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Vertebrate reproductive science and technology
REVIEW

Update on advanced semen-processing technologies and their application for in vitro embryo production in horses

Lee H. Morris https://orcid.org/0000-0001-9821-4506 A B and Lisa J. Maclellan A
+ Author Affiliations
- Author Affiliations

A EquiBreed NZ, 399 Parklands Road, Te Awamutu 3879, New Zealand.

B Corresponding author. Email: lee@equibreed.co.nz

Reproduction, Fertility and Development 31(12) 1771-1777 https://doi.org/10.1071/RD19301
Submitted: 25 July 2019  Accepted: 29 August 2019   Published: 23 October 2019

Abstract

The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable and practical improvements in the results from equine ICSI.


References

Aitken, R. J., Finnie, J. M., Muscio, L., Whiting, S., Connaughton, H. S., Kuczera, L., Rothkirch, T. B., and De Iuliis, G. N. (2014). Potential importance of transition metals in the induction of DNA damage by sperm preparation media. Hum. Reprod. 29, 2136–2147.
Potential importance of transition metals in the induction of DNA damage by sperm preparation media.Crossref | GoogleScholarGoogle Scholar | 25141857PubMed |

Aitken, R. J., Gibb, Z., Baker, M. A., Drevet, J., and Gharagozloo, P. (2016). Causes and consequences of oxidative stress in spermatozoa. Reprod. Fertil. Dev. 28, 1–10.
Causes and consequences of oxidative stress in spermatozoa.Crossref | GoogleScholarGoogle Scholar | 27062870PubMed |

Anzalone, D. A., Palazzese, L., Iuso, D., Martino, G., and Loi, P. (2018). Freeze-dried spermatozoa: an alternative biobanking option for endangered species. Anim. Reprod. Sci. 190, 85–93.
Freeze-dried spermatozoa: an alternative biobanking option for endangered species.Crossref | GoogleScholarGoogle Scholar | 29397252PubMed |

Barkalina, N., Jones, C., and Coward, K. (2016). Nanomedicine and mammalian sperm: lessons from the porcine model. Theriogenology 85, 74–82.
Nanomedicine and mammalian sperm: lessons from the porcine model.Crossref | GoogleScholarGoogle Scholar | 26116055PubMed |

Bedford, S. J., Kurokawa, M., Hinrichs, K., and Fissore, R. A. (2003). Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa. Reproduction 126, 489–499.
Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa.Crossref | GoogleScholarGoogle Scholar | 14525531PubMed |

Bedford, S. J., Kurokawa, M., Hinrichs, K., and Fissore, R. A. (2004). Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse. Biol. Reprod. 70, 936–944.
Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.Crossref | GoogleScholarGoogle Scholar | 14656727PubMed |

Bezard, J., Magistrini, M., Duchamp, G., and Palmer, E. (1989). Chronology of equine fertilisation and embryonic development in vivo and in vitro. Equine Vet. J. 21, 105–110.

Carnevale, E. M. (2016). Advances in collection, transport and maturation of equine oocytes for assisted reproductive techniques. Vet. Clin. North Am. Equine Pract. 32, 379–399.
Advances in collection, transport and maturation of equine oocytes for assisted reproductive techniques.Crossref | GoogleScholarGoogle Scholar | 27726987PubMed |

Choi, Y. H., and Hinrichs, K. (2017). Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program. Theriogenology 87, 48–54.
Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program.Crossref | GoogleScholarGoogle Scholar | 27634397PubMed |

Choi, Y. H., Love, C. C., Love, L. B., Varner, D. D., Brinsko, S., and Hinrichs, K. (2002). Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen–thawed spermatozoa. Reproduction 123, 455–465.
Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen–thawed spermatozoa.Crossref | GoogleScholarGoogle Scholar | 11882023PubMed |

Choi, Y. H., Love, C. C., Varner, D. D., and Hinrichs, K. (2006). Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze–thaw cycles. Theriogenology 65, 808–819.
Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze–thaw cycles.Crossref | GoogleScholarGoogle Scholar | 16095679PubMed |

Choi, Y. H., Varner, D. D., Love, C. C., Hartman, D. L., and Hinrichs, K. (2011). Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction 142, 529–538.
Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse.Crossref | GoogleScholarGoogle Scholar | 21846810PubMed |

Choi, Y. H., Velez, I. C., Macias Garcia, B., Riera, F. L., and Hinrichs, K. (2013). Effect of medium and medium glucose concentration on equine embryo development after intracytoplasmic sperm injection. Clin. Theriogenology 5, 405.

