| In this study,the small antral follicular oocytes were collected from follicles(＜2mm)by using cutting method,and the oocytes of A and B grade were used for in vitro maturation(IVM),in vitro fertilization(IVF)and in vitro culture(IVC).The objective of this paper was conducted to establish an optimum culture system and methods for IVM,IVF and IVC of Nanyang cattle,and some affect factors were also involved.The results as follow:1.Small antral follicular oocytes were collected by using cutting method,peel-off method,tissue-breaking method respectively,and the obtained oocytes used for IVM. The results showed that the number of oocytes of the three method were 8.63,10.96,12.04 and there were no significant difference amomg them(p＞0.05),the matured rate(MR)and fertilized rate(FR)of cutting method was the highest among the three methods(28.57%,26.28%),and there was no significant difference between cutting method and peel-off method(25.71%,21.24%)(p＞0.05),but there was significant difference between cutting method and tissue-breaking method(18.57%, 19.17%)(p＜0.05).2.Maturation medium added with 0.0%BFF(10%FCS),10%BFF,20%BFF,30% BFF respectively for IVM.The results showed that the MR and FR of 10%BFF group were significantly higher than 30%BFF group(p＜0.05).The MR and FR of 20%BFF group were both lower than 10%BFF group,but there were no significant difference between them(p＞0.05).3.Small antral follicular oocytes were IVM for 24～26h,26～28h,28～30h,30～32h.The results showed that the MR and FR of 26～28h group were higher than other three groups(25.00%,VS 23.33%,21.67%,20.00%)(22.11%VS 19.17%,21.11%, 19.48%),but there were no significant difference among them(p＞0.05).The FR of 24～26h group was lower than other three groups,but there were no significant difference among them(p＞0.05).4.Maturation medium added with 10%FCS,20%FCS,10%OCS,20%OCS respectively for IVM.The results showed that in the FCS group and OCS group respectively,the MR and FR of 10%group(OCS:31.67%,28.00%)(FCS:28.33%, 27.50%)were higher than 20%group(OCS:23.33%,23.08%)(FCS:20.00%,20.95 %)(p＞0.05),So the effect of OCS was better than FCS.5.Maturation medium added with A:10%FCS+GCM,B:10%OCS+GCM, C:10%OCS+LIF+GCM,D:10%OCS+LIF+BOECM for IVM.The results showed that the MR and FR of C,D groups(35.00%,34.21%)(32.50%,32.56 %)were significantly higher than A group(22.50%,23.00%)(p＜0.05).The MR and FR of C group were higher than D group,but there no significant difference(p＞0.05).6.Three washing methods(percoll method,swimming up method,direct washing method)were used in this study.The results showed that the FR and cleavage rate(CR) of swimming up method(31.11%,28.57%)were higher than percoll method(26.56 %,25.00%),but there were no significant difference between them(p＞0.05).The FR and CR of direct washing method(21.21%,14.28%)were significantly lower than swimming up method(p＜0.05),and its CR was also significantly lower than percoll method(p＜0.05).7.The fertilization medium(mTyrode's)added with 10mg/l,20mg/l,30mg/l, 40mg/l,50mg/l respectively for in vitro capacitation of sperm.The results showed that the FR of five groups increased(20.45%,21.57%,23.81%,26.47%,27.91%), but there were no significant difference among them(p＞0.05).The CR of five groups also increased,but there were no significant difference among 10rag/l,20rag/l,30mg/l groups(p＞0.05),the CR of 40mg/l and 50mg/l group were significantly higher than 10mg/l group(p＜0.05).8.The GCM and BOECM were used for IVC.The results showed that the FR(28.99%)and 4-cell development rate(7.14%)of BOECM group were higher than GCM group,but there no significant difference between them(p＞0.05).So the GCM and BOECM both can used on in vitro culture of embryos. |
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