Effect Of Melatonin On Bovine Semen Quality,Oocyte In Vitro Maturation And Embryo Development

In vitro fertilization(IVF) is an important pathway for in vitro embryo production(IVP). Improving the efficiency of IVP can provide greater number and lower cost sources of embryos for livestock husbandry. In this study, we investigated the effect of melatonin on semen quality, oocyte in vitro maturation and the subsequent embryo development, and the possible mechanism of the protective role of melatonin was also discussed. The results were as follows:1. Experiment I was conducted to study the effect of different concentrations of melatonin(0、10-7、10-5、10-3 M) on frozen semen quality and the subsequent embryo development. The results showed that:(1) the intracellular ROS level was significantly decreased in 10-5 and 10-3 M melatonin treatment group(9.9, 6.5 vs15.0),(P﹤0.05). The semen characteristics, including plasma membrane integrity,mitochondrial membrane integrity and acrosome integrity were higher in these two groups than those in the control group(61.3% vs 45.3%, 65.5% vs 47.2%, 70.9% vs45.8%),(P ﹤ 0.05), which indicated that the semen quality was significantly improved;(2) the blastocyst formation rate using spermatozoa treated with 10-3 M melatonin was significantly increased compared with the control group(43.0% vs34.3%),(P﹤0.05). Although the average number of total cells in the blastocysts was not significantly different in each treatment group(P ﹥ 0.05), the incidence of apoptotic nuclei in the blastocyst in 10-3 M melatonin treatment group decreased markedly(3.7% vs 6.0%),(P﹤0.05);(3) mechanistic explorations revealed that 10-3M melatonin treatment during sperm preparation significantly down-regulated the expressions of pro-apoptotic caspase-3 and Bax(P﹤0.05), while the expressions of anti-apoptotic BCL-2, XIAP and anti-oxidative CAT were significantly elevated in the resulted embryos(P﹤0.05).2. Experiment II studied the effect of different concentrations of melatonin(0、10-11、 10-9 、10-7 M) on maturation of bovine oocytes and the subsequent embryo development. The results showed that:(1) compared with the control group, at the concentration of 10-11 and 10-9 M, melatonin significantly improved the polar body formation rate(82.3%、79.3% vs 74.7%),(P﹤0.05). The proportion of oocytes with homogeneous distribution of mitochondria was significantly higher than that in the control and 10-11 M melatonin treatment group(74.1% vs 60.0%, 64.8%),(P﹤0.05).The percentage of oocytes with peripheral distribution of cortical granule was marked increased in all melatonin treatment groups, and the highest rate was observed in 10-9M melatonin group(69.1% vs 54.0%),(P ﹤ 0.05);(2) melatonin supplementation remarkably promoted the blastocyst formation rate in the resulted embryos(38.6%,42.5%, 37.3% vs 32.8%),(P ﹤ 0.05). Apoptotic assay showed that melatonin treatment did not change the total cell number in blastocysts, but the apoptosis index was significantly decreased, and the lowest rate was seen in 10-9 M melatonin group(4.5% vs 7.5%),(P ﹤ 0.05);(3) Gene expression analysis showed that 10-9 M melatonin treatment significantly reduced the expression of the pro-apoptotic caspase-3, while the expression of the anti-apoptotic BCL-2, XIAP and theanti-oxidative CAT, GPx4 were significantly up-regulated in the resulted embryos(P﹤0.05).