Effects Of Alpha-lipoic Acid On The Quality Of Goat Oocyte Maturation And Development

In the animal embryo engineering and technology research and production of animalembryo engineering, oocytes in vitro maturation is an important technical link. In Oocyte invitro maturation culture project, oxidative injury is an inevitable problem, and it affect theefficiency of oocyte maturation and the quality of matured oocytes, then influence theeffective use of oocytes. Alpha lipoic acid as a powerful antioxidant capacity are widely usedin cell culture and disease treatment. In This research the alpha-lipoic acid is added in oocytematuration culture medium,to study the optimum adding dose of LA in oocyte maturationculture medium and the effect of LA on he rate of oocyte maturation and quality of maturatedoocytes and the development of parthenogenetic embryos. the main results are as follows:1. In the oocyte in vitro maturation medium supplemented with500μM LA, almost halfof oocyte is dead, and oocyte quality was obviously decreased, so the median lethal dose ofLA in oocytes in vitro culture maturation system for oocytes is500μM.Taking500μM as amax added concentration limit progressively decrease, adding25μM LA in the oocyte invitro maturation medium,the oocytes in vitro maturation rate is highest and parthenogeneticembryos develop better,so25μM for LA in oocytes matured in vitro culture system is thebest concentration.2. In the oocyte in vitro maturation medium supplemented with25μM LA,the oocyte invitro maturation rates is significantly higher than that group without adding LA(67.5%vs59.66%)(P <0.05). And the blastocyst rate of parthenogenetic embryos issignificantly higher than the group without adding LA(23.51%vs18.8%)(P <0.05). Althoughthe early cleavage rate of two groups have no significant difference (P>0.05), but the earlycleavage embryo quality of adding LA group is significantly better than the group without LAsupplment. Based on the parthenogenetic blastocysts CDX2immuostaining, theparthenogenetic blastocyst inner cell mass proportion of LA adding group is significantlyhigher than the group without LA supplment (30.87%vs27.81%)(P <0.05).As is said above,adding25μMLA during oocyte maturation can improve the maturation rate of the oocytesand the development rate and quality of parthenogenetic embryo.3. In the oocyte in vitro maturation medium supplemented with25μMLA thenparthenogenetic activate the oocytes and develop the embryos to blastocysts. According to the TUNEL staining results of blastocyst apoptosis of we found that the day eighth blastocysts’apoptosis rate of LA adding group is significantly lower than the group without adding LA(2.51%vs3.68%)(P<0.05). Taking β-actin as the internal control gene,the quantitativedetection results of the apoptotic genes Bax,Bad, Caspase-3, Caspase-8, CytC andanti-apoptosis gene Bcl-2show that the apoptotic genes Bax, Bad, Caspase-3, CytC of the LAadding group express significantly lower than the group without LA supplment (P <0.05), butthe anti-apoptosis gene Bcl-2expression and apoptosis gene Caspase-8expression of twogroups are not significantly different (P>0.05).4. In the oocyte in vitro maturation medium supplemented with25μ MLA thenparthenogenetic activate the oocytes and develop the embryos to blastocysts. The GSHcontent in matured oocytes cytoplasm of LA adding group is sharply significantly higher thanthe group without LA supplment (9.3pmol/oocyte vs5.83pmol/oocyte)(P <0.01).However,the GSH contents in parthenogenetic blastocysts cytoplasmic of two groups have nosignificantly difference (P>0.05).In the two groups,GSH in parthenogenetic blastocystscytoplasm is significantly lower than that in matured oocyte cytoplasm (P <0.05).Taking β-actin as the internal control gene, t the expression of SOD and GPX4in matured oocytes andparthenogenetic blastocysts are quantitativly detected.The results show that,the SODexpression in matured oocytes of LA adding group was significantly higher than the groupwithout LA supplment (P <0.05), but the SOD expression in parthenogenetic blastocystshave no significant differences between the two groups (P>0.05). GPX4expression level inmatured oocytes of LA adding group is sharply significantly higher than the group withoutadding LA (P <0.01), in the parthenogenetic activation blastocysts, the GPX4expression ofLA adding group is significantly higher than the group without LA supplment (P <0.05).This paper researches on the supplment of alpha lipoic acid in oocyte in vitro maturationmedium provide reference for the application of LA in the oocytes in vitro cultivation, whileoptimizing the oocyte maturation system. This resarch has important significance on theimprovment of oocyte in vitro maturation rate and mature quality.