EFFECT OF ORGANIC SUPPLEMENTATION ON MALE QUAIL REPRODUCTION

A THESIS BY

MD. MAHMUDUL HASAN Examination Roll No. 10 AHPS. JJ 23 M Registration No. 31409 Semester: January – June 2011 Session: 2004-2005

MASTER OF SCIENCE (M S) IN POULTRY SCIENCE

DEPARTMENT OF POULTRY SCIENCE AGRICULTURAL UNIVERSITY MYMENSINGH

MAY 2011 EFFECT OF ORGANIC SELENIUM SUPPLEMENTATION ON MALE QUAIL REPRODUCTION

A THESIS BY

MD. MAHMUDUL HASAN Examination Roll No. 10 AHPS. JJ 23 M Registration No. 31409 Semester: January – June 2011 Session: 2004-2005

Submitted to the Department of Poultry Science, Bangladesh Agricultural University, Mymensingh in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE (M S) IN POULTRY SCIENCE

DEPARTMENT OF POULTRY SCIENCE BANGLADESH AGRICULTURAL UNIVERSITY MYMENSINGH

MAY 2011 EFFECT OF ORGANIC SELENIUM SUPPLEMENTATION ON MALE QUAIL REPRODUCTION

A THESIS BY

MD. MAHMUDUL HASAN Examination Roll No. 10 AHPS. JJ 23 M Registration No. 31409 Semester: January – June 2011 Session: 2004-2005 Approved as to style and content by

………………………………………………………………………………. …………………………………………………………………………….. (Associate Prof. Dr. Md. Shahidur Rahman) (Associate Prof. Dr. Shubash Chandra Das) Supervisor Co‐supervisor

…………………………………………………………………………… (Dr. Md. Shawkat Ali) Chairman, Defence Committee And Head, Department of Poultry Science Bangladesh Agricultural University Mymensingh

MAY 2011 ACKNOWLEDGEMENTS

First of all, the author expresses his sincere gratitude to Almighty Allah, the supreme of the universe for His never ending blessings for the successful completion of the present research work and in the preparation of this thesis. The author feels immense proud privilege to express his heartiest appreciation and deepest sense of gratitude, indebtedness and profound respect to his teacher and research Supervisor, Associate Professor Dr. Md. Shahidur Rahman, Department of Poultry Science, Bangladesh Agricultural University, Mymensingh for his cordial guidance, responsible supervision, sympathetic cooperation, priceless suggestions, inspiration, constant affectionate feelings and constructive criticisms throughout the research work and successful completion of this manuscript. The author desires to express his profound appreciation veritable gratefulness and heart- felt thanks to his Co-supervisor, Associate Professor Dr. Suvash Chandra Das and senior Professor Dr. M. A. R. Howlider for his scholastic guidance, magnanimous help and helpful advice and constant instruction in the way to accomplish this thesis. The author extended his cordial thanks to Associate professor Dr. MD. Shawkat Ali, Head, Department of Poultry Science, Bangladesh Agricultural University, Mymensingh for his help and cooperation during the research work, and in preparing this manuscript. The author feels proud to my sincere appreciation and boundless gratitude, best regards and respect to my honorable class teachers Professor Dr. S. M. Bulbul (Rtd), Professor Dr. Safiuddin Ahmed (Rtd), Professor M. A. Wahid (Rtd), Professor Dr. S. D. Chowdhury, Professor Dr. Ashraf Ali, Associated Professor Dr. Fowzia Sultana, Assistant Professor Mr. Musabbir Ahmed, Department of Poultry Science, Bangladesh Agricultural University, Mymensingh for their generosity and suggestions, keen interest, encouragement, constant inspiration and constructive comments and valuable advice in carrying out this research work. The author expresses his thanks to Md. Shajahan Bhai, Md. Mohammad Ali Bhai, Md. Ruhul Amin Bhai, Nasir, Kabir, Rahman and all other officers and staffs, Department of Poultry Science, Bangladesh Agricultural University, Mymensingh for their continuous help and kind assistance, while undergoing this research work.

The author gratefully admires to his classmates and friends specially ,Maidul, Tainur,Tareq, Nebash, Yousuf, Jewel, Litu Bhai, Mukul Bhai, Kabir Bhai and Akhirul for their kind co- operation and affectionate encouragement to carryout the research work. The author would like to acknowledge his heartiest gratitude to his beloved parents, respectable dear elder sister Selina Akter and Afroza Akter and all well wisher for their never ending prayer, affection, support, sacrifice, inspiration, encouragement and continuous blessings in the long process of building my academic career which can never be repaid.

The author

ABSTRACT A trial on dietary organic selenium (Se) supplementation with locally manufactured compound feed was conducted to evaluate sperm motility, sperm morphology, sperm concentration, sperm hole,fertility performance and testis weightof Japanese Quail. A total of 24 adult males and 60 breeder females were randomly and equally allocated to 25, 2.5, 0.25, and 0.0 ppm organic Se (Sel-Plex) supplemented diets having 3 replications in each. Results demonstrated that the semen quality was reduced (p<0.05) at 25 ppm Se supplemented groups without altering the feed intake, body weight and survivability of the bird. sperm motility, sperm morphology, sperm concentration, were found to be significantly higher (p<0.05) in the semen of 0.25 ppm organic Se supplemented birds. The sperm hole number were found to be markedly higher (p<0.05) at the 0.25 and 2.5 ppm organic Se supplementation. . The fertility was also higher (p<0.05 ) in 0.25 ppm and 2.5 ppm Se supplemented eggs in comparison to that of control and 25 ppm Se supplementation. The weight of testis in the Se supplemented breeder quail did not differ significantly (p>0.05). The present study suggested that the compound breeder feed manufactured in Bangladesh is deficient of Se and its fortification with 0.25 ppm exogenous Sel-Plex is recommended for its use in breeder quail to ensure the optimum performance. However, an extensive trial on organic Se supplementation covering a range of poultry species and the available branded compound poultry feed is needed to generalize the present findings for the breeders of all poultry species.

