Viability of Pony Stallion Semen in Different Temperature and Dilution

Janislene Mach Trentin, Murilo Farias Rodrigues, Gilson Antonio Pessoa, Mariani Farias Fiorenza, Ricardo Olimpio Schenatto, Laurence Boligon de Araujo, Karine Vargas Aires, Mara Iolanda Batistella Rubin

Abstract


Background: Artificial insemination and transport of cooled semen has been routinely used in equine industry in the past 20 years. However, more investigations are needed regarding the methods for long time storage in pony stallion semen. The effect of dilution and cooling temperature on pH, sperm motility, membrane integrity and mitochondrial activity were investigated before and after cooling of stallion semen.

Materials, Methods & Results: Two ejaculates each from nine Brazilian ponies were diluted in a nonbuffered powder milk extender cooled at 5°C or 15°C for 48 h using three different dilutions (1:1, 1:2 or 1:3). Data were assessed by analysis of variance and the rate comparison was performed using the Duncan test. Samples diluted 1:1 at 5oC or 15°C showed higher pH values (7.63 ± 0.34 e 7.57 ± 0.27) and lower progressive motility (10.3 ± 11.05, 17.08 ± 9.95). All samples cooled at 15°C also showed lower incidence of morphologically altered spermatozoa (1:1 = 55.84%; 1:2 = 51.84%; 1:3 = 49.95%) [P < 0.01]. Mitochondrial activity was higher on the 1:3 dilution (0.86 ± 0.19 nm) at 5°C and on the 1:1 (0.89 ± 0.23 nm), 1:2 (0.93 ± 0.2 nm) and 1:3 (0.92 ± 0.2 nm) dilutions at 15°C. Progressive motility was higher when semen was diluted 1:3 and cooled at 15°C (42.22 ± 12.38; P < 0.05). Considering mitochondrial activity, similar results were observed when different dilutions of semen were used (P > 0.05) despite time and temperature. The pH, progressive motility, mitochondrial activity and membrane integrity remained similar (P > 0.05) on fresh semen samples independent of the dilution grade used. The best results were obtained when semen was diluted 1:3 and cooled at 15°C. All dilution grades were safe for fresh semen and pH wasincreased when semen was diluted and cooled for 48 h.

Discussion: The methodology used to collect and process equine semen and semen from ponies is practically the same. Equine semen when sent for artificial insemination is usually cooled to 5°C. Our results showed that cooling reduces sperm viability, which has also been demonstrated by other studies. In contrast, the best cooling temperature was at 15°C. However, it is easier to keep the temperature at 5°C during transport, due to the large temperature oscillation that may occur during transportation. The semen of ponies can tolerate cooling at both 5 and 15°C. The 1:3 dilution cooled to 15°C provided better viability of pony sperm, and more stable pH during 48 h of cooling. Dilution 1:1 should not be used for cooling in powdered skim milk extender.


Keywords


concentration; cooling; equine; extender; semen.

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References


Aurich J.E. & Aurich C. 2006. Developments in European horse breeding and consequences for veterinarians in equine reproduction. Reproduction in Domestic Animals. 41: 275-279.

Aziz D.M., Ahlswede L. & Enbergs H. 2005. Application of MTT reduction assay to evaluate equine sperm viability. Theriogenology. 64: 1350-1356.

Ball B.A. 1998. Evaluation and use of transported equine semen. In: Equine Assisted Reproductive Technology Workshop (Davis, USA). pp.18-24.

Brinsko S.P., Rowan K.R., Varner D.D. & Blanchard T.L. 2000. Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa. Theriogenology. 53: 1641-1655.

Bogart R. & Mayer D.T. 1950. The effects of egg yolk on the various physical and chemical factors detrimental to spermatozoa viability. Journal of Animal Science. 9: 143-152.

Crespilho A.M., Spizziri B.E., Meyers M. & Graham J.K. 2013. The effect of cholesterol addition, buffer, and pH on equine sperm stored at 5°C. Journal of Equine Veterinary Science. 33(8): 663-666.

Gibb Z. & Aitken R.J. 2016. The Impact of Sperm Metabolism during In Vitro Storage: The Stallion as a Model. BioMed Research International. 2016: 1-8.

Gottschalk M., Sieme H., Martinsson G. & Ottmar D. 2016. Analysis of breed effects on semen traits in light horse, warmblood, and draught horse breeds. Theriogenology. 85: 1375-1381.

Harrison R.A.P. & White I.G. 1972. Glycolytic enzymes in the spermatozoa and cytoplasmic droplets of bull, boar and ram, and their leakage after shock. Journal of Reproduction and Fertility. 30: 105-115.

Hayden S.S., Blanchard T.L., Brinsko S.P., Varner D.D., Hinrichs K. & Love C.C. 2015. The “dilution effect” in stallion sperm. Theriogenology. 83: 772-777.

Heckenbichler S., Deichsel K., Peters P. & Aurich C. 2011. Quality and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology. 75: 849-856.

Jasko D.J., Hathaway J.A., Schaltenbrand V.L., Simper W.D. & Squires E.L. 1992. Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology. 37: 1241-1252.

Katila T. 1997. Procedures for handling fresh stallion semen. Theriogenology. 48: 1217-1227.