Choi, Y. H., Gibbons, J. R., Canesin, H. S., and Hinrichs, K. (2016a). Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection. Theriogenology 86, 1782–1788.
Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 27377209PubMed |

Choi, Y. H., Velez, I. C., Macias-Garcia, B., Riera, F. L., Ballard, C. S., and Hinrichs, K. (2016b). Effect of clinically-related factors on in vitro blastocyst development after equine ICSI. Theriogenology 85, 1289–1296.
Effect of clinically-related factors on in vitro blastocyst development after equine ICSI.Crossref | GoogleScholarGoogle Scholar | 26777560PubMed |

Clulow, J. R., Buss, H., Sieme, H., Rodger, J. A., Cawdell-Smith, A. J., Evans, G., Rath, D., Morris, L. H., and Maxwell, W. M. (2008). Field fertility of sex-sorted and non-sorted frozen–thawed stallion spermatozoa. Anim. Reprod. Sci. 108, 287–297.
Field fertility of sex-sorted and non-sorted frozen–thawed stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar | 17977675PubMed |

Clulow, J. R., Evans, G., Morris, L. H., and Maxwell, W. M. (2009). Factors influencing the ‘sortability’ of stallion spermatozoa into X- and Y-chromosome bearing populations. Anim. Reprod. Sci. 113, 220–228.
Factors influencing the ‘sortability’ of stallion spermatozoa into X- and Y-chromosome bearing populations.Crossref | GoogleScholarGoogle Scholar | 19022595PubMed |

Clulow, J. R., Evans, G., Maxwell, W. M., and Morris, L. H. (2010). Evaluation of the function of fresh and frozen–thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay. Reprod. Fertil. Dev. 22, 710–717.
Evaluation of the function of fresh and frozen–thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay.Crossref | GoogleScholarGoogle Scholar | 20353730PubMed |

Clulow, J. R., Buss, H., Evans, G., Sieme, H., Rath, D., Morris, L. H., and Maxwell, W. M. (2012). Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation. Reprod. Domest. Anim. 47, 1–7.
Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation.Crossref | GoogleScholarGoogle Scholar | 22053724PubMed |

Cochran, R., Meintjes, M., Reggio, B., Hylan, D., Carter, J., Pinto, C., Paccamonti, D., and Godke, R. A. (1998). Live foals produced from sperm-injected oocytes derived from pregnant mares. J. Equine Vet. Sci. 18, 736–740.
Live foals produced from sperm-injected oocytes derived from pregnant mares.Crossref | GoogleScholarGoogle Scholar |

Colleoni, S., Spinaci, M., Duchi, R., Merlo, B., Tamanini, C., Lazzari, G., Mari, G., and Galli, C. (2009). ICSI of equine oocytes with sex-sorted frozen–thawed semen results in low cleavage rate but normal embryo development and pregnancies. Reprod. Fertil. Dev. 21, 228–229.
ICSI of equine oocytes with sex-sorted frozen–thawed semen results in low cleavage rate but normal embryo development and pregnancies.Crossref | GoogleScholarGoogle Scholar |

Coy, P., Garcia-Vazquez, F. A., Visconti, P. E., and Aviles, M. (2012). Roles of the oviduct in mammalian fertilization. Reproduction 144, 649–660.
Roles of the oviduct in mammalian fertilization.Crossref | GoogleScholarGoogle Scholar | 23028122PubMed |

Dell’Aquila, M. E., De Felici, M., Massari, S., Maritato, F., and Minoia, P. (1999). Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro. Biol. Reprod. 61, 533–540.
Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.Crossref | GoogleScholarGoogle Scholar | 10411537PubMed |