LIST OF ABBREVIATIONS, SYMBOLS AND ACRONYMS

Abbreviations Elaboration wt Weight LBW Live body weight cm Centimeter S Sex ANOVA Analysis of Variance BAU Bangladesh Agricultural University Contd. Continued CRD Completely Randomized Design e.g. For example et al. And others FCR Feed conversion ratio Fig. Figure H Hour i.e. That is Kg Kilogram LSD Least Significant Difference Ltd. Limited mg. Milligram No. Number NS Non-significant oC Degree Celsius BW Body Weight P Probability Pm Post meridian

LIST OF ABBREVIATIONS, SYMBOLS AND ACRONYMS

Abbreviations Elaboration ppm Parts per million RH Relative humidity % Percentage * 5% level of significant ** 1% level of significant / Per : Ratio @ At the rate of + Plus < Less than > Greater than ± Plus minus

= Equal

CONTENTS CHAPTER TITLE PAGE No.

ACKNOWLEDGEMENTS i ABSTRACT iii LIST OF ABBREVIATIONS, SYMBOLS AND iv ACRONYMS CONTENTS vi-ix LIST OF TABLES ix LIST OF FIGURES x LIST OF APPENDICES Xi

Chapter I INTRODUCTION 1-3

Chapter II REVIEW OF LITERATURE 4-9 2.1 Sources and forms of selenium 4 2.2 Metabolism of Se 4 2.3 Symptoms of Se deficiency 5 2.4 Toxicity of Se 6 2.5 Effects of Se sources on reproductive 7 performance of breeder bird 2.6 Effects of Se sources on feed intake, 7

growth and performance of broiler

2.7 Effects of Se sources on body maintenance 8

and survivability

2.8 Effect of Selenium on Fertility of bird 8

Research gap and scope of present 2.9 9 investigation

CONTENTS(contd.) CHAPTER TITLE PAGE No.

Chapter III MATERIALS AND METHODS 10-16 3.1 Birds, chemicals and statement of the 10 experiment 3.2 Methods 10 3.2.1 Housing of the bird 10 3.2.2 Mixing of organic Se in feed and 10 methods of feeding the bird 3.2.3 Water management and other practices 12 3.3 Comparison of fertility performance 12 3.3.1 Collection and storing of egg 12 3.3.2 Incubation of eggs for fertility 13 3.3.3 Determination of fertility 13 3.4 Counting of sperm hole concentration on 13 egg perivitelline membrane 3.5 Semen collection and evaluation 14 3.5.1 Semen collection 14 3.5.2 Semen evaluation 14 3.5.2.1 Mass motility(%) 14 3.5.2.2 Sperm concentration determination 15 (million/ml) 3.6 Dissection of bird and observation of 16 different organs 3.7 Statistical analysis 16

CONTENTS(contd.) CHAPTER TITLE PAGE. No.

Chapter IV RESULTS 17-23 4.1 Effect on feed intake and body 17 maintenance 4.2 Semen quality parameters 17

4.3 Sperm holes concentration on egg 19 perivitelline membrane 4.4 Fertility rate 21 4.5 Reproductive system and other vital organs 21

Chapter V DISCUSSION 24-25 Chapter VI SUMMARY AND CONCLUSION 26-27

Chapter VII REFERENCES 28-35

APPENDICES 36-37

LIST OF TABLES

Table Title Page 1 Composition of the basal feed supplied to the 12 experimental quail 2 Feed intake and body maintenance of Japanese 17 quail fed on diet supplemented organic Se 3 Semen quality of Japanese quail fed on diet 18 supplemented with organic Se 4 The effect of organic selenium on sperm holes 20 of eggs from breeder quails. 5 Fertility rate in 4 dietary levels of organic Se 21 supplementation in quail 6 Reproductive and other vital organs weight in 23 Se supplemented quail

LIST OF FIGURES

Figure Title Page 1 View of mixing organic Se with mash feed 11 2 Storing of eggs in earthen pot 13 3 View of dissecting the birds 16 4 Sperm concentration at 4 different levels of dietary 19 Se supplementation 5 Sperm hole concentration in perivitelline membrane 20 of eggs at 4 dietary level of Se supplementation (arrow heads are the sperm holes)

6 Size of the testis of quail fed on 4 dietary levels of Se 22 supplementation

LIST OF APPENDICES

APPENDIX TITLE PAGE I NRC Nutrient requirement of breeder Japanese 36 quail II Body maintenance of breeder quail fed on organic 36 Se supplemented diet III Semen quality of breeder quail fed on diet 37 supplemented with organic Se IV Sperm hole on previtelline membrane of egg from 37 breeder quail fed on diet supplemented with organic Se V Fertility performance from breeder quail fed on 37 diet supplemented with organic Se

