Marked blastomere herniation is the only post-thaw morphological characteristic associated with a reduced pregnancy rate for in vitro produced equine blastocysts.

It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). Morphological characteristics (shape, extent of presumptive trophectoderm layer, % extruded blastomeres, expansion and % herniated cells) of frozen-thawed, biopsied (n = 36) and non-biopsied (n = 444) IVP blastocysts were recorded prior to transfer, and pregnancy outcome was recorded. A binomial multivariate generalized linear model was used to identify factors influencing pregnancy outcome. The overall pregnancy rate was 73.3 %, but was significantly affected by the interval between ICSI and confirmation of blastocyst formation, with lower pregnancy rates for later developing embryos (p < 0.01). Furthermore likelihood of pregnancy was significantly influenced by the stallion used for ICSI (p < 0.01) and the biological mother (p = 0.02), although no single biological sire or dam could be identified as contributing significantly. The biopsy procedure per se did not compromise pregnancy outcome (p = 0.59). On the other hand, pregnancy was less likely (p = 0.02) for blastocysts with more than 10 % herniated cells post-thaw (39 %; n = 13) than for those with fewer than 10 % (69 %; n = 166) or no (77 %; n = 301) herniated cells. No other post-thaw morphological characteristic affected pregnancy outcome. In conclusion, the sporadic herniation of a large part of the embryo through the ICSI-derived hole in the ZP during thawing negatively influences pregnancy outcome.