Domínguez, E., Moreno-Irusta, A., Ramírez Castex, H., Flores Bragulat, A., Ugaz, C., Clemente, H., Giojalas, L., and Losinno, L. (2018). Sperm sexing mediated by magnetic nanoparticles in donkeys, a preliminary in vitro study. J. Equine Vet. Sci. 65, 123–127.
Sperm sexing mediated by magnetic nanoparticles in donkeys, a preliminary in vitro study.Crossref | GoogleScholarGoogle Scholar |

Edmond, A. J., Brinsko, S. P., Love, C. C., Blanchard, T. L., Teague, S. R., and Varner, D. D. (2012). Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm. Theriogenology 77, 959–966.
Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm.Crossref | GoogleScholarGoogle Scholar | 22115813PubMed |

Esteves, S. C., Sharma, R. K., Thomas, A. J., and Agarwal, A. (2000). Effect of swim-up sperm washing and subsequent capacitation on acrosome status and functional membrane integrity of normal sperm. Int. J. Fertil. Womens Med. 45, 335–341.
| 11092705PubMed |

Farini, V. L., Camano, C. V., Ybarra, G., Viale, D. L., Vichera, G., Yakisich, J. S., and Radrizzani, M. (2016). Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles. J. Biotechnol. 229, 33–41.
Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.Crossref | GoogleScholarGoogle Scholar | 27164256PubMed |

Feugang, J. M. (2017). Novel agents for sperm purification, sorting, and imaging. Mol. Reprod. Dev. 84, 832–841.
Novel agents for sperm purification, sorting, and imaging.Crossref | GoogleScholarGoogle Scholar | 28481043PubMed |

Foss, R., Ortis, H., and Loncar, K. D. (2018). Effect of artificial activation of equine oocytes on cleavage and blastocyst production following ICSI. J. Equine Vet. Sci. 66, 167.
Effect of artificial activation of equine oocytes on cleavage and blastocyst production following ICSI.Crossref | GoogleScholarGoogle Scholar |

Galli, C., Crotti, G., Turini, P., Duchi, R., Mari, G., Zavaglia, G., Duchamp, G., Daels, P., and Lazzari, G. (2002). Frozen–thawed embryos produced by ovum pick up of immature oocytes and ICSI are capable to establish pregnancies in the horse. Theriogenology 58, 705–708.

Galli, C., Colleoni, S., Duchi, R., and Lazzari, G. (2016). Male factors affecting the success of equine in vitro embryo production by ovum pick up-intracytoplasmic sperm injection in a clinical setting. J. Equine Vet. Sci. 43, S6–S10.
Male factors affecting the success of equine in vitro embryo production by ovum pick up-intracytoplasmic sperm injection in a clinical setting.Crossref | GoogleScholarGoogle Scholar |

Garner, D. L., and Seidel, G. E. (2008). History of commercializing sexed semen for cattle. Theriogenology 69, 886–895.
History of commercializing sexed semen for cattle.Crossref | GoogleScholarGoogle Scholar | 18343491PubMed |

Gibb, Z., Morris, L. H., Maxwell, W. M., and Grupen, C. G. (2011). Use of a defined diluent increases the sex-sorting efficiency of stallion sperm. Theriogenology 75, 610–619.
Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.Crossref | GoogleScholarGoogle Scholar | 21144575PubMed |

Gibb, Z., Butler, T. J., Morris, L. H., Maxwell, W. M., and Grupen, C. G. (2013a). Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm. Theriogenology 79, 1001–1009.
Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.Crossref | GoogleScholarGoogle Scholar | 23453253PubMed |

Gibb, Z., Morris, L. H., Maxwell, W. M., and Grupen, C. G. (2013b). Dimethyl formamide improves the postthaw characteristics of sex-sorted and nonsorted stallion sperm. Theriogenology 79, 1027–1033.
Dimethyl formamide improves the postthaw characteristics of sex-sorted and nonsorted stallion sperm.Crossref | GoogleScholarGoogle Scholar | 23453787PubMed |

Gibb, Z., Grupen, C. G., Maxwell, W. M., and Morris, L. H. (2017). Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm. Equine Vet. J. 49, 160–166.
Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm.Crossref | GoogleScholarGoogle Scholar | 27060979PubMed |

Gonzalez-Castro, R. A. (2018). Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. Ph.D. Thesis, Colorado State University, Fort Collins, CO, USA.

