EFFECT OF FEEDING ORGANIC ZINC (Zn) AND SELENIUM (Se) ON THE PERFORMANCE OF MALE PARENTAL LINES OF GIRIRAJA AND SWARNADHARA VITTAL SUBHAS.

Transcription

1 EFFECT OF FEEDING ORGANIC ZINC (Zn) AND SELENIUM (Se) ON THE PERFORMANCE OF MALE PARENTAL LINES OF GIRIRAJA AND SWARNADHARA VITTAL SUBHAS. KOUJALAGI DEPARTMENT OF POULTRY SCIENCE VETERINARY COLLEGE, BANGALORE KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR SEPTEMBER, 2012 i

2 EFFECT OF FEEDING ORGANIC ZINC (Zn) AND SELENIUM (Se) ON THE PERFORMANCE OF MALE PARENTAL LINES OF GIRIRAJA AND SWARNADHARA Thesis submitted to the KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR In partial fulfilment of the requirements for the award of the degree of MASTER OF VETERINARY SCIENCE in POULTRY SCIENCE By VITTAL SUBHAS. KOUJALAGI DEPARTMENT OF POULTRY SCIENCE VETERINARY COLLEGE, BANGALORE KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR SEPTEMBER, 2012 ii

3 KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR DEPARTMENT OF POULTRY SCIENCE VETERINARY COLLEGE, BANGALORE CERTIFICATE This is to certify that the thesis entitled EFFECT OF FEEDING ORGANIC ZINC (Zn) AND SELENIUM (Se) ON THE PERFORMANCE OF MALE PARENTAL LINES OF GIRIRAJA AND SWARNADHARA submitted by Mr. VITTAL SUHAS. KOUJALAGI, I.D. No. MVHK 939 in partial fulfillment of the requirements for the award of MASTER OF VETERINARY SCIENCE in POULTRY SCIENCE of the Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar is a record of bonafide research work carried out by him during the period of his study in this University under my guidance and supervision, and the thesis has not previously formed the basis for the award of any degree, diploma, associate ship, fellowship or other similar titles. Bangalore, September, 2012 Dr. T. Munegowda Associate Professor Dept. of Poultry Science Approved by: Chairman : Dr. T. MUNEGOWDA Members : 1. Dr. H.N. NARASIMHA MURTHY 2. Dr. JAYANAIK 3. Dr. B. UMAKANTHA 4. Dr. M. NARAYANA SWAMY iii

4 Affectionately Dedicated To, My Beloved Parents Brother, Sisters and Poorva iv

5 ACKNOWLEDGEMENTS I place on the record of my deep sense of gratitude with heartfelt respects to Dr. T. Munegowda, Associate Professor, Department of Poultry Science, Veterinary College, Hebbal, Bangalore and chairperson of my Advisory committee, for his expert guidance, constant encouragement, kindness and keen interest throughout the period of study. I remain grateful to him for having taken great pains during preparation of this thesis. It s my immense pleasure to express my deep sense of gratitude to Dr. Venkat Reddy, Professor and Head, Department of Poultry Science, Veterinary College, Hebbal, Bangalore, for his valuable suggestions and timely help during the course of my experiment. It is indeed, an immense pleasure to express my sense of gratitude and indebtedness to Dr. B. Umakantha Associate Professor, Department of Poultry Science, Veterinary College, Hebbal, Bangalore, for his guidance, constant encouragement, constructive suggestions and timely help during the time of my whole PG life. I also express my sincere thanks to Dr. H. N. Narasimha Murthy, Dr. Jayanaik, Professor, Department of Poultry Science and Dr. M. Narayana Swamy, Professor and Head, Department of Physiology, Veterinary College, Hebbal, Bangalore, for having served as members of my advisory committee and for their valuable guidance and suggestions in carrying out this investigation and for the critical scrutiny of manuscript. I also extend my sincere thanks to Dr. Ruben, Dr. Naveen and Dr. Nithin Prabhu, Teaching faculty, Veterinary College Hassan, for their valuable suggestions and guidance during the course of my study. I would like to offer my special thanks to Provimi Animal Nutrition India Pvt. Ltd., Bangalore and Pristine Organics Pvt. Ltd., Bangalore for free supply of Organic Zinc and Selenium for the trials. I express my indebtedness to my mother Manjula, father Subhas B. Koujalagi, my brother Viranna and my sisters Aruna and Pratibha for their patience, moral support, encouragement and cooperation, without which it could not have been possible for me to attain this stage. I feel inadequacy of words to express my heartfelt thanks to my friends Drs. Sudharshan, Sandip, Vinod, Prasad Nagpure, Pradeep Bawane, Sudhir, Suttu, Bharamappa sir and Anil, for there untiring help and moral support throughout the pursuit of this trial. I express my regards to my seniors, Dr. Satish K. G., Dr. Rajkumar, Dr. Shashidhar M. D., Dr. Venugopal, Dr. Mahesh Patil and Dr. Vishal, for their timely help, guidance and encouragement. v

6 My sincere thanks to my friends Naveen, Raghu, Shivaprasad, Ranganath for their immense help and support during the course of my study. I extend my indebtedness to my Parents, Sisters and my brother for their moral support and help during the time of my whole PG life in Bangalore. I extend my sincere thanks to my senior colleagues of our Department Drs. Mahesh Patil, Sreekanta, Prabhudeva, Shivappa Nayak and my junior colleagues Shashi, Chethan, Rajesh and Dawood for their immense support during the course of my study. My sincere thanks to my junior friends Shravan, Suryakanth, Pampa And all others, for their immense help and support during the course of my study. My sincere thanks are also due to Mahesh, Siddegowdru, Krishnamurhy, Murthy, Shankar, Ramchandra and Ms. Radha and employees of our Department for their help and co-operation during my course of my study. At last I submit my record of gratitude to all the persons who have helped me in one or the other way for the successful completion of this piece of work. Any unintentional omission in this brief acknowledgement does not mean lack of gratitude. Bangalore September, 2012 (VITTAL SUBHAS. KOUJALAGI) vi

7 CONTENTS CHAPTER TITLE PAGE No. I INTRODUCTION 1-3 II REVIEW OF LITERATURE 4-24 III MATERIALS AND METHODS IV RESULTS V DISCUSSION VI SUMMARY VII BIBLIOGRAPHY VIII ABSTRACT 117 IX APPENDICES vii

