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Studies on reproductive characteristics and artificial insemination in captive Mallard Ducks (Anas platyrhynchos) Stunden, Carolyn Elizabeth 1996

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STUDIES ON REPRODUCTIVE CHARACTERISTICS AND ARTIFICIAL INSEMINATION IN CAPTIVE MALLARD DUCKS (Anas platyrhynchos) by CAROLYN ELIZABETH STUNDEN B.Sc, The University of B r i t i s h Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1996 © Carolyn Elizabeth Stunden, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date Parch DE-6 (2/88) 11 ABSTRACT Several s t u d i e s were conducted to evaluate the use of a r t i f i c i a l insemination (AI) as a means to improve the reproductive success of hand-reared w i l d - s t r a i n M a l l a r d s (Anas platyrhynchos). The e f f e c t s of age, mate choice and i s o l a t i o n on reproductive parameters of c a p t i v e M a l l a r d s were s t u d i e d . Eighteen a d u l t and y e a r l i n g hens were allowed p h y s i c a l , v i s u a l - a u d i t o r y or a u d i t o r y only contact w i t h drakes. In a d d i t i o n , another 28 y e a r l i n g hens were placed (17 random, 15 self-chosen) w i t h y e a r l i n g drakes. Age of males was a key determinant i n M a l l a r d r e p r o d u c t i v e success, s p e c i f i c a l l y egg f e r t i l i t y . Self-chosen p a i r s performed b e t t e r than randomly assigned p a i r s and i s o l a t i o n of hens from drakes d i d not a f f e c t egg production. The data suggests that a r t i f i c i a l breeding i s needed to supplement n a t u r a l mating and increase egg f e r t i l i t y i n y e a r l i n g s . Since egg f e r t i l i t y was g e n e r a l l y low i n cap t i v e held y e a r l i n g M a l l a r d s , comparisons between c a p t i v e and f r e e - f l y i n g M a l l a r d drakes were made to assess d i f f e r e n c e s i n gonadal development. Measurements of numerous parameters i n d i c a t e d that, gonadal development was suppressed i n c a p t i v e drakes of both age cl a s s e s . For the c a p t i v e drakes, seasonal p r o f i l e s of testosterone (T) l e v e l s and semen c h a r a c t e r i s t i c s were determined i n y e a r l i n g s and a d u l t s . Plasma T concentrations increased from b a s a l l e v e l s i n March, peaked i n A p r i l and decreased to b a s a l l e v e l s i n May. The decrease i n T concentr a t i o n to basa l l e v e l occurred two weeks e a r l i e r i n y e a r l i n g s compared to that of a d u l t s . S u r p r i s i n g l y , I l l semen volume and semen concent r a t i o n were not d i f f e r e n t between the two age groups. M a l l a r d s were a r t i f i c i a l l y inseminated by modifying a technique developed f o r domesticated p o u l t r y . Egg f e r t i l i t y obtained with A l was 73%. Thus, these r e s u l t s suggest that A l may indeed be used s u c c e s s f u l l y i n propagating w i l d M a l l a r d s . Two p o u l t r y semen extenders [ B e l t s v i l l e P o u l t r y Semen Extender (BPSE) and Lake's P o u l t r y Semen Extender (LAKE)] were compared with respect to t h e i r e f f e c t s ..on sperm s u r v i v a l and sperm f e r t i l i z i n g c a p a c i t y . BPSE and LAKE worked e q u a l l y w e l l to maintain the semen f e r t i l i z i n g c a p a c i t y during short-term storage. Last of a l l , e f f e c t s of various concentrations of cry o p r o t e c t a n t s [ g l y c e r o l , ethylene g l y c o l (EG), propylene g l y c o l (PG) and sucrose] on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa were t e s t e d . The a d d i t i o n of sucrose to EG or PG on the v i a b i l i t y of frozen-thawed chicken semen was determined. F e r t i l i t y d e c l i n e d with i n c r e a s i n g concentrations of g l y c e r o l and sucrose, but not with i n c r e a s i n g concentrations of EG and PG. The a d d i t i o n of sucrose (0.2 M) d i d not improve freeze-thaw p r o t e c t i o n of EG and PG. i v TABLE OF CONTENTS Abstract i i Table of Contents. i v L i s t of Tables v i i i L i s t of Figures x L i s t of Appendices x i Acknowledgements . x i i i GENERAL INTRODUCTION 1 1.0 FACTORS AFFECTING REPRODUCTIVE PERFORMANCE IN CAPTIVE MALLARD DUCKS 6 1.1 Abstract 6 1.2 Introduction 6 1.3 Materials and Methods 9 1.3.1 Housing and Management 9 1.3.2 Egg C o l l e c t i o n and Incubation 11 1.3.3 Se l f - chosen versus Random P a i r s 12 1.3.4 S t a t i s t i c a l Analyses 12 1.4 Results 13 1.5 Discussion 25 2.0 COMPARATIVE GONADAL DEVELOPMENT BETWEEN CAPTIVE AND FREE-FLYING MIGRATORY MALLARD DRAKES 29 2.1 Abstract 29 2.2 Introduction 29 2.3 Materials and Methods 30 2.3.1 Drake C o l l e c t i o n 30 2.3.2 Age Determination 30 V 2.3.3 Histology 31 2.3.4 Sperm Counts 32 2.3.5 S t a t i s t i c a l Analysis 32 2.4 Results 32 2.5 Discussion 37 3.0 PLASMA TESTOSTERONE PROFILES, SEMEN CHARACTERISTICS AND ARTIFICIAL INSEMINATION IN CAPTIVE MALLARDS 38 3.1 Abstract 38 3.2 Introduction 38 3.3 Materials and Methods 43 3.3.1 Breeding Stock .43 3.3.2 Care and Management of Birds 43 3.3.3 Semen Co l l e c t i o n 44 3.3.4 Semen Evaluation 45 3.3.5 Laboratory Analyses 45 3.3.6 Insemination 47 3.3.7 Egg Co l l e c t i o n and Incubation 47 3.3.8 Blood C o l l e c t i o n 48 3.3.9 Radioimmunoassay (RIA) 48 3.3.10 S t a t i s t i c a l Analyses 49 3.4 Results 50 3.5 Discussion 57 4.0 COMPARISON OF TWO POULTRY SEMEN EXTENDERS 61 4.1 Abstract 61 4.2 Introduction 61 4.3 Materials and Methods 65 4.3.1 Breeding Stock 65 v i 4.3.2 In Vitro Sperm Survival 65 4.3.3 Laboratory Analyses 66 4.3.4 In Vivo Sperm F e r t i l i z i n g Capacity . . . . 68 4.3.5 Egg C o l l e c t i o n and Incubation 69 4.3.6 S t a t i s t i c a l Analyses 70 4 . 4 Results 71 4.4.1 In Vitro Sperm Survival 71 4.4.1.1 Morphological Abnormalities . . .71 4.4.1.2 Live Sperm Percentage 71 4.4.1.3 Sperm M o t i l i t y 72 4.4.2 In Vivo Sperm F e r t i l i z i n g Capacity . . . . 72 4.5 Discussion 81 5.0 EVALUATION OF VARIOUS CRYOPROTECTANTS FOR THE CRYOPRESERVATION OF AVIAN SEMEN 8 6 5.1 Abstract 86 5.2 Introduction 86 5.3 Materials and Methods 90 5.3.1 Semen C o l l e c t i o n and Evaluation 90 5.3.2 Cryoprotectant Concentration 91 5.3.3 Sucrose Addition 91 5.3.4 Freezing 92 5.3.5 Thawing and Glycerol Removal 92 5.3.6 Insemination 93 5.3.7 Egg Co l l e c t i o n and Incubation 93 5.3.8 S t a t i s t i c a l Analyses 93 5.4 Results 94 5.4.1 Glycerol Concentration 94 V l l 5.4.2 Ethylene Glycol and Propylene Glycol Concentration 95 5.4.3 Sucrose Concentration 95 5.4.4 Sucrose Addition 95 5.5 Discussion 104 Conclusions 107 Bibliography 109 Appendix 119 V l l l LIST OF TABLES Table Page 1. Body weights of yearling and adult Mallard hens over the breeding season 16 2. Body weights of yearling and adult Mallard drakes over the breeding season 17 3. Comparison of reproductive c h a r a c t e r i s t i c s of yearling and adult captive Mallard hens 18 4. Comparison of egg volume (ml) for yearling and adult captive Mallard 19 5. Comparison of reproductive parameters of d i f f e r e n t Mallard pair combinations 20 6. E f f e c t of mate choice on reproductive parameters of captive Mallard hens 22 7. E f f e c t of contact with drake on reproductive parameters of captive Mallard hens 24 8. Results of preliminary laboratory evaluation of captive Mallard semen c h a r a c t e r i s t i c s 34 9. Comparison of gonadal c h a r a c t e r i s t i c s between f r e e - f l y i n g and captive Mallard drakes 35 10. Comparison of c h a r a c t e r i s t i c s between yearling and adult f r e e - f l y i n g and captive Mallard drakes 36 11. Semen c h a r a c t e r i s t i c s of adult and yearling captive Mallard drakes 52 12. E f f e c t of Mallard drake age on plasma testosterone concentrations 53 13. F e r t i l i t y and h a t c h a b i l i t y of i n d i v i d u a l Mallard hens after a r t i f i c i a l insemination 55 14. E f f e c t of drake age (yearling verse adult) on f e r t i l i t y , h a t c h a b i l i t y and embryo mortality rates of Mallard eggs . . 56 15. E f f e c t of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE)on percentage of abnormal sperm as a function of storage time at 5°C . . . .73 ix Table Page 16. E f f e c t of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE) on percentage of dead sperm as a function of storage time at 5°C 75 17. E f f e c t of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE) on sperm m o t i l i t y score as a function of storage time at 5°C 77 18. Percent f e r t i l i t y over four weeks of fresh (Oh) and stored (24h) chicken semen di l u t e d i n B e l t s v i l l e Poultry Semen Extender (BPSE) or Lake's Poultry Semen Extender (LAKE). . .79 19. E f f e c t of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lakes Poultry Semen Extender (LAKE) on f e r t i l i t y a f t e r inseminations with fresh (Oh) and stored (24h) chicken semen 80 20. E f f e c t of d i f f e r e n t concentrations of gly c e r o l i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa 97 21. E f f e c t of d i f f e r e n t concentrations of propylene gl y c o l (PG) and ethylene glycol (EG) i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa 99 22. E f f e c t of d i f f e r e n t concentrations of sucrose i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa 101 23. E f f e c t of d i f f e r e n t combinations of cryoprotectants i n BPSE and various stages of the freezing process on the m o t i l i t y score of chicken semen 103 X LIST OF FIGURES Figure Page 1. Egg f e r t i l i t y and h a t c h a b i l i t y of y e a r l i n g - y e a r l i n g (YY), y e a r l i n g - a d u l t (YA), a d u l t - y e a r l i n g (AY) and a d u l t - a d u l t (AA) p a i r s 21 2. Egg f e r t i l i t y of c a p t i v e M a l l a r d s (17 random and 15 s e l f -chosen p a i r s ) 23 3. Plasma testosterone concentration i n r e l a t i o n to drake age 54 4. Percentage abnormal sperm as a f u n c t i o n of storage time at 5°C 74 5. Percentage dead sperm as a f u n c t i o n of storage time at 5°C 7 6 6. Sperm m o t i l i t y score as a f u n c t i o n of storage time at 5°C 78 7. E f f e c t of d i f f e r e n t concentrations of g l y c e r o l on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa (days 2-15 post-AI) 98 8. E f f e c t of d i f f e r e n t concentrations of EG and PG on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa (days 2-15 post-AI) . . . 100 9. E f f e c t of d i f f e r e n t concentrations of sucrose on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa (days 2-15 post-AI) 102 x i L I S T O F A P P E N D I C E S Appendix Page 1. D i s t r i b u t i o n of treatment groups f o r determining drake access and age e f f e c t s on reproductive performance . . . 119 2. Composition of B e l t s v i l l e (BPSE) and Lakes (LAKE) P o u l t r y Semen Extenders 120 3. A n a l y s i s of variance f o r c l u t c h s i z e f o r M a l l a r d p a i r s of d i f f e r e n t ages w i t h p h y s i c a l , v i s u a l - a u d i t o r y , or a u d i t o r y only contact 121 4. A n a l y s i s of variance f o r days to f i r s t egg from s t a r t of t r i a l f o r M a l l a r d p a i r s of d i f f e r e n t ages wi t h p h y s i c a l , v i s u a l - a u d i t o r y , or a u d i t o r y only contact 122 5. A n a l y s i s of variance f o r down deposited as a percentage of c l u t c h s i z e f o r M a l l a r d p a i r s of d i f f e r e n t ages with p h y s i c a l , v i s u a l - a u d i t o r y , or a u d i t o r y only contact . . .123 6. A n a l y s i s of variance f o r plasma testosterone c o n c e n t r a t i o n of y e a r l i n g and adult M a l l a r d drakes 124 7. A n a l y s i s of variance f o r arc sine transformed percentage of abnormal sperm a f t e r storage at 5°C i n B e l t s v i l l e or Lakes P o u l t r y Semen Extender i n comparison to neat semen. . . .125 8. A n a l y s i s of variance f o r arc sine transformed percentage of dead sperm a f t e r storage at 5°C i n B e l t s v i l l e or Lakes P o u l t r y Semen Extender i n comparison to neat semen. . . .126 9. A n a l y s i s of variance f o r sperm m o t i l i t y score a f t e r storage at 5°C i n B e l t s v i l l e or Lakes P o u l t r y Semen Extender i n comparison to neat semen 127 10. Repeated measures a n a l y s i s of variance f o r percent f e r t i l i t y of semen (fres h or stored) i n B e l t s v i l l e or Lakes P o u l t r y Semen Extender i n comparison to neat semen 128 11. A n a l y s i s of variance f o r e f f e c t of d i f f e r e n t concentrations of g l y c e r o l i n BPSE on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa 129 12. A n a l y s i s of variance f o r e f f e c t of ethylene g l y c o l and propylene g l y c o l c oncentration on chicken egg f e r t i l i t y . 131 X l l Appendix Page 13. A n a l y s i s of variance f o r e f f e c t of sucrose c o n c e n t r a t i o n on chicken egg f e r t i l i t y 133 X l l l ACKNOWLEDGEMENTS This p r o j e c t was made p o s s i b l e by funding from the North American W i l d l i f e Foundation through the D e l t a Waterfowl Research S t a t i o n and I am g r a t e f u l f o r t h e i r f i n a n c i a l support. I would l i k e to thank my supe r v i s o r , Dr. R. Rajamahendran f o r h i s guidance of t h i s p r o j e c t ; Dr. K. Cheng f o r a s s i s t a n c e w i t h s t a t i s t i c s (which was always c a r r i e d out with good humour); and the other members of my sup e r v i s o r y committee, Drs. C. Bluhm and R. Fitzsimmons f o r t h e i r comments and guidance. A d d i t i o n a l l y I would l i k e to thank: the s t a f f of the Delta Waterfowl Research S t a t i o n , the San Rafael Research A v i a r y and the U n i v e r s i t y of B r i t i s h Columbia Avian Research Center, p a r t i c u l a r l y C. N i c h o l s and K. Shekhtman, f o r t h e i r e x c e p t i o n a l care and management of the b i r d s ; the Department of Animal Science l a b o r a t o r y t e c h n i c i a n s and graduate students who gave of t h e i r time; M. MacCluskie f o r sharing s e l f - c h o s e n M a l l a r d p a i r data; and Dr. F. Rohwer f o r h a r v e s t i n g drakes. F i n a l l y I would l i k e to acknowledge my fa m i l y whose support, patience and encouragement have been paramount i n the completion of t h i s p r o j e c t . x i v In Memoria Kathleen Marie Stunden "An o b s t a c l e i s o f t e n an unrecognized opportunity." 1 GENERAL INTRODUCTION The M a l l a r d duck (Anas platyrhynchos) i s a popular game b i r d which i s widely d i s t r i b u t e d i n North America. Loss of i t s n a t u r a l h a b i t a t has lead to a slow d e c l i n e i n the po p u l a t i o n of t h i s species (Gottschalk and Studholm, 1970; Johnson and Shaffer, 1987; Greenwood et a l . , 1995). Although the l a r g e s c a l e r e l e a s e of game-farm M a l l a r d s have been proposed as a means to r e p l e n i s h w i l d stocks (Cheng et a l . , 1978), there i s concern that game-farm Ma l l a r d s are g e n e t i c a l l y d i f f e r e n t from the w i l d p o p u l a t i o n (Cheng et a l . , 1980). To help r e e s t a b l i s h the w i l d p o p u l a t i o n , the Delta Waterfowl and Wetlands Research S t a t i o n began i n 1990 an extensive program of c a p t i v e propagation and release of w i l d - s t r a i n M a l l a r d s . However, w i l d - s t r a i n ducks o f t e n do not achieve t h e i r f u l l r e p roductive p o t e n t i a l under c a p t i v e c o n d i t i o n s (Cheng et a l . , 1980; P h i l l i p s and van Tienhoven, 1960; P h i l l i p s , 1964; Bluhm et a l . , 1983). The primary focus of t h i s t h e s i s , t h e r e f o r e , i s to evaluate the use of a r t i f i c i a l insemination (Al) as a means to improve the reproductive success of ca p t i v e M a l l a r d s . A l i s a technique by which semen i s c o l l e c t e d from the male, evaluated, processed, and deposited i n the female's reproductive t r a c t . Since the development of a simple and e f f e c t i v e semen c o l l e c t i o n technique by Burrows and Quinn (1937), A l has been p r a c t i c e d i n the commercial breeding of domesticated fowl such as the turkey, chicken and duck. Reports of A l i n w i l d ducks are c u r r e n t l y not a v a i l a b l e . The i n i t i a l concerns r e l a t e d to designing an A l program f o r 2 hand-reared wild-stain ducks were: 1) determining the age of maximum response, 2) the ef f e c t of i s o l a t i o n of the sexes on egg production, 3) pair bonding, and 4) the ef f e c t of c a p t i v i t y on reproduction. Mallards w i l l breed as yearlings (Batt and Prince, 1978; Krapu and Doty, 1979), but productivity of yearlings i s less than that of adults (C. Bluhm personal communication). Poor f e r t i l i t y i n yearling pairs i s often attributed to copulatory inexperience (Curio, 1982; Bruggers et a l . , 1981), which could be over come by the use of AI. The monogamous relationship of pair-bonding i s a feature of the Mallard mating system (McKinney, 1985; Bluhm, 1985). When the present study was i n i t i a t e d , i t was not known what e f f e c t i s o l a t i o n of the sexes would have on egg production. In Canvasback ducks, hens only lay eggs after they have formed a strong pair-bond (Bluhm, 1985) and domesticated turkey hens w i l l lay fewer eggs when is o l a t e d from males (Jones and Leighton, 1987). For th i s work therefore, i t was hypothesized that females who are only v i s u a l l y exposed to or completely, i s o l a t e d from drakes would lay fewer eggs than hens with physical exposure to drakes. As well, hens that were placed with self-chosen drakes would be more productive than hens placed randomly with drakes. Failure of P i n t a i l and Mallard hens to reproduce i n c a p t i v i t y has previously been reported ( P h i l l i p s and van Tienhoven, 1960). P h i l l i p s and van Tienhoven (1960) found a lack of ovarian development i n captive hens, which was related to i n h i b i t e d p i t u i t a r y gonadotropin output. No work was done on Mallard drakes. 