CRYOBIOLOGY
15, 355-357
(1978)
Characteristics
and Cryopreservation JOHN
American
of Stallion
Spermcttozoa
J. SULLIVAN
Breeders Service, DePorest, Wisconsin
53532
vided a maximal number of spermatozoa per collection day and was considered a practical ejaculation frequency for longterm preservation of stallion semen (36). A myriad of extenders and packaging systems have been used in the cryopreservation of stallion semen (l-6, 8-21, 28-35). Spermatozoa1 post-thaw survival has been satisfactory, mares have been inseminated, and normal foaIs have been produced. In general, fertility has been low and the fertility trials have been plagued by small numbers of mares and inadequate experimental controls for a fair evaluation of the fertility of frozen semen. The fertilizing capacity of spermatozoa of some stallions is more adversely affected than that of other stalIions (2, 22). The majority of mares inseminated with frozen-thawed semen conceived when inseminations occurred within 12 hr of ovulation (22). As of now, no completely satisfactory cryoprotectant has been found. Recent data indicate that glycerol depresses fertility of spermatozoa in Tris-yolk and creamgelatin extenders ( 7, 22). Cream-gelatin and skim milk extenders without ,glycerol provide optimal fertility only when the extended semen is not cooled and stored at 5°C (7, 23). Insemination of large numbers of motiIe spermatozoa masks these Received October 21, 1977; accepted Decrmdetrimental effects and offers some comber 2, 1977. pensation for an inferior extender (24, 25, Synopsis paper presented at the 14th Annual Meeting of the Society for Cryobiology, August 27). Therefore, to evaluate properly a 1977,Minneapolis,Minnesota. seminal extender, the number of motile
In North America, stallion seminal characteristics vary with the seasonof the year. Fur stallions from which were collected two ejaculates per day, once per week, total volume (including gel) from winter to spring increased about 38% for the first ejaculate and 21% for the second ejaculate. Gel-free semina1volume for first ejaculates ranged between 44 and 80 ml, and for second ejaculates, between 41 and 63 ml, depending upon season. Ejaculates containing gel were less frequent during the winter. More spermatozoa were produced in Iate spring and early summer than in the winter. Throughout the year, first ejaculates ranged between 10 and 22 x lo” spermatozoa, and second ejaculates, between 5 and 11 x IO” spermatozoa (26). When chasing an ejaculation frequency, consideration must be given to the schedule which most efficiently provides the maximum number of spermatozoa and most conveniently adapts to the program for which the stallions are maintained. One ejacuIate collected on alternate days proved to be the most effective and practical ejaculation frequency for stallions maintained in an artificial insemination program without semina1 storage. Two ejaculates per day, 2 days per week, pro-
355 0011~!2240/78/0153-0355$02.00/O All
Copyright Q 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
JOHir; J. SULLIVAN
356 spermatozoa per insemination exceed 100 x 10B. REFERENCES
1. Aliev, A. An improved technique of semen freezing, Konecod. Konnyi Sport 7, 30 (1975); Anim. Bmed. Ah&. 44, No. 18 (1976).
2. Baczynski, J., BieIanski, W., Bilik, K., Dram, S., and Zapletal, Z. Artificial insemination of horses. VI. Preliminary results of inseminating mares with frozen semen. Zeszyty Problemowe Postepow Nauk Rolniczych 124, 131-1313 ( 1972). Anim. Breed. Abstr. 41, 3.
4.
5.
6. 7.
8.
9.
No.
9 (1973).
Bader, H. Deep-freezing of stallion semen in pellet form. In. “VI Congress of Animal Reproduction and Artificial Insemination,” pp. 985-988, 1968. Bader, H., and MahIer, R. Experiments of freezing stallion semen by the pellet method, and its insemination. Zuchthygiene 3, 6-13 (196s). Anim. Breed. Abstr. 36, No. 2275 (1988). Barker, C. A. V., and Gandier, J. C. C. Pregnancy in a mare resulting from frozen epididymal spermatozoa. Canad. J. Camp. Med. 21, 47-51. (1957). Buell, J. R. A method for freezing stallion semen and tests of its fertility. Vet. Rec. 75, 900-902 ( 1963 ) . Demick, D. S., Voss, J. L., and Pickett, B. W. Effect of cooling, storage, glycerolization and spermatozoa1 numbers on equine fertility. 1. Anim. Scl. 43, 633-637 ( 1976). ElIery, John C., Graham, E. F., and Zemjanis, R. Artificial insemination of pony mares with semen frozen and stored in liquid nitrogen. Amer. J. Vet. Res. 32, 16931698 (1971). Klug, E., Treu, H., Hillman, H., and Heinze, H. Results of insemination of mares with fresh and frozen stallion semen. In “Proceedings of the Ist International Symposium on Equine Reproduction,” pp. 107-110, 1975.
