Improvement of cryopreservation protocol in both purebred horses including Spanish horses
Abstract
There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio.Downloads
References
Alvarenga MA, Papa FO, Landim-Alvarenga FC, Medeiros AS, 2005. Amides as cryoprotectants for freezing stallion semen: a review. Anim Reprod Sci 89: 105-113. https://doi.org/10.1016/j.anireprosci.2005.07.001
Aurich C, 2008. Recent advances in cooled-semen technology. Anim Reprod Sci 107: 268-275. https://doi.org/10.1016/j.anireprosci.2008.04.015
Bamba K, 1988. Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosin-nigrosin stain. Theriogenology 29: 1245-1251. https://doi.org/10.1016/0093-691X(88)90004-0
Bedford SJ, Varner DD, Meyers SA, 2000. Effects of cryopreservation on the acrosomal status of stallion spermatozoa. J Reprod Fertil Suppl 56: 133-140.
Benito D, Alvarez A, Crespo F, Mateos E, Gómez-Cuétara C, Serres C, 2003. Análisis computerizado del semen de caballo de Pura Raza Española. IV Congreso Ibérico de Reproducción Animal, Las Palmas (España), Jul 11-12. p: 90.
Brinsko SP, Crockett EC, Squires EL, 2000. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 54: 129-136. https://doi.org/10.1016/S0093-691X(00)00331-9
Brum AM, Sabeur K, Ball BA, 2008. Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation. Theriogenology 69: 1041-55. https://doi.org/10.1016/j.theriogenology.2008.01.014
Cochran JD, Amann RP, Squires EL, Pickett BW, 1983. Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws. Theriogenology 20: 735-741. https://doi.org/10.1016/0093-691X(83)90194-2
Cochran JD, Amann RP, Froman DP, Pickett BW, 1984. Effects of centrifugation, glycerol level, cooling to 5°C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 22: 25-38. https://doi.org/10.1016/0093-691X(84)90470-9
Dell'Aqua JA, Papa FO, Alvarenga MA, Zahn FS, 2001. Effect of centrifugation and packing system on sperm parameters of equine frozen semen. Anim Reprod Sci 68: 324-325.
Gallardo Bolanos JM, Bolao da Silva CM, Martín Muñoz P, Morillo Rodríguez A, Plaza Dávila M, Rodríguez-Martínez H, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ. 2014. Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. Reproduction 148: 221-235. https://doi.org/10.1530/REP-13-0191
Gibb Z, Aitken RJ, 2016. Recent developments in stallion semen preservation. J Equine Vet Sci 43: 29-36. https://doi.org/10.1016/j.jevs.2016.06.006
Gutiérrez-Cepeda L, Fernández A, Crespo F, Gosálvez J, Serres C, 2011. Simple and economic coloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure. Anim Reprod Sci 124: 85-89. https://doi.org/10.1016/j.anireprosci.2011.02.001
Hidalgo M, Ortiz I, Dorado J, Morrell JM, Gosálvez J, Consuegra C, Díaz-Jiménez M, Pereira B, Crespo F, 2017. Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation. Anim Reprod Sci 185: 83-88. https://doi.org/10.1016/j.anireprosci.2017.08.005
Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, Schauwer C, De Kruif A, De Vliegher S, 2011. Sperm selection using single layer centrifugation prior to cryopreservation can increase thawed sperm quality in stallions. Equine Vet J 43 (Supp 40): 35-41. https://doi.org/10.1111/j.2042-3306.2011.00489.x
Janett F, Thun R, Bettschen S, Burger D, Hassig M, 2003. Seasonal changes of semen quality and freezability in Franches–Montagnes stallions. Anim Reprod Sci 77: 213-221. https://doi.org/10.1016/S0378-4320(03)00039-3
Janett F, Sacher K, Hassig M, Thun R, 2012. Quality of raw and of cold-stored semen in Icelandic stallions. J Equine Vet Sci 32: 590-595. https://doi.org/10.1016/j.jevs.2012.02.004
Johanisson A, Morrell JM, Thoren J, Jönsson M, Dalin A, Rodríguez-Martínez H, 2009. Colloidal centrifugation with Andocoll-ETM prolongs stallion sperm motility, viability and chromatin integrity. Anim Reprod Sci 116: 119-128. https://doi.org/10.1016/j.anireprosci.2009.01.008
Keeney RM, Bergman RV, Cooper WL, Morse GW, 1975. Minimal contamination techniques for breeding mares: technique and preliminary finding. Am Assoc Equine Pract, 327-336.
Loomis PR, Amann RP, Squires EL, Pickett BW, 1983. Fertility of stallion spermatozoa frozen in an EDTA-lactose-egg-yolk extender and packaged in 0.5-mL straws. J Anim Sci 56: 687-693. https://doi.org/10.2527/jas1983.563687x
Loomis PR, Graham JK, 2008. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 105: 119-128. https://doi.org/10.1016/j.anireprosci.2007.11.010
Macias Garcia B., Morrell JM, Ortega-Ferrusola C, Gonzalez-Fernandez L, Tapia JA, Rodriguez-Martinez H, Pena FJ, 2009. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen. Anim Reprod Sci 114: 193-202. https://doi.org/10.1016/j.anireprosci.2008.08.025
Madeira P, 2017. El Pura Raza Española, el valor de marca en el mundo. El caballo español PRE 237: 34-38.