3 In the present study, i t was hypothesized that c a p t i v i t y a lso prevents the gonads of M a l l a r d drakes from maturing normally. AI i n b i r d s has been r e s t r i c t e d to the use of f r e s h l y d i l u t e d semen. This i s p r i m a r i l y because the current methods of f r e e z i n g semen r e s u l t i n poor sperm s u r v i v a l . Experience w i t h domesticated p o u l t r y has shown that u n d i l u t e d , f r e s h semen h e l d at ambient temperatures w i l l d e t e r i o r a t e w i t h i n the f i r s t hour of semen c o l l e c t i o n depending on i n i t i a l q u a l i t y (Garren and Shaffner, 1952; Bootwalla and M i l e s , 1992). The physio-chemical nature of extenders are designed to ensure an i d e a l environment in vitro that w i l l m a intain semen f e r t i l i z i n g a b i l i t y . In general, some b a s i c c r i t e r i a f o r extenders i n c l u d e : proper pH, adequate b u f f e r i n g , and an energy source f o r the s u r v i v a l of spermatozoa (Renganathan, 1982). Despite the f a c t that extensive work has been c a r r i e d out towards p e r f e c t i n g an i d e a l p o u l t r y semen extender (Sexton et a l . , 1980; Tajima et a l . , 1989; Lake and Ravie, 1979), l i t t l e work has been reported on d i r e c t comparisons of d i f f e r e n t extenders. The a p p l i c a t i o n of AI makes p o s s i b l e the use of frozen semen. Cryopreservation, the f r e e z i n g of semen at extremely low temperatures, provides greater f l e x i b i l i t y i n handling of semen, allows the c r e a t i o n of gene banks, and f a c i l i t a t e s breeding between g e o g r a p h i c a l l y separated i n d i v i d u a l s . These aspects of c r y o p r e s e r v a t i o n would be b e n e f i c i a l to a program of captive propagation. Many researchers have reported research i n t o the most s u i t a b l e cryoprotectant f o r p o u l t r y semen (Sexton, 1975; Lake and Ravie, 1984; Maeda, 1984; P h i l l i p s , 1993). The s e l e c t i o n of a s u i t a b l e cryoprotectant i s the most c r i t i c a l aspect towards the s u r v i v a l of spermatozoa during the process of c r y o p r e s e r v a t i o n . The use of g l y c e r o l at a c o n c e n t r a t i o n of 8% i s most common and provides good freeze p r o t e c t i o n . However, the c o n c e n t r a t i o n of g l y c e r o l must be reduced to l e s s than 2% a f t e r thawing and before insemination otherwise f e r t i l i t y i s s i g n i f i c a n t l y reduced (Lake and Ravie, 1984). The c o n s t r a i n t s of time and equipment make the r e d u c t i o n of g l y c e r o l concentration i m p r a c t i c a l under f i e l d c o n d i t i o n s . A cryoprotectant i s needed that provides equal f r e e z i n g p r o t e c t i o n , but doesn't lower sperm f e r t i l i z i n g c a p a c i t y . Propylene g l y c o l (PG) and ethylene g l y c o l (EG) have been suggested as a l t e r n a t i v e s to g l y c e r o l , but they do not p r o t e c t as w e l l against freeze damage (Lake and Ravie, 1984; Maeda, 1984). Many reports have claimed that the a d d i t i o n of c e r t a i n carbohydrates, such as sucrose, p r o t e c t b i o l o g i c a l membranes of cryoprotected mammalian embryos (Schneider and Mazur, 1984; Takeda et a l . , 1987; S a i t o et a l . , 1994). The e f f e c t of sucrose i n combination w i t h ethylene g l y c o l or propylene g l y c o l on the f r e e z i n g of avian semen has not p r e v i o u s l y been i n v e s t i g a t e d . The general layout of t h i s scheme of research i s as f o l l o w s : Chapter one focuses on f a c t o r s r e l a t e d to the design of an A l program f o r ducks, s p e c i f i c a l l y age, mate choice and i s o l a t i o n of the sexes. Comparative gonadal development between c a p t i v e and f r e e - f l y i n g w i l d M a l l a r d drakes i s o u t l i n e d i n chapter two, w i t h an o b j e c t i v e to determine whether c a p t i v i t y a f f e c t s reproduction. Chapter three centres on plasma testosterone p r o f i l e s and semen 5 c h a r a c t e r i s t i c s of the ca p t i v e drakes. This chapter a l s o i n v e s t i g a t e s the use of AI as an a l t e r n a t i v e management technique i n the r e a r i n g of cap t i v e M a l l a r d s . Chapter four compares two commonly used p o u l t r y semen extenders, B e l t s v i l l e P o u l t r y Semen Extender and Lake's P o u l t r y Semen Extender, w i t h respect to t h e i r e f f e c t s on sperm s u r v i v a l and sperm f e r t i l i z i n g c a p a c i t y . I t i s hoped that one of these extenders could be modified f o r futu r e use wi t h wild-type duck semen. F i n a l l y , chapter f i v e examines the e f f e c t of various concentrations of g l y c e r o l , EG, PG and sucrose on the f e r t i l i z i n g a b i l i t y of chicken spermatozoa and the e f f e c t of sucrose a d d i t i o n to PG or EG i n BPSE on the s u r v i v a l of frozen-thawed chicken spermatozoa. 6 1.0 FACTORS AFFECTING REPRODUCTIVE PERFORMANCE IN CAPTIVE MALLARD DUCKS 1.1 ABSTRACT Propagation of w i l d - s t r a i n Mallards (Anas platyrhynchos) i n c a p t i v i t y i s hindered by low egg f e r t i l i t y . Therefore, the effects of age, mate choice and i s o l a t i o n on reproductive parameters of captive Mallards were studied. Eighteen adult and yearling hens were allowed physical, visual-auditory or auditory only contact with drakes. In addition, 28 yearling hens were placed (17 random, 15 self-chosen) with yearling drakes. Yearling hens weighed less than adult hens at the beginning of the breeding season, but no differences were found between t h e i r i n i t i a l clutch size (13.2 ± 1.9, 12.9 + 1.9), i n i t i a l egg volume (49.46 ± 0.40, 49.24 + 0.34) and clutch number (1.1 ± 0.2, 1.5 ± 0.2). Yearling pairs had lower egg f e r t i l i t y (7%) than adult pairs (80%) . Egg f e r t i l i t y was higher (51% vs. 21%) i n self-chosen pairs than i n randomly assigned pa i r s . Such random pairs d i d not reconcile t h e i r differences over the reproductive season. Isolation of hens did not influence egg production. The data suggest that a r t i f i c i a l insemination i s needed to supplement natural mating and increase egg f e r t i l i t y i n yearlings. 1.2 INTRODUCTION The North American Mallard duck population has been declining in recent years, due to loss of habitat and/or human a c t i v i t i e s 7 (Greenwood et a l . , 1995; Johnson and Shaffer, 1987). In the early 1980's, the Mallard breeding population was at i t s lowest l e v e l since population surveys began i n 1955 (Greenwood et a l . , 1995). This prompted the Delta Waterfowl and Wetlands Research Station i n 1990 to begin an extensive program of captive propagation and release of wild Mallards onto vacant restored or enhanced habitat. In contrast to domesticated farm animals, most of the emphasis on reproducing wild birds i n c a p t i v i t y has been on natural breeding. Unfortunately, even i n c a r e f u l l y controlled breeding environments where birds achieve f u l l reproductive condition, many f a i l to reproduce (Gee and Temple, 1978). Failure to reproduce i n c a p t i v i t y i s often linked to the f a i l u r e of wild-caught females held in c a p t i v i t y to lay eggs ( P h i l l i p s and van Tienhoven, 1960). However, in the case of hand-reared ducks hatched from eggs taken i n the wild, the f a i l u r e i s linked to poor male f e r t i l i t y (Cheng et a l . , 1980) . Often a pair w i l l engage i n a l l aspects of courtship behaviour, but s t i l l f a i l to lay f e r t i l e eggs (Gee and Temple, 1978) . Although age can play a role i n reproductive performance (Batt and Prince, 1978), low f e r t i l i t y i n yearling pairs i s often attributed to lack of experience or delayed physical maturation (Curio, 1982). Bruggers et a l . (1981) found the pre- and post-copulatory behaviour of yearling Mandarin ducks was more variable than adults. As well, he found that the mounting time was twice as long for yearling drakes due to t h e i r i n a b i l i t y to position themselves properly. It was therefore postulated that reproductive 8 performance would be improved by p a i r i n g experienced adult drakes with inexperienced yearling hens. In addition, i t was hypothesized that birds that are allowed to choose t h e i r own mate would be more successful than randomly assigned p a i r s . In captive Canvasback ducks, i t has been found that females are highly discriminatory i n t h e i r mate preferences and egg-laying i s dependent on allowing hens free choice of mate (Bluhm, 1985). A r t i f i c i a l insemination (Al) has been suggested as a means to improve w i l d - s t r a i n Mallard egg f e r t i l i t y . A l i s a technique by which semen i s c o l l e c t e d from the male, evaluated, processed and deposited within the female's reproductive t r a c t . A l i s used i n the commercial production of poultry, e s p e c i a l l y turkeys, allowing females to be housed separately from males. Wild Mallards are monogamous (McKinney, 1985) and v i s u a l and auditory displays are an important component of avian courtship (Lehrman, 1959). However, i t was not known what eff e c t i s o l a t i o n of the sexes would have on reproductive performance. Studies concerning the influence of s o c i a l displays on ovarian development i n captive Mallards are li m i t e d . Desforges (1971) found the sight of a drake was not necessary for ovulation to occur; however, a l l Mallard hens used had previously been through at least one laying cycle. Other studies on the effects of i s o l a t i o n of the sexes i n highly domesticated species also have c o n f l i c t i n g r e s u l t s . One study found that egg production i n turkeys was s i g n i f i c a n t l y higher for females under natural mating conditions than for those v i s u a l l y exposed to or completely i s o l a t e d from males (Jones and Leighton, 1987). 9 However,, i n two other reports studying chickens, the presence of sexually active males did not af f e c t egg production (Tarapovski, 1977 and Renden et a l . , 1982). Thus, i f AI i s to be practiced i n wild Mallards, one should know the e f f e c t of the separation of hens from drakes during the breeding season would have on egg laying and nesting behaviour. Age effects, i f present, and incompatible temperaments could also be overcome by AI. In summary, thi s study examined whether ce r t a i n factors, p o t e n t i a l l y important to the implementation of an AI program, influence reproductive performance i n captive Mallards. Sp e c i f i c objectives of this study were to determine i f : 1. Reproductive performance of yearling hens i s improved by p a i r i n g with adult drakes. 2. Self-chosen pairs are more successful than randomly assigned p a i r s . 3. Physical, visual-auditory, or auditory only contact with drakes affects reproductive performance of hens. 1.3 MATERIALS AND METHODS 1.3.1 Housing and Management Genetically wild Mallards used i n t h i s study were hatched and hand-reared from eggs salvaged from a g r i c u l t u r a l f i e l d s i n south central Manitoba. Birds were reared and maintained using standard procedures outlined by Bluhm et a l . (1993) at the Delta Waterfowl Research Station (50°11'N, 98°19'W) i n Delta, Manitoba. A l l birds were kept.over the winter within same age groups with exposure to 10 n a t u r a l d a y l i g h t . Two weeks before the expected onset of egg l a y i n g (May 1), t h i r t y - s i x hens (18 y e a r l i n g s and 18 adults) were randomly assigned to p h y s i c a l , v i s u a l - a u d i t o r y or a u d i t o r y only treatment groups. P a i r s were f u r t h e r d i v i d e d i n t o adult (?) - a d u l t (d") , adult ( ? ) -y e a r l i n g ( c f ) , y e a r l i n g (?)-adult (o") , and y e a r l i n g ( ? ) - y e a r l i n g (o*) p a i r s to examine age e f f e c t s on reproduction (Appendix 1). Ducks were weighed to the nearest 10 grams at: (1) the s t a r t of the t r i a l , (2) the end of each c l u t c h and (3) the end of the t r i a l u sing a Pesola s p r i n g s c a l e . Hens i n the p h y s i c a l contact group were placed i n i n d i v i d u a l breeding pens with a drake. The i n d i v i d u a l breeding pens were approximately 2 m x 2.5 m; o n e - t h i r d swimming water and two-thirds dry concrete base. I n d i v i d u a l pens f o r v i s u a l - a u d i t o r y contact groups were f u r t h e r d i v i d e d with an inner w a l l of nylon mesh. This allowed the hen and drake to see and hear each other, but prevented p h y s i c a l contact. Pens f o r the a u d i t o r y only groups were d i v i d e d w i t h s o l i d wood preventing p h y s i c a l and v i s u a l contact between the hen and drake. A l l ducks were fed ad libitum, a high p r o t e i n commercially a v a i l a b l e duck chow, supplemented w i t h wheat, g r i t and oyster s h e l l . Hens were given access to s t r a w - f i l l e d wooden nest boxes. At the time of breeding, many adult hens had l a i d eggs the previous year. In c o n t r a s t , y e a r l i n g hens had no previous breeding experience. 11 1.3.2 Egg c o l l e c t i o n and Incubation Nest boxes were checked each morning f o r new eggs. New eggs w i t h i n a c l u t c h were numbered and the length and diameter measured wit h a Mututoyo v e r n i e r metric c a l i p e r then returned to the nest. Egg volume was c a l c u l a t e d using the formula from Romanoff and Romanoff (1949) where egg volume equals 0.526(length)(diameter) 2. Clutches were l e f t i n the nest box u n t i l no new eggs were l a i d f o r f i v e consecutive days. At t h i s time, the eggs were removed and the nest box m a t e r i a l was replaced. Some hens were continuous l a y e r s . In such cases, when. more than 15 eggs were l a i d , the newest s i x eggs were l e f t i n the nest and the remaining eggs removed. The appearance of down w i t h i n the nest was a l s o monitored and recorded as an i n d i c a t i o n of n e s t i n g behaviour. A f t e r completion of the f i r s t c l u t c h , a l l hens were allowed p h y s i c a l access to a drake. Eggs from hens w i t h p h y s i c a l contact w i t h drakes, were washed i n a c h l o r i n a t e d egg washing s o l u t i o n (Kleenegg), candled and incubated h o r i z o n t a l l y i n a Humidaire forced d r a f t incubator. Eggs were candled on day 7, 14 and 23 to determine whether development was proceeding. On Day 23, v i a b l e eggs were t r a n s f e r r e d to i n d i v i d u a l hatching compartments of a Humidaire hatcher. At the time of f i r s t c a n d l i n g , eggs l a c k i n g development were opened and c l a s s i f i e d as e a r l y dead or i n f e r t i l e , using the technique o u t l i n e d by Fant (1957). This method i s based on the observation that the yolks of i n f e r t i l e eggs incubated f o r seven days remain c l e a r and unblemished, while a dead embryo i n the g a s t r u l a t i o n stage causes the yolk to mottle. Dead embryos detected during a d d i t i o n a l 12 candlings were recorded, aged by the technique of Caldwell and Snart (1974) and c l a s s i f i e d as early-dead (<7 days), mid-dead (8-18 days) or late-dead (>19 days). M o r t a l i t i e s were also c l a s s i f i e d into one of seven malposition groups (Fant, 1957). 1.3.3 Self-chosen versus Random Pairs An additional 64 yearlings were placed i n i n d i v i d u a l breeding pens as self-chosen pairs (15) or randomly assigned pairs (17). Eggs were removed from the nest boxes d a i l y and replaced with p l a s t i c dummy eggs to maintain the normal laying pattern. Pulled eggs were used by another researcher for a d i f f e r e n t study. Therefore, only date to f i r s t egg, clutch size and percent f e r t i l i t y were analyzed. 1.3.4 S t a t i s t i c a l Analyses Data for drake contact and pairs of d i f f e r e n t ages (date to f i r s t egg from sta r t of t r i a l , clutch size, and down deposited as a percent of clutch size) were analyzed by two-way analysis of variance for unbalanced data sets ( L i t t e l l , 1991). The lea s t -squares method i n the General Linear Models, S t a t i s t i c a l Analysis 6.04 ( L i t t e l l , 1991) was used to test for differences between main e f f e c t s . The two-way c l a s s i f i c a t i o n model was: Yljk = u + Ts + + TAsj + e i j k where i - 1...3; j = 1..4 Y i j k = equals the kth observation for the ( i , j ) t h c e l l . u = population mean. T i = e f f e c t of drake contact. 13 Aj = e f f e c t of p a i r age. TAi:J = i n t e r a c t i o n of main e f f e c t s . e i j k = random e r r o r a s s o c i a t e d with i n d i v i d u a l observations. T-tests were used to compare t r a i t means between self-chosen and random p a i r s , and adult and y e a r l i n g M a l l a r d s . Percent f e r t i l i t y was defined as the number of f e r t i l e eggs d i v i d e d by the t o t a l number of eggs incubated. Percent h a t c h a b i l i t y was defined as the number of eggs hatched d i v i d e d by the number of f e r t i l e eggs s e t . Contingency t a b l e s i n chi-square a n a l y s i s were used to t e s t e f f e c t s on f e r t i l i t y . Due to the l i m i t e d number of observations, h a t c h a b i l i t y and embryo m o r t a l i t i e s were not s t a t i s t i c a l l y analyzed. Unless otherwise s t a t e d , a l l comparisons were made at the 5% l e v e l of s i g n i f i c a n c e (P<0.05). 1.4 RESULTS At the s t a r t of the breeding season, adu l t hens weighed s i g n i f i c a n t l y (P<0.05) more than y e a r l i n g hens; adu l t hens averaged lOOg heavier than y e a r l i n g s (Table 1). This trend was al s o present when comparing only l a y i n g y e a r l i n g hens w i t h l a y i n g a d u l t hens. By the end of the f i r s t c l u t c h , the weight d i f f e r e n c e was no longer s i g n i f i c a n t and remained so through to the end of the breeding season. Y e a r l i n g drakes weighed the same as adul t drakes (Table 2). The average number of clutches f o r y e a r l i n g and adult p a i r s was 1.1 and 1.5 cl u t c h e s , r e s p e c t i v e l y (Table 3). I n i t i a l c l u t c h s i z e , down deposited and d u c k l i n g hatch mass d i d not d i f f e r s i g n i f i c a n t l y between y e a r l i n g and ad u l t p a i r s . Egg volume (Table 4) d i d not d i f f e r between y e a r l i n g and adult 14 hens (P>0.05). However, pair-wise comparisons between clutches revealed that second clutch (B) eggs were s i g n i f i c a n t l y bigger than f i r s t clutch (A) and t h i r d clutch (C) eggs. LSMeans and analysis of variance on clutch size are presented i n Table 5 and 7 and Appendix 3. I n i t i a l clutch sizes l a i d by pairs of d i f f e r e n t ages were not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). A l l hens that f a i l e d to lay eggs were yearlings. Egg production for hens with physical contact with drakes was not s i g n i f i c a n t l y d i f f e r e n t from hens with visual-auditory or auditory only contact (P> 0.05). LSMeans and analysis of variance on reproductive parameters of Mallard pair combinations of d i f f e r e n t ages with physical, visual-auditory or auditory only contact are presented i n Tables 5 and 7, Figure 1 and, Appendix 4 and 5. Adult hens did not lay e a r l i e r than yearling hens. In addition, hens with physical contact did not i n i t i a t e egg laying sooner than hens with v i s u a l -auditory or auditory only contact. No s i g n i f i c a n t differences were observed between treatment groups for down deposited as a percentage of clutch s i z e . Figure 1 summarizes f e r t i l i t y data on 193 i n d i v i d u a l eggs from d i f f e r e n t pair combinations. Chi-square tests performed on data indicated s i g n i f i c a n t differences between expected and observed values [%2 - 52.28, N = 193). Yearling pairs had lower f e r t i l i t y than adult pairs; however, f e r t i l i t y could be improved dramatically by pa i r i n g a yearling hen with an adult drake. Figure 2 summarizes egg f e r t i l i t y data for self-chosen and randomly assigned yearling p a i r s . Yearling hens placed with s e l f -chosen mates had f e r t i l i t y r a tes twice as high as random p a i r s (%2 = 27.09, N = 295) . Random p a i r s d i d not r e c o n c i l e t h e i r d i f f e r e n c e s over the reproductive season. Egg f e r t i l i t y was s i g n i f i c a n t l y lower i n second cl u t c h e s (x 2 = 21.29, N = 160) . I n i t i a l body weights, days to f i r s t egg, c l u t c h s i z e s and t o t a l eggs l a i d were not s i g n i f i c a n t l y d i f f e r e n t (P>0.05) (Table 6). 16 TABLE 1. Body weights of yearling and adult Mallard hens over the breeding season Time Y e a r l i n g Adult S t a r t of t r i a l * ( a l l hens) S t a r t t r a i l * ( l a y i n g hens) End of f i r s t c l u t c h End of t r i a l * ( l a y i n g hens) 958 ± 20 (18) 960 ± 25 (13) 938 ± 14 (13) 999 + 21 (13) 1058 ± 18 (18) b 1058 ± 18 (18) b 950 ± 22 (18) a 1021 ± 16 (18) a Values are presented as mean ± SEM (n) " T r i a l s t a r t e d May 1 T r a i l ended J u l y 9 Means in the same row with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P < 0 .05) . Di f ferences in n are due to m o r t a l i t i e s or hens that d i d not lay . 17 TABLE 2 . Body weights of yearling and adult Mallard drakes over the breeding season Time Yearling Adult Start of t r i a l * 1097 ± 32 (18) a 1137 ± 32 (18) a End of t r i a l " 1081 ± 20 (16) a 1134 ± 17 (15) b Values are presented as mean + SEM (n) " T r i a l s t a r t e d May 1 T r a i l ended J u l y 9 Means in the same row with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P < 0 .05) . D i f f erences in n are due to m o r t a l i t i e s , or drakes pa ired to hens that d i d not l a y . 18 TABLE 3. Comparison of reproductive characteristics of yearling and adult captive Mallard hens Age Parameter Yearling Adult Number of clutches 1.1 ± 0 .2 (18) a 1.5 ± 0 .2 (18) a I n i t i a l clutch size" 13.2 ± 1 .9 (13) a 12.9 + 1 .9 (18) a Down deposited* 65.7 + 5 .5 (10) a 64.0 + 5 .5 (16) a Duckling hatch wt.(g) 35.91 + 0 74 (12~)a 36.16 + 0 40 (45 A) a Values are presented as means ± SEM (n). Means w i t h i n rows with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P > 0 .05) . "laying hens on ly • as a percentage of c l u t c h s i z e , "n = hatched duck l ing sample s i z e . 