4.5 Article

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

Journal

BIOLOGY OF REPRODUCTION
Volume 107, Issue 6, Pages 1551-1564

Publisher

OXFORD UNIV PRESS INC
DOI: 10.1093/biolre/ioac172

Keywords

horse; in vitro fertilization; sperm capacitation; protein tyrosine phosphorylation; fertilization; oocyte activation

Funding

  1. clinical ICSI program of the Penn Equine Assisted Reproduction Laboratory, University of Pennsylvania
  2. Clinical Equine ICSI Program, Texas AM University

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This study demonstrates that pre-incubation of fresh equine sperm with penicillamine, hypotaurine, and epinephrine can greatly improve the success rates of in vitro fertilization and the production of viable blastocysts. The use of extended semen and 22 hours of pre-incubation resulted in high rates of fertilization and blastocyst formation. This is the first report of successful standard in vitro fertilization in horses and the production of blastocysts and foals through this technique.
Pre-incubation of fresh equine sperm for 22 h in the presence of penicillamine, hypotaurine and epinephrine supports high rates of in vitro fertilization and production of viable blastocysts. 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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