Ifllllllllzllllfllllllljlllllsllll will R A R y 87 Michigan State University This is to certify that the thesis entitled EFFECTS OF SPERM CELL NUMBERS, ROUTE OF INSEMINATION, AND DILUTION OF SEMEN ON FERTILITY IN THE DOMESTIC FOWL presented by Ronald James DeMeritt has been accepted towards fulfillment of the requirements for Ph.D. degree in Poultry Science V _// . .A 1 U I L A ‘ “Pm Major professor D Date III/37!??? 0-7539 ovmmuz FINES ARE 25¢ PER DAY _ PER mm Return to book drop to remove this checkout from your record. EFFECTS OF SPERM CELL NUMBERS, ROUTE OF INSEMINATION, AND DILUTION OF SEMEN ON FERTILITY IN THE DOMESTIC FOWL By Ronald James DeMeritt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1978 "7. ’) C; [. OAK if i v ABSTRACT EFFECTS OF SPERM CELL NUMBERS, ROUTE OF INSEMINATION AND DILUTION OF SEMEN ON FERTILITY IN THE DOMESTIC FOWL BY Ronald James DeMeritt By using intravaginal artificial insemination (I.V.A.I.), intramag- nal artificial insemination (I.MQA.I.) and genetic markers, three separate experiments were conducted on the domestic fowl to examine the following: (1) the effect of sperm cell numbers and route of insemination on fer- tility, hatchability and duration of fertility; (2) the effect of semen dilution on fertility, hatchability and duration of fertility; and (3) movement of spermatozoa into and out of the host glands located at the uterovaginal junction (U.V.J.). In Experiment I various volumes of viable spermatozoa equivalent to (1) 12.5 million, (2) 25 million, (3) 50 million and (4) 100 million from Single Comb White Leghorn (S.C.W.L.) or Barred Plymouth Rock (B.P.R.) males were inseminated I.V.A.I. or I.MMA.I. into S.C.W.L. hens. The results indicated that S.C.W.L. spermatozoa was superior to B.P.R. sperma- tozoa's fertilizing capacity at all four spermatozoa number levels of in- semination. Fertility measured during the first week of Experiment I in- dicated that hens inseminated I.M.A.I. with 12.5 million and 25 million viable spermatozoa produced eggs with higher fertility than I.V.A.I. inseminated hens. Inseminations with 50 million and 100 million viable spermatozoa I.V.A.I. resulted in superior fertility during week one when compared with 50 million and 100 million spermatozoa inseminated I.M.A.I. During weeks two and three following a single insemination, fertility achieved with I.M.A.I. was higher than with I.V.A.I. at all four levels of insemination. Duration of fertility with I.M.A.I. was longer than with I.V.A.I. to a highly significant degree. The S.C.W.L. spermatozoa provided a significantly longer duration of fertility than the B.P.R. spermatozoa. A linear trend indicated a longer duration of fertility with increased numbers of viable spermatozoa occurred with I.V.A.I. for both breeds and I.M.A.I. for the B.P.R. only. Intramagnal artificial insemination with the S.C.W.L. spermatozoa did not have a linear trend because the 12.5 million and 25 million level of insemination had long durations of fertility similar to the higher levels of insemination. In Experiment II, in which only S.C.W.L. were used, the results suggested higher fertility was gained during the first week after I.V.A.I. as the neat (pure semen) was diluted with Beltsville Extender to a 1:4 (semen:diluent) ratio. The opposite appeared to be true with I.M.A.I. as indicated by decreased fertility during the first week as the semen was diluted up to 1:4. During the second and third weeks of Experiment II, hatchability was significantly higher at the 1:1 and 1:4 dilution ratios than with neat semen. Duration of fertility was longest when diluted semen, par- ticularly 1:1 dilution, was inseminated. In Experiment III, New Hampshire (N.H.) females were inseminated. S.C.W.L., B.P.R. or N.H. Sires were identified by progeny phenotypes. Therefore, the sequence of inseminations with 25 million or 75 million viable spermatozoa I.V.A.I. could be compared to the pattern of sperm cell release from the U.V.J. host glands. All possible two breed combin- ations were made with inseminations at 0-1 hour or 0-24 hour intervals while all possible three breed combinations were made at 0-24-48 hour intervals. In Experiment III a definite breed effect on percent fertility, du- ration of fertility and hatchability was shown with the S.C.W.L. being superior followed by B.P.R. and N.H., respectively. The fertility and duration of fertility were superior with 75 million viable spermatozoa compared to 25 million viable spermatozoa for both the two breed and three breed combinations. Also, in both the two breed and three breed combinations, semen from the last insemination regardless of breed, fa- cilitated higher fertility and a longer duration of fertility than semen from the previous insemination(s). These data suggested the sequence of spermatozoa release from the host glands was opposite to the sequence of introduction. Therefore, the U.V.J. host glands can regulate or contri- bute to the release of viable spermatozoa as indicated by the lack of complete randomization of the resulting progeny. To my wife Nancy 11 ACKNOWLEDGEMENT I wish to express my sincere appreciation to Dr. H. C. Zindel for his support and many words of wisdom during my stay at Michigan State University; to Dr. T. H. Coleman for his guidance in the preparation of this dissertation; and to Dr. B. J. Marquez for his research assistance. A special thank you to Terry Wing for his aid in the statistical analysis and to my wife for her encouragement and enthusiastic support. iii List of Tables List of Figures . Introduction. . . TABLE OF CONTENTS Experiment I. . . . . . . . . . . . . . . . . . . vi Experiment II . . . . . . . . . . . . . . . . . viii Experiment III . . . . . . . . . . . . . . . . ‘ 2 Review of Literature . . . . . . . . . . . . . . . . . . . . . . . 3 Method and Materials . . . . . . . . . . . . . . . . . . . . . . . 12 Results Discussion Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . 9: Appendix A Appendix B Appendix C Appendix D Appendix E Experiment I. . . . . . . . . . . . . .i. . . . . 19 Experiment II . . . . . . . . . . . . . . . . . . 24 Experiment III . . . . . . . . . . . . . . . . . 2? Experiment I. . . . . . . . . . . . . . . . . . . 38 Experiment II . . . . . . . . . . . . . . . . . . 44 Experiment III. . . . . . . . . . . . . . . . . . 46 Practical Layer Ration. . . . . . . . . . . . . .102 Technique for Measuring Optical Density of Diluted semen I O O O I O O O O O O O O O O I I O O O O 103 Linear Regression for Estimation of Sperm-Cell Numbers 0 O O O O O O O I O O O I I O O I O O O 104 Macroscopic Identification of Undeveloped Fertile Eggs. 0 O O O O O O O O O O O O O O O O O O O O 105 Phenotypic Identification of Progeny from Parent cross 0 O O O O O O O O O O O O O C O I O O O I 106 iv Appendix F Appendix G Bibliography. . . TABLE OF CONTENTS (Continued) Page Parameters Analyzed in Experiment III, Trials IIIA’ IIIB. O O O O O O O O O O O I O O O O O .107 Parameters Analyzed in Experiment III, Trials IIIC’ IIIDO O O O O O O C I O O O O O O O I O .108 .109 Table LIST OF TABLES Experiment I l} 2. 10. 11. 12. 13. 14. Title Page EXPERIMENTAL DESIGN OF TRIAL IA . . . . . . . . . . . . . . 51 EXPERIMENTAL DESIGN OF TRIAL IB. . . . . . . . . . . . . . . 51 EXPERIMENTAL DESIGN OF TRIAL IC . . . . . . . . . . . . . . 51 ANALYSIS OF VARIANCE OF TRIAL IA . . . . . . . . . . . . . . 52 ANALYSIS OF VARIANCE OF TRIAL IB . . . . . . . . . . . . . . 53 ANALYSIS OF VARIANCE OF TRIAL IC . . . . . . . . . . . . . . 54 BREED EFFECT* WITH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-l OF FERTILITY.55 INTERACTION* BETWEEN ROUTE 0F INSEMINATION (INTRAMAGNAL - I.M. 0R INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED DURING WEEK-1 0F FERTILITY o o o o o o o o o o o o o o o o o 55 INTERACTION** BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) SEMEN OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE ROUTE OF INSEM- INATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) DURING WEEK-2 OF FERTILITY . . . . . . . . . . . . . . . . . . . . . . . . 56 INTERACTION* BETWEEN ROUTE 0F INSEMINATION (INTRAMAGNAL - I.M. 0R INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED DURING WEEK-2 OF FERTILITY o o o o o o o o o o o o o o o o o 56 BREED EFFECT** WITH SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-3 OF FERTILITY.57 DILUTION EFFECT* WITH PURE SEMEN (NEAT) AND DILUTED SEMEN DURING WEEK-1 0F HATCHABILITY o o o o o o o o o o o o o o o o o o o 57 ROUTE EFFECT** WITHIN DILUTED SAMPLES WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTES OF INSEMINATION DURING WEEK-1 0F HATCMBILIW O O O O O C O C O O O O O O C C C O O . C O O S 7 INTERACTION** DURING WEEK-3 OF HATCHABILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . 58 vi LIST OF TABLES (Continued) Table 15. 16. 17. 18. _19. 20. 21. 22. 23. 24. 25. 26. Title Page INTERACTION* BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE NUMBER OF SPER- MATOZOA INSEMINATED ON DURATION OF FERTILITY . . . . . . . . 58 INTERACTION* BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) ON DURATION OF FERTILITY. . . . . . . . . . . . . . . . . . . . . . . . . . 59 INTERACTION* DURING WEEKrl OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVACINAL - I.V.) o o o o o o o o o o o o o o o o o o o o o o o o o o 59 INTERACTION" DURING WEEK-2 OF FERTILITY BETWEEN ROUTE OF INSEM- INATION (INTRAMAGNAL - I.M. OR.INTRAVAGINAL - I.V.) AND THE NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . 6O ROUTE EFFECT** DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTES OF INSEMHNATION . . . 6O BREED EFFECT* WITH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-3 OF FERTILITY.60 INTERACTION* DURING WEEK-1 OF HATCHABILITY BETWEEN INTRAMAGNAL (I.M.) OR INTRAVAGINAL (I.V.) ROUTES 0F INSEMINATION AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . . . . 61 BREED EFFECT* WITH THE BARRED PLYMOUTH ROCK (B.P.R.) AND SINGLE COMB WHITE LEGHORN (S.C.W.L.) SEMEN DURING WEEK-1 OF HATCH- ABILITY . O O O O I O I O O O O O O O O O O I C O C O O I C 61 TREND DURING WEEK-3 OF HATCHABILITY WITH BARRED PLYMOUTH ROCK (B.P.R.) SEMEN INDICATING INCREASED HATCHABILITY WITH INCREASED NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . 62 INTERACTION** ON DURATION OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . 62 ROUTE EFFECT* WITH THE INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTE 0F INSEMNATION O O O O O O O O O O O O .1 O O O O I O 63 TREND DURING WEEK-3 OF FERTILITY WITH BARRED PLYMOUTH ROCK SEMEN INDICATING INCREASED FERTILITY WITH INCREASED NUMBERS OF SPER- MTOZOA IN s MNATED O O O I O I O O O C O O O O O I O O O O O 6 3 vii 29. 30. 31. 32 Ex LIST OF TABLES (Continued) Table 27. 28. 29. 30. 31. 32. Title Page INTERACTION** DURING WEEK-1 OF FERTILITY BETWEEN INTRAVAGINAL (I.V.) OR INTRAMAGNAL (I.M.) ROUTES OF INSEMINATION AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . . . . 64 ROUTE EFFECT* DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTES OF INSEMINATION FOR BOTH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) S MN I I I I I I I I I I I I I I;: I I I I I I I I I I I I I 64 BREED EFFECT** DURING WEEK-3 OF FERTILITY AND HATCHABILITY WITH SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (BIPIRI) SEEN I I I I I I I I I I I I I I I I I I I I I I I 64 INTERACTION** DURING WEEK-2 0F HATCHABILITY BETWEEN ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . 65 INTERACTION* ON DURATION OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - IIVI) I I I I I I I I I I I I I I I I I I I I I I I I I I I 65 NUMBER EFFECT* ON DURATION OF FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . . . . 66 Experiment II 33. 34A. 34B. 35. 36. 37. 38. EXPERIMENTAL DESIGN OF TRIALS IIA, IIB . . . . . . . . . . . 66 ANALYSIS OF VARIANCE OF TRIAL IIA . . . . . . . . . . . . . 67 ANALYSIS OF VARIANCE OF TRIAL IIB . . . . . . . . . . . . . 68 INTERACTION* DURING WEEK-l OF FERTILITY BETWEEN ROUTE OF INSEMF INATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND DILUTION OF SEMEN (PURE SEMEN - NEAT, OR DILUTED 1:1 OR 1:4 WITH BELTS- VILLE EXTENDER) I I I I I I I I I I I I I I I I I I I I I I I 6 9 ROUTE EFFECT** DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTES OF INSEMINATION . . . . . . . 69 NUMBER EFFECT* DURING WEEK-2 OF FERTILITY WITH INCREASED NUMBERS 0F SPERMTOZOA INSEMNATED I I I I I I I I I I I I I I I I I 69 NUMBER EFFECT* DURING WEEK-3 OF FERTILITY WITH INCREASED NUMBERS OFSPERMATOZOAINSEMINATED................. 7O viii LIST OF TABLES (Continued) Table 39. 40. 41. 42. 43. 44. 45I 46. 47. 48. 49. 50. 51. 52. Title Page INTERACTION* DURING WEEK-1 OF HATCHABILITY BETWEEN ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . 7O DILUTION EFFECT* ON HATCHABILITY DURING WEEK-2 WITH SEMEN DILUTED 1:1 AND 1:4 WITH BELTSVILLE EXTENDER . . . . . . . . 7O DILUTION EFFECT* ON HATCHABILITY DURING WEEK-3 WITH SEMEN DILUTED 1:1 AND 1:4 WITH BELTSVILLE EXTENDER . . . . . . . . 71 ROUTE EFFECT* ON DURATION OF FERTILITY WITH INTRAMAGNAL INSEM- INATION (I{M.) COMPARED TO INTRAVAGINAL INSEMINATION (I.V.) . 71 NUMBER EFFECT** ON DURATION OF FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . . . . 71 DILUTION EFFECT* ON DURATION OF FERTILITY AS THE SEMEN WAS DILUTED 1:1 AND 1:4 WITH BELTSVILLE EXTENDER . . . . . . . . 72 INTERACTION* DURING WEEK-1 OF FERTILITY BETWEEN ROUTE OF INSEM- INATION (INTRAMAGNAL - I.Mw OR INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED . . . . . . . . . . . .'. . . . . 72 ROUTE EFFECT** DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) COMPARED TO THE INTRAVAGINAL (I.V.) INSEMINATIONS . . . . . . 72 NUMBER EFFECT* DURING WEEK-2 OF FERTILITY ON FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED . . . . . . . . 73 INTERACTION* DURING WEEK-2 OF FERTILITY BETWEEN ROUTE OF INSEMr INATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND DILUTION OF SEMEN INSEMINATED AS PURE SEMEN (NEAT) OR DILUTED 1:1 OR 1:4 WITH BELTSVILLE EXTENDER . . . . . . . . . . . . . . . . . . 