19 TABLE 4. Comparison of egg volume (ml) for yearling and adult captive Mallards Age C l u t c h Y e a r l i n g A d u l t C l u t c h A 49. 46 + 0.40 (171) a 49. 24 + 0.34(233) a C l u t c h B 52. 46 ± 0.64 (59) b 53. 18 ± 0.56(58) b C l u t c h C 51. 91 ± 0.90(12) a 52. 60 ± 0.88 (13) a Values are presented as means + SEM (n). Means w i th in rows and columns with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P > 0 .05) . o\o o\o 1—1 M ^ H-ft) fD rt s LT rt l-S H-ft) H- &> hi O rt ft) rt a i-s tr H- h-1 fD i h-J o fD r-1 1 a <^ a fD O D, fD 1 H- I—1 CD ft) a o C OJ a ro rt a ft> B O a o t r H ri- CO ft) p-1—1 N fD rr o o o o o o o CO LO o U) o 1—> o NJ CO CO co o o o o o o o O O r-" \ \ o O O h-» — o ^1 NJ Cn Cn o • • O O o CO h-1 o >fc» .fc. .fc. *•—* > o <x> O CO O Cn Cn Cn ~— .fc. CO .fc. NJ a co rt O fD XJ iX) iX) r-> CO 1+ 1+ NJ NJ .fc. Cn IX) - J NJ IX) Cn o CT> IX) h-' o co 1+ 1+ . . —~ NJ NJ —1 \ o .fc. XJ h-' o CO _ ^ ^ CO X) cr ai f—> 1—> co Cn o • • • o IX> 1+ 1+ „—^ o 1—1 NJ NJ o • • .fc. NJ o cn . -<X) <X> OJ -— — tr 1—1 1—1 CO NJ Cn .fc. t—» • • • NJ o co 1—1 1+ 1+ NJ NJ .fc. NJ IX) VX> TJ ft) l-S ft) fD rt fD -< K fD fD ft) ft) i-i 1-5 I— !_. H . 3 X5 XJ -to q. fD ft) > l-i CL I—' £ H-I—1 3 rtX} fD a ft) C i-i M h-1 rt H-!3 +o X) q. ^ > c i—1 I—1 rt rt 40 q. CD l-S O i-3 S W O 0 0) H H-to O 3 O 21 FIGURE 1. Egg fertility and hatchability of yearling-yearling (YY), yearling-adult (YA), adult-yearling (AY) and adult-adult (AA) pairs. YY YA AY AA PAIR COMBINATIONS (hen,drake) Bars with different letters are different (P<0.05) Hatchability = fertilie eggs only 22 TABLE 6. Effect of mate choice on reproductive parameters of captive Mallard hens Parameter Random Self-chosen Hen i n i t i a l body weight (g) Days to f i r s t egg* Number of clut c h e s F i r s t c l u t c h s i z e T o t a l eggs l a i d 991 ± 17 (17) a 14.4 ± 1.7 (14) 1.4 ± 0.2 (17) 10.1 ± 1.7 (17) 16.8 ± 3.0 (17) 994 ± 18 (15) a 13.10 + 2.1 (10) 1.2 ± 0.3 (15) 8.3 ± 1.7 (15) 13.5'± 3.0 (15) Values are presented as group means ± SEM (n) . . * Days from s t a r t of t r i a l . Means w i th in rows with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P > 0 .05) . 23 FIGURE 2. Egg fertility of captive Mallards (15 self-chosen and 17 randomly assigned pairs) ^9 H Z LU o rr LU Q. E H 1st Clutch • 2nd Clutch Self-Chosen Random MATE CHOICE Bars with different letters are different (P<0.05) 24 o I u - — - ro . — - CM . — . H CO >1 o I~1 4-) Cs] Cs] Ul • rr 11 +1 +1 +1 Cs] LO cn OO rH sD tn ro ro i—1 Cs] O i—1 i—l i—i rd O rH • • • CO Cs] Cs] -H > +1 +1 +1 CT) rH m ^ cn •«—i rH sD 10 ro ro t-H Cs] o i—1 i—1 rH i—1 rd O O i—I •H • • • CO Cs] CM r— >1 +1 +1 +1 CM cn cn o CO cn rH rH sO *Cr> T i cn CU 4-> CD -H N CO M I—1 •H O O) CO -U o CO 0) 4-) 4=: TS g o rd CO U >i •3-rd rd i—1 O CM Q CJ Q 25 1.5 DISCUSSION Mallards usually breed as yearlings (Batt and Prince, 1978; Krapu and Doty, 1979). However, several studies have reported that yearlings do not reproduce as well as older more experienced birds (Batt and Prince, 1978; Blohm, 1979). The findings of t h i s study confirm that age i s a key determinant to reproductive success, s p e c i f i c a l l y egg f e r t i l i t y . In the present study, both adult hens and yearling hens paired with yearling drakes exhibited low f e r t i l i t y . Reproductive performance was improved dramatically by p a i r i n g yearling hens with adult drakes; however, f e r t i l i t y rates for yearling hens paired with adult drakes did not increase to the degree obtained with adult hens. Poor reproductive output therefore, i n yearling birds cannot be contributed s o l e l y to the yearling drake. Conventionally, poor reproductive performance by yearlings i s attributed to inexperience, delayed physical development, or both (Curio, 1982; Bruggers et a l . , 1981; Blohm, 1979; Hamann et a l . , 1987). If lack of experience and f a i l u r e to copulate contributed to the poor reproductive performance of yearlings, then under conditions of AI, f e r t i l i t y rates of yearling hens inseminated with viable semen should be improved. A l t e r n a t i v e l y , i f sexual maturity i s delayed i n yearlings, t h i s should be r e f l e c t e d i n c i r c u l a t i n g androgen levels as the timing and extent of drake gonadal development i s correlated to c i r c u l a t i n g testosterone concentrations (Paulke and Haase, 1978). A delay i n sexual maturity may help optimize l i f e t i m e reproduction (Curio, 1982; Hamann et a l . , 1987). This aspect of the 26 reproductive physiology i n Mallards warrants further consideration. Body condition of the hen i s an important factor influencing reproductive success. Blohm (1979) found the tendency of free-f l y i n g female Gadwalls to renest i s greater i n adults. He speculated that t h i s tendency to renest was related to i n i t i a l differences i n breeding condition. Exhaustion of energy reserves during the i n i t i a l laying period may have discouraged further breeding attempts i n yearling hens. The results of the present study do not support Blohm's hypothesis i n captive Mallards. At the beginning of the breeding season, adult hens averaged lOOg heavier than yearling hens, but no difference i n clutch number or clutch size were observed. The results, however, do agree with the findings of Batt and Prince (1978) who found that under captive conditions where food i s available ad libitum, yearling Mallard females were capable of the same reproductive p o t e n t i a l as older birds weighing s i g n i f i c a n t l y more at the onset of breeding. The number of eggs l a i d by captive Mallards was found by Krapu and Doty (1979) to be d i r e c t l y related to the quantity and qu a l i t y of food available during the laying period. In the f i e l d , yearling hens entering the breeding season i n suboptimal physical condition may be less able to compete with older birds for prime nesting habitat and high qu a l i t y food sources (Batt and Prince, 1978; Dzubin, 1969) . Thus the reproductive potential of yearling hens may be reduced under these conditions. In the present study, a l l hens f a i l i n g to lay eggs during the breeding season were yearlings. Similar observations have been made 27 by Hamann et at. (1987) and Curio (1982) who speculated that some yearlings may r e f r a i n from entering the breeding population to be i n better condition upon t h e i r f i r s t breeding attempt and to enhance t h e i r prospects of future reproduction. E a r l i e r nesting by adults has been observed i n Mallards (Coulter et a l . , 1968), Wood ducks (Bellrose et a l . , 1964/ Rohwer et a l . , 1991) and Gadwalls (Blohm, 1979) and i s thought to contribute to larger clutch s i z e . No differences i n the date to f i r s t egg or clutch size were observed between adult and yearling hens i n t h i s study. It cannot be c e r t a i n that adult hens did not begin egg laying sooner than yearlings as egg laying by individual birds was not monitored u n t i l after birds were placed i n individual outdoor breeding pens. This make comparisons of c l u t c h s i z e between adult and yearling hens suspect. Down generally appear i n the nest after 65% of a clutch was l a i d . This agrees with work done by Caldwell and Cornwell (1975) who found that Mallards hens w i l l incorporate down plucked from t h e i r breast into the nest bowl between the fourth and sixth egg. They found that down was an important i n s u l a t i n g source for maintaining egg temperature when the nest was unattended. Ducklings hatched from eggs l a i d by yearling hens weighed the same as those hatched from eggs l a i d by adult hens. This i s i n contrast to the results of Calogeros (1996) who found that ducklings hatched from eggs l a i d by yearling hens weighed less than those hatched from eggs l a i d by adults, even aft e r correction for egg s i z e . 28 The results demonstrate that self-chosen pairs perform better than randomly assigned pairs during the breeding season i n terms of egg f e r t i l i t y . Although, Bluhm (1985) found that only Canvasback females of self-chosen pairs l a i d eggs, i n Mallards i n i t i a t i o n of egg laying was not dependent on allowing free-choice of mate. There was no e f f e c t of physical, visual-auditory or auditory only hen contact with drakes on egg production. This i s consistent with results reported by Desforges (1971) for Mallards, and Renden and Pierson (1982) for chickens. These observations suggest that hens can be housed separately from drakes to simplify AI, without any adverse effects on egg production. This i s i n contrast to Canvasbacks, where i t was found that courtship of hens by t h e i r self-chosen mates was important i n stimulating ovarian development and egg laying (Bluhm, 1985). In view of the a b i l i t y to r e a d i l y reproduce i n c a p t i v i t y without the need for male courtship, i t i s easy to understand why the Mallard i s the ancestor of a l l 17 breeds of domesticated duck except the Muscovy {Cairina moschata) (Batt and Prince, 1978). In summary, data presented i n this chapter suggests that there i s a need for the use of AI to supplement natural mating i n yearling pairs and that hens can be housed separately from drakes to f a c i l i t a t e easier handling of birds under an AI program. 29 2.0 COMPARATIVE GONADAL DEVELOPMENT BETWEEN CAPTIVE AND FREE-FLYING MIGRATORY MALLARD DRAKES 2.1 ABSTRACT Comparisons between ca p t i v e and f r e e - f l y i n g M a l l a r d drakes {Anas platyrhynchos) were made to assess d i f f e r e n c e s i n gonadal development. Body weight, t e s t e s s i z e , t e s t e s weight, sperm number and gonad h i s t o l o g y were determined i n y e a r l i n g and adult drakes. Captive drakes had smaller t e s t e s , lower sperm numbers and h i s t o l o g i c a l l y immature t e s t e s . This study provides f u r t h e r support that gonad development i s suppressed i n ca p t i v e drakes. 2.2 INTRODUCTION Reproduction i n w i l d ducks i s of t e n i n h i b i t e d by c a p t i v i t y ( P h i l l i p s and van Tienhoven, 1960; P h i l l i p s , 1964) . P h i l l i p s and van Tienhoven (1960) found that the gonads of female M a l l a r d s held i n c a p t i v i t y f a i l e d to mature normally. They speculated that constant anx i e t y or fear was the main cause of the i n h i b i t i o n . S u r p r i s i n g l y , l i t t l e or no work was done on gonadal development i n cap t i v e drakes. In p r e l i m i n a r y s t u d i e s of ca p t i v e M a l l a r d semen c h a r a c t e r i s t i c s (Table 8), i t was apparent that sperm counts f o r the c a p t i v e M a l l a r d s were lower than comparable f i g u r e s a v a i l a b l e f o r p o u l t r y and domesticated ducks (Lake and Stewart, 1978a; Gvaryahu et a l . , 1984; Gee, 1983). Low f e r t i l i t y i n captive M a l l a r d s , e s p e c i a l l y y e a r l i n g ' p a i r s , i s a major problem that r e t a r d s progress i n duck r a i s i n g . I t i s important, t h e r e f o r e , to 30 determine i f the gonads of M a l l a r d drakes h e l d i n c a p t i v i t y are i n h i b i t e d by manipulated environmental or b e h a v i o r a l r e s t r a i n t s . 2.3 MATERIALS AND METHODS 2.3.1 Drake Collection Twenty w i l d , f r e e - f l y i n g M a l l a r d drakes were harvested i n Minnedosa, Manitoba between May 26 and June 7, 1994 to get q u a n t i t a t i v e i n f o r m a t i o n on gonadal development and determine an appropriate benchmark f o r cap t i v e M a l l a r d s . S i x ca p t i v e M a l l a r d drakes were used f o r comparisons. 2.3.2 Age Determination A l l c o l l e c t e d carcasses were brought back to the la b o r a t o r y f o r examination and recording of various data. Harvested b i r d s were c a t e g o r i z e d as y e a r l i n g or adult s based on a i r - d r i e d feather measurements ( G a t t i , 1983) . Measurements taken on feathers were: sha f t diameter of primary V (DPV); t o t a l l e n g t h of primary IX (LPIX); t o t a l length of greater secondary covert nine (L9C); bottom vane width of greater secondary covert f i v e (BW5C) and top vane width of greater secondary covert f i v e (TW5C). Lengths of pr i m a r i e s and greater secondary coverts (to 0.5 mm) were measured from the t i p of the i n f e r i o r umbilicus to the t i p of the r a c h i s , while s t r a i g h t e n e d . Primary shaft diameter (to 0.02 mm) was measured with a v e r n i e r c a l i p e r taken i n the plane of the feather vane with the su p e r i o r umbilicus centred between the instrument t i p s ( G a t t i , 1983). The equation (D) used to d i s c r i m i n a t e between y e a r l i n g and adults was: D = 0.684(TW5C)+ 2.527(DPV) + 0.073(LPIX) - 0.221(L9C) + 0.268(BW5C) - 24.335. where adult i s D > 0.03 and yearling i s D < 0.03. 2.3.3 Histology Drakes were weighed using a Pesola spring scale. Birds were dipped i n a solution of soapy water before dissection to keep feathers down. Testi c u l a r length and width (measured i n the dorso-ventral plane) i n millimetres were obtained with vernier c a l i p e r s . Combined weight of testes was determined on a Sartorius type 2205 scale. Testes used for h i s t o l o g i c a l work were f i x e d i n 10% formalin and imbedded i n p a r a f f i n . Preparations (3 microns thick) were stained with G i l l III haematoxylin and counterstained with a c i d i f i e d 1.0% eosin. Sections were viewed under low magnification (400x) and c l a s s i f i e d , based on Johnson's (1961) h i s t o l o g i c a l development of the testes as follows: Stage 1 = (inactive t e s t i s ) , lumen bordered by clear cytoplasmic material, peripheral row of spermatogonia and few spermatocytes. Stage 2 = increased number of spermatocytes, many of which are i n synapsis. Stage 3 = majority of spermatocytes i n synapsis, and a few secondary spermatocytes present. Stage 4 = secondary spermatocytes, some spermatids and immature spermatozoa. Stage 5 = large number of spermatids, and moderate numbers of spermatozoa. Stage 6 = ( f u l l breeding condition) large number of mature spermatozoa, some present i n large central lumen. 32 Stage 7 = (regression) large numbers of degenerating spermatozoa and c e l l s i n tubular lumen. 2.3.4 Sperm Count The phallus was removed by cutting along i t s l i n e of attachment adjacent to the cloacal wall and weighed. The right and l e f t vas deferens were s u r g i c a l l y excised from t h e i r point of entry at the cloaca to the i n f e r i o r edge of the kidneys. The vas deferens was flushed with a warmed Lakes solution (Lake and Ravie, 1979) toward the cloacal end. The contents were c o l l e c t e d i n to a small test tube and stored at room temperature. Test tubes were centrifuged at 2500 rpm for fi v e minutes i n a Damon IEC HN-S centrifuge. The supernatant was removed and replaced with 250 u l of Lakes solution. Sperm were resuspended. A sample of sperm solution was taken and di l u t e d appropriately with spermicide (Lakes plus a few drops of 90% ethyl alcohol) to get an accurate sperm count using a haemocytometer (see Materials and Methods Chapter Three). 2.3.5 S t a t i s t i c a l Analysis T-tests were used to compare t r a i t means between wild and captive drakes and, yearling and adult drakes ( L i t t e l l , 1991). Unless otherwise stated, a l l comparisons were made at the 5% l e v e l of s i g nificance (P<0.05). 2.4 RESULTS Comparisons of captive and f r e e - f l y i n g migratory Mallard drake gonadal development i s summarized i n Table 9 and Table 10. Testis 33 and p h a l l u s weight were s i g n i f i c a n t l y higher i n f r e e - f l y i n g M a l l a r d drakes than i n ca p t i v e drakes (P<0.01) . However, i t should be noted that the c a p t i v e M a l l a r d sample s i z e was s m a l l . As w e l l , t e s t i s s i z e was s i g n i f i c a n t l y l a r g e r i n f r e e - f l y i n g M a l l a r d drakes (P < 0 . 0 1 ) . In both f r e e - f l y i n g and c a p t i v e drakes, the l e f t t e s t i s was l a r g e r . Captive drakes tended to be i n the t r a n s i t o r y stages (3 and 4) between the i n a c t i v e c o n d i t i o n and f u l l breeding c o n d i t i o n . Very l i t t l e v a r i a b i l i t y was found i n the m a t u r i t y of t e s t e s of f r e e - f l y i n g drakes. 34 TABLE 8. Results of preliminary laboratory evaluation of captive Mallard semen characteristics Mean ± SEM (n) 0.05 + 0.02 (12) 8.2xl0 7 ± 2.4xl0 7 (3) 3.0 ± 0.3 (15) 1.0 ± 0.3 (12) Parameter Volume (ml) Concentration (sperm/ml) Appearance M o t i l i t y 35 TABLE 9. Comparison of gonadal characteristics between free-f l y i n g and captive Mallard drakes Parameter Free Captive Body weight (g) 1140 + 13a (20) 1108 ± 66a (6) Testes weight* (g) 21.9 ± 1.0 1 (20) 8.3 ± 3.2" (6) Distribution of spermatogenic stages 6(20) 3(2)), 4(2), 6(2) Right testis length (mm) 44.9 ± 1.0" (20) 27.2 ± 3.7b (6) Right testis width (mm) 22.6 + 0.5a (20) 15.4 + 2.4b (6) Left testis length (mm) 46.8 ± 1.2" (19) 29.1 ± 3.5b (6) Left testis width (mm) 23.1 ± 0.5a (19) 15.5 + 2.2b (6) Phallus weight (g) 2.1 + 0.1a (20) 1.3 + 0.2b (6) Total sperm number 5.6xl08 ± 1.3xl08a (12) 8.6xl07 ± 7.6xl07b (5) Data presented as mean ± SZM (n). Values wi th in rows with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05). Combined weight of r ight and l e f t t e s t i s . TABLE 10. Comparison of character i s t i c s between year l ing and adult f r e e - f l y i n g and captive Mal lard drakes Parameter Y e a r l i n g A d u l t F r e e - f l y i n g : Body weight (g) 1150 ± 36a (5) 1137 ± 13a (15) Testes weight* (g) 21.9+ 1.1' (5) 21.8 ± 1.3" (15) Captive: Body weight (g) 1175 ± 100a (2) 1075 ± 91" (4) Testes weight* (g) 2.3 + 0.8a (2) 11.3 + 4.2a (4) Data presented as mean ± SEM (n). Values w i t h i n rows with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P>0.05). Combined weight of r ight and l e f t t e s t i s . 37 2.5 DISCUSSION Comparisons of t e s t i c u l a r weight and size between captive and f r e e - f l y i n g Mallards suggest that gonadal development i s suppressed i n captive drakes. Several factors can influence gonad development, pri m a r i l y daylength (Haase, 1983; Donham, 1979), ambient temperature and n u t r i t i o n (see review by Bluhm, 1992). These factors appear to be adequate for reproductive development i n th i s case, as Mallard hens held under the same conditions l a i d eggs. P h i l l i p s and van Tienhoven (1960) found that wild-caught P i n t a i l ducks held i n c a p t i v i t y f a i l e d to show ovarian development and bioassays of t h e i r p i t u i t a r y glands revealed a lack of gonadotropin a c t i v i t y . It was speculated that c a p t i v i t y i n some way in h i b i t e d normal gonadotropin secretion. It i s highly probable that t h i s i s also the case i n Mallard drakes held i n c a p t i v i t y . Further work i s needed i n th i s area. 38 3.0 PLASMA TESTOSTERONE PROFILES, SEMEN CHARACTERISTICS AND ARTIFICIAL INSEMINATION IN CAPTIVE MALLARDS 3.1 ABSTRACT Testosterone (T) p r o f i l e s and semen c h a r a c t e r i s t i c s were determined on y e a r l i n g and adult hand-reared w i l d M a l l a r d (Anas platyrhynchos) drakes. M a l l a r d hens were a r t i f i c i a l l y inseminated by modifying a technique developed f o r domesticated p o u l t r y . In both adult and y e a r l i n g drakes, there was a change i n the c o n c e n t r a t i o n of c i r c u l a t i n g plasma T during the reproductive season. T concentrations increased from b a s a l l e v e l s i n March, peaked i n A p r i l and decreased to b a s a l l e v e l s i n May. The decrease i n T c o n c e n t r a t i o n to basal l e v e l was two weeks e a r l i e r i n y e a r l i n g s compared to a d u l t s . The decrease i n T c o n c e n t r a t i o n was a s s o c i a t e d w i t h the onset of p o s t n u p t i a l molt. Semen volume (0.04 to 0.08 ml) and semen concentration (~1.32xl0 9 spermatozoa per ml) were not d i f f e r e n t between adult and y e a r l i n g drakes. F e r t i l i t y obtained w i t h a r t i f i c i a l insemination was 73%. These r e s u l t s suggest that a r t i f i c i a l insemination may be used s u c c e s s f u l l y i n propagating w i l d M a l l a r d s . 