PSXIII-15 Functional and molecular responses of cultured bovine granulosa cells to increased concentrations of ammonia in vitro

Abstract Animals in agricultural production settings are often fed high levels of protein to maximize their performance (e.g., growth, lactation). However, animals that consume diets that are excessively high in protein often suffer from reduced reproductive efficiency. The causes behind this decreased reproductive performance are not fully understood. It is well established that increased dietary protein leads to increased concentrations of both ammonia and urea in the blood and other fluids of the body. These increased metabolite concentrations may be a possible link to the decline in reproductive success. The goal of this study is to characterize the molecular response of cultured bovine granulosa cells to various levels of ammonia. Our hypothesis is that increasing concentrations of ammonia in the culture medium will be associated with greater concentrations of intracellular reactive oxygen species (ROS), changes in gene expression, and reduced steroidogenesis, particularly 17-β estradiol production. The cells used for these experiments were harvested from abattoir-derived bovine ovaries. After the cells were established in culture for 48 h, they were exposed to media containing 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or 7 mM ammonia for 24 h. The cells were then evaluated for viability, proliferation rates, gene expression, oxidative stress, and 17-β estradiol production. There were significant changes in proliferation between the control and the 6 mM and 7 mM ammonia groups (2.71 vs. 1.96 vs 1.92 population doublings per 72 h culture period, respectively; P < 0.05). Cell viability was also statistically different between control (97.8% ± 0.3%) and 7 mM ammonia-treated (93.3% ± 1.2%) cells (P < 0.05). Ten out of 25 genes involved in steroidogenesis, redox reactions, or solute transport from our analysis showed significant changes (P < 0.05) and at least ± 50% difference in relative transcript abundance in at least one of the pairwise comparisons between treatment groups, but no obvious, consistent trends regarding dose-dependent effects of ammonia on granulosa cell gene expression were noted. There were no significant changes in 17-β estradiol production (P > 0.05) or ROS (P > 0.05) in response to ammonia treatment. The concentrations of ammonia that induced the changes noted are above normal physiological concentrations but are achievable in vivo depending on the amounts of protein in the diet of an animal. This study shows that granulosa cells are in some ways impacted by varying concentrations of ammonia. Future studies will reveal if and how these and other changes to granulosa cells under similar conditions might impact the ovarian follicular microenvironment, subsequent oocyte maturation and embryo development, and reproductive performance in general.