8 LIST OF TABLES Table No. Title 3.1 Experimental Design Percent ingredient composition of the experimental diet Calculated nutrient composition of experimental diets Effect of Feeding Inorganic and Organic Zinc and Selenium on Hen Day Egg Production of birds of Different Treatment Groups Effect of inorganic and organic source of Zn and Se on hen housed egg production of birds of different treatment groups Effect of Feeding Inorganic and Organic Zinc and Selenium on Body Weight Gain of birds of Different Treatment Groups Effect of Feeding Inorganic and Organic Zinc and Selenium on Feed Consumption of birds of Different Treatment Effect of Feeding Inorganic and Organic Zinc and Selenium on Survivability of birds of Different Treatment Groups Effect of inorganic and organic source of Zn and Se on fertility percentage of eggs of birds of different treatment groups Effect of Feeding Inorganic and Organic Zinc and Selenium on Hatchability percentage on Total Eggs Set of birds of Different Treatment Groups Effect of Feeding Inorganic and Organic Zinc and Selenium on Hatchability percentage on Fertile Eggs of birds of Different Treatment Groups Effect of inorganic and organic source of Zn and Se on Egg weight of birds of different treatment group Effect of inorganic and organic source of Zn and Se on shape index of eggs of birds of different treatment group Effect of inorganic and organic source of Zn and Se Albumen index of eggs of birds of different treatment group Effect of inorganic and organic source of Zn and Se yolk colour score of eggs of birds of different treatment group Effect of inorganic and organic source of Zn and Se yolk index of eggs of birds of different treatment group Effect of inorganic and organic source of Zn and Se shell thickness of eggs of birds of different treatment group Effect of inorganic and organic source of Zn and Se on Net profit of birds of different treatment groups Page No viii

9 LIST OF FIGURES Figure No Title Effect of inorganic and organic source of Zn and Se on hen day egg production of different treatment groups Effect of inorganic and organic source of Zn and Se on hen housed egg production of different treatment groups Effect of inorganic and organic source of Zn and Se on Body Weight Gain of birds of different treatment groups Effect of inorganic and organic source of Zn and Se on Feed Consumption of birds of different treatment groups Effect of inorganic and organic source of Zn and Se on Survivability of birds of different treatment groups Effect of inorganic and organic source of Zn and Se on fertility percentage of eggs of birds of different treatment groups Effect of inorganic and organic source of Zn and Se on hatchability percentage of total eggs set of birds of different treatment groups Effect inorganic and organic source of Zn and Se on hatchability on fertile egg set of different treatment groups Effect of inorganic and organic source of Zn and Se Egg weight of hens of different treatment group Effect of inorganic and organic source of Zn and Se Egg shape index of eggs of hens of different treatment group Effect of inorganic and organic source of Zn and Se Albumen index of eggs of hens of different treatment group Effect of inorganic and organic source of Zn and Se yolk colour score of eggs of hens of different treatment group Effect of inorganic and organic source of Zn and Se yolk index of eggs of hens of different treatment groups Effect of inorganic and organic source of Zn and Se shell thickness of eggs of hens of different treatment groups Effect of inorganic and organic source of Zn and Se on Net profit of hens of different treatment groups Page No ix

10 LIST OF PLATES Plate No. Title Page No. 3.1.A Mass candling of eggs B Setting of eggs in incubator A Scoring of yolk colour by Roche yolk colour fan B Measuring the height of yolk by using Ames Haugh unit spherometer A Measuring the width of albumen by using vernier caliper B Measuring the width of yolk by using vernier caliper 36 x

11 LIST OF ABBREVIATION AI ANOVA BW BIS o C Cr Cu df DCP DM Fig g GSH-Px HDEP HHEP h HU Kcal Kg Mn MnAA MSS Albumin Index Analysis of Variance Body Weight Bureau of Indian Standards Degree(s) centigrade Chromium Copper Degree s of Freedom Dicalcium Phosphate Dry Matter Figure gram Glutathione peroxidase Hen day egg production Hen housed egg production Hour Haugh unit Kilocalorie Kilogram Manganese Manganese amino Acid Mean sum of square xi

12 ME mg mm ppm Metabolizable energy Milligram Millimeter Parts per million % Percentage P Period ± Plus or minus SE Se SeM SI SY YCS YI Zn ZnAA Zn met ZnO ZnSO 4 Standard Error Selenium Selenomethionine Shape Index Seleno-yeast Yolk color score Yolk Index Zinc Zinc Amino Acid Zinc methionine Zinc oxide Zinc Sulphate xii

13 Introduction

14 I. INTRODUCTION Indian poultry industry has remarkably progressed from backyard activity to an organized scientific and vibrant industry. During the past two decades the poultry industry has been one of the most dynamic and ever expanding sectors in the world. In poultry farming, feed alone accounts for more than 60 to 70 per cent of production cost. In order to reduce the cost of feeding by means of better feed conversion ratio, pellet feeding, automatic feeding systems and purchase of feed ingredients of high quality is a must. In the compound livestock feed manufacturing along with major feed ingredients, feed additives also play an important role. These feed additives include vitamins, minerals, antibiotics etc. Among minerals, trace minerals are essential in all animals including poultry for wide variety of physiological processes. Hence, trace minerals are essential in poultry diets as they participate in biochemical processes required for normal growth and development. Several hundred enzymes require the presence of one or more trace minerals for their activity. As such, most diets are supplemented with inorganic and/ or organic forms of trace minerals. Inorganic trace minerals such as sulfates and oxides form the bulk of trace minerals supplementation, but these forms are more prone to dietary and environmental antagonisms and under such circumstances they are less bioavaliable than organic trace minerals. Indeed, use of organic trace minerals has shown to enhance mineral uptake and reduce mineral excretion. Therefore, there is currently an increasing interest in studying factors that improve absorption and metabolization of these trace elements. Organic minerals have also been shown to improve egg production,

15 2 reproductive performance and decrease mortality and stress, as well as chelated minerals can potentially save resources and reduce pollution, which enhances utilization efficiency (Downs et al., 2000 and Guo et al., 2001). The supplementation of parent stock s feed with micro-nutrients like essential vitamins, trace elements and substances like antioxidants, coccidiostats and organic acids are essential for maximum hatching egg production and hatchability. Use of organic trace minerals in broiler feeds had shown promising results in improving live performance, bird s health, processing yield and meat quality characteristics. The most commonly used organically-complexed minerals include zinc, manganese, selenium, copper and iron. Organic trace minerals particularly zinc, manganese and selenium, show improved bioavailability over inorganic sources for commercial poultry and are being used to improve health and processing performance in broilers and breeders. Interest is also building up in using organic trace minerals to substitute inorganic mineral supplements in the feed in order to get maximum growth and health with lower levels of mineral intake, thus lowering the amount of minerals excreted from the birds (Bao et al., 2005). Zinc plays an important role in poultry, particularly for layers, as a component of a number of metalloenzymes such as carbonic anhydrase which is essential for eggshell formation in the hen s shell gland. Other important zinc metalloenzymes in the hen include carboxypeptidases and DNA polymerases. These enzymes play important role in