3.2 INTRODUCTION A r t i f i c i a l insemination (AI) i s a technique by which semen i s c o l l e c t e d from the male, evaluated, processed and deposited w i t h i n the female's reproductive t r a c t . C o l l e c t e d semen can be processed f o r short-term ( f r e s h ; < 24 hours) or long-term (frozen; 39 indef i n i t e ) storage i n vitro (Hafez, 1987). AI has been practiced routinely i n the commercial breeding of domesticated fowl, such as the turkey, since the development of a simple and e f f e c t i v e procedure by Burrows and Quinn (1937). AI has also been successfully applied to the breeding of domesticated ducks such as the Muscovy (Tan, 1980; Watanabe, 1957), Peking (Tai, 1983) and Golden (Kim, 1980). Several researchers have noted the periodic use of AI i n the breeding of wild ducks, but no d e t a i l e d studies have been reported. Many domesticated ducks are polygamous and drakes w i l l mount any hen to the point of ejaculation (Gvaryahu et a l . , 1984). In such cases i t i s possible to use an a r t i f i c i a l vagina for the c o l l e c t i o n of semen. Wild Mallards are monogamous and, as was shown in Chapter one, hens are very tenacious i n t h e i r choice of a mate. It can be assumed, therefore, that wild Mallards would not copulate in the presence of attendants to enable the use of an a r t i f i c i a l vagina. However, i t may be possible to c o l l e c t semen by adapting the forced or massage method commonly used i n the poultry industry. Due i n recent years to unprecedented population declines, e f f o r t s to breed endangered species i n c a p t i v i t y have increased (Ballou, 1992). This i s a worthy cause since breeding individuals of some waterfowl are at c r i t i c a l l y low lev e l s (NAWF, 1991) It i s therefore a matter of urgency that we seek to improve our understanding of the basic breeding biology of waterfowl and to increase our control over the breeding process by the application of special techniques of a r t i f i c i a l breeding. AI may be one such 40 technique. F a i l u r e to reproduce i n c a p t i v i t y i s o f t e n l i n k e d to a f a i l u r e of w i l d caught females h e l d i n c a p t i v i t y to l a y eggs ( P h i l l i p s , 1960) . However, i n the case of hand-reared b i r d s hatched from eggs taken from nests i n the w i l d , the f a i l u r e to reproduce tends to be due to low egg f e r t i l i t y (personal o b s e r v a t i o n ) . Often a p a i r w i l l engage i n a l l aspects of c o u r t s h i p behaviour but s t i l l f a i l to l a y f e r t i l e eggs (Gee and Temple, 1978). In cases where f a i l u r e to copulate i s due to inexperience, incompatible temperaments or l a c k of a mate, i t may be worthwhile to use AI to supplement n a t u r a l mating. Reduced genetic v i a b i l i t y due to inbreeding i s a problem i n any small w i l d or c a p t i v e p o p u l a t i o n . For example, the New Zealand Brown Teal, when bred i n c a p t i v i t y at the Wildfowl Trust had shown l o s s of reproductive e f f i c i e n c y ; i n p a r t i c u l a r i n male i n f e r t i l i t y (Kear, 1978) . Consequently, outbreeding became necessary to maintain the po p u l a t i o n . With the advent of AI i t would be p o s s i b l e to introduce genetic v a r i a b i l i t y i n t o small c a p t i v e or w i l d populations by t r a n s p o r t i n g semen from a d i s t a n t p o p u l a t i o n . A c c o r d i n g l y , the r i s k of i n t r o d u c i n g p a r a s i t e s and diseases a s s o c i a t e d w i t h f o r e i g n b i r d s and the problems connected w i t h the t r a n s p o r t of l i v e b i r d s i s reduced. Work has been done to c h a r a c t e r i z e the annual reproductive c y c l e i n Ma l l a r d s (Donham, 1979; Paulke and Haase, 1978; Haase, 1983); however, v i r t u a l l y no s t u d i e s have compared the r e l a t i o n s h i p between age and blood testosterone l e v e l s i n drakes. The major f u n c t i o n s of the t e s t e s are the production of spermatozoa and the s e c r e t i o n of t e s t o s t e r o n e . Testosterone s t i m u l a t e s development and maintenance of the reproductive t r a c t , spermatogenesis and sexual d i s p l a y s ( P h i l l i p s and McKinney, 1962; Bluhm, 1992). In M a l l a r d s exposed to n a t u r a l l i g h t i n g , the breeding season i s r e s t r i c t e d to the s p r i n g . During the winter b i r d s become p h o t o s e n s i t i v e and i n t h i s s t a t e respond to i n c r e a s i n g photoperiod w i t h gonadal growth and i n c r e a s i n g plasma testosterone l e v e l s (Haase, 1983). C i r c u l a t i n g t estosterone l e v e l peak and the t e s t e s are f u l l y developed during the breeding season i n A p r i l and May (Paulke and Haase, 1978; Haase et a l . , 1985). In l a t e s p r i n g and e a r l y summer, b i r d s become p h o t o r e f r a c t o r y as reproductive a c t i v i t y i s not maintained by long days. This corresponds to a r a p i d d e c l i n e i n c i r c u l a t i n g concentrations of testosterone and r e g r e s s i o n of the t e s t e s (Haase et a l . , 1985). Decline i n testosterone i s c o r r e l a t e d w i t h p o s t n u p t i a l molt at the end of which drakes are rendered f l i g h t l e s s f o r two to three weeks due to l o s s of t h e i r primary f l i g h t feathers (Johnson, 1961) . A f t e r p o s t n u p t i a l molt, the drake can no longer be d i s t i n g u i s h e d from the hen on the b a s i s of plumage colour since the drakes c h a r a c t e r i s t i c green head feathers are replaced by brown fe a t h e r s . When the growth of new f l i g h t feathers i s completed, the p r e n u p t i a l molt begins (Johnson, 1961) . The t e s t e s appear to be completely regressed by the time that complete p r e n u p t i a l plumage i s acquired (Johnson, 1961) and the drakes have returned to t h e i r c h a r a c t e r i s t i c breeding plumage. A second peak i n testosterone occurs i n l a t e f a l l , but u n l i k e 42 the f i r s t i s not accompanied by an increase i n t e s t e s s i z e ; r ather, i t i s thought to be r e l a t e d to p a i r bond formation (Paulke and Haase, 1978). Blood l e v e l s of avian testosterone have a l s o been shown to e x h i b i t a d i u r n a l rhythm and p u l s a t i l e r e l e a s e (Johnson, 1986) . Although M a l l a r d drakes breed as y e a r l i n g s , they show reduced p r o d u c t i v i t y compared wi t h t h e i r o l d e r counterparts. This reproductive i n f e r i o r i t y of young b i r d s i s o f t e n a t t r i b u t e d to lack of reproductive experience, lack of p h y s i c a l development or both (Curio, 1982). In a study on Gadwalls (Blohm, 1982) a d u l t males were i n v o l v e d i n 74% of p a i r bonds while y e a r l i n g males were i n v o l v e d i n only 26%. I t was suggested that age r e l a t e d d i f f e r e n c e s i n neuroendocrine development or c o u r t s h i p behaviour may have bestowed a competitive advantage to the o l d e r more experienced males during e a r l y c o u r t s h i p , thereby e x p l a i n i n g the preponderance of a d u l t drakes i n p a i r bonds. C i r c u l a t i n g androgen l e v e l s are a r e f l e c t i o n of the t i m i n g and extent of gonadal development. Therefore, a comparison of t e s t o s t e r o n e p r o f i l e s of y e a r l i n g and adult drakes may determine i f the poor reproductive success of y e a r l i n g drakes i s due to inexperience or lack of t e s t i c u l a r development. S p e c i f i c o b j e c t i v e s of t h i s study were t o : 1. Compare semen c h a r a c t e r i s t i c s of y e a r l i n g and a d u l t M a l l a r d drakes 2. Compare the plasma testosterone p r o f i l e s of y e a r l i n g and adult M a l l a r d drakes. 43 3. Determine i f AI could be used as an a l t e r n a t i v e management technique i n the r e a r i n g of cap t i v e w i l d M a l l a r d s 3.3 MATERIALS AND METHODS 3.3.1 Breeding Stock G e n e t i c a l l y w i l d M a l l a r d s used i n t h i s study were reared from eggs salvaged from a g r i c u l t u r a l f i e l d s i n south c e n t r a l Manitoba. Eggs were hatched, and b i r d s were reared and maintained using standard procedures o u t l i n e d by Bluhm et a l . (1993) at the Delta Waterfowl Research S t a t i o n i n Del t a , Manitoba. In January 1995, adult (two years old) and y e a r l i n g (hatched summer 1994) M a l l a r d s were transported by a i r from D e l t a , Manitoba to the U n i v e r s i t y of B r i t i s h Columbia's San Raf a e l Research A v i a r y i n Surrey, B r i t i s h Columbia. Although D e l t a has f a c i l i t i e s f o r housing c a p t i v e b i r d s , the San Rafael l o c a t i o n o f f e r e d the advantage of being c l o s e to s p e c i a l i z e d equipment (such as a gamma counter), and due to warmer climate and e a r l i e r s p r i n g , the p e r i o d a v a i l a b l e f o r the t r a i n i n g of drakes f o r semen c o l l e c t i o n was extended. 3.3.2 Care and Management of Birds On Feb. 14, 1995 twenty-three drakes were assigned to outdoor c l u s t e r pens ( s i b l i n g s and brood mates were separated) . Drakes were separated by age and kept approximately s i x to a pen. Each pen (approximately 2.4 m by 3 m) contained areas of open water and dry 44 land (gravel). A 1.2 m plywood wall around the base of the pen, shielded the birds from outside disturbances. On March 28, hens were i n d i v i d u a l l y assigned to pens (approximately 2.4 m x 1.5 m) adjacent to drakes. Birds i n separate pens had v i s u a l and auditory exposure to each other, but were p h y s i c a l l y separated by chain-link fencing. A l l birds were fed ad libitum, with commercially available duck p e l l e t s , supplemented with wheat and oyster s h e l l . Hens were given access to straw f i l l e d wooden nest boxes. Before introduction to t h e i r respective pens, hens were weighed with a spring scale. 3.3.3 Semen Collection Collections were done between 1300 and 1500h, within drake pens. Each drake was caught by hand. The method of semen c o l l e c t i o n follows the massage procedure outlined by Lake and Stewart (1978a) with the following modifications. Semen was c o l l e c t e d by a single seated technician, with the drake held with his head toward the technician and his wings held between the technicians knees. The tube, used for catching the semen, was held between the fingers of the hand used to massage the abdomen. The opposite hand gently stroked the back toward the t a i l ending with the thumb and forefinger on each side of the cloaca. Gentle pressure was sometimes applied around the cloacal region to extrude the phallus. It was found preferable to obtain the semen before complete extrusion, i n which case the semen was l o s t as i t flowed down the spiral-shaped phallus. It was also sometimes h e l p f u l to c l i p the 45 feathers around the vent. Twenty-three drakes used f o r AI were t r a i n e d twice a week f o r about three weeks before inseminations were begun. This t r a i n i n g i n v o l v e d handling and attempting to e j a c u l a t e the b i r d s . A f t e r that, drakes were r o u t i n e l y c o l l e c t e d from twice a week. Care was taken to minimize contamination of semen w i t h u r i c a c i d waste. Semen was pooled by age group. 3.3.4 Semen Evaluation The parameters to assess and compare semen c h a r a c t e r i s t i c s of i n d i v i d u a l y e a r l i n g and a d u l t M a l l a r d drake semen samples in c l u d e d volume, concentration, v i s u a l appearance, m o t i l i t y , l i v e sperm percentage, and abnormal sperm percentage. 3.3.5 Laboratory Analyses The f o l l o w i n g l a b o r a t o r y procedures were performed to determine semen q u a l i t y and c o n c e n t r a t i o n . (1) Appearance - A small drop of u n d i l u t e d semen was placed on a microscope s l i d e . Semen appearance and c o l o u r were scored as f o l l o w s : 1 = watery or c l e a r semen 2 = watery w i t h white streaks 3 = medium 4 = t h i c k white 5 = very viscous and chalky white 46 (2) M o t i l i t y - After evaluating semen appearance, the drop of semen was covered with a coverslip and viewed under low magnification (lOOx) to award a score as follows: 0 = No m o t i l i t y . 1 = 1 - 20% motile, l i t t l e movement. 2 = 20-40% motile, no waves 3 = 40-60% motile, slow eddies or waves. 4 = 60-80% motile, waves and eddies of movement. 5 = 80-100% motile, extremely rapid eddies and movement. (3) Semen volume (cc) - V i a l s were pre-weighed before semen c o l l e c t i o n and again after c o l l e c t i o n using an e l e c t r o n i c scale. The s p e c i f i c gravity of semen i s approximately equal to one; therefore, one gram of semen i s approximately equal one ml of semen. (4) Concentration - Ten u l of undiluted semen was added to 1990ul of 3% NaCl solution (200x d i l u t i o n ) . Two counting chambers of a clean dry haemocytometer were covered with a coverslip and f i l l e d with approximately lOul of sample. Spermatozoa were allowed to s e t t l e and then f i v e squares of the large central g r i d were counted. Sperm concentration per ml was calculated as follows: 5 squares x 200 d i l u t i o n x 10,000 x average number of spermatozoa i n 2 chambers. 47 3.3.6 Insemination A one ml tuberculin syringe was used for inseminations d i r e c t l y into the oviduct of laying b i r d s . About 0.1 ml of a i r was f i r s t drawn into the syringe to be able to push out the whole volume of semen at the time of insemination. To prevent injury, insemination was not attempted u n t i l each hen had l a i d at least one egg. It was v i r t u a l l y impossible to evert the oviduct, as i s commonly done with domestic chickens and turkeys; therefore, the syringe was gently inserted on the l e f t side of the cloaca and somewhat do r s a l l y u n t i l the oviduct was discovered. The syringe was then gently inserted one or two centimetres and the semen deposited. 3.3.7 Egg Collection and Incubation Nest boxes were examined once a day. Eggs were removed from the nest boxes as they were l a i d and replaced with dummy chicken eggs to maintain the normal egg laying pattern. Dummy clutches were removed f i v e days after the l a s t egg was l a i d . Collected eggs were stored for one week i n a cool cupboard during which they were rotated d a i l y . Eggs were washed with an a n t i b a c t e r i a l solution just before incubation i n a Humidaire Model 20 incubator at 37.6°C and 21.1°C wet bulb temperature. Eggs were automatically rotated hourly through 90°. Eggs were candled at seven days to determine whether development was proceeding. Questionable eggs were removed from the incubator and opened to ascertain whether they were f e r t i l e , using 48 the technique outlined by Fant (1957). Two days before the expected hatch date, f e r t i l e eggs were placed into a hatching tray at the bottom of the incubator. At hatch, ducklings were weighed and sexed by cloacal exposure (Ward and Batt, 1973). Dead embryos detected during additional candlings were recorded, aged by the technique of Caldwell and Snart (1974) and c l a s s i f i e d as early-dead (<7 days), mid-dead (8-18 days) or late-dead (>19 days). M o r t a l i t i e s were also c l a s s i f i e d into one of seven malposition groups (Fant, 1957). 3.3.8 Blood Collection Blood was c o l l e c t e d every two weeks between February and May 1995 from the twenty-three drakes (10 yearlings, and 13 adults) used for AI. Blood samples (1-1.5 ml) were c o l l e c t e d between 1200 and 1600 hours from the medial wing vein using s t e r i l e 3 cc syringes and 22 gauge needles. Samples were transferred to i n d i v i d u a l 3 ml heparinized vacutainer tubes (Becton Dickson, Vacutainer Systems, Rutherford, New Jersey, USA) and centrifuged at 1250 X g for f i v e minutes within 30 minutes of c o l l e c t i o n ; the plasma was aspirated and stored at -20°C u n t i l assayed for testosterone. 3.3.9 Radioimmunoassay (RIA) A commercial radioimmunoassay k i t (Coat-A-Count, Diagnostic Products Corporation, Los Angeles, CA, USA) was used for testosterone assay. Samples were run i n duplicate. The k i t uses a solid-phase radioimmunoassay based on polypropylene tubes coated 49 with antibodies to testosterone. 1 2 5 I - l a b e l l e d testosterone competes for a fixed period of time with testosterone i n the sample for antibody s i t e s . Once the supernatant i s decanted (to separate bound testosterone from free), the tube i s counted i n a gamma counter to y i e l d counts per minute. These counts were converted to the concentration of testosterone i n the sample by way of a c a l i b r a t i o n curve. The Coat-A-Count k i t i s equipped with human serum-based c a l i b r a t o r s ready to use having testosterone values ranging from 0 to 16 ng/ml. The assay can detect as l i t t l e as 0.04 ng/ml 3.3.10 S t a t i s t i c a l Analyses T-tests were used to compare t r a i t means between yearling and adult drakes. Plasma testosterone values were analyzed as repeated measures analysis for a s p l i t - p l o t design. Differences in treatment means were separated by least squares analysis of variance procedure using General Linear Models procedure of SAS Instit u t e Inc. ( L i t t e l l , 1991). The model used was: Y 1 ] k = u + As + D5 (AJ + Tk + AT i k + e i j k i = 1...2 (age); j = 1...8 (time) Y i j k = equals the kth observation for the ( i , j ) t h c e l l . u = population mean. A4 = e f f e c t of drake age. D^AJ = drake nested within age group. Tk = ef f e c t of sampling time. AT i k = int e r a c t i o n of age with sampling time. e l j k = random error associated with i n d i v i d u a l observations. The drake(age) mean square was used as error term to test age mean square. The other effects [drake(age), sampling time and int e r a c t i o n of age with sampling time] were tested using the random 50 e r r o r mean square. Chi-square a n a l y s i s with Yates c o r r e c t i o n f o r c o n t i n u i t y on 2 x 2 contingency t a b l e s was used to t e s t f o r drake e f f e c t s on egg f e r t i l i t y . F e r t i l i t y was expressed as the percentage of v i a b l e embryos on day seven of i n c u b a t i o n . H a t c h a b i l i t y was defined as the percentage of eggs hatched per f e r t i l e eggs incubated. Due to the l i m i t e d number of observations, h a t c h a b i l i t y and embryo m o r t a l i t i e s were not s t a t i s t i c a l l y analyzed. Unless otherwise s t a t e d , a l l comparisons were made at the 5% l e v e l of s i g n i f i c a n c e . 3 . 4 RESULTS Semen was c o l l e c t e d from 18 (nine y e a r l i n g s and nine adults) out of 24 drakes. The a b i l i t y to o b t a i n semen from i n d i v i d u a l b i r d s was h i g h l y v a r i a b l e . I f semen d i d not appear a f t e r 60 seconds of massage of any drake, i t was u n l i k e l y that f u r t h e r massage would r e s u l t i n an e j a c u l a t i o n . In which case, he was r e l e a s e d i n h i s pen and t r i e d another day. Mean semen c h a r a c t e r i s t i c s of adult and y e a r l i n g c a p t i v e M a l l a r d drakes are presented i n Table 11. There were no s i g n i f i c a n t (P>0.05) d i f f e r e n c e s w i t h respect to volume, appearance, m o t i l i t y or concentration between the two age groups. LSMeans, p r o f i l e s and a n a l y s i s of variance of plasma tes t o s t e r o n e concentrations are presented i n Table 12, Figure 3 and Appendix 6. Testosterone LSMean concentrations ranged from 0.02 to 1.59 ng/ml during the 4-month c o l l e c t i o n p e r i o d . Testosterone concentrations were not s i g n i f i c a n t l y d i f f e r e n t between y e a r l i n g and a d u l t drakes (P>0.05), but there was considerable v a r i a t i o n i n 51 t e s t o s t e r o n e concentrations between i n d i v i d u a l b i r d s . Testosterone concentrations were s i g n i f i c a n t l y i n f l u e n c e d by time (P<0.01), i n c r e a s i n g from a low of 0.13 ng/ml i n February to a high of 1.26 ng/ml i n e a r l y A p r i l . In May, testosterone concentrations d e c l i n e d . On A p r i l 25, y e a r l i n g drakes had s i g n i f i c a n t l y lower plasma testosterone concentrations than adult drakes (P<0.01). The r e s p e c t i v e LSMeans f o r plasma testosterone concentrations were 0.55 ± 0.28 ng/ml and 1.59 ± 0.25 ng/ml f o r y e a r l i n g and adult drakes. By May 9, plasma testosterone concentrations f o r y e a r l i n g (0.61 + 0.29 ng/ml) and adult (0.99 ± 0.27 ng/ml) drakes were not d i f f e r e n t (P>0.05) . The f e r t i l i t y and h a t c h a b i l i t y data i s presented i n Table 13 and Table 14. The mean f e r t i l i t y was 72.6%, ranging from 25 to 100%. This was comparable to f e r t i l i t y of matings between adult M a l l a r d s i n 1994 (Figure 1) . The mean h a t c h a b i l i t y f o r 1995 was 53%, ranging from 0 to 67%. F e r t i l i t y of eggs from hens inseminated w i t h pooled y e a r l i n g or adult drake semen was 59% and 76%, r e s p e c t i v e l y . There was no s i g n i f i c a n t (P>0.05) d i f f e r e n c e between egg f e r t i l i t y r a t e s f o r the two age groups. 