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Veterinary sciences2024; 11(2); doi: 10.3390/vetsci11020065

Freezing Stallion Semen-What Do We Need to Focus on for the Future?

Abstract: Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation.
Publication Date: 2024-02-02 PubMed ID: 38393083PubMed Central: PMC10893461DOI: 10.3390/vetsci11020065Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article discusses the challenges and potential improvements related to freezing horse semen for artificial insemination (AI) procedures. It proposes advancements in areas such as stallion nutrition, semen collection, and the identification of biomarkers for better sperm freezability.

Artificial Insemination and the Use of Frozen Semen

  • The article begins by highlighting the frequent use of artificial insemination (AI) in sport horses breeding, not including Thoroughbreds. However, most AI procedures use cooled semen over frozen semen.
  • This preference is mainly due to challenges in developing a universally-effective freezing protocol for all ejaculates, and the short life of thawed spermatozoa, necessitating insemination close to ovulation.

Advantages of Using Frozen Semen

  • Increased use of frozen semen could potentially improve biosecurity and offer greater flexibility in semen deliveries to the stud. It would also allow for more stallion choices.
  • Because of the smaller volume required for AI compared to cooled semen, the use of frozen semen could also potentially reduce the amount of antibiotics used in semen extenders.

Challenges with the Use of Frozen Semen

  • The article acknowledges that there are considerable variations in the cryosurvival – the ability of living organisms to withstand freezing conditions – of spermatozoa from different stallions. This results in stallions being classified as either good or bad freezers.
  • Stallion sperm membranes are considerably prone to lipid peroxidation, a process which can damage cell structures.

Potential Improvements in the Process

  • Improvements in freezing stallion semen could be achieved through various means, including optimizing stallion nutrition, refining the semen collection regimen, improving the extender used, increasing the removal of seminal plasma, and bettering the cooling protocol.
  • Research into antioxidants has yet to determine an additive that would benefit all stallions universally, suggesting the need for individualized approaches or further research in this area.
  • In future, identifying biomarkers for sperm freezability could help in choosing which ejaculates are best suited for cryopreservation, an area that currently faces challenges due to significant variability between different stallions.

Cite This Article

APA
Al-Kass Z, Morrell JM. (2024). Freezing Stallion Semen-What Do We Need to Focus on for the Future? Vet Sci, 11(2). https://doi.org/10.3390/vetsci11020065

Publication

ISSN: 2306-7381
NlmUniqueID: 101680127
Country: Switzerland
Language: English
Volume: 11
Issue: 2

Researcher Affiliations

Al-Kass, Ziyad
  • Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, SE-75007 Uppsala, Sweden.
  • Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul 41002, Iraq.
Morrell, Jane M
  • Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, SE-75007 Uppsala, Sweden.