16 3 hormone production (testosterone and corticosteroids). Classic deficiency symptoms of zinc in poultry could include poor feathering, infertility and poor shell quality. Selenium (Se) is an essential trace element that was discovered in 1817 by Berzelius in Sweden. Selenium (Se) is an essential micronutrient required for normal growth and maintenance in poultry. In poultry females selenium deficiency was shown to decrease egg production and hatchability (Cantor and Scott, 1974). Trace mineral supplementation may improve eggshell quality and egg production. Fakler et al. (2002) reported that the production of marketable eggs and egg efficiency were improved in hens supplemented with organic Zn. Khajaren et al. (2002) reported that organic Zn and Mn improved egg production, egg quality, and shell quality in hens. Hudson et al. (2004) reported that hens diets supplemented with a combination of ZnAA and ZnSO 4 had greater settable egg production, highest hen day egg production, improved specific gravity and enhanced egg shell quality. Hence, the present study has been designed with the following objectives: 1. To evaluate the performance of male parental lines of Giriraja and Swarnadhara fed with organic zinc and selenium. 2. To determine the economic impact of feeding organic zinc and selenium in the diets of male parental lines of Giriraja and Swarnadhara.

17 Review of Literature

18 II. REVIEW OF LITERATURE Most of the minerals are added to the poultry feed in an inorganic form, such as sulphates and oxides. The problem with these minerals is that they dissociate during digestion to form cations (positively charged) and anions (negatively charged). Being positively and negatively charged, these chemicals interact with other minerals in the gut, which decreases their absorption and availability into the bird s bloodstream. On the other hand, organic minerals are neutrally charged, so they do not dissociate and there is no interaction with other minerals. Since, these organic minerals are mostly chelated to amino acids and/or short chain peptides, they are directly available for absorption; in addition, less minerals are excreted into the environment. 2.1 Effects of chelated mineral supplementation in poultry diets The minerals are protected in the form of metal aminoacid chelates, metal proteinate and metal polysaccharide complexes. The protected minerals may be more available than inorganic minerals and may not react with digesta due to their both chemical (electrically neutral, ligand and metal make up) and physical structures (size and ligand source). Mineral proteinate usually contain aminoacids, dipeptides, tripeptides or proteins, and are thought to enhance digestibility and availability (90 to 95 per cent) of the mineral sequestered by the ligand. Improved digestibility may be due to better solubilization, greater stability in the lumen and/or perhaps the ligand serves as an efficient carrier for the mineral across the brush border than those provided by inorganic salts (Wedekind et al., 1992). They also provide readily bioavaliable amino acids (Aoyagi and Baker, 1993). These metal complexes may improve egg production and

19 5 decrease mortality and stress, as well as reduce the excretion of potentially contaminant minerals in environment, as they are absorbed and retained in the poultry body. Chelated mineral can potentially save resources and reduce pollution, which enhances utilization efficiency (Downs et al., 2000 and Guo et al., 2001). Proteinate, therefore seem to be an ideal choice in formulating diets containing minimum levels of trace minerals. 2.2 Bioavailability of trace minerals The trace minerals such as selenium and zinc are important in poultry diets for growth, bone development, enzyme structure and function and eggshell formation. Zinc involved in the production, storage and secretion of reproductive hormones and affects receptor sites (McDowell, 1992). Commercial corn-soybean diet may not provide enough trace minerals to meet the requirements for birds because of non availability of the phytase enzyme to break the phytate bound minerals present in natural plant sources. Therefore, supplemental trace minerals such as Zn, Mn, and Cu are often added to poultry diets in the form of sulfates, oxides, chelates, proteinates and polysaccharides (Wedekind et al., 1992) Inorganic trace minerals Selenium Ort and Latshaw (1978) reported that supplementation of female chicken diet with sodium selenite (0, 0.1, 1.0, 3.0 and 5.0 ppm) had no effect on egg production, egg weight or fertility, but hatchability of fertile eggs decreased at 5.0 ppm level.

20 6 Todorovic et al. (1999) conducted study on broilers by supplementing sodium selenite at a level of 2, 5, 10, 20 and 30 mg/kg diet and reported 2 mg/kg had no influence on the live weight, 5 mg/kg decreased daily weight gain and 15, 20 and 30 mg/kg resulted in up to 80% mortality. Edens et al. (2001) reported no significant difference in body weight or feed efficiency in broiler chickens fed diets containing 0.2 ppm se from sodium selenite. Ryu et al. (2005) reported that feeding higher concentrations (1 to 8 ppm) of dietary Se from an inorganic source did not affect the BW of broilers Zinc Sandoval et al. (1997) reported that ZnO had a bioavailability ranging from 61 to 77 per cent relative to Zn sulfate. Batal et al. (2001) reported that the two feed grade sources of Zn widely used in industry were ZnO and Zn sulfate and their bioavailability were 72 and 36 per cent Zn, respectively Organic trace minerals Organic sources of trace minerals have been reported to have greater bioavailability due to the ability of organic compounds such as amino acids bind strongly to divalent minerals under physiological ph conditions (Kidd et al., 1996). This strong bond between the mineral and its organic carrier prevented phytate from binding to the metals in the gastrointestinal tract while still being water soluble, thus facilitating mineral

21 7 uptake into the mucosa of the small intestine (Cao et al., 2000). Organic trace minerals, particularly zinc, manganese and selenium, show improved bioavailability over inorganic sources for commercial poultry and are being used to improve health and processing performance in broilers and breeders Selenium inconsistent. Reports of organic mineral bioavailability in livestock and poultry are Cantor et al. (1982) reported that organic Se such as selenocysteine, selenomethionine (SM), or Se-enriched yeast (SY), as supplemental sources of Se is more bioavailable than Se in sodium selenite. Sevcikova et al. (2006) suggested Se-yeast is capable of increasing the activity of the selenoenzymes. Its bioavailability was found to be higher than that of inorganic sources. Yoon et al. (2007) suggested a higher bioavailability of Se from the organic source compared with inorganic sodium selenite and that the difference in bioavailability may exist between organic sources of Se Zinc Pimentel et al. (1991) reported that Zn source (ZnO or Zn-Methionine) had no effect on growth or tibia and liver. But the broilers fed with Zn-Met had higher levels of pancreatic Zn.

