Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes

Abstract

The present study was undertaken to study the effect of metabolic stressors like elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and β-hydroxybutyric acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles, hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100-250 μm) and large pre-antral follicles (LPFs, 250-450 μm) were isolated from slaughterhouse ovaries by a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7 days respectively and examined for their growth, survival and growth rates of enclosed oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300 and 400 μM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70 μM): stearic acid, SA (10 μM)+Palmitic acid, PA(20 μM)+oleic acid, OA(40 μM), b) Medium combo (140 μM): SA (20 μM)+ PA(40 μM)+ OA(80 μM), c) High combo (210 μM): SA (30 μM)+PA(60 μM)+OA(120 μM), d) Very high Combo (280 μM): SA(40 μM)+PA(80 μM)+OA(160 μM)] and BHB (0, 0.5, 0.75, and 1 μM). Results indicated that ammonia, urea, NEFA and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes at the levels of 300 μM, 8 mM, high combo level of NEFA and 0.75 μM respectively. Our study may contribute to the identification of the mechanisms involved in decline of fertility due to metabolic and nutritional stress in ruminants.