Studies On Maturation And Fertilization Of Wapiti (Cervus Elaphus) Oocytes In Vitro

Along with the development of embryo engineering technique, it changes more and more urgent and essential to begin to study in vitro embryo production of deer in our country. We studied in vitro maturation and fertilization of Wapiti(Cervus elaphus) ovary oocytes. The aim of the study is to upbuild a set of perfect protocol of in vitro oocytes culture that is feasible to deer in the practice, so as to give theoretic basis, experience and methods to the deep study of deer IVF.The results of our studies are as followings:l.Wapiti(Cervus elaphus) superovulation with Folltropin-V from Canada can increase the number of ovary oocytes derived from abattoir material. The program is as follows: After being treated with CIDR device for 8 d, all hinds received a four-day twice-per-day injection of 120~140mg, 80~100mg, 60mg, 40mg, Folltropin-V, or a single injection of 500IU, PMSG in place of Folltropin-V at the fourth day.2.The number of COCs collected by first aspirated and then deeply cut from the abattoir-derived deer ovaries can be increased. The method of deeply cut can make up for the shortage of single aspiration and have made the number of COCs increase to twice of single aspiration. The total operating time is within 3minutes.3.Gently washing the follicular fluid, compared with no washing and direct checking oocytes, has more advantage: convenient, the protein difficultly coacervating,'fastly moving ooocytes and saving time.4.The COCs can be divided into four degrees:grade A is with fully cumulus cells and have four layers of comulus cells at least; grade B have 1-3 layers fully cumulus cells; grade C is hemi-nude egg with sectional cumulus cells; grade D are nude eggs> deformed oocytes and degenerative oocytes. The COCs of grade A and grade B have higher maturation rates.5.The in vitro maturation rate of oocytes is obviously higher under 38.5℃ than under 37.0'C(p<0.05).6.Medium 199 is the culture medium of in our experiments.. The following culture systems are all available and can make the in vitro maturation rate reach 78.9%, 84.2%: (l)M199+10%FBS+0.2mM Na-pyruvate+lmM antibiotic+10mg/ml GnRH +10mM Hepes +lmg/ml E2; (2)M199+20%FBS+0.2mM Na-pyruvate+lmM antibiotic+lOmg/ml GnRH.7.The method of swim-up is effective for Wapiti(Cervus elaphus) sperm washing and capitation in vitro. The two centrifugalizing speeds in washing sperm being 2200rpm, 2000 rpm is best. The effects of operating in TALP sperm washing or fertilization medium(2) and in BO sperm washing or fertilization medium(2) are all better than in TALP sperm washing or fertilization medium(l) and in BO sperm washing or fertilization medium(l). The time of sperm living in vitro can reach to 24 hour when sperm cultured in BO fertilization medium(2), and the time in TALP-fertilization medium(2) is only 9h.8.In vitro fertilization with BO or TALP can all make the ovum culturing to cleavage stage, and the fertilization rate are 66.7%, 70.6%. The effects of fertilizing in the two mediums are unobviously(p>0.05).9.The early embryo of Wapiti(Cerv玸 elaphus) developed in embryo culture madium M199. The rate of the cells that have developed to 2-cell can reach to 50%.