22 8 Wedekind et al. (1992) reported that the bioavailability of Zn-Met when compared to Zn sulfate was decreased from 206 to 117 per cent in a conventional cornsoybean diet versus a purified amino acid diet in broilers. They attributed this to the chelating effects of phytic acid in the more complex corn soybean diet and the ability of organic complexes to compete more effectively against phytic acid in the small intestine of the animal, while Zn present in Zn sulfate forms such a strong bond with phytic acid in the small intestine that it is unavailable to the animal for absorption. Rojas et al. (1995) showed increased bioavailability of Zn-lysine and Zn methionine, over inorganic Zn sources, when supplemented to sheep. Kidd et al. (1996) stated that Zn-Met differed from inorganic Zn, Zn proteinates, and Zn polysaccarides complexes because it was a specific amino acid complex. Cao et al. (2000) reported that only one commercial Zn proteinate product was more available than ZnSO4 with an RBV of 130 per cent. All other forms of organic Zn, Zn-Met, Zn-Lys, Zn polysaccharide, out of four Zn chelated products, and two other Zn proteinate products were not different than ZnSO 4 in bioavailability. 2.3 Growth performance Effect of Zinc and Selenium Mohanna and Nys (1999) reported that body weight gain, feed intake and feed conversion in broilers were not influenced by supplementation either with Zn sulfate or Zn-Met as a Zn source.

23 9 Swain et al. (2000) reported maximum daily weight gain and highest feed conversion in chickens that received 0.50 mg Se/ kg and 300 IU/ kg Vit E. Edens et al. (2001) reported no significant difference in body weight or feed efficiency in broiler chickens fed diets containing 0.2 ppm se from sodium selenite or selenium yeast. Hess et al. (2001) reported improvements in broiler growth rate and/or feed conversion with organic zinc sources. Spears et al. (2003) reported no difference in gain or feed conversion efficiency of broilers fed diets containing 0, 0.05, or 0.15 ppm Se from sodium selenite or selenomethionine. Choct et al. (2004) reported feed intake and mortality were not influenced by the level or source of selenium. Choct et al. (2004) reported that increased dietary selenium content markedly increased feed conversion ratio (FCR) as result of significantly lowered feed intake of birds while maintaining the similar weight gain. Bou et al. (2005) conducted a study on broilers by supplementing with Zn (0, 300, or 600 mg/kg), and Se (0 or 1.2 mg/kg as Se-enriched yeast) and reported that final body weight, feed intake, feed conversion and mortality were not affected by dietary treatment.

24 10 Papazyan et al. (2006) conducted a study on broilers by replacing sodium selenite with organic selenium in the form of Sel-Plex in their diet and observed better growth, improved FCR and decreased mortality. Fernandes et al. (2008) conducted a study to evaluate the effect of organic minerals (Zn, Mn and Se) at two different levels (0.250 and ppm). They observed uniform initial and final body weights in laying hens and found no influence on feed intake or feed conversion ratio. Shyam Sunder et al. (2008) reported that supplementing Zn at 10, 20, 40, 80, 160 or 320 ppm using ZnSO 4.7H 2 O had no influence on body weight gain, feed intake or feed efficiency. Maysa et al. (2009) reported live body weight significantly increased in females and males fed Sel-Plex supplementation, while no significant effect on feed consumption. Live body weight of females was significantly increased with increase of hens age, but no significant effect on feed consumption as the age of birds increased. El-Sheikh et al. (2010) showed that selenium supplementation as selenium yeast especially at 0.2 and 0.3 mg/ kg diet significantly (P 0.05) increased live body weight of Bandarah chicks during the experimental period. Also, live body weight of males was significantly (P 0.05) heavier than females. Osman et al. (2010) reported no significant effect in feed conversion values for hens fed diet containing 100 or 200mg/kg diet as Sel-plex during overall experimental periods.

25 Production performance Egg production Effect of Zinc and Selenium Holder and Huntley (1978) conducted an experiment to study the effects of dietary calcium level (2.5 or 3.5 per cent) and the addition of zinc (130 mg/kg) on the performance of Leghorn hens for 12 periods of 28 days. They reported non-significant differences in egg production due to added trace minerals or to calcium level. Kienholz (1992) showed that feeding zinc chelate to layers submitted to heat stress, associated with low calcium intake, and maintained egg size, whereas inorganic zinc supplementation reduced egg size. Balnave and Zhang (1993) compared the responses of laying hens on dietary supplementation with various zinc compounds viz. Zn-methionine (Zinpro 200; 0.5 g/kg), Zn sulfate (ZnSO 4.7H 2 O; 46 g/kg), or chelated Zn-EDTA (0.54 g/kg) to supply the same concentration of Zn (0.1 g/kg). They reported that the hens receiving the saline drinking water without any dietary Zn supplement produced significantly (P<0.05) more eggs with shell defects than hens receiving the town water. Abdallah et al. (1994) studied the effect of removing supplemented iron, copper, zinc, manganese or all of them from the diet of laying hens for 10 weeks and reported that the withdrawal of Zn, Fe and Cu had no effect on egg production. Lima et al. (2000) reported that the addition of Zn and Mn in the organic or inorganic form did not improve egg production.

26 12 Paik (2001) evaluated the utilization of organic zinc, copper and manganese, either individually or in combination, and observed improvement in productive performance of birds fed organic copper and the combination of these three metals in an organic complex. However, no performance improvement was observed with the use of either organic zinc or the combination of copper and zinc chelates. Edens (2002) conducted a study by feeding sodium selenite or selenomethionine at the level of 0.3 ppm and reported improved egg production, fertility, daily numbers of settable eggs and hatchability. Fakler et al. (2002) reported that the production of marketable eggs and egg efficiency were improved in hens supplemented with organic Zn. Guo et al. (2002) reported that providing diets with supplemental zinc (80 ppm) from a mixture of ZnAA and ZnSO4 rather than solely ZnSO4 reduced the incidence of cracked eggs in laying hens. Synergistic effects of ZnAA and ZnSO4 may enhance mechanisms involved in calcification of eggshells. Khajaren et al. (2002) studied the effect of zinc and manganese amino acid complexes on broiler breeder production and immunity and reported an improvement in egg production and number of settale eggs and egg quality, and shell quality in the second phase of egg production (38 to 65 wk of age) in layers fed Zn and Mn organic complexes. Klecker et al. (2002) reported that supplementation of Manganese and Zinc chelates in layer diets increased egg production.

