Effect of Sperm Numbers on Fertility of Frozen Bull Spermatozoa in Skim Milk Diluent 1 J. O. ALMQUIST Dai,ry Breeding Research Center Department of Dairy Science The Pennsylvania State University University Park 16802 Abstract
Insemination doses of 10, 15, and 20 million progressively motile spermatozoa prior to freezing were compared in a split-ejacaflate fertility experiment involving 4,603 first service inseminations to 34 semen samples from six Angus bulls. Based on 60- to 90-day nonreturn rates, fertility was significantly lower ~or inseminating doses of 10 and 15 X 10e than 20 X 106 mottle sperm (61, 62, and 70%). The 1-ml doses of skim milk diluted semen were frozen in glass ampules. T h e post-thaw number of motile sperm per ampule after freezing and storage for 3 wk in liquid nitrogen averaged 5.5, 8.4, and 12.4 X l0 s for corresponding pre~freeze numbers of 10, 15, and 20 X 10e motile sperm/ml and ranged from 4.0 to 15.7 X 10~/anl. Frozen semen packaged in ampules should contain more than 8.4 X 10e motile cells post-thaw per ampule.
termine the fertility of cows inseminated with 1 ml of skim milk diluted semen containing 10, 15, or 20 X 10~ progressively motile spermatozoa prior to freezing. Experimental Procedure
Thirty-seven semen samples (some first and second ejaculates were pooled) from six relatively fertile 6-yr-old Angus bulls housed at this center were frozen between September 8 and November 13, 1969. The semen quality averaged 66% progressively motile sperm and 1,096 X l0 s sperm/ml. Each sample was divided into three equal portions and diluted to provide a final concentration of 10, 15, or 20 X l0 s progressively motile sperm/ml. Each portion was diluted initially at 35 C to 20, 30, or 40 X 106 progressively motile sperm/ml of fresh heated, unfortified skim milk (1,000 units of penicillin a n d 1 mg of dihydrostreptomycin/ml ddluent). After cooling to 5 C in 4 h, an equal volume of skim milkglycerol-fructose diluent was added in four equal portions at 15-rain intervals so that the final diluted semen contained 11% glycerol, Introduction 1.25% fructose, 1,000 units of penicillin, 1 mg One approach to maximizing the usefulness of dihydrostreptomyein, and 10, 15, or 20 X of genetically outstanding sires is to reduce the 106 motile sperm/ml. As soon as glycerolization was completed, number of sperm per insemination. However, the reduction must be consistent with mainte- the diluted semen was dispensed in 1.0 ml nance of optimum fertility. With frozen semen, quantities into ampules. The sealed ampules in addition to differences of bull and e~aeu/ate, were placed on aluminum canes, equilibrated Sulh'van (6) suggested that the optimum num- for 18 -+- 1 h and frozen in a Linde BF-3-2 ber of sperm is ir~uenced by many factors, in- freezer. The semen was cooled at 1 C/rain eluding the kind of diluent, processing system, from + 5 to --15 C (or crystallization) and freezing rate, storage conditions, volume of the 4 C/min from --15 to --60 12. After reachinseminating dose, and type of inseminating ing --60 C, ampules were cooled rapidly to equipment: Most recent reports (2, 3, 7) have --110 C and transferred to liquid nitrogen for concerned the effect of sperm number on fer- storage. After storage for 3 wk, dt~plieate coded amtility of semen diluted in egg yolk diluents but pules were thawed in 5 C water, and the pernot milk diluents. The objective of this experiment was to de- centage of progressively motile sperm at 37 C were estimated with a phase-contrast microscope. The recorded value for post-thaw Received May 16, 1974. XAuthorized for publication on April 9.2, 1974 as motility was the average of independent estiPaper No. 4678 in the journal series of The Penn- mates of two observers each using a different ampule from the same sample. Only 3 of the sylvania Agricultural Experment Station. 420
SPEtI1VI NUMBERS AND FEBTILITY