Conflict of Interest Statement

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

This article includes 106 references
  1. Givens M.D.. Review: Risks of disease transmission through semen in cattle.. Animal 2018;12((Suppl. S1)):s165–s171.
    doi: 10.1017/S1751731118000708pubmed: 29665869google scholar: lookup
  2. Mulu M., Moges N., Adane M.. Review on process, advantages and disadvantage of artificial insemination in cattle.. Int. J. Vet. Sci. Anim. Husb. 2018;3:8–13.
  3. Aurich J.E.. Artificial insemination in horses—More than a century of practice and research.. J. Equine Vet. Sci. 2012;32:458–463.
  4. Katila T.A., Reilas T., Nivola K., Peltonen T., Virtala A.-M.. A 15-year survey of reproductive efficiency of Standardbred and Finnhorse trotters in Finland—Descriptive results.. Acta Vet. Scand. 2010;52:40.
    doi: 10.1186/1751-0147-52-40pmc: PMC2907380pubmed: 20546559google scholar: lookup
  5. Vidament M., Dupere A.M., Julienne P., Evian A., Noue P., Palmer E.. Equine frozen semen: Freezability and fertility field results.. Theriogenology 2008;48:907–917.
    doi: 10.1016/S0093-691X(97)00319-1pubmed: 16728182google scholar: lookup
  6. Blottner S., Warnke C., Tuchscherer A., Heinen V., Torner H.. Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season.. Anim. Reprod. Sci. 2001;65:75–88.
    doi: 10.1016/S0378-4320(00)00214-1pubmed: 11182510google scholar: lookup
  7. Peña F.J., O’Flaherty C., Ortiz Rodríguez J.M., Martín Cano F.E., Gaitskell-Phillips G.L., Gil M.C., Ferrusola C.O.. Redox regulation and oxidative stress: The particular case of the stallion spermatozoa.. Antioxidants 2019;8:567.
    doi: 10.3390/antiox8110567pmc: PMC6912273pubmed: 31752408google scholar: lookup
  8. Kuisma P., Andersson M., Koskinen E., Katila T.. Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods.. Acta Vet. Scand. 2006;48:14–21.
    doi: 10.1186/1751-0147-48-14pmc: PMC1564023pubmed: 16987393google scholar: lookup
  9. Reyes-Perea A.D., Diaw M., Guerrero-Netro H.M.. Assisted Reproductive Technologies in Horses: A Systematic Review.. Biomed. J. Sci. Tech. Res. 2023;49:41098–41102.
  10. Colenbrander B., Gadella B.M., Stout T.A.E.. The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility.. Reprod. Domest. Anim. 2003;38:305–311.
  11. Loomis P.R.. Advanced methods for handling and preparation of stallion semen.. Vet. Clin. N. Am. Equine Pract. 2006;22:663–676.
    doi: 10.1016/j.cveq.2006.07.002pubmed: 17129794google scholar: lookup
  12. Loomis P.R., Graham J.K.. Commercial semen freezing: Individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.. Anim. Reprod. Sci. 2008;108:119–12813.
  13. Vašíček J., Baláži A., Svoradová A., Vozaf J., Dujíčková L., Makarevich A.V., Bauer M., Chrenek P.. Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.. Int. J. Mol. Sci. 2022;23:5920.
    doi: 10.3390/ijms23115920pmc: PMC9180808pubmed: 35682598google scholar: lookup
  14. Prien S., Iacovides S.. Cryoprotectants & Cryopreservation of Equine Semen: A Review of Industry Cryoprotectants and the Effects of Cryopreservation on Equine Semen Membranes.. J. Dairy Vet. Anim. Res. 2016;3:63.
  15. Amann B.W., Pickett B.W.. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa.. J. Equine Vet. Sci. 1987;7:145–173.
  16. Aurich J., Kuhl J., Tichy A., Aurich C.. Efficiency of Semen Cryopreservation in Stallions.. Animals 2020;10:1033–1045.
