| China is a traditional horse-raising country,Although horses are in large stock,but We are not developed countries in horse industry,there were no horse can reach the international competitive arena of modern equestrian sports.The shortage of modern horsemanship horses has become an important constraint factor for the development of horse industry in China.Embryo transfer technology has been mature and applied in cattle,sheep and other livestock breeding,but horse embryo transfer technology is not yet fully mature,and related research and application are relatively backward.Therefore,The search was utilizable for the technical needs of the development of horse industry in China,to discuss the key technologies of horse embryo transfer,such as estrus identification,artificial insemination,non-surgical embryo collection and embryo transfer.On the basis of this research,we commissioned a study on the in vitro construction technology of equine embryos,including ovaries transport,oocytes collection,oocyte maturation in vitro,establishment of somatic cell lines,cell fusion and somatic cell nuclear transfer.The results are as follows.1.Accurate identification of estrus in a mare.We explored three different acquisition methods,and the success rate of B-ultrasound examination for estrus of the mare was 100%,which wassignificantly higher than that of external observation(47.3%)and rectal touch(85%),P<0.05.2.An effective method for inducing estrus and synchronized estrus in mares was established in this experiment.Effective induction of estrus of mare.The oestrus rate was 63.2%after intramuscular injection of 0.2 mg PGF2? on the 5th day of ovulation,higher than that of 1500 IU PMSG and natural oestrus(47.2%,0),P<0.05.The estrus cycle of 6 breeds of horses was significantly shortened,which was 11.7±3.1,12.2±2.8,11.5±1.9,12.8±2.1,11.8±2.5 and 12.7±2.3 d,respectively.The estrus rate of the horses injected with 0.2 mg progesterone and PGF2? was 76.4±2.6%,significantly higher than that of the group injected with progesterone alone(62.8±2.1%)and the control group(16.7±1.2%),P<0.05.3.This experiment established an effective method for regulating ovulation in mares.The key lies in the follicle development and the selection of hormone types and measurements.Effective regulation of female ovulation.Follicle diameter ?30 mm,ovulation rate within 48 h after intravenous injection of 1500 IU hCG was 84.3%,significantly higher than that of FSH+LH group(31.6%)and control group(15.8%),P<0.05,ovulation rate of 6 breeds of horses was 41.2±2.3,43.9±2.7,42.3±1.6,39.7±3.1,40.9±2.9 and 39.8±2.4 h,respectively.It was found that the ovulation rate of the left ovary was higher than that of the right ovary,59.5%of ovulation occurred on the left side,compared with 40.5%on the right(P<0.05).4.Preservation and fertilization of horse semen.The methods of preservation of horse semen at room temperature,low temperature and ultra-low temperature were established.Semen was stored at room temperature(22?)for 20 min.The sperm motility of the skim milk group was 53.5±3.2%,lower than that of the HN-SD(lab-prepared)and INRA96(IMV commercial)groups(65.6±2.3%and 68.6±1.2%),P<0.05.The rates of HN-SD and INRA96 groups were 65.5±2.3%and 67.7±2.1%,respectively,when stored at low temperature(0?4?)for 24 h,higher than skim milk group(49.5±3.4%),P<0.05.After 1 day of semen preservation with liquid nitrogen(-196),the sperm motility of the HN-SD group was 36.2±2.3%,higher than skim milk group(21.3±1.8%)and lower than the INRA96 group(44.3±2.2%)(P<0.05).In this study,the optimum insemination site was found,and the different insemination sites had different effects on the conception rate of mares.The conception rate of uterus horn transference was 85.4%,significantly higher than cervix(78.4%)and natural mating(25%),P<0.05.The conception rate of fresh semen was 61.1%,lower than 84%of fresh semen,but higher than 40%of frozen semen,P<0.05.5.Non-surgical collection of equine embryos.In this experiment,a non operative method for collecting embryos from horses was established.The embryo was taken as embryo blush and the embryo was taken from 5-8 d after conception.The success rate of embryo collection was 72.8%,with the highest