52 TABLE 11. Semen characteristics of adult and yearling Mallard drakes Parameter Yearling Adult Volume (ml) 0.04 ± 0 .01a (23) 0.08 ± 0 .02a (26) Appearance 3.1 + 0 .2a (22) 3.3 + 0 .2a (20) M o t i l i t y 2.8 ± 0 .3a (21) 3.5 ± 0 .3a (19) Concentration (xlOVml) 1.32 ± 0. 2 9a (18) 1.32 ± 0. 31a (21) Values are means of nine y e a r l i n g and nine adult drakes subjected to nine c o l l e c t i o n s p e r i o d s . Values wi th in rows with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P>0.05). 53 Table 12. Effect of Mallard drake age on plasma testosterone concentrations * Age Date Yearling Adult February 14 0.09 + 0.28 0.18 ± 0.24 February 28 0.32 ± 0.28 0.42 + 0.24 March 14 1.01 ± 0.28 0.77 + 0.27 March 28 1.28 + 0.31 0.92 + 0.24 A p r i l 11 1.34 + 0.28 1.19 ± 0.24 A p r i l 25 0.55 ± 0.28a 1.59 ± 0.25b May 9 0. 61 + 0.29 0.99 ± 0.27 May 23 0.19 + 0.29 0.02 + 0.28 Values are presented as LSMeans ± SEM. Values w i th in a row with d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05). 54 FIGURE 3. Plasma testosterone concentration in relation to drake age Feb14 Feb28 Marl4 Mar28 April Apr25 May 9 May 23 D A T E 55 TABLE 13. F e r t i l i t y and hatchability of individual Mallard hens after a r t i f i c i a l insemination Hen Hen age Drake age Laying sequence after f i r s t and subsequent inseminations* % F e r t i l e % Hatch of f e r t i l e B 1 2 AI -O-FFF-F 80.0 0.0 B 1 2 AI NFOO-F-F 66.7 50.0 C 1 2 AI --FFFFF- 100.0 n/a F 2 1 AI FO-00 25.0 0.0 F 2 1 AI NFFFOOO- 57.0 50.0 J 2 2 AI FF-F- 100.0 n/a J 2 2 AI O-FF—FO 60.0 66.7 J 2 2 AI 0-FOFF— 60.0 0.0 K 2 2 AI OOFF-FF- 66.7 50.0 L 2 2 AI FF 100.0 0.0 M 1 1 AI OF-FF-FF 83.0 0.0 Mean 72.6 53.3 *0 = i n f e r t i l e egg; F = f e r t i l e egg; - = no egg l a i d ; n/a = f e r t i l e eggs not incubated to hatch; N = eggs l i k e l y f e r t i l i z e d by previous inseminat ion . 56 TABLE 14. Effect of drake age (yearling verse adult) on f e r t i l i t y , hatchability and embryo mortality rates of Mallard eggs Parameter Yearling Adult Total " O F e r t i l e 58.8 (10/17) t a 75. 7 (28/37)a 70. 4 (38/54) Q . " O Hatched 20.0 (2/10) a 33. 3 (6/18) a 28 .6 (8/28) Q . Mortality: Early-dead* 50.0 (4/8) 33 3 (4/12) 40 .0 (8/20) Late-dead A 50.0 (4/8) 66 .7 (8/12) 60 .0 (12/20) Ten out of 17 eggs f e r t i l e . Values w i th in rows with common s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P>0.05). M o r t a l i t i e s <7 days of incubat ion . M o r t a l i t i e s >19 days of incubat ion . 3 . 5 DISCUSSION 57 This i s the f i r s t known report of s u c c e s s f u l AI i n hand-reared w i l d M a l l a r d s . The o v e r a l l percentage f e r t i l i t y obtained w i t h AI was comparable w i t h that reported f o r n a t u r a l l y mated p a i r s (Chapter 1; Lo, 1994; Batt and P r i n c e , 1978) . The production of l i v e ducklings s t r o n g l y suggests that AI can be used s u c c e s s f u l l y to supplement n a t u r a l mating i n c a p t i v e M a l l a r d s . A feature of the hens reproductive t r a c t i s the a b i l i t y to s t o r e semen i n a v i a b l e s t a t e f o r extended periods of time a f t e r c o p u l a t i o n or insemination. This allows f o r the f e r t i l i z a t i o n of c o n s e c u t i v e l y l a i d eggs between inseminations. F e r t i l e eggs were obtained as much as eleven days a f t e r a s i n g l e insemination. This agrees w i t h r e s u l t s reported by others (Elder and Weller, 1954; Lake, 1983) . The r e s u l t s i n d i c a t e that the p e r i o d i n c l u d e d from the t h i r d to seventh day a f t e r an insemination i s most l i k e l y to c o n t a i n f e r t i l e eggs. I t i s apparent that w i t h the spermatozoa co n c e n t r a t i o n used i n t h i s study, hens need to be inseminated at weekly i n t e r v a l s to maintain maximum f e r t i l i t y u sing A I . One disadvantage of AI might be the f a i l u r e to f e r t i l i z e those eggs l a i d before insemination. The i r r e g u l a r i t y of the M a l l a r d hens reproductive c y c l e makes i t d i f f i c u l t to p r e d i c t the date upon which an egg w i l l be l a i d . Often hens w i l l s k i p a day between egg l a y i n g , or stop l a y i n g completely f o r up to two weeks between c l u t c h e s . I t may be p o s s i b l e to over come t h i s problem by inseminating before egg-laying begins, as i s done w i t h s e v e r a l other avian species (Berry, 1972). N e s t - b u i l d i n g o f t e n occurs a 58 few days before ovulation of the f i r s t egg and has been correlated to a readiness to copulate i n many avian species (Lehrman, 1959) . Therefore, nest-building could possibly be used as an i n d i c a t i o n of reproductive maturation. It i s u n l i k e l y that the poor h a t c h a b i l i t y rates obtained with AI i s a res u l t of the use of i n f e r i o r semen or eggs. Postmortem examinations, revealed that the bulk of m o r t a l i t i e s occurred during the f i n a l stages of incubation and the majority of these dead embryos were i n abnormal positions. T y p i c a l l y the duckling was either rotated so that i t s beak was away from the a i r c e l l or the beak was over the right wing instead of under the wing. Deaths caused by malpositions that make hatching extremely d i f f i c u l t are usually attributed to unfavourable incubation conditions such as abnormal temperature or an excess of atmospheric carbon dioxide (Romanoff, 1949) . No such conditions were noted i n t h i s study, although incubator humidity readings did fluctuate. The results of t h i s study do not support a hypothesis that differences i n semen c h a r a c t e r i s t i c s or neuroendocrine development contribute to the low reproductive success of yearling drakes. Semen c o l l e c t e d from yearlings and adults did not d i f f e r with respect to the number of spermatozoa per ml, sperm m o t i l i t y or semen volume. It i s generally accepted that semen c h a r a c t e r i s t i c s a f f e c t f e r t i l i t y (Allen and Champion, 1955). Comparisons of ejaculates from wild Mallards with those reported by various authors for domesticated duck species (Gvaryahu et a l . , .1984; Kamar, 1962), suggest that quantitative 59 c h a r a c t e r i s t i c s are lower f o r semen obtained from w i l d M a l l a r d s . Kamar (1962) recorded a mean value of 3.63xl0 9 spermatozoa per ml i n Sudani drakes and 5.85xl0 9 spermatozoa per ml i n Pekin drakes. In c o n t r a s t , Gvaryahu et a l . (1984) reported a co n c e n t r a t i o n of 1.35xl0 9 sperm c e l l s per ml f o r Muscovy drakes, which i s s i m i l a r to the c o n c e n t r a t i o n obtained i n t h i s study. I t i s p o s s i b l e that the lower co n c e n t r a t i o n found i n the present study maybe due to the a d d i t i o n of ejaculatory-groove region f l u i d during c o l l e c t i o n . This region i s considered an accessory reproductive organ i n the drake ( F u j i h a r a et a l . , 1976). At c o p u l a t i o n , f l u i d s i m i l a r i n composition to lymph, i s released and mixes wi t h the semen eje c t e d from the vas deferens. Such s e c r e t i o n s have been found to have favourable e f f e c t s on the f e r t i l i t y of f r e s h spermatozoa from the drake ( F u j i h a r a et a l . , 1976). As w e l l , domesticated ducks have been s e l e c t e d f o r high reproductive performance and t h i s may have a l t e r e d the semen c h a r a c t e r i s t i c s . D i f f e r e n c e s i n degree of adaptation to c a p t i v i t y , breed, and method of c o l l e c t i o n between t h i s experiment and that of the others, cannot be r u l e d out as sources of v a r i a t i o n . Seasonal changes i n the concentration of testosterone i n the p e r i p h e r a l blood of M a l l a r d drakes observed i n t h i s study agree wi t h previous f i n d i n g s (Paulke and Haase, 1978; Donham, 1979). M a l l a r d drakes e x h i b i t e d a steep decrease i n the concent r a t i o n of testosterone i n May, ass o c i a t e d w i t h the onset of the p h o t o r e f r a c t o r y p e r i o d . I t should be appreciated t h a t testosterone concentrations i n drakes tended to be q u i t e v a r i a b l e . 60 One of the most s t r i k i n g features of these r e s u l t s was the p a t t e r n of c i r c u l a t i n g t e s t osterone concentrations decreasing to basa l l e v e l s two weeks e a r l i e r i n y e a r l i n g drakes than i n adult drakes. I t appears that adulthood delayed the onset of the p h o t o r e f r a c t o r y p e r i o d . The r e s u l t s showed no trend f o r an o v e r a l l i n c rease i n c i r c u l a t i n g t e s t osterone concentrations i n o l d e r b i r d s , which i s a p a t t e r n observed i n many farm animals (Gunarajasingam et a l . , 1985). Results presented provide evidence that AI may be used to supplement n a t u r a l mating i n M a l l a r d p a i r s not w i l l i n g or unable to mate n a t u r a l l y i n c a p t i v i t y . Furthermore, the p r a c t i c e of semen c o l l e c t i o n and insemination should provide v a l u a b l e i n s i g h t s i n t o the reproductive physiolo'gy of w i l d M a l l a r d s . F i n a l l y , a l o g i c a l e xtension of an AI program would be to preserve semen to allow the exchange of genetic m a t e r i a l between d i s t a n t populations and the establishment of gene banks. 61 4.0 COMPARISON OF TWO POULTRY SEMEN EXTENDERS 4.1 ABSTRACT L i t t l e work has been reported on d i r e c t comparisons of d i f f e r e n t p o u l t r y semen extenders. B e l t s v i l l e P o u l t r y Semen Extender (BPSE) and Lake's P o u l t r y Semen Extender (LAKE) were compared. Leghorn hens (6-7 per treatment) were inseminated weekly wit h approximately 1.2 x 108 spermatozoa before and a f t e r 24 hours storage at 5°C. No s i g n i f i c a n t d i f f e r e n c e was found i n egg f e r t i l i t y when semen was f r e s h l y d i l u t e d and inseminated (BPSE 83%; LAKE 90%; u n d i l u t e d 92%) . The f e r t i l i z i n g c a p a c i t y of sto r e d semen was s i g n i f i c a n t l y reduced; however, semen s t o r e d i n e i t h e r BPSE (30%) or LAKE (32%) performed b e t t e r than the u n d i l u t e d c o n t r o l (3%) (P<0.05). From these r e s u l t s i t appears that BPSE and LAKE work e q u a l l y w e l l to maintain the semen f e r t i l i z i n g c a p a c i t y during short term storage. 4.2 INTRODUCTION Short-term storage of semen i s a p r a c t i c e by which semen i s c o l l e c t e d from the male, d i l u t e d with a b u f f e r e d s a l t s o l u t i o n ( r e f e r r e d to as an extender or d i l u e n t ) , and then h e l d at 5°C u n t i l insemination. The a b i l i t y to store semen f o r a s e v e r a l hours o f f e r s f l e x i b i l i t y i n the tim i n g of inseminations and may allow the tr a n s p o r t of semen between farms and even c o u n t r i e s . Semen extenders serve two main purposes: F i r s t , extenders provide an i d e a l environment in vitro to s u s t a i n sperm v i a b i l i t y 62 during short-term storage. The c a p a b i l i t y of maintaining semen at temperatures above freezing has been c r u c i a l to the success of a r t i f i c i a l insemination i n farm animals. Secondly, extenders maximize the number of hens that can be inseminated from a single ejaculate. Avian semen has low volume, but i s highly concentrated. Therefore, i t i s common for extenders to be used to increase semen volume and enable a proper dose to be delivered during insemination. The extender used for domesticated ducks i s an egg yolk sodium c i t r a t e buffer developed by Watanabe (1961). However, this extender has a holding time of less than 30 minutes and Sexton and Fewless (1978) found that the addition of egg yolk to sperm always resulted i n a decline' i n f e r t i l i z i n g capacity. Due to disease prevention protocols, egg yolk acquired from chicken or domesticated duck eggs would not be a desirable component of an extender for w i l d - s t r a i n Mallard semen. Egg yolk could be a po t e n t i a l vector i n the spread of salmonella and other avian diseases to wild populations. Early work to develop diluents to maintaining poultry sperm f e r t i l i z i n g capacity concentrated on the use of simple diluents containing inorganic ions (Schindler et a l . , 1955; Wilcox et a l . , 1958) . Since then several complex diluents have been developed (see reviews by Bootwalla et a l . , 1992; Bakst, 1990). In contrast to simple extenders, complex extenders provide (a) buffers to protect against changes i n pH, (b) agents to maintain osmotic balance, (c) an energy source and (d) chelating agents to protect against toxic 63 ions. Glutamate and potassium phosphate are components common to most poultry semen extenders. Glutamic acid has been reported to be a major non-protein nitrogen constituent of poultry seminal plasma and i t may help to maintain the osmotic pressure of semen (Lake and Mclndole, 1959) and/or serve as a chelator (Ogasawara and Ernst, 1975). The role of glutamate i n B e l t s v i l l e Poultry Semen Extender was examined by Sexton and Fewlass (1978), who reported a s i g n i f i c a n t decline i n the f e r t i l i z i n g capacity of spermatozoa when i t was removed from the extender. High potassium lev e l s are necessary for semen survival since potassium i s r e a d i l y l o s t from the spermatozoa during s t r e s s f u l conditions such as d i l u t i o n or storage (Renganathan, 1982). Sugar additives are commonly used as energy sources i n poultry semen extenders. Sexton (1974) found that spermatozoa u t i l i z e fructose much more e f f i c i e n t l y than glucose and r e l y primarily on the g l y c o l y t i c pathway for survival in v i t r o . Hence replacing fructose with glucose could reduce sperm metabolic a c t i v i t y and f e r t i l i z i n g capacity. Thus, i t i s hypothesized that extenders containing d i f f e r e n t energy sources may d i f f e r i n t h e i r a b i l i t y to maintain sperm f e r t i l i z i n g a b i l i t y . Experience with domesticated species has shown that undiluted (neat) semen held at ambient temperatures w i l l deteriorate within 30 to 35 minutes of c o l l e c t i o n ; however, extended semen can be maintained for up to 24 hours at 5°C without a s i g n i f i c a n t loss i n f e r t i l i z i n g capacity (Garren and Shaffner, 1952; Bootwalla and 64 Miles, 1992). Extensive work has been c a r r i e d out perfecting the id e a l extender. Many have been developed and used successfully by researchers and commercial poultry breeders (Austin and Natarajan, 1991; Chaudhuri and Lake, 1988; Lake and Ravie, 1979). The most widely accepted and commercially available extender i s B e l t s v i l l e Poultry Semen Extender (BPSE)(Sexton, 1977a). High f e r t i l i t y l e v e l s (>88%) have been achieved by Sexton i n hens inseminated weekly with semen d i l u t e d i n BPSE and containing as few as 20 m i l l i o n sperm. Lake and Ravie (1979) also developed an acceptable poultry semen extender (LAKE) by modifying a simple unbuffered s a l t solution. They have also reported high f e r t i l i t y (>87%). Long-term preservation of d i l u t e d semen i s achieved through freezing and thawing of a semen sample suspended i n an extender containing a cryoprotectant. The successful cryopreservation of avian semen would enable the transport of semen, and the establishment of gene banks (gene pools) . L i t t l e e f f o r t has been made to formulate an extender e s p e c i a l l y for use with frozen semen; yet, choice of an extender i s one component c r i t i c a l to the survival of spermatozoa during cryopreservation. The problem of finding a feasible method of freezing poultry semen i s therefore dependent on choosing the "best" extender available. There are, however, few reports of di r e c t comparisons between d i f f e r e n t poultry semen extenders. This study was conducted to compare two commonly used poultry semen extenders (BPSE and LAKE) with respect to t h e i r e f f ects on sperm sur v i v a l and sperm f e r t i l i z i n g capacity. Adaption of one of these inorganic poultry semen extenders for future use with captive 65 M a l l a r d semen may be p o s s i b l e . 4.3 MATERIALS AND METHODS 4.3.1 Breeding Stock L i m i t a t i o n s imposed by the short breeding season of w i l d -s t r a i n M a l l a r d s f o r s t u d i e s on semen extenders l e d to the use of chickens as a model f o r developing a technique of semen p r e s e r v a t i o n . The stock used was a commercial White Leghorn s t r a i n . A l l b i r d s were housed i n d i v i d u a l l y , fed an 18% p r o t e i n breeders r a t i o n ad libitum and maintained on 15 hours l i g h t and 9 hours dark a f t e r 18 weeks of age. Handling of r o o s t e r s began at 23 weeks of age to habituate the b i r d s to e j a c u l a t i o n procedures. Semen was c o l l e c t e d r o u t i n e l y three time per week (Monday, Tuesday and Friday) between 1230 and 1330h by abdominal massage (Lake and Stewart, 1978a). T r i a l s commenced when r o o s t e r s and hens were 33 weeks of age. Approximately eight males from a p o p u l a t i o n of 11 males, were used f o r semen c o l l e c t i o n . 4.3.2 In Vitro Sperm Survival This study was conducted to t e s t f o r d i f f e r e n c e s i n the a b i l i t y of each extender to maintain spermatozoa in vitro. A f t e r c o l l e c t i o n , i n d i v i d u a l semen samples, free of contamination with u r i c a c i d wastes and dust e t c . , were pooled and d i v i d e d i n t o three a l i q u o t s : neat, d i l u t i o n (1:1) w i t h BPSE or d i l u t i o n (1:1) w i t h LAKE (Appendix 2). Both semen and extenders were at room temperature (17-20°C) before mixing. A f t e r p i p e t t i n g g e n t l y to mix 66 samples, 200 u l of dil u t e d semen (or 200 u l of neat semen) was placed i n separate tubes for 0, 8, 24, or 48 hours storage at 5°C. Tubes were l i g h t l y covered with p a r a f f i n . Laboratory analyses were performed to determine sperm quality before and afte r storage. 4.3.3 Laboratory Analyses The following laboratory procedures were performed to determine semen qual i t y and concentration. (1) Appearance - A small drop of undiluted semen, placed on a microscope s l i d e . Semen appearance and colour was scored as follows: 1 = watery or clear semen 2 = watery with white streaks 3 = medium 4 = thick white 5 = very viscous and chalky white (2) M o t i l i t y - After evaluating semen appearance, the drop of undiluted semen was covered with a coverslip and viewed under low magnification (lOOx) to award a score as follows: 0 = No m o t i l i t y . 1 = 1 - 20% motile, l i t t l e movement. 2 = 20-40% motile, no waves 3 = 40-60% motile, slow eddies or waves. 4 = 60-80% motile, waves and eddies of movement. 5 = 80-100% motile, extremely rapid eddies and movement. 