Chapter I INTRODUCTION Selenium is an essential trace element which occurs in cereals and primarily in the form of selenomethionine. Selenium (Se) is an essential mineral for a range of organisms including birds (Schwarz and Foltz, 1957). It can be found as a part of at least 25 selenoproteins in living bodies (Brown and Arthur, 2001). Therefore, it is unsurprising that selenoproteins are considered to be involved in the regulation of a variety of physiological functions including reproduction, immunity, growth and development (Surai, 2002). Selenium has effectiveness on the poultry reproduction. The role of Se in body’s antioxidant defense system as a constituent part of glutathione peroxidase (GSH-Px), thioredoxin reductase and methionine sulphoxide reductase B has received considerable attention in last few years. The efficiency of vitamin E was found to be increased in chicks fed diet containing Se. Moreover, Se alone even in absence of vitamin E reported to prevent the development of exudative diathesis in birds (Sing and panda, 1988). In man, an association between low Se status and Keshan disease, a cardiomyopathy endemic to part of China, was documented in 1979 (Keshan Disease Research Group, 1979). Brown et al.,(1986) described an association between muscle weakness and severe Se deficiency in a female patient with sort-bowel syndrome who was maintained on parental nutrition at home. Syndromes included inability to rise from a squatting position, rapid tiring when climbing stairs, and difficulty lifting heavy objects. There are two main sources of Se in the diet; organic and inorganic Se. Among organic Se, seleno-methionine (SeMet) is an integral part of many feed or food ingredients, whereas selenite or selenate is the sources of inorganic Se. Both of the forms of Se can be added into the animal and poultry diets. In wild, animals normally receive Se in the form of SeMet (Combs and Combs, 1986). The plant absorbs Se from the soil in the form of selenite or selenate and synthesizes seleno- amino acids of which SeMet is the major seleno-compound in cereal grains, grassland legumes and soybeans (Whanger, 2002). In maize, rice, wheat and soybean SeMet comprises 46 to 82%, 55 to 87%, 50 to 81% and 63 to 72% of total Se, respectively (Yang et al1997). SeMet is also the major seleno-compound in Se- enriched yeast (Polatajko et al2005), which is used as a natural form of Se for dietary supplementation. Yeast cells can take up Se in the form of selenite or selenate from media and synthesise selenoamino acids. However, data accumulated over the last decade clearly indicate that there are fundamental differences between two forms of Se and the so-called organic form provides more Se reserves in the body and become efficiently transferred into the egg, colostrums and milk (Surai, 2002). Se concentration in the soil of Bangladesh is low (Oldfield, 2002). For this reason, plant cannot absorb the proper amount of Se from soil. As a result, natural feedstuffs such as cereal grains like maize, rice, wheat and soybean contain lower levels of Se .As a consequence, farmer supply the commercial organic selenium ( e.g., Sel-plex) with natural feedstuffs to fulfill the bird’s requirement. Approximately, 100% of the organic Se is imported from abroad that increase the feed cost and ultimately the production cost. Until today, no data on optimum level of se supplementation in the breeder ration at Bangladesh condition is available. As a result, farmers are using the Sel-plex or other organic form of Se in poultry diets, particularly in breeder rations, depending upon their imagination only. Sometimes the trader’s exaggerated advertisement in favor of organic Se misleads the farmers to use the organic Se at over doses that results poor performance of the birds and cause an unexpected loss in the business. However, the identification of appropriate level of organic selenium supplementation in breeder diets that is mainly manufactured from locally available ingredients is still lacking. The present study was conducted to investigate the effects of organic Se supplementation on- z To determine the effect of dietary organic Se supplementation at various levels on the semen quality of Japanese quail, z To know the effect of organic Se supplementation on fertilization capability of male quail, and z To know the optimum level of organic Se supplementation in the breeder male quail ration for better reproductive performance.

Chapter II REVIEW OF LITERATURE

2.1 Sources and forms of selenium Selenium (Se) is widely distributed over the surface of the earth. It is about 70th in order of abundance of minerals in the earth. Overall, it occurs at a concentration of about 50 to 200 µg/kg in rocks and soil, but in some places, depending on geological and other factors, it can be present in greater or lesser amounts (Reilly, 1995). Selenium that present in soil enters into the food chain through plants, grains and seeds (Brown and Arthur, 2001; Gibson, 1990). In industry, selenium is obtained as by-product of others metal production, principally electrolytic refining of copper. Several compounds of selenium are commercially available, including ferro- and -, , cadmium sulfoselenide, selenium diethyldithiocarbomate, sodium selenite, and sodium selenate. There are two main sources of Se in the diet, i.e., organic Se (mainly SeMet) and inorganic Se like selenite or selenate (Combs and Combs, 1986). Whanger (2002) reported that plant absorbs Se from soil in the form of selenite or selenate and synthesizes selenoamino acids, with SeMet being the major selenocompound in cereal grains, grassland legumes and soybeans. In yeast, SeMet is the major selenocompound in which is used as a natural form of Se for dietary supplementation (Polatajko et al., 2005).

