Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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Animal Feed Science and Technology xxx (2008) xxx–xxx

Contents lists available at ScienceDirect

Animal Feed Science andTechnology

journal homepage: www.elsevier.com/locate/anifeedsci

Influence of sunflower meal based diets supplemented withexogenous enzyme and digestible lysine on performance,digestibility and carcass response of broiler chickens

T. Mushtaqa,∗, M. Sarwara, G. Ahmadb, M.A. Mirzaa,T. Ahmadc, U. Noreend, M.M.H. Mushtaqe, Z. Kamrana

a Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad 38040, Pakistanb Alwatnia Poultry, Buraidah 51431, Saudi Arabiac Faculty of Veterinary and Animal Sciences, PMAS University of Arid Agriculture, Rawalpindi 46000, Pakistand Department of Zoology and Fisheries, University of Agriculture, Faisalabad 38040, Pakistane Department of Food and Nutrition, Faculty of Animal Production and Technology,University of Veterinary and Animal Science, Lahore 54000, Pakistan

a r t i c l e i n f o

Article history:Received 21 September 2007Received in revised form 20 May 2008Accepted 26 June 2008Available online xxx

Keywords:Enzyme supplementationBroilersCarcass responseDigestible lysineSunflower meal

a b s t r a c t

Response of broiler chickens to 2 levels of endo-1,4-� xylanase(EC 3.2.1.8) and endo-1,3-� glucanase (EC 3.2.1.6) cocktail (withand without), 3 levels of digestible lysine (8.0, 9.0 and 10.0 g kg−1

with the applicability of ideal protein concept), and 2 levels ofsunflower meal (SFM; 200 and 300 g kg−1) were evaluated in thepresent experiment. The factorial design of 2 × 3 × 2 using 2448one-day-old straight-run Hubbard broiler chicks was used to eval-uate the effect of two levels of enzymes, 3 levels of digestible lysineand 2 levels of SFM in practical vegetable based mash diets hav-ing 11.51 MJ ME and 190 g CP kg−1 from 1 to 42 d of age. The BWgain was improved linearly (P≤0.001) with the increasing level ofdigestible lysine during 1–21 d and 22–42 d of age. The gain:feedwas improved linearly (P≤0.001) with the increasing digestiblelysine during 1–21 d and a quadratic effect of digestible lysinewas noted during 1–42 d where 9.0 and 10.0 g digestible lysinekg−1 of diet responded similar for gain:feed. Enzyme supplementa-tion at 300 g SFM kg−1 improved BW gain (P≤0.008) and gain:feed(P≤0.048) during 1–21 d. When 10.0 g digestible lysine kg−1 of diet

∗ Corresponding author at: D-35, K.D.A. Scheme No. 1, Miran Muhammad Shah Road, Karachi 75350, Pakistan.Tel.: +92 21 4543579/4543785; fax: +92 21 4543763.

E-mail addresses: tmmirza@fsd.paknet.com.pk, tmmirza@ghazibrothers.com (T. Mushtaq).

0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.anifeedsci.2008.06.008

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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was used at 2 levels of SFM, gain:feed was improved (P≤0.013) dur-ing 1–21 d. However, this interaction was not significant on otherperformance parameters either at 21 d or at 42 d of age. The enzymesupplementation decreased the feed intake (P≤0.019) by 4.67%and improved gain:feed by 5.53% (P≤0.012) during 1–42 d withoutaffecting BW gain. The main effect of SFM was not significant forlive performance or carcass response at any stage. The depressionin breast weights at 42 d due to 300 g SFM or 8.0 g digestible lysinekg−1 of diet was compensated by the enzyme addition (a signifi-cant enzyme × SFM effect). No significant effect of enzyme, SFM,digestible lysine or their interactions was observed on apparentmetabolizable energy of the diets. Coefficient of total tract apparentdigestibility of nitrogen was improved by the addition of enzyme to10.0 g digestible lysine kg−1 of diet or addition of digestible lysineto high SFM. The results of the present study revealed a positiveresponse of enzyme addition in low nutrient density and high SFMdiets. Moreover, digestible lysine is not suggested to be loweredthan 10.0 g during 1–21 d and it may be reduced to 9.0 g kg−1 if asingle diet having high level of SFM is planned to be offered during1–42 d.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Sunflower (Helianthus annus) is high oil-yielding seed crop, that adapts very well to a wide range ofclimatic and soil conditions. Sunflower meal (SFM), a by-product of sunflower oil extraction, is availablein considerable quantities for use in animal feeds (Afifi, 1972; Green et al., 1987; Ibtisam et al., 1990;Klain et al., 1956; Leeson et al., 1987; Villamide and San Juan, 1998). Rad and Keshavarz (1976) reportedthat SFM could be included up to 175 g kg−1 of diet without adversely affecting the chick performanceprovided diet was supplemented with lysine. No effect on weight gain, feed consumption and gain:feedwas reported when high oil sunflower seeds were included up to 200 g kg−1 of the broiler diet (Selvarajand Purushothaman, 2004).

