Vitrificação em fluido folicular bovino.

Abstract

Vitrification is one of the methods used for immature and mature bovine oocytes. However, it needs improvement regarding hearing competence after warming. The use of bovine follicular fluid (BFF) is known in in vitro production systems, but unexplored in vitrification media. The aim of this study was to vitrify oocytes in a solution supplemented with bovine follicular fluid (BFF) or fetal bovine serum (FBS) to investigate post-warming survival through nuclear maturation and analysis of HSP70 gene expression. The study was divided into two experiments: In experiment 1, immature cumulus oophorus complexes (COCs) were distributed into three experimental groups. In experiment 2, three experimental groups were used for mature oocytes and three experimental groups for immature cumulus oophorus complexes. The control group of oocytes (without vitrification) in immature oocytes were directly subjected to in vitro maturation. Both experiments used, in group 1, (TCM 199 medium + 20% fetal bovine serum (FBS) + cryoprotectants), and in group 2, vitrification in (TCM 199 medium + 20% bovine follicular fluid (BFF) + cryoprotectants), followed by in vitro maturation of immature oocyte complexes (OCCs). In experiment 1, all immature oocytes from the experimental groups, after maturation, were stained with acetic orcein for structural visualization. Meanwhile, in experiment 2, matured and immature oocytes, after warming, were subjected to gene expression analysis of the heat shock protein HSP70. Survival rates of oocytes that were matured to assess meiotic stage II (MII) were expressed as percentages and evaluated by analysis of variance (ANOVA) and Bonferroni post-hoc test. For statistical analysis, SigmaPlot® 14.5 software was used with a significance level of P<0.005. The statistical analysis of gene expression was performed using two-way ANOVA in the Sigmaplot program with a significance level of 0.05. In the maturation analysis of immature oocytes, no significant differences were found between the groups vitrified with FBS or FFB (p>0.001), and the maturation rate was lower (p>0.001) than the control group. Regarding experiment 2, oocytes from the vitrified matured group, the gene expression analysis of the HP70 protein showed no difference between the groups vitrified with FBS and FFB, while the granulosa cells of immature oocytes, FFB, and FBS also differed from each other and from the control (p < 0.05). These results indicate that follicular fluid can be used to replace FBS supplementation in vitrification media of bovine oocytes.