157 Effects of high temperature within physiological range on the amino acid metabolism of in vitro growing oocyte-granulosa cell complexes derived from bovine early antral follicles

Summer heat stress impairs the developmental competence of bovine oocytes during the growing phase; however, the detailed mechanisms of this are still unclear. Amino acid metabolism in matured oocytes and early stage embryos is associated with their developmental competence. In addition, the supplementation of specific amino acids into culture medium can improve the developmental competence of oocytes. We hypothesised that heat exposure may alter the amino acid metabolism in follicles, which results in low oocyte competence. Therefore, we evaluated the effects of high temperature within physiological range on the amino acid metabolism of oocyte-granulosa cell complexes (OGCs) derived from early antral follicles using an in vitro growth (IVG) culture system. OGCs (n = 138) were collected from early antral follicles (0.5–1 mm) and cultured for 12 days. OGCs in the heat shock group (n = 69) were cultured under a temperature cycle of 38.5°C for 5 h, 39.5°C for 5 h, 40.5°C for 5 h, and 39.5°C for 9 h, while those in the control group (n = 69) were cultured at a constant temperature of 38.5°C for 24 h. The viability and antrum formation of OGCs were evaluated every four days, and half of the culture medium was replaced simultaneously. Among the OGCs that survived after IVG culture, those showing antrum formation and a similar size between groups in each replicate (n = 6; two samples per replicate) were selected for amino acid analysis. The diameters of the selected OGCs were measured every four days. Free amino acids and their metabolites in the spent culture medium were measured by the post-column ninhydrin method with an amino acid analyser. The rates of viability and antrum formation were analysed using the chi-squared test. The effect of group and culture period on the OGC diameters and the concentrations of amino acids and metabolites were evaluated using two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The rates of viability and antrum formation, and the diameter of the OGCs did not differ between the groups. 24 amino acids and their metabolites were detected. The concentrations of 12 amino acids decreased (P < 0.05), and those of alanine and ammonia increased during IVG culture (P < 0.05). The concentrations of leucine (control vs heat shock group; 409 vs 400 µM), isoleucine (266 vs 260 µM), aspartic acid (199 vs 194 µM), glutamine (261 vs 249 µM), and ornithine (35 vs 34 µM) were lower in the heat shock group than in the control group (P < 0.05), while that of alanine (291 vs 302 µM) was higher in the heat shock group (P < 0.05). Changes in alanine and glutamine metabolism have also been reported in matured oocytes and early stage embryos with low developmental competence. Therefore, we conclude that heat exposure can impair oocyte competence through metabolic changes in amino acids. Some treatments targeting amino acid metabolism in follicles may improve the quality of oocytes collected in summer.