Melo CM, Zahn FS, Martin I, Orlandi C, Dell'Aqua Jr, JA, Alvarenga MA, Papa FO, 2007. Influence of semen storage and cryoprotectant on post-thaw viability and fertility of stallion spermatozoa. J Equine Vet Sci 27: 171-175. https://doi.org/10.1016/j.jevs.2007.02.008
Metcalf ES, 2007. The efficient use of equine cryopreserved semen. Theriogenology 68: 423-428. https://doi.org/10.1016/j.theriogenology.2007.04.039
Miró J, Taberner E, Rivera M, Peña A, Medrano A, Rigau T, Peñalba A, 2009. Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen. Theriogenology 72: 1017-1022. https://doi.org/10.1016/j.theriogenology.2009.06.012
Morató R, Souza-Soares J, Orero G, Mogas T, Miró J, 2013. Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion. Anim Reprod Sci 139: 62-68. https://doi.org/10.1016/j.anireprosci.2013.04.005
Morillo Rodríguez A, Balao da Silva C, Macías-García B, Gallardo Bolanos JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ, 2012. Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. Reprod Domest Anim 47: 995-1002. https://doi.org/10.1111/j.1439-0531.2012.02005.x
Morrell JM, Johannisson A, Strutz H, Dalin A, Rodriguez-Martinez H, 2009. Colloidal centrifugation of stallion semen: Changes in sperm motility, velocity, and chromatin integrity during storage. J Equine Vet Sci 29: 24-32. https://doi.org/10.1016/j.jevs.2008.11.006
Morrell JM, Rodríguez-Martínez H, Johanisson A, 2010. Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large simple size. Equine Vet J 42: 579-585. https://doi.org/10.1111/j.2042-3306.2010.00101.x
Morrell JM, Lagerqvist A, Humblot P, Johanisson A, 2017. Effect of single layer centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen. Reprod Fertil Dev 29: 1039-1045. https://doi.org/10.1071/RD15440
Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colembrander B, Agüero A, 2003. Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation. Theriogenology 59: 1693-1705. https://doi.org/10.1016/S0093-691X(02)01231-1
Peña FJ. García BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO, 2011. Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols? Theriogenology 76: 1177-1186. https://doi.org/10.1016/j.theriogenology.2011.06.023
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K, 2007. Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro. Theriogenolgy 68: 560-566. https://doi.org/10.1016/j.theriogenology.2007.04.057
Rota A, Magelli C, Panzani D, Camilo F, 2008 Effect of extender, centrifugation and removal of seminal plasma on cooled-preserved Amiata donkey spermatozoa. 69: 176-185.
Samper JC, Vidament M, Katila T, Newcombe J, Estrada A, Sargeant J, 2002. Analysis of some factors associated with pregnancy rates of frozen semen: a multi-center study. Theriogenology 58: 647-650. https://doi.org/10.1016/S0093-691X(02)00754-9
Serres C, Rodríguez A, Alvarez AL, Santiago I, Gabriel J, Gómez-Cuétara C, Mateos E, 2002. Effect of centrifugation and temperature on the motility and the plasma membrane integrity of Zamorano-Leonés donkey semen. Theriogenology 58: 329-332. https://doi.org/10.1016/S0093-691X(02)00876-2
Sieme H, Martinsson G, Rauterberg H, Walter K, Aurich C, Petzoldt R, Klug E, 2003. Application of techniques for sperm selection in fresh and frozen-thawed stallion semen. Reprod Domest Anim 38: 134-140. https://doi.org/10.1046/j.1439-0531.2003.00416.x
Sieme H, Katila T, Klug E, 2004. Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions. Theriogenology 61: 769-784. https://doi.org/10.1016/S0093-691X(03)00251-6
Sieme H, Harrison RAP, Petrunkina AM, 2008. Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim Reprod Sci 107: 276-292. https://doi.org/10.1016/j.anireprosci.2008.05.001
Varner DD, Love CC, Brinsko SP, Blanchard TL, Hartman DL, Bliss SB, Carroll BS, Eslick MC, 2008. Semen processing for the subfertile stallion. J Equine Vet Sci 28: 677-685. https://doi.org/10.1016/j.jevs.2008.10.012
Varner DD, Gibb Z, Aitken RJ, 2015. Stallion fertility: a focus on the spermatozoon. Equine Vet J 47: 16-24. https://doi.org/10.1111/evj.12308
Vázquez E, 2017. 525 años del Pura Raza Española en América. El caballo español PRE 238: 42-46.
© CSIC. Manuscripts published in both the printed and online versions of this Journal are the property of Consejo Superior de Investigaciones Científicas, and quoting this source is a requirement for any partial or full reproduction.
All contents of this electronic edition, except where otherwise noted, are distributed under a “Creative Commons Attribution 4.0 International” (CC BY 4.0) License. You may read here the basic information and the legal text of the license. The indication of the CC BY 4.0 License must be expressly stated in this way when necessary.
Self-archiving in repositories, personal webpages or similar, of any version other than the published by the Editor, is not allowed.