The use of appropriate feed enzymes offers an opportunity to overcome some of the potentiallimitations imposed by exclusive vegetable protein-based diets including lower digestibility of proteinand starches, etc. The effect of feed grade enzyme has been reported to improve in vitro digestibility ofstarch and acid soluble nitrogen fraction of autoclaved high fibre sunflower seed cake (Swain and Johri,1999a,b). Similarly, Kocher et al. (2000) reported a positive effect of multi-enzyme supplementationto SFM based diets.

Broiler feed formulation based on ideal protein concept may be a better option than based on CPor total amino acid (AA). Most non-soy vegetable protein sources used in poultry diet formulationsare moderate to low in lysine contents; hence supplementation with lysine is inevitable in growingbroilers to ensure rapid growth and optimum efficiency of feed utilization (Corzo et al., 2006; Ahmadet al., 2007).

The SFM has 230 g insoluble NSP and 45 g soluble polysaccharides (Irish and Balnave, 1993). Kocheret al. (2000) reported 780 g insoluble, 40 g soluble and 180 g free sugars kg−1 of total fibre in SFMhaving 260 g CP kg−1. More specifically they reported more than 375 g arabinose and xylose kg−1 oftotal fibre. Other studies have also shown high acid detergent and neutral detergent fibres in SFM(Villamide and San Juan, 1998; San Juan and Villamide, 2001). Nutritionists usually avoid high fibreingredients in poultry diets because they have low energy values. High levels of fibre also reduce thetime of feed passage throughout the digestive system (Connell, 1981). Furthermore, fibre may causeabrasion of the intestinal mucosa and thus increase the AA requirements of the synthesis of mucosacells (Parsons et al., 1983).

In countries where soybean meal (SBM) is not cultivated, the SFM may be an economical feedingredient for poultry diets. High fibre contents and high supplementation cost of crystalline lysine

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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are the main hurdles of its use in broiler diets. With the increase in feed prices, there is a general trendto formulate diets to meet specifically the requirements of the birds without offering extra nutrients.Thus, most commercial feed mill and integrators are switching to low nutrient concentration diets foreconomical broiler production. The study therefore, was conducted to evaluate the exogenous enzymesupplementation and three AA concentration in low nutrient density broiler diets having high SFM.The ultimate objective of the study was to provide cushion in formulation to cope with the increasingingredient prices.

2. Materials and methods

2.1. Bird husbandry

A total of 2448 one-day-old straight-run Hubbard broiler chicks were obtained from a local hatcheryand were randomly allotted to one of 48 floor pens (51 birds in each replicate) with rice straw asbedding material over a concrete floor. A floor space of 0.074 m2 (0.80 feet2) per bird was allowed inan open sided house with sidewall curtains. Each pen was equipped with a separate tube feeder anda manual drinker. On 11 d, the tube feeders were replaced with round bottom feeders. The manualdrinkers were replaced with automatic drinkers on 15 d. The house temperature was maintained at32 ◦C during the first week of age. A weekly reduction of 3 ◦C was practiced till a house temperature of25 ◦C was attained. Birds were vaccinated against infectious bronchitis at 2 d, Newcastle disease at 4 dand infectious bursal disease at 8 d of age. Birds were re-vaccinated against Newcastle disease at 11 and32 d and against infectious bursal disease at 14 d. A 24 h’ light was provided throughout the experiment.

2.2. Ingredients and experimental diets

Two levels of SFM, i.e., 200 and 300 g kg−1 were used with three levels of digestible lysine, i.e., 8.0, 9.0and 10.0 g kg−1 in a factorial design of 2 × 3 resulting in 6 dietary combinations in isonitrogenous andisocaloric diets (Table 1). Total and digestible AA were calculated from analyzed DM and CP contentsof each ingredient using AminoDatTM 2.0 (Degussa AG, Germany). The feed formulation was based ondigestible AA. The ME value of each ingredient was calculated by regression equation of NRC (1994).Diets were formulated by linear formulation method using WinFeed 2.8 (WinFeed Ltd., Cambridge, UK).The nutrient specifications other than AA used in the experiment were similar to those of commercialbroiler diets of this country. The digestible AA met or exceeded ideal AA ratio as suggested by Bakerand Han (1994) in which digestible lysine was used as reference AA against which other essential AAwere balanced (Table 2). As soybean meal is substantially high in lysine and inclusion of soybean mealas sole protein source leads to dietary digestible lysine to about 12.0 g kg−1 which was out of the scopeof the study so a corn-soy meal based positive control diet was not included in the study. Enzyme(RovabioTM Excel AP 10%; Adisseo, Asia Pacific Pte Ltd., Singapore) at 0.5 g kg−1 of finished feed wasmixed through vitamin and mineral premix. Endo-1,4-� xylanase activity was assayed with 1% oat splitxylan as the substrate at 60 ◦C by the method described by Baily et al. (1992). Each of the experimentaldiets (total of 12 dietary combinations) was offered to four replicates in mash form throughout theexperimental period of 42 d as suggested by Ahmad et al. (2007) for low nutrient concentration diets.Feed and water were offered at will throughout the experiment.