37 semen samples frozen were discarded because less than one-third of the pre-freeze number of progressively motile sperm had survived freezing, storage of 3 wk at --196 C, and thawing at 5 C. Thus, 34 samples were shipped to Atlantic Breeders Cooperative from JanualT, 1970 to June, 1971 for use in commerical artificial insemination. Distribution of ampules to individual technicians was at the discretion of the breeding organization. Teehnieiaus were instructed to inseminate the semen as soon as possible after thawing the ampule for 8 rain in 5 C water. Each technician was required to record the semen collection number and bull number on the breeding receipt. Breeding receipt information was recorded on punch cards, and all second services were matched to first services by ear tag or registration number. Receipts with conflicting bull codes and semen collection numbers were discarded. Fertility data representing services to dairy cows, for the most part, were summarized for each portion of each split ejaculate. For statistical analysis, teehnician differences were ignored and 60- to 90-day nonreturn percentages were determined for each ejaeulate~ treatment class on the basis of first services. Analysis of variance and Duncan's New Multiple Range Test were used for data interpretation. Results and Discussion
Post-thaw progressive motility (Table 1) was higher for the portion of each ejaculate diluted to 20 X l0 s motile sperm than for the portions diluted to 10 or 15 X 106 motile sperm prior to freezing. This finding generally supports previous reports (1, 2) that sperm TABLE 1. Effect of number of progressively motile sperm per insemination on fertility. Item
Pre-free'zenumber of motile sperm/m] 10× 10' 15X 106 20X 106
Avg post-thaw progresslve motility, ~; Avg post-thaw no. motile sperm (10'/ml) No. first inseminations
Nonrctttm rate, T
36 5.5
37 8.4
41 12.4
1,576 1,488 1,539 61.1" 61.5" 69.5b
,.b Means followed by a different letter are sigmL fieantly different at 1~; proba~lity.
421
freezability in ampules improved as sperm concentration increased. Three weeks after freezing, the numbers of progressively motile sperm in ampules representing the three treatments ranged from 4.0 to 8.3, 5.4 to 11.1, and 10.0 to 15.7 X 106/rrd; 55, 56, and 62% of the pre-freeze values. While the experimental design specified reduction in pre-freeze motile sperm numbers of 25% between 20 and 15 X 106 sperm and 33.3% between 15 and 10 X 106 sperm, the reduction after freezing averaged 32 and 35%, respectively. Thus, based on post-freeze evaluation, there was a reduction of about one third in motile sperm between successive treatments. There was a decrease (P < .01) in fertility when a motile sperm dose prior to freezing of 15 X 106/ml was used rather than 20 X 106/ml (Table 1), but there was 11o significant difference between doses of 10 and 15 X l0 s motile sperm. When each insemination was weighted equally, the nonrettwn rates averaged 60.2, 62.8, and 68.5% for inseminating doses of 10, 15, and 20 X 10~ motile sperm. Fertility varied (P < .01) among bulls, but the interaction of bulls and treatments was not significant, indicating that the six bulls in this study responded similarly in fertility to changes in sperm dosage. Sullivan and Elliott (7), however, found that bulls of high, medium, and low fertility responded differently to changes in sperm dosage. R e d u c i n g motile sperm numbers prior to freezing from 20 to 15 X l0 s in skim milk diluent resulted in a highly significant impairment of fertility. In contrast, Foote (3) reported no significant difference in fertility between 12 and 24 X 106 sperm per ampule before freezing using either Tris-fructose-yolk or citrate-yolk diluent. Pickett et al. (5) reported no significant difference in fertility between 12 and 22 X 10~ motile sperm per ampule post-thaw but Goffanx (4) found that fertility was significantly higher for 18.3 than 11.0 X 10e motile sperm post-thaw. My finding that fertility was significantly lower for 5.5 and 8.4 than for 12.4 X 106 motile sperm per ampule pest-thaw agrees with Sullivan and Elliott (7). Post-thaw numbers of motile sperm per ampule for individual one-third portions of samples ranged from 4.0 to 15.7 X l0 s. The correlation between these numbers and percent nonreturns was .43 (P < .01). Thus, only 18% of the variation in nonreturn rate is accounted for by differences in sperm numbers JOURNAL O1: DAIRY SCIENCe, VOL. 58, NO. ~.