73 ROUTE EFFECT** DURING WEEK-1 OF HATCHABILITY WITH INTRAVAGINAL (I.V.) AND INTRAMAGNAL (I.M.) ROUTES OF INSEMINATION . . . . 73 ROUTE EFFECT* DURING WEEK-2 OF HATCHABILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTES OF INSEMINATION . . . . 74 ROUTE EFFECT** ON DURATION OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) INSEMENATIONS . . . . . . . . . . . . 74 NUMBER EFFECT* ON DURATION OF FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED. . . . . . . . . . . . . . . . . . 74 ix S7. 58. 59. 60. 61. 62. 63. LIST OF TABLES (Continued) Table Experiment III 53. 54A. 54B. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. Title Page EXPERIMENTAL DESIGN OF TRIALS IIIA & IIIB . . . . . . . . . . 75 ANALYSIS OF VARIANCE OF TRIAL IIIA. . . . . . . . . . . . . . 76 ANALYSIS OF VARIANCE OF TRIAL IIIB. . . . . . . . . . . . . . 77 EXPERIMENTAL DESIGN OF TRIALS IIIC & IIID . . . . . . . . . . 78 NUMBER EFFECT* DURING WEEK-1 OF FERTILITY WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSEMINATION . . . . . . . . . . . . 78 NUMBER EFFECT** DURING WEEK-2 OF FERTILITY WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSEMINATION . . . . . . . . . . . . 79 NUMBER EFFECT* DURING WEEKrZ OF HATCHABILITY WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSEMINATION . . . . . . . . . . . . 79 NUMBER EFFECT** ON DURATION OF FERTILITY WITH 75 MILLION VIABLE SPERMATOZOA COMPARED TO THE 25 MILLION LEVEL OF INSEMINATION. 79 RATIOS OF CHICKS HATCHED DURING WEEK-1 FROM THE FIRST BREED INSEMINATED SHOWING THE INTERACTIONS* BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMP- SHIRE (N.H.) SEMEN AND NUMBER OF VIABLE SPERMATOZOA AT VARIOUS INTERVALS OF INSEMINATION . . . . . . . . . . . . . . . . . . 80 RATIOS OF CHICKS HATCHED DURING WEEK-2 FROM THE FIRST BREED INSEMINATED SHOWING THE INTERACTIONS* BETWEEN SEMEN OF THE SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND THE NUMBER OF VIABLE SPERMATOZOA AT VARIOUS INTERVALS OF INSEMINATION. . . . . . . 81 BREED EFFECT* ON RATIOS OF FIRST BREED CHICKS HATCHED FOR THE TOTAL TRIAL WHEN SINGLE COMB WHITE LEGHORN (S. C. W. L. ), BARRED PLYMOUTH ROCK (B. P. R. ) OR NEW HAMPSHIRE (N. H. ) SEMEN WAS USED FOR THE SECOND BREED INSEMINATED. . . . . . . . . . . . . . . 82 NUMBER EFFECT* ON RATIOS OF FIRST BREED CHICKS HATCHED WITH 25 MILLION OR 75 MILLION VIABLE SPERMATOZOA INSEMINATED . . . . 82 INTERVAL EFFECT** ON RATIOS OF FIRST BREED CHICKS HATCHED WHEN INSEMINATED AT THE 0-1 HOUR INTERVAL COMPARED TO THE 0-24 HOUR INTERVAI‘ I I I I I I I I I I I I I I I I I I I I I I I I I I 8 3 LIST OF TABLE (Continued) Table 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. Title Page RATIOS OF CHICKS HATCHED DURING WEEK-l FROM THE SECOND BREED INSEMINATED SHOWING THE INTERACTIONS* BETWEEN SEMEN OF SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND NUMBER OF VIABLE SPERMATOZOA AT VARIOUS INTERVALS OF INSEMINATION . . . . . . . . . . . . . . 83 RATIOS OF CHICKS HATCHED DURING WEEK-2 FROM THE SECOND BREED INSEMINATED SHOWING THE INTERACTION* BETWEEN SEMEN OF SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND NUMBER OF VIABLE SPERMATOZOA AT VARIOUS INTERVALS OF INSEMINATION . . . . . . . . . . . . . . 84 RATIOS 0F CHICKS HATCHED FOR THE TOTAL TRIAL FROM THE SECOND BREED INSEMINATED SHOWING THE INTERACTION* BETWEEN SEMEN OF THE SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) 0R NEW HAMPSHIRE (N.H.) AND VARIOUS INTERVALS OF INSEMINATION. 85 NUMBER EFFECT* FOR TOTAL TRIAL ON RATIOS OF SECOND BREED CHICKS HATCHED WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA PER INSEMINATION I I I I I I I I I I I I I I I I I I I I I I I I 85 NUMBER EFFECT* ON DURATION OF FERTILITY WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA PER INSEMINATION . . . . . . . . . 86 NUMBER EFFECT* DURING WEEK-1 OF FERTILITY WITH 25 MILLION AND 75 MILLION SPERMATOZOA INSEMINATED. . . . . . . . . . . . . . . . 86 NUMBER EFFECT* DURING WEEK-2 0F FERTILITY WITH 25 MILLION AND 75 MILLION SPERMATOZOA INSEMINATED . . . . . . . . . . . . . . . 86 BREED EFFECT** DURING WEEK-2 OF HATCHABILITY WHEN SINGLE COMB WHITE LEGHORN (S.C.W.L.), NEW HAMPSHIRE (N.H.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN WAS USED AS THE SECOND BREED INSEMINATED . 87 NUMBER EFFECT* DURING WEEK-2 OF HATCHABILITY WITH.25 MILLION AND 75 MILLION VIABLE SPERMATOZOA . . . . . . . . . . . . . . . . 87 INTERACTION* ON DURATION OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMP- SHIRE (N.H.) SEMEN AND VARIOUS INTERVALS OF INSEMINATION . . . 88 NUMBER EFFECT** ON DURATION OF FERTILITY WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSEMINATION . . . . . . . . . . . . . . 88 xi 79 8C 81 82 83 84. as, 86. LIST OF TABLES (Continued) Table 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. Title Page RATIOS OF CHICKS HATCHED DURING WEEK-1 FROM THE FIRST BREED INSEMINATED WITH THE DOMINATING BREED EFFECT* OF SINGLE COMB WHITE LEGHORN (S.C.W.L.) SEMEN OVER BARRED PLYMOUTH ROCK (B.P.R.) AND NEW HAMPSHIRE (N.H.) SEMEN . . . . . . . . . . . 89 RATIOS OF CHICKS HATCHED DURING WEEK-2 FROM THE FIRST BREED INSEMINATED WITH THE INTERACTIONS* BETWEEN SEMEN OF SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND VARIOUS NUMBERS OF SPERMATOZOA INSEMINATED 89 RATIOS OF CHICKS HATCHED FOR THE TOTAL TRIAL FROM THE FIRST BREED INSEMINATED WITH THE INTERACTIONS* BETWEEN SEMEN OF SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND VARIOUS INTERVALS OF INSEMINATION .90 BREED EFFECT** ON RATIOS OF SECOND BREED CHICKS HATCHED DURING WEEK-1 WHEN THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) WAS USED AS THE SECOND BREED INSEMINATED, COMPARED TO BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) SEMEN . . . . . . . . . 9O RATIOS OF SECOND BREED CHICKS HATCHED DURING WEEK-1 WITH THE INTERACTION* BETWEEN NUMBER OF SPERMATOZOA INSEMINATED AND INTERVAII 0F INSEMINATION I I I I I I I I I I I I I I I I I I I 91 RATIOS OF SECOND BREED CHICKS HATCHED DURING WEEK-2 WITH THE INTERACTION*BETWEEN SEMEN FROM SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND VARIOUS NUMERS 0F VIABLE SPERMATOZOA INSEMINATED . 91 BREED EFFECT** ON RATIOS OF SECOND BREED CHICKS HATCHED FOR THE TOTAL TRIAL WHEN SEMEN FROM SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) WAS INSEM- INATED AS THE SECOND BREED . . . . . . . . . . . . . . . . . . 92 RATIOS OF SECOND BREED CHICKS HATCHED FOR THE TOTAL TRIAL WITH THE INTERACTION* BETWEEN NUMBER OF VIABLE SPERMATOZOA INSEMIN- ATED AND VARIOUS INTERVALS OF INSEMINATION . . . . . . . . . . 92 NUMBER EFFECT** ON DURATION OF FERTILITY WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSEMINATION. . . . . . . . . . . . . 93 INTERACTION* ON DURATION OF FERTILITY OF SEMEN FROM SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) OR NEW HAMPSHIRE (N.H.) AND INTERVAL OF INSEMINATION . . . . . . . . 93 NUMBER EFFECT* DURING WEEK-1 OF FERTILITY WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA INSEMINATED. . . . . . . . . . . 93 xii LIST OF TABLES (Continued) Table Title Page 87. NUMBER EFFECT* DURING WEEK-2 OF FERTILITY WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA INSEMINATED . . . . . . . . . . 94 88. BREED EFFECT** ON DURATION OF FERTILITY IN TRIALS IIIC AND IIID WITH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) AND NEW HAMPSHIRE (N.H.) SEMEN, RESPECTIVELY. . 94 89. NUMBER EFFECT** ON FERTILITY DURING WEEK-2 WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA PER INSEMINATION. . . . . . . . 94 90. NUMBER EFFECT* ON DURATION OF FERTILITY FOR THE FIRST BREED INSEMINATED WITH THE 25 MILLION AND 75 MILLION LEVELS OF INSMNATION I I I I I I I I I I I I I I I I I I I I I I I I I 95 91. BREED EFFECT** ON DURATION OF FERTILITY FOR THE FIRST BREED INSEMINATED WITH SINGLE COMB WHITE LEGHORN (S.C.W.L.), BARRED PLYMOUTH ROCK (B.P.R.) AND NEW HAMPSHIRE (N.H.) SEMEN IN BOTH TRIALS IIIC AND IIID . . . . . . . . . . . . . . . . . . . . 95 92. NUMBER EFFECT** ON DURATION OF FERTILITY FOR THE SECOND BREED INSEMINATED WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA PER INSEMINATION I I I I I I I I I I I I I I I I I I I I I I I 95 93. NUMBER EFFECT** ON DURATION OF FERTILITY FOR THE TOTAL TRIAL WITH 25 MILLION AND 75 MILLION VIABLE SPERMATOZOA PER INSEMNATION I I I I I I I I I I I I I I I I I I I I I I I I I 96 94. RATIOS OF CHICKS HATCHED DURING WEEK-2 FROM THE FIRST BREED INSEMINATED INDICATING A BREED EFFECT** WITH BARRED PLYMOUTH ROCK (B.P.R.), SINGLE COMB'WHITE LEGHORN (S.C.W.L.) AND NEW HAMPSHIRE (N.H.) SPERMATOZOA . . . . . . . . . . . . . . . . 96 95. RATIOS OF CHICKS HATCHED DURING WEEK-2 FROM THE THIRD BREED INSEMINATED INDICATING A BREED EFFECT** WITH BARRED PLYMOUTH ROCK (B.P.R.), SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND NEW HAMPSHIRE (N.H.) SPERMATOZOA . . . . . . . . . . . . . . . . 96 xiii Experiment I II II Figure LIST OF FIGURES Title 1 Percent Fertility (Weekly) S.C.W.L.(ma1es) x S.C.W.L.(females) Percent Fertility (Weekly) B.P.R.(ma1es) x S.C.W.L.(females) Percent Fertility (Weekly) S.C.W.L.(ma1es) vs. B.P.R.(ma1es) Duration of Fertility S.C.W.L.(ma1es) x S.C.W.L.(females) Duration of Fertility B.P.R.(ma1es) x S.C.W.L.(females) Route and Number Interaction Percent Fertility (Weekly) Route and Dilution Interaction Percent Fertility (Weekly) xiv Page 115 116 117 118 119 120 121 INTRODUCTION The use of artificial insemination (A.I.) has been shown to elicit excellent fertility, hatchability and duration of fertility in the do- mestic fowl. Uterovaginal host gland tubules and infundibular crypts facilitate sperm storage for intravaginal artificially inseminated (I.V.A.I.) semen and intramagnal artificially inseminated (I.M.A.I.) semen, respectively. By use of Beltsville Extender in dilutions of 1:1 to 1:4 semen to diluent ratios with 100 million spermatozoa, optimum fertility and dura- tion of fertility are achieved with I.V.A.I. (Sexton, 1977). This is a direct reflection of the storage capabilities of the uterovaginal tubules when fresh semen is used. However, the dilution and number of spermato- zoa necessary for peak fertility and duration of fertility using I.M.A.I. have not been determined. With the advent of new frozen-thawed semen storage techniques, more information is needed about the storage sites and their relationships to sperm numbers and dilution. The purpose of this research was three fold. The research was divided into three separate experiments accordingly. Experiment I was designed to gain insight as to the capability of the infundibular crypts to handle various numbers and/or volumes of Single Comb White Leghorn or Barred Plymouth Rock spermatozoa during intramagnal inseminations, when compared to the I.V.A.I., which served as a control. Experiment II was designed to validate Experiment I data and add the effect of different dilutions of viable spermatozoa from S.C.W.L. males on the storage capabilities of the infundibular crypts as measured by fertility and duration of fertility. Experiment III was designed to examine sperm cell introduction, storage, release and transport in the oviduct with special emphasis on the role of the U.V.J. host glands. The area of research involving the relationships between sperm cell numbers, dilution of semen, route of insemination, fertility, hatchability, duration of fertility and the role of the U.V.J. host glands and infundi- bular crypts is just beginning to be understood. With the advent of new freeze-thaw techniques, new avian wildlife breeding programs, extinction of rare birds, etc., groundwork concerning the previously mentioned relationships needs to be done. The purpose of this dissertation is to examine these relationships and relate them in such a way as to be advantageous to the poultry indus- try and other concerns. REVIEW OF LITERATURE In the domestic fowl (Callus domesticus), the passage of spermatozoa up the female reproductive tract to the site of fertilization has received special attention by researchers in past years. Although great strides have been made, more questions about sperm transport have been left un- answered than have been resolved. The early works in avian reproduction emphasized the importance of proper environmental conditions to obtain high levels of fertility and hatchability. Floor type matings were utilized with different male to female ratios. Brantos et a1. (1972) found ratios of 15 females/male for light breeds and 12 females/male for heavy breeds to be satisfactory. This floor mating system was an area of the poultry industry which had drawbacks in disease control, feed efficiency, space utilization and low fertility. Hayes and Sanborn (1939) working with Rhode Island Reds indi- cated that the infertile matings were caused by the males, because a change in males resulted in 93% of the infertile matings becoming fertile. This emphasized the need for male selection based on semen quality and possibly the need for another type of mating system. Artificial insemination (A.I.), along with cage rearing of birds, has been shown to overcome some of these problems. While working with turkeys, Stotts and Darrow (1955) achieved increased fertility and there- by increased economical gain when artificially inseminated cage birds were compared with floor matings. Compqring 31,000 hens from 21 flocks, 3 they found an increase of 7% in high fertility flocks (from 80-85% fertil- ity to 87-92% fertility) and an increase of 12% in low fertility flocks (from 65-70% fertility to 77-822 fertility). Practical application and increased fertility utilizing artificial insemination in the chicken was demonstrated by Burrows and Quinn (1939), Warren and Gish (1943), Rainford (1956) and Perek (1966). Although the actual outcome of A.I. was higher fertility and hatchability, the physio- logical events which lead to these outcomes are just beginning to be understood. Chicken semen was collected as early as 1914 when Payne collected spermatozoa from the cloaca of a hen following mating. Hutt (1929) col- lected directly from the male with a watch glass during a mating attempt. One of the more practical and expedient techniques