Gonzalez-Castro, R. A., and Carnevale, E. M. (2018). Association of equine sperm population parameters with outcome of intracytoplasmic sperm injections. Theriogenology 119, 114–120.
Association of equine sperm population parameters with outcome of intracytoplasmic sperm injections.Crossref | GoogleScholarGoogle Scholar | 30006126PubMed |

Gonzalez-Castro, R. A., and Carnevale, E. M. (2019). Use of microfluidics to sort stallion sperm for intracytoplasmic sperm injection. Anim. Reprod. Sci. 202, 1–9.
Use of microfluidics to sort stallion sperm for intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 30655027PubMed |

Gonzalez-Castro, R. A., Trentin, J. M., Carnevale, E. M., and Graham, J. K. (2019). Effects of extender, cryoprotectants and thawing protocol on motility of frozen–thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses. Theriogenology 136, 36–42.
Effects of extender, cryoprotectants and thawing protocol on motility of frozen–thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses.Crossref | GoogleScholarGoogle Scholar | 31242456PubMed |

Guthrie, H. D., Johnson, L. A., Garrett, W. M., Welch, G. R., and Dobrinsky, J. R. (2002). Flow cytometric sperm sorting: effects of varying laser power on embryo development in swine. Mol. Reprod. Dev. 61, 87–92.
Flow cytometric sperm sorting: effects of varying laser power on embryo development in swine.Crossref | GoogleScholarGoogle Scholar | 11774379PubMed |

Herrera, C., Dufourq, P., Freije, M., Morikawa, I., Centeno, J. E., Aristi, V., Menghini, L., and Sporleder, C. (2012). Selection of stallions for in vitro embryo production by ICSI in a commercial program. Journal of Equine Veterinary Science 32, 409.
Selection of stallions for in vitro embryo production by ICSI in a commercial program.Crossref | GoogleScholarGoogle Scholar |

Hinrichs, K., Love, C. C., Brinsko, S. P., Choi, Y. H., and Varner, D. D. (2002). In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer. Biol. Reprod. 67, 256–262.
In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.Crossref | GoogleScholarGoogle Scholar | 12080025PubMed |

Ishijima, S. A., Okuno, M., and Mohri, H. (1991). Zeta potential of human X- and Y-bearing sperm. Int. J. Androl. 14, 340–347.
Zeta potential of human X- and Y-bearing sperm.Crossref | GoogleScholarGoogle Scholar | 1794918PubMed |

Johnson, L. A., Flook, J. P., Look, M. V., and Pinkel, D. (1987). Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res. 16, 1–9.
Flow sorting of X and Y chromosome-bearing spermatozoa into two populations.Crossref | GoogleScholarGoogle Scholar | 3506896PubMed |

Landim-Alvarenga, F. C., Alvarenga, M. A., Seidel, G. E., Squires, E. L., and Graham, J. K. (2001). Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187. Theriogenology 56, 937–953.
Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187.Crossref | GoogleScholarGoogle Scholar | 11665894PubMed |

Lazzari, G., Crotti, G., Turini, P., Duchi, R., Mari, G., Zavaglia, G., Barbacini, S., and Galli, C. (2002). Equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen–thawed spermatozoa from semen of different fertilities. Theriogenology 58, 709–712.
Equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen–thawed spermatozoa from semen of different fertilities.Crossref | GoogleScholarGoogle Scholar |