422
~Q~S't
within this range. This supports Foote (3), who used from 3 to 23 X 106 motile sperm per ampule post-thaw in three experiments and found that sperm numbers accounted for only 3 to 18% of the variation in nonreturn rate. Acknowledgments The author wishes to thank D. L. Yoder and his staff of Atlantic Breeders Cooperative, Lancaster, PA for their assistance and cooperation; and Amber Keene, Helen Homan, and Teresa Shirk for able technical assistance. Partial financial support by Atlantic Breeders Cooperative, Lancaster, PA, Northeastern Breeders Association and Sire Power, Inc., Tunkhannoek, PA, are gratefully acknowledged. References (1) Benson, R. W., T. J. Sexton, B. W. Pickett, J. J. Lueas, and M. R. Gebauer. 1968. Influence of processing techniques and dilution rates on survival of frozen bovine spermatozoa. Connecticut Agr. Exp. Sta. Res. Rep. 28.
JOURNAL OF DAIRy SCIENCE, VOL. 58, NO. 3
(2) Dosjardins, C., and H. D. I-Iafs. 1962. Motility and fertility during post-thawing storage of bovine spermatozoa frozen c~neentrated, thawed, mad re-extended. J. Dairy Sei. 45:1242. (3) Foote, B. H. 1970. Influence of extender, extension rate, and glyeerolating technique on fertility of frozen bull semen. J. Dairy Sei. 53:1478. (4) Goffaux, M. 1965. Effect of the number of motile spermatozoa used per insemination on the fertilizing ability of frozen bull semen. Ann. Zooteeh. 14:409. (Citation from Animal Breed. Abstr. 35:78. 1967.) (5) Pickett, B. W., R. C. Hall, Jr., J. J. Luoas, and E. W. Gibson. 1064. Influence of sperm number on fertility of frozen bovine semen. J. Dairy Sei. 47:916. (6) Sullivan, J. J. 1970. Sperm numbers required for optimum breeding efficiency in cattle. Page 36 in Proe. Third N.A.A.B. Teeh. Conf. Artff. Insem. Repro& (7) Stdlivan, J. J., and F. I. Elliott. 1968. Bull fertility as affected by an interaction between motile spermatozoa concentration and fertility level in artificial insemination. VIth Inter. Congr. Anita. Repr~t. Artff. Insern. II: 1307.
Insemination doses of 10, 15, and 20 million progressively motile spermatozoa prior to freezing were compared in a split-ejacaflate fertility experiment involving 4,603 first service inseminations to 34 semen samples from six Angus bulls. Based on 60- to 90-day nonreturn rates, fertility was significantly lower ~or inseminating doses of 10 and 15 X 10e than 20 X 106 mottle sperm (61, 62, and 70%). The 1-ml doses of skim milk diluted semen were frozen in glass ampules. T h e post-thaw number of motile sperm per ampule after freezing and storage for 3 wk in liquid nitrogen averaged 5.5, 8.4, and 12.4 X l0 s for corresponding pre~freeze numbers of 10, 15, and 20 X 10e motile sperm/ml and ranged from 4.0 to 15.7 X 10~/anl. Frozen semen packaged in ampules should contain more than 8.4 X 10e motile cells post-thaw per ampule.
