Studies On Production Of Bovine Sexed Embryos Using Ovum Pick-Up And In Vitro Fertilization Technologies

The objective of this study was to investigate the methods of ovum pick-up(OPU)and in vitro fertilization(IVF)with flow cytometrically-sorted X and Y chromosome-bearing sperm so as to improve the efficiency of bovine embryos production by OPU-IVF system.This thesis is arranged in two parts,partⅠincludes chapter 1 and chapter 2, which is a review of literatures;partⅡincludes chapter 3-5,which deals with the experimental work.Chapter 3 was to determine the effect of dominant follicle in the donor cow,extro-gonadotropin and a single injection of FSH dissolved in a solution of polyvinylpyrrolidone(PVP)on OPU.In Experiment 1, Eight Holstein cows were randomly divided into 4 groups(2 cows/group). The dominant follicle with diameter≥10 mm was aspirated or left intact, and cows were injected with or without FSH,OPU was performed on 72h after injection of FSH by Ultrasound-guided Transvaginal.This experiment was repeated 10 times.In Experiment 2,Six Holstein cows were divided into 3 groups(2 cows/group)and were injected with FSH, PMSG and control respectively.OPU was performed as described above in experiment 1.This experiment was repeated 6 times.In Experiment 3, Eight Holstein cows were divided into 4 groups(2 cows/group)using 4×4 Latin Square design and were given a single injection of FSH dissolved in saline(control group),and in 15%and 30%PVP,or were given multiple intramuscular injections of FSH every 12h interval for 2d(conventional group)respectively.Blood samples were collected from each cow to determine FSH levels in serum during FSH treatment.Cows were treated once a month and this experiment was conducted for four months. The results showed that(1)The mean numbers of follicles aspirated and oocytes recovered were significantly improved(p＜0.05)with treatment of dominant follicle punctured compared to the intact one before injection of FSH,but if no FSH was injected,there was no significant difference (p＞0.05)between the dominant follicle punctured and the intact one.(2)The mean numbers of follicles aspirated and oocytes recovered were significant higher(p＜0.05)in FSH group than PMSG group and control group,but there was no significant difference(p＞0.05)between PMSG group and control group.(3)The mean numbers of aspirated follicles and usable oocytes collected from 30%PVP group were significantly higher(p＜0.05)than the control group and 15%PVP group. But there was no significant difference(p＞0.05)in the mean numbers of aspirated follicles and usable oocytes between 30%PVP and the conventional group,however,30%PVP group and the conventional group could maintain relatively high FSH concentration in serum,and the changing curves were also similar.(4)Continually OPU could injure the ovaries of donor cows,but after stopping OPU for 2-3 months,the pregnant rate was no significant difference between cows with or without OPU.Chapter 4 includes three experiments,the objective was to investigate the IVF results of oocytes by OPU from donor cows treated with or without hormones,oocytes from OPU and slaughterhouse ovaries,and oocytes from slaughterhouse ovaries kept in vitro for different time.In Experiment 1, oocytes derived from OPU of donor cows were treated with or without hormone(control),and the oocytes were matured,inseminated and subsequently cultured in vitro,then the cleavage and blastocyst rates were recorded on 48h and day 7-8d after insemination respectively.Three blastocysts derived from OPU-IVF were transferred to three synchronized recipients after artificial insemination.In Experiment 2-3,oocytes from slaughterhouse ovaries were matured for 22-24h and inseminated with frozen sperm following by co-culture on granulose cell monolayers,the IVF efficiencies of oocytes from OPU or slaughterhouse ovaries were compared. The oocytes from slaughterhouse ovaries were kept in vitro for Oh,3h,6h and 9h respectively,and then were matured,inseminated and subsequently cultured in vitro.The results showed that(1)The rates of cleavage and blastocyst of oocytes were not significantly different(p＞0.05)between hormones pretreatment and the control group,calf was born after transplantation of embryo derived from OPU-IVF.(2)The rates of cleavage, blastocyst and frozen-blastocyst of oocytes derived from OPU and slaughterhouse ovaries were not significantly different respectively.