67 (3) Live Percentage - The method for preparing the s t a i n and examining the smears was as described by Lake and Stewart (1978). Nigrosin and eosin were dissolved i n a sodium glutamate solvent. Two drops of semen were added to nigrosin/eosin s t a i n at the end of a glass s l i d e and gently mixed. After two minutes a smear was made and allowed to dry. A t o t a l of 200 spermatozoa were examined and d i f f e r e n t i a t e d into l i v e (unstained) or dead ( p a r t i a l l y or t o t a l l y stained) using an o i l immersion objective. Two s l i d e s were viewed per sample and the percentage l i v e sperm calculated. (4) Morphological Abnormalities - stained smears were prepared as above. A t o t a l of 200 spermatozoa were counted and d i f f e r e n t i a t e d into normal or abnormal (absent head, coiled, bent, etc.) under the o i l immersion lens. Two sl i d e s were viewed per sample and the percentage t o t a l abnormal sperm- calculated. (5) Concentration - Ten u l of undiluted pooled semen was added to 2990 ul of 3% NaCl solution (300x d i l u t i o n ) . Two counting chambers of a clean dry haemocytometer were covered with a coverslip and f i l l e d with 10 u l of sample. After f i v e minutes, f i v e squares of the large central g r i d were counted. Sperm concentration per m i l l i l i t r e was calculated as follows: 5 squares x 300 d i l u t i o n x 10,000 x average number of spermatozoa i n two chambers. 68 4 . 3 . 4 In Vivo Sperm F e r t i l i z i n g Capacity The purpose of t h i s study was to t e s t f o r d i f f e r e n c e s i n the a b i l i t y of each extender to preserve sperm f e r t i l i z i n g c a p a c i t y . A f t e r c o l l e c t i o n , i n d i v i d u a l semen samples fr e e of contamination were pooled and t r a n s f e r r e d i n t o three v i a l s : (1) no d i l u t i o n (2) d i l u t e d 1:1 wit h BPSE, and (3) d i l u t e d 1:1 w i t h LAKE. Both semen and extender were at room temperature (17-20°C) when mixed. Some of each a l i q u o t was removed f o r storage i n covered three ml v i a l s at 5°C f o r 24 hours. Each separate pooled sample represented a r e p l i c a t e f o r each treatment. Sperm q u a l i t y before and a f t e r storage was determined by l a b o r a t o r y a n a l y s i s as above. The remaining volume was inseminated w i t h i n one hour of c o l l e c t i o n . Forty-two hens ( s i x per treatment) were randomly placed i n i n d i v i d u a l l a y i n g cages assigned to one of s i x treatments (BPSE-0, BPSE-24, LAKE-0, LAKE-24, NEAT-0, NEAT-24) . Hens were inseminated weekly f o r four weeks wi t h approximately 1.2 x 108 spermatozoa (0.1 ml d i l u t e d or 0.05 ml ne a t ) . D i f f e r e n t semen treatments were inseminated a l t e r n a t e l y at each s e s s i o n , to el i m i n a t e any p o s s i b l e e f f e c t of d i f f e r e n t h o l d i n g times. Just before insemination, semen was loaded i n t o one ml t u b e r c u l i n s y r i n g e s . Stored samples were mixed g e n t l y before use. Before drawing semen i n t o the syringe, the plunger was withdrawn s l i g h t l y to provide an a i r space so that greater pressure could be a p p l i e d to help force the semen out of the syringe and up the reproductive t r a c t . Hens were inseminated by eversion of the ovid u c t . Hens were h e l d between the operators thighs a l l o w i n g both 69 hands to be free to evert the oviduct by app l y i n g gentle pressure to each side of the vent and the abdominal muscles. As a second operator i n s e r t e d the syringe i n t o the oviduct, the pressure on the abdominal muscles was rele a s e d and the plunger depressed to d e l i v e r the sperm. This helped to t r a n s f e r the semen up the reproductive t r a c t . 4.3.5 Egg Collection and Incubation I n d i v i d u a l records were kept on each hen. The eggs l a i d on the day of insemination and the day a f t e r were discarded. Eggs l a i d from day 2 to 15 were c o l l e c t e d d a i l y , i d e n t i f i e d by hen number and date and placed i n an egg cooler at 13°C and 36% r e l a t i v e humidity. Eggs, having been allowed to a c c l i m a t i z e to room temperature, were set once a week i n a conventional forced d r a f t Robbins incubator at 37.2°C dry bulb temperature and 22.2°C wet bulb temperature. F e r t i l i t y was assessed on the seventh day of i n c u b a t i o n by c a n d l i n g . A l l apparently i n f e r t i l e and questionable eggs were broken open and examined ma c r o s c o p i c a l l y f o r evidence of embryonic development using the method of Fant (1957) . Percent f e r t i l i t y was de f i n e d as the number of f e r t i l e eggs d i v i d e d by the t o t a l number of eggs incubated. F e r t i l i t y was computed f o r three periods: (1) i n i t i a l f e r t i l i t y as determined by the percentage of f e r t i l e eggs l a i d during Days 2-8, (2) f i n a l f e r t i l i t y as determined by the percentage of f e r t i l e eggs l a i d during Days 9-15 and (3) d u r a t i o n of f e r t i l i t y as i n d i c a t e d by the percentage of f e r t i l e eggs l a i d during Days 9-15. 70 4.3.6 S t a t i s t i c a l Analyses The i n vitro study data was analyzed by two-way A n a l y s i s of Variance using the General Linear Models procedure of S t a t i s t i c a l A n a l y s i s System ( L i t t e l l , 1991) . Percentage data was transformed to arc sine V% p r i o r to a n a l y s i s to normalize the d i s t r i b u t i o n (Steele and T o r r i e , 1960). Main e f f e c t s were conducted with respect to extender (NEAT, LAKE, and BPSE) and storage time (Oh and 24h) and extender-storage time i n t e r a c t i o n . S i g n i f i c a n t d i f f e r e n c e s between means f o r extender and storage time were separated by l e a s t squares means. U n i v a r i a t e Repeated Measures A n a l y s i s of Variance ( L i t t e l l , 1991) was used to compare in vivo study f e r t i l i t y data over four one week pe r i o d s . Percentage data was transformed to arc sine -i% p r i o r to a n a l y s i s to normalize the d i s t r i b u t i o n (Steele and T o r r i e , 1960) . S i g n i f i c a n t d i f f e r e n c e s i n treatment means were separated by l e a s t squares a n a l y s i s of variance procedure using General Linear Models procedure of SAC I n s t i t u t e Inc. The model used was: Y i j k l = U + T t + S j + T S ; j + 6 l j + W k + T W i k + S W j k + T S W i j k + e i j k l where: i = 1...3 treatment; j = 1...2 storage time; k = 1...4 week. Y i j k = equals the observation f o r the 1 t h hen, on i t h treatment, on the j t h storage time, on the kth week, u = po p u l a t i o n mean. T i = e f f e c t of treatment. S j = e f f e c t of storage time. T S i ; j = i n t e r a c t i o n of treatment w i t h storage time. W k = e f f e c t of weekly insemination. T W i k = i n t e r a c t i o n of treatment with week. S W j k = i n t e r a c t i o n of storage time w i t h week. T S W i j k = i n t e r a c t i o n of treatment w i t h storage time w i t h week. e i j k l = random e r r o r a s s o c i a t e d with i n d i v i d u a l observations, e ij = sampling e r r o r a s s o c i a t e d repeated measures. 71 The sampling e r r o r mean square was used to t e s t repeated measures e f f e c t s of weekly insemination. The between subject e f f e c t s (treatment, storage time and i n t e r a c t i o n of treatment with storage time) were t e s t e d using the experimental e r r o r . S t a t i s t i c a l r e s u l t s were the same f o r transformed and non-trans formed f e r t i l i t y data, so values are expressed as non-transformed l e a s t square means. Unless otherwise s t a t e d , a l l comparisons were made at the 5% l e v e l of s i g n i f i c a n c e (P<0.05). 4.4 RESULTS 4.4.1 In Vitro Sperm Survival 4.4.1.1 Morphological A b n o r m a l i t i e s : Maintenance of semen morphology was a f f e c t e d (P<0.01) by treatment,, storage time and the i n t e r a c t i o n of treatment by storage time (Appendix 7). The i n i t i a l mean percentage of abnormal spermatozoa was 3.1% (Table 15). D i l u t i o n of semen i n BPSE and LAKE maintained semen morphology during the f i r s t e i g h t hours of storage. Regardless of storage time, neat semen r a p i d l y degenerated when compared to extended semen (P<0.01)(Figure 4). 4.4.1.2 L i v e Sperm Percentage: The p r o f i l e s , LSMeans and a n a l y s i s of variance f o r semen v i a b i l i t y are presented i n Table 16, Figure 5, and Appendix 8, r e s p e c t i v e l y . Storage time and treatment s i g n i f i c a n t l y a f f e c t e d the v i a b i l i t y of spermatozoa. Treatment by storage time was not s i g n i f i c a n t . The percentage of dead spermatozoa increased a f t e r 72 eight hours of storage without extension (P<0.01). There was no s i g n i f i c a n t difference between semen stored i n BPSE and LAKE over the 48 hour storage period (P>0.05). 4.4.1.3 Sperm M o t i l i t y : Maintenance of semen m o t i l i t y was affected (P<0.01) by treatment, storage time and the in t e r a c t i o n of treatment by storage time (Appendix 9). I n i t i a l m o t i l i t y (Oh) averaged 3.6 for fresh semen. At 24 hours of storage, mean scores for m o t i l i t y for NEAT, BPSE, and LAKES were 0.8, 3.0 and 2.8, respectively (Table 17). It was evident that d i l u t i o n of semen i n BPSE or LAKE, resulted in consistently better maintenance of m o t i l i t y than neat semen (Figure 6) . M o t i l i t y of neat semen s i g n i f i c a n t l y decreased during the f i r s t 8 hours of storage (P<0.05). 4.4 .2 In Vivo Sperm F e r t i l i z i n g Capacity The LSMeans and analysis of variance for egg f e r t i l i t y are presented i n Table 18 and 19, and Appendix 10. F e r t i l i t y was affected by storage time, week and the interactions of treatment by storage time and week by treatment. Treatment effects were not s i g n i f i c a n t . F e r t i l i t y following AI with fresh neat semen was 81% or higher (Table 19) . In contrast, f e r t i l i t y following AI with stored neat semen was less than 7%. Regardless of extender treatments, inseminations with unstored semen resulted i n superior f e r t i l i t y to stored semen (P<0.01). 73 TABLE 15. Effect of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE) on percentage of abnormal sperm as a function of storage time at 5 °C Time (h) n* NEAT BPSE LAKE 0 5 3 . 1 ± 1 . 7 a 3 . 1 ± 1 . 7 a 3 . 1 ± 1 . 7 a 8 3 5 4 . 2 ± 2 . 2 d 5 . 1 + 2 . 2 a b 6 . 7 ± 2 . 2 a b 24 3 8 5 . 2 ± 2 . 2 e 9 . 4 ± 2 . 2 b 1 5 . 0 ± 2 . 2 C 48 3 9 0 . 7 ± 2 . 2 e 1 1 . 3 + 2 . 2 b 1 6 . 2 ± 2 . 2 C Values ace presented as nontransformed LSMeans ± SEM (n). *n = number of r e p l i c a t e s per treatment. S t a t i s t i c a l analyses were based on least square means of arc s ine transformed data . Non-transformed LSMeans with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y ( P < 0 . 0 5 ) . 74 FIGURE 4. Percentage abnormal sperm as a function of storage time at 5C. 100i 1 T ~i 1 1— 0 8 24 48 TIME (hours) 75 TABLE 16. Effect of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE) on percentage of dead sperm as function of storage time at 5 °C Variable Dead (%) Time (h) 0 15 4.0 ± 0.7a 8 15 5.0 + 0.7ab 24 15 6.5 + 0.7b 48 15 8.7 ± 0.7C Treatment NEAT 20 7 7 + 0. 6a BPSE 20 5 1 + 0. 6b LAKES 20 5 2 + 0. 6b Values are presented as nontransformed LSMeans ± SEM (n). *n = number of r e p l i c a t e s per treatment. S t a t i s t i c a l analyses were based on least square means of arc s ine transformed data . Non-transformed LSMeans with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05). 76 FIGURE 5. Percentage dead sperm as a function of storage time at 5C . 0 8 24 48 TIME (hours) 77 TABLE 17. Effect of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender (LAKE) on sperm mo t i l i t y score as a function of storage time at 5 °C Time (h) n* NEAT BPSE LAKE 0 5 3.6 ± 0.3a 3.6 ± 0.3a 3.6 ± 0.3a 8 5 2.0 + 0.3C 3.0 ± 0.3ab 3.0 ± 0.3ab 24 . ' 5 0.8 ± 0.3d 3.0 ± 0.3ab 2.8 + 0. 3 a b c 48 5 0.2 ± 0.3d 2.2 + 0.3bc 2.8 + 0. 3 a b c Values are presented as LSMeans + SEM (n). "n = number of r e p l i c a t e s per treatment. LSMeans with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05) 78 FIGURE 6. Sperm motility score as a function of storage time at 5C. 4 l : 1 QJ , , , r-0 8 2 4 4 8 TIME (hours) 79 TABLE 18. Percent f e r t i l i t y over 4 weeks of fresh (Oh) or stored (24h) chicken semen diluted i n B e l t s v i l l e Poultry Semen Extender (BPSE) or Lakes Poultry Semen Extender (LAKE) Storage Time Extender 0 Hours 24 Hours NEAT 92 ± 4.8a (6) 3 ± 4.8b (7) BPSE 83 ± 4.8a (6) 30 ± 4.5C (7) LAKE 90 ± 4 . 5a (7) 32 ± 4.6C (6) Values are presented as nontransformed LSMeans ± SEM (n) . n = number of hens per 0 hour and 24 hour groups, r e s p e c t i v e l y . D i f f erences in n are due to m o r t a l i t i e s and hens which where not inseminated for the f u l l four week p e r i o d . " c S t a t i s t i c a l analyses were based on l eas t squared means of arc s ine transformed data . Non-transformed LSMeans fo l lowed by d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05). 80 Table 19. Effect of B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender ( LAKE) on f e r t i l i t y after inseminations with fresh (Oh) or stored (24h) chicken semen Week* Storage NEAT Di l u e n t s BPSE LAKE 1 2 3 4 0 81 ± 8.4a (6) 0 95 ± 8.3a (6) 0 94 ± 10.4a (6) 0 98 ± 11.7a (6) 88 ± 8.3a (6) 79 + 8.3a (6) 93 ± 10.4a (6) 72 ± 11.7ac (6) 75 ± 7.7a (7) 94 ± 7.7a (7) 93 ± 9.6a (7) 96 ± 10.8a (7) 1 2 3 4 24 24 24 24 7 ± 8.4b(6) 3 ± 8.3b(6) 0 ± 10.4b(6) 0 ± 11.7b(6) 25 ± 7.7b (7) 24 ± 7.7b(7) 49 ± 9.6C(7) 24 ± 10.8b(7) 6 + 8.3b(7) 14 ± 8.3b(7) 54 ± 10.4C(6) 60 ± 11.7C(7) Week represents days 2 - 8 fo l lowing each A I . Values are presented as nontransformed LSMeans ± SEM (n) n = number of hens per groups. D i f f erences in n are due to m o r t a l i t i e s and hens which d i d not lay any eggs. fl"b S t a t i s t i c a l analyses were based on least squared means of arc s ine transformed data . Non-transformed LSMeans for weeks fol lowed by d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P<0.05) 81 4 .5 D I S C U S S I O N On the study s l i d e s , 97% of a l l c e l l s appeared to have normal morphology and 96% were a l i v e . These are s i m i l a r to values reported i n the l i t e r a t u r e f o r good q u a l i t y samples (Lake and Stewart, 1978a; A l l e n and Champion, 1955) . There was an increased number of c o i l e d spermatozoa i n smears of stored neat semen i n comparison to smears of unstored neat semen. Spermatozoa morphological ab n o r m a l i t i e s can be c l a s s i f i e d according to t h e i r o r i g i n . Primary ab n o r m a l i t i e s occur during spermatogenesis and secondary abn o r m a l i t i e s occur a f t e r the sperm has l e f t the t e s t e s , i e . those defects that a r i s e during or a f t e r semen c o l l e c t i o n . As soon as semen i s c o l l e c t e d from the male, a p r o p o r t i o n of the spermatozoa begin to n a t u r a l l y lose t h e i r i n t e g r i t y and the process continues during storage i n vitro (Lake, 1983). C o i l i n g , a secondary abnormality, i s an i n d i c a t i o n that sperm are i n the l a t e stages of d i s i n t e g r a t i o n (Lake and Stewart, 1978a). I t i s u n l i k e l y that such spermatozoa would have a chance to ascend the oviduct to e f f e c t f e r t i l i z a t i o n . Saeki (1960) found that an increase i n the p r o p o r t i o n of abnormal spermatozoa during the storage of u n d i l u t e d semen r e s u l t e d i n low f e r t i l i t y . In h i s study, the incidence of secondary ab n o r m a l i t i e s reached a rat e of 49% a f t e r nine hours of storage. Neat semen could not be stored (even f o r 24 hours) without s i g n i f i c a n t l o s s i n m o t i l i t y . This i s s i m i l a r to the f i n d i n g s reported by Ax and Lodge (1975) . The d e c l i n e i n m o t i l i t y i s l i k e l y a s s o c i a t e d with sperms l i m i t e d metabolic pathways and the 82 production of metabolic-by-products that may accumulate and reach toxic l e v e l s (Wishart, 1989). D i l u t i o n of chicken semen with both BPSE and LAKE prolonged sperm m o t i l i t y during storage. The addition of extenders s t a b i l i z e s pH and osmolality, protect against toxic ions and i n other ways extends the l i f e of the semen samples (Bottwalla and Miles, 1992). One noticeable feature i n a comparison between neat semen before and after 24 hours storage, i s the decline i n egg f e r t i l i t y (from 92% to 3%). Similar observations have been documented by other researchers (Schindler et a l . , 1955; Sexton, 1977b). Stored neat semen's decline i n f e r t i l i t y r e f l e c t s i t s poor keeping quality and the need for extenders. In the present study, the f e r t i l i z i n g capacity of unstored semen was not d i f f e r e n t when inseminated aft e r d i l u t i o n i n BPSE or LAKE (83% and 90%, respectively). As well, the f e r t i l i z i n g capacity of stored semen was not d i f f e r e n t when inseminated aft e r d i l u t i o n and storage for 24 hours i n BPSE or LAKE (30% and 32%, res p e c t i v e l y ) . These results are i n general agreement with those reported by others for semen di l u t e d i n BPSE and LAKE (Sexton, 1977b; Lake and Ravie, 1979). Sexton (1977b) stored poultry semen for 24 hours i n BPSE and found the f e r t i l i z i n g capacity of semen was s i g n i f i c a n t l y lower than that of unstored controls. Sexton (1974) presented evidence that fructose, present i n BPSE (Appendix 2), i s used much more e f f i c i e n t l y by spermatozoa as an energy source than glucose in v i t r o . It was hypothesized i n the present study, therefore, that differences may occur i n sperm f e r t i l i z i n g 83 capacity due to differences i n the energy source of each extender. The results did not support t h i s hypothesis. The highest stored semen f e r t i l i t y rate obtained i n th i s study was 60% for semen di l u t e d i n LAKE. Lake and Ravie (1979) reported f e r t i l i t y rates of up to 87% for semen stored for 24 hours i n LAKE. The high f e r t i l i t y obtained by Lake and Ravie may be a resu l t of a higher insemination dose, 0.06 ml i n contrast to 0.05 ml for the present study, or the selection of hens producing a succession of f e r t i l e eggs over a period of several weeks. It i s highly probable in the present study that i f the insemination dose had been increased or inseminations occurred more frequently, f e r t i l i t y would have be improved, perhaps to the l e v e l of unstored samples. In t h i s study, the sperm number was not taken into consideration at time of insemination. This p o t e n t i a l l y could have led to reduced f e r t i l i t y because the number of spermatozoa i n a semen sample i s l i k e l y to vary during the course of a breeding period. Variations i n sperm numbers can arise from variations i n the amount of transparent f l u i d present i n the c o l l e c t e d semen samples. During ejaculation, lymphatic folds within the cloaca of the fowl become swollen i n response to the massage stimulus and from these folds a variable a mount of transparent f l u i d i s obtained when the copulatory organ i s squeezed to obtain semen (Lake, 1983) . S i g n i f i c a n t week effects on f e r t i l i t y might r e f l e c t undetected procedural differences or "learning" rather than difference i n sperm numbers between inseminations as there appeared to be no co r r e l a t i o n between the f e r t i l i t y trends and the number of 84 sperm inseminated. As well, according to Lake and Stewart (1978a) the minimum number of sperm inseminated (9.6 x 107) should have been s u f f i c i e n t to ensure maximum f e r t i l i t y . It i s f r u s t r a t i n g that while chicken semen d i l u t e d i n extenders and cooled to 5°C to slow catabolic and degradation processes, may only retain t h e i r f e r t i l i z i n g capacity for 24 hours in vitro, spermatozoa can survive i n the oviduct of the hen at body temperature for at least f i v e to 11 days before they f e r t i l i z e an egg (Lake, 1983) . Clearly, future work i n thi s area needs to try to mimic the environment to which the sperm are exposed i n the sperm storage tubules. The sperm storage tubules (SST) are located near the junction of the uterus and vagina. After each egg i s l a i d , sperm are released from the SST and are transported to the infundibulum where f e r t i l i z a t i o n takes place. In commercial breeding, i t i s important to maintain a constant l e v e l of high f e r t i l i t y . To achieve th i s , a s u f f i c i e n t number of f u l l y viable sperm must be inseminated (Bakst, 1989) . The f e r t i l i t y of hens inseminated with stored semen i n the present study, was much lower than that obtained with fresh semen which indicates that the number of spermatozoa remaining viable after in vitro storage i s small. It has been suggested that the SST are sperm-selective (Zavaleta, 1987). Thus, the non-viable population of spermatozoa within the inseminate are u n l i k e l y to enter the sperm storage tubules, thereby decreasing the number of spermatozoa available for f e r t i l i z a t i o n . Subsequent inseminations may augment the spermatozoa populating the SST. This i s r e f l e c t e d i n the increase i n weekly f e r t i l i t y rate 85 for spermatozoa stored i n LAKE. Also, the presence of dead sperm i n the inseminate has been shown to have detrimental effects on f e r t i l i t y , possibly due to the release of i n t r a c e l l u l a r p r o t e o l y t i c enzymes and toxic organic peroxidases (Sexton, 1988). With increased storage time, the number of spermatozoa that degenerate and become non-viable increases. The magnitude and speed of this degeneration depends on d i l u t i o n of the semen. Therefore, a dose of unstored semen containing s u f f i c i e n t sperm may have to be increased afte r storage i n an undiluted state. In general, the results of this study indicate that sperm f e r t i l i z i n g capacity i s reduced aft e r storage in vitro, and extension of semen helps to maintain sperm v i a b i l i t y . Both BPSE and LAKE worked equally well to maintain poultry sperm f e r t i l i z i n g capacity after short-term storage in vitro. As BPSE i s available commercially, selection of the "best" extender can be made based on cost and a v a i l a b i l i t y of extender components and personal preference. More work i s needed to determine i f one of these extenders can be adapted for future use with wild Mallard semen. 