termine the fertility of cows inseminated with 1 ml of skim milk diluted semen containing 10, 15, or 20 X 10~ progressively motile spermatozoa prior to freezing. Experimental Procedure
Thirty-seven semen samples (some first and second ejaculates were pooled) from six relatively fertile 6-yr-old Angus bulls housed at this center were frozen between September 8 and November 13, 1969. The semen quality averaged 66% progressively motile sperm and 1,096 X l0 s sperm/ml. Each sample was divided into three equal portions and diluted to provide a final concentration of 10, 15, or 20 X l0 s progressively motile sperm/ml. Each portion was diluted initially at 35 C to 20, 30, or 40 X 106 progressively motile sperm/ml of fresh heated, unfortified skim milk (1,000 units of penicillin a n d 1 mg of dihydrostreptomycin/ml ddluent). After cooling to 5 C in 4 h, an equal volume of skim milkglycerol-fructose diluent was added in four equal portions at 15-rain intervals so that the final diluted semen contained 11% glycerol, Introduction 1.25% fructose, 1,000 units of penicillin, 1 mg One approach to maximizing the usefulness of dihydrostreptomyein, and 10, 15, or 20 X of genetically outstanding sires is to reduce the 106 motile sperm/ml. As soon as glycerolization was completed, number of sperm per insemination. However, the reduction must be consistent with mainte- the diluted semen was dispensed in 1.0 ml nance of optimum fertility. With frozen semen, quantities into ampules. The sealed ampules in addition to differences of bull and e~aeu/ate, were placed on aluminum canes, equilibrated Sulh'van (6) suggested that the optimum num- for 18 -+- 1 h and frozen in a Linde BF-3-2 ber of sperm is ir~uenced by many factors, in- freezer. The semen was cooled at 1 C/rain eluding the kind of diluent, processing system, from + 5 to --15 C (or crystallization) and freezing rate, storage conditions, volume of the 4 C/min from --15 to --60 12. After reachinseminating dose, and type of inseminating ing --60 C, ampules were cooled rapidly to equipment: Most recent reports (2, 3, 7) have --110 C and transferred to liquid nitrogen for concerned the effect of sperm number on fer- storage. After storage for 3 wk, dt~plieate coded amtility of semen diluted in egg yolk diluents but pules were thawed in 5 C water, and the pernot milk diluents. The objective of this experiment was to de- centage of progressively motile sperm at 37 C were estimated with a phase-contrast microscope. The recorded value for post-thaw Received May 16, 1974. XAuthorized for publication on April 9.2, 1974 as motility was the average of independent estiPaper No. 4678 in the journal series of The Penn- mates of two observers each using a different ampule from the same sample. Only 3 of the sylvania Agricultural Experment Station. 420
SPEtI1VI NUMBERS AND FEBTILITY
37 semen samples frozen were discarded because less than one-third of the pre-freeze number of progressively motile sperm had survived freezing, storage of 3 wk at --196 C, and thawing at 5 C. Thus, 34 samples were shipped to Atlantic Breeders Cooperative from JanualT, 1970 to June, 1971 for use in commerical artificial insemination. Distribution of ampules to individual technicians was at the discretion of the breeding organization. Teehnieiaus were instructed to inseminate the semen as soon as possible after thawing the ampule for 8 rain in 5 C water. Each technician was required to record the semen collection number and bull number on the breeding receipt. Breeding receipt information was recorded on punch cards, and all second services were matched to first services by ear tag or registration number. Receipts with conflicting bull codes and semen collection numbers were discarded. Fertility data representing services to dairy cows, for the most part, were summarized for each portion of each split ejaculate. For statistical analysis, teehnician differences were ignored and 60- to 90-day nonreturn percentages were determined for each ejaeulate~ treatment class on the basis of first services. Analysis of variance and Duncan's New Multiple Range Test were used for data interpretation. Results and Discussion
Post-thaw progressive motility (Table 1) was higher for the portion of each ejaculate diluted to 20 X l0 s motile sperm than for the portions diluted to 10 or 15 X 106 motile sperm prior to freezing. This finding generally supports previous reports (1, 2) that sperm TABLE 1. Effect of number of progressively motile sperm per insemination on fertility. Item
Pre-free'zenumber of motile sperm/m] 10× 10' 15X 106 20X 106
Avg post-thaw progresslve motility, ~; Avg post-thaw no. motile sperm (10'/ml) No. first inseminations
Nonrctttm rate, T
36 5.5
37 8.4
41 12.4
1,576 1,488 1,539 61.1" 61.5" 69.5b
,.b Means followed by a different letter are sigmL fieantly different at 1~; proba~lity.
