1, _l‘nm—‘v<-“-“V‘V‘. TVA L»! .__ . K ‘A ' A I 3 "14-. n. t. View: u I?» r,‘ r "I "4. .__ .‘ -x-.- ‘ - $7.}; ‘ > ‘ - ‘ ’29 \! (0‘ JR 4' : . ~ n'.‘ 'FV‘ 1"" CI, .‘1. $3: 21-;naq.;.." 7 ‘ 221' . . 31‘???” .mh.‘ a“) ' ’ r 6% "'I' ' I ’(N'4 ’ 1", 9331*“ A ‘K ‘ A a ' - v‘: '1': 14' a" ’9 " . A gnu" ....' 1" ‘29 t" -‘ ‘V!.“.'..' f. . a . .'.Vn‘:'u'1/'l'|\‘ ‘4"! 1 “up-,1,“ ' ”lug ‘ l “I. ’ ~ .9 A “ .s -. J‘b’t" I. Wt“? "1‘” 14¢ ll '9 v-tfix .. Mag; . . u: 4 ' 1 ' $11: 1;“ 4. ’3 ’1 . :~t-f'l<' ‘ 21‘ u_\._..‘a_- ‘ _ ”.7‘ — {)8 . X: i h ' ,' -‘. n ‘ .. “4“‘1',|;‘x'r " ‘ » ' .v' .- . O‘E-al. ' A L. D E H “ ' ‘ u l "‘ ‘ H" ‘ _ . ‘ 'n;t.| 13“? . . . 1 .-‘-‘;:;‘fl‘rl~'\3"-"‘~,' gt! . t.) '‘w‘ _l- ‘ 9 I1 «Infii Al ' 1'.) .‘1,5 ‘ ""19 I. if V. 1... '1‘. r This is to certify that the thesis entitled COMPETITIVE FERTEIZATION IN ogus DOMESTICUS presented by Cleveland J. Allen has been accepted towards fulfillment of the requirements for Doctor of Philosophy. degree iansbandry egg—6% ' Major professor Date 5 O unb O 1 COMPETITIVE FERTILIZATION IN GALLUS DOMESTICUS BY CLEVELAND JAMES ALLEN a...» . 1 \\7 ‘~ A THESIS ' Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department; of Poultry Husbandry 1953 ‘3 vs- ‘-"~ 7:! 4‘ 3.1: 'C‘ ‘ V" V vyOE‘N‘.‘ THESE T-\ uepartntett a Piiyfloin :7 ESPCVE Ana er‘ T'vfl‘lv usduJ .. A partmen’. of I ACKNOW LEDGMEN TS The writer wishes to express his appreciation to Dr. Lloyd R. Champion of the Department of Poultry Husbandry for his aid in conducting this experiment, for his counseling throughout this inves- tigation, and for his assistance in the preparation of this manuscript. Grateful acknowledgment is also given to Dr. K. J. Arnold, Department of Mathematics, and to Dr. J. Meites, Department of Physiology, for their assistance and guidance. Especial acknowledgment is made of the advice, materials, and equipment made available by Professor C. G. Card of the De- partment of Poultry Husbandry. ii 329540 COMPETITIVE FERTILIZATION IN GALLUS DOMESTICQS BY Cleveland Jame s Allen AN ABS TRAC T Submitted to the School of Grad'date Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Poultry Husbandry Year 195 3 Approv ~ /€%~t t t. 1. ... . . . in l 4 v Aw V n l. Paw .. . UK ,> A y. IN N . e p s . . ,_ V. u l . . - . - Aw] ,( C. . .... t,» .. t1 u. .w. F‘s .i .1 vi l. ,1 l .HL FV. t rr.. W. wt ulu o1 CLEVELAND JAMES ALLEN ABSTRACT Investigational reports in the literature on selective fertili- zation in the domestic fowl present conflicting results. Certain studies involved the pooling of equal volumes of semen from dif- ferent breeds of males, injecting the mixture into females, and trac- ing the resulting progeny to their respective sires. Other research procedures used the technique of alternate matings. To test whether-the spermatozoa from nearly related indi- viduals showed a special affinity for the ova of that breed, a series of experimental matings were conducted. Semen was collected from males of the following breeds: S. C. White Leghorn, White Wyan- dotte, New Hampshire, and Barred and White Plymouth Rock. Fe— males of all these breeds excepting the Barred Plymouth Rock were used. In matings designed to give clear—cut genetic differentiation of the resulting progeny, equal concentrations of spermatozoa were pooled, thoroughly mixed, and injected into the females at weekly intervals. Total semen volumes obtained, microscopic counts of spermatozoa contained therein, initial motility, and percentages of morphologically defective spermatozoa were noted. Weekly settings of all hatching eggs were made. The chicks were classified CLEVELAND JAMES ALLEN ABSTRACT phenotypically for plumage coloration and/or comb types at one day, two weeks, and four weeks of age to facilitate assigning them to their respective sires. Comparisons were made between males based upon the number of chicks sired. Further, the effects of initial spermatozoa motility and the incidence of defective sperma~ tozoa on fertilizing capacity under competitive conditions were de— termined. The following ranges of semen volumes (cc.) and spermatozoa concentrations (millions per cu. mm.) were found: White Wyandotte males, 0.39 to 1.78 cc., 1.38 to 3.12 millions; White Leghorn males, 0.42 to 1.32 cc 1.43 to 2.76 millions; White Plymouth Rock males, 0.42 to 1.30 cc., 1.34 to 2.90 millions; Barred Plymouth Rock males, 0.39 to 1.30 cc., 1.98 to 3.83 millions; and New Hampshire Inales, 0.42 to 1.82 cc., 1.36 to 3.12 millions. Spermatozoa motility ratings ranged from two to five in all males tested, with the semen from the White Wyandotte males nearer the lower limit of the range. The ranges in percentages of morphologically defective sperm- atozoa were: White Wyandotte, 3 to 20 percent; White Leghorn, 5 CLEVELAND JAMES ALLEN ABSTRACT to 18 percent; White Plymouth Rock, 2.5 to 15.5 percent; Barred Plymouth Rock, 7 to 15 percent; and New Hampshire, 3.5 to 15 per— cent. A highly significant positive correlation of 0.72 :I: 0.018 was found between spermatozoa motility rating and the number of prog- eny resulting. Therefore, the higher the motility rating of the spermatozoa, the greater the number of chicks sired by an individ— ual male. A significant negative correlation of - 0.55 :I: 0.027 was found between the percentage of defective spermatozoa and fertiliz- ing capacity. Therefore, the greater the proportion of morphologically defective spermatozoa, the lower the fertilizing capacity of an indi- vidual male. A significant negative correlation of - 0.31 :I: 0.035 was found between the motility rating of the spermatozoa and the percentage of abnormal spermatozoa in the semen. Therefore, the greater the proportion of abnormal spermatozoa, the lower the mo- tility rating of the spermatozoa of an individual male. From the results obtained, the following conclusions are warranted: 1. Affinity of spermatozoa from males of a particular breed fiiii‘il 5 ”Air .' CLEVELAND JAMES ALLEN ABSTRACT for ova of females of the same breed does not occur in the breeds and strains of chickens used in this investigation. 2. A type of selectivity based on spermatozoa possessing certain advantageous variations reflected in a high degree of motility and a low incidence of abnormalities is suggested as occurring in competitive fertilization in the domestic fowl. 3. The technique of competitive fertilization is suggested as a method of testing relative degrees of fertility in birds and mul— tiparous animals, where suitable genetic markers can be found. TABLE OF CONTENTS White Leghorn .............................. White Wyandotte ............................. White Plymouth Rock ......................... Barred Plymouth Rock ........................ New Hampshire ............................. Yield of Semen ............................. Sperm Concentration and Number of Spermatozoa ..... Morphology of Spermatozoa ..................... Motility of Spermatozoa ....................... MATERIALS AND METHODS ...................... RESULTS ................................... Experiment 1 .......... . ..................... Semen production .......................... Sperm concentration and numbers of spermatozoa Motility ................................. iii ~l 10 ll 13 14 16 18 20 31 31 31 34 35 Morphology Competitive Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 Experiment 10 Expe riment 11 of spe rmato zoa ................... fe rtilization ..................... OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ooooooooooooooooooooooooooooooo 0000000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOO » I O O O I O Motility and Morphology of Spermatozoa in Relation to Competitive-Fertilizing Capacity ............... DISCUSSION . . . SUMMARY AND CONCLUSIONS .................... BIBLIOGRAPHY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO iv 51 62 71 89 90 91 92 93 94 95 104 105 111 116 Table LIS T OF TABLES Evaluation of semen characteristics in three males: WW (0099), WL (9000), and NH (0040) The results of analysis of variance of semen yields (Expe riment 1) ...................... The results of analysis of variance of sperm concentrations (Expe riment l) ................ Classification of abnormal spermatozoa found in the three males; White Wyandotte (0099), White Leghorn (9000), and New Hampshire (0040) ................................. Weekly semen pools affording to each female equal concentrations of spermatozoa from each of three male 3 ........................... Sequence of progeny from New Hampshire and White Leghorn females inseminated with equal concentrations of spermatozoa collected from White Wyandotte, White Leghorn, and New Hamp- shire males ............................. The progeny of New Hampshire females insem- inated with equal concentrations of Spermatozoa collected from White Wyandotte), White Leghorn, and New Hampshire males .................. The progeny of White Leghorn females insem- inated with equal concentrations of spermatozoa collected from White Wyandotte, White Leghorn, and New Hampshire males .................. 32 34 35 37 39 41 49 51 Table Page 9. Semen evaluation for White Plymouth Rock male (7491) ............................. 53 10. The results of analysis of variance of semen yields (Experiment 2) ...................... 54 11. The results of analysis of variance of sperm concentrations (Experiment 2) ................ 54 12. Weekly semen pools affording to each female equal concentrations of spermatozoa from each male .................................. ‘ 55 13. Sequence of progeny from White Plymouth Rock females inseminated with equal concentrations of spermatozoa collected from White Wyandotte, White Plymouth Rock, and New Hampshire males . . 57 14. Progeny of White Plymouth Rock females in- seminated with equal concentrations of sperma- tozoa collected from White Wyandotte, White Plymouth Rock, and New Hampshire males ....... 61 15. Evaluation of semen characteristics in three males: WW (5278), WL (1113), NH (1180) ....... 63 16. The results of analysis of variance of semen yields (Experiment 3) ...................... 65 17. The results of analysis of variance of sperm concentrations (Experiment 3) ............ 66 18. Sequence of progeny from New Hampshire and White Leghorn hens inseminated with equal concentrations of spermatozoa collected from White Wyandotte, White Leghorn, and New Hampshire males ..................... 67 vi Table 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. The progeny from New Hampshire and White Leghorn females inseminated with equal concentrations of spermatozoa col- lected from White Wyandotte, White Leg- horn, and New Hampshire males ........... Evaluation of semen characteristics in three males: WW (0099), BR (2000), WL (1113) ........................... The results of analysis of variance of semen yields (Experiment 4) .............. The results of analysis of variance of sperm concentrations (Experiment 4) ........ Sequence of progeny of New Hampshire females inseminated with equal concentra- tions of spermatozoa collected from White Wyandotte, White Leghorn, and Barred Plymouth Rock males ................... The progeny of New Hampshire females inseminated with equal concentrations of spermatozoa collected from White Wyan- dotte, Barred Plymouth Rock, and White Leghorn males ........................ Evaluation of semen characteristics ......... The progeny of New Hampshire females ...... The progeny of New Hampshire females ...... The progeny of New Hampshire females ...... The progeny of White Leghorn females ....... The progeny of White Rock females ....... i. . vii Page C 70 73 75 75 77 80 81 89 90 91 92 93 Table 31. 32. 33. The progeny of White Rock females ............ 94 The progeny of White Wyandotte females ........ 95 Sequence of progeny from females insem- inated with equal concentrations of sperma- tozoa from different males .................. viii INTRODUCTION One of the major economic losses in the field of poultry hus- bandry is the incidence of infertile eggs among eggs produced for hatching purposes. In the endeavor to reduce this loss, numerous investigations have been directed toward a better understanding of the mechanism of fertility and the many factors affecting it; yet many unexplored and imperfectly explored areas are still to be found. One such area providing rich opportunity for exploratory research is that of selective fertilization and sperm competition. In a preliminary survey of the available literature relating to competitive fertility of fowl spermatozoa, conflicting reports were found to exist. Dunn (1927) found that the spermatozoa from males more nearly related to the females fertilized a significantly higher percentage of eggs than did spermatozoa from nonrelated males. Curtis and Lambert (1929) mated White Plymouth Rock and Rhode Island Red males to White Plymouth Rock females and found no evi- dence of selective fertilization. Using Rhode Island Red females, Bonnier and Trulsson (1939) found that the semen from .a Rhode Island Red male was more effective in fertilizing ova than was the semen from a White Leghorn male. In experiments using mixed semen obtained from New Hampshire, Barred Plymouth Rock, and White Leghorn males injected into New Hampshire females, Parker, McKenzie, and Kempster (1942) observed that about equal numbers of chicks were sired by the Barred Plymouth Rock and New Hamp- shire males, but conspicuously fewer chicks were sired by the White Leghorn males. Bohren, Garrick, and Andrews (1945) placed semen collected from carotenoid-free males in competition with se— men obtained from normal males and found that the spermatozoa from the carotenoid-free males fertilized fewer ova than did the spermatozoa from males fed basal diets. Ferrand and Bohren (1948) mixed semen from high-carotenol White Plymouth Rock males with semen from high-carotenol Barred Plymouth Rock males and found that a significantly greater percentage of chicks were sired by the Barred Plymouth Rock males. In semen pools obtained from high- carotenol Barred Plymouth Rock males and high-carotenol New Hamp- shires, significantly fewer chicks were sired by the Barred Plymouth Rock males. These researchers ranked the males in order of their sperm-competitive ability as follows: New Hampshires, Barred Plymouth Rocks, and White Plymouth Rocks, and concluded that sperm competition can and does occur. The techniques employed in studies of competitive fertiliza- tion involved either the combining of equal volumes of semen col- lected from males of different breeds of chickens (Bonnier and Trulsson,'l939; Parker, McKenzie, and Kempster, 1942; Bohren, Carrick, and Andrews, 1945; Ferrand and Bohren, 1948), or using the method of alternation of males of different breeds of the domestic fowl (Dunn, 1927; Curtis and Lambert, 1929; Warren and Kilpatrick, 1929). On one hand, the data were interpreted to indicate the af— finity of spermatozoa from a male of a specific breed for ova pro- duced by females of the same breed, whereas other data were in- terpreted as suggesting no evidence for the theory of selective fer- tilization. That fertility depends upon no single factor, but rather upon a number of factors probably intercorrelated through devious ways, suggests a reason for the widely varying reports. It appears logical to assume that the characteristics of spermatozoa produced by a particular male are not similar to those of any other male. No doubt the difference between an infertile male and one that is fertile lies in the difference between their reproductive states, which are undoubtedly reflected in the characteristics of the spermatozoa produced by these males. While fertility is generally accepted as being influenced by inherent qualities in the Spermatozoa, some of which are measurable, it has been conclusively demonstrated that the concentration of sperm- atozoa is an important consideration. Munro (1938b) demonstrated that fertility was reduced to a highly significant degree when the number of spermatozoa inseminated into females was below one hundred million. Further, when the concentration of spermatozoa in the seminal fluid was less than one million, no fertile eggs were obtained. Confirmation of these data was presented by Parker, McKenzie, and Kempster (1942). In sperm-competition studies, if competing males are made to contribute equally to a concentration conducive to normal fertility, and comparative fertility is determined, much valuable information can be gained. In the investigation herein reported, 519323323 of spermatozoa were equalized—-not volumes--in view of the variations with respect to sperm concentrations known to occur among males. Furthermore, the characteristics of spermatozoa motility and morphology were measured and related to each male's respective fertility. There is full awareness that a number of uncontrolled factors were still operative in the experiments. Failure to measure them cannot invalidate the relationships which have been found, but certainly obscures many causal factors underlying the fertility phe- nomenon. This must be the case until more and exact methods of semen evaluation are available. REVIEW OF THE LITERATURE In sperm-competition studies it is an experimental necessity to provide for the mating of individuals having suitable genetic mark- ers. Therefore, a brief review of the genes known to affect plumage colors of the breeds used in this investigation is highly desirable. White Leghorn Bateson (1902) demonstrated that the white of White Leghorns was due to a hereditary factor which acted according to Mendelian principles, and functioned by inhibiting the production of melanic pigment. White Leghorns mated with Indian Games produced F1 chicks which had white or dingy white down ticked with black. The F2 progeny did not depart from the expected 3:1 ratio with respect to down color. These findings were later confirmed by Bateson and Punnett (1905, 1906), Hurst (1905), Davenport (1906), and Hadley (1914, 1915). Hadley (1913) suggested the symbol "I" for the dom- inant gene which inhibits color in White Leghorns. That the gene is incompletely dominant to red (gold) is suggested from crosses of White Leghorns. and New Hampshires or other gold breeds. The F1 progeny produced by individuals of this mating exhibit flecking, which demonstrates that in the heterozygous state the gene, 1, is incapable of complete suppression of color. Danforth (1933) reported that Gene 1 was dominant to black'. Hadley (1913, 1915) demonstrated that White Leghorns carry the sex-linked gene, B, for barring. This gene reduces melanic pigment in the shanks and plumage. From the results of many dif~ ferent experimental matings it is generally agreed that the White Leghorn is a barred fowl, but completely white in coloration when the factor I is in the homozygous state. The Single Comb White Leghorns used in this study were found to be of the following genotypes: males, IICCBB; females, IICCB -. White Wyando tte Most White Wyandottes are typical examples of recessive whites, lacking either Gene C (chromogen) or O (oxidase). Bate- son and Punnett (1906) demonstrated the complementary nature of these two genes where a 9:7 ratio (coloredzwhite) is obtained in the F2 progeny when the two genes are brought together. Platt (1921) and Robinson (1924) reported that the White Wyan- dotte was a mutation, or "sport," from the silver-laced variety. Certain strains of White Wyandottes have been known to carry sev- eral hidden characters, or cryptomeres. Crew (1933) found White Wyandottes which carried genes for barring and black. Quinn (1936) and Jeffrey (1947) also found certain White Wyandottes which carried barring. It is not uncommon to find White Wyandottes with the dominant inhibiting gene, I. Dryden (1916) and Lippincott (1921) reported the presence of Gene I in certain strains of White Wyandottes. (Punnett (1923) was of the opinion that White Leghorns may have been used at one time or another to improve the egg-laying qualities of White Wyandottes, thereby contributing the I gene which is occasionally found in this particular breed. Quinn (1936) tested four strains of White Wyandottes and confirmed these data. The genotypes of the White Wyandottes used in this study are as follows: males, iiccSSbbee; females, iiccS-b-ee. White Plymouth Rock Most strains of White Plymouth Rocks are recessive--white individuals carrying Gene c (lack of chromogen), and, therefore, any other color genes which they may carry cannot be expressed. As previously indicated, Gene C is complementary to Gene O in the production of color. Quinn (1936) reported that reciprocal crosses of White Plymouth Rocks and White Wyandottes produced white chicks, demonstrating that the White Plymouth Rocks were recessive (cc) for the color-producing gene, C. When White Plymouth Rocks are crossed with colored varieties, the F progeny are colored. If the plumage-color inheritance were 1 dominant, the F individuals produced by matings of White Plymouth 1 Rocks with colored breeds would be predominantly white (Jaap, 1943). However, there are some strains of White Plymouth Rocks today which carry the dominant white inhibitor, I (Lippincott, 1921; Jerome and Cavers, 1952). When dominant-white White Plymouth Rock fe- males were crossed with a New Hampshire male (iiCCeess), ap- proximately one-half of the chicks produced were heterozygous for dominant white, and one-half of the chicks lacked dominant white. Smokiness or gray in the down color of White Plymouth Rock chicks has not been explained. Jaap (1943) concluded that smokiness in the down occurred only when Gene E (extension of black) was carried by the chick. Jeffrey (1947) found that 10 per cent of the chicks produced by a strain of White Plymouth Rocks homozygous L 10 for silver carried red tints in the down. He therefore suggested that the red tints in the down of recessive-white chicks apparently had no relationship with the gene for gold. Jerome and Cavers (1952) could not substantiate Jeffrey's (1947) findings. These researchers mated nine females of the genotypes IicceeS- and two females of the geno- type iicceeS- with an iicceeSS genotype male. The percentage of smoky chicks obtained was 27.6. Dams of the genotype ii produced 61 per cent of smoky chicks. It was suggested that the presence of dominant white might not allow smokiness to be expressed. Analyses of two strains of White Plymouth Rocks for color genes by Jerome and Cavers (1952) revealed the following genotypes among the females: iiccees-B-, 62 per cent; iicceeS-B-, 29 per cent; iiccEes-B—, 6 per cent. In the study herein reported, the White Plymouth Rocks used were of the following genotypes: males, iiccEeSsBB; females, iicceeS-B and iiccEes -B -. Barred Plymouth Rock The Barred Plymouth Rock is the classical example demon- strating the effect of the sex-linked barring gene, B. This barring gene is also carried, but not expressed, in White Plymouth Rocks, White Leghorns, Delawares, and some strains of White Wyandottes. 