in the retina results in electrical changes in the tissue that can be recorded as a mass potential called the electroretinogram. Each retinal ganglion cell relays that information to higher visual centers. The visual evoked potentials however, is a gross electrical potential recorded from the visual cortex in response to a visual stimulus from the retina. That is a visual stimulus results in the excitation of many cells in the cortex and the summed activity of these cells is recorded as the VEP on the scalp 14.

In a study, which was given Ringer’s solution to the anterior chamber of the rabbit eyes to increase IOP, changes in VEP parameters were monitored 15. In VEP recordings, decrease in N1 wave amplitude with elongation of implicit time was determined in treated eyes. In the present study, postoperative first day at which IOP is significant, decrease in the amplitude of both waves, as well as elongation in the implicit time was concluded. The time periods in which increase IOP is significant, as a result of increase in pressure of retinal cells damages occurs, and it was thought that in ganglion cells electrical stimulation is not transmitting in a normal limit. In this experiment, the persistent change in ERG was not in the same line with VEP except for amplitude of N1 wave.

In conclusion, marked elevation of IOP until the 1st postoperative week was in consistence with alterations in USG, ERG and VEP (N1 amplitudes) parameters. Even

though the increasing IOP came back to the normal limits, ERG parameters and measurements between C-ALC and PLC-R did not return to normal limits. As a result, in clinical cases, even if IOP return to normal limits after treatment, intraocular structures and the retina should be evaluated and treatment should be advanced in this direction.

Acknowledgements

I gratefully thank Prof. Dr. Bahattin KOÇ and Prof. Dr. Perran GÖKÇE for their supports and comments of the manuscript and Assoc. Prof.Dr. Safa GÜRCAN for his contributions to statistical analysis.

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7. Lim KS, Wickremasinghe SS, Cordeiro MF, Bunce C, Khaw PT: Accuracy of intraocular pressure measurements in New Zealand white rabbits. Invest Ophthalmol Vis Sci, 46, 2419-2423, 2005.

8. Aihara M, Lindsey JD, Weinreb RN: Experimental mouse ocular hypertension: establishment of the model. Invest Ophthalmol Vis Sci, 44, 4314-4320, 2003.

9. Nyland TG, Mattoon JS: Ocular ultrasonography. In, Nyland TG, Mattoon JS (Eds): Veterinary Diagnostic Ultrasound. 1st ed., pp. 178-197. W.B. Sounders Company Ltd, Philadelphia, 1995.

10. Grozdanic SD, Betts DM, Sakaguchi DS, Kwon YH, Kardon RH, Sonea IM: Temporary elevation of the intraocular pressure by cauterization of vortex and episcleral veins in rats causes functional deficits in the retina and optic nerve. Exp Eye Res, 77, 27-33, 2003.

11. Mittag TW, Danias J, Pohorenec G, Yuan HM, Burakgazi E, Redman RC, Podos SM, Taton WG: Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat model of glaucoma. Invest Ophthalmol Vis Sci, 41 (11): 3451-3459, 2000.

12. Kiel JW, Shepherd AP: Autoregulation of choroidal blood flow in the rabbit. Invest Ophthalmol Vis Sci, 33 (8): 2399-2410, 1992.

13. Nissirios N, Chanis R, Johnson E, Morrison J, Cepurna WO, Jia L, Mittag T, Danias J: Comparison of anterior segment structures in two rat glaucoma models: An ultrasaund biomicroscopic study. Invest Ophthalmol Vis Sci, 49 (6): 2478-2482, 2008.

14. Sawada A, Neufeld AH: Confirmation of the rat model of chronic, moderately elevated intraocular pressure. Exp Eye Res, 69, 525-531, 1999.

15. Okuno T, Oku H, Sugiyama T, Yang Y, Ikeda T: Evidence that nitric oxide is involved in autoregulation in optic nerve head of rabbits. Invest Ophthalmol Vis Sci, 43 (3): 784-789, 2002.


SummaryThe aim of this case report is to describe the clinical, ultrasonographic, and surgical findings in an 8-week-old female Holstein calf

with right abomasal displacement and ulceration. Auscultation/percussion, auscultation/ballottement, and ultrasound examination were used to identify displacement of the abomasum. Right-flank laparotomy was used for repositioning of the displaced abomasum. The abomasum was greatly distended between the rumen and right body wall. Approximately 7 L of abomasal content was discharged from the abomasum. A few ulcerative areas in the mucosal surface of abomasum were observed. After surgery, antimicrobial and antiulcerative therapy were given. The calf discharged was followed with telephonically. As a result, right displacement of the abomasum is very rarely seen in calves. Right flank laparotomy gives good result in calves with right displaced abomasum, and ultrasound examination helps to distinguish right displaced abomasum from other metabolic disorders of the digestive tract in calves.

Keywords: Calf, Right abomasum displacement, Ulceration, Ultrasonography

Bir Buzağıda Abomazum Ülseri ve Sağa Deplasmanı

ÖzetBu olgu sunumunun amacı, 8 haftalık Holstein ırkı dişi bir buzağıda karşılaştığımız abomasum ülseriyle birlikte abomazun sağa

deplasmanı olgusunun klinik, ultrasonografik ve operatif bulgularını tanımlamaktır. Hastalığın teşhisi oskültasyon/perküsyon, oskültasyon/çalkantı ve ultrason aracılığıyla konulduktan sonra tedavisi sağ karın duvarından yaklaşımla operatif olarak yapıldı. Operasyon sırasında abomazumdan yaklaşık 7 L içerik boşaltıldı. Abomazum içeriği boşaltıldıktan sonra abomazum mukozasında bir kaç ülserli alana rastlandı. Postoperatif olarak buzağıya antibiyotik ve ülsere yönelik tedavi uygulandı. Taburcu edilen buzağının telefonla yapılan takibinde herhangi bir komplikasyonla karşılaşılmadığı belirlendi. Sonuç olarak, buzağılarda abomazumun sağa deplasmanı çok nadir gözlenen bir durumdur. Tedavide sağ karın duvarından yaklaşarak abomazumun normal yerine getirilmesi ile gayet başarılı sonuçlar elde edilebilir. Bununla beraber ultrasonografik muayene, buzağılarda abomazumun sağa deplase olduğu olguların ayırıcı tanısında güvenle kullanılabilecek bir teşhis aracı olabilir.