    doi: 10.3390/ani10061033pmc: PMC7341245pubmed: 32545785google scholar: lookup
  17. Bazzano M., Laus F., Spaterna A., Marchegiani A.. Use of nutraceuticals in the stallion: Effects on semen quality and preservation.. Reprod. Domest. Anim. 2021;56:951–957.
    doi: 10.1111/rda.13934pmc: PMC8360198pubmed: 33772909google scholar: lookup
  18. Brinsko S.P., Varner D.D., Love C.C., Blanchard T.L., Day B.C., Wilson M.E.. Effect of feeding a DHA-enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen.. Theriogenology 2005;63:1519–1527.
  19. Aurich C.. Recent advances in cooled-semen technology.. Anim. Reprod. Sci. 2008;107:268–275.
  20. Schmid-Lausigk Y., Aurich C.. Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter.. Theriogenology 2014;81:966–973.
  21. Rodrigues P.G., de Moura R.S., Rocha L.G.P., Bottino M.P., Nichi M., Maculan R., Bertechini A.G., Souza J.C.. Dietary polyunsaturated fatty acid supplementation improves the quality of stallion cryopreserved semen.. J. Equine Vet. Sci. 2017;54:18–23.
  22. Van Dorland A., Janett F., Bruckmaier R., Wach-Gygax L., Jeannerat E., Bollwein H., Sieme H., Burger D.. Herbal yeast product, Equi-Strath®, alters the antioxidant status of stallion semen.. Anim. Reprod. Sci. 2019;208:106119.
  23. Gee E.K., Bruemmer J.E., Siciliano P.D., McCue P.M., Squires E.L.. Effects of dietary vitamin E supplementation on spermatozoal quality in stallions with suboptimal post-thaw motility.. Anim. Reprod. Sci. 2008;107:324–325.
  24. Freitas M.L., Bouéres C.S., Pignataro T.A., Gonçalves de Oliveira F.J., de Oliveira Viu M.A., de Oliveira R.A.. Quality of fresh, cooled, and frozen semen from stallions supplemented with antioxidants and fatty acids.. J. Equine Vet. Sci. 2016;46:1–6.
  25. Freitas M.L., Pignataro T.A., Pivato I., Cunha A.M., Oliveira R.A.D.. Quality of frozen-thawed semen from stallions supplemented with nutraceutical; Proceedings of the 30th Annual Meeting of the Brazilian Embryo Technology Society (SBTE); Foz do Iguaçu, PR, Brazil.. 25–27 August 2016; pp. 549–564.
  26. Gerlach T., Aurich J.E.. Regulation of seasonal reproductive activity in the stallion, ram and hamster.. Anim. Reprod. Sci. 2000;58:197–213.
    doi: 10.1016/S0378-4320(99)00093-7pubmed: 10708895google scholar: lookup
  27. Aurich C., Burgmann F., Hoppe H.. Opioid regulation of LH and prolactin release in the horse—Identical or independent endocrine pathways?. Anim. Reprod. Sci. 1996;44:127–134.
  28. Aurich C.. Seasonal Influences on Cooled-Shipped and Frozen-Thawed Stallion Semen.. J. Equine Vet. Sci. 2016;43:S1–S5.
  29. Crespo F., Wilson R., Díaz-Jimenez M., Consuegra C., Dorado J., García Barrado B., Gosálvez J., Louis Smit R., Hidalgo M., Johnston S.. Effect of season on individual stallion semen characteristics.. Anim. Reprod. Sci. 2020;223:106641.
  30. Waheed M.M., Ghoneim I.M., Abdou M.S.S.. Morphometric Characteristics of Spermatozoa in the Arabian Horse With Regard to Season, Age, Sperm Concentration, and Fertility.. J. Equine Vet. Sci. 2015;35:244–249.
  31. Mislei B., Bucci D., Malama E., Bollwein H., Mari G.. Seasonal changes in ROS concentrations and sperm quality in unfrozen and frozen-thawed stallion semen.. Theriogenology 2020;144:89–97.
  32. Janett F., Thun R., Niederer K., Burger D., Hässig M.. Seasonal changes in semen quality and freezability in the Warmblood stallion.. Theriogenology 2003;60:453–461.