11 Spillman (1908) was possibly the first to report that barring was caused by a sex-linked gene dominant to self-colored plumage. This observation was confirmed by Goodale (1909, 1910), Pearl and Surface (1910), and many other workers. The barring gene tends to reduce melanic pigment of the feathers in certain areas in a def- inite rhythm, thereby producing an alternate condition of white and black barring. A dosage effect--i.e., each gene has a graded effect, with the presence of two BB genes being more effective than the presence of only one B gene in reducing melanic pigment--has been demonstrated. Barred Plymouth Rocks also carry Gene S for silver (allelo- morphic to 5, gold) and the gene for extended black, E. Plymouth Rocks homozygous for the restrictor of black (ee) to the neck, wings, and tail are also found. The genotype of the Barred Plymouth Rock male used in this investigation was iiCCEESSBB. New Hampshire New Hampshires are classified among the red breeds of chick- ens, as are the Rhode Island Reds, Red Sussex, and Red Caps. How- ever, the only true breed of red chickens is the Red Leghorn. More genetic information is available concerning the Rhode Island Red than 12 the New Hampshire. Nevertheless, the genes affecting plumage color and patterns have been shown to be the same in both breeds. Hutt (1949) reported that red breeds and varieties of chickens have genes for both red and black, the latter color being restricted to the wings, neck, and tail. Hutt has pointed out that one evidence in favor of the presence of e in Rhode Island Reds is found in a cross popular in England, that of Light Sussex females x Rhode Island Red males. Typical Columbian males result from this mating, which indicates that both breeds carry ee. Earlier reports (Dunn, 1922) had indicated that the gene for extension of black (E) was absent in Rhode Island Reds and buff varieties, but present in Barred Ply- mouth Rocks and White Plymouth Rocks. New Hampshires are known to carry the gene for gold (5), which is the allele of the dominant sex-linked gene for silver (S). In the popular cross referred to previously, gold males (53) are mated to silver females (5-), allowing sex identification at hatching time. The males are genetically silver, appearing white or silvery gray, while the females are genetically gold, and show buff down color. The recessive allelomorphs of the respective genes for ex- tension of black (E), barring (B), and silver (S) are the only genes which need be considered here in accounting for plumage color and pattern in the New Hampshire breed of poultry. l 5 The genotypes of the New Hampshires used in this study are: males, iiCCeessbb; females, iiCCees-b-. Yield of Semen The yield of semen has been found to vary between different individuals of the same breed, between individuals of different breeds, and with the frequency of collections. The male fowl produces from 0.1 to 1.5 milliliters of semen per ejaculate (Lambert and McKenzie, 1940), the average volume reported to be approximately 0.6 milliliter. Parker, McKenzie, and Kempster (1942) used a semen collector, and obtained volumes ranging from 0.05 to 1.00 milliliter, with a mean value of 0.36 milliliter. Larger volumes of semen have been obtained with the technique of abdominal massage than with a semen collector. Seasonal variation in semen production has also been noted. In gen— eral, the volume of semen ejaculated appears to be rather closely correlated with the season of the year. Warren and Gish (1943) found little difference in yield of semen when intervals between stimulations varied from one-half hour to eight hours. These workers demonstrated that two stimu- lations could be made at an interval as short as thirty minutes, and as much semen could be obtained as that obtained after an interval ~5DIP‘UG m5: 4 “in“ 14 of several hours. Martinez (1947) determined semen volumes twice weekly for three males over a period of sixty-two days, and found that volumes of semen collected ranged from 0.20 to 0.51 milliliter per collection. Wilwerth (1948) found that, over a period of nineteen days, a group of males averaged 0.40 to 0.68 milliliter, the volume ranging from 0.22 to 0.85 milliliter. It is generally agreed that the yields of semen when collec- tions are made once per day are almost as high as those yields obtained when semen is collected twice per day. For maximum in- dividual yields it is recommended that semen be collected every other day. Sperm Concentration and Number of Spermatozoa It is not inconceivable that sperm concentration and total num- bers of spermatozoa will vary between males of the same or of dif— ferent breeds, with frequency of collection and many other factors. Penquite, Craft, and Thompson (1930) found that White Leghorn cock- erels produced a less-concentrated semen that contained a smaller percentage of live spermatozoa in the fall of their first year than they did the following spring. Sampson and Warren (1939) reported a tendency for sperm concentration to decrease with succeeding 15 ejaculates when males were stimulated to ejaculate at hourly intervals during the day. Fluctuations in density in different samples from the same male and among males in general were also observed. Parker, McKenzie, and Kempster (1942) determined sperm concentration in semen from seventy-two collections obtained from thirteen different males. The majority of the collections had concentrations of 2.00 to 3.99 million spermatozoa per cubic millimeter. There was a ten- dency for the concentration of spermatozoa to increase when the period of sexual rest prior to ejaculation was lengthened. Wilwerth (1948) determined sperm concentration in five young males over a period of fifty-four days. The highest average concentration of sperm- atozoa obtained was 2.2 million per cubic millimeter, with the total number being 1.732 million. The lowest concentration was 1.67 million per cubic millimeter, with the total number being 501 million. The minimum volume of semen and the number of spermatozoa required for fertilization have been suggested by a number of inves- tigators. Hutt (1929) found no correlation between sperm concentration and the male's ability to fertilize ova when the number of spermatozoa per cubic millimeter of semen was between 825,000 and 7,000,000. Burrows and Quinn (1938) reported that 0.1 cubic centimeter of un- diluted semen injected into females at weekly intervals resulted in 16 80 to 95 per cent fertility. Fertility, as previously stated (Munro, 1938b), was found to be adversely affected when the number of sperm- atozoa inseminated fell below one hundred million. Moreover, no fertile eggs were obtained when the number was below one million. Parker, McKenzie, and Kempster (1942) found that when the total number of spermatozoa per insemination exceeded one hundred mil- lion, the sperm concentration of the inseminating fluid appeared to have little influence on fertility. It is apparent from these reports that the emphasis is on the concentration of spermatozoa required for fertilization rather than on a stated minimum volume. This is readily understandable by reason of the fact that the sperm concen— tration in the seminal fluids varies from male to male. Morphology of Spermatozoa Avian spermatozoa differ from mammalian spermatozoa in having slender, cylindrical heads which are capped anteriorly with distinct, sharp structures called "acrosomes" (Bradley, 1915; Warren and Kilpatrick, 1929; and Parker, McKenzie, and Kempster, 1942). Fatty acid materials have been observed in the head of the avian spermatozoon. Adamstone and Card (1934) suggested that these globules might serve as a reserve food supply. 17 In any normal male, spermatozoa are continually being pro- duced in the testes and are moving down the tract. Thus, if they are not emitted they must undergo degeneration and resorption. If spermatozoan degeneration is a continual process, it is expected that a number of abnormal spermatozoa is always present in the seminal fluids. Milovanov (1936) felt that the appearance of patho- logical forms should be judged not only from the viewpoint of a reduction in the total number of spermatozoa capable of fertilization, but mainly as a symptom of the pathological condition of the genital apparatus of the male. Investigations concerning the relationship between the mor- phology of spermatozoa and fertility in the domestic fowl are some- what limited. Sampson and Warren (1939) demonstrated that the number of males producing spermatozoa having a relatively high incidence of obvious abnormalities was relatively small. One male which produced semen with a large number of defective spermatozoa was found to be sterile. Parker, McKenzie, and Kempster (1942) observed eleven types of abnormal spermatozoa in avian semen; namely, coiled tail, broken tail, tailless, coiled head, hooked head, ruptured head, small spermatozoa, blunt head, swollen head, balloon head, and filiform mid-piece. A negative correlation between abnormal O WM“. -v-a 18 spermatozoa and fertility was found to exist. Largely on the basis of the significant correlations found between spermatozoa morphology and fertility in mammals, more studies in chickens are warranted in order to determine the possible relationship between these vari— ables in avian species. Motility of Spermatozoa If a spermatozoon is to penetrate an ovum successfully, and effect fertilization, tremendous distances must be covered. Without active movement on the part of the