86 5.0 EVALUATION OF VARIOUS CRYOPROTECTANTS FOR THE CRYOPRESERVATION OF AVIAN SEMEN 5.1 ABSTRACT Several s t u d i e s were conducted to determine the e f f e c t of: a) var i o u s concentrations of g l y c e r o l , ethylene g l y c o l (EG), propylene g l y c o l (PG) and sucrose on the f e r t i l i z i n g c a p a c i t y of unstored chicken spermatozoa and b) the a d d i t i o n of sucrose to EG or PG on the v i a b i l i t y of frozen-thawed semen. Levels of 4% g l y c e r o l , 16% EG or PG and 0.3, 0 .4 , and 0.5M sucrose s i g n i f i c a n t l y reduced f e r t i l i t y (P<0.05) . No d i f f e r e n c e s were observed between EG and PG. The treatment with the highest post-thaw m o t i l i t y score was g l y c e r o l . The presence of sucrose during f r e e z i n g d i d not improve avian semen freeze-thaw recovery a f t e r c r y o p r e s e r v a t i o n . 5.2 INTRODUCTION Cryopreservation of semen i s a process by which a sample i s suspended i n a d i l u e n t c o n t a i n i n g a cryoprotectant and maintained frozen at -196°C i n a v i a b l e s t a t e f o r long periods of time. The main ob s t a c l e to the success of developing a technique to freeze semen i s i d e n t i f y i n g a cryoprotectant that w i l l maintain good semen f e r t i l i z i n g a b i l i t y . Simply f r e e z i n g and thawing sperm i n bu f f e r e d s a l t s o l u t i o n s , r e s u l t s i n c e l l death. The l e t h a l i t y of freeze-thawing i s a s s o c i a t e d w i t h the formation of i n t r a c e l l u l a r i c e and i n c r e a s i n g s o l u t e concentration, r a t h e r than t h e i r a b i l i t y to endure storage at sub-zero 87 temperatures (Saacke, 1982). These damaging processes have two opportunities to occur i n the c e l l , once during freezing and once during thawing. As freezing begins, the water component of the extender i s frozen out of solution and transformed into i c e . This leaves the sperm bathed i n increasing concentrations of the soluble components of the extender. The increasing e x t r a c e l l u l a r solute concentration causes water to be drawn out of the sperm. If freezing i s slow, this water contributes to the exclusive formation of e x t r a c e l l u l a r ice (Saacke, 1982). But i f cooling i s too rapid, the c e l l i s not able to osmotically lose water fast enough to prevent the formation of damaging i n t r a c e l l u l a r ice (Szell and Shelton, 1986; Mazur, 1984). Cooling rates slow enough to prevent the formation of i n t r a c e l l u l a r ice can also be damaging. The cause of t h i s damage i s s t i l l unclear. As c e l l s lose water during the freeze process the become dehydrated and shrink. Maryman (1974) suggested that the c e l l s may shrink below a minimum volume where s t r u c t u r a l i n t e g r i t y i s jeopardized. In his theory cryoprotectants protect by decreasing the amount of the c e l l ' s water that needs to leave during freezing. In contrast, Mazur (1980) effects injury to changes i n size of the unfrozen portion and suggests that freezing becomes damaging whenever the unfrozen percentage of the sample drops below 8 to 12%. As ice expands outside the c e l l , i t puts constraints on the shapes that can be assumed by the c e l l s , r e s u l t i n g i n c e l l deformation and damage (Mazur, 1984). Cryoprotectants are compounds that are added to extenders to 88 help reduce the detrimental effects of freezing and thawing. Cryoprotectants can be c l a s s i f i e d as permeating or non-permeating based on t h e i r a b i l i t y to enter the c e l l . Permeating cryoprotectants such as gl y c e r o l , ethylene g l y c o l , propylene g l y c o l and other alcohols penetrate into the c e l l and lower the temperature at which water i n i t i a t e s ice c r y s t a l formation (Mazur, 1984) . As a result, there i s a decrease i n the amount of ice formed and a decrease in the solute concentration to which the c e l l s are exposed at the same sub-zero temperature (Saacke, 1982). Non-permeating cryoprotectants such as sucrose, glucose, and other sugars are added to the cryoprotectant medium to regulate water movement across the c e l l membranes. Sucrose, which i s impermeable to c e l l s , has been found to have a s t a b i l i z i n g effect on b i o l o g i c a l membranes by minimizing dramatic changes i n osmotic pressure (Takeda et al.', 1987; Schneider and Mazur, 1984). This helps lower the amount of cryoprotectant that penetrates the c e l l , thus reducing possible toxic e f f e c t s (Szell and Shelton, 1987) and helps prevent rapid swelling as the permeating cryoprotectant moves out of the c e l l and water flows i n during thawing (Takeda et a l . , 1987) . Many researchers have investigated the use of cryoprotectants for fowl semen (see reviews by Graham et a l . 1984; Lake, 1986; Hammerstedt et a l . , 1992; Bellagamba et a l . , 1993). Good f e r t i l i t y has been obtained under certain circumstances with semen that has been frozen and stored i n the presence of glycerol (Lake and Stewart, 1978b). However, t h e i r success was probably augmented by 89 using only males that produced high q u a l i t y semen at insemination and depositing the frozen-thawed semen into the hen by deep i n t r a -vaginal inseminations near the sperm storage tubules. The consensus i s that under current protocols rooster semen survives poorly with only 1.6% of spermatozoa retaining t h e i r f e r t i l i z i n g a b i l i t y after freezing and thawing (Wishart, 1985) . Unlike mammalian spermatozoa, which must f e r t i l i z e a single ova within 24 hours of insemination, avian spermatozoa must survive i n the oviductal sperm storage tubules to f e r t i l i z e multiple ova over a period of several days. One concern with glycerol, i s i t s contraceptive e f f e c t i n the female. Glycerol concentration must be reduced aft e r thawing and before insemination (Lake and Ravie, 1984) . Glycerol concentrations can be lowered from the cryoprotectant l e v e l (8%) to a more tolerable l e v e l (less than 2% v/v) by slow d i l u t i o n or d i a l y s i s (to minimize osmotic-induced swelling). However, these techniques frequently cause damage to already weakened c e l l s and are impractical under routine f i e l d conditions. The mechanism by which glycerol exerts the contraceptive action has not yet been i d e n t i f i e d (Bellagamba et a l . , 1993). Alternative cryoprotectants that do not depress sperm f e r t i l i z i n g a b i l i t y when inseminated at concentrations necessary to protect spermatozoa during freezing are needed. Ethylene glycol and propylene gl y c o l have been examined as alternative cryoprotectants (Lake and Ravie, 1984; Maeda, 1984; Bellagamba, 1993) but neither has been found better than glycerol at protecting against freeze i n j u r i e s . It may be possible to improve the 90 protective a b i l i t y of propylene gly c o l (PG) or ethylene gly c o l (EG) by the addition of sucrose to the cryoprotectant. To determine the effectiveness of sucrose addition on freeze-thaw recovery, i t i s f i r s t necessary to know the e f f e c t these cryoprotective agents have on the f e r t i l i z i n g a b i l i t y of fresh semen. Obj ectives: To determine the e f f e c t of: 1) Various concentrations of glycerol, propylene g l y c o l , ethylene glyc o l , and sucrose on the f e r t i l i z i n g capacity of fresh semen. 2) Sucrose addition to the cryoprotectant medium on m o t i l i t y and f e r t i l i z i n g capacity of frozen-thawed semen. 5.3 MATERIALS AND METHODS To test these concepts, B e l t s v i l l e Poultry Semen Extender (BPSE) (Sexton, 1977) was selected as a base to which cryoprotectants were added. The following general protocol was used: 5.3.1 Semen Collection and Evaluation Roosters and hens were maintained as described under Materials and Methods of Chapter four. Semen was c o l l e c t e d from roosters by the abdominal massage method (Lake and Stewart, 1978a), inspected to eliminate samples contaminated with debris and pooled for use. 91 5.3.2 Cryoprotectant Concentration Pooled semen was d i v i d e d i n t o equal q u a n t i t i e s and placed i n t o three ml glass tubes c o n t a i n i n g treatments o u t l i n e d below. Semen was d i l u t e d at a r a t e of 1:1 wi t h treatment s o l u t i o n s at ambient temperature. (1) G l y c e r o l c o n c e n t r a t i o n - Four concentrations of g l y c e r o l were used f o r f e r t i l i t y t r i a l s . The four concentrations of the g l y c e r o l were 0, 1, 2 or 4% by volume of BPSE ( f i n a l c o n c e n t r a t i o n 0.5%, 1.0% or 2%, r e s p e c t i v e l y ) . (2) Ethylene g l y c o l and propylene g l y c o l c o n c e n t r a t i o n - The e f f e c t of ethylene g l y c o l and propylene g l y c o l on f e r t i l i t y of ro o s t e r semen were t e s t e d . There were f i v e treatment groups of 0% EG and PG, 8% EG, 16% EG, 8% PG or 16% PG by volume of BPSE ( f i n a l c o n c e n t r a t i o n 0% EG and PG, 4% EG, 8%EG, 4% PG and 8% PG, r e s p e c t i v e l y ) . (3) Sucrose conce n t r a t i o n - The e f f e c t of sucrose c o n c e n t r a t i o n (0, 0.1, 0.2, 0.3, 0.4, 0.5m; f i n a l c o n c e n t r a t i o n 0.0, 0.5, 1.0, 1.5, 2.0, 2.5M sucrose, r e s p e c t i v e l y ) i n BPSE on f e r t i l i t y of rooster semen were t e s t e d . 5.3.3 Sucrose Addition Pooled semen was taken to a c o l d room (13°C) and allowed to e q u i l i b r a t e f o r ten minutes. Semen (500 ul) was placed i n a t e s t tube and mixed wi t h BPSE c o n t a i n i n g e i t h e r : (1) 0% EG and PG, (2) 8% PG, (3) 8% EG, (4) 8% PG + 0. 2M sucrose, (5) 8% EG + 0.2M sucrose or (6) 8% g l y c e r o l . Volumes were adjusted to give an 92 insemination dose of 1.0 x 108 sperm/0.15 ml. The aliquot containing 0% EG and PG was inseminated immediately. The remaining aliquots of d i l u t e d semen were frozen and then inseminated as outlined below. 5.3.4 Freezing The procedure for preparing, freezing and thawing the semen was s i m i l a r to that of Lake and Stewart (1978b) . Sperm were allowed to e q u i l i b r a t e for 10 minutes before being drawn up into 0.5 cc straws. Loaded straws were sealed with polyvinyl propylene powder (PVP) and placed i n a programmable freezing chamber (Bio-cool II Model 1A000, FTS Systems Ltd., Stone Ridge, NY, USA) at 5°C. The temperature was allowed to drop to -40°C at 1°C per minute. Straws were held at -40°C for f i v e minutes, removed from the freezing chamber and quickly plunged into l i q u i d nitrogen. Each hen was inseminated once with 0.15 ml (1.0 x 108 sperm) of frozen-thawed semen. Sperm m o t i l i t y was scored before and aft e r freezing using the procedures outlined i n Chapter four. 5.3.5 Thawing and Cryoprotectant Removal After storage for one week i n l i q u i d nitrogen, straws were thawed i n a 13°C water bath. The contents of two straws were emptied and pooled. Straws requiring deglycerolization, were emptied into a test tube. The glycerol concentration was reduced by the addition of 0.16, 0.44, 0.80, 1.46, 3.0, and 3.8 ml of BPSE at three minute i n t e r v a l s . Samples were held at 13°C. Samples were 93 c e n t r i f u g e s at 1000 x g f o r 15 minutes. The supernatant was decanted and the spermatozoa resuspended to the o r i g i n a l volume by g e n t l y mixing w i t h BPSE. 5 . 3 . 6 Insemination White Leghorn hens (approximately 26 weeks of age) were randomly assigned to s i n g l e l e v e l l a y i n g cages. Hens f o r each treatment were inseminated once (at approximately 1400h) wi t h 0.01 ml of d i l u t e d semen unless otherwise s t a t e d . Each treatment c o n s i s t e d of two r e p l i c a t e s of s i x hens each. AI was c a r r i e d out as described under M a t e r i a l s and Methods of Chapter four. 5 . 3 . 7 Egg Collection and Incubation Egg c o l l e c t i o n and i n c u b a t i o n was c a r r i e d out as o u t l i n e d i n M a t e r i a l s and Methods of Chapter four. 5 . 3 . 8 S t a t i s t i c a l Analyses F e r t i l i t y was computed f o r three p e r i o d s : (1) i n i t i a l ; f e r t i l i t y as determined by the percentage of eggs l a i d during days 2-8 a f t e r insemination, (2) f i n a l ; f e r t i l i t y as determined by the percentage of eggs l a i d during days 9-15 a f t e r insemination and, (3) d u r a t i o n of f e r t i l i t y as determined by the percentage of f e r t i l e eggs l a i d during days 2-15 of a given t r a i l . The percentage of f e r t i l i z e d eggs l a i d by each hen was transformed to arc sine V"% p r i o r to a n a l y s i s to normalize the d i s t r i b u t i o n (Steele and T o r r i e , 1960) . 94 Data f o r Days 2-8, 9-15 and 2-15 were subjected to a n a l y s i s of variance t e s t i n g main e f f e c t s of cryoprotectant c o n c e n t r a t i o n and r e p l i c a t e ( g l y c e r o l a d d i t i o n - 4 x 2 , EG and PG a d d i t i o n - 5 x 2 and sucrose a d d i t i o n - 6 x 2 ) using the General L i n e a r Models (GLM) procedure of SAS ( L i t t e l l , 1991). Each d a i l y pooled semen sample (n = 15) c o n s t i t u t e d a r e p l i c a t e f o r each treatment. A f t e r o b t a i n i n g a s i g n i f i c a n t F-value, means were separated by l e a s t squares mean at the 5% l e v e l of s i g n i f i c a n c e (P<0.05). 5.4 RESULTS 5.4.1 Glycerol Concentration The f e r t i l i t y r e s u l t s obtained w i t h semen f r e s h l y d i l u t e d i n BPSE c o n t a i n i n g various concentrations of g l y c e r o l are presented i n Table 20, Figure 7 and Appendix 11. F e r t i l i t y was a f f e c t e d by g l y c e r o l c o n c e n t r a t i o n (0, 1, 2 or 4%) and r e p l i c a t e (1 or 2) . Day 2 to 8 LSMeans were 95.3 + 7.3, 90.3 ± 7.3, 76.8 ± 7.3, and 31.4 ± 7.3 f o r sperm t r e a t e d with 0, 1, 2 or 4% g l y c e r o l , r e s p e c t i v e l y . F e r t i l i t y was s i g n i f i c a n t l y (P<0.01) reduced w i t h a f i n a l g l y c e r o l c o n c e n t r a t i o n of 4%. There was no s i g n i f i c a n t d i f f e r e n c e i n i n i t i a l f e r t i l i t y (day 2-8) among the 0, 1, and 2% g l y c e r o l treatment groups. The number of sperm per insemination dose ranged from 2.9 x 10 8 f o r r e p l i c a t e one and 2.0 x 108 f o r r e p l i c a t e two. There was no s i g n i f i c a n t treatment by r e p l i c a t e i n t e r a c t i o n . 95 5.4.2 Ethylene Glycol and Propylene Glycol Concentration The f e r t i l i t y r e s u l t s of hens inseminated w i t h semen d i l u t e d i n v a r i o u s concentrations of propylene g l y c o l or ethylene g l y c o l are presented i n Table 21, Figure 8 and Appendix 12. A c o n c e n t r a t i o n of 8% PG or EG had l i t t l e i n f l u e n c e on i n i t i a l egg f e r t i l i t y when compared to the c o n t r o l . PG and EG at any c o n c e n t r a t i o n t e s t e d , s i g n i f i c a n t l y decreased d u r a t i o n of f e r t i l i t y from a high of 92.6 + 8.8 percent'(0% EG and PG) to a low of 39.3 ± 8.2 percent (16% PG). 5.4.3 Sucrose Concentration The LSMeans, p r o f i l e s and a n a l y s i s of variance f o r egg f e r t i l i t y a f t e r inseminations with various concentrations of sucrose are presented i n Table 22, Figure 9 and Appendix 13. F e r t i l i t y was a f f e c t e d by sucrose c o n c e n t r a t i o n (0.0, 0.1, 0.2, 0.3, 0.4, or 0.5 M) and r e p l i c a t e (1 or 2). D i l u t i o n with BPSE c o n t a i n i n g greater than 0.2M sucrose r e s u l t e d i n s i g n i f i c a n t l y (P<0.01) lower egg f e r t i l i t y than f o r lower sucrose concentrations. Only 4.1% of eggs l a i d between day 2-8 were f e r t i l e when 0.4M sucrose was used. The number of sperm per insemination dose was approximately 1.0 x 108 f o r r e p l i c a t e one and two. There was no s i g n i f i c a n t treatment by r e p l i c a t e i n t e r a c t i o n . 5 .4.4 Sucrose Addition M o t i l i t y data are summarized i n Table 23. Sperm i n f r e s h l y c o l l e c t e d semen samples had a m o t i l i t y score of 4.5 (based on an 96 a r b i t r a r y scale of 0-5). D i l u t i o n i n BPSE containing EG or PG did not aff e c t m o t i l i t y . It was evident that the prefreeze m o t i l i t y of semen was decreased by the addition of sucrose to cryoprotectant solutions. Freezing always reduced spermatozoa m o t i l i t y when compared with m o t i l i t y of neat or pre-freeze semen. Post-thaw, the m o t i l i t y score of semen stored with various cryoprotectants ranged between 0.5 and 2. The treatment with the highest post-thaw m o t i l i t y was gl y c e r o l . No f e r t i l e eggs were obtained post-insemination with frozen-thawed semen stored with any cryoprotectant combinations tested (not presented). 97 TABLE 20. Effect of different concentrations of glycerol i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa Variable F e r t i l i t y (%) Days 2-8 Days 9-15 Days 2-15 Glycerol 0% 95 3 ± 7 3a 75 .2 ± 7 .3a 86 .4 + 6 .3a 1% 90 3 + 7 3a 66. 7 + 6. gab 78 0 ± 6. 3 a b 2% 76 8 + 7 3a 54 2 ± 6 9 b 64 .9 + 6 3b 4% 31 4 ± 7 3b 10 .5 ± 6 .9C 21 .1 ± 6 .3° Replicate 1 65 1 ± 5 2a 39 .0 ± 5 .0a 52 . 6 + 4 . 4a 2 81 8 + 5 2 b 64 .3 + 4 .9b 72 . 6 + 4 .4b LSMeans in columns and v a r i a b l e s with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y {P < 0.05}. Data for g l y c e r o l group are LSMeans for 12 hens and r e p l i c a t e for 48 hens. 98 FIGURE 7. Effect of different concentrations of glycerol on the fertilizing capacity of unstored chicken spermatozoa (days 2-15 post-AI) 1 0 0 8 0 ^ 6 0 4 0 2 0 0 a d B " —_ S . a Replicate 1 \ cd • Replicate 2 d 0 1 2 Glycerol (%) Points with different letters are different (P<0.01) 9 9 TABLE 21. Effect of different concentrations of propylene glycol (PG) and ethylene glycol (EG) i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa Variable F e r t i l i t y (%) Days 2 - 8 Days 9 - 1 5 Days 2 - 1 5 Cryoprotectant 0 % E G & P G 9 7 . 4 ± 1 0 . 0 a 7 9 . 0 ± 1 0 a 9 2 6 ± 8 8 a 8 % E G 7 2 . 