    doi: 10.1016/S0093-691X(03)00046-3pubmed: 12763159google scholar: lookup
  33. Janett F., Thun R., Bettschen S., Burger D., Hassig M.. Seasonal changes of semen quality and freezability in Franches–Montagnes stallions.. Anim. Reprod. Sci. 2003;77:213–221.
    doi: 10.1016/S0378-4320(03)00039-3pubmed: 12695055google scholar: lookup
  34. Magistrini M., Chanteloube P., Palmer E.. Influence of season and frequency of ejaculation on production of stallion semen for freezing.. J. Reprod. Fertil. 1987;35:127–133.
    pubmed: 3479568
  35. Wach-Gygax L., Burger D., Malama E., Bollwein E., Fleisch A., Jeannerat E., Thomas S., Schuler G., Janett F.. Seasonal changes of DNA fragmentation and quality of raw and cold-stored stallion spermatozoa.. Theriogenology 2017;99:98–104.
  36. Morrell J.M., Winblad C., Georgakas A., Stuhtmann G., Humblot P., Johannisson A.. Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation.. Anim. Reprod. Sci. 2013;140:62–69.
  37. Sieme H., Troedsson M.H., Weinrich S., Klug E.. Influence of exogenous GnRH on sexual behavior and frozen/thawed semen viability in stallions during the non-breeding season.. Theriogenology 2004;61:159–171.
    doi: 10.1016/S0093-691X(03)00205-Xpubmed: 14643870google scholar: lookup
  38. Murphy B.A., Walsh C.M., Woodward E.M., Prendergast R.L., Ryle J.P., Fallon L.H., Troedsson M.H.. Blue light from individual light masks directed at a single eye advances the breeding season in mares.. Equine Vet. J. 2014;46:601–605.
    doi: 10.1111/evj.12153pubmed: 23909505google scholar: lookup
  39. Sieme T., Katila E., Klug E.. Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions.. Theriogenology 2004;61:769–784.
    doi: 10.1016/S0093-691X(03)00251-6pubmed: 14698065google scholar: lookup
  40. Lindeberg H., Karjalainen H., Koskinen E., Katila T.. Quality of stallion semen obtained by a new semen collection phantom (Equidame®) versus a missouri® artificial vagina.. Theriogenology 1999;51:1157–1173.
    doi: 10.1016/S0093-691X(99)80019-3pubmed: 10729034google scholar: lookup
  41. Troedsson M.H.T., Loset K., Alghamdi A.M., Dahms B., Crabo B.G.. Interaction between equine semen and the endometrium: The inflammatory response to semen.. Anim. Reprod. Sci. 2001;8:273–278.
    doi: 10.1016/S0378-4320(01)00164-6pubmed: 11744271google scholar: lookup
  42. Hidalgo M., Ortiz J., Dorado I., Morrell J.M., Gosálvez J., Consuegra C., Diaz-Jimenez M., Pereira B., Crespo F.. Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation.. Anim. Reprod. Sci. 2017;185:83–88.
  43. Aziz D.M., Enbergs H.. Effect of Seminal Plasma Removal on Cell Membrane, Acrosomal Integrity and Mitochondrial Activity of Cooled Stallion Semen.. J. Adv. Vet. Res. 2012;2:148–152.
  44. Brinsko S.P., Crockett E.C., Squires E.L.. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage.. Theriogenology 2000;54:129–136.
    doi: 10.1016/S0093-691X(00)00331-9pubmed: 10990354google scholar: lookup
  45. Waite J.A., Love C.C., Brinsko S.P., Teague S.R., Salazar J.L., Jr., Mancill S.S., Varner D.D.. Factors impacting equine sperm recovery rate and quality following cushioned centrifugation.. Theriogenology 2008;70:704–714.
  46. Barrier-Battut I., Bonnet C., Giraudo A., Dubois C., Caillaud M., Vidament M.. Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa.. Reprod. Domest. Anim. 2013;48:64–71.
