Aquaculture Research, 1996, 27, 847-852

Induced spawning in perch, Perca fluviatilis L. usingcarp pituitary extract and HCG

D Kucharczyk, R Kujawa, A Mamcarz & A SkrzypczakDepartment of Fisheries, Olsztyn University of Agriculture and Technology, Olsztyn-Kortowo, Poland

E WyszomirskaDepartment of Chemical Analysis, Drug Institute, Warsaw, Poland

Correspondence: Dr Dariusz Kucharczyk, Department of Fisheries, Olsztyn University of Agriculture & Tbchnoiogy, Oczapowskiego 5.

10-718 Olsztyn-Kortowo, Poland

Abstract

Induced spawning in Eurasian perch, Perca fluviatilisL., was studied using human chorionic gonado-trophin (hCG) or acetone-dried common carppituitary with or without hCG, The doseadministered to fish was 5700 IU hCG kg"' or 4,0mg kg~^ carp pituitary with or without 500-700IU hCG kg~i for females and 2850 IU hCG kg"' or2,0 mg kg~' carp pituitary with or without 250-350IU hCG kg"^ for males. There were no statisticallysignificant differences in quantity of milt in treatedand control groups, although the best result wasobserved when males were treated with a tripleinjection of hCG and carp pituitary extract, Malespermiating success, expressed as a quantity ofmilt, was negatively correlated with fish weight. Allfemales treated in this experiment had oocytes atthe same division, so time of ovulation was highlysynchronous. Spawning success, expressed as aspawning eflectivity coefficient (S^), was highest infish treated with the triple injection. Spawningmethods described in this paper were successful,even though hormones from another order of fishwere used.

Introduction

One of the main problems in aquaculture is obtainingmature gametes. Most fish from cultured and wildstocks are incapable of spontaneous maturationunder artificial conditions (Ahmad Tajuddin, Chuah,Haron & Zahari 1988; Glubokov, Motloch & Sedova

1991). In some fish species there are problems ofsynchrony in time of spawning (Dabrowski,Ciereszko, Ramseyer, Culver & Kestemont 1994),

The most commonly used method for stimulationof maturation in fish is the administration of fishpituitary extracts (Okoniewsi & Klodzinska 1975;Kozlowski 1994). However, this simple method isnot always successful. In many cases, much betterresults in controlled reproduction are obtained whenpure or synthetic hormones are used for hormonalstimulation (Takashima, Weil, Billard, Crim & Fostier1984; Crim, Glebe & Scott 1986; Glubokov etal.1991; Drori, Ofir, Levavi-Sivan & Yaron 1994; Yaron1995), On the other hand, extract of carp pituitarycombined with other hormones, mainly with hCG(human chorionic gonadotrophin), has provedsuccessful (Ahmad Tajuddin et al. 1986; Thalathiah,Ahmad & Zaini 1988; Kozlowski 1994),

The Eurasian perch, Perca fluviatilis L, is one ofthe most important freshwater species in Poland,Recent years have seen growing interest in perch,not only by fishermen but also by anglers andimportant developments in aquaculture of perch inEurope (Fontaine, Tamazouzt & Capdeville 1995;Goubier 1995; Kestemont, Melard, Fiogde, Vlavonou& Masson 1995),

Perch is a species that can be spawned easilyunder artificial conditions. Our earlier observationsshowed that perch spawned spontaneously in tanks,but this was a long process. However, it is importantin aquaculture to obtain mature gametes artificially,especially for genetic engineering (Tave 1990) andfor cryopreservation (Babiak, Glogowski, Luczynski,Kucharczyk & Luczynski 1995). In this paper, we

© 1996 Blackwell Science Ltd. 847

Induced spawning in Eurasian perch D Kucharczyk et al. Aquaculture Research, 1996. 27, 847-852

T^ble 1 Hormonal treatment applied to induce artificial spawning in perch, Perca fluviatilis: CP, carp pituitary; hCG,human chorionic gonadotrophin. Doses are expressed as IU hCG and as mg CP kg" '

Group of fish Type ofhormones

Dose of primingInjection (0 h)

Females Males

Dose of stimulatory- Injection (24 h)

Females Males

Dose of resolvinginjection (48 h)

Females Males

Contror

2

3

4

5

CP

HCG

HCGCP

HCGCP

200

200

100

100

0.4

500

5000.4

5000.4

—

250

250-

250...

3.6

5000

—

3.6

_

36

2.0

2500

2.0

_

2.0

"Injection of 0.9% NaCl, given at time(s) marked +.

