72 Effect of incubation temperature and of CO₂ concentration during early cleavage on equine in vitro embryo production

Abstract

Intracytoplasmic sperm injection (ICSI) is currently being used for equine in vitro embryo production for both research and clinical purposes. However, some basic parameters for in vitro embryo production, such as the optimum incubator temperature and the optimum CO₂ concentration/pH of medium for early embryo development, have not yet been established in the horse. The incubation temperature used by many laboratories for equine in vitro embryo production is 38.2°C, whereas the range of normal rectal temperature in the horse is 37.2 to 38.3°C. In Exp. 1, we evaluated maturation, cleavage, and blastocyst rates under 3 different culture temperatures. Cumulus-oocyte complexes were recovered from slaughterhouse-derived ovaries and shipped overnight at room temperature. Oocyte maturation was performed concurrently in separate incubators set to 37.2, 37.7, or 38.2°C. Mature oocytes were subjected to ICSI, then cultured in mixed gas (6% CO₂, 5% O₂, and remainder N₂) at the same temperature at which they were matured. Embryo culture media used were a commercial human medium (global) for Days 0 to 5, then DMEM/F-12 from Day 5 to 10, both with 10% fetal bovine serum (FBS). In Exp. 2, we evaluated 3 different CO₂ concentrations (6, 6.5, or 7% CO₂) in mixed gas for the Day 0 to 5 culture in global + FBS, at 38.2°C. Cumulus-oocyte complexes were recovered from live mares by transvaginal aspiration and held overnight at room temperature; all other parameters remained the same as for Exp. 1. Data were analysed by Fisher’s exact test. In Exp. 1, a total of 280 oocytes were utilised; the outcomes for the 37.2, 37.7, and 38.2°C treatments were, respectively: maturation rates of 33, 38, and 42%; cleavage rates of 84, 86, and 88%; and blastocyst rates per injected oocyte of 35, 44, and 44%. There were no significant differences among the 3 temperature treatments for any parameter (P > 0.2). In Exp. 2, the pH of the global + FBS medium was 7.38, 7.35, and 7.3 for 6, 6.5, and 7% CO₂, respectively. A total of 106 mature oocytes underwent ICSI; the outcomes for the 3 CO₂ atmospheres, respectively, were cleavage rates of 68, 80, and 70% and blastocyst rates per injected oocyte of 42, 54, and 27%. The blastocyst rate for the 7% CO₂ treatment was significantly lower than that for the 6.5% CO₂ treatment (P < 0.05). These results indicate that equine in vitro embryo production is equally effective throughout the range of normal equine body temperature, and that equine blastocyst production is sensitive to small changes in CO₂ atmosphere/pH of medium during early cleavage-stage development.

Research was supported by the Clinical Equine ICSI Program, Texas A&M University, the Link Equine Research Endowment, Texas A&M University, and Fonds en Santé Équine (FSÉ), Faculté de Médecine Vétérinaire, and Université de Montréal.