  47. Ramires Neto C., Monteiro G.A., Soares R.F., Pedrazzi C., Dell’aqua J.A., Papa F.O., Castro-Chaves M.M., Alvarenga M.A.. New seminal plasma removal method for freezing stallion semen.. Theriogenology 2013;79:1120–1123.
  48. Vigolo V., Gautier C., Falomo M.E., Aurich C.. Selection of frozen–thawed stallion semen by microfluidic technology.. Reprod. Domest. Anim. 2023;58:443–449.
    doi: 10.1111/rda.14305pubmed: 36510754google scholar: lookup
  49. Al-Essawe E., Johannisson A., Wulf M., Aurich C., Morrell J.M.. Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma.. Cryobiology 2018;81:145–152.
  50. Al-Essawe E.M., Johannisson A., Wulf M., Aurich C., Morrell J.M.. Addition of seminal plasma to thawed stallion spermatozoa did not repair cryoinjuries.. Anim. Reprod. Sci. 2018;196:48–58.
  51. Al-Essawe E., Wallgren M., Wulf M., Aurich C., Macías-García B., Sjunnesson Y., Morrell J.M.. Seminal plasma influences the fertilizing potential of cryopreserved stallion sperm.. Theriogenology 2018;115:99–107.
  52. Neuhauser S., Dörfel S., Handler J.. Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa.. Andrology 2015;3:536–543.
    doi: 10.1111/andr.12003pubmed: 25755119google scholar: lookup
  53. Liu Z., Foote R.H., Brockett C.C.. Survival of Bull Sperm Frozen at Different Rates in Media Varying in Osmolarity.. Cryobiology 1998;37:219–230.
    doi: 10.1006/cryo.1998.2117pubmed: 9787067google scholar: lookup
  54. Khalil W.A., El-Harairy M.A., Zeidan A.E.B., Hassan M.A.E., Mohey-Elsaeed O.. Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights.. Int. J. Vet. Sci. Med. 2017;22:S49–S56.
    doi: 10.1016/j.ijvsm.2017.11.001pmc: PMC6161860pubmed: 30761321google scholar: lookup
  55. Sieme H., Martinsson G., Rauterberg H., Walter K., Aurich C., Petzoldt R., Klug E.. Application of techniques for sperm selection in fresh and frozen-thawed stallion semen.. Reprod. Domest. Anim. 2003;38:134–140.
  56. Macías García B., Morrell J.M., Ortega-Ferrusola C., González-Fernández L., Tapia J.A., Rodriguez-Martínez H., Peña F.J.. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen.. Ani. Reprod. Sci. 2009;114:193–202.
  57. Gloria A., Carluccio A., Wegher L., Robbe D., Befacchia G., Contri A.. Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates.. J. Anim. Sci. Biotechnol. 2016;7:30.
    doi: 10.1186/s40104-016-0088-6pmc: PMC4858911pubmed: 27158492google scholar: lookup
  58. Colleoni S., Lagutina I., Lazzari G., Rodriguez-Martinez H., Galli C., Morrell J.M.. New Methods for Selecting Stallion Spermatozoa for Assisted Reproduction.. J. Equine Vet. Sci. 2011;31:536–541.
  59. Umair M., Henning H., Stout T.A.E., Claes A.. A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index.. Animals 2021;11:1973.
    doi: 10.3390/ani11071973pmc: PMC8300244pubmed: 34359101google scholar: lookup
  60. Morrell J.M., Nunes M. M. Practical guide to Single Layer Centrifugation of stallion semen.. Equine Vet. Educ. 2018;30:392–398.
    doi: 10.1111/eve.12658google scholar: lookup
  61. Malaluang P., Wagner L.H., Spergser J., Aurich C., Morrell J.M.. Colloid centrifugation as an alternative to antibiotics in stallion semen preparation.. J. Equine Vet. Sci. 2023;125:104597.
  62. Squires E.L., Keith S.L., Graham J.K.. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa.. Theriogenology 2004;62:1056–1065.
