| Embryo sex identification and sex control technology are key techniques in mammalian biotechnology, and the optimization of them have great value in the widespread application of biotechnology. In this study, we(1) Obtained high titer H-Y antibody using different immunization methods and screened female embryos;(2) Optimized procedures of in vitro embryo production, including conditions of in vitro oocyte maturation, capacitation methods of sex-sorted sperm, and selections of in vitro embryo culture medium, et al.(3) Studied the effects of ovary transportation temperature on oocyte recovery, maturation, in vitro fertilization, embryo development and Hsp70.1and Bax expression in bovine blastocyst;(4) Studied the influence of sex control technology, combined with embryo cryopreservation, on in vitro development, embryo quality and birth rate of bovine embryos.In this study,(1) H-Y antigen and antibody was obtained and co-cultured with early embryos to screen female embryos, and results showed that the accurate sex of4-8cell embryos could not be identified by H-Y antigen inhibition, but at morula stage PCR validation showed85%embryos selected by H-Y antigen inhibition were correct, indicating that selectively inhibiting embryo development was a reliable method for selecting female embryos;(2) combined application of EGF and IGF-I promoted in vitro maturation of bovine oocytes and enhanced in vitro development ability of in vitro fertilized embryos; development competence of embryos using different spermatozoa obtained by Percoll density gradient centrifugation and swim-up capacitation had no significant difference(P>0.05); optical spermatozoa density was0.5×106/ml, which facilitated the utilization of sex-sorted semen; mSOF and G1/G2were suitable for the culture of in vitro fertilized embryo derived from sex-sorted semen and as a commercial culture medium, G1/G2performed very stable; at optimized condition,we compared the IVF embryo development between sex-sorted semen and control semen and found cleavage rate and blastocyst rate showed no significant difference. Female rate of IVF embryos derived from sex-sorted semen was89.9%(62/69) whereas the female/male ratio of IVF embryos derived from control semen was47.9/52.1. (3) as to sex-sorted semen, ovary transportation temperature had significant influence on in vitro fertilization rate and embryo development. Storage at35℃for3-4h significantly decreased recovery rate of A and B grade oocytes, oocytes maturation rate, fertilization rate and blastocyst rate. Meanwhile, the apoptosis rate of day7blastocyst and the expression level of Hsp70.1and Bax increased significantly. Fertilization rate of oocytes derived from ovaries stored at25℃was significantly higher than other two groups.(4) compared with control group, the revival rate and24h hatching rate of IVF embryos derived from sex-sorted semen showed no significant difference after thawing, but the48h hatching rate of control group was higher than sex-sorted semen group. Apoptosis rate between two groups was not significantly different. Inner cell mass number/trophoblast cell number ratio of sex-sorted semen and control group was0.26±0.13and0.24±0.09, respectively, showing no significant difference. After embryo transfer, the pregnancy rate, birth rate and sex controlling efficiency also had no significant difference between sex-sorted and control groups. |
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