27 13 Sahin et al. (2002) reported that layers fed supplemental Cr and Zn, either individually or together, had an increased egg production or improved feed efficiency. Hassan (2003) investigated some productive and physiological responses by using dietary zinc oxide (0, 5,000, 10,000 and 20,000 ppm) on laying hens and reported that adding 5,000, 10,000 and 20,000 ppm zinc oxide decreased the egg number and rate of egg production. Lim and Paik (2003) studied the effects of supplementary mineral methionine chelates each at the level of 100 ppm (Zn, Cu and Mn) on the performance and eggshell quality of brown shelled eggs from hens of 96 weeks old and reported that 100 ppm Cu in Cu - methionine chelate showed highest hen day and hen housed (72.57 per cent in both) egg production compared with control (67.13 and per cent). They also reported that soft shelled eggs were lowest in Cu - methionine chelate (0.311 per cent) while broken eggs were lowest in control (3.808 per cent). Egg weight was heaviest in Zn - methionine chelate (70.77 g) while lightest in Cu - methionine chelate (69.33 g). Hudson et al. (2004b) reported that broiler breeders fed zinc amino acid complex Produced 2.3 extra chicks per hen housed when compared to those fed broiler breeders either a sulfate control diet containing 160 ppm Zn. Hudson et al. (2004c) reported that hens provided diets supplemented with ZnAA and ZnSO4 had the highest hen-day egg production and settable eggs per hen housed, but the incidence of mortality was not influenced by dietary zinc source.

28 14 Kout El- Kloub et al. (2004) studied the effect of different sources (Zn oxide vs. Zn - methionine) and levels (50,100 and 150 mg/kg) of zinc on the performance of laying hens from 32 to 48 weeks and results showed that the source and levels of Zn (except ZnO at 150mg) significantly improved egg number, egg weight and feed conversion. Chandra Deo et al. (2005b) investigated the calcium (2.5 and 3.0 per cent) and zinc (30, 50 and 80 mg/kg) levels in laying hens diet on egg production performance from 31 to 47 weeks of age and the results indicated that the hen day egg production did not differ significantly due to either main effects or interaction of various combinations of calcium and zinc levels in the diet. Further, they concluded that a dietary level of 3.0 per cent calcium and 30 mg/kg zinc was found optimum for egg production performance in naked neck x White Leghorn layers. Utterback et al. (2005) reported supplementation of organic selenium yeast may affect metabolism and production because it is essential for the synthesis of active thyroid hormones, while, no differences in egg production, egg weight, feed intake or mortality by using organic selenium yeast which is very effective for increasing the Se content of eggs. Sechinato et al. (2006) did not find any effects of the supplementation of organic selenium, zinc, and manganese combinations on the performance and egg production of 48 to 60 week-old layers as compared to inorganic sources. Fernandes et al. (2008) reported that eggs from hens fed with diet containing ppm organic minerals (Zn, Mn and Se) were significantly heavier (P<0.05) and observed significant difference in the egg weight.

29 15 The study conducted by Jim et al. (2008) on white layers indicated that egg production, feed intake, feed conversion (kg/doz or kg /kg egg), egg weight, egg mass and egg quality traits were not affected by supplementation of organic selenium, however highest body weight found in layers supplemented with 0.05 ppm of organic selenium compared to control group. Lesson et al. (2008) conducted an experiment using 3 sources of Se (selenium selenite, Se- yeast and B-TRAXIM-Se) on forty eight, 50 wk old broiler breeders at two levels (0.01 ppm and 0.03 ppm) and reported higher egg production at 0.03 ppm level and egg weight was unaffected by either dietary Se level or source (P>0.05). Mohiti-Asli et al. (2008) reported that supplementing laying hens diet with Selenium selenite or Se- yeast (0.4 mg kg -1 ) and/or Vit E had no significant difference in egg production, egg weight, egg mass, feed consumption and feed conversion ratio. Sara et al. (2008) and Hanafy et al (2009) reported that addition of organic Sel- Plex to laying hens diets significantly (P<0.05) increased egg production (%) during the period ranged from wk of age. Maysa et al. (2009) reported egg production percentage, egg weight, egg quality (Haugh unit, egg yolk index and shell thickness) and selenium content in yolk and albumen were significantly increased for hens fed diet supplemented with Sel-Plex supplementation compared with those in control group. Reis et al. (2009) evaluated the effect of source and levels of Se in broiler breeder diets on egg production and Se concentration in eggs. Results of the study revealed that

30 16 during the first period, the hens fed with 0.30 ppm of organic Se produced more eggs (P< 0.05), whereas no difference (P > 0.05) in egg production was found in the second period. Period evaluation was found no difference in egg weight (P > 0.05), whereas specific gravity decreased (P < 0.05) and Se concentration in eggs increased (P < 0.05) in second period regardless of Se source. EL-Mallah et al. (2011) conducted a study using two hundred seventy Hi- sex Brown layers at 25 weeks of age to determine the effect of adding vitamin E (Vit. E) and/or selenium as seleno-yeast (SY) in layer diets on the performance, egg quality and some blood constituents of laying hens and reported that dietary organic SY had a significant effect on egg production but not on egg weight compared to control diets. 2.5 Hatchability and fertility Kienholz et al. (1961) found that laying hens which were fed an isolated soyprotein based diet that contained 10 ppm Zn had lower than normal egg production and hatchability compared to hens which were fed the same diet with additional Zn. Further, they also reported decrease in hatchability of eggs, grossly impaired development, and high mortality in chick embryos from the eggs of Zn-deficient hens. Palafox and Ho-a (1980) found that provision of 20,000 mg/kg DM in layer diets resulted in decreased hatchability and fertility of laying pullets. Stahl et al. (1986) reported that supplementation Zinc was not required to maximize fertility and showed that dietary supplementation of 28 ppm of zinc through corn-soy laying diet, was not necessary for maintaining normal hatchability.

