Heat stress can impair ovarian function and reduce fertility in cattle. It is correlated with oxidative stress and can induce granulosa cell apoptosis and reduce steroid production. Effects of heat stress can be reversed by melatonin, but studies with ovarian cells have only been evaluated under atmospheric oxygen tension. Here we cultured granulosa cells (GCs) from antral follicles in fibronectin-coated 12-well plates in M199 for up to 144h under physiological (5%) oxygen tension. Treatments commenced after 48h of culture and consisted of two incubation temperatures (37.5 vs 40.0°C) and four melatonin concentrations (0, 20, 200, 2000 pg/ml) in a factorial arrangement. Cell number, steroidogenesis and ROS generation were assessed. Cell number decreased by 144 h of culture (P=0.028) at 40.0°C. Melatonin reversed the deleterious effect of high temperature on cell number. However, BAX mRNA expression was greater (P<0.009) in GCs cultured at 40.0°C than at 37.5°C by 144 h. Culture temperature did not affect ROS, but melatonin reduced (P<0.001) ROS generation at all concentrations tested. Oestradiol (E2 pg/105 cells) production increased with time (P<0.001) and was not affected by temperature. In contrast, high temperature caused a reduction in progesterone production (P4 ng/105 cells P<0.001) at 144 h of culture. Similarly, the effect of melatonin treatment depended on temperature melatonin linearly increased P4 production at 37.5°C whilst reducing P4 in cells cultured at 40.0°C. In summary, high temperature reduced cell viability and P4 production by GCs. Under low oxygen melatonin mitigated the negative effect of heat stress on cell number, reduced ROS generation, increased P4 production by GCs cultured at 37.5°C, but reduced P4 production by cells cultured at 40.0°C. The results of this study contribute to our understanding of the effects of heat stress on ovarian function and seasonal variation in cow fertility.
*PhD supported by Kurdistan Regional Government, Iraq.
11 Jul 2016 - 11 Jul 2016