Anahtar sözcükler: Buzağı, Abomazumun sağa deplasmanı, Ülser, Ultrasonografi

The Right Displacement of Abomasum with Ulceration in A Calf [1]

Semih ALTAN * Fahrettin ALKAN * Yılmaz KOÇ *

[1]

*

This case report was presented as poster presentation in I. European Buiatrics Forum, 1-3 December 2009, Marseille/France and supported by SUBAPK (The coordinaton of scientific research projects University of Selçuk, Project number 09701575)University of Selçuk, Faculty of Veterinary Medicine, Department of Surgery, TR-42075 Selçuklu, Konya - TURKEY


The left or right displacement of the abomasum (LDA or RDA) is a very important metabolic disorder of the digestive tract in cattle and is often seen especially in high performing milk breeds 1-3. Moreover, right displacement and dilatation of the abomasum is a subacute disease, which generally occurs in adult cattle. However, in calves, reports related to RDA are very rare 2,4. Clinical signs of LDA or RDA in calves are anorexia, poor weight-gain, recurrent

tympani, depressive behavior, and diarrhea 1,4. The cause of recurrent tympani is accumulation of gas and fluid in the abomasum due to obstruction of the forestomach, and abomasal atony due to its content 5. In calves, diagnosis of the RDA is characterized by auscultation/percussion, auscultation/ballottement, and ultrasonography 6,7. In simultaneous auscultation, the tympanic resonance (a “ping” sound) centered over the 10th to 13th ribs is

INTRODUCTION


CASE REPORT

344The Right Displacement of ...

the primary diagnostic sign of RDA 1. Ultrasonography is a very useful diagnostic method in indecisive cases of right abomasal displacement 8,9.

The purpose of this case report is to describe the clinical, ultrasonographic, and surgical findings in an 8-week-old female Holstein calf with right abomasal displacement and ulceration.

CASE HISTORY

An 8-week-old female Holstein calf was referred to the clinic of Veterinary Hospital of the University of Selcuk on April 07, 2009, owing to anorexia and absence of defecation. These complaints had continued for 2 days. The owner of the calf said that the calf had eaten adult-cow forage.

RDA with dilatation was diagnosed using auscultation/percussion, auscultation/ballottement, and ultrasound examination. Ultrasound examination was performed with

a 3.5-MHz sector transducer and real-time scanner (Pie-Medical scanner 250). The 10th and 13th intercostal spaces on the right side and area was examined ventrally to dorsally with 3.5-MHz transducer held ventro-cranial to the ribs. In ultrasonography, the displaced abomasum was seen on the right abdominal area with its hypoechogenic ingesta (Fig. 1), and the liver was not seen its normal location. Blood sample was taken from the jugular vein. Laboratory results are given in Table 1.

The right flank was clipped and prepared routinely. A right-flank laparotomy was performed under local infiltration anesthesia. The abomasum was greatly distended with gas and abomasal content, and the abomasum was

identified between the rumen and right body wall (Fig. 2-A). The abomasum was punctured at its highest point with a needle attached to a tube to allow the release of accumulated abnormal gas (Fig. 2-B). The abomasum was incised about 4 cm in length, and approximately 7 L abomasal contents were discharged from the abomasum (Fig. 2-C). A few ulcerative areas in the mucosal surface of the abomasum were observed. Then, the abomasum was sutured in the usual way and returned to normal position. The muscle layers and skin were closed in the usual manner (Fig. 2-D).

After surgery, antimicrobial therapy with Dipenisol (Penicillin-Streptomycin, Bayer - Istanbul) 1 ml/25 kg body weight was given by intramuscular injection daily for 5 days. For the abomasal ulceration, Ulcuran (Ranitidine 25 mg/2 ml, Abfar, Istanbul) Antepsin (Sucralfate 1 g, Bilim, Istanbul), and diet were prescribed. For the postoperative analgesia, Meloxicam 0.5 mg/kg body weight was given (Maxicam, 5 mg/ml, Sanovel, Istanbul) by the subcutaneus injection a single dose. Sutures were removed after

Fig 1. The abomasum and its hypoechogenic ingesta by ultrasound examination

Şekil 1. Ultrason muayenesinde abomasum ve abomasum içeriğinin hipoekojenik görü-nümü

Table 1. Results of blood gas evaluation performed before surgical intervention

Tablo 1. Cerrahi müdahaleden önce ölçülen kan gazı sonuçları

Calf Measured Rate Referents Rate

pH 7.424 7.3-7.5

Na+ 132 mmol/L ↓ 134-146

K+ 3.30 mmol/L ↓ 3.40-4.50

Cl- 104 mmol/L 97-111

pO2 24.8 mmHg ↓ >40

pCO2 36.2 mmHg ↓ 42-46

HCO3 23.2 mmol/L 22.4-25.5

345ALTAN, ALKAN, KOÇ

healing of operation wound. Follow-up information about the calf discharged was obtained telephonically.