was developed by Burrows and Quinn (1937). By a series of stimulations in the latero-ab- dominal regions, and pressure above the vent, seminal fluid was forced from the posterior vas deferens into a collection tube. Van Wambake (1976) studied two different methods of semen collection to investigate the influence of a slight contamination of semen with trans- port fluid resulting from a simplified semen collection technique upon fertility and hatchability. He concluded that transport fluid was not detrimental to fertility. Fecal contamination of semen by careless collection also decreases semen quality. Boone and Hughes (1970) illustrated the relationships between increased contamination of the sample and decreased sperm motility and vigor, thereby decreasing fertility. The time and frequency of collection of semen from the males appears to affect its quality. Parker (1949) suggests a collection every five days to maintain adequate numbers of spermatozoa. The actual time of artificial insemination or natural mating also has been shown to affect fertility. Moore and Byerly (1942) utilizing artificial insemination noted lower fertility when hens with a hard- shelled egg in the uterus were inseminated compared to the higher fertil- ity of hens inseminated with no egg in the oviduct. Gracewski and Scott (1943) found higher fertility when males were permitted to mate only during the afternoon. Parker (1945) found similar results and showed its relationship to time of lay, indicating inseminations made prior to lay- ing resulted in lower fertility. Johnston and Parker (1970), adding to this information, concluded fertility was lowest when inseminations were made one hour after or four hours before oviposition. They indicated that the best time for A.I. was in the late afternoon or after the majority of eggs were laid. Christensen and Johnston (1975) found similar results working with turkeys. When studying the effect of time of day in which inseminations were made on fertility, they concluded fertility was highest when inseminations were made at 6:00 P.M. and lowest when inseminations were made at 1:00 P.M. or during a period ten hours prior to oviposition. The volume, dilution and type of extender to be inseminated became an important factor for economic reasons. Munro (1938) studied the effect of the dilution and density of semen on the fertilizing capacity of fowl semen suspensions. He found fertility decreased as the dilution ratio was increased from 1:2 to 1:128 with seminal plasma and Milanov's solu- tion. Lake (1960) performed studies on the dilution and storage of fowl semen indicating the importance of fructose in the extender. Clark (1969) noted increased fertility and hatchability with a diluted semen sample compared to neat semen in turkeys. Clark combined fertility data from two strains of turkeys and showed that the use of diluent to store semen was not very successful. However, the use of diluent at 0 hours enhanced not only egg fertility but also the hatchability. The overall production of first quality poults, associated with the use of diluent, was enhanced by 13.2 percent. Allen and Bobr (1955) used glycerol as a diluent and measured fertil- ity when inseminations were made intrauterine and intravaginally. They found intrauterine inseminations made with semen containing 15% glycerol resulted in higher fertility (73%) compared to the intravaginal insemina- tiors with semen containing the same diluent resulting in 0% fertility. Wilcox and Clark (1962) studied the use of a diluent during storage . of chicken Spermatozoa. An optimum dilution ratio of 1:2 and 1:4 resulted in higher fertility than dilution ratios of 1:10 or greater which resulted in zero fertility. The storage of spermatozoa outside the fowl (in 31:59) has generated interest. The use of freezing techniques to preserve sperm until a fur- ther date has. been studied by Allen (1958), who reviewed the work by Polge which indicated the following: (1) the early successes in the field of deep freezing were carried out on fowl semen and were reported by Polge (1951); (2) the incorporation of glycerol in the diluent per- mitted complete resumption of motility on thawing the sperm from frozen condition, but rendered the sperm infertile even if they had been frozen; (3) the osmotic damage to the sperm in the oviduct was one major cause of the loss of fertilizing power of glycerolized semen. Allen (1958) main- tained factors as yet unknown (other than osmotic shock) were involved in the low fertility. Graham and Pace (1967 a, b) and Harris and Sweeney (1971) studied biochemical changes and protein concentration in spermatozoa due to freez— ing while noting their relationships to decreased fertilizing capabilities of the frozen-thawed sperm cells. Lake (1967) made a reappraisal of A.I. which demonstrated the need for further research in the area of frozen-thawed semen because of the problem of maintaining sufficient spermatozoa in a condition enabling them to survive in the oviduct for long periods after being stored at sub-zero temperatures. Harris (1968) studied fertility of chickens inseminated intraperitone- ally with semen preserved in liquid nitrogen. He noted a decrease in fertility from preserved semen when compared to similar inseminations with fresh semen. Harris et a1. (1973) noted changes in the ultrastructure of fowl spermatozoa due to rapid freeze-thaw. He noted several changes in mor- phology attributed to rapid freeze-thaw as follows: (1) extensive di- vergence of the cytoplasmic membrane throughout the entire length of the spermatozoa; (2) complete destruction of the cytoplasmic membrane sur- rounding the acrosomal cap and (3) release of mitochondria from the midpiece. Lake (1968, 1972) stored fowl spermatozoa in liquid nitrogen and found that not only are individual hens different in their ability to sustain and permit thawed spermatozoa to fertilize eggs within their oviduct, but that males differ in the ability of their semen to withstand freezing. Lake (1972) also noted glycerol at 2% or lower to be the best cryoprotective agent for the spermatozoa to survive thawing and achieve any fertility after insemination. The work done with frozen avian semen has expanded greatly over recent years. Marquez and Ogasawara (1973) found greater fertility using intramagnal artificial insemination compared to I.V.A.I. of frozen-thawed turkey semen. Freezing semen has great potential in the poultry industry. Further work is required not only in the types of extender, holding temperature, freezing and thawing, but on the actual site of the insemination using frozen-thawed semen in both the turkey and chicken. In order to fulfill the goal of successful insemination, more work needs to be done on the optimum numbers of spermatozoa to be inseminated I.M.A.I. or I.V.A.I.; the proper dilutions to be used (1:1, 1:4, etc); and the introduction, transport and release of spermatozoa into and out of the storage sites within the oviduct. Two spermatozoa storage sites have been identified in the domestic fowl hen. The first is the infundibular crypts described by Van Drimmelen (1946). The second storage site is the uterovaginal host glands discussed by Bobr et a1. (1962, 1964 a, b). The importance of the tubules located in the host glands was noted when spermatozoa were uniformly found in the tubules of the uterovaginal junction but rarely in the infundibulum after natural mating. These observations suggest that the uterovaginal junction is the primary site of storage of spermatozoa and that a few spermatozoa ascend to the site of fertilization immediately prior to ovulation. Mero and Ogasawara (1970) studied the anatomy of the host glands and described the sperm storage tubules as being enlarged or nonenlarged with similar lengths (approximately 500 microns) and similar neck widths (approximately 38 microns). They suggested that tubular enlargement was associated with sperm release. Gilbert et a1. (1968) studied the innervation and vascular supply of the uterovaginal sperm host glands. This work indicated that nerves were not associated with these glands, although nerves were demonstrated in other regions of the oviduct. The glands have a complex blood supply. This information stimulated questions concerning storage potentials of each area of storage. That is, which site of storage, the infundibulum or the U.V. junction, plays the major role in storage and release of viable spermatozoa? A new method of insemination (I.M.A.I.) was necessary to study the storage potentials of the U.V. junction and infundibulum. Intraperitoneal insemination in the chicken was done by Van Drimmelen (1951), and Gilbert et al. (1965). This technique, either intraperitoneal or intramagnal artificial insemination (I.M.A.I.), allowed for the introduction of spermr atozoa into the infundibular storage area without first passing by or through the uterovaginal junction. One problem researchers found to exist with I.M.A.I. was the delay of oviposition. Rothchild and Fraps (1945) found that the incidence of follicular atresia and the time before resumption of ovulation following operation were inversely proportional to the rate of ovulation preceeding the operation. Bobr et a1. (1965) noted inhibition of ovulation in the domestic hen when intrauterine inseminations were performed. Olsen (1952), using a series of intraovarian inseminations, provided evidence that fertilization of mature chicken ova occurred in the oviduct and not the ovary. Allen and Bobr (1955) studied the role of the uterovaginal junction in limiting the numbers of spermatozoa that ascend the oviduct. Ogasawara et a1. (1962) expanded upon this idea by inseminating semen from low fecundity cocks, characterized by poor motility, abnormal spermatozoa and 10 poor fertilizing capabilities when inseminated by standard techniques, and obtained moderate fertility when semen was deposited in the uterus. Fujii and Tamura (1963) studied the location of spermatozoa in the oviduct of the domestic fowl with special reference to storage of sperm in the vaginal gland. They indicated the majority of sperm are found in the uterovaginal host gland after insemination. Burke et a1. (1969) found sperm transport from the U.V. junction to the site of fertilization occurred even with inhibition of ovulation and oviposition by hormones, although the rate of movement was reduced. Burke et a1. (1972) noted no evidence of a cyclic secretion of material into the lumen of tubules corresponding with the movement of ova in the oviduct and with the time of oviposition. Harris (1968) studied the effects of cooling rate, equilibration time, and addition of turkey egg yolk to a glutamate diluent, on the fertility of frozen chicken spermatozoa. He stated the highest fertility (77%) was obtained with intraperitoneally inseminated semen that was equilibrated 0.5 hours in a turkey egg yolk diluent and cooled at a rate of 6°C. per minute. The intraperitoneal insemination resulted in a higher fertility than the intravaginal insemination. Ogasawara et a1. (1969) stored semen at 5°C. for various lengths of time and noted fertility after intravaginal (I.V.A.I.) and intramagnal artificial insemination (I.MJA.I.). It was found that I.MQA.I. hens had equally high fertility but for a longer duration, indicating that the uterovaginal junction is an effective barrier to handicapped spermatozoa, which yet are capable of fertilizing eggs if deposited close to the site of fertilization. The study of fertility in the domestic fowl had been specialized to 11 the degree that the actual numbers of spermatozoa inseminated affected the fertility, hatchability and duration of fertility. Specialized tech- niques for estimating the numbers of spermatozoa collected had to be de- veloped. Shaffner and Andrews (1943) used the packed cell volume to estimate semen density. Techniques have been described by Salisbury et al. p (1943) for bull semen and used for studying fowl and turkey semen by Jones and Lamoreux (1942), Gowe (1949), Howes (1951), Carson et a1. (1955), Kosin and Wheeler (1956), van Tienhovan et a1. (1958) and McCartney and Brown (1959). These authors used pooled semen samples from several cocks at known dilutions to determine the relationship between the optical density of semen and the direct count of spermatozoa made by the hemacytometer. Allen and Champion (1955) noted the relationship between abnormal spermatozoa and competitive fertilization utilizing different breeds of chickens. They studied competitive fertilization using distinct genetic markers. In their study, they found that the number of viable spermatozoa inseminated was different from the actual number of spermatozoa inseminated. Earlier work involving genetic markers in the poultry industry is volumi- nous. Goodale (1909) (1910), Pearl and Surface (1910), Dryden (1916), Dunn (1922), Hutt (1949) and Jerome and Cavers (1952) laid the groundwork when they studied the genes for black pigment and barring. Rosin and Wakely (1950) and Harper and Parker (1952), with the aid of genetic markers studied the overlapping of progeny from changing mates in turkey matings. Warren and Kilpatrick (1929), and warren and Gish (1943) did similar work with chickens. Crew (1926) and Dunn (1927) in- dicated that artificial insemination twice within a three day interval with semen from replacement males would decrease the male changeover time. METHOD AND MATERIALS Experiment I One hundred and sixty Single Comb White Leghorn (S.C.W.L.) laying hens, fifteen mature S.C.W.L. males, and fifteen mature Barred Plymouth Rock (B.P.R.) males were housed with thirteen hours of continuous light, fed layer-breeder ration (Appendix A), and given clean water ed libitum. All hens in individual cages were shown to be infertile prior to insemination. Semen samples were collected weekly from each male, and tested for quality by the method developed by Ernst and Ogasawara (1970). Trial IA - The one hundred and sixty hens were divided into twenty groups containing eight birds per group. Twelve groups were inseminated intra- vaginally (I.V.A.I.) and eight groups were inseminated intramagnally (I.M.A.I.) using pooled semen from S.C.W.L. males or B.P.R. males. In- seminations encompassing different volumes were used equivalent to the following number of viable sperm cells: (1) 12.5 million diluted 1:1; (2) 25 million diluted 1:1; (3) 50 million diluted 1:1; (4) 50 million neat; (5) 100 million diluted 1:1; (6) 100 million neat (Table l). Belts— ville Extender was used as the diluent. Trial IB - In Trial 18 (Table 2) the experimental design was the same as in‘Trial IA with the addition of two more groups intramagnally insem- inated with 100 million spermatozoa from S.C.W.L. or B.P.R. males. Trial IC - Trial IC (Table 3) was the same as Trial IB with the elimina- tion of 50 million spermatozoa (neat) inseminated intravaginally. 