39 The effect of the supplementation of type III antifreeze protein on cryotolerance of bovine in vitro-produced blastocysts

The number of calves produced by bovine cryopreserved in vitro-produced embryo transfer has been increasing with the spread of ovum pickup (OPU)-IVF. However, the conception rate of cryopreserved embryos transfer is lower than that of fresh embryos. One possible reason for the low conception rate of cryopreserved embryos is a damage to cells and organelles caused by ice crystal formation during freezing. We hypothesised that if ice crystal formation could be suppressed, embryo damage could be improved and fertility could be increased. Antifreeze protein (AFP) from fish is known to protect cells from freezing damage by inhibiting ice crystal formation during freezing. In this study, we evaluated the effect of the supplementation of AFP to embryo freezing medium on cryotolerance. Cumulus–oocyte complex (COC) collected from the ovaries obtained from the local abattoir were matured in vitro at 38.5°C 5% CO₂ in air for 22 h and then fertilized with frozen–thawed sperm at 38.5°C 5% CO₂ in air for overnight. The cumulus cells removed putative zygotes were cultured for 7 days at 38.5°C 5% O₂, 5% CO₂ in serum-free SOF-BE1 culture medium. After blastocysts rate was confirmed on Day 7, blastocysts were pre-incubated with SOF-BE1 supplemented with type III AFP 0 (control), 0.1, 0.5, 1.0 and 5.0 µg/mL for 1 h at 38.5°C 5% O₂ 5% CO₂. Subsequently, total of 23 to 37 blastocysts (replicates = 4) were subjected to slow freezing was containing the same concentrations of AFP in a freezing solution (5% ethylene glycol, 6% propylene glycol, 0.1 M sucrose and 4 mg/mL BSA in mPBS) by the program freezer. After storage in LN₂ for at least 72 h, the embryos were thawed and cultured with 20% fetal bovine serum, 100 µM β-mercaptoethanol in M-199 at 38.5°C 5% O₂, 5% CO₂ for recovery. After 24 h, embryo viability was determined by embryo morphology (presence of blastocoel, cell mass shrink), the rate of blastocysts expanded was analysed with ANOVA. For control and AFP 1.0 µg/mL group, which showed the highest survival rate, 6 to 7 viable all stage blastocysts each with 5 replicates were sampled 24 h after thawing, and analysed for expression of genes related to quality (POU5F1, NANOG, CDX2, IFNT), apoptosis (CASP3) and stress (HSPA1A) using RT-quantitative polymerase chain reaction. The Student’s t-test for gene expression were performed for statistical analysis. No changes in embryo morphology were observed due to AFP preincubation. Embryo viability at 24 h after thawing tended to be higher only in AFP 1.0 µg/mL (80.4%) than that of control (63.3%, P = 0.059). But expanded rates were not different. The expression of trophectoderm specific gene CDX2 was significantly lower in control than that in AFP group (1.00 vs 1.48, P < 0.05). HSPA1A, a stress gene, was significantly suppressed in the AFP group (1.00 vs 0.69, P < 0.05). These results suggest that the embryo viability, expanded, and hatched rates were not statistically significant due to the treatment. But the supplementation of 1.0 µg/mL AFP to cryopreservation medium improves the stress level and that the trophectoderm located outside of the blastocysts may be less susceptible to the damage from cryopreservation.

This research was financially supported by JSPS KAKENHI grant number 20K06370.