DISCUSSION

According to literature, right displacement of the abomasum is very rarely seen in calves. Generally, calves between 6 and 14 weeks old are more sensitive for abomasal diseases 10,11. Consistent with this knowledge, in the present case the calf was 8-weeks-old. According to reports, majority of abomasal displacements in calves happen in male calves and especially in fleshy male calves 12,13. However, it was seen also in female calves 4. In the present case, the calf was a femal e and not fleshy as well.

The diagnosis of right displacement of the abomasum and dilatation is consistent with authors 1,4 who reported that in diagnosis of abomasal displacement, simultaneous auscultation and ballottement as well as auscultation and percussion give the best results followed by auscultation, external palpation, and observation. However, ultrasonographic examination is very useful in suspect cases 2,8. We used ultrasonography as well as methods that were mentioned previously in diagnosis. In animals with right displacement of the abomasum, the liver is displaced from the abdominal wall and cannot be distinguished in an ultrasonography image because the abomasum is seen where the liver would normally be in the right abdominal cavity 9. In ultrasonographic examination of the 10th and 13th intercostal space in the right side and adjacent area, the abomasum was placed between the rumen and right abdominal wall. We did not see where the liver would normally be on the right abdominal

wall by ultrasound because of right displacement of the abomasum. The abomasum on the right abdominal wall could be clearly differentiated from the adjacent organs by ultrasound. Because the abomasal contents seemed as a heterogeneous and moderately echogenic structure with echogenic stippling, the displaced abomasum was seen in a dilated form 8. Our ultrasonographic findings were consistent with these informations.

Abomasal displacements in calves generally have been seen with pneumonia, diarrhea, and abomasal torsions to perforating ulcers 2. When we incised the abomasum, we observed a few ulcerative areas in the abomasal mucosa. In calves, hypochloremic metabolic alkalosis may occur because of hydrochloric acid sequestration within the abomasum. Such metabolic changes are well substantiated in dairy cattle 5,14. However, in this calf there was no metabolic alkalosis. Calves with RDA had increased blood pH, HCO3, and sodium, and decreased chloride and potassium 4. In the present case, in contrast to this knowledge, blood pH, HCO3, and chloride had not changed but sodium and potassium had decreased.

In conclusion, the RDA is seen adult cattle rather than calves. However, sometimes it could be seen in calves, which is wrongly fed. This digestive tract disturbance of calves can treat easily with adult cattle RDA operation.

Acknowledgment

We thank to the Prof. Dr. Mahmut OK (staff of the Medicine of the Faculty of Veterinary) for the ultra-sonographic examination.

Fig 2. A- Distended abomasum the right abdominal wall, B- Discharging of abomasal gas with a needle attached to a tube, C- Discharging of abomasal content with rubber tube, D- Closed skin incision

Şekil 2. A- Sağ karın duvarında şişkin abomasum, B- Bir boruya takılmış kanül vasıtasıyla abomasum içindeki gazın boşaltılması. C- Kauçuk bir boruyla abomazum içeriğinin boşal- tılması. D- Deri ensizyonun kapatıl-ması

346The Right Displacement of ...

REFERENCES

1. Alkan F, Koc Y, Calim KH : Right flank omentopexy for correction of left displaced abomasum in cattle. Indian Vet J, 81, 1387-1389, 2004.

2. Trent AM: Abomasal disease. Surgery of the calf gastrointestinal system. In, Fubini SL, Ducharme NG (Eds): Farm Animal Surgery. pp. 461-466, Elsevier, USA, 2004.

3. Karakurum MC, Albay MK, Şahinduran Ş, Sezer K: Coagulation parameters in cattle with left displacement of abomasum. Kafkas Univ Vet Fak Derg, 15 (2): 293-296, 2009.

4. Cruz MM, Roblesgil AP, Escamilla MRG, Rubio MS: Description of abomasal displacements in dairy calves. Bov Pract, 25, 95-98, 1990.

5. Sahinduran S, Albay MK: Haematological and biochemical profiles in right displacement of abomasum in cattle. Revue Méd Vét, 157, 352-356, 2006.

6. Aslan V, Turgut K, Koc Y: The operative treatment of the caecum dilatation and right abomasal displacement case in a weaning calf. J Fac Vet Med Univ Selcuk, 2, 143-148, 1986.

7. Hull BL: Correction of Abomasal Displacement. 80th Western Veterinary Conference. Las Vegas, Nevada, USA. 17-21 February 2008.

8. Ok M, Arican M, Turgut K: Ultrasonographic findings in cows with left and right displacement of abomasum. Revue Méd Vét, 15, 15-18, 2002.

9. Braun U: Ultrasonography in gastrointestinal disease in cattle. Vet J, 166, 112-124, 2003.

10. Gingerich DA, Murdick PW: Paradoxic aciduria in bovine metabolic alkalosis. JAVMA, 166, 227-230, 1975.

11. Dirksen G: Left abomasal displacement in calves. Bov Pract, 17, 75-79 1982.

12. Dennis R: Abomasal displacement and tympany in a nine-week-old calf. Vet Rec, 114, 218-219, 1984.

13. Dirksen G, Doll K: Ileus and subileus in the young bovine animal. Bov Pract, 21, 38-40, 1986.

14. Yılmaz Z, Seyrek-Intas D, Senturk S, Golcu E, Ilcol Y, Gorgul S. Evaluation of biochemical parameters in the pre-and post-operative periods of cows with right-and left displacement of the abomasum. JTVS, 8, 20-26, 2002.


