Studies On Effects Of Vitamin C And E And Serum Types On Buffalo Early Embryo Development And Apoptosis In Vitro

The objectives of the present study were to assess the effects of vitamin C(vit c)and vitamin E(vit e)on in vitro early embryo development and apoptosis in buffalo,and test the effects of estrus buffalo serum(EBS)and estrus cattle serum(ECS)on the oocytes maturation by investigating in vitro early embryo development in buffalo,based on this study,to subsequently optimize the culture system for a better in vitro early embryo development in buffalo.The thesis is arranged in three chapters:chapterⅠis a review of literatures;chapterⅡandⅢdeal with the experimental work.The objective in chapterⅡwas to assess the effects of the supplementations of vit c or/and vit e in culture medium on in vitro early embryo development and apoptosis in buffalo.Oocytes were aspirated from 2～6 mm follicles on the buffalo ovaries from slaughterhouse and matured in vitro. After maturation,oocytes were fertilized with buffalo sperm for 18～20 h and then the presumptive zygotes were cultured with granulose cells in culture medium supplemented with different concentration vit c or/and vit e.Cleavage and blastocyst rates were recorded on D2 and D6～D9 after insemination, respectively.Apoptosis,necrosis and total cells of each blastocyst were counted with fluorescent microscope after staining with Annexin V-FITC Apoptosis Derection Kit(KGA1032).The results showed that:ⅰ)supplemented with 100μmol/L or 200μmol/L vit c in culture medium,the cleavage rate,blastocyst rate and cell numbers of blastocyst were improved to a certain extent,while the D7 blastocyst rate and the hatching rate lower than that of control,but there was no significant difference between the treatment groups and the control group(P＞0.05).there was a benefic effect on the development of early embryo and the decrease of apoptosis rate and necrosis rate when the concentration of vit c in culture medium was 200μmol/L,but there was no significant difference(P＞0.05);ⅱ)supplemented with different concentrations of vite in cul ture medium (20μmol/L,100μmol/L or 200μmol/L),with the increasing of vit e concentration,both the cleavage rate and blastocyst rate were increased,but there were no significant differences among the treatments and the control group (P＞0.05).supplemented with 100μmol/L vit e,both the D7 blastocyst rate (80.00 VS 77.22)and hatching rate(67.20 VS 65.17)were higher than the control's,but the differences of them were not significant,cultured in the medium supplemented with different concentrations of vit e,both the apoptosis and necrosis rates of blastocyst cells were decreased(P＞0.05);ⅲ)the combination of supplementation with different concentrations(20μmol/L,100 μmol/L or 200μmol/L)of vit c and vit e,the cleavage rate and blastocyst rate were not significant differences among the treatment and control groups,but their blastocyst qualities was better than the controls,the combination of supplementations with 20μmol/L or 200μmol/L of vit c and vit e,both the apoptosis and necrosis rate of blastocyst cells were low than that of control,but there were not significant differences between them(P＞0.05);ⅳ)the apoptosis and necrosis cells could be distinguished from normal cells in blastocyst by using Annexin V-FITC apoptosis Derection Kit(KGA1032).The results indicated that:1)Early embryo development and apoptosis,necrosis decrease in blastocyst cells could be promoted in some extent by supplementing with 200μmol/L vit c in culture medium based on TCM-199;2)It helped to promote the development of buffalo early embryo and decrease apoptosis rate and necrosis rate in blastocyst cells supplemented with vit e in culture medium based on TCM-199,the effect was best when the concentration of vit e supplemented in culture medium was 100μmol/L;3)A combination of supplement with vit c and vit e in culture medium based on TCM-199 had no significantly effect on the promotion for buffalo early embryo development but it helped to reduce apoptosis rate and necrosis rate in blastocyst cells.The objective in chapterⅢwas to test the effects of EBS and ECS supplemented in the oocytes maturation culture medium to promote early embryo development of buffalo in vitro.After maturation in medium supplemented with 10%EBS or ECS,oocytes were fertilized with buffalo sperm for 18～20 h and then the presumptive zygotes were cultured with granulose cells in culture medium.The results showed that:supplemented 10%EBS in TCM-199 culture medium,a similar blastocyst rate(20.59%VS 21.09%)with the ECS and higher blastocyst rate on D7 and hatching rate(proportions of total blastocysts)than the ECS were got(P＜0.05),but the average cell number of blastocyst on D8 was significantly lower than the ECS's(P＜0.05)(78.31 VS 108.87).The results indicated that EBS could be used on in vitro maturation of buffalo oocytes system.