Efecto del KnockOut™ Serum Replacement y del suero fetal bovino en el cultivo y criotolerancia de embriones bovinos producidos in vitro

Abstract

The objective of this research was to evaluate the effect of KnockOut™ Serum Replacement as a substitute for fetal bovine serum in the production and freezing of bovine embryos produced in vitro. Ovaries were collected postmortem and transported to the Singamia laboratory where oocyte-cumulus complexes (COCs) were obtained. The COCs (n=1798) were matured and fertilized in vitro (IVF), and the presumptive embryos were incubated for 6 days in culture medium supplemented with 5% KnockOut™ Serum Replacement (KSR; n=589), or 5% Fetal Bovine Serum (SFB; n=555), or 0.6% bovine serum albumin (BSA; n=654). On day 7 after IVF, embryos were sorted and quality 1 embryos were frozen and stored in liquid nitrogen. One group of embryos was thawed and subjected to fluorescent double staining (propidium iodide and Hoechst) to determine the total number of blastomeres, and the number of intact and plasma membrane disrupted blastomeres. Another group of embryos was thawed and incubated for 72 hours to determine embryo survival at 4, 24, 48 and 72 hours post-thawing. Embryos cultured with SFB (P=0.0214) and KSR (P=0.0681) produced a higher percentage of blastocysts and a lower proportion of morulae and early blastocysts than BSA (P<0.05). From the SFB group, a higher percentage of regular blastocysts was produced than KSR and BSA. After thawing, embryos cultured with KSR had a lower number and proportion of altered cells than SFB and BSA (P<0.05). The reexpansion and hatching rate at 4, 24, 24, 48 and 72 hours was significantly lower in embryos cultured with SFB and KSR than with BSA, with KSR embryos achieving the lowest survival rate. In conclusion, the addition of KSR or SFB resulted in a higher proportion of blastocysts and a lower proportion of late stage embryos than BSA. Although a lower proportion of altered cells was observed in KSR, this group and SFB experienced lower post-thaw survival rate than BSA.