2.3. Performance study

At the end of 21 and 42 d, birds and residual feed were weighed. Mortality was recorded on dailybasis. Birds that were removed or died during the experiment were weighed and used to adjust thefeed intake (FI) and gain:feed. On 43 d, two birds from each replicate were selected at random forcarcass evaluation (Mushtaq et al., 2005).

2.4. Digestibility study

At 37 d, diets mixed with 3 g chromic oxide (Cr2O3) kg−1 of feed as digestibility marker were offeredto the birds for 5 d. After 5 d, ileal digesta from each of the pen was collected by the method describedby Scott and Boldaji (1997). Four birds from each replicate were killed by cervical dislocation. The

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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Table 1Ingredient composition of experimental diets (g kg−1)a

Ingredients 200 g SFM 300 g SFM

Digestible lysine (g kg−1 of diet)

8.0 9.0 10.0 8.0 9.0 10.0

Sunflower meal, 320 g CP kg−1 200 200 200 300 300 300Maize 584 582 587 533 535 532Maize gluten meal, 560 g CP kg−1 40 40 40 40 40 40Soybean meal, 445 g CP kg−1 99.8 95.7 91.4 39.3 34.8 29.8Rice, polishing 7.7 6.8 – – – –l-Lysine HCl 2.5 3.9 5.3 3.4 4.8 6.2dl-Methionine – 0.4 1.2 – 0.3 1.1l-Tryptophan – – 0.1 – – 0.1l-Threonine – 0.4 1.2 – 0.7 1.4Sunflower oil – – – 22.9 22.8 23.2Cane molasses 35.1 40.0 40.0 30.0 31.2 35.8Limestone (ground) 9.0 9.0 10.0 8.5 8.5 8.4NaCl .1.70 1.4 1.3 1.3 0.9 0.4NaHCO3 – – 0.1 0.6 1.2 1.5Dicalcium phosphate 15.1 15.1 17.4 15.2 15.2 15.3Vitamin and mineral premixb 5.0 5.0 5.0 5.0 5.0 5.0

a Each diet was divided into two aliquots, one aliquot was fed as such whereas second one was supplemented with (RovabioTM

Excel AP 10%), 0.50 g kg−1 of finished feed through premix. The supplemental enzyme activities reported by the manufacturerwere 2200 visco units g−1 (1400 xylanase units g−1) for endo-1,4-� xylanase (EC 3.2.1.8) and 200 glucanase units g−1 for endo-1,3-� glucanase (EC 3.2.1.6). One visco unit of endo-1,4-� xylanase is defined as the amount of enzyme which hydrolyzes thesubstrate, reducing the viscosity of the standard wheat arabioxylan solution to give a change in relative fluidity of 1 (dimension-less unit) min−1 mg−1 of enzyme whereas one xylanase unit is defined as the release of oligomers from chromophore-boundxylan which are not precipitable by ethanol, equivalent to an absorbance of 1.23 units at 590 nm. One unit of endo-1,3-� glu-canase is defined as the release of oligomers from a chromophore-bound glucan which are not precipitable by ethanol, equivalentto an absorbance of 0.820 units at 590 nm.

b Supplied per kilogram of finished diet: vitamin A (as vitamin A acetate), 15,000 IU; vitamin D3 (as cholcalciferol), 3000 IU;vitamin E (as dl-�-tocopheryle acetate), 54 IU; vitamin K3 (as menadione nicotinamide bisulfite), 3 mg; thiamine (as thiaminemononitrate), 2.94 mg; riboflavin, 8 mg; B6 (as pyridoxine hydrochloride), 3.92 mg; B12, 20 �g; folic acid, 1.52 mg; calcium-d-pantothenate, 15 mg; niacin (as nicotinic acid), 60 mg; biotin, 200 �g; choline chloride 60%, 600 mg; Se, 0.2 mg; Zn (asZnSO4·H2O), 110 mg; Cu (as CuSO4·7H2O), 10 mg; Fe (as FeSO4·H2O), 60 mg; Mn (as MnSO4·H2O), 200 mg; I (as KI), 0.92 mg;furazolidon, 60 mg; salinomycin, 60 mg.

intestinal tract of each bird was excised and the contents of the tract from Meckel’s diverticulum to40 mm above the ileo-caecal junction were gently squeezed directly into 200 mL plastic cups and keptin an ice container. During digesta collection, few drops of formalin were also added to stop bacterialgrowth. The digesta samples within a pen were pooled and dried in hot air oven at 65 ◦C until aconstant weight was obtained. They were ground to pass through 0.5-mm sieve and stored at −10 ◦Cuntil analyses.