31 17 Badway et al. (1987) suggested a positive relationship between zinc concentration of egg contents and hatchability. Inadequate transmission of zinc from the hen to the hatching eggs was likely responsible for low hatchability and poor chick quality when dietary zinc intake was insufficient. Stahl et al. (1990) reported that supplementation of practical breeder diets with up to 2000 mg/kg zinc had little or no effect on hatchability. Kidd et al. (1993) indicated no significant differences in hatchability, but fertility data showed significant increase in fertility in Hatch 2 for birds fed a diet supplemented with Zn-Met when compared to those fed the control diet. When hatches 2, 3, and 4 were combined, per cent fertility in Zn-Met was increased over the control. Further, they concluded that zinc has been demonstrated to play a vital role in the reproduction of the chicken. Wilson (1997) opined that low level of Zinc is necessary in breeder diet to obtain normal hatchability. Further he revealed that zinc deficiency in breeder diet resulted in decreased hatchability, increased embryonic mortality and impaired development of skeleton and feathers. Edens (2002) has shown that Sel-plex fed roosters had higher sperm quality as compared to those fed diet containing no additional or additional inorganic Se. It is important that Se as component of GSH-PX, helps to protect lipid rich membrane of the sperm.

32 18 Rutz et al. (2003) indicated that the addition of Sel-plex and other organic minerals to commercial breeder diet increased the number of chicks per hen housed from to Durmuş et al. (2004) suggested that the requirements for zinc of brown parent stock layers are higher than suggested Zn concentration, 65 mg Zn kg -1 DM for parent hens (Underwood and Suttle, 1999). When various production and hatchability traits are considered together, the diets of brown parent stock layers should include 180 mg Zn kg -1 DM zinc for optimal performance. Kout El- Kloub et al. (2004) studied the effect of different sources (Zn oxide vs. Zn - methionine) and levels (50,100 and 150 mg/kg) of zinc on the performance of laying hens from 32 to 48 weeks old and observed that the addition of Zn-Met at levels of either 100 or 150 mg, significantly increased fertility percentages and both source of Zinc did not affect hatchability percentages. Renema et al. (2004) reported that breeder hens fed with Sel-plex showed higher number of sperm holes at the site of fertilization in the perivitelline membrane, as compared to hens fed with inorganic selenium. Selenium supplementation increases the hatchability of fertile eggs and number of hatched chicks (Davtyan et al., 2006 and Petrosyan et al., 2006). As a result, Se has an important role in poultry fertility and embryonic development.

33 19 Papazyan et al. (2006) indicated that replacement of sodium selenite with organic selenium in the form of Sel-Plex in the breeder diet (0.04 ppm) improves fertility, hatchability and early chick viability. Pappas et al. (2006) reported that broiler breeder diets supplemented with organic Se (Sel-plex) at < 0.1 mg/kg of diet increased fertility, hatchability and decreased embryonic mortality as the hens age increased. Lesson et al. (2008) conducted an experiment using 3 sources of Se (selenium selenite, Se- yeast and B-TRAXIM-Se) on forty eight, 50 wk old broiler breeders at two levels (0.01 ppm and 0.03 ppm) and concluded that hatchability was affected by interaction between dietary selenium level or source (P<0.05). 2.6 Egg quality characteristics External quality characteristics Kienholz (1992) showed that feeding zinc chelate to layers submitted to heat stress, associated with low calcium intake, and maintained size egg, whereas inorganic zinc supplementation reduced egg size. Moreng et al. (1992) reported that dietary zinc methionine (0.2 g and 0.5 g/kg diet) plus 2 g NaCl/L in the drinking water significantly improved shell breaking strength over those birds on the 2 g NaCl/L with no zinc methionine supplementation in 60 weeks old laying hens. This same pattern occurred for shell weight, shell weight per unit of surface area, and percentage of shell defects. There were no significant differences in

34 20 eggshell quality measures for birds on the town water with or without the dietary zinc methionine. No statistical difference among treatments was observed in egg weight. Balnave and Zhang (1993) reported that hens receiving supplemental Zn had decreased eggshell defects and an increased concentration of calcium-binding protein and carbonic anhydrase activity, compounds that aid in eggshell mineralization. Dale and Strong (1998) reported non significant improvement of egg quality by supplementing either organic or inorganic trace mineral blends. However, when the supplementation of organic trace minerals was replaced by inorganic sources, significantly lower egg specific gravity was obtained. Ceylan and Scheideler (1999) demonstrated that organic zinc was associated with higher activity of carbonic anhydrase and in turn with improved shell quality. The fact that zinc is a co-factor of this enzyme makes both activity and proper function of this enzyme potentially sensitive to trace elements, their interactions and availability. The dietary concentration of zinc needed to meet daily zinc requirements ranges from 40 to 60 mg/kg of feed. Lima et al. (2000) reported that supplementation of 40 ppm Zn-methionine chelate in single or in combination with 40 ppm Mn-methionine chelate increased specific gravity of eggs, eggshell strength and eggshell thickness. Siske et al. (2000) reported increase in eggshell thickness when organic manganese, zinc, and selenium replaced 50% of inorganic presentations of these trace minerals.

35 21 Klecker et al. (2002) reported an increase in laying hen performance, eggshell quality, eggshell strength, and eggshell weight when 20 per cent and 40 per cent of supplemental inorganic Mn and Zn were replaced with the organic forms. Guo et al. (2002) reported that providing diets with supplemental zinc (80 ppm) from a mixture of ZnAA and ZnSO4 rather than solely ZnSO4 reduced the incidence of cracked eggs in laying hens. Synergistic effects of ZnAA and ZnSO4 may enhance mechanisms involved in calcification of egg shells. Mabe et al. (2003) showed that addition of Zn, Mn and Cu to a basal cornsoybean meal diet at 60, 60 and 10 mg/kg, respectively increased Zn, Mn, and Cu concentrations in egg yolk and improved the eggshell quality and did not affect eggshell percentage, eggshell index or eggshell stiffness.. Hudson et al. (2004) reported that supplementing diets with a combination of ZnAA and ZnSO4 resulted in superior reproductive performance of caged broiler breeder hens. Adding zinc from ZnAA and ZnSO 4 to the diet reduced the incidence of cracked eggs. The percentage of cracked eggs was inversely related to percentage of settable eggs. Eggs from hens given diets with supplemental zinc from ZnAA or ZnAA and ZnSO 4 had improved shell quality as measured by higher specific gravity than eggs laid by hens fed diets supplemented with ZnSO4. Chandra Deo et al. (2005c) reported that the main effect of zinc did not significantly influence the shell thickness and shell weight.

