34 Influence of fetal bovine serum addition post-thaw in the survival of cryopreserved embryos

In vitro embryo production (IVP) is successfully applied in the bovine industry as a strategy to accelerate genetic gain. Cryopreservation of IVP blastocysts is important to provide flexibility for producers, and this technique is also used to evaluate embryo quality. Re-expansion (restoration of the blastocoel) and hatching (partial or complete rupture of the zona pellucida) are considered indicators of the recovery status for frozen-thawed blastocysts. The goal of this research was to compare the survival (re-expansion) and hatching of frozen-thawed embryos, cultured for 48 h post-thawing in two different media, supplemented with or without 2.5% fetal bovine serum (FBS, Cripion). For this study, only Day 7 in vitro-produced blastocysts classified as grade 1 according to the IETS guidelines were used. Three replicates with a total of 289 blastocysts were evaluated. Embryos were submitted to slow freezing using 1.5 M ethylene glycol, 0.1 M sucrose, and 0.4% bovine serum albumin (BSA) and placed in a freezing machine using the standard slow-freezing protocol at a ramp of 0.5°C min⁻¹. For thawing, the straws were removed from liquid nitrogen and exposed in air for 5 s before being warmed in a water bath at 30°C for 30 s. After thawing, embryos were cultured at 38.5°C and 5% CO₂ in air for 48 h in either (1) medium supplemented with 2.5% FBS (n = 155; FBS group) or (2) medium supplemented with 2.5% of FBS for 2 h and then transferred to culture medium with BSA alone for the remaining 46 h (n = 134, BSA group). The embryos were evaluated at 24 and 48 h post-thaw for re-expansion and hatching. Data were analysed by one-way ANOVA followed by Tukey test with JMP 14 software (SAS Institute Inc.). Results revealed that embryos cultured in both FBS and BSA successfully re-expanded. Blastocysts cultured after thawing in medium with FBS undergo hatching significantly more often than those cultured with BSA (Table 1). In conclusion, these results demonstrated that FBS positively influences blastocyst hatching after freeze-thaw and culture of bovine embryos. Further studies need to be conducted to explore the mechanism by which FBS affects embryo growth, metabolism, and/or zona hardening to positively affect hatching.

Table 1.  Mean and standard error of re-expansion and hatching rates of post-thaw bovine embryos