Effect of Heat Shock and Cysteamine During In Vitro Fertilization on Preimplantation Development of Bovine Embryo.

Abstract

Summer heat stress is well known to decrease the conception rate in domestic animals by affecting oocyte quality, maturation, fertilization and early embryo development. Under heat stress condition, both of sperm and oocytes are exposed to heat stress during the fertilization. But there are few reports about the effect of heat shock during fertilization. In this study, we investigated the effect of heat shock on membrane damage of sperm and stress levels in zygotes, fertilization rate, embryo development. In addition the effect of cysteamine, an antioxidant thiol, on sperm damage and embryo development under heat-schocked condition was also evaluated. Oocytes derived from the local slaughter house and matured at 38°C for 20 h were used for experiments. Frozen sperm were thawed and live sperm were prepared by 90% percoll and BO solution. Experiment 1: Oocytes and sperm were fertilized at 40°C for 6 h under 5% CO2 as heat shock group. After in vitro fertilization (IVF), putative zygotes removed cumulus cells were cultured at 38.5°C 5% CO2 5% O2 in air. Day 2 and day 7 after IVF, cleavage and blastocysts rates were evaluated, respectively. Sperm damage was evaluated by PI/FDA staining after IVF. Stress status of putative zygotes was analyzed by detecting the level of reactive oxygen species with DCHFDA and hsp70 mRNA expression level. Rates of fertilization and polyspermy were measured at 9 h after IVF by staining the pronuclei with Hoechst 33342. All results of heat shocked group were compared with control group fertilized at 38°C for 6 h. Experiment 2: Sperm and oocytes were fertilized at 40 °C for 6 h with 0, 1, 5 and 10 µM cysteamine to IVF medium (HS). After IVF, putative zygotes removed cumulus cells were cultured at 38.5°C. Sperm damage was evaluated after IVF. Cleavage rate on day 2 after IVF and blastocysts rates on day 7 were also evaluated. Total cell number of day 7 blastocysts was also counted. All results were compared with sperm and embryos fertilized at 38°C for 6 h. All data were analyzed by Student t-test. A P value of less than 0.05 denoted a statistically significant difference. Experiment 1: Heat shock significantly increased the number of PI positive sperm. Fertilization rate was not significantly different, but the rate of polyspermy tended to be increased by heat shock. The levels of reactive oxygen species and hsp70 mRNA expression of zygotes were significantly increased by heat shock. Besides, both cleavage and blastocysts rate were decreased significantly by heat shock. Experiment 2: Under heat shocked condition, supplementation of 10 µM cysteamine to IVF medium significantly decreased the number of PI positive sperm and improved the blastocysts rates compared to HS group without cysteamine. There were no significant differences of blastocysts total cell number. These results indicate that 1) heat shock during IVF inhibits the normal fertilization by increasing the membrane damage of sperm and oxidative stress of oocytes and 2) cysteamine protects the sperm and oocytes by possible reduction of heat-shock induced oxidative stress. (poster)