Lee, S., Lee, Y. S., Lee, S. H., Yang, B. K., and Park, C. K. (2016). Effect of methyl-beta-cyclodextrin on the viability and acrosome damage of sex-sorted sperm in frozen–thawed bovine semen. J. Biol. Res. (Thessalon.) 23, 5.
Effect of methyl-beta-cyclodextrin on the viability and acrosome damage of sex-sorted sperm in frozen–thawed bovine semen.Crossref | GoogleScholarGoogle Scholar |

Leemans, B., Gadella, B. M., Stout, T. A., De Schauwer, C., Nelis, H., Hoogewijs, M., and Van Soom, A. (2016). Why doesn’t conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization. Reproduction 152, R233–R245.
Why doesn’t conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization.Crossref | GoogleScholarGoogle Scholar | 27651517PubMed |

Leemans, B., Stout, T. A. E., De Schauwer, C., Heras, S., Nelis, H., Hoogewijs, M., Van Soom, A., and Gadella, B. M. (2019). Update on mammalian sperm capacitation: how much does the horse differ from other species? Reproduction 157, R181–R197.
Update on mammalian sperm capacitation: how much does the horse differ from other species?Crossref | GoogleScholarGoogle Scholar |

Leisinger, C. A., Pinto, C. R. F., Cramer, E., Love, C. C., and Paccamonti, D. L. (2017). Effects of repeated partial thaw and refreeze on post-thaw parameters of stallion semen cryopreserved in cryovials. J. Equine Vet. Sci. 49, 19–24.
Effects of repeated partial thaw and refreeze on post-thaw parameters of stallion semen cryopreserved in cryovials.Crossref | GoogleScholarGoogle Scholar |

Li, X., Morris, L. H., and Allen, W. R. (2000). Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection. J. Reprod. Fertil. 119, 253–260.
Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection.Crossref | GoogleScholarGoogle Scholar | 10864837PubMed |

Li, X., Morris, L. H., and Allen, W. R. (2001). Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). Reproduction 121, 925–932.
Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI).Crossref | GoogleScholarGoogle Scholar | 11373179PubMed |

Libbus, B. L., Perreault, S. D., Johnson, L. A., and Pinkel, D. (1987). Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting. Mutat. Res. 182, 265–274.
Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting.Crossref | GoogleScholarGoogle Scholar | 3309639PubMed |

Mari, G., Bucci, D., Love, C. C., Mislei, B., Rizzato, G., Giaretta, E., Merlo, B., and Spinaci, M. (2015). Effect of cushioned or single layer semen centrifugation before sex sorting on frozen stallion semen quality. Theriogenology 83, 953–958.
Effect of cushioned or single layer semen centrifugation before sex sorting on frozen stallion semen quality.Crossref | GoogleScholarGoogle Scholar | 25542457PubMed |

McCue, P. M., Moore, A. I., and Bruemmer, J. E. (2004). Refreezing stallion spermatozoa for assisted reproduction. Reprod. Fertil. Dev. 16, 176–177.
Refreezing stallion spermatozoa for assisted reproduction.Crossref | GoogleScholarGoogle Scholar |

McKinnon, A. O., Lacham-Kaplan, O., and Trounson, A. O. (2000). Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen–thawed spermatozoa into in vivo matured mare oocytes. J. Reprod. Fertil. Suppl. 56, 513–517.

Morotti, F., Sanches, B. V., Pontes, J. H., Basso, A. C., Siqueira, E. R., Lisboa, L. A., and Seneda, M. M. (2014). Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle. Theriogenology 81, 696–701.
Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle.Crossref | GoogleScholarGoogle Scholar | 24412681PubMed |

Morris, L. H. (2005). Challenges facing sex preselection of stallion spermatozoa. Anim. Reprod. Sci. 89, 147–157.
Challenges facing sex preselection of stallion spermatozoa.Crossref | GoogleScholarGoogle Scholar | 16084041PubMed |

Morris, L. H. A. (2018). The development of in vitro embryo production in the horse. Equine Vet. J. 50, 712–720.
The development of in vitro embryo production in the horse.Crossref | GoogleScholarGoogle Scholar |