36 22 Zamani et al. (2005) conducted an experiment to evaluate the effects of supplementing the diet of laying hens with a combination of Zn and Mn on egg shell quality by preparing the basal diet containing 50 mg/kg Zn and 30 mg/kg Mn supplemented with 0-0, 0-30, 0-60, 0-90, 50-0,50-30, 50-60, 50-90, 100-0, , , , 150-0, , and mg/kg of Zn and Mn, respectively. They reported that addition of Zn increased eggshell thickness, concentration of Ca in eggshell and eggshell index but did not affect eggshell percentage, stiffness, elastic modulus, breaking strength and fracture toughness and egg weight. Papazyan et al. (2006) reported that replacement of sodium selenite with Sel-Plex in the laying hen diet has shown to improve FCR, shell quality and improve egg freshness during storage. Fernandes et al. (2008) reported that supplementation of organic minerals (Zn, Mn and Se) at two levels (0.250 and ppm) showed positive effect on percentage cracked eggs and shell thickness Internal quality characteristics Siske et al. (2000) reported increased eggshell thickness when organic manganese, zinc, and selenium replaced 50 per cent of inorganic presentations of these trace minerals Guo et al. (2002) reported that zinc concentration of the egg yolks from laying hens was increased when they were provided diets supplemented with ZnAA rather than ZnSO4.

37 23 Hassan (2003) investigated some productive and physiological responses for using dietary zinc oxide (0, 5,000, 10,000 and 20,000 ppm) on laying hens and reported that adding 5000, 10,000 and 20,000 ppm zinc oxide decreased eggshell thickness. Lim and Paik (2003) studied the effects of supplementary mineral methionine chelates each at the level of 100 ppm (Zn, Cu and Mn) on the performance and eggshell quality of brown shelled eggs from hens of 96 weeks age and reported that specific gravity of eggs and eggshell strength were higher in Cu - methionine chelate treatment. Albumen height, Haugh unit and eggshell thickness were not significantly affected by treatments. Mabe et al. (2003) showed that addition of Zn, Mn and Cu to a basal cornsoybean meal diet at 60, 60 and 10 mg/kg, respectively increased Zn, Mn, and Cu concentrations in egg yolk. Kout El- Kloub et al. (2004) studied the effect of different sources (Zn oxide vs. Zn - methionine) and levels (50, 100 and 150 mg/kg) of zinc on the performance of laying hens from 32 to 48 weeks old and results showed that zinc had no effect on egg shape, yolk index and Haugh unit but slightly improved shell thickness. Chandra Deo et al. (2005c) investigated the calcium (2.5 and 3.0 per cent) and zinc (30, 50 and 80 mg/kg) levels in laying hens diet on egg quality traits from 31 to 47 weeks of age and reported that the main effect of zinc did not significantly influence the egg weight, albumen index, yolk index, shape index and Haugh unit. The calcium x zinc interaction significantly affected the albumen index (P<0.01), yolk index (P<0.05) and

38 24 Haugh unit. They concluded that a dietary level of 3.0 per cent calcium and 30 mg/kg zinc was found optimum for egg quality traits in naked neck x White Leghorn layers. Payne et al. (2005) carried out experimental studies comparing inorganic and organic Se sources showed that both Se presentations increased Se egg content, but the organic form was more efficient. Payne et al. (2005) reported that albumen quality of eggs stored at 22.2 o C was preserved in eggs from hens fed SS, but not in the eggs stored at 7.2 o C. Mohiti-Asli et al. (2008) observed no effect on egg yolk, shell weights (P>0.05) and yolk color by supplementing laying hens diet with Selenium selenite or Se- yeast (0.4 mg kg -1 ) and/or Vit E. 2.7 Economics Dirk Fremaut (2005) reported that organic mineral levels in feed were reduced to 30 per cent of normal levels in pigs due to their better availability.

39 Materials and Methods

40 III. MATERIALS AND METHODS The present experiment was conducted to study the effect of feeding organic zinc and selenium on the productive performance of male parental lines of Giriraja and Swarnadhara. The experimental procedures followed are presented in this chapter, 3.1 Biological trial A biological trial was conducted at the Department of Poultry Science, Veterinary College, KVAFSU, Hebbal, Bangalore, using 35 week old 128 male parental lines of Giriraja and Swarnadhara (64 each) maintained at the same department. The experiment was conducted for three periods each with 28 days duration and designated as PI, PII and PIII periods Experimental birds One hundred twenty eight male parental lines of Giriraja and Swarnadhara (64 each) of uniform body weight were selected for the experiment and were randomly distributed into four treatment groups under each line, with two replicates under each treatment and 8 birds per replicate, which were maintained in 16 pedigree pens with 1:8 (male: female) ratio. All the birds were reared under deep litter system with paddy husk as a litter material, each pedigree pen was fitted with plastic tubular feeder and continuous running water channel type of watering system to provide clean and fresh water. The birds were kept under 16 L: 8 D lighting programme. The experimental birds were maintained on 20% protein level upto six weeks of age followed by 16% protein

41 26 level upto twenty one weeks and on 18% protein level upto thirty five weeks of age and thereafter feed with experimental diets Experimental diets Basal diet was formulated using maize, soybean meal and feed additives. The breeder rations were formulated as per BIS (1992) except for the selenium and zinc levels. Initially, the birds were fed with a conventional breeder diet containing adequate levels of all nutrients as recommended by BIS (1992). Four experimental diets were formulated and allotted to each treatment as detailed in Table (3.1). The experimental diets were introduced at the end of 35 th week and were fed for a period of 12 weeks ie. upto 47 weeks. All the birds were randomly assigned to pedigree pens that have been previously assigned. The experimental birds were fed as follows, i. T1-Basal diet (Control) with 100 mg/ kg diet inclusion of Zn mg /kg diet Se from inorganic source, ii. T2- Basal diet with 100 mg/ kg diet Zn mg /kg diet Se from organic source, iii. T3- Basal diet with 80 mg/ kg diet Zn mg /kg diet Se from organic source, iv. T4- Basal diet with 60 mg/ kg diet Zn mg /kg diet Se from organic source. The inorganic supplementation was ZnSO 4 and SeSO 4. The organic supplementation consists of Zinc and Selenium procured from Vet care Bangalore. Ingredient and nutrient composition of experimental diets is given in Table 3.2 and Housing and management At the age of 21 st week, all the experimental birds were dewormed using mg/kg BW through feed and periodical deworming was practiced throughout the experimental period. On attaining 22 week of age, all the birds were

42 27 vaccinated against New Castle disease and Infectious Bursal disease using inactivated vaccine (0.5 ml per bird) through subcutaneous route. All the birds were fed 150 g of feed on daily basis. All the birds were provided with 16 hours of light during laying period. Utmost care was taken to maintain uniform managemental and health condition during experimental period (35 to 47 weeks) which was divided into three periods with 28 days each and accordingly results were recorded. 3.2 Growth performance Feed Consumption The daily amount of the concerned diet was weighed and offered to the eight birds replicate group. The feed consumption in each replicate was recorded daily Body Weight Gain Individual body weight was recorded at the beginning of the experiment and further body weight was recorded at the end of experiment to monitor the pattern of body weight changes. Group wise average body weights under different treatments were arrived. The weighing of the birds was done in the early hours of the day before feeding Survivability Survivability was calculated after every 28 days interval for the birds under each treatment groups.

