A Preliminary Study On The Effect Of Vitamin A On The Maturation Of Yak Oocytes In Vitro And Its Mechanism Of Action

The yak,known as the "boat of the plateau",is an important economic source for local herders.However,yaks are less sexually mature and have lower reproductive capacity than cattle on the plains,so it is important to improve yak reproductive capacity through embryo engineering technology.In vitro culture of oocytes is a fundamental part of embryo engineering,but the low maturation rate of yak oocytes in vitro and the poor embryonic development ability will limit the development of yak embryo engineering technology.Therefore,it is important to improve the in vitro maturation of yak oocytes to further improve the application of yak oocyte embryo engineering technology.Vitamin A can promote the meiosis of oocytes in mice,pigs,cows and sheep,and further promote the maturation of oocytes.However,the effect of vitamin A on the maturation of yak oocytes in vitro has not been reported in the literature.Therefore,in this experiment,four different concentrations of vitamin A(2,5,10 and 20 μM)were added to yak oocytes to examine its effects on the maturation and developmental ability of yak oocytes,and to verify the potential signaling pathway node genes,so as to provide reference materials for further improving the maturation of yak oocytes in vitro.The experimental results were as follows.1.The maturation rates of oocytes were counted by observing the expulsion of first polar bodies from yak oocyte complexes(Cumulus-oocyte complexes,COCs)in different vitamin A concentration groups(0,2,5,10 and 20 μM).The results showed that the maturation rate of control group was 63.60% at 0 concentration,and the difference was significant(85.77%,71.68%,71.44%)at 2,5 and 10 μm concentrations compared with the control group(p < 0.05),and the maturation rate of oocytes was the highest at 2 μM concentration of vitamin A,which was significantly different compared with other groups(p < 0.05).While 20 μM The maturation rate was 57.20% for the 20 μM concentration,which was the lowest and significantly different compared to the other groups(p < 0.05).2.Mature yak oocytes containing different concentrations of vitamin A in each group were further activated by solitary females and their oocytosis rates were counted.The results showed that the oocyte cleavage rate in the control group was 52.03%,and the three concentrations of 2,5 and 10 μM were significantly different(76.80%,64.85% and61.22%)compared with the control group(P < 0.05),and the highest oocyte cleavage rate was achieved at the concentration of 2 μM of vitamin A,which was significantly different compared with all groups(p < 0.05),while the concentration of 20 μM The blastocyst rate was 24.79% in the control group,and 65.61%,40.63% and 33.96% at 2,5 and 10 μM,respectively,which were significantly higher than the control group(p < 0.05)and the highest rate at 2 μM.This further demonstrated that 2 μM of vitamin A promoted the maturation of yak oocytes.3.The effect of different concentrations of vitamin A on the expression of genes related to the maturation of yak oocytes was examined by RT-q PCR.The nodal genes RXRA,RXRB,RXRG,RARA,RARB,RARG,STRA8 in the typical signaling pathway of oocytes and MEK and ERK1/2 in the atypical signaling pathway were selected and examined.The results showed that RARA,RXRA and STRA8 were expressed at different concentrations of vitamin A.The rest of the genes were not expressed,demonstrating that vitamin A mainly affects yak oocytes through the typical signaling pathway.The expression of the genes RARA,RXRA and STRA8 was significant at 2 μM compared to the control group,further demonstrating that the addition of 2 μM of vitamin A had a beneficial effect on the maturation of yak oocytes.On this basis,the dynamic expression of RARA,RXRA and STRA8 genes at 2 μM vitamin A concentration was examined in yak oocytes during the activation period of GV,MI,MII and solitary females.The results showed that the expression of these three genes gradually increased from the GV stage and peaked at the MII stage,with significant differences compared with the control group.The expression of STRA8 gene was the highest,which further demonstrated that vitamin A promoted the maturation of yak oocytes at the molecular level.In conclusion,we optimized the concentration of vitamin A in the in vitro culture of yak oocytes and found for the first time that the addition of 2 μM vitamin A could promote the maturation of yak oocytes during the in vitro maturation of yak oocytes and significantly increase the rate of oocyte cleavage activated by late solitary females.