| AbstractIn this paper, cattle ovaries collected from Xi'an slaughterhouse were used, ability of bovine 2-8mm 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 were also investigated. The results were as follows:⒈ The corresponding maturation rate ,cleavage rate of oocytes and in vitro development rate of morula or blastocyst had no significant differences(P>0.05) between in U-shaped glass service,which volume is 400μl and no mineral oil overlaided and in traditional droplet,which was overlaided by mineral oil and volume is 50μl. The results indicated that U-shaped glass service of which volume is larger than conventional culture service can simplify the procedure of in vitro production of bovine embryos and may be used as a kind of medium for culturing oocytes and embryos under steady cultural circumstance. ⒉ Single addition of 10%D0ECS(V/V) in maturation medium obtained higher maturation rate (80.67%),cleavage rate(57.07%) and development rate of morula or blastocyst(30.00%) .The results indicated further that single addition of 10% D0ECS is practicable,and and is fit for applying spreadly.⒊ Comparative experiments were carried out among swimming-up method, Percoll method and Washing 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 shows that there was not any significant differences among cleavage rate (P>0.05) of fertilized eggs treated by frozen-semen from swimming-up method, Percoll method and Washing method respectively, there was not any significant differences of in vitro development rate of morula or blastocyst between treatment of swimming-up method and of Percoll method; between treatment of Percoll method and of Washing method(29.10%和28.47% :28.47%和22.41%；respectively, P>0.05),but there was significant differences of in vitro development rate of morula or blastocyst between treatment of swimming-up method and of Washing method(29.10%and 22.41%,respectively,P<0.05).The result indicated that swimming-up method is more suitable for applying in IVP of bovine embryos than Percoll method and Washing method under circumstance of considering cost. ⒋ Compared with 18-20h' oocyte-sperm interaction when m--Tyrode's was used as in vitro fertilization medium ,6-8h' oocyte-sperm interaction has not significant effect on the cleavage rate of zygote,in vitro development rate of 8-cell and morula or blastocyst(P>0.05).This result indicated that conventional 18-20h'oocyte-sperm interaction can be substituted by 6-8h'oocyte-sperm interaction so as to accord further practicality of IVP of bovine embryo. ⒌ Embryonic culture medium of human,that is IVC-ONETM(0-8cell stage)and Quinn's D3+HTF(8cell-morula or blastocyst developmental stage)were used tentatively for culturing bovine embryo, and gained the same effect as bovine culture medium --TCM-199 (IVP rate of 8-cell and morula or blastocyst were 60.63%,64.83%and 26.60%,32.76% respectively between treatment of human's embryonic culture medium and of bovine culture medium.).This result implied that research of both composition of human's embryonic culture medium and its mechanism to embryo can contribute to optimizing in vitro culture system of bovine embryo.⒠Comparatively feasible and effective IVM-IVF-IVC procedure of bovine follicular oocytes was established as follows: Bovine cumulus-enclosed oocytes were matured in tissue culture medium-199(with Earle's salts)buffered with 25mM Hepes and supplemented with 10%(v/v)ECS, which solution filled in U-shaped glass service and volume is 400μl and no mineral oil overlaided. After 23h of IVM, matured oocytes and sperms which come from frozen-semen treated by swimming-up method were put in IVF medium contained 6mg/ml bovine serum albumin(BSA) , 10μg/ml heparin and 5mM caffeine. Oocytes and sperms were incubated all together for 6-8h, then presumed... |
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