Improved cryotolerance of bovine blastocysts by 1α,25-dihydroxyvitamin D3 supplementation during in vitro culture and post-thaw recovery

In vitro embryo production (IVP) is a valuable technique for advancing cattle breeding and reproductive biotechnology. Nevertheless, embryos produced in vitro often display reduced developmental competence and cryotolerance compared with their in vivo counterparts. Vitamin D is known to regulate many cellular functions, including growth and antioxidant defence. This study evaluated the impact of vitamin D supplementation during in vitro culture on bovine embryo development and post-thaw recovery. Oocytes were matured and fertilised in vitro and presumptive zygotes were cultured with 0 (control), 25, or 50 ng/ml of 1α,25-dihydroxyvitamin D3. Supplementation of 25 ng/ml vitamin D did not significantly alter cleavage or blastocyst rates; however, it increased the expression of IGF2R and SOD2. In contrast, 50 ng/ml impaired embryonic development. the VD/VD group showed higher blastocyst hatching rates, lower mitochondrial reactive oxygen species (ROS) levels, increased IGF2R expression and reduced BAX expression and BAX/BCL2L1 ratio (P < 0.05). Overall, these findings suggest that the low-dose vitamin D supplementation may support embryonic recovery from cryo-induced stress, potentially through the modulation of the oxidative balance and growth-related pathways, rather than by enhancing the pre-freezing developmental competence.