spermatozoon, and particularly movement of a space-gaining type, it is unlikely that fertilization will occur. Initial motility is therefore regarded as important evi- dence of viability, although it is not necessarily a definite indication that the spermatozoon possesses normal fertilizing powers. On the other hand, there can be no doubt that the absence of motility is an indication that fertilizing capacity has been lost. Fresh semen of high fertilizing power, when examined micro- scopically, shows millions of spermatozoa in rapid and vigorous movement. Spermatozoa move by unilateral strokes of the tail in one plane, and rotate simultaneously around their longitudinal axis (Milovanov, 1934). This movement has been suggested as being due 'Jh' C!" "'h ‘. rm‘h 19 to the asymmetrical structure of the head, which ensures rectilinear progressive movement. In addition to this progressive movement, Walton (1933) described a rotary motion, whereby the spermatozoa move in circles, the diameter of which are about equal to their own length. A third kind of motility, an oscillatory or pendular motion that is not propulsive in nature, was also described. Munro (1938a) found that motility of chicken spermatozoa was lost at body temperature when suspended in physiological saline, Ringer's, Baker's, or Milanov's solution, and in fluids from the infundibulum and magnum. At lower temperatures, however, a high degree of motility was observed. Parker gt__a_1_. (1942.) found a sig- nificant relationship between the motility of seminal fluids and fer- tility. In estimating motility, investigators have found it expedient to use some arbitrary scale for denoting the type of movement and the proportion of spermatozoa possessing that movement. Obviously, the method is highly subjective, but it has been found satisfactory, especially for routine work. MA TERIALS AND ME THODS The experimental animals used in this study consisted of year- ling males of the New Hampshire, White Plymouth Rock, White Wyan- dotte, Single Comb White Leghorn, and Barred Plymouth Rock breeds of poultry. The maternal parents were yearling females of the same breeds, excepting the Barred Plymouth Rock. These birds belonged to the Department of Poultry Husbandry at Michigan State College. Throughout the experimental period, the females were confined in individual cages in laying batteries equipped with raised wire bot- toms. The males were also maintained in individual cages in the same building. The ration fed consisted of a specially formulated battery breeding mash known to contain all essential nutrients. All individuals were fed ad libitum, and a supply of fresh running water was available at all times. Two important preliminaryaspects of this investigation con- sisted of determining the genotypes of all birds to be used in this Study and testing the fertility of the males and females involved. It was necessary to determine the essential color and/or comb geno- tYPBS of all sires and dams to facilitate the accurate identification 20 21 of the sires of the chicks when the semen from different breeds of males was pooled for the sperm-competitive study. The two pre- liminary studies were made simultaneously. "All matings were ac- complished by means of artificial insemination as described by Bur- rows and Quinn (1937), with minor modifications. The following genetic considerations were met by test matings: l. Homozygosity of the dominant inhibitor gene, I, in all White Leghorns. 2.. The absence of the I gene in the White Plymouth Rocks and White Wyandottes. 3. The presence of the complementary color-producing genes (C-chromogen, O-oxidase) in all White Leghorns. 4. The presence of the genes for barring (BB in the Males and B- in the females) in all White Leghorns. 5. The absence of one of the complementary color-producing genes (C or O) in White Plymouth Rocks and all White Wyandottes. 6. Homozygosity of the gene for rose comb, R, in all White Wyandottes. All birds used in these experiments had their respective genotypes for plumage color and/or comb types assigned from data obtained on a minimum of eight chicks. The experiments were 22. designed, therefore, to facilitate the accurate identification of the chicks. This was of fundamental importance because the superiority of a male's spermatozoa is determined only on the basis of signifi- cantly larger numbers of offspring sired by that particular male. Preliminary studies on fertility revealed that all males in- volved were fertile, ranging in fertility from 92 to 99 per cent. The differences in fertility were not significant. It was noted, how- ever, that the White Wyandotte males were nearer the lower limit of the fertility range. At the culmination of some of the experiments involving pooled semen, it was observed that certain males sired significantly fewer chicks. To eliminate any possibility that the particular males involved were sterile, further checks on their fer—- tility we re made. One additional pilot experiment was performed to determine the possible day-to-day variations in semen volumes and in spermatozoa concentrations of the males under study. Thus, if no significant dif- ferences could be established between successive ejaculates of a given male, then indices of sperm concentration and other measure- ments of semen could be determined. These indices were to be used in analyses of sperm-competition data. However, it was found that wide differences existed in successive ejaculates of these males. 23 Therefore, no reasonable index sufficiently devoid of errors could be devised. The question of seasonal variation and other environ- mental factors was also considered. Accordingly, it was decided to measure semen characteristics on the actual day of insemination. A total of eleven experiments were conducted in the sperm- competition phase of this study. Nine of these experiments (Experi- ments 1 to 9, inclusive) involved the pooled semen of males of three different breeds of chickens. Experiments 10 and 11 involved the use of pooled semen collected from males representing two different breeds. The classification of matings (WW = White Wyandotte, WL White Leghorn, WR = White Rock, NH = New Hampshire, and BR Barred Rock) by experiments is as follows: Experiment 1 Experiment 2. Meg: .5393;le Males. 5333312: NH(0040) NH(0040) NH WW(0099) X WW(OO99) X WR WL WL(9000) WR(7491) Experiment 3 Males Femalei ———.———-—— NH(1180) WL WW(5278) x NH WL(1113) Experiment 5 M2192 $212222 BR(2000) WW(SZ78) x NH WL(1113) Expe rimen t 7 £21.12: Females WW(5283) WL(1113) x NH NH(1180) Experiment 9 Males Females WW(5283) WR(1151) X WR NH(1180) Z4 £32598 rimsrltjl Make Essflles BR(ZOOO) WW(0099) X NH WL(1113) Expe riment 6 Mags Esmales WW(5283) WL(lll3) x NH BR(ZOOO) Expe_riment 8 Males Eemales WW(5283) WL(1113) X WL NH(1180) Expe riment 10 Males Eema‘l‘es WW(5283) X WR NH(1180) 25 Expe riment ll Males. Eagles ww:<* Highly significant. Table 11. The results of analysis of variance of sperm concentra— tions (Experiment 2). Source of Variation d/f SS MS F Total ..................... 29 5.06 Between males ............... 2 0.30 0.15 Within males ................ 27 4.76 0.176 0.852 The volumes of semen from the males making up the weekly pools are shown in Table 12. The indicated volumes contained equal numbers of spermatozoa. The White Wyandotte male contributed the largest volumes to the pooled semen (mean of 0.477 cc.), followed by the White Plymouth Rock male, with a mean semen volume of 0.440 cubic centimeters. The New Hampshire male contributed the smallest volumes of semen (mean of 0.429 cc.). Table 12 . 55 Weekly semen pools affording to each female equal con- centrations of spermatozoa from each male. Vol. Cipgtcen- Volumes per Male Insem- 10“ No. Tom inated 0f f W O k P 1 s Wee WW WR NH ((2:0) per p:rm Fe- (0099) (7491) (0040) ° Female p 1' males (cc) Female ° (millions) 1 0.479 .548 .296 1.32 0.10 182 10 2 0.449 .297 .354 1.10 0.11 240 10 3 0.485 .369 .303 1.16 0.08 166 10 4 0.567 .533 .479 1.58 0.10 152 9 5 0.559 .442 .442 1.44 0.10 167 9 6 0.548 .377 .491 1.42 0.10 169 9 7 0.417 .494 .588 1.50 0.10 160 9 8 0.479 .457 .485 1.42 0.10 169 9 9 0.442 .597 .435 1.47 0.10 163 9 10 0.343 .290 .417 1.05 0.10 229 9 Mean 0.477 .440 .429 1.35 0.10 180 56 The progeny from each dam classified by sires are found in Table 13. Although evidence of periodic dominance by a male is not as marked as in Experiment 1, it is not altogether absent. The total numbers of chicks sired by each male are summarized in Table 14. Again, the total number of chicks sired by the White Wyandotte male was extremely small when compared with the total number of chicks sired by the New Hampshire male (approximately one White Wyandotte chick to every seven New Hampshire chicks). The White Plymouth Rock male sired a total of approximately 5%: times as many chicks as the White Wyandotte male. From these results it is suggested that there is no evidence for selectivity of White Plymouth Rock spermatozoa for White Plymouth Rock ova. Conversely, the inter- pretation might be made that selective fertilization is slightly in favor of the New Hampshire spermatozoa. These findings, together with those of Experiment 1, demonstrate the decided inferiority of the White Wyandotte spermatozoa. That the fertility of the White Wyandotte male may have be- come impaired in the course of these experiments could not be ig- nored (original fertility index was 92.3 per cent). Therefore, the White Wyandotte male was tested for fertility by mating him to White Plymouth Rock females. The fertility index was found to be 57 Table 13. Sequence of progeny from White Plymouth Rock females inseminated with equal concentrations of spermatozoa col— lected from White Wyandotte, White Plymouth Rock, and New Hampshire Males (first insemination, December 10, 1952; last insemination, February 19, 1953). WR Date _ Fe— males Dec . 11 12 13 14 15 16 17 18 19 20 21 9816 NH 1 9817 WW 9818 I NH NH 9819 I I WR WR 9820 WW NH 9821 9822 WW 4367 4376 WR 4378 I WR NH WR Dec. Jan. 22 23 24 25 26 27 28 29 30 31 1 9816 I WR NH 9817 NH 1 NH 9818 I WR NH NH WR NH 9819 WR NH NH WR NH NH NH 9820 NH NH 1 I 9821 WR WR NH WR 9822 WR WR NH WR WR NH NH 4367 4376 4378 WR WR NH WR WR NH Table 13 (Continued) 58 Date WR Fe- Jan. males 2 3 4 5 6 7 8 9 10 11 12 9816 I 9817 I 9818 I I I 1 WR WR 9819 I I I I I NH 9820 WR WW I I I WR 9821 9822 WW I I I NH NH 4367 NH NH 4376 I 1 WR WR NH 4378 WR NH WR 1 I NH NH NH NH Jan. 13 14 15 16 17 18 19 20 21 22 23 9816 9817 I 9818 I I WR 9819 NH NH NH WW 1 NH 9820 WR 9821 9822 WR NH I I 4367 WR WR I WR 1 I 4376 4378 WR WR WR NH NH NH I 59 Table 13 (Continued) WR K Date Fe- Jan. Feb. males 24 25 26 27 28 29 30 31 1 2 3 9816 9817 I I 1 NH NH 1 1 9818 WR WR WR NH NH NH 1 9819 NH WR WR I I WW NH NH 9820 WR 1 1 NH 1 9821 9822 NH I 1 WR 1 4367 NH NH WR NH NH 4376 NH WW 4378 NH WR WR NH I NH NH WW Feb. 4 5 6 7 8 9 10 11 12 13 14 9816 9817 1 WR NH I 1 1 NH 9818 I D2 1 I 1 WR 9819 NH NH NH NH NH 9820 1 I I D1 WR 9821 9822 WW NH 1 NH 1 4367 NH NH NH NH WR WR I NH 4376 NH NH NH NH NH 4378 NH 1 NH 1 D2 1 60 Table 13 (Continued) Date WR fl # Fe- Feb. males 15 16 17 18 19 20 21 22 23 24 25 9816 9817 WR NH WR NH WR WR WR 1 1 9818 WR 1 WR WR 1 1 1 1 9819 I 9820 1 1 1 WR WR 9821 9822 WR 1 WR 1 I 4367 WR WR . 