• cooling
• cooling media.
• DAPI
• embryo
• horse

The aim of this study was to compare the number of embryo dead cells after 6 and/or 24 h of cooled preservation in three different media. Day 8 embryos were collected by uterine flushing with Ringer Lactate at 37°C in an Ez-Way Filter (PETS, Canton, TX, USA) from 17 Standardbred mares artificially inseminated with fresh or frozen semen. Embryo quality, stage and diameter were evaluated before embryos were randomly allocated to one of these treatments: Emcare Holding Solution (ICPbio Repro- duction, USA), 24 h (EHS24; n = 6), using the standard protocol (control); Emcare Flushing Solution (ICPbio Reproduction, USA), 6 h (EFS6; n = 6) and 24 h (EFS24; n = 4); Ringer Lactate, 6 h (RL6; n = 7). In EFS’s and RL6 groups, the recovered embryos were kept in the filter and rinsed with 1 l of EFS or RL, which replaced the medium flushed from the uterus. Fluid filled filters were then placed in the Equitainer® and after cooling for the due time, the embryos were incubated with the cells stain DAPI and the dead cells were counted using epifluorescence UV- illumination on a Leica DM LB microscope. Embryo’s total cell number was estimated using the correlation:
n = 0.0106d2 + 2.0542d-375.28 (n = cells number, d = embryo diameter in μm) (Moussa et al., 2004;160). The effects of treatment and diameter group (<300, 300–1000, >1000μ) on the percentages of dead cells/estimated total embryo cells were evaluated by ANOVA (GLM). All the recovered embryos were excellent or good quality blastocysts.
No differences in proportion of dead cells were observed between diameter and treatment. Holding for 24 h or in RL for 6 h, 8 days old, good quality equine embryos in EFS didn’t increase the proportion of dead cells compared to cooling in EHS for 24 h. If in vivo studies will confirm the absence of major embryo damage, these protocols will simplify equine embryo cooling and shipping, before transfer.