| This study was designed to investigate the effect of L-carnitine on the in vitro maturation (IVM) of oocytes in buffalo, meanwhile, the effect of L-carnitine on the change of the lipid utilization efficiency and antioxidant capacity of oocytes was also detected, so as to explain the mechanism that L-carnitine impacts the maturation of buffalo oocytes.Firstly, the effects of L-carnitine on the maturation of oocytes in buffalo were investigated. The buffalo cumulus-oocyte complexes (COCs) were respectively cultured in the maturation medium supplemented with different concentrations of L-carnitine (1μg/mL, 10μg/mL or 1000μg/mL) for 24h, and the maturation rate of oocytes was statistics respectively. The result showed that adding with 1μg/mL or 10μg/mL or 100μg/mL L-carnitine into the maturation medium, there were significant differences in the maturation rate of oocytes compared with the Oμg/mL control group (64.44%±1.93%,66.17%±2.76%, 63.27%±1.19%vs 56.60%±1.56%, P<0.05), furthermore, the developmental rate of blastocyst in 1 μg/mL group was significantly improved than that of the control group (34.37%±3.34% vs 21.62%±2.89%, P<0.05). The relative expression of cumulus expansion related gene ptx3 and has2 in buffalo cumulus cells were detected with the method of real-time PCR, and found that the relative expression of has2 in each L-carnitine treatment groups was significantly increased compared with the control group(P<0.05), among of them, the relative expression in 10μg/mL group was highest. Moreover, the relative expression of ptx3 in each L-carnitine treatment group was all increased compared with the control group, but it was significantly higher in the both of 10μg/mL and 100μg/mL groups than that of the control group (P<0.05).Secondly, the effect of L-carnitine on the lipid utilization efficiency of buffalo oocytes was detected. The change of lipid droplet content in buffalo oocytes during the maturation progress in vitro was detected by oil red O staining method, and the result revealed that there was a phenomenon that the lipid utilization efficiency of buffalo oocytes was very low during the maturation progress in vitro. However, when buffalo COCs were respectively cultured in the maturation medium supplemented with different concentrations of L-carnitine μg/mL,10μ/mL or 100μg/mL) for 24h, the lipid droplet content in buffalo oocytes of each treatment group was significantly lower than the control group(P<0.05). The enzyme linked immunosorbent assay method was used to analyse the triglyceride content of buffalo oocytes, and found that the triglyceride content in oocytes of the 10μg/mL and 100μg/ml treatment groups were significantly lower than that of the control group (2.601nM,2.113nM vs 3.542nM, P<0.01).The relative expression of lipid metabolism related gene cptla, atgl and hsl were respectively detected with the method of real-time PCR, the results showed that comparing with the control group, the relative expression of these genes in each L-carnitine treatment group were increased, among of them, the expression of cptla in μg/mL and 10μg/mL groups, the expression of atgl in 10μg/mL group and the expression of hsl in 100μg/mL group were significantly higher than that of the control group (P<0.05).Lastly, the effect of L-carnitine on the antioxidant capacity of buffalo oocytes was investigated. The malondialdehyde content in buffalo oocytes during the maturation progress in vitro was detected by the enzyme linked immunosorbent assay method, and found that there was a phenomenon that the buffalo oocytes were constantly threated by the active oxygen during IVM. However, when the buffalo COCs were respectively cultured in the maturation medium supplemented with different concentrations of L-carnitine (1μg/mL, 10μg/mL or 100μg/mL) for 24h, the malondialdehyde content in oocytes of 1μg/mL,10μg/mL and 100μg/mL treatment groups were significantly lower than that of the control group (0.125797μM,0.1252170μM,1241552μM vs 0.142609μM, P<0.01). The data from detecting the reactive oxygen content in buffalo oocytes also showed that the reactive oxygen content of treatment groups (1μg/mL,10μg/mL or 100μg/mL) were significantly lower than that of the control group (P<0.05). The RT-PCR results showed that the relative expression of antioxidative genes gpx4 in buffalo oocytes in each L-carnitine treatment group were increased than that of the control group, and there was a significant difference in 10μg/mL and 100μg/mL groups compared with the control group (P<0.05).These results indicated that:(1) During the maturation progress in vitro of buffalo oocytes, oocytes are damaged by the active oxygen and its lipid utilization efficiency was lower. (2) Adding with an appropriate concentration of L-carnitine into the buffalo maturation medium can improve the lipid utilization efficiency and antioxidant capacity of oocytes, so as to promote the oocyte maturation. |
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