1 NH NH NH 1 4376 1 WW 4378 WW 1 WW 1 WR NH NH 1 Feb. Mar. 26 27 28 1 2 3 4 5 6 7 8 9816 9817 WR WR WR 1 I I 1 I 9818 1 1 1 1 1 1 1 9819 i i i 1 1 9820 1 1 I 1 1 1 1 9821 1 9822 WR 1 1 1 1 4367 WR 4376 4378 1 1 dead New I = infertile; D1 = dead embryo during first week; D2 embroy during second week; WR = White Plymouth Rock; NH Hampshire; WW = White Wyandotte. Table 14. Progeny of White Plymouth Rock females inseminated 61 with equal concentrations of spermatozoa collected from White Wyandotte, White Plymouth Rock, and New Hamp- shire males. WR g“ Males A Females NH (0040) WR (7491) WW (0099) 9816 Z 1 ' 9817 8 9 1 9818 18 12 ' 9819 18 6 Z 9820 4 7 Z 9821 1 3 ' 9822 9 9 3 4367 14 8 ' 4376 7 3 Z 4378 19 14 3 Total 90 72' 13 62 87.9 per cent. Although this fertility figure was slightly lower than the value obtained four months earlier, the question of sterility was ruled out. Experiment 3 The results obtained in Experiments 1 and 2 suggested the necessity of studying the semen from different males of the same breeds under similar competitive conditions. In addition, the esti- mated numbers of spermatozoa inseminated were not to vary from week to week, but, rather, an estimated 180 million spermatozoa (60 million from each of three males) were to be injected weekly into each female. Fertility for the new males was as follows: 95.6 per cent for the White Wyandotte male (5278), 99.0 per cent for the White Leghorn male (1113), and 96.4 per cent for the New Hampshire male (1180). The differences in fertility, once fertility is above 90 per cent, suggest no significant disparity among the males. Data pertaining to the three males are presented in Table 15. The mean semen yield of the White Wyandotte male was 1.16 cubic centimeters, with a range in volume of 0.60 to 1.62 cubic centimeters; the mean semen yield of the White Leghorn male was 63 Table 15. Evaluation of semen characteristics in three males: WW (5278), WL (1113), NH (1180). V01. (1:22:2' Total Pc t. v01. of , , N0. M0- Abnor- for Week (millions . , Semen er cu Sperms tility mal Pool. (cc.) P ' (billions) Sperms (cc.) mm.) WW (5278) 1 0.60 3.18 1.9080 4 20.5 0.453 2 1.62 1.56 2.5272 4 10.5 0.923 3 1.10 1.98 2.1780 3 11.0 0.515 4 1.30 1.81 2.3530 3 7.0 0.530 5 1.20 1.71 2.1240 2 3.0 0.407 Means 1.16 2.06 2.2180 3.2 10.4 0.566 WL (1113; 1 0.73 2.45 1.7885 5 15.5 0.589 2 0.86 2.20 1.8920 5 15.0 0.654 3 1.28 2.18 2.7904 4 16.5 0.468 4 1.32 2.60 3.4320 3 18.0 0.369 5 0.56 2.48 1.3888 4 7.0 0.290 Means 0.95 2.38 2.2583 4.2 14.4 0.474 NH (1mg 1 0.62 3.12 1.9344 5 7.5 0.461 2 1.40 1.88 2.6320 5 6.5 0.766 3 0.42 2.94 1.2348 4 4.5 0.347 4 0.56 2.92 1.6352 4 5.0 0.329 5 1.42 2.74 3.8908 5 10.0 0.263 Means 0.88 2.72 2.2654 4.6 6.7 0.433 64 Table 15 (Continue d) Concen- . Vol. No. tration Total Insem- Week of per , Pool inated Hens Female ( ) (millions) CC. 1 24 1.50 180 0.063 2 24 2.34 180 0.098 3 17 1.33 180 0.078 4 16 1.23 180 0.077 5 12 0.96 180 0.080 Means 19 1.47 180 0.079 0-95 cubic centimeter, with a range in volume of 0.56 to 1.32 cubic centimeters; and the New Hampshire male had a mean semen volume of 0.88 cubic centimeter, with a range in volume of 0.42 to 1.42 cubic centimeters. The differences between these mean volumes were not significant (Table 16). The density of spermatozoa in the White Wyandotte male's semen ranged from 1.56 to 3.18 million per cubic millimeter, with a mean concentration of 2.06 million per cubic millimeter. For the White Leghorn male, the range in density of spermatozoa was 2.18 to 2.60 million per cubic millimeter, with a mean concentration of 2.38 million spermatozoa per cubic millimeter. For the New Hamp- shire male the range in density of spermatozoa was 1.88 to 3.12 65 Table 16. The results of analysis of variance of semen yields (Ex- periment 3). Source of Variation d/f SS MS F Total ...................... 14 2.16 Between males ................ 2 0.21 0.105 Within males ................. 12 1.95 0.1625 0.646 million per cubic millimeter, with a mean of 2.72 million sperma— tozoa per cubic millimeter. The differences between these mean concentrations of spermatozoa were not statistically significant (Table 17). The lowest spermatozoa motility rating observed was in the semen of the White Wyandotte male (mean of 3.2); the highest sperm- atozoa motility rating was observed in the semen of the New Hamp- shire male (mean of 4.6). The semen of the White Leghorn male had a mean motility rating of 4.2. A t test of significance applied to motility ratings showed that the motility rating of the spermatozoa collected from the White Wyandotte male did not differ significantly from the spermatozoa motility rating of the White Leghorn male (t = 2.08). However, a significant difference in motility rating was found between the spermatozoa of the White Wyandotte male and the 66 Table 17. The results of analysis of variance of sperm con- centrations (Experiment 3). Source of Variation d/f SS MS F Total ...................... 14 3.84 Between males ................ 2 1.01 0.505 Within males ................ 12 2.83 0.236 2.14 New Hampshire male (t = 345*). There were no statistically sig- nificant differences between the New Hampshire male and the White Leghorn male in the motility ratings of their semen (t = 0.987). The percentages of abnormal spermatozoa observed in the semen of the different males were as follows: White Leghorn male, 14.4; White Wyandotte male, 10.4; New Hampshire male, 6.7. The volumes of semen containing equal numbers of sperma- tozoa from each male are shown in Table 15. The White Wyandotte male contributed the largest volumes to the pooled semen (mean of 0.566 cc.). The mean volumes of semen from the White Leghorn male and the New Hampshire male were 0.474 and 0.433 cubic centi- meter, respectively. The resulting progeny obtained after five inseminations are pre- sented on a day-to-day basis in Table 18 and summarized by dams in Table 19. 67 Table 18. Sequence of progeny from New Hampshire and White Leg- horn hens inseminated with equal concentrations of sperm- atozoa collected from White Wyandotte, White Leghorn, and New Hampshire males (first insemination, March 12, 1953; last insemination, April 9, 1953). NH Date Fe- Mar. males 15 16 17 18 19 20 21 22 23 24 1021 9824 NH NH NH NH NH WL NH NH 9825 WL WL NH WL NH I WL I 726 NH NH I 100 3 NH WL WW WL NH WL 1007 102 1 WL NH Mar. Apr. 26 27 28 29 30 31 1 2 3 4 9824 WL NH WL NH WL NH NH NH 9825 NH NH NH WL I I 726 WL 100 3 WL WL WL WL NH WL 1007 NH WW WL I 102 1 Apr. 6 7 8 9 10 11 12 13 14 15 9824 WL WW WW I NH I WL WL 9825 WL NH WL WL WL 726 WL I NH NH NH WL 100 3 WL NH NH WL NH 1 1007 Table 18 (Continued) 68 NH % * Date Fai- Apr. m es 16 17 18 19 20 21 22 23 24 25 26 9824 1 NH NH NH 1 WL NH 1 9825 W1. 1 1 WL 726 WL WL 1 NH NH NH WL WL WL 1003 NH WL W1. NH WW 1007 WL WL 1 I NH WL 1021 WL WL NH NH Apr. 27 28 29 30 9824 WL NH 1 I 9825 WL 726 NH 1 1 1003 NH 1007 WW 1021 WL g Date p 1281-5 Mar. m e 14 15 16 17 18 19 20 21 22 23 24 903 W1. 954 NH NH WL WW WL NH WL 958 WL NH 1 .WL NH 973 I WL WW 1 WW NH NH WL Table 18 (Continued) 69 973 Dt WL r—fi 3.8 W Fael-s Mar. Apr. m e 25 26 27 28 29 30 31 1 2 3 4 903 954 WW NH 958 973 NH NH WL NH NH NH Apr. 5 6 7 8 9 10 .11 12 13 14 15 903 D1 954 958 973 W1. NH Apr. 16 17 18 19 20 21 22 23 24 25 26 903 WL NH NH WW 954 WL NH 958 973 I Apr. 27 28 29 30 903 WL 1 1 954 WL 1 I 958 Table 19. The progeny from New Hampshire and White Leghorn females inseminated with equal concentrations of sperm- atozoa collected from White Wyandotte, White Leghorn, and New Hampshire males. NH Males Females ww (5278) WL (1113) NH (1180) 9824 Z 9 18 25 12 6 726 8 9 1003 2 13 9 1007 Z 4 2 1021 3 3 Total 9 48 47 VVL. Females 903 l 3 2 954 5 5 89555 2 4 958 2 2 973 2 4 8 Total 5 16 21 Grand Total 1 1 64 68 71 The results show no evidence of selective affinity of New Hampshire or White Leghorn spermatozoa for the ova of their re- spective breeds. The paucity of White Wyandotte chicks is again observed, but the numbers of New Hampshire and White Leghorn Chicks are approximately equal (Table 19). The motility ratings and percentages of abnormal spermatozoa in this experiment (Ex- periment 3) and in Experiments 1 and 2 are further links in the chain of evidence showing the influence of motility and abnormal spermatozoa on competitive fertilization. Experiment 4 The White Wyandotte male used in Experiments 1 and 2 was further tested in another competitive fertilization study in which the semen from a Barred Plymouth Rock male was substituted for the semen of the New Hampshire male. No breed selectivity, in the sense of like for like, was to be demonstrated here, but it was con- sidered desirable to observe the behavior of spermatozoa from this White Wyandotte male under the competitive conditions afforded by the use of semen from the Barred Plymouth Rock male. The Barred Plymouth Rock male had a fertility index of 99.8 per cent. 72 The data for the males used in this experiment are shown in Table 20. The White Wyandotte male (0099) produced the largest volumes of semen (mean volume of 1.35 cc., range of 1.20 to 1.60 cc.). The mean volume of semen from the White Leghorn male was 0.79 cubic centimeter, with a range of 0.44 to 1.32 cubic centimeters. The Barred Plymouth Rock male produced a mean volume of 0.66 cubic centimeter, with a range of 0.39 to 1.38 cubic centimeters. An analysis of variance revealed highly significant differences be- tween these values (Table 21). The highest spermatozoa density was found in the semen of the Barred Plymouth Rock male (2000), a mean density of 2.89 mil- lion spermatozoa per cubic millimeter, with a range of 2.09 to 3.83 million. The mean density of spermatozoa in the semen of the White Leghorn male was 2.67 million per cubic millimeter, with a range of 2.20 to 3.00 million. The White Wyandotte male had a mean den- sity of 2.05 million spermatozoa per cubic millimeter, with a range of 1.82 to 2.39 million. An analysis of variance revealed no signifi- cant differences between these males in the concentrations of sperm- atozoa in their semen (Table 22). In tests for significant differences between motility ratings of spermatozoa in semen obtained from these males, it was found 73 Table 20. Evaluation of semen characteristics in three males: WW (0099), BR (2000), WL (1113). Concen- Vol. , Total Pct. Vol. tration of . , N0. M0- Abnor- for Week (millions , , Semen er cu Sperms tility mal Pool (cc.) p ' (billions) Sperms (cc.) mm.) WW (0099) 1 1.60 2.33 3.7280 3 15.5 0.180 2 1.38 1.88 2.5944 3 15.0 0.223 3 1.20 1.93 2.3160 2 19.0 0.218 4 1.30 1.98 2.5740 3 18.0 0.212 5 1.30 2.39 3.1070 2 17.0 0.176 6 1.29 1.82 ' 2.3478 3 11.5 0.231 Means 1.35 2.05 2.7779 2.7 16.0 0.207 BR (2000) 1 0.60 2.09 1.2540 4 7.5 0.201 2 1.38 3.83 5.2854 5 8.0 0.110 3 0.42 2.65 1.1130 5 9.5 0.158 4 0.52 3.02 1.0504 4 15.0 0.139 5 0.64 3.17 2.0288 3 7.5 0.132 6 0.39 2.59 1.0101 5 10.5 0.162 Means 0.66 2.89 1.9569 4.3 9.7 0.150 WL (1113) 1 1.01 2.80 2.8280 5 15.0 0.150 2 0.44 3.00 1.3200 4 14.5 0.140 3 0.40 2.97 1.1880 3 13.5 0.141 4 0.73 2.45 1.7885 5 15.5 0.171 5 0.86 2.20 1.8920 5 15.0 0.191 6 1.32 2.60 3.4320 3 18.0 0.162 Means 0.79 2.67 2.0747 4.2 15.2 0.159 Table 20 (Continue (1) 74 C .. oncen Vol. No tration Total Insem- Week of per , Pool inated Hens Female ( ) (millions) CC' 1 7 0.531 180 0.076 2 7 0.473 180 0.068 3 7 0.517 180 0.074 4 7 0.522 180 0.075 5 7 0.499 180 0.071 6 7 0.555 180 0.079 0.516 0.074 Me ans 75 Table 21. The results of analysis of variance of semen yields (Ex- periment 4). Source of Variation d/f SS MS F Total ..................... 