Effect Of Melatonin On Buffalo Oocyte Maturation And Their Subsequent Embryo Development In Vitro

The reactive oxygen species (ROS) were considered as aggressive molecules against oocyte maturation and embryo development in vitro. Embryos cultured in vitro encountered higher oxygen toxicity elicited by ROS than their in vivo counterparts. Melatonin could improve oocyte quality and protects oocyte from oxidative stress. The process of melatonin and its metabolites (?) successively scavenging ROS is referred as the free radical scavenging cascade. Melatonin differs from other conventional antioxidants because the cascade reaction is a novel property. Mitochondria are an important target of melatonin.This study was carried out to investigate the effect of different concentrations of melatonin supplemented in vitro maturation and culture media on buffalo oocyte maturation and embryo development in vitro.In experiment1, immature oocytes were cultured for24h in vitro in maturation medium supplemented with different concentrations of melatonin (10-10mol/L,10-9mol/L,10-8mol/L) and without melatonin as control. After24hr, oocytes with first polar body were counted as matured ones. The result showed that supplementation of melatonin at10-9mol/L concentration in the maturation medium resulted in similar maturation rate to other treatments (46.28±1.74%vs 43.15±5.24%,43.40±3.12%, P>0.05), but significantly higher than the control (46.28±1.74%vs32.90±2.21%, P<0.05).In experiment2, only culture medium was supplemented with various concentrations of melatonnin (10-8mol/L,10-9mol/L,10-10mol/L), the cleavage rate of10-8mol/L treatment was similar to the other groups (59.6%vs53.6%,49.5%,48.0%, P>0.05), but the blastocyst rate was significantly higher than the control (34.3%vs20.6%, P<0.05). Based on the results of experiments1, the maturation medium was supplemented with the most optimal concentration of melatonin (10-9mol/L) and the culture medium was supplemented with three threatments (10-8mol/L,10-9mol/L,10-10mol/L) and control. The cleavage rate of10"9mol/L treatment was similar to the other groups (70.0%vs64.8%,65.2%,60.9%, P>0.05). And the blastocyst rate of10-9mol/L treatments was similar to the other groups (28.9%vs15.4%,26.1%,25.3%, P>0.05), however, only significantly higher than control (28.9%vs15.4%, P<0.05).In experiment3, buffalo oocytes were matured in the presence or absence of melatonin for24hr. The quantity of ROS produced by the oocytes was measured using2’,7’-dichlorodihydrofluorescein diacetate. The oocytes without the first polar body that matured in the absence of melatonin was the control. Compared with the control, melatonin-treated (10-9mol/L) oocytes had significantly lower levels of ROS than did the control oocytes.In experiment4, Mitochondria copies were measured by real-time polymerase chain reaction (RT-PCR). Buffalo oocytes were matured with 10"9mol/L melatonin and without melatonin for24hr. There is no difference in the melatonin-treatment group and control. The result indicated that melatonin had no effects on the Mitochondria copies.These data suggest that melatonin could increase the maturation and blastocyst rates of buffalo oocyte in vitro and may have important implications for scavenging ROS.