SummaryA male red deer (Cervus elaphus) of unknown age was admitted to Istanbul University, Faculty of Veterinary Medicine, Department

of Surgery with an open fracture at the level of the right proximal third metacarpal bone. The leg was amputated and two months later he received an prosthetic leg and the clinical outcome was discussed.

Keywords: Red deer, Cervus elaphus, Amputation, Prosthetic limb

Yabani Bir Kızıl Geyikte (Cervus elaphus) Parsiyel Ön Bacak Amputasyonu ve Protez Bacak Uygulaması

Özetİstanbul Üniversitsi Veteriner Fakültesi Cerrahi Anabilim Dalı’na sağ metakarpus III’ün proksimalinde açık kırık şikayetiyle getirilen,

yaşı bilinmeyen erkek bir kızıl geyiğin (Cervus elaphus) sağaltımında bacağın amputasyonunu takiben 2 ay sonra protez bacak uygulanması ve klinik sonuçları değerlendirilmiştir.

Anahtar sözcükler: Kızıl geyik, Cervus elaphus, Amputasyon, Protez bacak

Partially Forelimb Amputation and Application of An Artificial Limb (Prosthetics) in A Free-Ranging Red Deer (Cervus elaphus)

DilekOLGUNERDİKMEN* SerhatÖZSOY* DidarAYDIN* HarisHASİMBEGOVİÇ* KaanDÖNMEZ*

* Istanbul University, Faculty of Veterinary Medicine, Department of Surgery, TR-34320 Avcılar, Istanbul - TURKEY


Farming of wapiti is becoming increasingly popular in North America and also in western Canada 1. In many parts of the world the meat (venison) from Red Deer is widely used as a food source 2.

In the literature, various disorders which has been seen in elks has been reported. These include ocular, pulmonary, musculoskletal, digestive, central nervous system disorders and neoplasms 1-8.

Capture myopathy is a life-threating syndrome due to stress of capture, restraint and transportation in wild mammals and birds. In mammals the syndrome is characterized clincally by muscular stiffness, incoordination, depression, paralysis, metabolic acidosis and death 9.

Musculoskeletal pathology, including long bone fracture and joint sepsis, have been the most documented

disorders in wapiti 1. Keppie and Naylor 1 reported that the common diagnosis was fractures, which were seen mostly traumatic long bone fractures among the other lesion or disorders. Several factors should be considered when choosing a treatment option for wildlife with fractured limbs. It has been reported that comminuted fractures of the metacarpus and radius in young and adult wapiti may be managed successfully by use of external skeletal fixation 3. Financial constraints and temperament of the animal may limit the number of options. Amputation of a limb with prosthesis application is principally a method of saving valuable animals for breeding purposes or animals of sentimental value. Therefore amputation can be a option in catastrophically injuired animals especially with open fractures with no financial support by an owner or corporation 2.

A prosthesis is an artificial device that replaces a missing

INTRODUCTION


CASE REPORT

348Partially Forelimb Amputation ...

part of the body. There is evidence of the use of prosthesis from the times of the ancient Eygptians. Prostheses were developed for cosmetic, function and sense of wholeness in human. Limb amputation and the fitting of a prosthesis is a well-established procedure in human. There are two basic structural types of the prosthesis: endoskeletal and exoskeletal. The exoskletal prosthesis are broadly called the socket-type devices. An exoskeletal limb prosthesis has three main parts: the interface, the components, and the cover. The prosthesis attaches to the body at the interface. The interface consists of a socket and a rigid frame. Components are the working parts of the prosthesis. They include terminal devices (artificial fingers, hands, feet, and toes) and joints (wrists, elbows, hips, and knees), as well as metal shafts, which act as bones. Prosthetic covers consist of foam shaped by the prosthetist to look like the missing limb. Attachable prosthetic devices have been used in different animal speices including horse, cattle, dog with variable success 10-13.

Endoskeletal prosthesis are also called permanently attached artificial limb or osseointegrated limb. Osseo-integration is a new method of attaching the artificial limb through the dermis into the bone 11,14.

CASE HISTORY

A male red deer (Cervus elaphus) of unknown age (estimated to 4-5 year-old) was found alive in a forest close to Tekirdağ, Turkey and admitted to the Istanbul Universty, Faculty of Veterinary Medicine Department of Surgery with an open fracture at the level of the right third metacarpal bone. The animal was in good condition on clinical examination but nervous about the capture. To aviod the capture myopathy the deer kept in a silence stall for a couple of hour for relaxing. It was decided to amputate the forelimb because of the severe soft tissue injuries, and the fracture was irreducible. To sum up the leg was attached only with a small part of skin to the body. But other than the injured leg, the deer was in good health, therefore, amputation was suggested as an alternative to euthanasia. After being fasted for 12 h the animal was sedated with xylazine HCl (Rompun®, Bayer, Germany) at 1.1 mg/kg BW, IM dosage and anesthesia was induced with isofloran first inhalation through a mask at an initial concenration of 5% together with 100% O2 then followed by entubation and maintained at a concentration of 2.5%. The limb was amputated proximal to the pathologic lesion. The incision location was at the level of the right antebrachiocarpal joint (desarticulation) with the surgeons’ personal preference and the amputation was done. The surrounding tissue were sutured with vicryl using matress suture to cover the bony stump. The skin was closed using prolen with interrupted sutures. Following surgery, the animal was given cefazolin sodium (Iespor, İ.E Ulagay, Turkey) at a dose of 11 mg/kg BW by