10. Kotjagina, V. RationaI utilisation of frozen semen. Koneuod. Konnyi sport 4, 27 (1974). Anim. Breed. Abstr. 42, No. 5176 (1974). Il. Kotjagina, V., Platov, E., and Romhe, S. The fertility of stallion semen preserved at a temperature of -78”. Ziuotnovodtio 25, 83 (1963). Anim. Breed. A&r. 36, No. 2718 12.
p&et
should not
(1968).
Krause, D., and Grove, E. Deep-freezing of jackass and stallion semen in concentrated
form. 1. Reprod. Fert.
14,
139-141
(1967).
13. Lapteva, T. New work in semen freezing. Koneuod. Konnyi Spurt 4, 13 ( 1975). Anim. Breed. Abstr. 43, No. 3887 ( 1975). 14. Merkt, H. Artificial insemination and horse breeding. In “VI International Congress on Animal Reproduction and Artificial Insemination,” Vol. 2, pp. 1581-1583, 1968. 15. Merkt, H. Equine artificial insemination. Vet. Rec. 99, 69-71 ( 1976). 16. Nagase, H., Soejima, S., Niwa, T., Oshida, H., Sagara, Y., Ishizaki, N., and Hoshi, S. Studies on the freezing storage of stallion semen. I. Fertility results of stallion frozen semen in concentrated pellet form. Jupan J. Anim. Reprod. 12, 48-51 ( 1966). 17. Nagase, H., Soejima, S., Tomizuka, T., Oshida, H., Mikawa, T., Sagara, Y., Hoshi, S., and Niwa, T. Studies on the freezing storage of stallion semen. Il. Factors affecting survival rates of stallion spermatoza after freezing and thawing and results of a fertility trial. Japnn 1. Anim. Reprod. 12, 52-57
(1966).
Nishikawa, Y. Studies on the preservation of raw and frozen horse semen. In “Proceedings of the 1st International Symposium on Equine Reproduction,” pp. 99-104, 1975. 19. Nishikawa, Y., Waide, Y., and Shinomiya, S. Studies on deep freezing of horse spermatozoa. In “VI International Congress on Animal Reproduction and Artificial lnsemination,” Vol. 2, pp. 1589-1591, 1968. 20. Oshida, H., Mikawa, T., Horiuchi, S., Takahashi, H., Tomizuka, T., and Nagase, H. Studies on the freezing of staIlion semen. III. PeIlet frozen semen preserved in liquid nitrogen. Japan. I. Anin. Reprod. 13, 13618.
140
(1967).
21. Oshida, H., Tomizuka, T., Masaki, J,, Hanada, A., and Nagase, H. Some observations on freezing of stallion semen. In “VII lnternational Congress on Animal Reproduction and Artificial Insemination (Summary),” p. 304, 1972. 22. Pace, M. M., and Sullivan, J. J. Effect of timing of insemination, numbers of spermatozoa and extender components on the pregnancy rate in mares inseminated with frozen stallion semen. J. Reprod. Pert. SwppE.
23, 115-121.
(1975).
23. Pickett, B. W. Persona1 communication ( 1977 ), 24. Pickett, B. W., Back, D. G., Burwash, L. D., and Voss, J. L. The effect of extenders, spermatozoa1 numbers and rectal palpation on equine fertility. In “Proceedings, 5th TechnoIogical Conference on Artificial Jn-
FROZEN
25.
26.
27.
28.
29.
30.
