Study Of Cryopreservation On Goat Oocyte And It's Effect

Owing to only a few preliminary researches has been carried out on cryopreservation of goat oocytes up to now, besides the still low efficiency of maturation, fertilization and development of goat oocytes in vitro, it is difficult or impossible for researchers to timely obtain much cheap, usable oocyte and/or early-stage embryo, to explore the developmental mechanism of oocyte, to research transgenic animal, and to apply it in practice. In order to optimize the series of mediums and technical systems, to prepare for the establishment of oocyte bank and the forthcoming study of bioreactor of mammary gland in transgenic goat, we carried out the study of cryopreservation on goat oocytes and it's effect. The main results are as follows: By aspiration method, usable oocytes(A grade and B grade) were recovered from slaughtered goat ovary follicles(ф>2mm) according to Hyttle's theory of oocytes "capacitation"and Pawshe's classified criteria of oocytes. Average usable oocytes per ovary during breeding and nonbreeding seasons are 4.37 and 2.86, without significant difference (p>0.05). To imitate the microenvironment of ooctyes development in vivo, the developmental potency of oocytes cultured in TCM199 mediums with gonadotropin, serum, goat follicle (ф>5mm) fluid (GFF) and/or epidermal growth factor (EGF) was compared scientifically. The results suggested that hormone, estrous goat serum (EGS), GFF and EGF improve the nucleus maturation and cytoplasm maturation of oocytes at different degree. According to the results, we consider the better medium for oocyte mature in vitro is TCM199+10ug/ml FSH +10ug/ml LH+1ug/ml E2 +50%GFF+10%EGS+50ng/ml EGF+100umol/L cysteamine. The maturation rate of oocytes in the system exceeded 70%. Proper concentration and constitute of cryoprotectants is the key to cryopreservation of oocytes. First, the toxicity of four protectants with different densities to goat GV oocytes was checked, after which interacted with oocytes for 20 minutes. The results demonstrated, 10% protectants at 25 ℃showed weak toxicity, however, the toxicity enhanced with the density and/or temperature increase, the toxicity to GV oocytes was the following sequence: Glycerol (GL)>Dimethyl sulfoxide (DMSO) >Propylene glycol (PROH)≈Ethylene glycol (EG) (greater symbols mean p<0.05, and approximate symbols mean p>0.05). In the experiment, GV and MII oocytes of goat were cryopreserved with EG, PROH or DMSO combined with sucrose by Open-pulled straw (OPS) method. The results showed, sucrose increased significantly the rates of fertilization and cleavage on MII oocytes, 37.0% vs. 28.4% and 15.7% vs. 9.5% respectively(p<0.05). To GV and MII oocytes, the cryopreservation effectiveness of EG and PROH was not significantly different, but better apparently than DMSO(p<0.05); EG group best, the rates of normal morphology, maturation and fertilization to GV oocyte were 60.3%, 44.7% and 13.1%, but only one 2-cell embryo was obtained; to MII oocyte, the rates of normal morphology, fertilization, cleavage and ≧8-cell embryo were 80.6%, 37.0%, 15.7% and 11.8%. The concentration of EG, the time of equilibration and vitrification showed different influences on the cryopreservation of MII oocytes. The results appeared, MII oocytes equilibrated with 6%EG solution for 10~15 min and vitrified for 20 sec experienced cryopreservation, and acquired better rates of normal morphology and cleavage, 84.6% and 21.2% respectively. MII oocyte is suitable for cryopreservation, but GV oocyte not. The better protocol: MII oocytes equilibrated with solution(TCM199 + 20%FCS +0.3mol/L sucrose +6%(v/v)EG )for10~15 min and vitrified with solution(TCM199 + 20%FCS +0.3mol/L sucrose+31%(v/v)EG) for 20 sec, is quickly immersed into liquid nitrogen by OPS vitrification cryopreservation method. 49 IVF-IVC embryos (2~4 cells) derived from cryopreserved MII oocytes were transferred to the oviduct tube of 11 recipient goats by laparotomy. Two goats gestated, only one gave birth of 1 lamb, the kid survived. Conclusions: 1. Many usable MII oocytes can be obtained by using the optimization IVM system. 2. Goat MII oocytes, not GV oocytes, are suitable for cryopreservation by OPS vitrification method with cryoprotectant of EG, sucrose, and ideal freezing effectiveness can be obtained, and cryopreserved oocyte can develope into individual.