intravenous administiration. The wound was cleaned daily and ten days after the surgery the sutures were removed. During this recovery period the animal showed diffuculty on standing (Fig. 1). Therefore the deer was mostly lying down. In the light of this situation it was decided to apply a prosthetic limb to make the deer more comfortable. The prosthesis has been constructed by the Prostheses Foundation for free of charge, which is a company that usually makes artificial limbs for human amputees. The socket type prosthesis used in this case consisted of a padded sleeve, a stump cover and the prostethic device that was made of carbon fibre and it was enclosed by a lifelike (hair) protective covering. The padded sleeve was placed on the residual limb and the stump cover placed over it and attached to the prosthetic device. After the artificial limb was being used, the deer became more comfortable. The balance of the body was very good (Fig. 2). Six months later the deer was discharged to a restricted national park. The deer had a good quality of life, a regular appetite and was in good body condition.

The result and the clinical outcome in this case indicate that a prosthetic limb may be used successfully even for the free-ranging animals to make their quality of life higher after limb amputation.

DISCUSSION

Farming of elks is becoming more popular in some countries 1. However in our country elk (Cervus elaphus) is not farmed for the industrial products or as a food source. In the last decade some negative environmental impacts

Fig 1. After the amputation of the leg

Şekil 1. Bacağın amputasyonundan sonraki görünüm

349

OLGUN ERDİKMEN, ÖZSOYAYDIN, HASİMBEGOVİÇ, DÖNMEZ

and as a result of poaching there is a rapid decrease in the number of red deer thus they are accepted to be endangered animals in Turkey. Thus, in our country it is really important to save a single deer’s life in the interest of preserving the speices.

In the literature, various disorders which has been seen in elks has been reported. These include ocular, pulmonary, musculoskletal, digestive, central nervous system disorders and neoplasms 1-8. As mentioned before farming wapiti is not common in Turkey therefore the certain prevelance of the disorders is notclear.

Capture myopathy is a life-threating syndrome in wild mammals and birds 9. In the present case, the animal seemed depressed about the transportation that lasted about 2 and half hour. To avoid capture myopathy the animal kept in a silence and dim stall for a couple of hours for relaxing before the examination. After the rest the deer seemed more stilly. Therefore it can be advised not to manupilate the deer immediately for clinical examination to aviod capture myopathy.

Musculoskeletal pathology, including long bone fracture and joint sepsis, have been the most documented disorders in wapiti and was reported that the common diagnosis was fractures, which were seen mostly traumatic long bone fractures among the other lesion or disorders 1. Several factors should be considered when choosing a treatment option for wildlife with fractured limbs. Financial constraints and temperament of the animal may limit the number of options. Amputation of a limb with prosthesis

application is principally a method of saving valuable animals for breeding purposes or animals of sentimental value. Therefore amputation can be a option in catastrophically injuired animals especially with open fractures with no financial support by an owner or corporation. In this case the leg was terribly injured and because the lack of an owner to pay for the treatment costs amputation was done.

Deer breeding season usually occurs in mid-September through October and in this period battles among competing males are common. Keppie et al.1 reported that forty-five percent of elk with fractures were admitted during August and September. In this case elk was admitted to our clinic at end of the September and probably was because of fighting associated with rutting season.

It has been reported that most fractures (81%) were seen in elk ≥ 2 months, no elk > 5 years was admitted for a fracture 1. It has been thought that bone strength in young animals is lesser than olders to be unable to withstand high forces generated during trauma. In contrast, in this case the age was unknown but estimated to 4-5 year-old.

There is evidence of the use of prosthesis from the times of the ancient Eygptians. Prostheses were developed for cosmetic, function and sense of wholeness in human 10. Butt et al. reported about complications including problems in standing and breakdown of the contralateral supporting limb after the amputation. According to the previous paper 2 the breakdown of the contralateral limb was seen mostly in the front limb possibly due to the increased proportion of weight on the front limbs. In this case, the affected limb was the front one and it was obviously clear that the deer was unable to stand without help. Therefore an artificial limb (prosthesis) usage was mandatory in this case.

There are two basic structural types of the prosthesis: endoskeletal and exoskeletal. The exoskeletal prosthesis are broadly called the socket-type devices. Prosthetic covers consist of foam shaped by the prosthetist to look like the missing limb 11. Our prosthesis was an exoskeletal type (socket-type) and enclosed by a lifelike (hair) protective covering and the deer seemed to be well tolerated.

Attachable prosthetic devices have been used in different animal speices including horse, cattle, dog with variable success 11-13. But to the authors’ knowledge this is the first report of prosthesis fitting in a deer in Turkey.

Complications associated with prosthetics are similar to those in human medicine. The microbial environment in the stump-socket is unnatural and can be detrimental to healthy tissues. Common stump prostheses problems include poor fit, pressure necrosis, pain, ulceration 11,12,14. The complications reported by Kelmer 12 were not seen in

Fig 2. The appereance after the prosthesis application

Şekil 2. Protez bacak takıldıktan sonraki görünüm

350Partially Forelimb Amputation ...

this case. This was probably due to fact that the prosthesis was applied two months after the amputation after complete healing of the wound.

The result and the clinical outcome in this case indicate that a prosthetic limb may be used successfully even for the free-ranging animals to make their quality of life higher after limb amputation. To the authors’ knowledge this is the first report of partial limb amputation and prosthesis fitting in a deer in Turkey. The deer had a good quality of life, a regular appetite and was in good body condition for six monts during the hospitalization. Documenting more cases is essential in order to try to talk about the prognosis and the patient’s quality of life and to provide experience for veterinary surgeons.