Diets and ileal digesta samples were analyzed for N by micro Kjehdahl method (AOAC, 2000) andgross energy (GE) by adiabatic oxygen bomb calorimeter (Parr Instrument CO; Moline, USA). The Cr2O3content in the ashed samples of the diets and digesta was analyzed spectrophotometrically after aciddigestion method (Divakaran et al., 2002). The apparent metabolizable energy (AME) of the diets wascalculated as follows:

AME(MJ/kg) = GEdiet −[

GEdigesta ×(

Cr2O3 in dietCr2O3 in digesta

)]

The coefficient of total tract apparent digestibility of nitrogen (CTTADN) was calculated by thefollowing equation (Ravindran et al., 1999):

CTTADN = (N/Cr2O3)diet − (N/Cr2O3)digesta

(N/Cr2O3)diet

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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Table 2Composition of experimental diets (g kg−1)

Components (g kg−1) 200 g SFM 300 g SFM

Digestible lysine (g kg−1 of diet)

8.0 9.0 10.0 8.0 9.0 10.0

CalculatedDMa 907 906 906 914 914 913MEN

a (MJ kg−1) 11.51 11.51 11.51 11.51 11.51 11.56CPa 190 190 190 190 190 190CFa 54.5 54.3 54.3 71.0 70.8 70.6EEa 26.2 25.9 25.9 22.9 22.7 22.5Ca 9.0 9.0 1.0 9.0 9.0 9.0Non-phytate P 4.5 4.5 5.0 4.5 4.5 4.5Na 2.6 2.6 2.6 2.5 2.6 2.6K 6.3 6.3 6.3 6.0 6.0 6.0Cl 2.0 2.1 2.3 2.0 2.0 2.0DEBb 219 218 210 210 210 210

Digestible amino acidsc

Methionine 3.4 3.8 4.5 3.6 3.8 4.6Cystein 2.7 2.7 2.7 2.7 2.6 2.6Met + cys 6.1 6.5 7.2 6.2 6.4 7.2Lysine 8.0 9.0 10.0 8.0 9.0 10.0Threonine 5.7 6.0 6.7 5.5 6.0 6.7Tryptophan 1.6 1.6 1.6 1.5 1.5 1.6Arginine 10.8 10.6 10.5 11.1 10.9 10.8Isoleucine 6.8 6.7 6.6 6.6 6.6 6.5Leucine 16.5 16.4 16.3 15.8 15.7 15.6Valine 7.9 7.8 7.7 7.8 7.7 7.6Histidine 4.2 4.2 4.1 4.1 4.1 4.0Phenylalanine 8.5 8.4 8.3 8.4 8.3 8.2

AnalysedDM 918 914 914 914 919 922CPa 179 179 182 170 175 181CFa 58.1 58.7 58.3 68.9 68.7 70.6EEa 26.2 27.3 24.8 28.4 24.5 24.0Ash 62.0 60.8 63.3 58.7 73.1 66.0Aflatoxins (mg kg−1)d 44 57 51 46 54 63

a DM is dry matter, MEN is nitrogen corrected metabolizable energy, CP is crude protein, CF is crude fibre, EE is ether extractand NFE is nitrogen free extract. MEN was calculated from the regression equations (NRC, 1994).

b DEB = dietary electrolyte balance calculated as [mequiv. kg−1 (Na + K − Cl)] described by Mushtaq et al. (2005).c Digestible amino acids were calculated using AminoDat 2 (Degussa AG, Germany) based on the analyzed DM and CP contents

of each ingredient. Digestible amino acids met or exceeded the ideal amino acid ratio (Baker and Han, 1994).d Aflatoxins were analyzed by VICAM Series-4 Flourimeter (Watertown, MA 02472).

where (N/Cr2O3)diet is the ratio of N to Cr2O3 in diet and (N/Cr2O3)digesta is the ratio of N to Cr2O3 indigesta.

2.5. Experimental design and data analyses

The experiment was conducted using completely randomized design with factorial struc-ture. Pen mean was an experimental unit. Effect of enzyme addition (with and without), SFM(200 and 300 g kg−1) and digestible lysine (8.0, 9.0 and 10.0 g kg−1) were statistically analyzedas 2 × 2 × 3 factorial design by GLM method of ANOVA using Minitab 14.1 (Minitab Inc., StateCollege, PA). All the linear and quadratic terms of main as well as interaction effects wereincluded in the model (Mead et al., 1993). The level of significance was 0.05 unless or otherwisestated.

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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3. Results

The analyzed nutrient composition is presented in Table 2. The aflatoxin contents ranged from44 to 63 �g kg−1 (Table 2). The analyzed enzyme activity was 3.91 �mol min−1 mg−1 for xylanase(1955 IU kg−1 diet).