2 Spermiation success of perch males. Groups of fish refer to the hormonal treatments described in Table 1

Group of fish No. of males No. of males spermiating Sperm quantity (ml kg i)

Range Mean ± SD

15.71-24.4018.22-24.8617.25-20.6718.60-27.7120.75-28.40

20.15 ± 4.3520.61 ± 3.6919.02 ± 1.7122.47 ± 4.7024.82 ± 3.85

present a method of artificially spawning wild,sexually mature perch.

Materials and methods

Perch spawners were caught in Lake Kortowskie(Olsztyn, Poland) in April 1995, Fish were selectedaccording to the following criteria: the belly offemales had to be fully distended and bulging, softand resilient to touch; males had to be spermiating.The selected males and females were kept in separate1000-1 tanks in a hatchery with controlledtemperature and photoperiod. After 1 day ofacclimation, the water temperature was raised from7°C to 10°C and kept oscillating between 10°C and11°C throughout the experiment. Five days later,the fish were individually marked and oocytes weretaken from females using the method described by

Kujawa & Kucharczyk (1996). Oocytes weresampled in vivo and placed in Serra's solution forclarification of the cytoplasm. After 5 min theposition of the oocyte nucleus was detertnined usinga four-division scale (Brzuska & Bieniarz 1977).For further experiment, only those females havingoocyte maturation between 2-3 and 3 divisionswere used. This is the best moment for hormonaltreatment in cyprinids (Brzuska & Bieniarz 1977;Kozlowski 1994). The fish were then treated withhormonal injections of hCG or carp pituitary extractwith or without hCG (Table 1). Pituitary extractwas prepared by homogenization with 0.9% NaCl.The hCG was dissolved in 0.9% NaCL Injections ofhCG were administered intramuscularly in the dorsalarea of the body. Injections of pituitary extract wereintraperitoneal at the base of the pelvic fin. The timeintervals between respective injections are presented

848 O 1996 Blackweii Science Ltd, Aquacuiture Research. 27, 847-852

Aquaculture Research, 1996, 27, 847-852 Induced spawning in Eurasian perch D Kucharczyk et al.

30

> 25

^S 15

1 ^ 10« 5

00.2 0.25 0.3 0.35 0.4 0.45 0.5

Weight (kg)

Figure 1 Relationship between quantity of milt and body weight in perch Pertafluviatilis males. CoefHcient of determinationR^ was 0.333. Number of males was 15.

= -29.32X +32.007

Ikble 3 Spawning success of perch females. Groups offish refer to the hormonal treatment described in Table 1. Groupswith common superscript were not significantly different by Duncan's multiple range test (P > 0.05)

Group of fish

1°

gab

3"

4ab

5"

Weight (kg)

0.30.40.450.350,4O.B0.250.350.50.30.40.350.50.50.350.450.50.40.50.3

Position

Before stimulation

32-32-32-32-32-32-3332-32-332-32-332-32-332-32-3

of nucleus*

After stimulation

333-4

3-43-4

Range

33.3-34.9

26.6-27.892.7-93.627.9-29.898.3-98.584.5-84.683.0-84.3

84.0-85.1 .78.6-91.928.03-31.041.4-42.678.1-79.881.2-82.647.6-51.577.9-78.6

Eyed eggs (%)

Mean ± SD

34.1 ± 1.13

27.1 ± 0.7193.1 ± 0.6428.8 i 1.3498.4 ± 0.1484.5 ± 0.0783.6 ± 0.92

84,5 ± 0.7885.2 ± 9.4029.5 ± 2.1242.0 ± 0.8578.9 ± 1.2081.9 ± 0.9949.5 ± 2.7678.2 4 0.49

'Scored following Brzuska & Bieniarz (1977).

in Tahle 1. After stimulatory itijection, the genitalpore of each female was sewn up to preventspontaneous shedding of eggs.

Females were checked every 30 min between 12and 18 h after resolving (the last) injections and

every hour between 18 and 24 h; they were alsochecked after 36 and 48 h. Eggs obtained from thefemales were fertilized with sperm pooled from threemales from the respective treated group, Oocytesamples were taken 48 h after the resolving

© 1996 Btackweli Science Ltd, Aquacuiturr Research. 27, 847-852 S49

Induced spawning in Eurasian perch D Kucharczyk et al. Aquaculture Research, 1996, 2 ^ 847-852

oCO

u.o ~

0.7

O.b

0.5

0.4

0.3

0.2

0.1

0 — 1—1— — 1 —

' -

• • .• • . '{'•'

No. of groups

Figure 2 Spawning success (S^) of perch, Perca fluviatilis. in control and treated groups. Number of groups of fish referto the hormonal treatment described in T^ble 1,

injection, from those few females in which ovulationwas not observed.

Two replicates of 400-500 eggs per trial wereincubated in special incubation chambers(Kucharczyk, Kujawa & Mamcarz unpublished data),at a temperature oscillating between 13°C and 16°C,which has been found optimal for perch embryonicdevelopment (Kokurewicz 1971). Eggs wereincubated up to the eyed egg stage (embryo eyeswere pigmented).