REFERENCES

1. Keppie NJ, Naylor JM: A retrospective study of the diagnoses and survival of elk admitted to a large animal referral clinic. Can Vet J, 46, 325-330, 2005.

2. Butt TD, Cruz AM, Bailey JV, Crawford WH: Outcome of limb amputations in wapiti: 13 cases (1995-2001). Can Vet J, 42, 936-939, 2001.

3. Toews AR, Bailey JV, Theoret C: External skeletal fixation for treatment of comminuted fractures in wapiti: 5 cases. Can Vet J, 39, 370-372, 1998.

4. Mills ML, Holland M, Cooper R: Use of a type-III trilateral external

skeletal fixation device in three deer with comminuted fractures. J Am Vet Med Assoc, 208, 559-561, 1996.

5. Quessada AM: Forelimb amputation in a red deer. Can Vet J, 34, 445-446, 1993.

6. Duncan C, Powers J, Davis T, Spraker T: Abomasal and uterine adenocarcinomas with ovarian metastasis in a captive elk (Cervus elaphus nelsoni). J Vet Diagn Invest, 19, 560-563, 2007.

7. Peters M, Wohlstein P: Anaplastic oligodendroglioma with meningeal infiltration in a free-ranging red deer (Cervus elaphus). J Comp Path, 138, 59-62, 2008.

8. Cruz AM, Bailey JV, Fretz PB: Managment of limb fractures in wapiti (Cervus elaphus): 22 cases (1993-1997). J Am Vet Med Assoc, 214 (12): 1829-1832, 1999.

9. Montane J, Marco I, Manteca X, Lopez J, Lavin S: Delayed acute capture myopathy in three roe red deer. J Vet Med Assoc, 49, 93-98, 2002.

10. Thurston AJ: Pare and prosthetics: the early history of artificial limbs. Anz Surg, 77, 1114-1119, 2007.

11. Drygas KA, Taylor R, Sidebotham CG, Hugate RR, Mcalexander H: Transcutaneous tibial ımplants: A Surgical procedure for restoring ambulation after amputation of the distal aspect of the tibia in a dog. Vet Surg, 37, 322-327, 2008.

12. Kelmer G, Steinman A, Levi O, Johnston DE: Amputation and prosthesis in a horse: Short- and long-term complications. Eq Vet Educ, 16 (5): 235-241, 2004.

13. St Jean G. Amputation and prosthesis. Vet Clin North Am: Food Anim Pract, 12 (1): 249-261, 1996.

14. Pendegrass CJ, Goodship AE, Gordon WE: Development of a soft tissue seal around bone-anchored transcutaneous amputation prostheses. Biomaterials, 27, 4183-4191, 2006.