6 ± 1 0 . gab 41. 3 ± 7 . Qbc 5 8 . 9 ± 8 . 2 be 8 % P G 8 4 . 7 + 1 0 . 0 a 5 5 . 5 + 7 0 b 7 0 1 ± 8 2 b 1 6 % E G 4 9 . 9 + 1 0 . 0 b 1 4 . 9 + 7 0 d 3 9 3 ± 8 2 C 1 6 % P G 6 7 . 7 ± 1 0 . Qab 2 9 . 3 + 7 0° 5 2 . 9 + 8 . 2 be Replicate 1 7 7 . 0 ± 6. 3 4 6 . 9 ± 4 .4 6 7 . 0 + 5 . 6 2 7 1 . 9 + 6. 3 4 1 . 1 ± 4 .4 5 8 . 5 + 5 . 0 Non-transformed LSMeans for columns and v a r i a b l e s with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y {P < 0.05) . Data for g l y c e r o l group are LSMeans for 12 hens and r e p l i c a t e for 60 hens. 100 FIGURE 8. Effect of different concentrations of EG and PG on the fertilizing capacity of unstored chicken spermatozoa (days 2-15 post-AI) 0 8 16 Cryoprotectant Concentration (%) Bars with different letters are different (P<0.01) 101 TABLE 22. Effect of different concentrations of sucrose i n BPSE on the f e r t i l i z i n g capacity of chicken spermatozoa Variable F e r t i l i t y (%) Days 2-8 Days 9 -15 Days 2 -15 Sucrose O.OM 91.7 ± 7.1a 68.0 + 6.3a 79.8 ± 5.9a 0.1M 94.1 ± 7.1a 64.7 ± 6.3a 78.7 ± 5.9a 0.2M 70.8 ± 7.1a 49.8 ± 6.3a 59.7 ± 5.9a 0.3M 31.9 ± 7.1c 7.3 + 6.3b 19.1 ± 5.9b 0.4M 4.1 ± 7.1d 2.8 ± 6.3b 3.5 ± 5.9b 0.5M 20.3 ± 7.1cd 2.4 + 6.3b 6.4 ± 5.9b Replicate 1 4"0.3 + 4.1a 26.7 ± 3.6a 33.3 + 3.4a 2 64.0 ± 4.1b ' 38.3 + 3.6b 49.0 ± 3.4b a-d Non-transformed LSMeans for w i th in columns and v a r i a b l e s with no common s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P < 0 .05) . Data for g l y c e r o l group are LSMeans for 12 hens and r e p l i c a t e for 48 hens. 102 Points with different letter are different (P<0.01) 103 TABLE 23. Effect of different combinations of cryoprotectants i n BPSE and various stages of the freezing process on the moti l i t y score of chicken semen Variable M o t i l i t y Score Neat Pre-freeze Post-thaw 8% Propylene glycol(PG) 4.5 4.5 0.5 8% Ethylene gly c o l (EG) 4.5 4.5 1.5 8% PG + 0.2M Sucrose 4.5 1.0 0.5 8% EG + 0.2M Sucrose 4.5 1.0 1.0 8% Glycerol 4.5 3.0 2.0 Non-transformed LSMeans for wi th in columns and v a r i a b l e s with no common s u p e r s c r i p t s d i f f e r s i q n i f i c a n t l v (P < 0 .05) . _ a y Data for g l y c e r o l group are LSMeans for 12 hens and r e p l i c a t e for 48 hens. 104 5 . 5 DISCUSSION From the results presented, i t i s apparent that glycerol concentrations greater that 2% by volume of semen extender were detrimental to the f e r t i l i z i n g capacity of unstored rooster semen. These results are i n agreement with those reported by Polge (1951), N e v i l l e et al.(1971) and Sexton, (1973). N e v i l l e et a l . (1971) determined that egg h a t c h a b i l i t y was not reduced by glycerol concentration, which suggested that glycerol does not exert harmful ef f e c t s of the developing embryo. In contrast to the methods of N e v i l l e et a l . (1971) f e r t i l e eggs i n t h i s study were terminated after seven days of incubation, rather than taken to hatch. However, there was no evidence of early embryonic development being affected by glycerol concentration. The number of early dead embryos was less than 5% of the f e r t i l e eggs observed per treatment group. The present study confirms the need to f i n d an alternative cryoprotectant i f cryopreservation of frozen avian semen i s going to be practiced routinely i n the f i e l d . Despite the acceptable f e r t i l i t y results obtained after insemination with fresh semen di l u t e d i n BPSE containing less than 2% glycerol, glycerol must be present at a concentration of 8% to successfully protect spermatozoa during the freeze-thaw process (Lake and Ravie, 1984). Post-thaw reduction of glycerol concentration using currently available methods would be impractical under f i e l d conditions. Increasing EG and PG concentrations were found not to reduce f e r t i l i t y i n comparison to g l y c e r o l . This i s i n agreement with 105 results reported by Lake and Ravie (1984), but i n contrast to res u l t s reported by Sexton (1973). Sexton (1973) found that the f e r t i l i z i n g capacity of washed spermatozoa resuspended i n a potassium phosphate buffer (KPB) was reduced by 4 and 8% EG when compared to the control. In the present study, the lack of reduced semen f e r t i l i z i n g capacity i n 8% EG might be explained by the use of BPSE as an extender, rather than KPB. M o t i l i t y of spermatozoa measured soon aft e r thawing was reduced compared with the fresh sample. This finding i s supported by other authors who have demonstrated declines i n m o t i l i t y of frozen-thawed semen by 30 to 60% compared to fresh (Westfall and Harris, 1975; Bakst and Sexton, 1979; Scott et a l . , 1980). Although EG and PG are known to act as cryoprotectants during the freezing of embryos and oocytes, i t was shown that i n comparison to glycerol they have reduced freeze-thaw protection as determined by thawed spermatozoa m o t i l i t y . Lake and Ravie (1984), and Westfall and Harris (1975) made similar observations. M o t i l i t y after freezing of spermatozoa i n BPSE containing glycerol, however, was not a good i n d i c a t i o n of f e r t i l i z i n g a b i l i t y . Although motile, the revived population did not appear to be f u l l y functional since the insemination of motile thawed spermatozoa did not res u l t i n any f e r t i l e eggs. F e r t i l i t y was found to decline with increasing concentrations of sucrose. This decline i n f e r t i l i t y agrees with the findings of Sexton (1975), who reported a s i g n i f i c a n t loss i n the m o t i l i t y and f e r t i l i z i n g capacity of spermatozoa d i l u t e d i n 4, 8, and 12% 106 sucrose. Although higher sucrose concentrations were found to aff e c t f e r t i l i t y , the author choose to use the maximum non-toxic concentration (0.2M) for freezing spermatozoa on the assumption that i t may be more e f f i c i e n t at cryoprotectant removal than lower concentrations. There was no profound b e n e f i c i a l e f fects i n spermatozoa freeze-thaw protection observed with the addition carbohydrate i n the form of sucrose (0.2M) to the di f f e r e n t cryoprotectants. The presence of carbohydrate during freezing does not appear to be a key element to improving avian semen cryopreservation. Under the conditions employed, glycerol s t i l l provides the best freeze protection for avian spermatozoa. While research on avian semen preservation has focused primarily on seeking alternative cryoprotectants to glycerol (Maeda et a l , 1984; Lake and Ravie, 1978) or on techniques of glycerol removal before f e r t i l i z a t i o n (Buss, 1993; Lake and Stewart, 1978b) , i t may be b e n e f i c i a l to di r e c t future work towards i d e n t i f y i n g the cause of glycerols contraceptive action in the female. Once the action of glycerol on the spermatozoa i s i d e n t i f i e d , corrective measures can be adopted, enabling the use of cryopreservation of avian semen to be routinely applied by game-farm breeders and the poultry industry. 107 C O N C L U S I O N S Propagation of Mallards i n c a p t i v i t y was found to be hindered by low egg f e r t i l i t y , e s p e c i a l l y i n yearling p a i r s . Comparisons of t e s t i c u l a r weight and size between captive and f r e e - f l y i n g Mallards suggest that gonadal development i s suppressed i n Mallard drakes under captive conditions. This suppression of gonadal development may p a r t i a l l y account for the poor f e r t i l i t y i n captive ducks. With regard to propagating wild avian species i n c a p t i v i t y by a r t i f i c i a l means, successful preliminary work has been done. This thesis has demonstrated the need for the use of AI to supplement natural mating i n yearling Mallard pairs and that AI can be used successfully. To f a c i l i t a t e easier handling of birds held under an AI program, i t was found that hens could be housed separately from drakes without any adverse effects on egg production. A l o g i c a l extension of an AI program would be to preserve semen to allow the exchange of genetic material between distant populations and the establishment of gene banks. In general, the re s u l t s of t h i s thesis indicate that sperm f e r t i l i z i n g capacity -is reduced af t e r storage in vitro, and d i l u t i o n of semen with extenders helps to maintain sperm v i a b i l i t y . B e l t s v i l l e Poultry Semen Extender (BPSE) and Lake's Poultry Semen Extender were compared and were found to work equally well to maintain poultry sperm f e r t i l i z i n g capacity after short-term storage. As BPSE i s available commercially, selection of the "best" extender can be made based on cost and a v a i l a b i l i t y of extender components and 108 personal preference. More work i s needed to determine i f one of these extenders can be adapted for future use with w i l d - s t r a i n Mallard semen. The presence of carbohydrate during freezing was found not to be the key element for improving long-term avian semen preservation. In th i s thesis, glycerol was found to s t i l l provide the best protection during cryopreservation of spermatozoa. While research on avian semen preservation has focused primarily on seeking alternative cryoprotectants to glycerol or techniques of glycerol removal before f e r t i l i z a t i o n , i t may be b e n e f i c i a l to di r e c t future work towards i d e n t i f y i n g the cause of glycerols contraceptive action i n the female rather than making observations of the spermatozoa reactions to cryoprotective agents. 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Distribution of treatment groups for determining drake access and age effects on reproductive performance P h y s i c a l V i s u a l -A u d i t o r y <? ? Au d i t o r y Only cr ? 3Y 3A 3Y 3A 3Y 3A 3Y 3Y 3Y 3Y 3Y 3Y 3A 3A 3A 3A 3A 3A 3A 3Y 3A 3Y 3A 3Y Y - Y e a r l i n g s , A - A d u l t s . 120 APPENDIX 2. Composition of B e l t s v i l l e (BPSE) and Lakes (LAKE) Poultry Semen Extenders Extender Function Compound BPSEa LAKEb Buffer Dipotassium phosphate.3H20 1.27 -Monopotassium phosphate 0.07 -BES* - 3.06 TES ' 0.20 -Chelator Sodium glutamate 0.87 1.52 Osmotic Sodium Acetate.3H 20 0.43 _ balance lN-Sodium hydroxide — 5.8 ml Potassium c i t r a t e 0.06 0.12 Magnesium acetate.4H 20 - 0.08 Magnesium chloride.6H 20 0.03 -Energy source Glucose - 0.60 Fructose 0.50 -D i s t i l l e d H20 100 ml 100 ml PH 7.5 7.05 Values are in grams unless otherwise s ta ted . • Adapted from Sexton ( 1977) . " Adapted from Lake and Ravie (1979). *BES = NN-Bis(2-hydroxyethyl) -2-aminoethane sulphonic a c i d . 'TES= N-tr is(hydroxymethyl)methyl-2-aminoethane s u l f o n i c a c i d . 121 APPENDIX 3. Analysis of variance for clutch size for Mallard pairs of different ages with physical, visual-auditory, or auditory only contact Dependent Variable: Clutch Size Source of Vari a t i o n df Sum of Squares Mean Square F-Value Inference Model 11 1177.56 107.05 2.05 ns Contact 2 206.72 103.36 1.98 ns Age 3 118.89 39. 62 0.76 ns Contact*age 6 851.94 142.00 2.72 ns Error 24 1250.67 52.11 Total 35 2428.22 ns = Not s i g n i f i c a n t P < 0.05. 122 APPENDIX 4. Analysis of variance for days to f i r s t egg from start of t r i a l for Mallard pairs of different ages with physical, visual-auditory, or auditory only contact Dependent Variable: Days to F i r s t Egg Source of df Sum of Mean F-Value Inference Va r i a t i o n Squares Square Model 11 317.02 28.82 0.68 ns Contact 2 96.74 48.37 1.15 ns Age 3 110.69 36.90 0.88 ns Contact*age 6 87.78 14.63 0.35 ns Error 19 800.33 42.12 Total 30 1117.35 ns - Not s i g n i f i c a n t P < 0.05. 123 APPENDIX 5. Analysis of variance for down deposited as a percentage of clutch size for Mallard pairs of different ages with physical, visual-auditory, or auditory only contact Dependent Variable: Clutch Size Source of df Sum of Mean F-Value Inference Vari a t i o n Squares Square Model 10 2157.15 215.71 0.41 ns Contact 2 462.56 231.28 0.44 ns Age 3 1151.64 383.88 0.73 ns Contact *age 5 541.59 108.32 0.21 ns Error 15 7903.52 526.90 Total 25 10060.66 ns = Not s i g n i f i c a n t P < 0.05. 124 APPENDIX 6. Analysis of variance for plasma testosterone concentration of yearling and adult Mallard drakes Dependent V a r i a b l e : Testosterone Concentration Source of V a r i a t i o n df Sum of Squares Mean Square F-Value Inference Model 36 65.62 1.82 2.39 * * Age 1 0.32 0.32 0.25 ns Drake(age) 21 26.93 1.28 1.68 * Time 7 30.27 4 . 32 5. 67 * * Time*age 7 7.39 1.06 1.38 ns E r r o r 35 103.00 0.76 T o t a l 71 168.62 = S i g n i f i c a n t at P < • S i g n i f i c a n t at P •; = Not s i g n i f i c a n t P • • 0 . 0 1 . 0 . 0 5 . < 0 . 0 5 . 125 APPENDIX 7. Analysis of variance for arc sine transformed percentage of abnormal sperm after storage at 5 °C i n B e l t s v i l l e or Lakes Poultry Semen Extender i n comparison to neat semen Dependent Variable: Arc sine Transformed Percent Abnormal Sperm Source of df Sum of Mean F-Value Inference Vari a t i o n Squares Square Model 11 17952.48 1632.04 128.84 Treat 2 9577.47 4788.74 378.06 Time 3 6105.82 2035.27 160.68 * * Treat*Time 6 4458.32 743.05 58.66 Error 30 380.01 12.67 Total 41 18332.49 ** - S i g n i f i c a n t at P < 0.01. 126 APPENDIX 8. Analysis of variance for arc sine transformed percentage of dead sperm after storage at 5 °C i n B e l t s v i l l e or Lakes Poultry Semen Extender i n comparison to neat semen Dependent V a r i a b l e : Arc sine Transformed Percent Dead Sperm Source of V a r i a t i o n df Sum of Squares Mean Square F-Value Inference Model 11 431.92 39.27 3.83 * * Treat 2 99.92 49.96 4.87 * * Time 3 266.92 88.97 8.67 * * Treat*Time 6 65.08 10. 85 1.06 ns E r r o r 48 492.68 10.26 T o t a l 59 924.60 ** = S i g n i f i c a n t at P < 0.01. ns = Not s i g n i f i c a n t P < 0.05. 127 APPENDIX 9. Analysis of variance for sperm m o t i l i t y score after storage at 5 °C i n B e l t s v i l l e or Lakes Poultry Semen Extender i n comparison to neat semen Dependent Variable: M o t i l i t y score Source of Varia t i o n df Sum of Squares Mean Square F-Value Inference Model 11 65.25 5. 93 12.06 * * Treat 2 24.40 12.20 24.81 Time 3 '28.58 9.53 19.38 * * Treat*Time 6 12 .27 2.04 4.16 * * Error 48 23.60 5.93 Total 59 88.85 ** = S i g n i f i c a n t a t P 0 . 0 1 . 128 APPENDIX 10. Repeated measures analysis of variance for percent f e r t i l i t y of semen (fresh or stored) i n B e l t s v i l l e or Lakes Poultry Semen Extenders Dependent V a r i a b l e : Percent F e r t i l i t y -Source of V a r i a t i o n df Sum of Mean F-Value Inference Squares Square Model Treat 2 0.53 0.26 2.48 ns Time 1 16.51 16.51 155.78 Treat*time 2 1.00 0.50 4.72 * E r r o r 32 3.39 0.11 Week 3 0.63 0.21 5.10 * * Week*treat 6 0.97 0.16 3.92 * * Week*store 3 0.17 0.06 1.39 ns Week*treat*store 6 0.45 0.08 1.82 ns E r r o r (week) 96 3.96 0.04 ** = S i g n i f i c a n t at P < 0.01. * = S i g n i f i c a n t at P < 0.05. ns = Not s i g n i f i c a n t P < 0.05. 129 APPENDIX 11. Analysis of variance for effect of different concentrations of glycerol i n BPSE on the f e r t i l i z i n g capacity of unstored chicken spermatozoa a) Dependent Variable: Percent F e r t i l i t y Source of Variat i o n df Sum of Squares Mean Square F-Value Inference Model 7 37672 .27 5381. 75 8 39 * * Treatment 3 30548 .46 10182 . 82 15 87 * * Replicate 1 3348 .35 3348. 35 5 22 * Treat*Repl 3 3775 .46 1258. 49 1 96 ns Error 40 25661 .13 641. 53 Total 47 63333 .40 N.B. Values used in the above a n a l y s i s were data obtained on Days 2-8. ** - S i g n i f i c a n t at P < 0.01. * = S i g n i f i c a n t at P < 0.05. ns = Not s i g n i f i c a n t P < 0.05. b) Dependent Variable: Percent F e r t i l i t y Source of df Sum of Mean F-Value Inference Var i a t i o n Squares Square Model 7 38337 .76 5476. 82 9. 52 * * Treatment 3 29143 .66 9714. 55 16. 89 * * Replicate 1 7525 .36 7525. 36 13. 08 * * Treat*Repl 3 1091 .85 363. 95 0. 63 ns Error 39 22435 .87 575. 28 Total 46 60773 . 64 N.B. Values used in the above a n a l y s i s were on data obtained on Days 9-15. * * = S i g n i f i c a n t at P < 0.01. ns - Not s i g n i f i c a n t P < 0.05. 130 c) Dependent Variable: Percent F e r t i l i t y Source of Vari a t i o n df Sum of Squares Mean Square F-Value Inference Model 7 37447 12 5349.59 11 42 * * Treatment 3 30387 80 10129.27 21 61 * * Replicate 1 4834 06 4834.06 10 32 * * Treat*Repl 3 2225 26 741.75 1 58 ns Error 40 18745 37 468.63 Total 47 56192 49 N.B. Values used in the above a n a l y s i s were on data obtained on Days 2-15. ** - S i g n i f i c a n t at P •: 0.01. ns = Not s i g n i f i c a n t P •: 0.05. 131 APPENDIX 12. Analysis of variance for effect of ethylene glycol and propylene glycol concentration on chicken egg f e r t i l i t y a) Dependent Variable: Arcsin Transformed F e r t i l i t y Source of Vari a t i o n df Sum of Squares Mean Square F-Value Inference Model 9 18133 .00 2014 .78 2 26 * Treatment 4 13227 .38 3306 .84 3 70 * * Replicate 1 148. 21 148. 21 0 17 ns Treat*Repl 4 4757 42 1189 .35 1 33 ns Error 50 44670 .07 893. 40 Total 47 62803 .07 N . B . Values used in the above a n a l y s i s were a r c s i n transformed data obtained on Days 2-8. ** = S i g n i f i c a n t at P •: 0.01. * = S i g n i f i c a n t at P •; 0.05. ns = Not s i g n i f i c a n t P 0.05. b) Dependent Variable: Arcsin Transformed F e r t i l i t y Source of Vari a t i o n df Sum of Squares Mean Square F-Value Inference Model 9 23102 .16 2566 .91 5. 82 * * Treatment 4 • 17897 .26 4474 .31 10 .14 Replicate 1 413. 44 413. 44 0. 94 ns Treat*Repl 4 . 4791 47 1197 .87 2. 71 * Error 50 22069 .65 441. 39 Total 59 • •45171 .81 N.B. Values used in the above a n a l y s i s were on a r c s i n transformed data obtained on Days 9-15. - S i g n i f i c a n t at P •-- 0.01. * = S i g n i f i c a n t at P < 0.05. , ' ns = Not s i g n i f i c a n t P - ; 0.05. 132 c) Dependent V a r i a b l e : A r c s i n Transformed F e r t i l i t y Source of V a r i a t i o n df Sum of Squares Mean Square F-Value Inference Model 9 12888 . 17 1432 .02 2 94 * * Treatment 4 10373 .70 2593 .42 5 32 * * R e p l i c a t e 1 586. 91 586. 91 1 20 ns Treat*Repl 4 2059 45 514. 86 1 06 ns E r r o r 44 21438 .20 487 . 23 T o t a l 53 34326 .37 N.B. Values used in the above a n a l y s i s were on a r c s i n transformed data obtained on Days 2-15. ** - S i g n i f i c a n t at P •: 0.01. ns = Not s i g n i f i c a n t P < 0.05. 133 APPENDIX 13. Analysis of variance for e f f e c t of sucrose concentration on chicken egg f e r t i l i t y a) Dependent Variable: A r c s i n Transformed F e r t i l i t y Source of Va r i a t i o n df Sum of Squares Mean Square F-Value Inferen ce Model 11 81638.22 7421.66 15.82 * * Treatment 5 68439.61 13687.92 29.19 Replicate 1 8062.38 8062.38 17.19 • * * Treat*Repl 5 5136.23 1027.24 2.19 ns Error 60 28139.71 4689.00 Total 71 109777.94 N . B . V a l u e s u s e d i n t h e a b o v e a n a l y s i s we re a r c s i n t r a n s f o r m e d d a t a o b t a i n e d on Days 2 - 8 . * * - S i g n i f i c a n t a t P < 0 . 0 1 . ns - No t s i g n i f i c a n t P < 0 . 0 5 . b) Dependent Variable: A r c s i n Transformed F e r t i l i t y Source of Va r i a t i o n df Sum of Squares Mean Square F-Value Inference Model 11 43165.76 3924.16 11.88 * * Treatment 5 40550.63 8110.13 24.55 * * Replicate 1 1913.74 1913.74 5.79 * Treat*Repl 5 701.38 140.28 0.42 ns Error 60 19824.68 330.41 Total 71 62990.44 N . B . V a l u e s u s e d i n t h e above a n a l y s i s we re on a r c s i n t r a n s f o r m e d d a t a o b t a i n e d on Days 9 - 1 5 . * * - S i g n i f i c a n t a t P •: 0 . 0 1 . * = S i g n i f i c a n t a t P •: 0 . 0 5 . ns - Not s i g n i f i c a n t P ••: 0 . 0 5 . 134 c) Dependent Variable: Arcsin Transformed F e r t i l i t y Source of Variat i o n df Sum of Squares Mean Square F-Value Inference Model 11 51250 .85 4659 19 15 28 * * Treatment 5 46154 .03 9230 81 30 27 * * Replicate 1 3358 .58 3358 58 11 01 Treat*Repl 5 1738 .24 347 65 1 14 ns Error 60 18296 .79 304 95 Total 71 69547 .65 N.B. Values used in the above a n a l y s i s were on a r c s i n transformed data obtained on Days 2-15. ** - S i g n i f i c a n t at P < 0.01. ns = Not s i g n i f i c a n t P 0.05. 

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