(3) The rates of cleavage and blastocyst from slaughterhouse ovaries oocytes kept in vitro for Oh and 3h were not significantly different(p＞0.05),but they were significantly decreased in the groups kept in vitro for 6h or 9h.Chapter 5 was to determine the effect of methods for removing the granulose cells from zygotes,and supplementation with different concentrations of insulin in culture medium,and bovine sorted sperm treated with two different treatments on the IVF results of oocytes.In Experiment 1-4,bovine oocytes aspirated from slaughterhouse ovaries follicles were matured in vitro and inseminated with sorted or unsorted sperm,which was treated with washing or Percoll method.Six to eight hours after insemination,granulose cells surrounding zygotes were removed by pipetting or vortexing,and these zygotes were transferred to the culture medium supplemented with 0,5 or 10μg/mL insulin.The rates of cleavage and blastocyst were recorded as described above.Blastocyst cell number was counted and the accuracy rate of sex of blastocysts was evaluated by a PCR procedure.In Experiment 5,oocytes from OPU were matured and inseminated with X-chromosome-bearing sperm in vitro,the hatching rate of frozen blastocysts was assessed,and frozen blastocysts from X sperm produced by OPU-IVF were transferred to five synchronized recipients.The results showed that(1)There was no significant difference in the rates cleavage and blastocyst of bovine oocytes,no matter granulose cells were removed by vortexing or pipetting after insemination with unsorted sperm,but after insemination with sorted sperm,the vortexing method had a positive effect on the development of early embryos compared to the pipetting method.(2)The rate and cell number of blastocysts cultured in medium supplemented with 5 and 10μg/mL insulin were significantly higher than those of the control group(p＜0.05),but there was no significant difference between 5μg/mL and 10μg/mL insulin.(3) There was no significant difference in sorted sperm motility and blastocyst rates between washing and Percoll treatment group,but efficiency of sorted sperm utilization with washing treatment was significantly higher than the Percoll treatment group.(4)The cleavage and blastocyst rates of oocytes inseminated with X or Y sperms were not significantly different,blastocyst cell number was also not significantly different between sorted and unsorted sperm,but cleavage and blastocyst rates were decreased in sorted sperm compared to unsorted sperm,The accuracy rates of sex of blastocysts from oocytes insemination with X or Y sperm were 90.63%and 86.67%respectively.(5)The rates of cleavage and blastocyst from oocytes collected by OPU insemination with X sperm were 46.15%and 13.85%, which were significant difference(p＜0.05)compared to that with unsorted sperm,and one pregnancy was obtained after transplant x-embryos produced by OPU-IVF.(6)The survival and hatching rates of frozen blastocyst inseminated with sorted or unsorted sperm were not significantly different.The above results demonstrate that(1)Aspiration of dominant follicles can significantly improve the result of OPU before injection of FSH.(2)The OPU result shows that injection FSH is much better than PMSG.(3)Single injection of FSH dissolved in 30%PVP can maintain a high FSH level in serum and improve the OPU efficiency.(4)OPU does not affect the reproductive competence of donor cows.(5)Embryos can be produced by OPU-IVF from donor cows treated with hormones,and the efficiency of embryo production of oocytes from OPU or slaughterhouse ovaries is not significantly different,and twin calves can be produced by transplantation of blastocyst from OPU-IVF to recipients.(6)Oocytes collected by OPU should be cultured within 3h from farm to lab.(7)Removment of granulose cells surrounding the oocytes by vortexing after insemination with sorted sperm,which has a positive effect on the development of early embryos. (8)Supplementation with 5μg/mL insulin in culture medium can significantly increase the rate and cell numbers of sexed blastocysts.(9)The efficiency of bovine sorted sperm utilization can be improved with washing treatment.(10)There is no significant difference IVF results inseminated with X sperm and Y sperm.(11)The OPU-IVF system with bovine sorted sperm is primarily established,and embryos from x sperm can be produced successfully by OPU-IVF,but the blastocyst rate is lower than the unsorted sperm.