43 Reproductive performance Hen day egg production (%) Hen day egg production in per cent was calculated by dividing the total number of eggs laid in each treatment group by number of hens survived during each day. No. of eggs laid on that day Hen day egg production % = X 100 No. of live birds available on that day Hen housed egg production (%) Hen housed egg production in per cent was calculated by dividing the number of eggs laid in each treatment on that day by number of birds at beginning of experiment in each treatment. No. of eggs laid on that day Hen housed egg production % = X 100 No. of birds at beginning of the experiment Fertility and Hatchability Eggs collected from the experimental hens were tested for their fertility as well as hatchability per cents set during each period. Each time, the eggs from different replicate under each treatment were collected for over seven days and were stored in a cold-storage room maintained at a temperature of 12.8 to 18.3 C. The replication wise eggs under each treatment were brought to the room temperature before setting in the incubator (Plate 3.1B). In the incubator the temperature and relative humidity were maintained at 37.6 to 37.9 C and 55 per cent, respectively. On 18 th day of incubation, eggs were subjected for mass candling

44 29 (Plate.3.1A), where the infertile eggs were discarded and only the fertile eggs were transferred into the Hatcher where the temperature was maintained at 36.1 to 36.7 C and with relative humidity of 65 per cent. The day-old chicks were removed from the hatcher on the 22 nd day. Number of dead in shell, killed/discard chicks and good chicks were recorded separately for each replication under each treatment. Fertility and hatchability per cents were determined replication wise using the following formulae. Number of fertile eggs Fertility % = X 100 Total number of settable eggs Number of chicks hatched out Hatchability % = X 100 (on total eggs set) Total number of eggs set Number of chicks hatched out Hatchability % = X 100 (on fertile egg set) Total number of fertile eggs set 3.4 Egg quality traits During the end of the each period two eggs were collected randomly from each replicate and a total of 32 eggs were used to study different egg characteristics of experimental eggs, which are enlisted here under, Egg weight (g) The eggs produced under each replicate were weighed to the nearest one gram accuracy on one occasions of every week (Tuesday) of the experimental period. The

45 30 weights so recorded on four occasions during a particular period (4 week) were pooled and the data was analysed as per treatments Shape index (SI) Average shape index of eggs (SI) was calculated from eggs produced in different replicate at every 28-day interval by measuring horizontal diameter as well as vertical diameter. The measurements were obtained by using Digital Calipers, Horizontal diameter (breadth) in cm SI = X 100 Vertical diameter (length) in cm Albumen index (AI) Albumen index was calculated by breaking open at the mid portion with a sharp knife and albumen along with yolk was carefully poured on a leveled glass slab. Then the albumen height was measured using Ames Haugh Unit Spherometer and diameter (Plate.3.3A) by Vernier Calipers. The Albumen Index (AI) was calculated as: Albumen height in mm AI = Albumen diameter in mm Yolk colour (YC) The score for colour of the egg of different replicates during each period was recorded by breaking open the eggs and by matching it with Roche yolk colour fan (Plate 3.2A) (Roche Company, 1969).

46 Yolk index (YI) Yolk index was calculated for the eggs produced from different replicates during each period. The yolk height was measured using Ames Haugh Unit Spherometer (Plate 3.2B) and diameter (Plate 3.3B) by Digital Calipers. The Yolk Index (YI) was calculated as Yolk height in mm Yl = Yolk diameter in mm Shell thickness After placing the entire contents of an egg on glass slab, the shell pieces were separated from shell membranes at broad end and narrow end and their thickness was measured using Digital Calipers and expressed in mm. 3.5 Economics Economical efficiency was calculated from the input-output analysis which was calculated according to the price of the experimental diets and saleable chicks. 3.6 Statistical analysis The experiment design was completely randomized (CRD) and the data pertaining to various parameters obtained during the biological trail of the present study was analyzed as per Snedecor and Cochran (1968). The data collected were analyzed statistically using GLM (GENERAL LINEAR MODEL) procedure of SAS (6.12), in which means showing significant difference were compared by Duncan multiple range test.

47 32 Table 3.1: Experimental Design Number of treatments: 4 Number of replicates per treatment: 2 Number of birds per replicates: 8 Number of birds per treatment: 32 Total number of birds: 128 TREATMENTS STRAINS T1 T2 T3 T4 Cross RHR TOTAL T1: inorganic Zn 100 mg / kg diet + inorganic Se 0.30 mg / diet CONTROL T2: organic Zn 100 mg / kg diet + organic Se 0.30 mg / diet T3: organic Zn 80 mg / kg diet + organic Se 0.20 mg / diet T4: organic Zn 60 mg / kg diet + organic Se 0.10 mg / diet

48 33 Table 3.2: Per cent ingredient composition of the basal diet Ingredient (Kg) (%) Corn 57 Soybean meal 21 Rice bran 3 Sunflower extraction 10 Mineral mixture* 3 DCP 0.5 Shell grit 5.5 Total 100 Additives (g) Vitamin mixture** 100 Salt (g) 400 Toxin binder (g) 100 Liver tonic(g) 100 Antibiotic(g) 50 * provided per Kilo gram of feed: Calcium 3%, Phosphorus 0.5%, Iodine 1 mg, Iron 90 mg, Copper 12 mg, and selenium 0.2 mg ** provided per Kilo gram of feed: Vit A 8000 IU, Vit D IU, Vit K 1 mg, Vit E 15 mg, Thiamine 3 mg, Riboflavin 8 mg, and Pyridoxine 8 mg. Table 3.3: Calculated nutrient composition of experimental diets Particulars Nutrient level ME Kcal/Kg 2692 Crude Protein% Calcium% 3.3 Phosphorus% 0.55 DL-Methionine% 0.4

49 34 Plate 3.1A. Mass candling of eggs Plate 3.1B. Setting of eggs in an incubator

50 35 Plate 3.2A. Scoring of yolk colour by Roche colour fan Plate 3.2B. Measuring the height of yolk by using Ames Haugh Unit Spherometer

51 36 Plate 3.3B. Measuring the width of yolk by using Vernier Calipers Plate 3.3A. Measuring the width of albumen by using Vernier Calipers