Cryopreservation of in vivo derived goat embryos: Influence of post-collection incubation methods on cellular stress and survival rates

Abstract

This study evaluated how post-collection conditions affect the survival of in vivo-derived (IVD) goat embryos after cryopreservation. A total of 223 IVD embryos (grades I and II) from three replicates were distributed into three groups: P0–3H, embryos maintained for 3 h at 37 °C in culture dishes with 2 mL of holding medium (HM); P6–8H, embryos held under the same conditions for 6–8 h; and T6–8H, embryos stored in cryotubes with 2 mL of HM at 37 °C for 6–8 h in a portable incubator. Osmolarity analysis showed increases of 15.3 % ± 3.4 and 48.8 % ± 8.5 in P0–3H and P6–8H, respectively, while T6–8H maintained osmotic stability (1.0 % ± 1.4). Developmental outcomes were better in T6–8H, with higher proportions of blastocysts and expanded blastocysts (27.5 %± 2.7 and 42.5 % ± 3.2) compared to P6–8H (14.1 % ± 1.4 and 34.6 % ± 3.0). However, post-thaw survival rates were similar across groups (P > 0.05). Gene expression analysis revealed increased stress (PRDX1, HSP70) and apoptosis (BAX) markers in P6–8H compared to T6–8H. The highest mitochondrial activity and ROS levels were observed in T6–8H, whereas GSH levels were elevated in P6–8H, matching P0–3H values. These findings suggest that the T6–8H condition—embryos stored in cryotubes with HM at 37 °C—preserved osmotic balance, supported better embryo development, and promoted a more favorable cellular environment. Therefore, this strategy may improve embryo quality before cryopreservation, contributing to more consistent post-thaw outcomes in goat embryo transfer programs.