Bovine Oocytes Maturation And Fertilization In Vitro

With the further studies on embryo engineering technique, commercialization and spread of it, the technique of bovine follicular oocytes maturation and fertilization in vitro have become one of important ways to solve the imminent problems of embryo production in vitro. Based on the experience and methods summed up by other researchers, the technique of cell culture was employed to study bovine follicular oocytes maturation, fertilization and the development of earlier embryo in vitro. The objective of this research is to upbuild and perfect a set of high-and-new-tech systems to obtain high-yield and high-quality bovine embryo, to provide technical operational regulations and ways and means to embryo engineering in practical production, to accumulate data for theoretical research. The results were as followings:1. The ovaries collected from slaughter houses were surface cut or aspirated, and then deeply cut so that the available numbers of oocytes were remarkably increased. The optimal preservation time for ovaries should be within 2h, and should not be more than 6h. The optimal in vitro maturation time for bovine superficial follicular oocytes was between 22~24h, but the in vitro culture time for bovine inner follicular oocytes should be prolonged by 1~2h. Consanguinity-distinct and genetic-strain-perfect embryo have been obtained by resorting to the technique of ovum pick up from live donors.2. The oocytes with highest maturation and cleavage rates collected fromovaries preserved in saline at 30~39 showed significant differences between the oocytes collected from ovaries preserved at 2~8 (16.7%, 0%) and the oocytes collected from ovaries preserved at 20~29 (54.1%, 31.7%), respectively(P<0.05).3. The corresponding maturation rate (61.1%vs62.9%) and cleavage rate (34.4%vs35.2%) of oocytes had no significant differences (P>0.05) between in 4-well plates containing 800 l culture medium and droplets containing50 l medium.4. Oocytes collected from ovaries in different estrous cycle(follicular phase, luteal phase) showed no influence on the in vitro maturation of oocytes(The maturation rate were 69.2%, 64.3%,respectively).5. The oocytes collected from follicular with diameters 2~6 nm showed highest maturation rate(72.1%) and significant differences between the oocytes from follicular with diameters less than 2 nm(58.4%), between 6~8 nnn(56.4%)and more than 8 nm(35.0%),respectively.6. The bovine oocyte in vitro maturation was accelerated by adding 10% fresh bovine follicular fluid (bFF) to maturation medium, but was inhibited if adding bFF with concentration of 20% or 30% to maturation medium.7. The quality of cumulus oocyte complexes showed positive influence on oocytes maturation in vitro, the maturation rate( 86.1%, 66.3%, 35.6%) and cleavage rate (42.8%, 31.9%, 10.5%) of the three-graded (A,B,C) oocytes were significantly different.8. Ml99 was the ideal medium for bovine oocytes in vitro maturation, M199 + 50IU/ml LH+ 100 IU/ml FSH + 1 g/ml 17P-E2 and M1 99 + 50IU GnRH + 1 g/ml 17P-E2 were ideal culture system for bovine oocytes in vitro maturation(maturation rate were 69.4%,67.5%; and cleavage rate were 35.4%,42.7%).9. BO swim-up and TALP Percoll were ideal methods for bovine sperm washing and capitation in vitro. The cleavage rates after fertilization were 56.1%, 57.6%, respectively, had no significant differences (P>0.05).10.Bovine oviduct epidermic cell was ideal co-culture cell for surmount bovine earlier embryo development block (blastocyst rate was 32.4%); parted-stage culture was suitable for the research of bovine earlier embryo development in vitro (8-cell development rate was 60.4%, blastocyst rate was 35.5%).