Embryo recovery on consecutive days from Day 6.5 to obtain small embryos for vitrification: Is it effective?

Abstract

To successfully vitrify equine embryos without aspiration or puncture of the blastocoele cavity, current methodology requires embryos ≤300µm. Such embryos are obtained by flushing mares shortly after the presumed time of entry of the embryo into the uterus (6–6.5 days post ovulation). Ovulation, and hence the timing of the flush, is usually calculated from expected ovulation times following administration of an induction agent. However, recovery rates tend to be lower at 6.5 days. A precise time for ovulation helps, but its determination is management intensive, and entry into the uterus varies by a reported 12h. The aim of this study was to determine if flushing donor mares on successive days after they failed to give an embryo on Day 6.5-7 increased recovery rates of embryos ≤300µm. During successive breeding seasons 442 inseminated cycles from donor mares, aged 3-21 years, were flushed for embryos on Day 6.5-7 with the aim of obtaining embryos ≤300µm for vitrification. Mares were flushed with Ringer's Lactate in a closed system and oxytocin given before the third flush. Filter contents were placed in a Petri dish prior to rinsing the filter with 10% PVA in Ringer's Lactate. If no embryo was found flunixin meglumine was administered and the mare re-flushed 24h later. From 442 first flushes 151 (34.2%) were positiveand 168 embryos were recovered with a median diameter of 170 µm (range; 140-320µm). The 292 negative mares, and 3 double-ovulating mares that yielded only one embryo at the first flush, were re-flushed the following day with 49 (16.6%) positive flushes yielding 51 embryos (180µm; 150-400µm). A subset of the negative mares from the second recovery attempt (n=35) were flushed the following day and 7 (20.6%) gave embryos with a median diameter of 170µm (range; 150-200 µm). Combining all flushing days, 226 embryos were recovered of which only 4 (1.8%) were ≥300µm. Although approaching significance, no difference existed in the size of embryos recovered at the first, second or third flush (P=0.051). Mare age did not appear to be a factor on the likelihood of embryo recovery on the first, second or third attempt (12 vs. 14 vs. 12 y.o., respectively; P=0.130). Therefore, when flushing donor mares to recover embryos ≤300µm, re-flushing negative mares on consecutive days results in recovery of embryos suitable for vitrification without the need for blastocoele collapse. From a physiological aspect and allowing for the lack of a precise time for ovulation in the study, there appears to be a wider margin than Day 6–6.5 for entry of the embryo into the uterus.