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Objective: To assess the effect of L-citine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes. Methods:L-citine (3.8 mM) was added to maturation medium and the effect was assessed in the quality (Experiment 1) and in the cleavage and 4-cells stage (Experiment 2). Besides, the effect of L-citine addition on maturation medium (3.8 mM) and culture medium (1.5 mM) on embryo rate production was assessed. In Experiment 1, bovine oocytes from abattoir were randomly separated into two groups (the control group and L-citine group) forin vitro maturation. Matured oocytes were examined for cumulus cells expansion as an indicator of maturation, and the content of the mitochondrial activity, the presence of lipid droplets, the reduced glutathione, and the reactive oxygen species were measured by using specific fluorochromes. In Experiment 2, oocytes were matured as performed in Experiment 1, afterward fertilized and cultured until day 3, and cleavage rate and 4-cells stage rate were determinated. In Experiment 3,in vitro maturation and fertilization were done as performed in Experiment 2, but at day 3 of culture, each group of embryos was separated into two new groups, and L-citine (1.5 mM) was added in culture media until day 8. The cleavage and embryo development rate were determined on the basis with the oocytes put on maturation. Hatching rate was calculated from cleaved embryos. Results: The cumulus expansion rate at gradeⅢ and mitochondrial activity were significantly higher in the L-citine group in comparison with the control group (P0.05). In addition, cleavage and the proportion of embryo development and hatching rate were similar for all groups (P>0.05). Conclusions:L-citine as a supplement in culture media improves the cumulus expansion and increases the mitochondrial activity during in vitro maturation process but has no apparent effect on the cleavage and development of bovine embryos. Further investigations of L-citine addition on in vitroculture are needed to test their effect on embryo quality.