Morris, L. H., de Haan, T., Landriscina, L. G., Wilsher, S., and Gibb, Z. (2018). The effects of nanoparticle semen purification on semen quality parameters in stallions. J. Equine Vet. Sci. 66, 75.
The effects of nanoparticle semen purification on semen quality parameters in stallions.Crossref | GoogleScholarGoogle Scholar |

Nomikos, M., Kashir, J., and Lai, F. A. (2017). The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation. Biochem. J. 474, 3659–3673.
The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation.Crossref | GoogleScholarGoogle Scholar | 29061915PubMed |

Palazzese, L., Gosalvez, J., Anzalone, D. A., Loi, P., and Saragusty, J. (2018). DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development. J. Reprod. Dev. 64, 393–400.
DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development.Crossref | GoogleScholarGoogle Scholar | 29973438PubMed |

Palmer, E., Bezard, J., Magistrini, M., and Duchamp, G. (1991). In vitro fertilization in the horse. A retrospective study. J. Reprod. Fertil. Suppl. 44, 375–384.
| 1795281PubMed |

Panarace, M., Pellegrini, R. O., Basualdo, M. O., Bele, M., Ursino, D. A., Cisterna, R., Desimone, G., Rodriguez, E., and Medina, M. J. (2014). First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program. Theriogenology 81, 520–525.
First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program.Crossref | GoogleScholarGoogle Scholar | 24360404PubMed |

Puglisi, R., Vanni, R., Galli, A., Balduzzi, D., Parati, K., Bongioni, G., Crotti, G., Duchi, R., Galli, C., Lazzari, G., and Aleandri, R. (2006). In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR. Reproduction 132, 519–526.
In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR.Crossref | GoogleScholarGoogle Scholar | 16940293PubMed |

Quan, G. B., Ma, Y., Li, J., Wu, G. Q., Li, D. J., Ni, Y. N., Lv, C. R., Zhu, L., and Hong, Q. H. (2015). Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen–thawed ram sperm. Cryobiology 70, 23–31.
Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen–thawed ram sperm.Crossref | GoogleScholarGoogle Scholar | 25481668PubMed |

Rader, K., Choi, Y. H., and Hinrichs, K. (2016). Intracytoplasmic sperm injection, embryo culture, and transfer of in vitro-produced blastocysts. Vet. Clin. North Am. Equine Pract. 32, 401–413.
Intracytoplasmic sperm injection, embryo culture, and transfer of in vitro-produced blastocysts.Crossref | GoogleScholarGoogle Scholar | 27726990PubMed |

Restrepo, G., Varela, E., Duque, J. E., Gómez, J. E., and Rojas, M. (2019). Freezing, vitrification, and freeze-drying of equine spermatozoa: impact on mitochondrial membrane potential, lipid peroxidation, and DNA Integrity. J. Equine Vet. Sci. 72, 8–15.
Freezing, vitrification, and freeze-drying of equine spermatozoa: impact on mitochondrial membrane potential, lipid peroxidation, and DNA Integrity.Crossref | GoogleScholarGoogle Scholar | 30929788PubMed |

Rodriguez-Martinez, H. (2007). Role of the oviduct in sperm capacitation. Theriogenology 68, S138–S146.
Role of the oviduct in sperm capacitation.Crossref | GoogleScholarGoogle Scholar | 17452049PubMed |

Roels, K., Smits, K., Ververs, C., Govaere, J., D’Herde, K., and Van Soom, A. (2018). Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration. Reprod. Domest. Anim. 53, 814–817.
Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.Crossref | GoogleScholarGoogle Scholar | 29498113PubMed |

Schmid, R. L., Kato, H., Herickhoff, L. A., Schenk, J. L., McCue, P. M., Chung, Y. G., and Squires, E. L. (2000). Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa. J. Reprod. Fertil. Suppl. 68, 519–525.