3.1. Performance during 1–21 d

The BW gain and gain:feed were improved linearly with increasing digestible lysine of the diets(Table 3). A significant enzyme × SFM interaction was observed for BW gain at 1–21 d (Table 4). Animprovement in BW gain and gain:feed was observed in enzyme supplemented diets at 300 g SFM kg−1

over the diet without enzyme at similar level of SFM whereas it depressed the BW gain at 200 g ofdietary SFM kg−1. No effect of enzyme × digestible lysine was noted on BW gain, feed intake, gain:feedor mortality. Adding digestible lysine significantly increased BW gain and gain:feed with both levelsof SFM. No effect of enzyme supplementation, levels of SFM or digestible lysine was observed on feedintake or mortality during 1–21 d. Enzyme × SFM × digestible lysine was also non-significant for anyresponse during 1–21 d.

3.2. Performance during 1–42 d

Enzyme supplementation during 1–42 d decreased the feed intake (3567 vs. 3408) and improvedgain:feed (432 vs. 398 g). Increase in dietary digestible lysine from 8.0 to 9.0 g kg−1 significantlyimproved BW gain and gain:feed (Table 4). Increase in dietary lysine to 10.0 g kg−1 did not resultin further improvement in BW gain or gain:feed (Table 3). Enzyme supplementation, levels of SFMand digestible lysine or their interactions were remained non-significant for any of the performanceresponses.

3.3. Carcass responses

Dressing percentage, breast, leg and abdominal fat weights are presented in Table 5. A depression inbreast weight noted with 300 g SFM or 8.0 g digestible lysine kg−1 was improved by enzyme addition(significant effects of enzyme × SFM, and enzyme × digestible lysine; Table 6). Dressing percent, legand abdominal fat weights were not affected by enzyme, SFM, digestible lysine or their interactions.

3.4. Digestibility

The AME determined by ileal digestibility method was not significantly different among the dietarytreatments. A significant effect of enzyme × digestible lysine was observed for CTTADN where increas-ing digestible lysine decreased CTTADN in the absence but not in the presence of enzyme (Table 5).Similarly, N was utilized efficiently when high lysine (10.0 g kg−1) was used with 300 g SFM kg−1.

4. Discussion

Although a substantial amount of fibre is needed for normal digestive function but still high fibreingredients are avoided in poultry diets mainly because of their low energy values. The acceptablerange of dietary CF is 45–55 g kg−1 in practical broiler diets. The crude fibre of SFM, depending on theextent of dehulling, appears to be the most crucial aspect in chicks’ diets (Senkoylu and Dale, 2006). Aninclusion level of 300 g SFM kg−1 in the experimental diets led to high dietary crude fibre (CF; Table 2).The high level of fibre is always associated with slow passage rate for feed in the digestive system(Connell, 1981) which may depressed performance of the birds. However, 10.0 g increase in dietaryCF kg−1 of diet by increasing dietary SFM to 300 g did not affect the BW gain, feed intake or gain:feedduring 1–21 d or 1–42 d in the present study (Table 3). El-Sherif et al. (1995, 1996) demonstratedthat 75% of SBM could be replaced with solvent-extracted SFM without adverse effects on BW gainor growth rate. Similarly Furlan et al. (2001) reported that dietary SFM at 150 g kg−1 could be fed to

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Table 3Effect of supplementation of exogenous multi-enzymes preparation on BW gain, feed intake, feed:gain and mortality of broilers during 1–21 d and 1–42 d of age fed sunflower meal baseddiets containing various digestible lysine levels

Itemsa 1–21 d 1–42 d

Enzyme SFM Lysineb BW gain (g) Feed intake (g) Gain:feed (g:kg) Mortality (%) BW gain (g) Feed intake (g) Gain:feed (g:kg) Mortality (%)

No 428 943 454 1.42 1417 3567 398 2.37Yes 427 938 456 0.98 1427 3408 421 2.36SEM 4.4 9.3 5.7 0.318 15.5 45.7 6.2 0.489

200 429 931 462 1.06 1416 3421 415 2.28300 425 949 449 1.06 1428 3553 404 2.45SEM 4.4 9.3 5.7 0.318 15.5 45.7 6.2 0.489

8.0 404 940 430 0.98 1358 3511 387 2.209.0 420 925 456 1.46 1435 3416 421 2.0710.0 458 957 480 0.74 1474 3533 420 2.82SEM 5.4 11.4 6.9 0.390 19.0 56.0 7.6 0.599

No 200 439 939 468 1.31 1426 3508 408 2.45300 417 946 441 0.98 1408 3626 387 2.28

Yes 200 420 924 457 0.81 1406 3334 423 2.11300 434 953 456 1.14 1448 3481 419 2.61

SEM 6.2 13.1 8.0 0.450 22.0 64.6 8.8 0.692

No 8.0 401 942 426 1.71 1322 3550 373 2.699.0 417 940 444 0.74 1422 3482 409 1.2310.0 466 946 493 0.98 1507 3668 412 3.19