For showing effectiveness of spawning success ineach group we used a new coefficient S^ (spawningeffectiveness coefficient), obtained from the formula:

= 0, X (1)

where Og is ovulating success (no, of ovulatingfemales/total no. of females) and }g is incubationsuccess (no. of live embryos/total no. of embryos).

Statistical differences between groups (spermiationsuccess and incubation success) were analysed withDuncan's multiple range test (P < 0,05),

Results and discussion

All males were ready to spermiate when transportedto the hatchery. Males from group 5 gave a higher

quantity of sperm (as ml kg ;̂ Table 2) than malesfrom other groups, but there was no significantdifference between groups. In yellow perch, Percaflavescens (Mitchill), a significant difference betweencontrol and hormonally induced groups wasobserved (Dabrowski et al. 1994), but fish were keptunder controlled conditions for a much longer time.In the present experiments, perch were treatedwith carp pituitary extract and human chorionicgonadotrophin. Kozlowski (1994) reported that inmany cases when hormones from fish of anotherorder were used for stimulation of maturation,spawning success was rather low.

The relationship between quantity of milt (Ymlkg~^) and body weight (X, kg) is described by theequation: Y = -29.32X + 32.007; R^ = 0.333.This negative relationship (Fig. 1) indicates thatsmaller males give relatively more milt. Males fromthe control group (mean weight 0.3 kg) gave a littlemore sperm than males in group 3 (mean weight0,35 kg) (Table 2),

In females from most groups, ovulation was highlysynchronous. Most females ovulated between 14and 16 h after the resolving (final) injection. Twofemales from group 3 ovulated between 48 and 80

850 © 1996 Biackweii Science Ltd, Aquaculture Research. 27 , 847-852

AquacultuTGResearch, 1996. 27, 847-852 Induced spawning in Eurasian perch D Kucharczyk et al.

h; two females from the control group ovulatedbetween 48 and 96 h (one female did not ovulate).This synchrony (range 2 h) was possible becauseall females had oocytes at the same division. Inyellow perch treated with LH-RH (Dabrowski et al.1994), the time between the final injection andovulation was much longer than in our experiment,Kozlowski (1994) reported that the time of ovulation(hours after the final injection) was generally longerwhen LH-RH (luteinizing-hormone-releasing hor-mone) or its analogues were used, rather thanpituitary extract.

The biological quality of eggs, expressed assurvival of embryos up to the eyed egg stage, washighest in group 3 and lowest in the control group(Table 3), Statistically significant differences betweensurvival of embryos up to the eyed egg stage showedbest results when perch females were treated withhCG alone (group 3) or with triple injection withhCG and carp pituitary (group 5) (Table 3). In manycyprinids the spawning success, expressed as apercentage of ovulated females and of hatching rate,was highest when triple injections with hCG andcarp pituitary were applied: bighead carp,Aristichthys nobilis (Rich,), silver carpHypophthalmichthys molitrix (Val.) (Thalathiah et al.1988; Kozlowski 1994); grass carpCtenopharyngodon idella (Val,) (Kozlowski 1994); andbream, Abramis brama (L.) (Kucharczyk, Kujawa,Luczynski, Glogowski, Babiak & Wyszomirskaunpublished data). Similar good results with thismethod have been observed in the riverine catfish,Pangasius sutchi Fowler (Thalathiah et al. 1988).

The spawning effectiveness coefficient (Se),combining results from ovulation and incubationsuccess, showed the best kind of hormonaltreatment. In our experiment, triple injection withhCG and carp pituitary (S^ = 0,722) proved best. Inother hormonal combinations; group 2, S^ = 0,619and group 4, S^ = 0,603. results were similar (Fig,2). For cyprinids, S^ values over 0,5 are regardedas good results (Ahmad Tajuddin etal 1986;Thalathiah etal. 1988; Kucharczyk, Kujawa etal.unpublished data).

We conclude that induced spawning techniquesfor wild mature perch as described in this paper aresuccessful and provide consistent results.

Acknowledgments

This study was supported by Project No, 5050.802financed by Olsztyn University of Agriculture &Technology, Olsztyn, Poland.

References

Ahmad TVijuddin A.Z,, Chuah H,P., Haron A, & ZahariA. (1986) Induced breeding techniques for some ofthe cyprinid foodfishes in Malacca. Proc. Intl. ConfDevelopment Management of Tropical Living AquaticResources. Serdang. Malaysia, 2-5 August 1993,pp. 44-51,

Babiak 1,, Glogowski ]., Luczynski M.]., Kucharczyk D.& Luczynski M. (1995) Cryopreservation of the miltof the northern pike. Journal of Fish Biology 46,819-828.