STALLION
semination and Reproduction,” NAAAB, pp. 47-58, 1974. Pickett, 3. W., Burwash, L. D., Voss, J. L., and Back, D. G. Effect of seminal extenders on equine fertility. J. Anim. Sci. 40, II361143 (1975). Pickett, B. W., Faulkner, L. C., Seidel, G. E., Jr., Berndtson, W. E., and Voss, J. L. Reproductive physiology of the stallion. VI. Seminal and behavioral characteristics. J. Anint. Sci. 43, 617-625 ( 1976). Pick&, B. W., Vass, J, L., and N&on, L. D. Factors influencing the fertility of stallion spermatozna in an A.I. program. In “VII International Congress on Animal Reproduction and Artificial Insemination.” pp. IO491052, 1976. Platov, E. M., Fomina, E. L., and Murav’eva, L. N. Freezing of stallion semen. Zhiootnovodstvo 8, 73-76 (1975); &aim. Breed. Abstr. 44, No. 1058 (1976). PIatov, E. M., and Fomina, E. L. Freezing stallion semen in the horsebreeding of the USSR. In “VIII International Congress on Animal Reproduction and Artificial Insemination,” Communication Abstract, p. 320, 1976. Platov, E. M., Kotjagina, V. A., and Roman’kova, N. K. The use of frozen stallion semen in trotter breeding. Nauch. Trudy. Vsesoyerznyi Nauchno-IssEedovQteI’skii Inst.
SEMEN
357
Konevod. 26, 84-87 Abe.
43,
(1973); No, 12 ( 1975). M., Pustovaja, E.
Anim.
Breed.
31. Platov, E. S., Kotjagina, V. A., and Roman’kova, N. K. Long-term semen. Veterinariya storage of stallion (Moscow) 47, 109-1111 (1970); Anlm. Breed. Abstr. 39, No. 1437 ( 1971). 32. Rajamannan, A. H. J. Freezing and fertility studies with stallion semen. In “VI International Congress on Animal Reproduction and Artificial Insemination,” pp. 16011604, 1968. 33. Rombe, S., Kotjagina, V., and Piler, N. On frozen semen. Koneood. Konnyi Sport 35, 8
(1965);
Anim
Breed.
Abstr.
34,
No.
991 (1966). 34. Rombe, S. M., and Kotjagina, V. A. Prolonged storage of stallion semen. In “VI International Congress on Animal Reproduction and Artificial Insemination,” Vol. 2, pp. 1605-1606, 1969. 35. Sukalic, M., Herak, M., Pavuna, H., and Sic., R. Artificial insemination in horses with deep frozen semen in straws. In “VIII International Congress an Animal Reproduction and Artificial Insemination,” Communication Abstract, p. 335, 1976. 36. Sullivan, J. J., and Pickett, B. W. Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa. J. Reprod. F&. SuppE. 23, 29-34 (1975).
15, 355-357
(1978)
Characteristics
and Cryopreservation JOHN
American
of Stallion
Spermcttozoa
J. SULLIVAN
Breeders Service, DePorest, Wisconsin
53532
vided a maximal number of spermatozoa per collection day and was considered a practical ejaculation frequency for longterm preservation of stallion semen (36). A myriad of extenders and packaging systems have been used in the cryopreservation of stallion semen (l-6, 8-21, 28-35). Spermatozoa1 post-thaw survival has been satisfactory, mares have been inseminated, and normal foaIs have been produced. In general, fertility has been low and the fertility trials have been plagued by small numbers of mares and inadequate experimental controls for a fair evaluation of the fertility of frozen semen. The fertilizing capacity of spermatozoa of some stallions is more adversely affected than that of other stalIions (2, 22). The majority of mares inseminated with frozen-thawed semen conceived when inseminations occurred within 12 hr of ovulation (22). As of now, no completely satisfactory cryoprotectant has been found. Recent data indicate that glycerol depresses fertility of spermatozoa in Tris-yolk and creamgelatin extenders ( 7, 22). Cream-gelatin and skim milk extenders without ,glycerol provide optimal fertility only when the extended semen is not cooled and stored at 5°C (7, 23). Insemination of large numbers of motiIe spermatozoa masks these Received October 21, 1977; accepted Decrmdetrimental effects and offers some comber 2, 1977. pensation for an inferior extender (24, 25, Synopsis paper presented at the 14th Annual Meeting of the Society for Cryobiology, August 27). Therefore, to evaluate properly a 1977,Minneapolis,Minnesota. seminal extender, the number of motile
In North America, stallion seminal characteristics vary with the seasonof the year. Fur stallions from which were collected two ejaculates per day, once per week, total volume (including gel) from winter to spring increased about 38% for the first ejaculate and 21% for the second ejaculate. Gel-free semina1volume for first ejaculates ranged between 44 and 80 ml, and for second ejaculates, between 41 and 63 ml, depending upon season. Ejaculates containing gel were less frequent during the winter. More spermatozoa were produced in Iate spring and early summer than in the winter. Throughout the year, first ejaculates ranged between 10 and 22 x lo” spermatozoa, and second ejaculates, between 5 and 11 x IO” spermatozoa (26). When chasing an ejaculation frequency, consideration must be given to the schedule which most efficiently provides the maximum number of spermatozoa and most conveniently adapts to the program for which the stallions are maintained. One ejacuIate collected on alternate days proved to be the most effective and practical ejaculation frequency for stallions maintained in an artificial insemination program without semina1 storage. Two ejaculates per day, 2 days per week, pro-
355 0011~!2240/78/0153-0355$02.00/O All
Copyright Q 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
JOHir; J. SULLIVAN
356 spermatozoa per insemination exceed 100 x 10B. REFERENCES
1. Aliev, A. An improved technique of semen freezing, Konecod. Konnyi Sport 7, 30 (1975); Anim. Bmed. Ah&. 44, No. 18 (1976).