  63. Papa F.O., Felício G.B., Melo-Oña C.M., Alvarenga M.A., De Vita B., Trinque C., Puoli-Filho J.N.P., Dell’Aqua J.A.. Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen.. Anim. Reprod. Sci. 2011;129:73–77.
  64. Macedo S., Bliebernicht M., Carvalheira J., Costa A., Ribeiro F., Rocha A.. Effects of two freezing methods and two cryopreservation media on post-thaw quality of stallion spermatozoa.. Reprod. Domest. Anim. 2018;53:519–524.
    doi: 10.1111/rda.13140pubmed: 29383772google scholar: lookup
  65. Nouri H., Shojaeian K., Samadian F., Lee S., Kohram H., Lee J.I.. Using Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality.. J. Equine Vet. Sci. 2018;70:18–25.
  66. Contreras M.J., Treulen F., Arias M.E., Silva M., Fuentes F., Cabrera P., Felmer R.. Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology.. Reprod. Domest. Anim. 2019;55:229–239.
    doi: 10.1111/rda.13611pubmed: 31868975google scholar: lookup
  67. Yeste M., Estrada E., Rocha L.G., Marin H., Rodriguez-Gil J.E., Miro J.. Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus.. Andrology 2015;3:395–407.
    doi: 10.1111/andr.291pubmed: 25294093google scholar: lookup
  68. Treulen F., Aguila L., Arias M.E., Jofré I., Felmer R.. Impact of post-thaw supplementation of semen extender with antioxidants on the quality and function variables of stallion spermatozoa.. Anim. Reprod. Sci. 2019;201:71–83.
  69. Vidament M., Yvon J.M., Couty I., Arnaud G., Nguekam-Feugang J., Noue P., Cottron S., Le Tellier A., Noel F., Palmer E.. Advances in cryopreservation of stallion semen in modified INRA82.. Anim. Reprod. Sci. 2001;68:201–218.
    doi: 10.1016/S0378-4320(01)00157-9pubmed: 11744265google scholar: lookup
  70. Scherzer J., Fayrer-Hosken R.A., Aceves M., Hurley D.J., Ray L.E., Jones L., Heusner G.L.. Freezing equine semen: The effect of combinations of semen extenders and glycerol on post–thaw motility.. Aust. Vet. J. 2009;87:275–279.
  71. Consuegra C., Crespo F., Bottrel M., Ortiz I., Dorado J., Diaz-Jimenez M., Pereira B., Hidalgo M.. Stallion sperm freezing with sucrose extenders: A strategy to avoid permeable cryoprotectants.. Anim. Reprod. Sci. 2018;191:85–91.
  72. Hernández-Avilés C., Love C.C., Serafini R., Ramírez-Agámez L., Friedrich M., Ghosh S., Teague S.R., LaCaze K.A., Brinsko S.P., Varner D.D.. Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage.. Theriogenology 2020;147:1–9.
  73. Neuhauser S., Bollwein H., Siuda M., Handler J.. Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm.. J. Equine Vet. Sci. 2019;79:1–8.
    doi: 10.1016/j.jevs.2019.05.002pubmed: 31405486google scholar: lookup
  74. Neuhauser S., Bollwein H., Siuda M., Handler J.. Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.. J. Equine Vet. Sci. 2019;82:102801.
    doi: 10.1016/j.jevs.2019.102801pubmed: 31732114google scholar: lookup
  75. Moura T.C.M., Arruda L.C.P., Araujo Silva R.A.J., Silva R.P.F., Oliveira A.S., Tobal L.F.M., Batista A.M., Carneiro G.F., Guerra M.M.P.. Diluent containing dimethylformamide added with sucrose improves in vitro quality after freezing/thawing stallion sperm.. J. Equine Vet. Sci. 2022;109:103825.
    doi: 10.1016/j.jevs.2021.103825pubmed: 34843891google scholar: lookup
  76. Rodríguez A.M., Ferrusola C.O., García B.M., Morrell J.M., Martínez H.R., Tapia J.A., Pena F.J.. Freezing stallion semen with the new Cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability.. Anim. Reprod. Sci. 2011;127:78–83.