Brzuska E, & Bieniare K. (1977) A method of in vivodesignating oocytes maturation of carp females withconnection of injections with common carp pituitaryextract. Broszura lnstytutu Rybactwa Srodlqdowego 105,1-27 (in Polish),

Crim W.L., Glebe B.D, & Scott A,P. (1986) The inOuenceof LHRH analog on oocyte development and spawningin female Atlantic salmon, Salmo salar. Aquaculture 56,139-149.

Dabrowski K,, Ciereszko A,, Ramseyer L., Culver D. &Kestemont P. (1994) Effects of hormonal treatmenton induced spermation and ovulation in the yellowperch (Perca flavescens). Aquaculture 120, 171-180,

Drori S., Ofir M,, Levavi-Sivan B, & Yaron Z, (1994)Spawning induction in common carp {Cyprinus carplo)using pituitary extract or GnRH superactive analoguecombined with metoclopramidae: analysis of hormoneprofile, progress of oocyte maturation and dependenceon temperature. Aquaculture 119, 393-407,

Fontaine P., Tamazouzt L. & Capdeville B, (1995) Fitnessof the common perch Perca fluviatilis L. for ongrowingin floating cage.s and in recirculated system. In:Workshop on Aquaculture of Percids. First Meeting of theEuropean Workgroup on Aquaculture of Pereids. Vaasa.Finland - August 23-24 (ed. by P, Kestemont &K, Dabrowski) pp, 38-41. Presses Universitaires deNemur, Belgium.

Glubokov A,I,, Motloch N.N, & Sedova M,A, (1991)Effect of a synthetic LHRH analogue and dopamineantagonists on the maturation of bream, Ahramisbrama L. Aquaculture 95 , 373-377.

Goubier, V. (1995) Reproduction of perch, control ofreproductive cycle and gamete quality. Workshop onAquaculture of Pereids. First Meeting of the EuropeanWorkgroup on Aquaculture of Percids, Vaasa - August23-24 (ed. by P. Kestemont & K. Dabrowski)pp. 5-7, Presses Universitaires de Nemur, Belgium.

Kestmont P., Melard C, Fiogde E., Vlavonou R. & MassonG. (1995) Larval and post-larval rearing of Europeanperch Perca fluviatilis: ontogeny of digestive system,growth and nutritional requirements. Workshop onAquaeulture of Percids. First Meeting of the EuropeanWorkgroup on Aquaculture of Percids. Vassa - August23-24 (ed, by P. Kestemont & Dabrowski),pp, 20-24. Presses Universitaires de Nemur, Belgium,

© 1996 Biackwell Science Ltd. Aquaculture Research. 27, 847-852 851

Induced spawning in Eurasian perch D Kucharczyk et al. Aquaculture Research. 1996, 27, 847-852

Kokurewicz B. (1971) Thermal conditions for spawningand development for chosen fish species, Broszuralnstytutu Rybactwa Srodladowego 47, 1-18 (in Polish).

Kozlowski B, (1994) Practise of hormonal stimulationof artificial spawning in cyprinid fishes, Broszuralnstytutu Rybactwa Srodladowego 162, 1-41 (in Polish).

Kucharczyk D., Kujawa R., Luczynski M., Glogowski J.,Babiak I. & Wyszomirska E. (1996a) Induced spawningin bream {Abramis brama L.) using bream andcarp pituitary extract and HCG, Aquacidture Research(In press).

Kucharczyk D., Kujawa R, & Mamcarz A. (1996b) Anew experimental incubation chambers for perch {Percafluviatilis) eggs. The Progressive Fish-Culturist (In press).

Kujawa R. & Kucharczyk D, (1996) A method of in vivodesignating oocytes from cyprinid and percid fish usingcatheter. Komunikaty Rybackie (in press) (in Polish),

Okoniewski Z. & Klodzinska H. (1975) Organization andmodification of artificial spawning methods of commoncarp. Broszura lnstytutu Rybactwa Srodladowego 91,1-32 (in Polish).

Takashima F,, Weil C, Billard R., Crim L,W, & FostierA. (1984) Stimulation of spermiaOon by LHRHanalogue in carp. Bulletin of the Japanese Society for

Scientific Fisheries 50, 1323-1329.

Tave D. (1990) Chromosomal manipulation. Aquaculture

Magazine 1, 62—65.Thalathiah S,, Ahmad A.O. & Zaini M.S. (1988) Induced

spawning techniques practised at Batu Berendam,Melaka, Malaysia, Aquaculture 74, 23-33,

Yaron, Z. (1995) Endocrine control of gametogenesisand spawning induction in the carp. Aquaculture 129,49-73,

852 C 1996 Blackwell Science Ud. Aquacuiture Research. 27, 847-852