12 l3 Pre—trial sperm cell numbers were determined from pooled semen by packed cell volume and optical density. The optical density was measured with the Fisher Electrophotometer set with the red filter, 350 millimi- crons, see Appendix B for technique. Once the optical density was known, the number of spermatozoa was counted by use of the hemacytometer. By plotting the actual counted num- ber against the optical density, a linear regression was made for the se- men from each breed. The S.C.W.L. and B.P.R. regressions were identical (Appendix C). Because time was a critical factor prior to the insemination of fresh semen, the actual number of viable spermatozoa to be inseminated was estimated by subtracting an estimated ten percent for dead and ab- normal spermatozoa from the number determined by optical density (Appen- dices B and C). This number of spermatozoa inseminated I.V.A.I. or I.M.A.I. was carefully calculated and volumes equivalent to the above numbers of viable sperm cells were measured and inseminated by use of the Hamilton Microliter Syringe. A live-dead stain was made at the time of insemination. The same samples utilized for optical density were later taken to the laboratory where counts were made with the hemacytometer. This num- ber minus the actual percent abnormals and dead spermatozoa as determined from the live-dead stain, Ernst and Ogasawara (1970), gave the real numr ber of viable sperm cells in the pooled sample. Differences in the esti- mated number and real number were insignificant during analysis of the data. Special care was taken during the insemination to utilize only fresh semen. If longer than ninety minutes were required for testing and in- semination, fresh samples were again collected and tested as shown earlier. 14 The I.V.A.I. was performed by introducing into the area of the U.V. junction, via plastic straw, different volumes of spermatozoa equal to the desired number of viable spermatozoa. This volume of spermatozoa was measured with the Hamilton Microliter Syringe. The I.M.A.I. was a bit more complex requiring: (l) anesthetic- Brevitol (trade name - Eli Lilly) or Sodium Pentabarbitol; (2) surgical opening of the left lateral wall exposing the magnum; (3) insemination by injecting spermatozoa with the Hamilton Microliter Syringe attached to an 18 gauge needle; and (4) suturing of the incision after insemination. This second route of insemination required the hen to have a hard-shelled egg in the uterus which allowed for easy exposure to the magnum, thus creating less stress on the bird. Both insemination methods were performed simultaneously. After inseminations were made, the eggs were collected and marked daily with the hen number and date of lay until infertility for five con- secutive days was established by examination of candled eggs. After the freshly laid eggs were collected and marked, they were stored between 50°-60°F. until setting which was done weekly. After seven days of incu- bation at 99.5°F., all eggs were candled for fertility. Those categorized as infertile were broken open for further examination of fertility as described by Kosin (1945). The remainder of the fertile eggs were incu- bated until the l8th day when they were transferred into the hatcher at 98°-99°F. and 93°F wet bulb reading until hatching. After twenty-one days incubation, all eggs set were categorized as the following: (1) hatched; (2) pipped live;.(3) pipped dead; (4) live in shell; (5) dead in shell; (6) early dead, as seen in Appendix D. Data from Experiment I Trials IA, 13, IC were analyzed by a modified 15 one way analysis of variance1 for fertility weeks 1, 2 and 3; hatchability weeks 1, 2 and 3; and duration of fertility. See Tables 1, 2, 3, 4, 5 and 6 for experimental format and analysis of variance tables, respectively. Because treatments were different in Trials IA, IB and IC, a separate analysis of variance on each set of data was performed after the arc-sin transformations were made. As a result of the shorter duration of fertility from I.V.A.I., only data for I.M.A.I. were analyzed for the third week of fertility and hatchability. Also it was assumed that any zero values for fertility, hatchability and duration of fertility were due to characteristics of the particular hen and not the technique used. Experiment II In this experiment one hundred and twenty S.C.W.L. hens in produc- tion and fifteen mature S.C.W.L. males were housed in conditions similar to those in Experiment I. The hens were divided into twenty-four groups that contained five birds per group. Twelve groups were inseminated via the I.V.A.I. route, and twelve groups were inseminated via the I.M.A.I. route using pooled semen from S.C.W.L. males. Twelve treatments encompassing different volumes were used equivalent to the following number of viable sperm cells: (1) 12.5 million neat; (2) 12.5 million diluted 1:1; (3) 12.5 million diluted 1:4; (4) 25 million neat; (5) 25 million diluted 1:1; ‘A modified one way analysis of variance was used for analyzing Experiments I, II and III. Further information on analysis can be obtained from Dr. D. L. Cox and Terry Wing, Iowa State University. 16 (6) 25 million diluted 1:4; (7) 50 million neat; (8) 50 million diluted 1:1; (9) 50 million diluted 1:4; (10) 100 million neat; (11) 100 million diluted 1:1; (12) 100 million diluted 1:4. Beltsville Extender was used as'the diluent. Sperm cell numbers were determined similar to Experiment I, with the optical density (Appendices B and C) being the sole indicator of sperm numbers. The eggs were collected, marked, incubated and hatched as done in Experiment I. Data from Experiment II, Trial IIA and IIB were analyzed with a modi- fied one way analysis of variance2 for fertility, hatchability and dura- tion of fertility similar to Experiment I. See Tables 33, 34A and 34B for format and analysis of variance outlined, respectively. As in Experiment I, a separate analysis of variance was performed on each trial. Experiment III Ninety-six New Hampshire (N.H.) hens in production, fifteen S.C.W.L. mature males, fifteen B.P.R. mature males, and fifteen N.H. mature males were housed in conditions similar to those in Experiment I. The hens were divided into twenty-four groups that contained four birds per group. Twelve groups were intravaginally inseminated with pooled semen equivalent to twenty-five million viable spermatozoa or seventy-five million viable spermatozoa diluted 1:1 with Beltsville Extender at time zero and one hour later. The other twelve groups were intravaginally in- seminated with pooled semen equivalent to twenty—five million viable spermatozoa or seventy-five million viable spermatozoa at time zero and 2Refer to Footnote 1 on Page 15. 17 twenty—four hours later. All possible breed combinations were utilized to assure that pooled semen used at time zero was not from the same breed used for the second insemination performed one hour or twenty—four hours later (Table 53). This experiment was repeated twice in Trials IIIA and IIIB, respectively. Viable sperm cell numbers were determined by the same method used in Experiment I with the optical density (Appendix B) being the sole indica- tor of sperm numbers. The eggs were collected, marked, incubated and hatched as in Experi— ment I. However, the experiment required special attention to assure proper identification of pedigree. A pre-trial test was done to establish the accuracy of the following color characteristics of the newly hatched chicks, from the threepossible matings: (l) N.H. male x N.H. female - rapid feathering, red color; (2) S.C.W.L. male x N.H. female - rapid feathering, white and yellow color; (3) B.P.R. male x N.H. female - slow feathering, black color. All of the offspring showed the respective genetic markings as de- scribed above. This pre-trial test was also used to check fertility, hatchability and duration of fertility of the three different crosses. Trials IIIA and IIIB were analyzed by a modified one way analysis of variances for characteristics which would lead to information con- cerning the introduction and release of spermatozoa from the uterovaginal host glands. See Tables 53, 54A and 54B for experimental design and analysis of variance. A list of parameters analyzed is in Appendix F. After the completion of the two breed cross, the experimental design °See Footnote 1 on Page 15. 18 was changed to facilitate a three breed cross (Table 55). This would give more insight into sperm introduction and release from the uterovaginal host glands. This experiment was also repeated twice in Trials IIIC and IIID, respectively. The experimental design (Table 55) again allowed for all possible breed combinations but eliminated the time interval as a variable. Three intravaginal inseminations were made consecutively twenty-four hours apart utilizing either twenty-five million or seventy-five million viable spermatozoa. See Appendix G for a list of parameters analyzed. RESULTS Experiment 1 Trial IA Fertility: Trial IA During the first week a comparison between 1:1 dilution and neat semen showed no dilution effect on fertility. There was no difference in fertility between various treatments using neat semen (Table 4). Inseminations of diluted semen resulted in a significant breed effect indicating the S.C.W.L. as having a higher mean fertility of 67.58% compared to the lower B.P.R. mean fertility of 54.66% (Table 7). The number of spermatozoa inseminated showed an interaction with the route of insemination. As indicated in Table 8, the mean fertility for 12.5 million, 25 million and 50 million viable spermatozoa inseminated was consistent while the 100 million was highest with the I.V. route of insemination. During the second week no difference in fertility was shown between neat and diluted semen, although within the diluted inseminations, the superiority of the I.MLA.I. route was highly significant. Also the breed and route interactions were highly significant. Table 9 shows the higher fertility of the S.C.W.L. for the I.M.A.I. as compared to the lower fer- tility with the I.V.A.I. The interaction occurred because the B.P.R. ‘breed did not elicit an increase in fertility with the I.M.A.I. The numbef and route interaction during the second week of fertility 19 20 (Table 10) showed a linear trend with the I.V.A.I. with 100 million being highest. The I.M.A.I. lacked this linear trend because of the lower fer- tility utilizing the 100 million spermatozoa. For the third week of fertility a highly significant breed effect was shown with the S.C.W.L. being superior regardless of the route or number of spermatozoa inseminated (Table 11). Hatchability: Trial IA During the first week there was a significant difference in hatcha- bility between the neat and 1:1 diluted semen (Table 12). This indicated that neat semen provided higher hatchability regardless of the route of insemination. Within the diluted samples inseminated, there was a highly significant route effect on hatchability (Table 13) with the I.V.A.I. providing greater hatchability. The second week showed no significant difference in hatchability from I.M.A.I. or I.V.A.I. In the third week (Table 14) of hatchability, data indicated a high- ly significant breed and number effect with the S.C.W.L. breed being superior to the B.P.R. Duration of Fertility: Trial IA Duration of fertility showed two breed interactions. The first was a breed and number interaction (Table 15) ihdicating the S.C.W.L. to be superior at 12.5 million, 25 million and 100 million spermatozoa insemin- ated. When 50 million spermatozoa were inseminated, the B.P.R. was superior. Secondly, the breed and route interaction (Table 16) indicated the 21 I.M.A.I. provided the longest duration of fertility for the S.C.W.L. while the I.V.A.I. provided longest duration of fertility for the B.P.R. Trial IB Fertility: Trial IB The first week of fertility showed a significant breed and route in- teraction (Table 17). The S.C.W.L. had higher percent fertility with the I.V.A.I. while the B.P.R. had higher percent fertility with the I.M.A.I. During the second week a significant route and number interaction with inseminations of neat semen was noted (Table 18). The I.V.A.I. pro- vided greater fertility than I.MgA.I. at the 100 million spermatozoa level, while the 50 million level I.V.A.I. gave higher fertility than the 100 million I.V.A.I. The second week of fertility was significantly better with the I.M. A.I. regardless of number or breed (Table 19). The third week of fertility showed a significant breed effect with the S.C.W.L. being superior to the B.P.R. regardless of number or route of insemination (Table 20). A significant linear trend in fertility utilizing B.P.R. semen was shown with the 100 million level of spermato- zoa inseminated being highest in fertility (Table 26). Hatchability: Trial IB Hatchability for the first week, within neat samples, showed a sig- nificant route and number interaction. One hundred million spermatozoa inseminated I.V. provided greater hatchability than 100 million level I.M.A.I. (Table 21), while the 50 million neat level I.V.A.I. was intermediate. 