Shengfa, F., and Liao, J. M. F. (2014). Lectin-functionalized magnetic iron oxide nanoparticles for reproductive improvement. JFIV Reprod. Med. Genet. 3, 2.
Lectin-functionalized magnetic iron oxide nanoparticles for reproductive improvement.Crossref | GoogleScholarGoogle Scholar |

Sielhorst, J., Hagen, C., Behrendt, D., Schuette, B., Burger, D., Martinsson, G., and Sieme, H. (2016). Effect of multiple freezing of stallion semen on sperm quality and fertility. J. Equine Vet. Sci. 40, 56–61.
Effect of multiple freezing of stallion semen on sperm quality and fertility.Crossref | GoogleScholarGoogle Scholar |

Simopoulou, M., Gkoles, L., Bakas, P., Giannelou, P., Kalampokas, T., Pantos, K., and Koutsilieris, M. (2016). Improving ICSI: a review from the spermatozoon perspective. Syst Biol Reprod Med 62, 359–371.
Improving ICSI: a review from the spermatozoon perspective.Crossref | GoogleScholarGoogle Scholar | 27646677PubMed |

Squires, E. L., Wilson, J. M., Kato, H., and Blaszczyk, A. (1996). A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro. Theriogenology 45, 306.
A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro.Crossref | GoogleScholarGoogle Scholar |

Suh, T. K., Schenk, J. L., and Seidel, G. E. (2005). High pressure flow cytometric sorting damages sperm. Theriogenology 64, 1035–1048.
High pressure flow cytometric sorting damages sperm.Crossref | GoogleScholarGoogle Scholar | 16125550PubMed |

Sutovsky, P., and Kennedy, C. E. (2013). Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle. Ind. Biotechnol. (New Rochelle N.Y.) 9, 24–30.
Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle.Crossref | GoogleScholarGoogle Scholar |

Takeshima, T., Yumura, Y., Kuroda, S., Kawahara, T., Uemura, H., and Iwasaki, A. (2017). Effect of density gradient centrifugation on reactive oxygen species in human semen. Syst Biol Reprod Med 63, 192–198.
Effect of density gradient centrifugation on reactive oxygen species in human semen.Crossref | GoogleScholarGoogle Scholar | 28332864PubMed |

Thomas, J. M., Locke, J. W. C., Vishwanath, R., Hall, J. B., Ellersieck, M. R., Smith, M. F., and Patterson, D. J. (2017). Effective use of SexedULTRA sex-sorted semen for timed artificial insemination of beef heifers. Theriogenology 98, 88–93.
Effective use of SexedULTRA sex-sorted semen for timed artificial insemination of beef heifers.Crossref | GoogleScholarGoogle Scholar | 28601161PubMed |

Wakayama, T., and Yanagimachi, R. (1998). Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotechnol. 16, 639–641.
Development of normal mice from oocytes injected with freeze-dried spermatozoa.Crossref | GoogleScholarGoogle Scholar | 9661196PubMed |

Xue, X., Wang, W. S., Shi, J. Z., Zhang, S. L., Zhao, W. Q., Shi, W. H., Guo, B. Z., and Qin, Z. (2014). Efficacy of swim-up versus density gradient centrifugation in improving sperm deformity rate and DNA fragmentation index in semen samples from teratozoospermic patients. J. Assist. Reprod. Genet. 31, 1161–1166.
Efficacy of swim-up versus density gradient centrifugation in improving sperm deformity rate and DNA fragmentation index in semen samples from teratozoospermic patients.Crossref | GoogleScholarGoogle Scholar | 25015033PubMed |

Yamanaka, M., Tomita, K., Hashimoto, S., Matsumoto, H., Satoh, M., Kato, H., Hosoi, Y., Inoue, M., Nakaoka, Y., and Morimoto, Y. (2016). Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms. J. Reprod. Dev. 62, 599–606.
Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms.Crossref | GoogleScholarGoogle Scholar | 27616283PubMed |

Yoisungnern, T., Choi, Y. J., Han, J. W., Kang, M. H., Das, J., Gurunathan, S., Kwon, D. N., Cho, S. G., Park, C., Chang, W. K., Chang, B. S., Parnpai, R., and Kim, J. H. (2015). Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development. Sci. Rep. 5, 11170.
Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development.Crossref | GoogleScholarGoogle Scholar | 26054035PubMed |