17 2.97 Between males ............... 2 1.59 0.795 Within males ................ 15 1.38 0.092 8.64** ** Highly signific ant. Table 22. The results of analysis of variance of sperm con- centrations (Experiment 4). ) Source of Variation d/f SS MS F Total ....................... 17 10.2 Between males ................. 2 2.39 1.195 Within males .................. 15 7.81 0.521 2.29 that the mean motility of 2.7 for the White Wyandotte male differed significantly from that of the Barred Plymouth Rock male (mean of 4.3; t = 4.57**), and significantly from the motility rating of the spermatozoa in semen collected from the White Leghorn male (mean of 4.2; t = 3370*). The difference of 0.01 in mean motility rating of spermatozoa in semen from the White Plymouth Rock male and White Leghorn male was not significant (t = 0.213). 76 The percentage of abnormal spermatozoa found in the semen of the Barred Plymouth Rock male (mean of 9.7) was markedly lower than the mean percentage of abnormal spermatozoa found in the semen of the White Wyandotte male (16.0 per cent), and also lower than that found in the semen of the White Leghorn male (15.2 per cent). Based on volumes containing equal numbers of spermatozoa, the White Wyandotte male contributed the largest quantity (mean vol- ume of 0.205 cc.). The White Leghorn male contributed a mean se- men volume of 0.159 cubic centimeter, and the Barred Plymouth Rock male contributed a mean semen volume of 0.150 cubic centimeter. The daily distribution of progeny during six weekly insemina- tions are shown in Table 23 and the data are summarized in Table 24. The results show an advantage of the White Leghorn spermatozoa over the spermatozoa from the Barred Plymouth Rock male and the White Wyandotte male in competing for the fertilization of New Hamp- shire ova. Spermatozoa from the White Wyandotte male fertilized only 18 per cent of the total number of ova, thus maintaining their inferiority under competitive conditions. In all the competitive -fertilization experiments reported herein, it was demonstrated that the spermatozoa of the particular White 77 Table 23. Sequence of progeny of New Hampshire females insem- inated with equal concentrations of spermatozoa collected from White Wyandotte, White Leghorn, and Barred Ply- mouth Rock males (first insemination, February 19, 1953; last insemination, April 2, 1953). NH Date ;:;8 Feb. Mar. 20 21 5:22 23 24 25 26 27 28 1 2 563 I BR WL 564 I I I 571 BR I BR BR I 573 BR BR BR D2 BR BR BR 577 I 578 I I I I I WL 579 BR WW BR BR BR WW Mar 3 4 5 6 7 8 9 10 11 12 13 563 WL WW 564 I I I I I WW WL WL 571 I WL I WL I I I I 573 BR WL BR 1 WL I 577 WL D2 I WL I D1 I WW 578 BR WW WW WL I I I I I 579 WL I I I I D2 3441!... ‘l 94'. )3". f «.4 Eel... 1812.! p. ‘ Table 23 (Continued) 78 NH Date Fe- Mar. males 14 15 16 17 18 19 20 21 22 23 24 563 W1. X WL WL WW 1 564 WL BR 1 WL 571 WL WL BR 1 573 I BR WL WL WL 577 BR WL WL WL WL WL 1 BR 578 WL 1 1 1 WL D1 . W1. I 579 WW BR WW WW WL 1 Mar. Apr. 25 26 27 28 29 30 31 1 2 3 4 563 WL W1. 1 564 W1. WW I I I I 1 1 BR 571 WL BR 57 3 BR 1 WL 1 1 I 577 W1. WL WL WL WW I WW BR 578 I WW WL WW 1 1 I D1 579 1 Apr. 5 6 7 8 9 1o 11 12 13 14 15 563 BR 564 D2 571 BR BR 1 x 573 BR WL WL WL I BR W1. WW 577 BR D2 BR 578 D1 WL D1 BR 1 1 WL 579 D2 D2 Table 23 (Continued) 79 579 NH _ Date F:l-s Apr. m e 16 17 18 19 20 21 22 23 24 25 26 563 564 1 I 1 571 D2 D2 1 I 573 WW D2 1 1 577 I 578 1 I 1 I 80 Table 24. The progeny of New Hampshire females inseminated with equal concentrations of spermatozoa collected from White Wyandotte, Barred Plymouth Rock, and White Leghorn males. NH # Males Females WW (0099) BR (2000) WL (1113) 563 2 2 7 564 2 2 5 571 7 5 573 2 12 10 577 3 5 11 578 4 2 8 579 5 5 2 Total 18 35 48 Wyandotte male (0099) were decidedly inferior to the spermatozoa of all competing males. Therefore, another series of experiments was designed to study the behavior of the spermatozoa obtained from two different White Wyandotte males. Semen evaluations for the males used in these experiments (Experiments 5 to 11, inclusive) are sum- marized in Table 25. 81 Table 25. Evaluation of semen characteristics. Concen- Vol. trati n Total Pct. Vol. of , ,0 N0. M0- Abnor— for Week (millions . , Semen er cu Sperms tility mal Pool (cc.) pmm ) ° (billions) Sperms (cc.) Experiment 5 - WW (5278) 1 0.82 2.27 1.8614 4.0 10.5 0.185 2 1.22 1.38 1.6836 2.0 10.5 0.304 3 1.41 2.08 2.9328 3.0 19.5 0.202 Means 1.15 1.91 2.1593 3.0 13.5 0.230 Experiment 5 - WL (1113) l 0.44 2.76 1.2144 5.0 10.0 0.152 2 0.84 2.50 2.1000 5.0 7.5 0.168 3 0.83 2.30 1.9090 4.0 8.0 0.183 Means 0.70 2.52 1.7411 4.6 8.5 0.168 Experiment 5 - BR (2000) 1 0.72 1.98 1.4256 5.0 11.5 0.212 2 0.65 1.98 1.2870 4.0 10.0 0.212 3 0.76 1.99 1.5124 5.0 4.0 0.211 Means 0.71 1.98 1.4083 4.6 8.5 0.212 Concen- No. Total tration 1:52; Week of Pool per , - inated Hens (cc.) Female (cc ) (millions) ' Experiment 5 1 7 0.549 180 0.078 2 7 0.684 180 0.098 3 7 0.596 180 0.085 Means 7 0.610 180 0.087 Table 2 5 (Continued) 82 Concen- Vol. tration Total Pct. Vol. of . . N0. M0- Abnor- for Week (millions , , Semen er cu Sperms tility mal Pool (cc.) p ° (billions) Sperms (cc.) mm.) Experiment 6 - WW (5283) 1 1.01 2.70 2.7270 4.0 15.5 0.155 2 1.10 2.58 2.8380 4.0 16.5 0.163 Means 1.06 2.64 2.7825 4.0 16.0 0.159 Emeriment 6 - WL (1113) 1 0.86 2.18 1.8748 4.0 7.5 0.193 2 0.84 2.38 1.9992 4.0 13.5 0.176 Means 0.85 2.28 1.9370 4.0 10.5 0.185 Experiment 6 - BR L2000) 1 0.79 2.10 1.6590 4.0 10.0 0.200 2 0.76 1.97 1.4972 5.0 9.0 0.213 Means 0.78 2.04 1.5781 4.5 9.5 0.207 Concen- Vol. No. Total tration Insem Week of P001 per inated- Hens (cc.) Female (c ) (millions) C. Emeriment 6 7 0.548 180 0.078 7 0.552 180 0.079 Means 7 0.550 180 0.079 Table 2 5 (Continue d) 83 Vol. 2:23:21- Total Pct. Vol. of . , N0. M0- Abnor- for Week (millions , , Semen Sperms tility mal P001 per cu. . . \ (cc.) mm ) (billions) Sperms (cc.) Experiment 7 - WWJ5283) 1 0.79 3.12 2.4648 4.0 10.5 0.192 2 0.72 2.80 2.0160 3.0 16.5 0.214 Means 0.76 2.96 2.2404 3.5 13.5 0.203 Experiment 7 - WL (1113) 1 0.84 2.50 2.1000 5.0 7.5 0.240 2 0.83 2.30 1.9090 4.0 8.0 0.261 Means 0.84 2.40 2.0045 4.5 7.8 0.251 Experiment 7 —NH (1180) 1 0.86 1.85 1.5910 5.0 11.0 0.324 2 1.22 2.01 2.4522 4.0 3.5 0.298 Means 1.04 1.93 2.0216 4.5 7.2 0.311 Concen- No. . Total tration 1:20:16 Week of P001 per 'n’iied- Hens (cc.) Female 1( ( ) (millions) CC. Eigeriment 7 1 10 0.756 180 0.076 2 10 0.773 180 0.077 Means 10 0.764 180 0.076 Table 25 (Continued) 84 V01. (1:226:15 Total Pct. vol. of . .0 No. Mo- Abnor- for Week (millions . . Semen er cu Sperms tility mal Pool (cc.) 13 ' (billions) Sperms (cc.) mm.) Experiment 8 - WW (5283) 1 1.01 2.70 2.7270 4.0 15.5 0.133 2 1.10 2.58 2.8380 4.0 16.5 0.140 Means 1.06 2.64 2.7825 4.0 16.0 0.137 Experiment 8 - WL (1113) 1 0.86 2.18 1.8748 4.0 7.5 0.165 2 0.84 2.38 1.9992 4.0 13.5 0.151 Means 0.85 2.28 1.9370 4.0 10.5 0.158 Experiment 8 - NH Q1801 1 1.10 2.22 2.4420 5.0 13.0 0.162 2 1.19 2.31 2.7489 5.0 14.0 0.156 Means 1.15 2.26 2.5955 5.0 13.5 0.159 C .. oncen Vol. No. Total tration I Week of P001 per 32:15:22- Hens (cc.) Female . . (cc.) (millions) Experiment 8 1 6 0.460 180 0.077 2 6 0.447 180 0.075 Means 6 0.454 180 0.076 flu.” . 0 4!. - ul). ,0 Table 2 5 (Continue (1) 85 V01. 2:226:11- Total Pct. Vol. of . .0 N0. M0- Abnor- for Week (millions , , Semen er cu Sperms tility mal Pool (cc.) p ' (billions) Sperms (cc.) mm.) Experiment 9 - WW @283) 1 1.01 2.70 2.7270 4.0 15.5 0.155 2 1.10 2.58 2.8380 4.0 16.5 0.163 Means 1.05 2.64 2.7825 4.0 16.0 0.159 Experiment 9 — WR (1151) l 0.42 2.00 0.8400 5.0 15.5 0.210 2 0.66 1.73 1.1418 5.0 11.5 0.243 Means 0.54 1.86 0.9909 5.0 13.5 0.277 Experiment 9 - NH (1180) 1 1.10 2.22 2.4420 5.0 13.0 0.189 2 1.19 2.31 2.7489 5.0 14.0 0.182 Means 1.15 2.26 2.5955 5.0 13.5 0.185 Concen- No. Total tration I :01' Week of P001 per 1:23:1- Hens (cc.) Female (cc ) (millions) ' Experiment 9 1 7 0.554 180 0.079 2 7 0.588 180 0.084 Means 7 0.521 180 0.081 Table 2 5 (Continued) 86 v61. (:23): Total Pct. vol. of . . N0. M0- Abnor- for Week (millions , , Semen er cu Sperms tility mal Pool (cc.) p ' (billions) Sperms (cc.) mm.) Experiment 10 - WW_L5283) 1 0.79 3.12 2.4648 4.0 10.5 0.173 2 0.72 2.80 2.0160 3.0 16.5 0.193 Means 0.76 2.96 2.2404 3.5 13.5 0.183 ExperimEept 10 - NH (1180) 1 0.86 1.85 1.5910 5.0 11.0 0.292 2 1.22 2.01 2.4522 4.0 3.5 0.269 Means 1.04 1.93 2.0216 4.5 7.8 0.281 Concen- V . No. Total tration Inszin Week of P001 per 'nated. Hens (cc.) Female 1 . . (cc.) (millions) 9 0.465 120 0.052 9 0.462 120 0.051 Means 9 0.464 120 0.051 Table 25 (Continued) 87 v61. (10:25.6? Total Pct. v01. of r. ID N0. M0- Abnor- for Week (millions , , Semen Sperms tility mal P001 per cu. . . (cc.) (billions) Sperms (cc.) mm.) Experiment 11 - WW (5283) 1 0.98 3.40 3.3320 4.0 10.0 0.158 2 1.01 2.70 2.7270 4.0 15.5 0.200 3 1.10 2.58 2.8380 4.0 16.5 0.209 Means 1.03 2.89 2.9657 4.0 14.0 0.189 Experiment 11 - NHJ1180j 1 0.79 2.80 2.2120 5.0 11.5 0.193 2 1.10 2.22 2.4420 5.0 13.0 0.243 3 1.19 2.31 2.7489 5.0 14.0 0.234 Means 1.03 2.44 2.4676 5.0 12.8 0.223 c - oncoen Vol. No. Total tration Insem Week of P661 per inated’ Hens (cc.) Female . . (CC-l (millions) 6 0.351 180 0.059 6 0.443 180 0.074 6 0.443 180 0.074 Means 6 0.412 180 0.069 88 In Experiments 5 to 11, the males are ranked on the basis of mean quantities of semen they contributed to the pooled volumes, and presented alongside the tabulated results for the particular mating (Tables 26 to 32). The sequence of progeny is presented in Table 33. It should be noted that Experiments 6 to 11 provide the first instances in which the quantities of White Wyandotte semen used in pools did not exceed the quantities from competing males. The White Wyandotte male maintained a high concentration of spermatozoa through- out the experiments. It may also be noted that the grades of motility and incidence of morphologically defective spermatozoa follow the same breed-trend observed before in which White Wyandotte sperm- atozoa seemed to be at a disadvantage. ‘1qu 4.7. I... .1 .51....5.’ ....lI-. u... ~ L. 4.11.. Ranking of males based on the quantities of semen contributed to the pooled volume. WW (5278) BR (2000) WL (1113) 0.230 cc. 0.212 cc. 0.168 cc. Expe riment 5 89 Table 26. The progeny of New Hamp- shire females. NH . Males Hens WW BR Wl. 563 - 1 1 564 2 2 4 .2. 570 571 1 3 8 573 2 5 2 577 1 2 _ 578 1 1 3 579 1 1 1 Total 7 14 19 90 Expe riment 6 Ranking of males based on the quantities Table 27. The progeny of semen contributed to the pooled volume. of New Hamp- shire females. BR (2000) 0.207 cc. WL (1113) 0.185 cc. NH - Males Hens WW (5283) 0.159 cc. WW WL BI: 564 1 5 3 571 - 4 3 573 - 1 _ 577 1 5 5 578 2 3 2 579 - 3 1 Total 4 21 14 Ranking of males based on the quantities of semen contributed to the pooled volume. N11(1180) 1Ni.(1113 WW (5283) 0.311 cc. 0.251 cc. 0.203 cc. Experiment 7 91 Table 28. The progeny of New Hamp- shire females. NH Male: Hens WW WL NH 9824 1 4 6 1003 - 5 3 1020 - 1 1 9825 - 2 - 9826 1 2 3 0726 - 2 1 Total 2 16 1 4 Ranking of males based on the quantities of semen contributed to the pooled volume. NH (1180) WL (1113) WW (5283) 0.159 cc. 0.158 cc. 0.137 cc. Experiment 8 92 Table 29. The progeny of White Leg- horn females. WL Males E Hens WW WL NH — 1001 - 4 3 1005 - 7 4 1009 - 8 4 903 2 6 4 954 - 6 7 Total 2 31 22 93 Expe riment 9 Ranking of males based on the quantities Table 30. The progeny of semen contributed to the pooled volume. of White Rock females. WR (1151) 0.277 cc. I Males WW WR NH _-——o—— NH (1180) 0.185 cc. WR Hens WW (5283) 0.159 cc. 9816 2 2 - 9818 - 1 3 9820 - 5 3 9822 — 1 1 4378 - 3 7 632 - 3 1 633 2 Z 6 Total 4 17 21 Expe riment 10 Ranking of males based on the quantities of semen contributed to the pooled volume. NH (1180) WW (5283) 0.269 cc. 0.193 cc. 94 Table 31. The progeny of White Rock females. WR E Males Hens WW ' NH 9816 5 4 9818 5 5 9820 5 6 9821 2 5 9822 3 8 4378 - 11 632 1 8 633 2 6 Total 2 3 5 3 95 Expe riment 11 Ranking of males based on the quantities Table 32. The progeny of semen contributed to the pooled volume. of White Wy- andotte females. NH (1180) 0.234 cc. WW (5283) 0.209 cc. WW _ Males H ens WW NH 718 4 5 719 3 6 726 l 2 728 1 5 947 3 4 948 2 7 Total 14 29 96 Table 33. Sequence of progeny from females inseminated with equal concentrations of spermatozoa from different males. Experiment 5 NH Days Following Inseminations Fe- —" males 2 3 4 5 6 7 8 9 10 11 12 563 WL BR 564 WL WW WL WW I 570 571 WL BR WL BR WL WL BR 573 WW BR BR BR BR WL 577 578 BR WL WL WL 579 WL 13 14 15 16 17 18 19 20 21 22 23 563 I 564 WL WL BR 570 571 WL WL WL WL 573 BR 577 I BR BR 1 I 578 I I WW 579 I I 24 25 26 27 28 29 563 I I I 564 BR 570 I 571 1 WW I I 573 WW WL I 577 WW I I 578 I I I I 579 D1 I I Table 33 (Continued) 97 Experiment 6 NH Days Following Inseminations Fe- males 2 3 4 5 6 7 8 9 10 11 12 564 W1. WW D1 WL WL “ 571 WL BR BR WL WL 573 WL 577 WL BR WL I BR BR I WL WW 578 WW WL WL WW BR WL 579 I WL WL .2, 13 14 15 16 17 18 19 20 21 564 WL BR I BR WL BR I I 571 WL BR 1 573 577 WL I BR BR WL I I 578 I BR 1 I 579 WL BR 98 Table 3 3 (Continue d) Expe riment 7 NH Days Following Inseminations Fe- males 2 3 4 5 6 7 8 9 10 11 12 9824 NH WL NH WW WL WL NH NH NH WL 1003 WL WL WL WL WL 1020 WL , I NH 9825 9826 NH WW WL I 50726 13 14 15 16 17 18 9824 I 1 NH 1003 NH NH NH 1020 9825 I WL WL 9826 WL NH NH 1 50726 I WL NH I WL a. . .. .1. .111.) 1114.134 g». Table 3 3 (Continued) 99 Experiment 8 WL Days Following Inseminations Fe- males 2 3 4 5 6 7 8 9 10 11 12 1001 WL WL WL 1002 1005 I NH NH WL NH WL WL WL 1009 NH D2 WL I WL WL WL WL NH 903 D2 I WL I WL WL NH 954 NH NH WL NH WL WL NH l3 14 15 16 17 18 19 20 21 22 23 1001 NH X NH WL NH 1002 1005 I NH WL X I WL I WL I 1009 ’ WL NH X I NH WL I 903 WL WW WW X WL NH NH 1 WL 954 I WL NH 1 WL NH WL 24 25 26 1001 1002 \ 1005 1 1 9 ‘ 1009 WL I \ 903 NH '\ 954 NH I 1 T Table 33 (Continued) 100 WR Fe- males Expe riment 9 Days Following Inseminations 5 6 7 8 9 10 11 12 9816 9818 9820 9822 4378 632 633 9816 9818 9820 9822 4378 632 633 NH NH NH NH NH WR 15 WR WR WW WW NH NH NH 16 NH WW NH NH WR 17 NH WW WR 18 WR NH NH WR WR WR 19 NH 20 WR WR NH WR 21 NH NH WR NH 22 WR WR NH 23 24 .25 26 9816 9818 9820 9822 4378 632 633 101 Table 33 (Continued) Expe riment 1 0 WR Days Following Inseminations Fe - "— “‘* mal e s 2 3 4 5 6 7 8 9 10 11 12 9816 NH NH WW NH I WW I 9818 NH NH I WW WW WW I I NH 9819 9820 WW WW NH NH NH NH NH 9821 NH NH NH WW NH I NH WW 9822 NH NH WW NH NH NH I NH I 4378 NH NH NH NH NH NH NH 6 32 NH NH N H NH NH NH 6 3 3 NH WW NH NH WW NH 13 14 15 16 17 18 19 20 21 22 9816 WW 1 1- NH WW WW 1 1 9818 WW WW 1 NH 1 NH 1 1 9819 9820 WW WW NH WW 1 1 9821 1 9822 NH 1 WW NH 1 WW 1 4378 NH NH I I NH NH 1 1 632 WW NH NH 1 1 633 184 DHi D1 1 1 1 Table 3 3 (Continued) 102 Expe riment 11 WW Days Following Inseminations Fe- -— —- ‘ males 2 3 4 5 6 7 8 9 10 11 12 '718 NH NH WW 719 NH NH 726 NH WW NH 728 NH 947 WW 948 WW N.I 13 14 15 16 17 18 19 20 21 22 23 718 NH NH NH WW WW 719 I WW NH NH WW WW NH 726 728 NH NH WW 947 NH NH I NH WW 948 I NH NH I NH WW 24 25 26 27 28 29 30 31 32 718 WW I I 719 I 1 NH I 726 D 728 NH X NH 1 947 WW NH I I 948 NH NH NH ' I 1 103 The data from these experiments furnish additional evidence that under conditions of sperm competition the equalizing of volumes of semen from competing males does not necessarily mean uniformity of conditions. From these results it is suggested that fertility is not as dependent upon volume of semen inseminated as it is upon the con- centration of spermatozoa in the semen. Of particular significance is the fact that the two White Wyan- dotte males duplicated the results obtained in the previous experi- ments in that they sired but few chicks. In Experiment 10 and 11, wherein White Wyandotte spermatozoa competed only with spermatozoa from a single male, more chicks were sired by the White Wyandotte male than when in competition with the spermatozoa from different breeds of males. When the dams were of the White Wyandotte breed, 33.3 per cent of the chicks produced were sired by the White Wyan- dotte male. When the dams were of the White Plymouth Rock breed, 30.3 per cent of the chicks were sired by the White Wyandotte male. Under the conditions of competitive fertilization used in this investigation, the over-all results suggest that the spermatozoa of the White Wyandotte males are unable to compete successfully with spermatozoa obtained from males of the White Leghorn, White Plymouth Rock, Barred Plymouth Rock, and New Hampshire breeds of chickens. 104 Motility and Morphology of Spermatozoa in Relation to Competitive —Ferti1izing Capacity The relationship between the motility of the spermatozoa from each particular male and the number of progeny resulting was deter- mined. Also, a determination was made of the relationship between the number of progeny sired by each male and the percentage of morphologically defective spermatozoa, as well as the percentage of morphological defects and motility. A highly significant positive ., correlation of 0.72 :h 0.018 was found between motility rating and fertility indices. From this correlation it is suggested that when equal concentrations of spermatozoa from two or three different males are mixed, the higher the motility rating, the more numerous are the progeny. A highly significant negative correlation of 0.55 :1: 0.027 was found between the morphology of the spermatozoa and the number of progeny sired. From this correlation it is suggested that the fewer the morphological defects observed in the spermatozoa, the more numerous are the progeny sired. A significant negative correlation of 0.31 :I: 0.035 was found between morphology and motility of the spermatozoa. It is suggested that semen which shows a rela- tively low motility may have a high incidence of morphologically defective spe rmato zoa. DISCUSSION Instead of following the method of studying competitive fertili- zation used by other investigators, in which they pooled equal volumes of semen from competing males, the experimental procedure reported herein is based upon equal numbers of spermatozoa from competing males. Differences in spermatozoa concentrations not revealed in mixing equal volumes of semen can be the cause of widespread dis- crepancies in results. This must necessarily be so because of the wide differences which exist among males in the concentrations of spermatozoa found in their semen. The ratio of offspring sired by competing males should be expected to depend upon the relative abundance of the males' spermatozoa, other things being equal. There- fore, the method of approach whereby concentrations of spermatozoa are equalized appears to be a logical procedure. But the countless other factors which enter into the delicate phenomenon of fertilization may not be so readily controlled. There can be no guarantee that the motility of competing spermatozoa will be alike, any more than can the qualification be met that exactly equal numbers of normal spermatozoa be contained in all contributing volumes. Variations 105 106 with respect to pH, spermatozoa viability, and factors within the fe- males themselves complicate the picture of fertilization. However, one approach to the problem consists of separating the factors which lend themselves to some kind of measurement, and relating them to the resulting frequencies of progeny. The stress laid upon one or the other of the available evaluations for semen with respect to fer- tility varies among different researchers, but there seems to be gen- eral acceptance that three of the most practical evaluations are con- centration, motility, and morphology of spermatozoa. Consideration has been given these factors in this study on selective fertilization. From the number of chicks sired by the different males used in these matings it is concluded that selective fertilization does occur; however, not necessarily selective fertilization in favor of the union of the germ cells from like breeds, but, rather, a type suggesting ”dis— crimination" against the ”weak and poorly adapted" spermatozoa. What causes the "weakness and poor adaptation'I cannot be definitely stated, but, on the basis of the measured relationships between fer- tility, abnormal spermatozoa, and motility, it may be concluded that these "weaknesses" are present. The results suggest inequalities in the sense that there are great variations amongcompeting sperm- atozoa. The fact that there is a preponderance of chicks sired by 107 the New Hampshire and White Leghorn males compared with the relatively few sired by the White Wyandotte males suggests that spermatozoa from the New Hampshire and White Leghorn males pos- sess advantageous variations. These advantages were well reflected in the semen evaluations made. On the strength of the low motility ratings, the high percent- ages of abnormal spermatozoa found in semen of the White Wyandotte males, and their conspicuously reduced fertility under competitive conditions, it is postulated that low motility and a high incidence of abnormal spermatozoa are factors associated with the poor fertility observed, as a rule, in Wyandottes. The influence of motility on fertility in the domestic fowl has not received the attention it deserves. Parker, McKenzie, and Kemp- ster (1942) found a positive correlation of 0.246 between motility and fertility in chickens. In cattle, Donham, Simms, and Shaw (1931) found a definite correlation between fertility and motility. These researchers suggested that semen containing less than 90 per cent of active spermatozoa should be regarded as abnormal, since it does not ensure satisfactory fertilization. The highly significant correlation found in this study (r = 0.72), between motility and fer- tility under competitive conditions, suggests no small relationship between these factors, in general. 