22 1 Within the diluted samples was found a significant breed effect (Table 22) indicating the B.P.R. to have greater hatchability regardless of number or route of insemination. During the third week, hatchability showed a linear trend for the B.P.R. when I.M.A.I. was utilized with the 100 million level having great- er hatchability for the neat and diluted samples (Table 23). Duration of Fertility: Trial IB There appeared to be a trend with the neat semen giving a longer duration of fertility of 17.81 days while the diluted semen gave a dura— tion of fertility of 15-19 days. Also with the neat semen, there was a significant route and number interaction in which the I.M.A.I. gave the longest duration of fertility while the I.V.A.I. 100 million was the shortest in duration of fertility. Within the diluted semen, there was a significant breed effect, highly significant breed and route interaction, and significant route interaction. The S.C.W.L. gave the longest duration of fertility at 50 million, and the shortest duration of fertility at 100 million level diluted 1:1 (Table 24). The B.P.R., although shorter in duration, showed a linear trend with the 100 million level being longest in duration (Table 24). The I.M.A.I. route was significantly longer in duration of fertility than the I.V.A.I. (Table 25). 23 Trial IC Fertility: Trial IC During the first week was shown a highly significant interaction of route and number (Table 27). The I.M.A.I. gave highest fertility at 12.5, 25 and 50 million spermatozoa levels with 100 million being the lowest in fertility. The I.V.A.I. provided highest fertility at the 50 million level and lowest at the 12.5 million level of viable spermatozoa inseminated. Fertility during the second week showed a significant breed and route effect. The breed and route interaction (Table 28) indicated that the S.C.W.L. were superior to the B.P.R. and the I.M.A.I. route was better than I.V.A.I. The third week also indicated the highly significant fertility of the S.C.W.L. (Table 29). Hatchability: Trial IC During the second week, there was a route and number interaction (Table 30) within the diluted semen inseminations. The I.M.A.I. hatcha- bility was high for the 12.5, 25 and 50 million spermatozoa levels, while the 100 million was the lowest level for the I.M.A.I. For the third week, a highly significant breed effect was shown with the S.C.W.L. being double the percent hatchability of the B.P.R. (Table 29). Duration of Fertility: Trial IC The duration of fertility for the third trial showed a significant 24 effect from breed, route of insemination and number inseminated. The breed and route interaction (Table 31) showed the superiority of the I.M.A.I. for both breeds with the S.C.W.L. having longer duration of fertility than the B.P.R. Also there was a linear trend with the 100 million giving the longest duration of fertility (Table 32). Experiment II Trial IIA Fertility: Trial IIA Data from Trial IIA during the first week of fertility showed a sig- nificant route and dilution interaction (Table 35). The I.M.A.I. had a definite linear trend as the dilution decreased with the neat samples having the highest fertility and the 1:4 dilution having the lowest fer- tility. The I.V.A.I. showed the opposite linear trend with the neat semen having the lowest fertility. During the second week of fertility, a highly significant route effect was evident (Table 36) along with a significant number effect (Table 37). The route effect indicated the increased fertility utilizing the I.M.A.I. regardless of the dilution or number. The number effect indi- cated a linear trend during the second week with 100 million spermatozoa giving the highest fertility. The fertility during the third week gave almost identical results for the number effect with only slightly smaller values (Table 38). Hatchability: Trial IIA During the first week, a significant route and number interaction was seen (Table 39). With the I.M.A.I. a trend indicated that as the 25 number of spermatozoa increased, the hatchability decreased. Just the opposite was true with the I.V.A.I. A definite linear trend indicated that as the number of spermatozoa increased, so did the hatchability increase. During the second week and third week (Tables 40, 41, respectively), a significant dilution effect was evident indicating a better hatchability with 1:1 and 1:4 diluted samples. Duration of Fertility: Trial IIA Data analyzed for Trial IIA showed three major effects. (1) The route effect was highly significant (Table 42) and pointed out the in— creased duration of seven days utilizing the I.M.A.I. (2) A highly significant number effect was noted with a linear trend of duration when increased numbers of spermatozoa were inseminated (Table 43). The 50 million level appeared to be largest in overall duration regardless of route of insemination. (3) The significant dilution effect (Table 44) suggested the diluted samples gave slightly longer duration of two days with no difference between the 1:1 and 1:4 diluted samples. Trial IIB Fertility: Trial IIB The first week of fertility had a significant route and number in— teraction (Table 45). The I.M.A.I. had a linear trend with decreasing fertility as the numbers of spermatozoa increased from 12.5 million sper- matozoa to 100 million spermatozoa. Just the opposite occurred with the I.V.A.I. The fertility increased as the numbers of spermatozoa inseminat- ed increased from 12.5 million to the 50 million level. There was a 26 slight drop in fertility at the 100 million level of spermatozoa inseminated. The second week of fertility showed three major effects. (1) The route effect was highly significant (Table 46) which indicated the I.M. A.I. route as giving the higher fertility. (2) A significant number effect (Table 47) showed 100 million as being better than the 12.5 million, 25 million and 50 million levels of spermatozoa inseminated, regardless of route used and dilution used. A significant route and dilution inter- action showed a reverse trend between the I.M.A.I. and I.V.A.I. (Table 48). With the I.M.A.I. a lower fertility was seen as the sample was more diluted. With the I.V.A.I. fertility increased as the sample was more diluted. Hatchability: Trial IIB The highly significant data on hatchability for the first week (Table 49) showed the I.V.A.I. route providing greater hatchability than the I.M.A.I. The second week of hatchability reversed this trend to a significant degree with the I.M.A.I. providing greater hatchability than the I.V.A.I. (Table 50). Duration of Fertility: Trial IIB There were two main effects concerning duration of fertility in Trial IIB. (1) The highly significant route effect (Table 51) again pointed out the increased duration of six days utilizing I.MQA.I. (2) The significant number effect (Table 52) indicated the increased duration of fertility with increased numbers of spermatozoa inseminated, regardless of dilution of spermatozoa or route of insemination. 27 Experiment'III Trial IIIA Fertility, Hatchability and Duration of Fertility The 75 million level of viable spermatozoa inseminated was superior during Trial III as follows: (1) fertility during the first week (Table 56); (2) fertility during the second week (Table 57); (3) hatchability during the second week (Table 58); (4) overall duration of fertility (Table 59). First Breed - First Week By looking at the ratios of chicks hatched from the first breed in- seminated during the first week, a significant breed effect was seen along with highly significant numbers, interval and breed—interval interactions. Also, a significant breed-number—interval interaction was seen (Table 60). Breed Effect - When N.H. spermatozoa was used as the second breed inseminated, regardless of the first breed, the first breed had the high— est ratio of chicks. No consistent correlation could be found concerning the second breed inseminated and the lower ratio of first breed chicks. Number Effect - The 25 million level of insemination gave the highest ratios for the first breed chicks at all breed combinations except the S.C.W.L.-N.H. and B.P.R.-S.C.W.L. in which the 75 million level gave the highest ratio during the first week. Within the 75 million level, the B.P.R.—S.C.W.L. combination showed the highest ratio for the B.P.R. at both the one hour interval and the twenty—four hour interval of insemina- tion. The one hour interval showed the highest ratio of the first breed chicks, regardless of number of spermatozoa inseminated. 28 First Breed - Second Week During the second week a significant breed effect along with a sig- nificant breed-number-interval.interaction was noted (Table 61). When looking at the chick ratios of the first breed inseminated, the data in- dicated the highest ratio of first breed S.C.W.l. chicks occurred when followed by N.H. as the second breed, regardless of the interval or num- ber of spermatozoa inseminated. The lowest number of first breed chicks occurred when the S.C.W.L. were followed by the B.P.R. First Breed - Total Trial IIIA The ratio of first breed chicks for the total length of the trial showed a significant breed and number effect along with a significant .interval effect. Breed Effect - Overall ratios indicated more first breed chicks were sired by the S.C.W.L. and B.P.R. when followed by a second insemination of N.H. spermatozoa (Table 62), regardless of number or interval. Number Effect - More of the first breed chicks, regardless of se- quence of breed, were obtained with the lower number of spermatozoa - 25 million per insemination (Table 63). Interval Effect - Over the total trial more first breed chicks oc- curred with the one hour interval (Table 64), regardless of the number of spermatozoa or breed inseminated. Second Breed - First Week ' During the first week the ratio of the second breed inseminated (Table 65) was as follows: (1) breed, significant; (2) number, highly significant; (3) interval, highly significant; (4) breed and interval 29 interaction, significant; (5) breed-interval-number interaction, signifi- cant. (1) Breed - The highest ratio during the first week for the second breed inseminated was seen when the second breed was the S.C.W.L. or B.P.R. Conversely, the lowest ratios occurred when the N.H. was used as the second breed. (2) Number - The higher number inseminated, 75 million, elicited the larger ratios of second breed chicks during the first week. Within the 75 million level the S.C.W.L. was good as the second breed, while the N.H. did poorly as the second breed of the one hour interval, but improved at the twenty-four hour interval. (3) Interval - The twenty-four hour interval showed the highest ratio of second breed chicks. Second Breed - Second Week During the second week the ratios of the second breed chicks showed a significant breed effect along with a significant breed-number-interval interaction (Table 66). The breed effect showed the lowest ratio of second breed chicks when the second breed sire was the N.H., regardless of the first breed. The higher ratio was the S.C.W.L. inseminated as the first breed and the B.P.R. as the second breed inseminated. Second Breed - Total Trial IIIA The combined data of the second breed ratios for the entire trial showed a significant breed effect, number effect, a highly significant interval effect and a significant breed-interval interaction (Table 67). I When the N.H. spermatozoa was used as the second sire, the ratio of 30 their offspring was lower, regardless of the number of spermatozoa in- seminated. The twenty-four hour interval of insemination showed a higher ratio of chicks hatched from the second breed than the one hour interval of insemination. The 75 million level of insemination gave a higher ratio of second breed chicks than the 25 million level (Table 68). Overall, the twenty-four hour interval showed more second breed chicks than the one hour interval (Table 67). Duration of Fertility The number of spermatozoa inseminated was significant in that 75 million spermatozoa showed a longer duration of fertility than 25 million spermatozoa inseminated (Table 69). Trial IIIB Fertility, Hatchability and Duration of Fertility Fertility data in Trial IIIB indicated that the 75 million level of spermatozoa inseminated was superior during the first week (Table 70) and second week (Table 71). i Hatchability data showed a highly significant breed effect (Table 72) and a significant number effect. When the N.H. semen was used as the second breed, the hatchability was lowest during the second week, whereas the highest hatchability occurred when S.C.W.L. semen was inseminated as the second breed. Inseminations with 75 million spermatozoa showed the highest hatchability, regardless of insemination sequence (Table 73). Duration of fertility showed a highly significant breed effect along with a significant breed-interval interaction (Table 74). 31 The number effect pointed out the longer duration of fertility with 75 million spermatozoa inseminated (Table 75). The breed-interval interaction suggested the twenty-four hour inter- val was superior in duration of fertility for all breed combinations, ex- cept those which involved the N.H. and B.P.R. together (Table 74). First Breed - First Week During the first week, the ratios of the first breed inseminated indicated a significant breed effect (Table 76). It appeared that when the S.C.W.L. was used as the second breed, the first breed chick ratios were low. Also, when the N.H. was used as the second breed inseminated, the first week ratios were high for both the S.C.W.L. and B.P.R. as first breed inseminations. First Breed - Second Week The first breed ratios for the second week indicated a highly signi- ficant breed effect, number effect and a significant breed-number inter- action (Table 77). When the N.H. was used as the first breed inseminated, the ratio of N.H. chicks hatched was lowest for the second week. When N.H. spermatozoa was used as the second breed, the ratios of chicks hatched for first breeds (S.C.W.L. or B.P.R.) inseminated were higher. The ratio of the first breed chicks during the second week was highest with 75 million spermatozoa inseminated. 