2.2 Metabolism of Se The metabolism of selenium is dynamic. Animals synthesize many different intermediary metabolites in the cause of converting inorganic selenium to organic forms, which can be enzymatically catalyzed (Ip, 1998). Hydrogen selenite is a key metabolite, formed from inorganic sodium selenite (oxidation state +4) via selenodiglutathione through reduction by thiols and NADPH-dependent reductases. Methylation is a major pathway for selenium metabolism in microbes, plants, and animals. The hydrogen selenite is generally regarded both as substrate for biosynthesis of by cysteine synthases and as molecule for the transformation into selenophosphate by selenophosphate synthetase, and both are required for synthesis of selenoproteins (Ganther, 1999; Birringer et al., 2002 ).

2.3 Symptoms of Se deficiency In man, an association between low selenium status and Keshan disease, a cardiomyopathy endemic to part of China, was documented in 1979 (Keshan Disease Research Group, 1979). Brown et al.,(1986) described an association between muscle weakness and severe selenium deficiency in a female patient with sort-bowel syndrome who was maintained on parental nutrition at home. Syndromes included inability to rise from a squatting position, rapid tiring when climbing stairs, and difficulty lifting heavy objects. Other selenium deficient disease is Kashin-Beck disease. The disease has observed in regions with soil low in selenium. Symptoms of the disease include joint swelling, pain, general malaise, short status (due to the effect of the disease on the growth plate of tubular bone), and secondary osteoarthritis (Sokoloff, 1988). The sign of selenium deficiency have also been reported in quail and chicken (Jensen, 1968; Thompson and Scott, 1969). It includes lost of body weight, poor feathering, impaired reproduction, reduced hatchability, and reduced viability. In ducklings, selenium deficiency reduced plasma glutathione peroxidase activity and body weight gain, increased mortality. Ducklings that succumb to selenium deficiency may exhibit necrosis of several tissues including the ventriculus, heart, skeletal muscle and smooth muscle of intestine, and show signs of hydropericardium and ascites (Dean and Combs, 1981). Chicks severely deficient in selenium exhibit poor growth and feathering, impaired fat digestion, pancreatic atrophy and fibrosis, and reduced activities of selenium dependent glutathione peroxidase in pancreas. Selenium deficiency depressed growth of broilers by inhibiting hepatic 5’-deiodinase activity, which causes lower plasma 3,5,3-triiodothyronine concentration (Klasing and Austic, 2003).

2.4 Toxicity of Se In livestock, interest in the toxic effect of selenium was obtained after the discovery in the early 1930’s by scientists from the U.S. Department of Agriculture and from South Dakota and Wyoming State Agricultural Experiment Stations that selenium was the toxic substance in forages and grains responsible for “blind staggers” and “alkali disease” which sometimes occurred in livestock in the certain areas of the American western plains (NRC, 1971). Chronic selenium toxicity in livestock occurs when animals consume seleniferous plants containing 3-20 ppm of selenium over a prolonged period. When it occurs in cattle and horses, it is often called alkali disease. Symtoms include lameness, loss of vitality, hoof malformations, loss of hair in the mane and tail, atrophy cirrhosis of the liver and chronic nephritis (NRC, 1983). Laboratory rats poisoned with selenium on long term basis exhibit growth depression and cirrhosis (Shike, 2005). Selenium toxicity had been reported to be a cause of death and deformities of embryos and chicks in aquatic birds within Kesterson area of California. Selenosis was caused by high concentration of selenium in the runoff which had bioaccumulated in the bird’s food chain by plants, invertebrates and fish (Ohlendorf et al., (988). Selenium in a diet of the mallard at 10 ppm as selenomethionine or 25 ppm as sodium selenite caused a 40-44 % decreased in the total number of eggs that hatched compared with control (Hoffman and Heinz, 1988).

2.5 Effects of Se sources on reproductive performance of breeder bird Forms of Se were found to affect the egg production and egg quality in birds. Rutz et al., (2004) found an improvement in egg production, egg weight and albumen weight in laying birds fed diets with organic selenium compared to that of inorganic form. Higher egg production on organic Se supplementation has been reported by Jang et al.(2006) but they did not find any benefit in daily egg mass, Haugh unit, yolk colour and eggshell breaking strength.

2.6 Effects of Se sources on feed intake, growth and performance of growing bird The relationship between the sources of Se and the overall performances of broiler is well documented. Edens et al., (2000) reported that organic Se was associated with better feathering in broilers but feed conversion ratios were not affected by selenium source. Wang-YanBo (2009) observed the improved daily weight gain and decreased feed conversion ratio in broiler fed both the sodium selenite and nano-Se. Choct et al., (2004) and Moreira et al.(2001) found the organic selenium (selenised yeast) to be superior to inorganic selenium (sodium selenite) for increasing feathering, eviscerated weight and breast yield. Improvement in broiler performance fed organic Se to Se deficient broiler were also reported by Stolic et al., (2002). Peric et al., (2006) investigated the effect of sel-plex and bioplex substituting inorganic mineral sources on broilers feathering, hock conditions and some other parameters and forecasted that the inorganic Se levels in broiler feed can be reduced to at least 1/3 of the original levels using organic forms (Bioplex, Sel-Plex) without compromising the performance.

However, some authors found similar effects on growth performance, carcass traits in both of inorganic and organic selenium supplemented broiler (Payne and Southern, 2005).