Yes 8.0 408 937 435 0.25 1395 3473 401 1.729.0 423 910 467 2.19 1447 3351 434 2.9210.0 450 968 466 0.49 1441 3399 428 2.45

SEM 7.6 16.1 9.8 0.551 26.9 79.1 10.8 0.848

200 8.0 400 947 421 1.23 1332 3494 381 2.709.0 431 907 461 1.46 1449 3346 434 1.9510.0 468 942 496 0.49 1468 3424 430 2.21

300 8.0 410 933 440 0.73 1384 3530 393 1.719.0 418 945 443 1.47 1420 3488 409 2.2010.0 449 968 463 0.98 1480 3643 410 2.73

SEM 7.6 16.1 9.8 0.551 26.9 79.1 10.8 0.848

a Means of 4 replicate having 51 birds at placement per enzyme × sunflower meal × digestible lysine combination.b The digestible lysine follows AA ratio (Baker and Han, 1994).

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Table 4Analysis of variance summary of the effects of supplementation of exogenous enzyme, sunflower meal (SFM), and digestible lysine on chicken performance during 1–21 and 1–42 d of age

Items d.f. 1–21 d 1–42 d

BW gain Feed intake Gain:feed Mortality BW gain Feed intake Gain:feed Mortality

Enzyme 1 0.885 0.735 0.729 0.713 0.626 0.019 0.012 0.994SFM 1 0.518 0.179 0.116 0.994 0.588 0.048 0.205 0.813Lysine 2 <0.001 0.146 <0.001 0.414 <0.001 0.304 0.002 0.647

LysineL 1 <0.001 0.297 <0.001 0.662 <0.001 0.783 0.003 0.473LysineQ 1 0.102 0.095 0.880 0.212 0.425 0.131 0.036 0.555

Enzyme × SFM 1 0.008 0.402 0.048 0.464 0.187 0.822 0.416 0.635Enzyme × lysine 2 0.250 0.288 0.044 0.056 0.045 0.461 0.605 0.232

Enzyme × lysineL 1 0.146 0.410 0.043 0.382 0.015 0.230 0.320 0.888Enzyme × lysineQ 1 0.415 0.179 0.072 0.055 0.650 0.761 0.994 0.091

SFM × lysine 2 0.156 0.210 0.013 0.674 0.338 0.517 0.159 0.464SFM × lysineL 1 0.056 0.193 0.008 0.378 0.466 0.256 0.115 0.201SFM × lysineQ 1 0.955 0.228 0.189 0.986 0.201 0.911 0.269 0.928

Enzyme × SFM × lysine 2 0.742 0.993 0.699 0.876 0.775 0.848 0.850 0.708Enzyme × SFM × lysineL 1 0.913 0.908 1.000 0.662 0.589 0.624 0.950 0.476Enzyme × SFM × lysineQ 1 0.447 0.983 0.401 0.789 0.644 0.772 0.574 0.675

Error 36Total 48

The values in this table are P values.L & Q are linear and quadratic effects, respectively.

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Table 5Effect of enzyme supplementation, sunflower meal (SFM) and digestible lysine on carcass, breast, legs, abdominal fat weights,apparent metabolizable energy (AME) and coefficient of total tract apparent digestibility of nitrogen (CTTADN) of broiler chickensat 43 d of age

Itemsa Carcassweightb

Breastweightc

Legweightc

Abdominalfatc

AME(kcal kg−1)

CTTADN

Enzyme SFM Lysined

No 633.5 317.8 400.1 0.18 11.28 0.87Yes 634.3 325.4 413.0 0.16 11.30 0.88SEM 6.71 4.11 8.15 0.010 0.090 0.003

200 628.3 321.2 411.6 0.17 11.26 0.86300 639.5 322.1 402.6 0.17 11.32 0.89SEM 6.71 4.11 8.15 0.010 0.090 0.003

8.0 635.8 312.0 411.1 0.17 11.29 0.889.0 629.3 325.6 401.0 0.18 11.40 0.8910.0 632.4 327.4 408.6 0.16 11.18 0.86SEM 8.15 5.06 10.04 0.013 0.111 0.004

No 200 628.9 323.7 411.6 0.19 11.26 0.86300 638.2 312.0 390.6 0.17 11.30 0.89

Yes 200 620.2 318.6 411.6 0.15 11.26 0.86300 640.8 332.2 414.4 0.16 11.35 0.89

SEM 9.49 5.91 12.27 0.015 0.128 0.005

No 8.0 631.6 291.8 408.6 0.12 11.23 0.899.0 642.0 337.8 386.3 0.19 11.36 0.8810.0 627.1 328.1 407.7 0.17 11.25 0.86

Yes 8.0 639.9 332.1 413.6 0.17 11.35 0.879.0 630.4 317.4 415.6 0.16 11.44 0.8910.0 637.8 326.8 409.6 0.15 11.12 0.87