32 First Breed — Total Trial IIIB The ratios of first breed chicks for the total experiment had a highly significant breed effect and a significant breed-interval inter- action (Table 78). During the first week the firstbreed which showed the lowest number of chicks was the N.H., particularly when followed by the S.C.W.L. as the second breed inseminated at both the one hour and twenty-four hour intervals. If N.H. spermatozoa was used as the second breed inseminated, the result was more first breed chicks at both the one hour and twenty-four hour intervals during the first week. Second Breed - First Week The first week ratios for the second breed inseminated showed a high- ly significant breed effect (Table 79) and a significant.number-interval interaction (Table 80). The S.C.W.L. was the highest while the N.H. showed the lowest ratios as the second breed inseminated. The one hour interval with 25 million spermatozoa inseminated gave the highest number of second breed chicks during the first week. Second Breed - Second Week The second week ratios for the second breed inseminated had a highly significant breed effect, number effect and a significant breed-number interaction (Table 81). When the S.C.W.L. spermatozoa was used as the second breed inseminated, the ratios were highest at the 25 million level of spermatozoa inseminated. The N.H. spermatozoa used as the second breed inseminated gave low ratios at both 25 million and 75 million levels of 33 spermatozoa inseminated. The best for both levels of 25 million and 75 million spermatozoa inseminated was the N.H. followed by the S.C.W.L. Within the 75 million level of insemination, the N.H. followed by the , B.P.R. gave the lowest ratio of chicks hatched. The 25 million level gave the highest number of second breed chicks during the second week compared to the lower number of chicks at the 75 million level (Table 81). Second Breed - Total Trial IIIB Overall, the second breed showed a highly significant breed effect and significant number-interval interaction (Tables 82 and 83). When N.H. was used as the second breed inseminated, the ratios were lowest, regardless of what the first breed inseminated was. When the S.C.W.L. was used as the second breed inseminated, the ratios were highest. The ratio of the second breed chicks was higher at the one hour in- terval of insemination particularly within the 25 million level of sper~ matozoa inseminated. Duration of Fertility The duration of fertility for the overall experiment, showed a sig- nificant breed effect, highly significant number effect (Table 84) and a significant breed-interval interaction (Table 85). When the S.C.W.L. was the second breed inseminated, the duration of fertility was longest. Duration of fertility was shortest when the N.H. was the second breed inseminated. The 75 million level of insemination gave the longest duration of fertility, regardless of the breed or interval. 34 When the S.C.W.L. was followed by an N.H. insemination one hour later, duration of fertility was very short; but duration of fertility was longer with the same breed sequence at the twenty-four hour interval. Trial IIIC Fertility, Hatchability and Duration of Fertility During both the first and second weeks (Tables 86 and 87L.the fertil— ity was significantly higher with 75 million spermatozoa inseminated. Duration of fertility for the second breed inseminated was highly significant indicating the S.C.W.L. as the breed giving the longest dura- tion of fertility and the N.H. having the shortest duration of fertility (Table 88). Duration of fertility for the third breed inseminated was significant having the same trend seen for the second breed inseminated. The S.C.W.L. had the longest duration of fertility when utilized as the third breed; B.P.R. was intermediate, and N.H. had the shortest duration of fertility when utilized as the third breed inseminated. The ratios of offspring sired by the second breed inseminated during the first week of fertility showed a highly significant breed effect. Second Breed - First Week When the S.C.W.L. spermatozoa was used as the second breed inseminated (twenty-four hours after the first insemination), there was a greater number of S.C.W.L. chicks in the first week of fertility than either B.P.R. or N.H. chicks. The B.P.R. was intermediate, and the N.H. was lowest in number of chicks produced. 35 Third Breed - First Week Also during the first week of fertility the ratios of chicks hatched from the third breed inseminated were highly significant. Again the S.C.W.L. ratio of chicks hatched was highest with the N.H. ratio of chicks hatched being lowest. Second Breed - Second Week During the second week of fertility, the ratios of chicks hatched for both the second and third breed inseminated were highly significant, indicating the S.C.W.L. as being superior to the B.P.R. and N.H., respectively. Second and Third Breed - Total Trial IIIC The ratios for the overall trial indicated the S.C.W.L. as being superior when inseminated either as the second or third breed. The B.P.R. was intermediate as the second or third breed inseminated. The N.H. had the lowest ratio of chicks hatched when the N.H. males were used for either the second or third breed inseminated. Trial IIID Fertility, Hatchability and Duration of Fertility The experimental design of Trial IIID was identical to Trial IIIC (Table 55). Fertility: Trial IIID Percent fertility showed a highly significant number effect for the second week (Table 89), which indicated the 75 million level of spermatozoa 36 as being superior to the 25 million level of spermatozoa inseminated. Duration of Fertility The first breed inseminated showed a significant number effect with 75 million having the longest duration of fertility of 5.8 days compared to the lower duration of 3.75 days for the 25 million level of spermatozoa inseminated (Table 90). It should be pointed out that duration of fertil- ity of the individual breeds was calculated from the day of insemination of that particular breed. The breed effect was highly significant for the duration of fertility of the first breed inseminated (Table 91). The N.H. showed the shortest duration of fertility while the S.C.W.L. and B.P.R. were longest. The second breed inseminated had a highly significant number effect (Table 92) indicating that the 75 million level was better for the second breed inseminated. The total duration of fertility of Trial IIID (Table 93) showed the 75 million level of spermatozoa inseminated being superior to the 25 mil- lion level. This number effect was highly significant. First Breed - Second Week Ratios of chicks hatched from the first breed inseminated showed a highly significant breed effect during the second week of Trial IIID (Table 94). When N.H. spermatozoa was the first breed inseminated, the smaller number of chicks sired during the second week was N.H. 37 Third Breed — Second Week For the second week, the ratios of chicks hatched for the third breed inseminated indicated the B.P.R. as being superior with the N.H. having the lowest ratios (Table 95). DISCUSSION Experiment 1 Trial IA Fertility: Trial IA Trial IA was the first of three trials. An outline of the experi- mental design and analysis of variance may be seen in Tables 1 and 4, respectively. The breed effect indicated the superiority of the S.C.W.L. spermato- zoa on fertility during the first and third weeks. S.C.W.L. hens had a higher percent fertility when mated with S.C.W.L. males as compared to the B.P.R. male and S.C.W.L. female cross. Perhaps this higher fertility is due to some physiological or anatomical factor which enhances the S.C.W.L. female predilection for S.C.W.L. spermatozoa or discrimination toward B.P.R. spermatozoa. Of more importance was the effect of sperm cell numbers and the route of insemination on the fertility, hatchability and duration of fertility, and the possible relationship to sperm storage areas, the uterovaginal host glands and the infundibular crypts. As seen in the linear trend in Table 8, during the first week of fertility, the uterovaginal junction had the ability to hold and support up to 100 million spermatozoa diluted 1:1. At the same time, the infun- dibular crypts (I.M.A.I.) appeared to reach their maximum potential with 50 million spermatozoa diluted 1:1. 38 39 The first week may be the most critical when relating capacity of sperm cell storage areas to numbers of spermatozoa inseminated because of the movement of the spermatozoa throughout tle oviduct. In other words, can we safely assume that spermatozoa from the I.M.A.I. were actually responsible for the fertility, hatchability and duration associated with that insemination? 0r had some of the spermatozoa migrated to the host glands where they were released for fertilization? This author assumed that transfer of spermatozoa from one storage site to another was negli- ble at best (Bobr et 31., 1964b). During the second week of fertility, there was no difference between the neat and diluted samples when the 50 million and 100 million I.V.A.I. were compared. This was as expected since Beltsville Extender has been found to have no effect on fertility, hatchability or duration of fertility as discussed in a personal communication with Dr. T. Sexton (1977). The second week showed a linear trend (Table 10) for the I.V.A.I. indicating the capability of the host glands located at the uterovaginal junction to adequately hold 100 million spermatozoa I.V.A.I. Conversely, the infundibular crypts following I.M.A.I. seemed to store the 50 million spermatozoa best with the highest fertility. The 100 million level appeared to overwhelm the crypts thus a lower fertility. The third week of fertility (Table 11) completed the highly signifi- cant pattern of S.C.W.L. spermatozoa being superior to the B.P.R. sperma- tozoa when inseminated in the S.C.W.L. regardless of route, I.M.A.I. or I.V.A.I., and number, 12.5 million, 25 million, 50 million or 100 million. ‘Hatchability: Trial IA During the first week, the neat samples resulted in a higher hatcha- ‘bility regardless of the route of insemination used. This was most likely 40 due to the increased numbers of birds inseminated with diluted semen (Table 1). Within the diluted samples, the intravaginal artificial insemr ination gave the higher hatchability than I.M.A.I. regardless of the number of spermatozoa inseminated. This seemed logical if the U.V.J. host glands had the ability to "filter out" abnormal spermatozoa. Theo- retically only viable spermatozoa were released for fertilizing past the U.V. junction. The opposite situation held for the infundibular crypts which evi- dently did not have the ability to segregate the normal from the abnormal spermatozoa. Thus a higher number of spermatozoa which were incomplete or having poor fertilizing capabilities may have been released resulting in poor hatchability. During the third week of hatchability the S.C.W.L. had greater hatch- ability than B.P.R. regardless of the route of insemination. This fur- ther added to the breed effect seen in fertility and duration of fertility. Possibly a reaction by the S.C.W.L. female limited the B.P.R. spermatozoa activity. Also there may have been some chemical factor within the S.C. W.L. female which limited or retarded the B.P.R. spermatozoa's capabilities particularly at the lower numbers (Table 14). Duration of Fertility: Trial IA There was no significant difference in hatchability due to the use of Beltsville Extender when used to dilute the samples. The tendency for the S.C.W.L. to be superior and elicit a longer duration of fertility was seen in this experiment (Table 15). Also the I.M.A.I. was the best for the S.C.W.L., but not for the B.P.R. This could be accounted for by referring to the raw data. Several of the hens 41 inseminated with B.P.R. spermatozoa I.M.A.I. failed to return to produc— tion. Because the number of females remained constant, any that did not V return to production were included in the data thus lowering duration for that group. Normally one would expect the I.M.A.I. to have a longer dura- tion than the I.V.A.I. (Ogasawara et al., 1969). Trial 13 Before discussing Trial 13, several points should be made. (1) The males were the same males used for Trials IA and IC. (2) The hens were determined to be infertile before insemination. (3) The technique for insemination was basically the same although improvements in technique for accuracy of numbers were made. The experimental design (Tables 2 and 5) was identical with Experi- ment I, Trials IA and IC for the 1:1 dilution, but for further comparison, the level of 100 million neat spermatozoa was added to the I.M.A.I. route for both S.C.W.L. and B.P.R. spermatozoa. Fertility: Trial IB Fertility for the first week showed an unusually low fertility for the S.C.W.L. when I.M.A.I. was used creating the breed and route inter- action (Table 17). This was due to a significantly large number of birds which failed to lay eggs during the first week but returned to production with fertile eggs during the second week of the trial. Thus the fertility was lowered for the first week. This trend was not significant during the second week (Table 19) of fertility because of the return to production of fertile eggs. Also the I.M.A.I. became highly significant regardless if either S.C.W.L. or B.P.R. 42 spermatozoa was used. How this might be related to the storage sites may be quite complicated or might be a simple matter of anatomical relation- ships of storage site to the site of fertilization. The storage site for I.M.A.I. is the infundibular crypts which are located adjacent to the site of fertilization providing little difficulty or distance in terms of spermatozoa travel. Remembering, of course, that only the I.M.A.I. gave a third week's fertility data, the S.C.W.L. had significantly higher fertility than the B.P.R. during the third week of fertility (Table 20). With the B.P.R. semen, the neat samples at the 100 million level had a fertilizing capa- city greater than the 1:1 dilution of 100 million spermatozoa (Table 26). Very possibly the extra volume added by the diluent had some effect on the spermatozoa's ability to fertilize during the third week. It could be that more spermatozoa, for example, 200 million 1:1, could elicit fer- tility equal to 100 million neat. Duration of Fertility: Trial 13 The mean fertility for diluted samples inseminated was slightly lower than with neat samples. This occurred because the extra levels (12.5 and 25 million) of spermatozoa were included into the data analysis for diluted samples and not the neat samples. The I.M.A.I. route through the infundibular crypts gave the longest duration for both neat and diluted samples. Unlike Trial IA, a definite linear trend was obvious (Table 24) for the B.P.R. samples diluted 1:1 with 100 million level of insemination having the longest duration of fertility. The linear trend was lacking with the S.C.W.L. samples due to a peaking in duration of fertility at 43 50 million along with the lowest duration of fertility at 100 million. This was most likely related to the filling and storage potentials of the S.C.W.L. host glands and infundibular crypts, discussed earlier along with their association to breeds. Trial IC Fertility: Trial IC The experimental design (Tables 3 and 6) was identical to Experiment II except for the deletion of 50 million I.V.A.I. for both breeds. The techniques were similar. Fertility the first week of Trial IC was almost identical to Trial IA. Compare Tables 7 and 27. The I.M.A.I. for the 12.5 million and 25 million was consistent for both trials, while a peak fertility was reached near the 50 million mark with a decline from 50 million to 100 million. In both cases the infundibular crypts appeared to be most functional when inseminated with 50 million spermatozoa diluted 1:1. Whereas data from inseminations of up to 100 million spermatozoa into the U.V. junction appeared to be linear in trend. The second week in Trial IC followed the same trend (Table 28) with the S.C.W.L. being superior and the I.M.A.I. dominating over the I.V.A.I. The third week (Table 29) confirmed week two data with S.C.W.L. spermato- zoa inseminated I.M.A.I. as the best combination. 44 Experiment II Trials IIA-IIB Because of the similarity, both Trials IIA and IIB will be discussed together. The experimental design (Table 33) and the analysis of variance (Table 34) were identical for both trials. The overall fertility in both trials indicated the I.M.A.I. route as being superior, regardless of the number of spermatozoa inseminated or dilution of sample. The trend for the I.M.A.I. indicated a higher fertility during the first week utilizing 12.5 million and 25 million spermatozoa inseminated (Table 45). Evidently these low numbers were compatible with the storage area available in the infundibular crypts. Higher numbers of spermatOzoa may have overwhelmed the crypts creating lower fertility during the first week. This trend in numbers was not maintained for the third week of fertility. The higher numbers of spermatozoa inseminated showed higher fertility by the third week (Table 38). The route and number interaction was not significant for weeks two and three in either trial. Therefore, the overloading of the storage area appeared to be only temporary. Thiscorresponded with the lower hatchability during the first week (Table 49) utilizing I.MaA.I. As discussed in Experiment I, the increased numbers of abnormal spermatozoa available for potential fertilization may have lowered the hatchability during the first week. This may have been the case (Table 49) for hatch- ability the first week which indicated lower hatchability with I.M.A.I. than during the second week (Table 50). Overall fertility, hatchability and duration of fertility during both trials showed the superiority of 45 the I.M.A.I. This supported the work of Van Krey et al. (1964) and Ogasawara and Lorenz (1966) that increased abnormal spermatozoa reaching the infundibular crypts with I.M.A.I. resulted in increased potential for decreased hatchability during the first week of the trials. This held true if, in fact, the U.V.J. host glands filtered out abnormal spermato- zoa and released basically only normal viable spermatozoa. The superiority of the I.M.A.I. could also be seen with the longer duration of fertility utilizing I.M.A.I. (Tables 42 and 51). This corre- sponded with Van Krey et a1. (1964) and Experiment I. The linear trend also was consistent with Experiment I in that the lower numbers gave slightly shorter duration of fertility and the higher numbers gave greater duration of fertility (Tables 43 and 52). The actual spread of the overall means of duration of fertility was not as great as first expected because of the higher duration of fertility at the lower numbers utilizing I.M.A.I. During the first trial the dilution effect was significant for batch- ability during the second week and duration of fertility during the entire trial (Tables 40 and 44) which was indicated by better hatchability and longer duration of fertility with the 1:1 and 1:4 dilution. The extender and technique used were the same as in Trial IIB, which showed no dilution effect. One possible explanation involved the concept of virgin hens. Trial IIA was done on hens which, although mature, had never been insem- inated either I.M.A.I. or I.V.A.I. This virgin condition could have play- ed some physiological role in the dilution effect. The trend might have been related to the virgin condition of the uterovaginal host glands, (Trial IIA) or the used condition of the host glands (Trial IIB). By comparing the above data in Tables 40 and 44 to Table 35, and 46 looking at just I.V.A.I., it could be hated that there was a similarity in values with the neat samples being lowest. This was opposite of the I.M.A.I. (Table 35) which suggested the diluent had a detrimental action on fertility during the first week by possibly over filling the infundi- bular crypts. It was just possible that the effect of extender was to aid in dispersion of the spermatozoa through parts of the host glands which cannot be reached by neat semen. There was no significant dilution and number interaction in either Trials IIA or IIB. Experiment III Trials IIIA and IIIB Trials IIIA and IIIB were exactly the same in experimental design (Table 53). All N.H. hens were in production and had infertile eggs prior to insemination. The three different breeds of males utilized (N.H., S.C.W.L. and B.P.R.) were handled at the same intervals prior to and during the semen collection. During the first week of fertility, which began on the third day following the first insemination, the breed effect was significant for the first breed inseminated. The data indicated the superiority or dom- inant influence of the S.C.W.L. spermatozoa in the second insemination over the N.H. and B.P.R. semen from the first insemination (Tables 60 and 76). This data appeared to be consistent throughout both Trials IIIA and IIIB. An attempt was made to rationalize this superior effect of the S.C.W.L. based on number of spermatozoa inseminated, but careful examina- tion of the insemination records indicated the equalized numbers of sper- matozoa were, in fact, equal (see Method and Materials). Any breed effect 47 was due to possible interaction of the different breeds of semen or some hen reaction to the semen. Because all inseminations were intravaginal, the role of the host glands may have been related to the breed effect, either directly or indirectly. A second aspect of the ratios of chicks hatched during the entire length of both Trial IIIA and Trial IIIB was the low numbers of N.H. chicks which hatched. This occurred both when N.H. spermatozoa was used as the first breed inseminated or the second breed inseminated. As a precautionary measure, a pre and post trial experiment was performed to determine the fertility, hatchability and duration of fertility of the three different breeds when crossed with the N.H. hens. When the N.H. males were crossed with the N.H. females, similar re- sults were found compared to the B.P.R. and S.C.W.L. matings with N.H. females. This ruled out the possibility of N.H. males being inferior to the other two breeds. Therefore, it could be assumed that the low number of chicks sired by the N.H. males was due either to the spermatozoa inter- action, seminal fluid interaction, hen and semen interaction or some in- fluence of the storage sites of the hen. The ratios of chicks hatched for the first breed inseminated over the total time was similar for both trials and may give some insight as to the introduction, release and transport of spermatozoa to the site of fertilization (Tables 62, 63, 64 and 78). During the first trial, more chicks were sired by the first breed inseminated at the one hour interval (Table 64) indicating the storage sites were more receptive to the first breed when the second insemination was only one hour later. Perhaps the shorter interval allowed for the U.V.J. host glands to regulate the order of storage and release of spermatozoa while the passage of the egg or 48 oviposition which occurred within the twenty-four interval altered this release of spermatozoa. During Trial IIIA the lower number of spermatozoa inseminated (25 million) was also favorable to the first breed inseminated (Table 63). This may indicate that the U.V. junction storage site may regulate the first breed introduced as the first breed released, at the lower number of spermatozoa. Whereas the higher number of spermatozoa (75 million) may overwhelm the U.V. junction and its ability to release spermatozoa in the same sequence as it was introduced. The second breed inseminated sired the highest number of chicks at the 75 million level (Tables 63 and 68) at the twenty-four hour interval. In both trials, the duration of fertility was higher at the 75 mil- lion level of insemination (Tables 69 and 84). No significant difference in duration of fertility was noted between the one hour interval and the twenty-four hour interval within each trial although the overall duration of fertility was longer in Trial IIIA. (Compare Tables 69 and 84). Be- cause the spermatozoa numbers were equalized after estimation of numbers, this difference was most likely due to some environmental or management stress on the birds and not due to lack of or decreased numbers of spermatozoa. Trials IIIC and IIID Both Trials IIIC and IIID had the same experimental design (Table 55). The males and females used were the same for each trial. The semen was collected, tested and inseminated in the same way. The only factors that may possibly have altered the results were those in managment which 49 were minimal. Special care was taken when collecting semen to use those males which had not been milked the previous day. This would hopefully cut down on the number of immature spermatozoa in the sample. One factor which may have affected the results was the slight de- crease in egg production during the start of each trial. This was be- lieved to be due to the stress of handling the hens three days in a row. In both trials, the 75 million level of spermatozoa inseminated was superior for (l) fertility (Tables 86, 87 and 89) and (2) duration of fer- tility (Tables 90, 92 and 93). Although the 75 million level of insemina- tion was not significantly longer for duration of fertility in Trial IIIA, the 75 million level of spermatozoa gave a longer duration of fertility for every breed combination and total duration of fertility. The duration of fertility for the first breed inseminated (Table 90) was quite low at both the 25 and 75 million level. This may be due to the masking effect of the second and third inseminations. When the duration of fertility of the first breed inseminated in Trials IIIA and IIIB was compared to the first breed inseminated in Trials IIIC and IIID, the duration of fertility was longer in Trials IIIA and IIIB. Possibly the third insemination in Trials IIIC and IIID had some deleterious effect thereby decreasing the ability of the first breed in- seminated to maintain its fertilizing capacity. Another explanation involved the overwhelming of the uterovaginal host glands with the third insemination. If as previously indicated, 100 million spermatozoa appeared to elicit the longest duration, then the additional insemination particularly at the 75 million level might mask the ability of the host glands to regulate the release of the spermatozoa 50 as inseminated. This resulted in the first and second breed duration of fertility as being shorter (Tables 90 and 92) when compared to the overall duration of fertility (Table 93) of Trial IIID and Trials IIIA and IIIB. 51 Table 1 EXPERIMENTAL DESIGN OF TRIAL IA‘ Inseminations with 2 Inseminations with Semen Diluted 1:1 Neat Semen S.C.W.L. B.P.R. S.C.W.L. B.P.R. I.V.- 50 I.V.- 50 I.Vo- 1205 I.M.- 12.5 I.V.- 1205 I.Mo_ 12.5 I.V.-100 I.V.-100 I.V.- 25 I.M.- 25 I.V.- 25 I.M.- 25 I.V.- 50 I.M.- 50 I.V.- 50 I.M.- 50 I.V.-100 I.M.-100 I.V.-100 I.M.-100 Table 2 EXPERIMENTAL DESIGN OF TRIAL IB1 Inseminations with Neat Semen2 Inseminations with Semen Diluted 1:1 S.C.W.L. B.P.R. S.C.W.L. B.P.R. I.V.- 12.5 I.M.- 12.5 I.V.- 12.5 II.M.- 12.5 I.M.-100 I.M.-100 I V.- 25 I.M.- 25 I.V.- 25 I.M.- 25 I.V.- 50 I.V.- 50 I.V.- 50 I.M.- 50 I.V.- 50 I.M.- 50 I.V.—100 I.V.-100 I V.-lOO I.M.-100 I.V.-100 I.M.-100 Table 3 EXPERIMENTAL DESIGN or TRIAL Ic1 Inseminations with Neat Semen2 Inseminations with Semen Diluted 1:1 S.C.W.L. B.P.R. . S.C.W.L. B.P.R. I.V.- 12.5 I.M.- 12.5 I.V.- 12.5 I.M.- 12.5 I.V.- 25 I.M.- 25 I.V.- 25 I.M.- 25 I.M.-100 I.M.-100 I.V.- 50 I.M.- 50 I.V.- 50 I.M.- 50 I.V.-100 I.V.-100 I.V.-100 I.M.-100 I.V.-100 I.M.-100 1Eight Single Comb White Leghorn (S.C.W.L.) females per group were artificially inseminated intramagnally (I.M.) or intravaginally (I.V.) with 12.5 million (12.5), 25 million (25), 50 million (50), or 100 mil- lion (100) viable spermatozoa. 