Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change.
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In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30-60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.