SEM 11.61 7.20 14.70 0.019 0.156 0.006

200 8.0 630.4 308.0 416.9 0.16 11.08 0.899.0 625.3 324.9 416.8 0.19 11.53 0.8710.0 624.2 330.7 401.4 0.16 11.18 0.83

300 8.0 641.2 316.0 405.4 0.18 11.50 0.879.0 636.6 326.3 395.5 0.17 11.28 0.9110.0 640.7 324.1 416.0 0.15 11.19 0.90

SEM 11.61 7.20 14.70 0.019 0.156 0.006

a Means of 8 samples for carcass responses and 4 for digestibility per enzyme × sunflower meal × digestible lysine combina-tion.

b g kg−1 of live weight.c g kg−1 of carcass weight.d The digestible lysine follows AA ratio (Baker and Han, 1994).

broilers from day of hatch with no adverse effects on growth and feed utilization in isoenergetic andisoaminoacid diets. Similarly, Mushtaq et al. (2006) did not report any harmful effects of adding SFMup to 300 g kg−1 in young chicks of 14 d age.

Diets formulated with higher levels of SFM may be severely deficient in lysine for broilers. Thedeficiency of lysine in high SFM diets could be overcome by supplementing with suitable lysine source.In present study, digestible lysine linearly improved BW gain and gain:feed during 1–21 d and 1–42 d.The results were consistent with those reported by Knowles and Southern (1998) who estimated10.0 and 11.0 g digestible lysine kg−1 of diet for maximum daily BW gain and gain:feed, respectively.The NRC (1994) recommended a level of 11.0 g lysine kg−1 of diet for broiler of 0–21 d of age (9.7 gdigestible lysine kg−1 assuming 0.88 digestibility in a maize-soy diet). Similarly, Han and Baker (1991)estimated the digestible lysine requirement of 0–3 weeks old chicks as 10.1 g kg−1 for maximum BWgain and feed intake and 12.1 g kg−1 for maximum feed efficiency. Also, total lysine recommendationof 11.5 g (10.2 g digestible lysine) and 12.6 g (11.2 g digestible lysine) per kg of diet have been reportedfor BW gain and feed efficiency, respectively (Baker and Han, 1994). The results of the present studydemonstrated similar lysine requirements for gain:feed and for BW gain during 1–21 d and 1–42 d. As

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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Table 6Analysis of variance summary of the effects of supplementation of exogenous enzyme, sunflower meal (SFM), and digestiblelysine on carcass, breast, legs, abdominal fat weights, apparent metabolizable energy (AME) and coefficient of total tract apparentdigestibility of nitrogen (CTTADN) of broiler chickens at 43 d of age

Items d.f. Dressing weight Breast weight Leg weight Abdominal fat AME CTTADN

Enzyme 1 0.632 0.179 0.260 0.125 0.873 0.853SFM 1 0.051 0.895 0.368 0.961 0.649 <0.001Lysine 2 0.590 0.061 0.777 0.658 0.394 0.001

LysineL 1 0.974 0.051 0.742 0.690 0.509 0.011LysineQ 1 0.307 0.348 0.530 0.412 0.235 0.004

Enzyme × SFM 1 0.572 0.033 0.220 0.556 0.876 0.139Enzyme × lysine 2 0.136 <0.001 0.662 0.766 0.649 0.002

Enzyme × lysineL 1 0.919 0.003 0.869 0.577 0.423 0.003Enzyme × lysineQ 1 0.057 0.005 0.373 0.640 0.731 0.033

SFM × lysine 2 0.870 0.552 0.358 0.421 0.122 <0.001SFM × lysineL 1 0.646 0.280 0.446 0.426 0.199 <0.001SFM × lysineQ 1 0.797 0.915 0.217 0.296 0.093 0.108

Enzyme × SFM × lysine 2 0.307 0.803 0.548 0.441 0.533 0.190Enzyme × SFM × lysineL 1 0.129 0.509 0.977 0.256 0.485 0.582Enzyme × SFM × lysineQ 1 0.850 0.976 0.275 0.561 0.383 0.084

Error 84 36 36Total 96 48 48

The values in this table are P values.L & Q are linear and quadratic effects, respectively.

the experimental diets were high in fibre (Table 2), which may cause abrasion of the intestinal mucosaand thus increase the AA requirements for the synthesis of mucosa cells (Parsons et al., 1983), it seemsthat AA requirements may be lowered in low nutrient density diets with low fibre contents.

Feed consumption was increased when dietary SFM was increased from 200 to 300 g kg−1 of dietduring 1–42 d in present study. Feed consumption in poultry is regulated to maintain constant caloricintake (Hill and Danski, 1954). If low energy diets are offered for birds, their feed intake is increased(Vieira et al., 1992). The high CF contents in simple stomach animals is generally laxative in nature andmay impair nutrient digestion due to high passage rate in the digestive tract. As the energy contentswere similar across the diets in the present diet, the increased feed intake may be due to the highdietary CF contents although the CTTADN were high in diets having 300 g kg−1. However, the fibre mayhave effects on the digestibility of other dietary components and this need further investigations.