2Semen was collected from Single Comb White Leghorn (S.C.W.L.) or Barred Plymouth Rock (B.P.R.) males and inseminated I.M. or I.V. fresh as pure semen (neat), or diluted with Beltsville Extender (1:1). Aaomvac .ooaoHum> mo mHmaHmco has moo an unmoHustHm hstHm I «« $0.va 628.32, no 393on .33 use .3 ”253::me I « 52 muHmuH you monDUm amoz mo mHm>H umoz zuHHHuuom no N I xmoz N I xmmz H I xooz H I xooa .m.a mouaom cOHumuaa muHHHnmsoumm zuHHHuuom zuHHHnmnuumm zuHHHuuom 53 Ho. use» mmoH sUHHHnmpoum «« no. amen maoH sUHHHnmnoum « mm~.om mes.oaH.H mwm.omm HHs.Hem sHm.som ooH House no Hmaanos som.om mmw.mmN.H smo.Ham «Ha.aHs snm.~s~ n m x z x a ~a~.m smm.sss HHs.sHs omm.sam.H Hmm.mHm.H m a x 2 HNO.@ nos.H~ amm.osm amm.ams.H «omo.mso.m H s x m «mmm.esH msH.~Hw .Iwas.omH.cH mna.osm.m smo.H H Hue aoHamaHammaH so mazes IIaaH.ooH oso.oHs snm.Hms can.onH.H ooH.mmo m z x a s~m.os amm.soa mHs.ass.H wHa.mss Hmm.me.H m sz sumam mo umaasz «ooN.Hmm «Hs.ow~ so~.~aH.~ IHoq.sss.s mms.smm.H H Hmv umoum vansHHo :HeUHs ~om.a aso.Hmm «as.sue sas.Hcm sue.Hms N 2m x m «wmo.ssH mHa.soo.H «ONH.mom.m a~m~.~oo.s mos.nHH.H N Hzmv uuaasz can muses mNH.H qu.sm~ NmH.o moo.~oH omm.~me H Ame amass umuz cHnqu smoo.soH Hmm.ss oHH.sa~.H ~ms.ms Nom.som H HHHHV emusHHe .> ummz suHHHuuum as N I saw: N I saw: H I saw: H I some .s.a mousom aoHuausn suHHHnmeuumm suHHHuumm suHHHnmnoumm suHHHuuom muHmuH you moumnmm com: mH H ho mHmMH uauz suHHHuumm No N I saw: N I some H I sum: H I some .N.n mouaom soHumuaa suHHHamaouum suHHHuuom suHHHnmnoum: suHHHuuum wuH pm. New mono—am. coo: UH H.388 “HO moz mo mHmWHdzH. o oHnma 55 Table 7 BREED EFFECT* WITH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-1 OF FERTILITY Breed of Semen Inseminated Mean Percent Fertility S.C.W.L. 67.58 B.P.R. 54.66 Table 8 INTERACTION* BETWEEN ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. 0R INTRA- VAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED DURING WEEK-1 OF FERTILITY Number of Spermatozoa Mean Percent Fertility Inseminated (millions) Route of Insemination I.V. I.M. 12.5 61.54a 62.95a 25 57.85a 62.74a 50 74.90ab 74.79ab 100 83.43b 69.85ab *Probability less than .05 a,b - Statistical significance utilizing Tukey's Test (probability less than .05) is denoted by different letters in rows or columns. Example: By utilizing Tukey's Test, mean value differences greater than 17.35 (Table 8) are significant at the .05 level. Therefore, 61.54a is significantly different from 83.43b but not significantly different from 74.90ab. Subscript a is always significantly different from subscript b but not subscript ab. 56 Table 9 INTERACTION** BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) SEMEN OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE ROUTE 0F INSEMINATION (INTRAMAGNAL - I.M. 0R INTRAVAGINAL - I.V.) DURING WEEK-2 0F FERTILITY Breed of Semen Mean Percent Fertility Inseminated I.V. I.M. S.C.W.L. 30.09a 69.12b B.P.R. 47.25ab 47.41ab Table 10 INTERACTION* BETWEEN ROUTE 0F INSEMINATION (INTRAMAGNAL - I.M. 0R INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED DURING WEEK -2 OF FERTILITY Number of Spermatozoa Mean Percent Fertility Inseminated (millions) I.V. I.M. 12.5 31.77a 59.63b 25 34.79ab 55.38b SO 38.62ab 78.10c 100 49.50b 39.97a *Probability less than .05 **Probability less than .01 a, b, c - Statistical significance utilizing Tukey's Test (probability less than .01 or .05, respectively) is denoted by different letters in rows or columns. 57 Table 11 BREED EFFECT** WITH SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-3 OF FERTILITY Breed of Semen Inseminated Mean Percent Fertility S.C.W.L. 39.03 B.P.R. 17.03 Table 12 DILUTION EFFECT* WITH PURE SEMEN (NEAT) AND DILUTED SEMEN DURING WEEK-l OF HATCHABILITY Dilution of Semen Inseminated Mean Percent Hatchability Neat 75.61 Diluted‘ 61.18 Table 13 ROUTE EFFECT** WITHIN DILUTED SAMPLES WITH INTRAMAGNAL (I.M.)AND INTRA- VAGINAL (I.V.) ROUTES 0F INSEMINATION DURING WEEK-1 OF HATCHABILITY Route of Insemination Mean Percent Hatchability I.V. 74.92 I.M. 47.43 *Probability less than .05 **Probability less than .01 1Semen diluted 1:1 with Beltsville Extender. 58 Table 14 INTERACTION** DURING WEEK-3 0F HATCHABILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE NUMBER OF SPERMATOZOA INSEMINATED Number of Spermatozoa Mean Percent Hatchability Inseminated (millions) S.C.W.L. B.P.R. 12.5 41.68c 7.73ab 25 39.51c 3.32a 50 30.43bc 32.10c 100 44.50c 27.69bc Table 15 INTERACTION* BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) 0R BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND THE NUMBER OF SPERMATOZOA INSEMINATED 0N DURATION OF FERTILITY Number of Spermatozoa Duration of Fertility (Days) Inseminated (millions) S.C.W.L. B.P.R. 12.5 17.17cd 13.42ab 25 13.33ab 12.423 50 15.17abc 17.50cd 100 18.92d 12.25a *Probability less than .05 **Probability less than .01 a,b,c,d - Statistical significance utilizing Tukey's Test (probability less than .05 or .01, respectively) is denoted by different letters in rows or columns. 59 Table 16 INTERACTION* BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) ON DURATION OF FERTILITY Breed of Semen Duration of Fertility (Days Inseminated Route - I.V. I.M. S.C.W.L. 12.19a 18.13b B.P.R. 16.06ab 12.81a Table 17 INTERACTION* DURING WEEK-1 OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) 0R BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE OF INSEMINA- TION (INTRAMAGNAL - I.M. 0R INTRAVAGINAL - I.V.) Breed of Semen Mean Percent Fertility Inseminated Route I.V. I.M. S.C.W.L. 68.88ab 52.34a B.P.R. 55.17ab 71.16b *Probability less than .05 a, b - Statistical significance utilizing Tukey's Test (probability less than .05) is denoted by different letters in rows or columns. 60 Table 18 INTERACTION* DURING WEEK-2 OF FERTILITY BETWEEN ROUTE OF INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND THE NUMBER OF SPERMATOZOA INSEMINATED Number of Spermatozoa Mean Percent Fertility Inseminated (millions) Route I.V. I.M. 50 78.53b ----- 100 66.83ab ----- 100 ----- 58.17a Table 19 ROUTE EFFECT** DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTE OF INSEMINATION Route of Insemination Mean Percent Fertility I.V. 33.65 I.M. 55.50 Table 20 BREED EFFECT* WITH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN DURING WEEK-3 OF FERTILITY Breed of Semen Inseminated Mean Percent Fertility S.C.W.L. 46.62 B.P.R. 30.60 *Probability less than .05 **Probability less than .01 a, b - Statistical significance utilizing Tukey's Test (probability less than .05) is denoted by different letters in rows or columns. 61 Table 21 INTERACTION* DURING WEEK-1 OF HATCHABILITY BETWEEN INTRAMAGNAL (I.M.) OR INTRAVAGINAL (I.V.) ROUTESOF INSEMINATION AND NUMBER OF SPERMATOZOA INSEMINATED Route of Insemination Number of Semen Mean Percent Inseminated Hatchability I.V. 50 Neat 75.70b I.V. 100 Neat 82.88b I.M. 100 Neat 48.06a Table 22 BREED EFFECT* WITH THE BARRED PLYMOUTH ROCK (B.P.R.) AND SINGLE COMB WHITE LEGHORN (S.C.W.L.) SEMEN DURING WEEK-l OF HATCHABILITY Breed of Semen Mean Percent Inseminated Hatchability S.C.W.L. 54.85 B.P.R. 68.96 *Probability less than .05 a, b - Statistical significance utilizing Tukey's Test (probability less than .05) is denoted by different letters in rows or columns. 62 Table 23 TREND DURING WEEK-3 OF HATCHABILITY WITH BARRED PLYMOUTH ROCK (B.P.R.) SEMEN INDICATING INCREASED HATCHABILITY WITH INCREASED NUMBER OF SPERMATOZOA INSEMINATED Number of Spermatozoa Mean Percent Hatchability , Inseminated (millions) 25-1:11 11.25 50-1:1 ' 39.38 lOO-l:1 59.34 lOO—Neat . 56.60 Table 24 INTERACTION** ON DURATION OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND NUMBER OF SPERMATOZOA INSEMINATED Number of Spermatozoa Duration of Fertility (Days) Inseminated (millions) S.C.W.L. B.P.R. 12.5 17.75def 10.923 25 15.08bcde 12.08ab ' 502 20.9lf 14.16abcd 100 13.25abc 17.4ldef **Probability less than .01 1Semen was diluted 1:1 with Beltsville Extender. 2Peak duration of fertility is reached with 50 million S.C.W.L. spermatozoa. a-f - Statistical significance utilizing Tukey's Test (probability less than .01) is denoted by different letters in rows or columns. 63 Table 25 ROUTE EFFECT* WITH THE INTRAMAGNAL (I.Mg) AND INTRAVAGINAL (I.V.) ROUTE OF INSEMINATION Route of Duration of Fertility Insemination (Days) I.V. 13.28 I.M. 16.16 Table 26 TREND DURING WEEK-3 OF FERTILITY WITH BARRED PLYMOUTH ROCK SEMEN INDI- CATING INCREASED FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED Number of Spermatozoa Mean Percent Fertility Inseminated (millions) 25-l:1‘ 13.44 50-l:l 18.91 100-1:l 38.90 loo-Neat 51.17 * Probability less than .05 1Semen was diluted 1:1 with Beltsville Extender. 64 Table 27 INTERACTION** DURING WEEK-1 OF FERTILITY BETWEEN INTRAVAGINAL (I.V.) OR INTRAMAGNAL (I.M.) ROUTES OF INSEMINATION AND NUMBER OF SPERMATOZOA INSEMINATED ‘ Number of Spermatozoa Mean Percent Fertility Inseminated (millions) I.V. I.M. 12.5 27.73a 67.9lcde 25 ' 52.54bc 60.54bcde 50 75.90e 55.18bcd 100 64.50cde 42.37ab Table 28 ROUTE EFFECT* DURING WEEK-2 OF FERTILITY WITH INTRAMAGNAL (I.M.) AND INTRAVAGINAL (I.V.) ROUTESOF INSEMINATION FOR BOTH THE SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN Route of Mean Percent Fertility Insemination S.C.W.L. B.P.R. I.V. 31.82a '26.99a I.M. ' _ 69.00b ' 31.36a Table 29 BREED EFFECT** DURING WEEK-3 OF FERTILITY AND HATCHABILITY WITH SINGLE COMB WHITE LEGHORN (S.C.W.L.) AND BARRED PLYMOUTH ROCK (B.P.R.) SEMEN Breed of Semen Mean Percent . Inseminated _ Fertility ~ Hatchability S.C.W.L. . 52.52 45.37 B.P.R. 9.22 20.48. *Probability less than .05 **Probability less than .01 a-f - Statistical significance utilizing Tukey's Test (probability less than .01 or .OSIrespectively) is denoted by different letters in rows or columns. 65 Table 30 INTERACTION** DURING WEEK-2 OF HATCHABILITY BETWEEN ROUTE 0F INSEMINATION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) AND NUMBER OF SPERMATOZOA INSEMINATED Number of Spermatozoa Mean Percent Hatchability Inseminated (millions) I.V. I.M. 12.5 11.25a 58.03bc 25 53.66b 52.76b 50 87.22d 49.49b 100 82.32cd 40.6lb Table 31 INTERACTION* ON DURATION OF FERTILITY BETWEEN SINGLE COMB WHITE LEGHORN (S.C.W.L.) OR BARRED PLYMOUTH ROCK (B.P.R.) SEMEN AND ROUTE 0F INSEMINA- TION (INTRAMAGNAL - I.M. OR INTRAVAGINAL - I.V.) Route of Duration of Fertility (Days) Insemination S.C.WQL. B.P.R. I.V. ll.06a 10.75a I.M. 20.18b 11.25a *Probability less than .05 **Probability less than .01 a-d - Statistical significance utilizing Tukey's Test (probability less than .01 or .05, respectively) is denoted by different letters in rows or columns. 66 Table 32 NUMBER EFFECT* ON DURATION OF FERTILITY WITH INCREASED NUMBERS OF SPERMATOZOA INSEMINATED Number of Spermatozoa Duration of Fertility Inseminated (millions) (Days) 12.5 11.41 25 13.45 50 14.88 100 16.71 Table 33 EXPERIMENTAL DESIGN OF TRIALS IIA, IIB1 Dilutions of Semen for Dilutions of Semen for Intravaginal Inseminations Intramagnal Inseminations Neat 1:1 1:4 Neat 1:1 1:4 Number of Spermatozoa Inseminated Number of Spermatozoa Inseminated (millions) (millions) 12.5 12.5 12.5 12.5 12.5 12.5 25 25 25 25 25 25 50 50 50 50 50 50 100 100 100 100 100 100 *Probability less than .05 1LFive female Single Comb White Leghorn hens per group were inseminated intravaginally (I.V.) or intramagnally (I.Ms) with 12.5 million, 25 million, 50 million, or 100 million viable spermatozoa. The inseminations were made with pooled semen samples as pure semen (neat) or diluted 1:1 or 1:4 with Beltsville Extender. 67 Ho. qua» ouuH NNHHHHuHoNN «I no. sues anoH NNHHHHuHoNH « mo.sm Na.NmH.H aN.Hco.H Ho.NNN «N.HNN NoH Hmuoa cauoouuoo No.NH om.osH.H mH.aNn NN.HNw NN.ooo «N Haschms NN.HH «n.0NN.H mn.cmm mm.NNm ms.ooo.H o a x z x a oH.N an.Nsn ao.mNm om.oas mo.sHo.H o n x z Hm.oN HN.N¢H.N NH.oHN nN.NN «sm.NNm.N N a x a «mm.oo aoN.NNN.N No.oNs.H mo.NsN NN.mas N an aOHuaHHn No.n aN.omo.H NN.NNN «HN.Nmo.N No.9oH N z x a «INH.NN wo.sNH IH¢.NNN.H NH.HN¢ NN.NNm n sz HBenz «INH.Nem.H nn.NmN «IHo.mNN.Ns oN.anm «H.Hoo.H H ANS muses NNHHHuNoN No NI so»: NI sums HI sums HI sums soHuousa suHHHaasuuum HNHHHuuum NNHHHHmeuumm suHHHuNuN mums m comm .m.a ouuaom so mHNMH no mHmeuuN=H Hn.mm NH.sNo mm.oso.H mm.NNN mo.NNN m z u N HoH.oNN «IoH.NmN.s IIoH.NmN.N mm.oNN.H «HH.oNN.N H sz uoaasz sm.NH so.mNo.H NH.N¢H.H sH.NNH.H mm.nmm m HNV noose NAUHHfiuHmh MO lemu3 leww3 Hlxwmww Hlxmuz . h . n— mouaow aoHuauaa suHHHsmauuam suHHHuNoN soHHHpmeuumm NuHHHuNom museum coo: mo mHme42¢ don oHan 77 Ho. cues ammH NuHHHHaHoNN II He. can» mmuH HHHHHsuHoHN I «N.NN HN.NNH.H om.Nmm mH.NHo.H NN.NoH.H mm Hoses souuuuuoo NN.mN He.oHN.H «N.HNN Nc.NHo.H sm.NHo.H NH HmseHmoe mm.e Hs.mmm os.NNN NN.NmH.H oN.NHN n H x z x N Ho.m Nq.¢Na.H No.9mH mm.NNm mm.msm.m H H x z «we.em Ne.NNs.H «N.Hmm.H os.on.H Hm.oom.H n H x H oN.NH mm.mHo.H nH.on Ho.mso N9.NN H HHV Hm>uau=H NH.N NN.mNm.N so.ssm oe.mos mo.NNN m z x N NIHH.NNN «0N.NHN.H «INN.oso.o we.mos.H «oH.Nmm.m H sz Noaasz IINH.NN ««mo.mNN.s NN.HHm.H mo.Nmn «H.4NH.H n Ame muons NNHHHuan Ho NIswuz NIxmm: HIsous HIsoos .H.n mousom :OHumuaa suHHHpmeuom: NHHHHuuoH suHHHHmauumm suHHHuuoN ouosvm use: mHHH H mo mHmMH> q n E .52. o E .42 d r H 5:8... I on .5266 .400 /m L on om om. - oo. mm 538 :6 Em A>wamgv >._._J_._.mwu_ Fzmommm N mung... 117 3.3%.... 33.05.03 00 mm m. H .3. N _ A N. 7%.... _. . Ho. 7. ‘5 ON __ Tom H I HH H H 19558... H ._ H Ion Booed H H .H H HHH . H, H .00 nHH H/ u ’ HH ION / ImH 0\\ H, H .8 (Y I 5. L00. .0 mam .m> :0 430m 915$ng >F_I=._.mw...H hzmommm m MKDGHu 118 Hmco==EH 63266..on 00. On mm 0d. u q ‘0’ H". 32. O HHH _<>_ 4 mm 330m 3.8338 Essen“. .10 292:8 _v maze—I... o N v m m -- o. N. w. o. m_ COP—0530 ow «zoo MN 3 mm mm on 119 HmcozzeH ooNoHoEcoaw 00. on mm 0N. _N. .4). H". 32. 3 526m 3. D Em KEEN... .10 29.5.8 0 MEG.“— ,0 N V 0 Q .o. N— @— w. Pose , om mace NN ¢N mm mm On 120 .2332: 0320.5QO 0 m . . ON .on .ov £58 28ch .on om . on L _.0m ..0m #00. 9.235 $3.53 hzmummm 20:04:32. mmmzaz oz< 258 m mane... 121 30:25 v; _ _; _ p._v.mm>>v >._._.:._.mw.... Pzwummm ZO_.—.o