2. Baczynski, J., BieIanski, W., Bilik, K., Dram, S., and Zapletal, Z. Artificial insemination of horses. VI. Preliminary results of inseminating mares with frozen semen. Zeszyty Problemowe Postepow Nauk Rolniczych 124, 131-1313 ( 1972). Anim. Breed. Abstr. 41, 3.
4.
5.
6. 7.
8.
9.
No.
9 (1973).
Bader, H. Deep-freezing of stallion semen in pellet form. In. “VI Congress of Animal Reproduction and Artificial Insemination,” pp. 985-988, 1968. Bader, H., and MahIer, R. Experiments of freezing stallion semen by the pellet method, and its insemination. Zuchthygiene 3, 6-13 (196s). Anim. Breed. Abstr. 36, No. 2275 (1988). Barker, C. A. V., and Gandier, J. C. C. Pregnancy in a mare resulting from frozen epididymal spermatozoa. Canad. J. Camp. Med. 21, 47-51. (1957). Buell, J. R. A method for freezing stallion semen and tests of its fertility. Vet. Rec. 75, 900-902 ( 1963 ) . Demick, D. S., Voss, J. L., and Pickett, B. W. Effect of cooling, storage, glycerolization and spermatozoa1 numbers on equine fertility. 1. Anim. Scl. 43, 633-637 ( 1976). ElIery, John C., Graham, E. F., and Zemjanis, R. Artificial insemination of pony mares with semen frozen and stored in liquid nitrogen. Amer. J. Vet. Res. 32, 16931698 (1971). Klug, E., Treu, H., Hillman, H., and Heinze, H. Results of insemination of mares with fresh and frozen stallion semen. In “Proceedings of the Ist International Symposium on Equine Reproduction,” pp. 107-110, 1975.
10. Kotjagina, V. RationaI utilisation of frozen semen. Koneuod. Konnyi sport 4, 27 (1974). Anim. Breed. Abstr. 42, No. 5176 (1974). Il. Kotjagina, V., Platov, E., and Romhe, S. The fertility of stallion semen preserved at a temperature of -78”. Ziuotnovodtio 25, 83 (1963). Anim. Breed. A&r. 36, No. 2718 12.
p&et
should not
(1968).
Krause, D., and Grove, E. Deep-freezing of jackass and stallion semen in concentrated
form. 1. Reprod. Fert.
14,
139-141
(1967).
13. Lapteva, T. New work in semen freezing. Koneuod. Konnyi Spurt 4, 13 ( 1975). Anim. Breed. Abstr. 43, No. 3887 ( 1975). 14. Merkt, H. Artificial insemination and horse breeding. In “VI International Congress on Animal Reproduction and Artificial Insemination,” Vol. 2, pp. 1581-1583, 1968. 15. Merkt, H. Equine artificial insemination. Vet. Rec. 99, 69-71 ( 1976). 16. Nagase, H., Soejima, S., Niwa, T., Oshida, H., Sagara, Y., Ishizaki, N., and Hoshi, S. Studies on the freezing storage of stallion semen. I. Fertility results of stallion frozen semen in concentrated pellet form. Jupan J. Anim. Reprod. 12, 48-51 ( 1966). 17. Nagase, H., Soejima, S., Tomizuka, T., Oshida, H., Mikawa, T., Sagara, Y., Hoshi, S., and Niwa, T. Studies on the freezing storage of stallion semen. Il. Factors affecting survival rates of stallion spermatoza after freezing and thawing and results of a fertility trial. Japnn 1. Anim. Reprod. 12, 52-57
(1966).