YAZIM KURALLARI

1- Yılda 6 (Altı) sayı olarak yayımlanan Kafkas Üniversitesi Veteriner Fakültesi Dergisi’nde (Kısaltılmış adı: Kafkas Univ Vet Fak Derg) Veteriner Hekimlik ve Hayvancılıkla ilgili (klinik ve paraklinik bilimler, hayvancılıkla ilgili biyolojik ve temel bilimler, zoonozlar ve halk sağlığı, hayvan besleme ve beslenme hastalıkları, hayvan yetiştiriciliği ve genetik, hayvansal orijinli gıda hijyeni ve teknolojisi, egzotik hayvan bilimi) orijinal araştırma, kısa bildiri, ön rapor, gözlem, editöre mektup, derleme ve çeviri türünde yazılar yayımlanır. Dergide yayımlanmak üzere gönderilen makaleler Türkçe, İngilizce veya Almanca dillerinden biri ile yazılmış olmalıdır.2- Dergide yayımlanması istenen yazılar Times New Roman yazı tipi ve 12 punto ile A4 formatında, 1.5 satır aralıklı ve sayfa kenar boşlukları 2.5 cm olacak şekilde hazırlanmalı ve resim, tablo, grafik gibi şekillerin metin içindeki yerlerine Türkçe ve yabancı dilde adları ve gerekli açıklamaları mutlaka yazılmalıdır.Dergiye gönderilecek makale ve ekleri (şekil vs) http://vetdergi.kafkas.edu.tr adresindeki online makale gönderme sistemi kullanılarak yapılmalıdır. Başvuru sırasında yazarlar yazıda yer alacak şekilleri (13 X 18 cm boyutlarından büyük olmamalı) online makale gönderme sistemine yüklemelidirler. Yazının kabul edilmesi durumunda tüm yazarlarca imzalanmış Telif Hakkı Devir Sözleşmesi editörlüğe gönderilmelidir. 3- Yazarlar yayınlamak istedikleri makale ile ilgili olarak gerekli olan etik kurulu onayı aldıkları kurumu ve onay numarasını Materyal ve Metot bölümünde belirtmelidirler. Yayın kurulu gerekli gördüğünde etik kurul onay belgesini ayrıca isteyebilir.4- Makale TürleriOrijinal Araştırma Makaleleri, yeterli bilimsel inceleme, gözlem ve deneylere dayanarak bir sonuca ulaşan orijinal ve özgün çalışmalardır.Türkçe yazılmış makaleler Türkçe başlık, Türkçe özet ve anahtar sözcükler, yabancı dilde başlık, yabancı dilde özet ve anahtar sözcükler, giriş, materyal ve metot, bulgular, tartışma ve sonuç ile kaynaklar bölümlerinden oluşur ve toplam (metin, tablo, şekil vs dahil) 10 sayfayı geçemez. Yabancı dilde yazılmış makaleler yabancı dilde başlık, yabancı dilde özet ve anahtar sözcükler, Türkçe başlık, Türkçe özet ve anahtar sözcükler dışında Türkçe makale yazım kurallarında belirtilen diğer bölümlerden oluşur. Türkçe ve yabancı dilde özetlerin her biri yaklaşık 200±20 sözcükten oluşmalıdır.Kısa Bildiri, konu ile ilgili yeni bilgi ve bulguların bildirildiği fakat orijinal araştırma olarak sunulamayacak kadar kısa olan yazılardır. Kısa bildiriler, orijinal araştırma makalesi formatında olmalı, fakat özetlerin her biri 100 sözcüğü aşmamalı, referans sayısı 15’in altında olmalı ve 4 sayfayı aşmamalıdır. Ayrıca, en fazla 4 şekil veya tablo içermelidir.Ön Rapor, kısmen tamamlanmış, yorumlanabilecek aşamaya gelmiş orijinal bir araştırmanın kısa (en çok 2 sayfa) anlatımıdır. Bunlar orijinal araştırma makalesi formatında yazılmalıdır.Gözlem, uygulama, klinik veya laboratuar alanlarında ender olarak rastlanılan olguların sunulduğu makalelerdir. Bu yazıların başlık ve özetleri orijinal makale formatında yazılmalı, bundan sonraki bölümleri giriş, olgunun tanımı, tartışma ve sonuç ile kaynaklardan oluşmalı ve 4 sayfayı geçmemelidir.Editöre Mektup, bilimsel veya pratik yararı olan bir konunun veya ilginç bir olgunun resimli ve kısa sunumudur ve 1 sayfayı geçmemelidir. Derleme, güncel ve önemli bir konuyu, yazarın kendi görüş ve araştırmalarından elde ettiği bulguların da değerlendirildiği özgün yazılardır. Bu yazıların başlık ve özet bölümleri orijinal araştırma makalesi formatında yazılmalı, bundan sonraki bölümleri giriş, metin ve kaynaklardan oluşmalı ve 10 sayfayı geçmemelidir.Çeviri, makalenin orijinal formatı dikkate alınarak hazırlanmalıdır.Yazarla ilgili kişisel ve kuruma ait bilgiler ana metin dosyasına değil, on-line başvuru sırasında sistemdeki ilgili yerlere unvan belirtilmeksizin eklenmelidir. 5- Makale ile ilgili gerek görülen açıklayıcı bilgiler (tez, proje, destekleyen kuruluş vs) makale başlığının sonuna üst simge olarak işaret konularak makale başlığı altında italik yazıyla belirtilmelidir. 6- Kaynaklar, metin içinde ilk verilenden başlanarak numara almalı ve metin içindeki kaynağın atıf yapıldığı yerde parantez içinde yazılmalıdır.Kaynak dergi ise, yazarların soyadları ve ilk adlarının başharfleri, makale adı, dergi adı (orijinal kısa ad), cilt ve sayı numarası, sayfa numarası ve yıl sıralamasına göre olmalı ve aşağıdaki örnekte belirtilen karakterler dikkate alınarak yazılmalıdır.Örnek: Gokce E, Erdogan HM: An epidemiological study on neonatal lamb health. Kafkas Univ Vet Fak Derg, 15 (2): 225-236, 2009.Kaynak kitap ise yazarların soyadları ile adlarının ilk harfleri, eserin adı, baskı sayısı, sayfa numarası, basımevi, basım yeri ve basım yılı olarak yazılmalıdır.Editörlü ve çok yazarlı olarak yayınlanan kitaptan bir bölüm kaynak olarak kullanılmışsa, bölüm yazarları, bölüm adı, editör(ler), kitap adı, baskı sayısı, sayfa numarası, basımevi, basım yeri ve basım yılı sırası dikkate alınarak aşağıdaki örneğe göre yazılmalıdır.Örnek: Mcllwraith CW: Disease of joints, tendons, ligaments, and related structures. In, Stashak TS (Ed): Adam’s Lameness in Horses. 4th ed. 339-447, Lea and Febiger, Philadelphia, 1988.Online olarak ulaşılan kaynaklarda web adresi ve erişim tarihi kaynak bilgilerinin sonuna eklenmelidir.Diğer kaynakların yazımında bilimsel yayın ilkelerine uyulmalıdır.Kaynak listesinde “et al.” ve “ve ark.” gibi kısaltmalar yapılmaz.7- Bakteri, virus, parazit ve mantar tür isimleri ve anatomik terimler gibi latince ifadeler orijinal şekliyle ve italik karakterle yazılmalıdır.8- Editörlük, dergiye gönderilen yazılar üzerinde gerekli görülen kısaltma ve düzeltmeleri yapabileceği gibi önerilerini yazarlara iletebilir. Yazarlar, düzeltilmek üzere yollanan yazıları online sistemde belirtilen sürede gerekli düzeltmeleri yaparak editörlüğe iade etmelidirler. Editörlükçe ön incelemesi yapılan ve değerlendirmeye alınması uygun görülen makaleler ilgili bilim dalından bir yayın danışmanı ve iki raportörün olumlu görüşü alındığı takdirde yayımlanır.9- Yayınlanan yazılardan dolayı doğabilecek her türlü sorumluluk yazarlara aittir.10- Yazarlara telif ücreti ödenmez.11- Resim ve baskı masrafları için yazarlardan ücret alınır. Ücret bilgileri http://vetdergi.kafkas.edu.tr/ adresinden öğrenilebilir. 12- Yazarlara 50 adet ayrı baskı ücretsiz olarak yollanır.