421
freezability in ampules improved as sperm concentration increased. Three weeks after freezing, the numbers of progressively motile sperm in ampules representing the three treatments ranged from 4.0 to 8.3, 5.4 to 11.1, and 10.0 to 15.7 X 106/rrd; 55, 56, and 62% of the pre-freeze values. While the experimental design specified reduction in pre-freeze motile sperm numbers of 25% between 20 and 15 X 106 sperm and 33.3% between 15 and 10 X 106 sperm, the reduction after freezing averaged 32 and 35%, respectively. Thus, based on post-freeze evaluation, there was a reduction of about one third in motile sperm between successive treatments. There was a decrease (P < .01) in fertility when a motile sperm dose prior to freezing of 15 X 106/ml was used rather than 20 X 106/ml (Table 1), but there was 11o significant difference between doses of 10 and 15 X l0 s motile sperm. When each insemination was weighted equally, the nonrettwn rates averaged 60.2, 62.8, and 68.5% for inseminating doses of 10, 15, and 20 X 10~ motile sperm. Fertility varied (P < .01) among bulls, but the interaction of bulls and treatments was not significant, indicating that the six bulls in this study responded similarly in fertility to changes in sperm dosage. Sullivan and Elliott (7), however, found that bulls of high, medium, and low fertility responded differently to changes in sperm dosage. R e d u c i n g motile sperm numbers prior to freezing from 20 to 15 X l0 s in skim milk diluent resulted in a highly significant impairment of fertility. In contrast, Foote (3) reported no significant difference in fertility between 12 and 24 X 106 sperm per ampule before freezing using either Tris-fructose-yolk or citrate-yolk diluent. Pickett et al. (5) reported no significant difference in fertility between 12 and 22 X 10~ motile sperm per ampule post-thaw but Goffanx (4) found that fertility was significantly higher for 18.3 than 11.0 X 10e motile sperm post-thaw. My finding that fertility was significantly lower for 5.5 and 8.4 than for 12.4 X 106 motile sperm per ampule pest-thaw agrees with Sullivan and Elliott (7). Post-thaw numbers of motile sperm per ampule for individual one-third portions of samples ranged from 4.0 to 15.7 X l0 s. The correlation between these numbers and percent nonreturns was .43 (P < .01). Thus, only 18% of the variation in nonreturn rate is accounted for by differences in sperm numbers JOURNAL O1: DAIRY SCIENCe, VOL. 58, NO. ~.
422
~Q~S't
within this range. This supports Foote (3), who used from 3 to 23 X 106 motile sperm per ampule post-thaw in three experiments and found that sperm numbers accounted for only 3 to 18% of the variation in nonreturn rate. Acknowledgments The author wishes to thank D. L. Yoder and his staff of Atlantic Breeders Cooperative, Lancaster, PA for their assistance and cooperation; and Amber Keene, Helen Homan, and Teresa Shirk for able technical assistance. Partial financial support by Atlantic Breeders Cooperative, Lancaster, PA, Northeastern Breeders Association and Sire Power, Inc., Tunkhannoek, PA, are gratefully acknowledged. References (1) Benson, R. W., T. J. Sexton, B. W. Pickett, J. J. Lueas, and M. R. Gebauer. 1968. Influence of processing techniques and dilution rates on survival of frozen bovine spermatozoa. Connecticut Agr. Exp. Sta. Res. Rep. 28.
JOURNAL OF DAIRy SCIENCE, VOL. 58, NO. 3
(2) Dosjardins, C., and H. D. I-Iafs. 1962. Motility and fertility during post-thawing storage of bovine spermatozoa frozen c~neentrated, thawed, mad re-extended. J. Dairy Sei. 45:1242. (3) Foote, B. H. 1970. Influence of extender, extension rate, and glyeerolating technique on fertility of frozen bull semen. J. Dairy Sei. 53:1478. (4) Goffaux, M. 1965. Effect of the number of motile spermatozoa used per insemination on the fertilizing ability of frozen bull semen. Ann. Zooteeh. 14:409. (Citation from Animal Breed. Abstr. 35:78. 1967.) (5) Pickett, B. W., R. C. Hall, Jr., J. J. Luoas, and E. W. Gibson. 1064. Influence of sperm number on fertility of frozen bovine semen. J. Dairy Sei. 47:916. (6) Sullivan, J. J. 1970. Sperm numbers required for optimum breeding efficiency in cattle. Page 36 in Proe. Third N.A.A.B. Teeh. Conf. Artff. Insem. Repro& (7) Stdlivan, J. J., and F. I. Elliott. 1968. Bull fertility as affected by an interaction between motile spermatozoa concentration and fertility level in artificial insemination. VIth Inter. Congr. Anita. Repr~t. Artff. Insern. II: 1307.
