Study On Bovine Oocyte In Vitro Maturation And In Vitro Fertilization

With the research development of bio-techniques and the requirement of market and profit, in Vitro Embryo Production (IVEP) system, which included in Vitro Maturation (IVM), in vitro fertilization (IVF), in vitro culture (IVC) and embryo cryo-preservation (EC), was improved a lot and turned to the stage of commercial application. IVF technique was called the third revolution in reproduction area, after artificial insemination (AI) and embryo transfer (ET). More research on bovine oocyte IVM and IVF is the important part of theoretic exploration and utilization of IVEP. This study research on IVM, sperm capacitation, IVF and IVC from the aspects of oocytes collection, sperm selection and sperm activity acceleration by extracellular ATP. The aim of this paper is optimizing the IVEP system, exploring the improving methods in IVEP procedure, so that accumulate experience for theoretic research and provide technique support.Firstly, this study investigated the efficiency of different oocyte collection ways. The result showed that among three methods, the efficiency of aspiration was the highest, better oocyes can be collected rapidly in this way, but some oocytes from the inner part of ovary maybe lost. Number of oocytes collected by mincing was the largest. Aspiration and mincing together not only more oocytes, but also can separate the oocyte by different quality, although the operation was complicated. The prior two methods were proper used when there were enough ovaries, the last way was proper to use when the number of ovaries was limited.During the bovine oocyte maturation, this research examined the possibility of serum-free maturation which 10%FBS was replaced with 0.3%PVP. As showed by result, the ratio of oocyte maturation treated with 0.3%PVP and 10%FBS were 70.08% and 79.75%, there were no difference between two groups. The fertility (49.19%, 50.78%)and morula and blastocyst(20.88%, 22.45%) rate were not different (P>0.05). This indicated that it was possible to replace serum by PVP in bovine IVM with some other improvement.Sperm selection is one of the most important steps n IVF procedures. This study made a contrast of swim-up and Percoll to deal with the ox semen. Result revealed that there were no difference between swim-up (52.14%) and percoll (54.29%) (P>0.05). The way of percoll is more convenient and efficient, so it could be the initial choice in utilization.At last, ATP and 0.9%NaCl was added to the thawed semen to examine if the extra cellular can improve the activity of sperm used in IVF. As it was revealed by the results, the fertility treated with ATP (55.74%) was remarkable higher than the contrast group(51.29%)(P<0.05). The cleavage rates were 51.64%(ATP) and 49.14%, the difference was not remarkable(P<0.05). Ratio of morula and blastocyst were 24.26% (ATP) and 24.36 %(P>0.05). The results suggested that extracellular ATP treatment to the sperm can improve the sperm activity and fertility.