2.7 Effects of Se sources on body maintenance and survivability Wang YanBo (2009) observed the improved survaival rate in commercial broiler fed on both the sodium selenite and nano-Se supplemented diet, whereas Korosi et al., (2005) had reported the lower mortality on male and female on Sel- PlexTM supplemented broiler parent stock. However, lower mortality rate in Se supplemented Japanese quail compared to control were also reported by Elaroussi et al., (2002). 2.8 Effect of Se on the fertility of bird Agate et al., (2001). Selenium improves the environment of the sperm storage tubules in the hen’s oviduct, allowing sperm to live longer. Feeding diets enriched with selenium (especially organic forms), can improve fertility. Selenium seems to play an important role in the maintenance of fertility in laying hens. Selenium supplementation could extend the life of the flock at higher production levels.

2.9 Research gap and scope of present investigation From the above literatures, it is clear that the supplementation of Se to poultry with appropriate doses is always favorable for better growth, reproduction and survivability, and the organic form was found to be superior to inorganic one in most of the cases. The organic form of Se is available in natural feed staffs including the cereal grains like maize, wheat, sorghum etc. However, Se content in feed ingredients depends upon the Se concentration in soil. The selenium concentration in Bangladeshi soil has been reported to be lower (Jason, 2004; Oldfield, 2002) than the standard. So, it is obvious that the feed grains grown on Bangladeshi soil will be deficient of Se. Therefore, there is a scope of investigating the necessity of Se supplementation in poultry as well as in other animals. But, until recently no work has been done to study the effects of Se supplementation with the appropriate form and levels in poultry rations formulated from locally available ingredients.

Chapter III MATERIALS AND METHODS

3.1 Birds, chemicals and statement of the experiment The experiment was conducted at Bangladesh Agricultural University (BAU), Mymensingh. Poultry Farm using wild type Japanese quail from July 2010 to Dec 2010. A total of 60 laying female and 24 adult male quails were equally and randomly allocated to 4 dietary treatments; 0.0, 0.25, 2.5 and 25 ppm organic Se supplemented diets. Each dietary treatment had 3 replications consisting of 5 female and 2 male. The birds were approximately 10-week-old at starting of the experiment having good health and similar body weight. The organic selenium (Se) used in this experiment was Sel-Plex manufactured by Altech, USA that contained 2 g organic Se in 1 kg.

3.2 Methods 3.2.1 Housing of the bird The birds were housed in mosquito-net made small pens on littered floor. The size of each pen was (110 X 85 X 85) cm3. The birds under different treatment were marked by different color on feathers. Lighting was maintained by a central 100 watt electric bulb at 1.5 m height for 4 pens. The house was well ventilated and protected from all sorts of predator and rodents.

3.2.2 Mixing of organic Se in feed and methods of feeding the bird Commercial layer mash feed of chicken was used to feed the laying quail (Table 1). The level of organic Se in 4 dietary treatments were 0.0 (control), 0.25, 2.5 and 25 ppm. To mix the Se in diet, 4 kg of feed were weighed by electronic balance and taken on a paper sheet. At first, 100 ppm of organic selenium were weighed and mixed well with about 1/8 of 4 kg basal mash feed. Then, this mixed feed was added to 1/4 of the total mash, which were gradually added to 1/2 and the total amount. The feed was mixed very carefully so that the Se became distributed homogenously over all parts of the feed. Thus, a 25 ppm organic Se supplemented feed had been prepared. The 90% basal mash feed was mixed with either 10% of 25 ppm Se mixed feed or 10% of 2.5 ppm Se mixed feed to prepare the 2.5 and 0.25 ppm Se containing feeds, respectively. The feed was supplied 3 times daily in the grill connected tube feeder (16 linear cm / bird).

Photograph 1. View of mixing organic Se with mash feed

Table 1. Composition of the basal feed supplied to the experimental quail Components Levels Moisture (maximum %) 12 Metabolizable energy (Kcal/kg) 2700 Crude protein (minimum %) 17 Crude fiber (maximum %) 5 Calcium (minimum %) 3.5 Available phosphorus (minimum 0.4 %) Lysine (minimum %) 0.8 Methionine (minimum %) 0.4

3.2.3 Water management and other practices Safe and cool drinking water was provided to the birds at all times in chick drinker (7 bird / drinker). The residual water was changed twice a day after cleaning the drinker. The droppings of quails were cleaned thoroughly from the litter at every alternative day. 3.3 Comparison of the fertility performance of the bird 3.3.1 Collection and storing of egg Eggs were collected from 4 different dietary treatment groups and labeled. At least 70 eggs / treatment were collected and stored at cool earthen pot for not more than 5 days (Fig 3).

Photograph 2. Storing of eggs in earthen pot 3.3.2 Incubation of eggs for fertility The eggs were incubated for 5 days. A constant 38.5 ºC temperature and 65 % relative humidity were maintained during setting periods. The automatic turner was switched to turn the setting tray at 1 hour interval.

3.3.3 Determination of fertility The percentage of fertility was calculated on the basis of total eggs set in the incubator.