Kankofer M., Kolm G., Aurich J. & Aurich C. 2005. Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5°C. Theriogenology. 63: 1354-1365.

Kenney R.M., Bergman R.V., Cooper W.L. & Morse G.M. 1975. Minimal contamination techniques for breeding mares: techniques and preliminary findings. In: Proceedings of the 21st Annual Convention of the American Association of Equine Practitioners (Boston, USA). pp.327-336.

Kiser A.M., Brinsko S.P., Love C.C., Varner D.D., Sudderth K. & Blanchard T.L. 2014. Relationship of Sperm Quality to Fertility after 4 Days of Cooled Storage of Equine Semen. Journal of Equine Veterinary Science. 34: 602-605.

Klug E. & Sieme H. 2003. Samenübertragungbeim Pferd in Theorie und Praxis. 5° volliguberarbeitete Auflage. Hannover: Verlag M. & H. Alfeld, 144p.

Lomeo A.M. & Giambersio A.M. 1991. ‘Water-test’: a simple method to assess sperm-membrane integrity. International Journal of Andrology. 14: 278-282.

Love C.C., Thompson J.A., Lowry V.K. & Varner D.D. 2001. The relationship between chromatin quality and fertility of chilled stallion sperm. In: Proceedings of the 47th Annual Convention of the American Association of Equine Practitioners (San Diego, USA). pp.229-231.

Malmgren L. 1998. Effectiveness of two systems for transporting equine semen. Theriogenology. 50: 833-839.

Monteiro G.A., Guasti P.N., Rocha A.S., Martin I., Sancler-Silva Y.F.R., Dell’Aqua C.P.F., Dell’Aqua Jr. J.A. & Papa F.O. 2013. Effect of storage time and temperature of equine epididymis on the viability, motion parameters, and freezability of epididymal sperm. Journal of Equine Veterinary Science. 33(3): 169-173.

Moran D.M., Jasko D.J., Squires E.L. & Amann R.P. 1992. Determinationof temperature and cooling rate which induce cold shock in stallionspermatozoa. Theriogenology. 38: 999-1012.

Neild D.M., Chaves M.G., Flores M., Miragaya M.H., Gonzalez E. & Agüerro A. 2000. The host test and its relationship to fertility in the stallion. Andrology. 32: 351-355.

Oliveira R.A., Scarlet D., Ille N. & Aurich C. 2017. Cooled-storage of equine semen does not induce major changes in sperm DNA methylation. Theriogenology. 89: 289-294.

Pagl R., Aurich J.E., Müller-Schlösser F., Kankofer M. & Aurich C. 2006. Comparison of an extender containing defined milk protein fractions with a skim milk-base extender for storage equine semen at 5°C. Theriogenology. 66: 1115-1122.

Pickett B.W. & Amann R.P. 1987. Extension and storage of stallion spermatozoa: a review. Journal of Equine Veterinary Science. 7: 289-302.

Plaza Davila J., Martin Muñoz P., Gallardo Bolaños J.M., Stout T.A.E., Gadella B.M., Tapia J.A., Balao da Silva C., Ortega-Ferrusola C. & Peña F.J. 2016. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation. Reproduction. 152: 683-694.

Price S., Aurich J., Davies-Morel M. & Aurich C. 2008. Effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15 degrees C. Reproduction in Domestic Animals. 43: 261-266.

Province C.A., Squires E.L., Pickett B.W. & Amann R.P. 1985. Cooling rates, storage temperatures and fertility of extended equine spermatozoa. Theriogenology. 23: 925-934.

Ramires Neto C., Silva Y.F.R.S., Resende H.L., Guasti P.N., Monteiro G.A., Papa P.M., Dell’Aqua Junior J.A., Puoli Filho J.N.P., Alvarenga M.A. & Papa F.O. 2015. Control methods an evaluation of bacterial growth on fresh and cooled stallion semen. Journal of Equine Veterinary Science. 35(4): 277-282.

Squires E.L., Pickett B.M., Graham J.K., Vanderwall D.K., Mccue P.M. & Bruemmer J.E. 1999. Cooled and frozen stallion semen. Animal Reproduction and Biotechnology Laboratory. Colorado State University Bulletin. 9: 1-38.

Varner D.D., Blanchard T.L., Love C.L., Garcia M.C. & Kenney R.M. 1988. Effects of cooling rate and storage temperature on equine spermatozoal motility parameters. Theriogenology. 29: 1043-1053.

Varner D.D., Blanchard T.L., Meyers P.J. & Meyers S.A. 1989. Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20ºC. Theriogenology. 32: 515-525.

Vidament M., Magistrini M., Le Foll Y., Levillain N., Yvon J.M., Duchamp G. & Blesbois E. 2012. Temperatures from 4 to 15°C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in inra96 extender. Theriogenology. 78: 297-307.




DOI: https://doi.org/10.22456/1679-9216.80492

Copyright (c) 2018 Janislene Mach Trentin, Murilo Farias Rodrigues, Gilson Antonio Pessoa, Mariani Farias Fiorenza, Ricardo Olimpio Schenatto, Laurence Boligon de Araujo, Karine Vargas Aires, Mara Iolanda Batistella Rubin

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