Studies On The In Vitro Fertilozation Of Follicular Oocyte In Jiaxian Red Cattle

In this paper, Jiaxian Red cattle ovaries collected from slaughterhouse were used, ability of bovine 2⁶mm follicular oocytes for in vitro maturation(IVM), in vitro fertilization(IVF), in vitro culture(IVC) of fertilized eggs were studied,and factors affecting IVM,IVF and IVC in Jiaxian Red cattle also were investigated,and a practicable IVP procedure of embryo of Jiaxian Red cattle has been established. The results were as follows: 1.The available numbers of oocytes which retrieved averagely from ovary A was lower than that of from ovary B(2.0 Vs 5.5).The in vitro maturation rate of oocyte from ovary A and ovary B(55.00%Vs80.00%) had significant differences(P< 0.05),and the cleavage rate (37.5%Vs 60.17%) had greatly significant difference(P<0.01).The results indicated that ovary quality is a very important factor affecting IVM and IVF on Jiaxian Red cattle. 2. When oocytes were respectively cultured <20hours,20hours, 24hours and 28hours, the corresponding maturation rate of oocytes was respectively 53.33% 77.77%, 78.57%, 74.43%, there was significant difference between group < 20h and group 20h,24h,28h (P <0.05).Cleavage rate of oocytes between group 20h (51.79%) and group 24h(58.16%) were appearantly higher than that of group <20h (38.24%) and 28h(36.98%)(P<0.05),but there was not significant difference between group 20h and group 24h (P>0.05) The results showed that the optimal in vitro maturation time for oocyte of Jiaxian Red cattle should be between 20²4h, if time cultured fell short of 20h, oocyte could not mature fully,if exceeded 28h, oocyte would become degraded and old, therefor cleavage rate will significantly decline. 3.Single addition of 10%ECS(V/V) or addition of 10%FCS and hormones(FSH,LH,E2) coordinately in maturation medium TCM-199 obtained comparable effect on maturation rate and cleavage rate.There were not significant difference of maturation rate (75.00%Vs73.33%) and cleavage rate(54.35%Vs 52.21%) under circumstance of addition of two different kinds of serum(P>0.05). The results indicated that addition of 10%FCS and hormones(FSH,LH,E2) coordinately in maturation medium TCM-199 can be substituted by Single addition of 10%ECS. 4.Maturation medium supplemented with hormones(FSH,LH,E2)was helpful for the in vitro maturation and fertilization of oocyte in Jiaxian Red cattle.Addition of 10μg/mlFSH +1μg/mlE₂(Group1)(maturation rate77.78%,cleavage rate64.37%)or addition of 10μg/mlFSH +1μg/mlE₂+10μg/mlLH(Group2)( maturation rate82.76%,cleavage rate67.26%) both obtained higher maturation rate and cleavage rate compared with that of control group(maturation rate52.29%,cleavage rate45.46%)(P<0.01).There was no obvious difference between Group1 and Group2(P>0.05). Compared Group1 with Group2, maturation rate and cleavage rate of Group2 respectively increased 5.02 percent and 2.89 percent. It was founded that when adding 10μg/mlLH in Group2 promoted further the in vitro maturation of oocyte, so this showed that there was good collaborational function among LH,FSH and E2. 5.Comparative experiments were carried out among Swimming-up method and Percoll method of treatment methods of frozen-semen in order to observe respective effect of these 3 methods on the in vitro development ability of fertilized eggs.The results showed that there was not any significant differences among cleavage rate(51.58%,52.83%,53.20%)(P>0.05) of fertilized eggs treated by frozen-semen from Washing method ,Swimming-up method and Percoll method,there was also not any significant differences on the in vitro development rate of morula or blastocyst between treatment of Percoll method(16.66%,10.18%) and Swimming-up method(17.85%,16.07%),between treatment of Percoll method and of Washing method(8.77%,7.01%)(p>0.05).But it can be seen from experimental results that in vitro development of morula and blastocyst of treatment of Swimming-up method had some enhance compared with that of treatment of Percoll method, there was significant differences of in vitro development rate of morula or blastocyst between treatment of Swimming-up method and of Washing method(P<0.05).This indicated that, compared these 3 methods, Swimming-up method pehaps is economical and simple method of treatment methods of frozen-semen . It can be applied for IVP of embryo of Jiaxian Red cattle. 6.Addition of caffeine in fertilization medium can promote activity of sperm.Cleavage rate or development rate of morula or blastocyst in the presence of caffeine were higher than that of in the absence of caffeine,there was best effect of capacitation on sperm. It showed that higher concentration of caffeine can maintance strong activity of sperm at comparative long period of time. 7.Embryonic co-culture system of mSOF +10%ECS+1%NEAA+bovine granulose -cell-monolayer (0⁸cell stage) and supplement of 2%(V/V)EAA(2ul/ml,50×)(after 8-cell stage) was tentatively used to culture fertilized eggs by two steps gained similiar effect as co-culture system of TCM-199+10%ECS+bovine granulose-cell-monolayer cultured fertilized eggs consecutively. IVD rate of 4⁸-cell and morula or blastocyst had no significant differences between two culture systems(P>0.05). The results showed that two culture systems are praticable for culturing early embryo of Jizxian Red cattle 8. Embryo-gene bank of Jiaxian Red cattle was established firstly by using IVF in china Yellow cattle, the feasible IVF program of Jiaxian Red cattle has been screened out ,which provide academic basis and new means for the protect and using in Jiaxian Red cattle and other bovine breed.