No. of fertile eggs x100 Fertility (%) = Total no. of eggs set 3.4 Counting of sperm hole concentration on egg perivitelline membrane Bramwell et al.,(1995) adapted the technique to use eggs for the determination of perivitelline sperm hole numbers.The sperm penetration assay of Bramwell et al., (1995) was used to quantify the perivitelline layer sperm holes. An approximately 1 cm2 section of the perivitelline layer above the germinal disc was cut free, cleaned, mounted to a microscope slide, fixed, and stained with Schiff’s Acid reagent to generate a contrast with the sperm holes. The holes were counted at 100X magnification. The raw numbers and change in numbers over time were used as a representation of quantifiable fertilization potential.

3.5 Semen collection and evaluation 3.5.1 Semen collection Semen from the birds of 4 different dietary groups were collected after slaughtering the birds and pooled at Genetics and Breeding Laboratory of BAU. 3.5.2 Semen evaluation The evaluation of fresh semen was done immediately after collection using the method described by Herman and Madden (1963).The volume, color, density, mass motility (%), sperm concentration (million/ml), live sperm count (%), normal sperm count (%), measurements of spermatozoa in fresh ejaculate of individual bull were recorded.

3.5.2.1 Mass motility (%) To observe the mass motility, one drop of semen was placed on a pre- warmed (37°C) slide and examined at low magnification (l0x) without cover slip. Mass motility was scored into 0 to 5 scales according to Herman and Madden :963): z Score 5= Excellent motility -80% or more of the spermatozoa are in very vigorous z Score 4 = very good motility - Approximately 70-80% of the spermatozoa are in vigorous rapid motion. Waves and eddies from and drop rapidly, but not so rapid as in excellent motility. z Score 3 = Good motility - About 50 to 70% of the spermatozoa are in motion. Motion is vigorous but waves and eddies formed moved slowly across the field, in the main. z Score 2 = Fair motility - From 20 to 50% of the sperm are in motion. The movements are largely vigorous but no waves or eddies are formed. z Score 1 = Poor motility - Less than 30% of the sperm are in motion. The motion in mostly weak and oscillatory, not progressive. z Score 0 = No motility discernible.

3.5.2.2 Sperm concentration determination (million/ml)

The concentration of spermatozoa was determined by haemocytometer method according to Herman and Madden (1963). Semen sample was drawn into a standard red cell dilution pipette up to 0.5 marks. Dilution fluid was drawn into the pipette up to 101 marks. Pipette was agitated for 3-5 minutes to ensure proper mixing by eight-knot motion. The first 4- to 5 drops were discarded to get properly diluted semen. A cover slip was placed over the ruled field of the counting chamber of the haemocytometer and a drop was allowed to run the cover slip. The count was made under low magnification approximately 25X objectives. Five large double ruled squares were counted over the field. This would give a total of small squares than the number of spermatozoa per ml of semen to be calculated. The concentration of spermatozoa per ml of semen was expressed as million (x 106)/ml. The following formula was used for calculating total number spermatozoa per ml of fresh semen. Total no. of spermatozoa per ml of semen: C x 4000 x DS where, C = Number of sperm counted in given number of small squares. S = No. of small squares counted. D = Dilution ratio.

3.6 Dissection of bird and observation of different organs At the end of experiment, 3 males from each dietary group were randomly chosen for dissection. The birds were dissected according to the procedures of Jones (1984) to observe the testis weight and deformities (if any) in the vital organs and reproductive system components

Photograph 3. View of dissecting the birds 3.7 Statistical analysis All data on semen quality and fertility performance parameters were analyzed by Genstat (Fifth edition, Lawes Agricultural Trust, Rothamsted Experimental Station, ) statistical package in computer. The data were compared by Analysis of Variance (ANOVA) under the Completely Randomized Design (Steel and Torrie, 1980). Least significant differences (LSD) were calculated and used to find out the significant difference in different treatment groups. SEDs (Standard error difference of the means) were calculated for non- significant difference. Significant difference were considered at p < 0.05.

Chapter IV RESULTS

4.1 Effect on feed intake and body maintenance Feed intake and weekly average body weight gain of the Se supplemented quail has been shown in Table 2. But, Se supplementation had no effect on feed intake and weekly average body weight gain of the birds. Up to the end of the experiment, all of the experimental birds were alive except in the control group, where 2 bird was died due to excessive cannibalism

Table 2. Feed intake and body maintenance of Japanese quail fed on organic Se supplemented diet

Level of Se (ppm) Feed intake/day (% to body Average BW gain (gm/week) weight) 25 14.44 7.63b 2.5 15.14 11.80b 0.25 14.35 21.72a 0.0 15.11 16.50ab LSD/ (SED) (0.50) 4.47 Level of sig NS *

*, p<0.05, when compared with 0.0 ppm (control) within the same row, NS= not significant (p>0.05), ppm = parts per million, BW = Body weight.

4.2 Semen quality parameters Results on the semen quality of the Se supplemented Japanese quail have been cataloged in Table 3. The motility %, normal sperm count %, sperm concentration were found to be significantly higher (p<0.05) in 0.25 and 2.5 ppm Se supplemented birds than that in control and 25 ppm groups.

Table 3. Semen quality of Japanese quail fed on diet supplemented with organic Se

Level of Se % Motility % Normal Concentration (ppm) sperm (Million/ml) 25 70.00b 82.00b 856.67b 2.5 75.00ab 87.50ab 945.00ab 0.25 83.00a 91.00a 1050.00a 0.00 75.67ab 89.00b 870.00b LSD 4.23 2.84 67.78 Level of ** * * significance *, p<.05, when compared with the values within the same row, values with uncomm on superscripts differ significantly, ppm = parts per million.