108 Complicating the picture, however, are the findings of Warren and Kilpatrick (1929), that sperms lose their flagellae within twenty- four hours after first entering the reproductive tract; yet, it is known that fowls will lay fertile eggs fifteen days or more after an initial insemination. The evidence is conflicting, for Walton and Whetham (1933) were unable to find sperms in the oviducts of females after mating, although fertile eggs were produced up to fifteen days there- after, and Van Drirnmelen (1945) found normal active spermatozoa in the infundibulum up to fourteen-days after insemination. If the flag- ellae of the spermatozoa are lost within twenty-four hours after introduction into the oviduct, it seems unlikely that motility could be of prime importance in effecting fertilization. However, the_ data of Warren and Kilpatrick (1929) indicated that if fresh spermatozoa are introduced at any time after initial mating they immediately take precedence over old spermatozoa. These workers postulated that one reason why fresh spermatozoa supersede old spermatozoa lies in their intact flagellae, the lashing of which provides the spermatozoa with their motility. It is known that the oviduct also aids in facili- tating the migration of spermatozoa to the infundibulum, the site of fertilization. Spermatozoa which avail themselves of this so-called ”pro-ovarian ciliary mechanism" plus the highest degree of their 109 own movements might be expected to excel those not so fortunate in their inherent motility, or even those which might have to depend solely on the ciliary mechanism, such as might be the case with stale spermatozoa. Whatever might be the causal factors, on the basis of the highly significant correlations found in this investigation between motility and fertility under competitive-sperm conditions, it is evident that these factors are intimately associated with motility. From the evidence presented herein it is further suggested that the .2- criterion of initial motility is one of the better methods for the eval- uation of semen. Let us now turn to the question of spermatozoa morphology. From the highly significant negative correlation found between mor- phological defects and competitive fertility (r = -0.55), it is suggested that the number of abnormal spermatozoa in the semen is a factor in fertility. If the concentration of spermatozoa is an important factor in fertility, high percentages of morphologically defective Spermatozoa can only mean a reduced number of virile spermatozoa present to effect fertilization. It should be realized, also, that the detrimental agency which brings about degeneration in spermatozoa will also affect other spermatozoa, the overt defects of which may not be readily seen under the microscope. These potentially defective spermatozoa may 110 be expected to have impaired performance, which becomes very ap- parent when it is necessary for them to compete with other sperm- atozoa. This might possibly be the case with the White Wyandotte males used in this investigation. The White Wyandotte males dem- onstrated a high fertility when mated singly to females of their own breed, as well as when mated to females of other breeds. When White Wyandotte spermatozoa were in competition with the sperma- tozoa from only one male, fertility was better than when in compe- tition with the spermatozoa of twoother males, each of a different breed. It is not unlikely that the characteristic ”weaknesses" seen in White Wyandotte spermatozoa could be hereditary and operating to give this breed the somewhat lower fertilizing capacity oftentimes observed. The evidence showed no special affinity of the spermatozoa of one breed of chickens for the ova of a similar breed; instead, condi- tions seem to favor spermatozoa more adequately prepared to reach the site of fertilization and to penetrate the female germ cell. In addition, the results suggest the possibility of sperm-competition technique for measuring relative differences between spermatozoa, providing that suitable genetic labels are used. 1.111401»! .130. Nullk. SUMMARY AND CONCLUSIONS In matings designed to make the identification of progeny pos— sible genetically, equal numbers of spermatozoa from three males, and in some cases two males, were pooled, thoroughly mixed, and injected into females by means of artificial insemination, the results of which have been presented. The study entailed the evaluation of semen for volumes pro- duced, concentrations of spermatozoa, motility ratings, and morpho- logical defects among spermatozoa. The effects of motility and inci- dence of morphologically defective spermatozoa on fertilizing capacity under competitive conditions were also determined. Semen volumes were found to range from 0.39 to 1.78 cubic centimeters in the White Wyandotte males, from 0.42 to 1.32 cubic centimeters in the White Leghorn males, from 0.42 to 1.30 cubic centimeters in the White Plymouth Rock males, from 0.39 to 1.30 cubic centimeters in the Barred Plymouth Rock males, and from 0.42 to 1.82 cubic centimeters in the New Hampshire males. 111 V gstuEllihfiilm 112 Spermatozoa concentrations ranged from 1.38 to 3.12 million per cubic millimeter in the semen collected from the White Wyan- dotte males, from 1.43 to 2.76 million spermatozoa per cubic milli- meter in the semen collected from the White Leghorn males, from 1.34 to 2.90 million spermatozoa per cubic millimeter in the semen collected from the White Plymouth Rock males, from 1.98 to 3.83 million spermatozoa per cubic millimeter in the semen collected from the Barred Plymouth Rock males, and from 1.36 to 3.12 mil- lion spermatozoa per cubic millimeter in the semen collected from the New Hampshire males. Motility ratings of semen (scaled 0 to 5) ranged from 2 to 5 in all males, with semen produced by the White Wyandotte males near the lower limit of the range. Morphologically defective spermatozoa in semen ranged from 3.0 to 20.0 per cent in the White Wyandotte males, from 5.0 to 18.0 per cent in the White Leghorn males, from 2.5 to 15.5 per cent in the White Plymouth Rock males, from 7.0 to 15.0 per cent in the Barred Plymouth Rock males, and from 3.5 to 15.0 per cent in the New Hampshire males. A highly significant positive correlation of 0.72 :1: 0.018 was found between the motility rating and the number of progeny resulting 113 when equal concentrations of competing spermatozoa from two or three males were inseminated into females. A significant negative coeffin cient of correlation of -0.55 :1: 0.027 was found between the percent- age of morphologically defective spermatozoa and fertilizing capacity. A significant negative correlation of -0.31 :1: 0.035 was also found between the morphology of the spermatozoa and motility rating. From the results obtained, the following conclusions were justified: 1. Wide differences existed between males in their semen- producing capacities. 2. Spermatozoa concentrations varied markedly among males, and even in Iday-to-day collections from the same individual. 3. For spermatozoa-competition studies, equal volumes of semen from different males did not yield equal concentrations of spermatozoa. 4. Ratings of semen obtained from the males of the White Plymouth Rock, White Leghorn, Barred Plymouth Rock, and New Hampshire breeds suggested no possible breed differences in motility. 5. White Wyandotte males showed consistently lower motility ratings in their semen, and differed to a significant degree from males of the New Hampshire, White Rock, White Leghorn, and Barred Plymouth Rock breeds in their characteristic motility. 114 6. The highest percentages of abnormal spermatozoa were demonstrated consistently in semen of the White Wyandotte male. 7. Where equal concentrations of spermatozoa from the New Hampshire, White Leghorn, and White Wyandotte males were injected into New Hampshire and White Leghorn hens, chicks sired by the New Hampshire and White Leghorn males far outnumbered those sired by the White Wyandotte male. Evidence was insufficient to suggest appreciable disparities between New Hampshire and White Leghorn sires. 8. Equal concentrations of spermatozoa from the Barred Ply- mouth Rock, White Leghorn, and White Wyandotte males inseminated into New Hampshire females resulted in conspicuously fewer chicks sired by the White Wyandotte male. The White Leghorn. invariably excelled the Barred Plymouth Rock, though not by margins as great as when compared with the White Wyandotte male. 9. Equal concentrations of spermatozoa from the New Hamp- shire, White Plymouth Rock, and White Wyandotte males, inseminated into White Plymouth Rock hens, resulted in significantly fewer chicks sired by the White Wyandotte male. No breed selectivity or affinity of White Plymouth Rock spermatozoa for White Plymouth Rock ova co uld be demonstrated. 115 10. Where equal concentrations of spermatozoa from the White Wyandotte and New Hampshire males were mixed and injected into White Wyandotte and White Plymouth Rock females, the number of chicks sired by the White Wyandotte male was approximately one -half the number sired by the New Hampshire male. 11. Selective fertilization in the sense of breed likeness could not be demonstrated, but it was suggested that there was a type of selectivity, based on spermatozoa (regardless of breed origin) possessing certain advantageous variations associated with or reflected in a high degree of motility and low incidence of abnormalities. 12. 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