  77. Heule M., Verstraete M., Blockx Z., De Blende P., Dini P., Daels P.. Slow Cooling is Beneficial for Storage of Frozen-Thawed Equine Spermatozoa.. J. Equine Vet. Sci. 2022;118:104132.
    doi: 10.1016/j.jevs.2022.104132pubmed: 36182047google scholar: lookup
  78. Choi Y.H., Varner D.D., Love C.C., Hartman D.L., Hinrichs K.. Production of live foals via intracytomplasmic injection of lyophilized soerm and sperm extract in the horse.. Reproduction 2011;142:529–538.
    doi: 10.1530/REP-11-0145pubmed: 21846810google scholar: lookup
  79. Olaciregui M., Luño V., Martí J.I., Aramayona J., Gil L.. Freeze-dried stallion spermatozoa: Evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays.. Andrologia 2016;48:988–994.
    doi: 10.1111/and.12530pubmed: 26804066google scholar: lookup
  80. Restrepo G., Varela E., Duque J.E., Gómez J.E., Rojas M.. Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity.. J. Equine Vet. Sci. 2019;72:8–15.
    doi: 10.1016/j.jevs.2018.10.006pubmed: 30929788google scholar: lookup
  81. Aurich C., Spergser J.. Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa.. Theriogenology 2007;67:912–918.
  82. Juan C., Ahmed T.. Disease transmission in horses.. Theriogenology 2006;66:551–559.
    pubmed: 16837034
  83. Cerny K.L., Littl T.V., Scoggin C.F., Coleman R.J., Mats H.T., Squires E.L.. Presence of bacteria on the external genitalia of healthy stallions and its transmission to the mare at the time of breeding by live cover.. J. Equine Vet. Sci. 2014;34:369–374.
  84. Ortega-Ferrusola C., González-Fernández L., Muriel A., Macías-García B., Rodríguez-Martínez H., Tapia J.A., Peña F.J.. Does the microbial flora in the ejaculate affect the freezeability of stallion sperm?. Reprod. Domest. Anim. 2009;44:518–522.
  85. Jasko D.J., Bedford S.J., Cook N.L., Mumford E.L., Squires E.L., Pickett B.W.. Effect of antibiotics on motion characteristics of cooled stallion spermatozoa.. Theriogenology 1993;40:885–893.
    doi: 10.1016/0093-691X(93)90356-Apubmed: 16727370google scholar: lookup
  86. Al-Kass Z., Spergser J., Aurich C., Kuhl J., Schmidt K., Johannisson A., Morrell J.M.. Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation.. Antibiotics 2018;7:1.
    doi: 10.3390/antibiotics7010001pmc: PMC5872112pubmed: 29267226google scholar: lookup
  87. Al-Kass Z., Spergser J., Aurich C., Kuhl J., Schmidt K., Morrell J.M.. Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen.. Reprod. Domest. Anim. 2018;54:342–349.
    doi: 10.1111/rda.13366pubmed: 30351456google scholar: lookup
  88. Johns I.C., Adams E.L.. Trends in antimicrobial resistance in equine bacterial isolates: 1999–2012.. Vet. Rec. 2016;179:330.
    doi: 10.1136/vr.102708pubmed: 25628448google scholar: lookup
  89. Morrell J.M., Malaluang P., Cojkic A., Hansson I.. The Global Antimicrobial Resistance Epidemic—Innovative Approaches and Cutting-Edge Solutions.. IntechOpen; London, UK: 2022.
    doi: 10.5772/intechopen.98171google scholar: lookup
  90. Malaluang P., Wilén E., Frosth S., Lindahl J., Hansson I., Morrell J.M.. Vaginal bacteria in mares and the occurrence of antimicrobial resistance.. Microorganisms 2022;10:2204.
  91. Malaluang P., Wilén E., Frosth S., Lindahl J.F., Hansson I., Morrell J.M.. Antimicrobial Resistance in Vaginal Bacteria in Inseminated Mares.. Pathogens 2023;12:375–395.
