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Home / Equine Research Bank / Biol Reprod / Successful in vitro fertilization in the horse: production o...
Biology of reproduction2022; 107(6); 1551-1564; doi: 10.1093/biolre/ioac172

Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†.

Abstract: Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.
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Publication Date: 2022-09-16 PubMed ID: 36106756PubMed Central: PMC9752964DOI: 10.1093/biolre/ioac172Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

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The research is about the successful application of a new method for in vitro fertilization in horses, where sperm is pre-incubated for capacitation, resulting in a high fertilization rate and the birth of healthy foals.

Methodology and Findings

  • The researchers investigated whether pre-incubating horse sperm would support successful in-vitro fertilization. They used fresh sperm which was pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 hours.
  • The fertilization of cumulus-oocyte complexes (COCs) after a 6-hour incubation was successful at a rate of 43%. Subsequently culturing the presumptive embryos yielded a 21% success rate in the formation of blastocysts.
  • The same procedure, but without the addition of PHE, was unable to fertilize oocytes, highlighting the critical role of PHE in this process.
  • When using extended semen in the system, the researchers managed to obtain a much higher blastocyst rate of 54%. This was the method used in subsequent experiments.

Further Experiments and Success Rates

  • As a part of these additional trials, they transferred three in-vitro fertilization-produced blastocysts to recipient mares. All three recipients successfully gave birth to healthy foals.
  • When sperm were pre-incubated for 22 hours, between 47% and 79% of oocytes were fertilized after just 1 hour of co-incubation.
  • If sperm was pre-incubated for shorter periods – either 15 minutes or 6 hours – no fertilization was achieved after 1 hour, implying that capacitation requires between 6 and 22 hours.
  • The researchers discovered that sperm displayed an increase in protein tyrosine phosphorylation after 15 minutes, 6 hours, or 22 hours of pre-incubation. They believe this pattern may denote functional equine sperm capacitation.

Overall Outcome

  • The final system, using extended semen, with a pre-incubation period of 22 hours for the sperm and a 3-hour co-incubation period for the COC, achieved a phenomenal 90% fertilization rate, with a blastocyst rate of 74%.
  • This research is the first of its kind to report a successful and repeatable standard in vitro fertilization in horses. Furthermore, it’s the first report of in vitro production of blastocysts and the subsequent birth of foals in horses.

Cite This Article

APA
Felix MR, Turner RM, Dobbie T, Hinrichs K. (2022). Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†. Biol Reprod, 107(6), 1551-1564. https://doi.org/10.1093/biolre/ioac172

Publication

Biology of reproduction
ISSN: 1529-7268
NlmUniqueID: 0207224
Country: United States
Language: English
Volume: 107
Issue: 6
Pages: 1551-1564

Researcher Affiliations

Felix, Matheus R
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA.
Turner, Regina M
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Dobbie, Tamara
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
Hinrichs, Katrin
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA.
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA.

MeSH Terms

  • Horses
  • Animals
  • Female
  • Male
  • Semen
  • Fertilization in Vitro / veterinary
  • Fertilization in Vitro / methods
  • Spermatozoa
  • Blastocyst
  • Sperm Capacitation
  • Oocytes
  • Penicillamine
  • Epinephrine

Grant Funding

  • Penn Equine Assisted Reproduction Laboratory, University of Pennsylvania

Conflict of Interest Statement

The authors have declared that no conflict of interest exists.

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