success rate of embryo collection in May(88.9%)and the lowest in October(42.9%).The success rate of morula collection was 70.6%.6.Non-surgical transfer of equine embryos.In this experiment,a non-surgical method of horse embryo transfer was established.Recipient horses from 4 to 7 days after ovulation were selected and IMV embryo preservation solution was selected for uterine transplantation.The success rates of embryo transfer in HN-EHM and IMV embryo preservation solutions were 80%and 83.3%respectively,higher than that in the test group(75%).After cryopreservation for 24 hours,the successful rate of transplantation was 60%.The successful rate of morula embryo transplantation was 90.9%.7.Transport of horse ovaries.The optimum temperature of preservation solution for horse ovaries in vitro transportation was obtained in this experiment,which was 33-37?in summer and 30-35? in winter.8.Collection of cumulus-oocyte complex(COCs).The success rate of aspiration and cutting method was the highest(73.3%),which was higher than that of cutting method(64.6%)and aspiration method(54.8%),P<0.05.The proportion of large,medium and small ovarian follicles in reproductive season were 20.9%,51%and 28.1%,respectively,and the proportion were 3.1%,51.9%and 45%in non-reproductive season,respectively.9.In vitro maturation of horse oocytes.The conditions of maturation of horse oocyte in vitro were explored.The maturation time of horse oocyte in vitro was 5%CO2,37?,saturated humidity,compact(Cp)oocyte culture for 32-36 h and extended(Ex)culture for 24-28 h.The results showed that the maturation rate of different types of COC was different,and the maturation rate of oocyte was different in different reproductive seasons.In breeding season,the mature rate of horse compacted(Cp)oocyte was 36.5%,lower than extended(Ex)oocytes(58.6%),P<0.05,and the mature rate was 29.1%and 49.2%in non-breeding season respectively,P<0.05.In breeding season,the maturation rate of donkey oocyte was higher than horse,P<0.05.ActA increased the maturation rate of compacted(Cp)oocytes to 47.3%and 42.2%in reproduction and non-breeding seasons respectively.10.Parthenogenetic activation of horse oocytes.The conditions of parthenogenetic activation of horse oocyte were explored.Ionomycin was selected for parthenogenetic activation in this experiment.ActA promoted the development of parthenogenetic activated embryos of horse compacted(Cp)oocytes,and the proportions of 2-cell(57.9%)and 4-cell(36.8%)were higher than the control group respectively,P<0.05.Different oocyte types had no effect on embryo development after parthenogenetic activation of oocyte.The proportion of Parthenogenetic Activated embryos to 2-cell stage,4-cell stage,8-cell stage and morula stage was 50%(42/80),35.7%(30/84),13.1%(11/84)and 6%(5/84)respectively in the breeding season of horses,which was higher than non-breeding season,P<0.05.In the non-reproductive season of horse,the proportion of Parthenogenetic Activated embryos co-cultured with granulosa cells was 41.7%and 25%respectively,which was significantly higher than that of control group,P<0.05,and 1 mulberry embryo(2.8%)was obtained.11.Construction of cloned embryos from horse somatic cells.The effects of different fusion voltage on the fusion rate of reconstructed embryos were explored.Cloned embryos were cultured in 38.5?,5%CO2 and saturated humidity incubators.The success rate of using 140 V,20 ?s and 2 fusion times was 72%.The cell fusion,2-cells,4-8 cells and morula rate of nuclear transfer by foal fibroblast were 77.4%,41.5%,33.9%and 15.1%respectively,which were higher than adult horse fibroblast(P<0.05).Cloned embryos transplantation of horse.No cloned horse was obtained after embryo transfer of 8 morula embryos.Through the above research,this study obtained a mature equine embryo transfer technology system,to achieve standardization,commercialization,market-oriented breeding of modern equestrian horses to provide technical solutions.On the basis of this,we studied the technology of constructing cloned embryos from mature oocytes in vitro and laid the foundation for breaking through the technology of equine cloning in China. |
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