Nishikawa, Y. Studies on the preservation of raw and frozen horse semen. In “Proceedings of the 1st International Symposium on Equine Reproduction,” pp. 99-104, 1975. 19. Nishikawa, Y., Waide, Y., and Shinomiya, S. Studies on deep freezing of horse spermatozoa. In “VI International Congress on Animal Reproduction and Artificial lnsemination,” Vol. 2, pp. 1589-1591, 1968. 20. Oshida, H., Mikawa, T., Horiuchi, S., Takahashi, H., Tomizuka, T., and Nagase, H. Studies on the freezing of staIlion semen. III. PeIlet frozen semen preserved in liquid nitrogen. Japan. I. Anin. Reprod. 13, 13618.
140
(1967).
21. Oshida, H., Tomizuka, T., Masaki, J,, Hanada, A., and Nagase, H. Some observations on freezing of stallion semen. In “VII lnternational Congress on Animal Reproduction and Artificial Insemination (Summary),” p. 304, 1972. 22. Pace, M. M., and Sullivan, J. J. Effect of timing of insemination, numbers of spermatozoa and extender components on the pregnancy rate in mares inseminated with frozen stallion semen. J. Reprod. Pert. SwppE.
23, 115-121.
(1975).
23. Pickett, B. W. Persona1 communication ( 1977 ), 24. Pickett, B. W., Back, D. G., Burwash, L. D., and Voss, J. L. The effect of extenders, spermatozoa1 numbers and rectal palpation on equine fertility. In “Proceedings, 5th TechnoIogical Conference on Artificial Jn-
FROZEN
25.
26.
27.
28.
29.
30.
STALLION
semination and Reproduction,” NAAAB, pp. 47-58, 1974. Pickett, 3. W., Burwash, L. D., Voss, J. L., and Back, D. G. Effect of seminal extenders on equine fertility. J. Anim. Sci. 40, II361143 (1975). Pickett, B. W., Faulkner, L. C., Seidel, G. E., Jr., Berndtson, W. E., and Voss, J. L. Reproductive physiology of the stallion. VI. Seminal and behavioral characteristics. J. Anint. Sci. 43, 617-625 ( 1976). Pick&, B. W., Vass, J, L., and N&on, L. D. Factors influencing the fertility of stallion spermatozna in an A.I. program. In “VII International Congress on Animal Reproduction and Artificial Insemination.” pp. IO491052, 1976. Platov, E. M., Fomina, E. L., and Murav’eva, L. N. Freezing of stallion semen. Zhiootnovodstvo 8, 73-76 (1975); &aim. Breed. Abstr. 44, No. 1058 (1976). PIatov, E. M., and Fomina, E. L. Freezing stallion semen in the horsebreeding of the USSR. In “VIII International Congress on Animal Reproduction and Artificial Insemination,” Communication Abstract, p. 320, 1976. Platov, E. M., Kotjagina, V. A., and Roman’kova, N. K. The use of frozen stallion semen in trotter breeding. Nauch. Trudy. Vsesoyerznyi Nauchno-IssEedovQteI’skii Inst.
SEMEN
357
Konevod. 26, 84-87 Abe.
43,
(1973); No, 12 ( 1975). M., Pustovaja, E.
Anim.
Breed.
31. Platov, E. S., Kotjagina, V. A., and Roman’kova, N. K. Long-term semen. Veterinariya storage of stallion (Moscow) 47, 109-1111 (1970); Anlm. Breed. Abstr. 39, No. 1437 ( 1971). 32. Rajamannan, A. H. J. Freezing and fertility studies with stallion semen. In “VI International Congress on Animal Reproduction and Artificial Insemination,” pp. 16011604, 1968. 33. Rombe, S., Kotjagina, V., and Piler, N. On frozen semen. Koneood. Konnyi Sport 35, 8
(1965);
Anim
Breed.
Abstr.
34,
No.
991 (1966). 34. Rombe, S. M., and Kotjagina, V. A. Prolonged storage of stallion semen. In “VI International Congress on Animal Reproduction and Artificial Insemination,” Vol. 2, pp. 1605-1606, 1969. 35. Sukalic, M., Herak, M., Pavuna, H., and Sic., R. Artificial insemination in horses with deep frozen semen in straws. In “VIII International Congress an Animal Reproduction and Artificial Insemination,” Communication Abstract, p. 335, 1976. 36. Sullivan, J. J., and Pickett, B. W. Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa. J. Reprod. F&. SuppE. 23, 29-34 (1975).