25 ppm 2.5ppm

0.25ppm 0.00 ppm Photograph 4.Sperm concentration at 4 different levels of dietary Se supplementation

4.3 Sperm holes concentration on egg perivitelline membrane The sperm hole concentration has been presented in Table 4. Data implied that the sperm hole number were higher (p<0.05 ) in 0.25 ppm Se supplemented birds than that of the control and other two groups.

On the other hand, sperm hole number were found to be markedly lower (p<0.05) in the 2.5 ppm organic Se supplemented groups .The males on the Sel-Plex® diet 0.25ppm level produced greater sperm hole, with an average of 25.2/egg compared to 20.6/egg in males on the control diet.

25 ppm 2.5ppm

0.25ppm 0.00 ppm Photograph 5. Sperm hole concentration in perivitelline membrane of eggs at 4 dietary level of Se supplementation (arrow heads are the sperm holes

Table 4. The effect of organic selenium on sperm holes of eggs from breeder quails. Level of Se (ppm) Sperm hole no/egg. 25 18bc 2.5 16.3d 0.25 25.2a 0.00 20.6b LSD 3.82 Level of significance ** **, p<.01, when compared the values within the same row. values with uncommon superscripts differ significantly

4.4 Fertility rate The rate of fertility has been presented in Table 5. Data implied that the rates of fertility and hatchability were higher (p<0.05 ) in 0.25 ppm and 2.5 ppm Se supplemented birds than that of the control and 25 ppm groups. On the other hand, the rate of fertility were found to be markedly lower (p<0.05) in the 25 ppm organic Se supplemented groups compared to control and 25 ppm groups during the whole period.The rate of fertility is similar in 2.5 Se supplemented and control group.

Table 5. Fertility rate in 4 dietary levels of organic Se supplementation in quail Level of Se (ppm) Fertility (%) 25 72.00c 2.5 83.33b 0.25 89.33a 0.00 83.33b LSD 4.86 Level of significance ** **, p<.01, when compared the values within the same row. values with uncommon superscripts differ significantly

4.5 Reproductive system and other vital organs The weights of liver, heart, gizzard and testis have been shown in Table 6. Se supplementation had no remarkable (p>0.05) effects on liver, gizzard and heart weight of the birds. The testis weight (Photograph 6) were found to be lower (p<0.05) in the 25 ppm organic Se supplemented groups compared to 2.5and 0.25 ppm groups. The weight of heart in the Se supplemented did not differ significantly (p>0.05). But the liver weights were found to be slightly higher (p>0.05) in 2.5 ppm and 25 ppm Se supplemented birds than that of control. The Gizzard weight were found to be slightly higher (p>0.05) in 0.25 ppm Se fed birds than the control and other two Se supplemented groups.

25 ppm 2.5ppm

0.25ppm 0.0 ppm Photograph 6.Size of the testis of quail fed on 4 dietary levels of Se supplementation

Table 6. Reproductive and other vital organs weight in Se supplemented quail

Level of Se Testis Wt Liver weight Gizzard weight Heart weight (ppm) (% to body (% to body (% to body weight) weight) weight) 25 1.44d 3.20 2.59 0.76 2.5 2.18ab 3.58 3.14 0.74 0.25 2.55a 2.98 3.46 0.82 0.00 1.98abc 2.82 2.84 0.72 LSD 0.43 0.58 0.51 0.06 Level of * NS NS NS significance *, p<.05, when compared with the values within the same row, values with uncommon superscripts differ significantly; NS, p>05, when compared the values within the same row.

Chapter V DISCUSSION Selenium (Se) is an essential trace mineral for poultry (Schwarz and Foltz, 1957) that can create toxicity at excessive dietary level. Recently, scientists have claimed the source related variability of Se-toxicity in birds, where the inorganic source has reported to exert more toxicity than the organic Se at similar level of administration (Surai, 2000). The current study has investigated the effects of organic Se incorporation in the locally made compound feed on the reproductive parameters of male breeder quail. Some major ingredients of poultry feed in Bangladesh are produced locally. But, there are reports that Bangladeshi soils are deficient of Se that might results in lower carry over to these feed ingredients (Oldfield, 2002). This assumption had been justified by better semen quality, sperm hole and fertility are found in 0.25 ppm and 2.5 ppm Se supplemented birds compared to that of basal diet (control) fed birds. The diminishing of positive effects due to Se supplementation at 25 ppm on the above parameters indicated that the critical level of organic Se supplementation in the locally made compound feed might be below 25 ppm. However, the favorable effects of organic Se supplementation on the reproductive parameters were agreed with the findings of Sotnikov and Trifonov (2003) and Hoffman and Heinz (1988). Actually, the dietary supplementation of Se in male bird exerts its effects on the reproductive parameters being altering the reproductive system functions. The favorable effects of Se supplementation to broiler on body weight gain, good feathering, carcass yield and survivability also had contradicted (Wang-YanBo, 2009) the equal survivability of all the Se supplemented birds maintaining statistically similar body weight that might be due the difference in age and genotype of the experimental birds. The testis weight and liver along with higher sperm motility%, normal sperm% and concentration at 0.25 ppm Se supplementation in spite of unchanged feed intake, body weight and survivability in the birds indicated that the Se effects was mostly oriented to the axis of reproduction than the growth and body maintenance of laying quails. The nearly similar effect of was also reported by NRC (1983) due to excess Se feeding in birds. From the present findings, it is clear that the locally made compound layer feed is needed to supplement with Se to use as the breeder ration for ensuring semen quality and sperm hole with enhanced fertility performance. The recommended dietary level of Se for poultry is 0.50 ppm (Sing and Panda, 1988). The best performance observed in the breeder quail in around 0.25 ppm Sel-Plex added ready made diet indicates that the locally available compound layer diet was deficient in Se content against the 0.50 ppm recommended level for poultry. However, the level of Sel-Plex supplementation as an organic source of Se in our locally made compound breeder diet should be below 25 ppm that is used for the breeder quail. CHAPTER VI