    doi: 10.3390/pathogens12030375pmc: PMC10058017pubmed: 36986297google scholar: lookup
  92. Morrell J.M., Klein C., Lundeheim N., Erol E., Troedsson M.H.T.. Removal of bacteria from stallion semen by colloid centrifugation.. Anim. Reprod. Sci. 2014;145:47–53.
  93. Guimarães T., Lopes G., Pinto M., Silva E., Miranda C., Correia M.J., Damásio L., Thompson G., Rocha A.. Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics.. Theriogenology 2015;83:186–191.
  94. Malo C., Gil L., Gonzalez N., Martínez F., Cano R., de Blas I., Espinosa E.. Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis). Cryobiology 2010;61:142–147.
  95. Yeste M., Morató R., Rodríguez-Gil J.E., Bonet S., Prieto-Martínez N.. Aquaporins in the male reproductive tract and sperm: Functional implications and cryobiology.. Reprod. Domest. Anim. 2017;52((Suppl. S4)):12–27.
    doi: 10.1111/rda.13082pubmed: 29052330google scholar: lookup
  96. Oberska P., Michałek K.. Aquaporins: New markers for male (in)fertility in livestock and poultry?. Anim. Reprod. Sci. 2021;231:106807.
  97. Bonilla-Correal S., Noto F., Garcia-Bonavila E., Rodríguez-Gil J.E., Yeste M., Miro J.. First evidence for the presence of aquaporins in stallion sperm.. Reprod. Domest. Anim. 2017;52((Suppl. S4)):61–64.
    doi: 10.1111/rda.13059pubmed: 29052325google scholar: lookup
  98. Luconi M., Cantini G., Baldi E., Forti G.. Role of a-kinase anchoring proteins (AKAPs) in reproduction.. Front. Biosci.-Landmark. 2011;16:1315–1330.
    doi: 10.2741/3791pubmed: 21196234google scholar: lookup
  99. Sergeant N., Briand-Amirat L., Bencharif D., Delehedde M.. The Sperm Specific Protein Proakap4 as an Innovative Marker to Evaluate Sperm Quality and Fertility.. Dairy Vet. Sci. J. 2019;11:555803.
  100. Blommaert D., Sergeant N., Delehedde M., Jouy N., Mitchell V., Franck T., Donnay I., Lejeune J.P., Serteyn D.. Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa.. Theriogenology 2019;131:52–60.
  101. Carracedo S., Briand-Amirat L., Dordas-Perpinyà M., Ramos Escuredo Y., Delcombel R., Sergeant N., Delehedde M.. ProAKAP4 protein marker: Towards a functional approach to male fertility.. Anim. Reprod. Sci. 2022;247:107074.
  102. Blommaert D., Sergeant N., Delehedde M., Donnay I., Lejeune J.P., Franck T., Serteyn D.. First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media.. Cryobiology 2021;102:133–135.
  103. Novak S., Smith T.A., Paradis F., Burwash L., Dyck M.K., Foxcroft G.R., Dixon W.T.. Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions.. Theriogenology 2010;74:956–967.
  104. Gaitskell-Phillips G., Martín-Cano F.E., Ortiz-Rodríguez J.M., Silva-Rodríguez A., Rodríguez-Martínez H., Gil M.C., Ortega-Ferrusola C., Peña F.J.. Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration.. Biol. Reprod. 2020;103:1275–1288.
    doi: 10.1093/biolre/ioaa153pubmed: 32857155google scholar: lookup
  105. Gaitskell-Phillips G., Martín-Cano F.E., Ortiz-Rodríguez J.M., da Silva-Álvarez E., Masot J., Redondo E., Gil M.C., Ortega-Ferrusola C., Peña F.J.. Seminal plasma proteins as potential biomarkers for sperm motility and velocities.. Theriogenology 2022;177:34–41.
  106. Ďuračka M., Benko F., Tvrdá E.. Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability.. Int. J. Mol. Sci. 2023;24:3379.
    doi: 10.3390/ijms24043379pmc: PMC9960060pubmed: 36834790google scholar: lookup