ABONE KOŞULLARI Yıllık: 60 TL Banka Hesap No: T.C. Ziraat Bankası Kars Şubesi, 26146825-5001 Nolu Hesap, Kars - TÜRKİYE IBAN: TR830001000147261468255001

INSTRUCTIONS FOR AUTHORS

1- The Journal of the Faculty of Veterinary Medicine, University of Kafkas (abbreviated title: Kafkas Univ Vet Fak Derg), published bi-monthly, covers original papers, short communication and preliminary scientific reports, case reports, observation, letter to the editor, review and translation on all aspects of veterinary medicine and animal science (clinical and paraclinical sciences, biological and basic sciences, zoonoses and public health, animal feeding and nutritional diseases, animal breeding and genetics, hygiene and technology of food from animal sources, exotic animal science). Manuscripts submitted for publication should be written in Turkish, English or German.2- The manuscripts submitted for publication should be prepared in the format of Times New Roman style, font size 12, A4 paper size, 1.5 line spacing and 2.5 cm margins of all edges. The legend or caption of all illustrations such as figure, table and graphic must clearly be written in both Turkish and foreign language and their appropriate position should be indicated in the text.The manuscript and its supplementary (figure etc.) should be submitted by using online manuscript submission system at the address of http://vetdergi.kafkas.edu.tr/During the submission, the authors should upload the figures of the manuscript (the dimensions must not to exceed 13 X 18 cm) to the online manuscript submission system. If the manuscript is accepted for publication, the copyright transfer agreement form signed by all the authors should be send to the editorial office.3- Authors should indicate the name of institute approves the necessary ethical commission report and the serial number of the approval in the material and methods section. If necessary, editorial board may also request the official document of the ethical commission report.4- Original (full-length) manuscripts are original and proper scientific papers based on sufficient scientific investigations, observations and experiments. Manuscripts written in Turkish consist of the title in Turkish, summary and keywords in Turkish, introduction, material and methods, results, discussion and references and it should not exceed 10 pages including text, tables and illustrations. Manuscripts written in a foreign language should follow the title in foreign language, summary and keywords in foreign language, title in Turkish, summary and keywords in Turkish and the remaining sections described above for the manuscripts written in Turkish.Summaries written in Turkish or foreign language should contain 200±20 words.Short communication manuscripts contain recent information and findings in the related topics; however, they are written with insufficient length to be a full-length original article. They should be prepared in the format of full-length original article but each of thesummaries should not exceed 100 words, the reference numbers should not exceed 15 and the length of the text should be no longer than 4 pages. Additionally, they should not contain more than 4 figures or tables.Preliminary scientific reports are short description (maximum 2 pages) of partially completed original research findings at interpretable level. These should be prepared in the format of full-length original articles. Case reports describe rare significant findings encountered in the application, clinic and laboratory of related fields. The title and summary of these articles should be written in the format of full-length original articles and the remaining sections should followintroduction, case history, discussion and references without exceeding the total of 4 pages.Letters to the editor are short and picture-documented presentations of subjects with scientific or practical benefits or interesting cases without exceeding 1 page.Reviews are original manuscripts gather the literature on current and significant subject along with the commentary and findings of the author on the particular subject. The title and summary of this manuscript should be prepared as described for the full-length original articles and the remaining sections should follow introduction, text and references without exceeding 10 page.Translations should be prepared based on the format of original document being translated.The information about author/s and institution/s should be added during the online submissoin and the main documant should be free of these information. 5- The necessary descriptive information (thesis, projects, financial supports etc) scripted as an italic font style should be explained below the manuscript title after placing a superscript mark at the end of title. 6- References should be listed with numerical order as they appear in the text and the reference number should be indicated inside the parentheses at the cited text place. References should have the order of surnames and initial letters of the authors, title of the article, title of the journal (original abbreviated title), volume and issue numbers, page numbers and the year of publication and the text formatting should be performed as shown in the example below.Example: Gokce E, Erdogan HM: An epidemiological study on neonatal lamb health. Kafkas Univ Vet Fak Derg, 15 (2): 225-236, 2009.If the reference is a book, it should follow surnames and initial letters of the authors, title of the book, edition number, page numbers, name and location of publisher and year of publication. If a chapter in book with an editor and several authors is used, names of chapter authors, name of chapter, editors, name of book, edition number, page numbers, name and location of publisher and year of publication and the formatting should be performed as shown in the example below.Example: Mcllwraith CW: Disease of joints, tendons, ligaments, and related structures. In, Stashak TS (Ed): Adam’s Lameness in Horses. 4th ed. 339-447, Lea and Febiger, Philadelphia, 1988.In the references can be reached online only, the web address and connection date should be added at the end of the reference information. The generally accepted scientific writing instructions must be complied with the other references. Abbreviations, such as “et al” and “and friends” should not be used in the list of the references.7- The Latin expression such as species names of bacterium, virus, parasite and fungus and anatomical terms must be written in italic character keeping their original forms.8- The editorial board has the right to perform necessary modifications and reduction on the manuscript submitted for publication and to express recommendations to the authors. The manuscripts sent to authors for correction should be returned to the editorial office within a month. After pre-evaluation and agreement of the submitted manuscripts by editorial board, the article can only be published after the approval of the publication advisor and two referees specialized in the particular field.9- All responsibilities from published articles merely belong to the authors.10- There is no copyright fee for the authors.11- A fee is charged from the authors to cover printing cost and other expenses. This payment information can be found at http://vetdergi.kafkas.edu.tr/12- Reprints (in multiples of 50) of the article are sent to the authors for free.

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