SUMMARY AND CONCLUSION

This experiment was conducted at Bangladesh Agricultural University (BAU), Mymensingh Poultry Farm using the wild type Japanese quail from July 2010 to Dec 2010. A total of 60 laying female and 24 adult male quails were equally and randomly allocated to 4 dietary treatments; 0.0, 0.25, 2.5 and 25 ppm organic Se supplemented diets. Each dietary treatment had 3 replications consisting of 5 female and 2 male. The birds were approximately 10-week-old at starting of the experiment having good health and similar body weight. The organic selenium (Se) used in this experiment was Sel-Plex manufactured by Altech, USA that contained 2 g organic Se in 1 kg. A trial on dietary organic selenium (Se) supplementation with locally manufactured compound feed was conducted to evaluate sperm motility, sperm morphology, sperm concentration, sperm hole, fertility performance and testis weight of Japanese quail.

The parameters evaluated on different dietary treatments were survivability of the birds, body weight gain and feed intake of the males and females together. The male reproductive capabilities were compared in terms of the sperm motility, normal sperm count and sperm concentration. The fertilizing capacity of the males was measured by counting the sperm holes on the perivitteline membrane of the eggs. The fertility rate was assessed in the eggs by incubating five days. The vital organs like liver, heart and gizzards weights were also evaluated in the Se supplemented male birds. The testis weights were measured after slaughtering the birds.

Results demonstrated that the semen quality was reduced (p<0.05) at 25 ppm Se supplemented groups without altering the feed intake, body weight and survivability of the bird. sperm motility, sperm morphology, sperm concentration, were found to be significantly higher (p<0.05) in the semen of 0.25 ppm organic Se supplemented birds. The sperm hole number were found to be markedly higher (p<0.05) at the 0.25 and 2.5 ppm organic Se supplementation. . The fertility was also higher (p<0.05 ) in 0.25 ppm and 2.5 ppm Se supplemented eggs in comparison to that of control and 25 ppm Se supplementation. The weight of testis in the Se supplemented breeder quail did not differ significantly (p>0.05).

The present study suggested that the compound breeder feed manufactured in Bangladesh is deficient of Se and its fortification with 0.25 ppm exogenous Sel- Plex is recommended for its use in breeder quail to ensure the optimum performance. However, an extensive trial on organic Se supplementation covering a range of poultry species and the available branded compound poultry feed is needed to generalize the present findings for the breeders of all poultry species.

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APPENDICES

Appendix I. NRC Nutrient requirement of breeder Japanese quail

Nutrient Requirement Protein (%) 23.00 Metabolizable energy (kcal/kg) 2900 Calcium (%) 2.40 Available phosphorus (%) 0.35 Lysine (%) 1.00 Methionine (%) 0.45 Methionine + Cystine (%) 0.75

Appendix II. Body maintenance of breeder quail fed on organic Se supplemented diet

Level of Se (ppm) Within Groups 0.837 6 0.14 Total 2.427 9 Between 1st wk BW Groups 264.061 3 88.02 3.452 0.092 Within Groups 152.98 6 25.497 Total 417.041 9 Between Last wk BW Groups 898.777 3 299.592 6.354 0.027 Within Groups 282.879 6 47.147 Total 1181.657 9 Between Difference Groups 269.199 3 89.733 5.962 0.031 Within Groups 90.299 6 15.05 Total 359.498 9

Appendix III. Semen quality of breeder quail fed on diet supplemented with organic Se

ANOVA Sum of Mean Squares df Square F Sig. %Motility Between Groups 203.433 3 67.811 5.044 0.044 Within Groups 80.667 6 13.444 Total 284.1 9 %N sperm Between Groups 119.5 3 39.833 6.548 0.025 Within Groups 36.5 6 6.083 Total 156 9 Concentration Between Groups 54493.33 3 18164.44 5.261 0.041 Within Groups 20716.67 6 3452.778 Total 75210 9

Appendix IV. Sperm hole on previtelline membrane of egg from breeder quail fed on diet supplemented with organic Se ANOVA

Spermhole Sum of Squares df Mean Square F Sig. Between Groups 403.255 3 134.418 8.598 0.000 Within Groups 422.1 27 15.633 Total 825.355 30

Appendix V. Fertility performance from breeder quail fed on diet supplemented with organic Se

ANOVA

Fertility Sum of Squares df Mean Square F Sig.

Between Groups 472 3 157.333 7.867 0.009

Within Groups 160 8 20

Total 632 11