The two unfavorable factors against SFM are the high fibre content and the accompanying low MEcontent, and addition of an enzyme mixture may facilitate the digestibility of arabinoxylan, arabi-nolactan, galactomanan and pectin substances in such diets and can increase BW and reduced feedcost per kg gain. In present study, enzyme supplementation had a negative effect on gain:feed at200 g SFM kg−1 of diet for 1–21 d (2.16 vs. 2.21). An improvement in gain:feed was observed during1–42 d by the enzyme supplementation. The results were in agreement with the Meng and Slominski(2005) who supplemented a cocktail of xylanase, glucanase, pectinase, cellulase, mannanase, andgalactanase, and observed improved gain:feed in broilers.

No effect of enzyme and SFM on AME was observed during the present study. The CTTADN wasimproved by 300 g SFM kg−1 of diet. Kocher et al. (2000) reported an improvement in NSP digestibilityat jejunum, and that of protein at ileal levels of birds fed SFM without any improvement in growth.Meng and Slominski (2005) and Tabook et al. (2006) have indicated that the addition of commerciallyavailable multiactivity enzyme products did not result in improved broiler performance in diets espe-cially having increased concentration of SFM (Irish and Balnave, 1993; Rebolé et al., 1999). Similarly,Wang et al. (2005) reported an increased apparent N digestibility and no effect on AME of the wheat-based diet when various levels of xylanase and �-glucanase were supplemented in wheat-based diets.However, they observed an improvement in the live performance with the increasing levels of enzymecombination. This may be due to high concentration of arabioxylans in wheat as compared to SFM. Theimproved CTTADN by SFM may be due to the high oil contents of the diets. Soybean oil supplementationhas been reported to increase the retention of dry matter, crude protein, ether extract, crude fibre, ash,

Please cite this article in press as: Mushtaq, T., et al., Influence of sunflower meal baseddiets supplemented with exogenous enzyme and digestible lysine on performance,digestibility. . . . Anim. Feed Sci. Technol. (2008), doi:10.1016/j.anifeedsci.2008.06.008

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organic matter and N-free extract (Azman and Seven, 2005). Considering diets with the same nutritivevalues, birds fed with rations containing oil present better performance than birds fed no oil (Baiãoand Lara, 2005). Similarly, an improved performance and nutrient utilization has been observed whencanola oil was added in diets having high canola meal (Meng et al., 2006).

Breast meat was improved when enzyme was supplemented with 300 g SFM kg−1 of diet. Also,enzyme addition at 8.0 g digestible lysine kg−1 of diet improved the breast meat. Carcass weight,breast, legs or abdominal fat was not affected by other dietary treatments (Table 5). Furthermore, bysplitting SFM cell walls, the enzyme product would be expected to give a better protein digestibility andthus greater breast meat yield. This was validated by an improvement in CTTADN by enzyme addition(Table 5) which in turn might improve the breast yield. NRC (1994) demonstrated a positive effect oflysine in breast meat of broilers. An improvement in breast yield has been observed by increasing sulfur-containing AA of the diets (Schutte and Pack, 1995). Wijtten et al. (2004) observed an improvement incarcass characteristics when lysine was increased with ideal protein concept rather increase in lysinealone.

No effects of SFM, enzyme addition or increasing levels of digestible lysine were noted on any of theother carcass characteristics in the present study. Urdaneta-Rincon and Leeson (2004) reported thatboth protein synthesis and breakdown increased at dietary lysine and CP levels above those requiredfor maximum growth. Similarly, Quentin et al. (2005) observed a linear effect of lysine on breastyield up to 7.6 g kg−1 of diet and not above that level in slow growing broilers. Corzo et al. (2006)observed a linear response of lysine on carcass and fillet weights in male broilers whereas the lackof lysine response was observed in female broilers during 42–56 d of age. The difference in carcassresponse to digestible lysine may be due to the difference in dietary structures, ME levels of the diets,environmental and other experimental structure. However, effect of other AA with increasing lysineon carcass characteristics is suggested in this regard.

5. Conclusions

Enzyme supplementation has a pronounced effect in low nutrient concentration and high SFM diets.The digestible lysine may not be reduced below than 10.0 g during 1–21 d but it could be reduced to9.0 g kg−1 diet if a single diet is fed during 1–42 d when diets have substantially high SFM.

Acknowledgements

The study was funded by the Higher Education Commission of Pakistan under title “Merit Scholar-ship for PhD Studies in Science and Technology”. Appreciation is expressed to Sadiq Brothers Poultry,Islamabad, Pakistan for providing material support to this study. The authors are grateful to Dr. Mahr-un-Nisa for his support during the course of studies.

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