Studies On Dairy Cattle Sexed Embryo Production In Vitro

Sexed embryo production in vitro (IVP) with oocytes collected from abattoir ovaries and sortedsemen of dairy bull could not only make the utmost of excellent genetic resources of obsolete dairycows, but also be an alternative resource of embryos in dairy cattle commercial embryo transfer (ET).However, the low outcome of transferable sexed embryos, the low pregnant rate of sexed embryotransfer, and the confused and unknowable pedigree information of sexed embryos from IVP affectedthe widely application of sexed embryo in vitro. The key factors involved in sexed embryos productionof dairy cattle were studied and some sexed embryos with clear pedigree were also produced in vitro inthis paper. The results are as follows:Experiment1: Effects of cumulus cells on bovine oocytes maturation in vitro and development afterparthenogenesisThe effects of bovine and mouse cumulus cells (CCs) monolayer on bovine oocytes maturation invitro (IVM) and development after parthenogenesis when bovine denuded oocytes (DOs) wereco-cultured with bovine and mouse CCs for22~24h during maturation in vitro were studied.The results showed that the nuclear maturation rate of bovine DOs could be improved significantlyby co-culture with bovine or mouse CCs [(71.00±4.27)%,(68.48±5.71)%vs (54.26±5.03)%](P<0.05)compared with the DOs group. Also, the relative expression of mRNA of BMP15, GDF9gene ofco-culture groups were higher than that of DOs group, but there were no significant difference ofglutathione (GSH) content and the parthenogenesis embryo development of MII stage DOs between theco-culture groups and the DOs group [(2.49±0.18) pmol/oocyte,(48.57±5.20)%;(2.48±0.24)pmol/oocyte,(44.19±4.03)%vs (2.40±0.17) pmol/oocyte,(43.47±7.34)%](P>0.05).Experiment2: Effects of FSH and LH supplemental ways during IVM on bovine oocytes maturationand fertilization in vitroThe concentrations of FSH, LH, E2and P4in maturation medium were determined on differentperiods during the COCs IVM in this experiment and the amount of them were all increased comparedwith control group. Supplementation of FSH, LH and FSH+LH to the media during bovine IVM withdifferent ways was carried out. The results showed that (1) the nuclear maturation rate of oocytes inFSH×2group was significantly higher than that of FSH×0group [(79.12±8.55)%vs(62.37±2.47)%](P<0.05), but the blastocyst rate of the two group were opposite [(36.92±4.42)%vs(32.00±5.82)%](P<0.05);(2)There were no significant effects of the changes of LH supplemental wayson nuclear maturation of oocytes and the cleavage rate of IVF, but the blastocyst rate of the threechanging medium groups were all higher than that of non-changing medium group (31.67±3.71)%;(3)The nuclear maturation rate, cleavage rate and blastocyst rate of IVF in changing (FSH+LH)×2werethe highest [(72.82±6.11)%,(94.28±3.18)%,(49.29±3.18)%] in all of the four groups.Experiment3: Effects of the apoptosis in sperms on bovine fertilization in vitroThe apoptosis of sorted X-semen from different bulls (A, B, C and D) was analyzed by flow cytometry and three kinds of fluorescent staining methods including Annexin/PI, JC-1/PI and TUNEL.Meanwhile, the effects of different apoptosis of sorted X-sperms from different bulls on IVF were alsostudied in this experiment.The results showed that the percentage of early apoptosis in sorted X-semen from bull A sortedX-semen was the lowest, and the cleavage rate and blastocyst rate after IVF of X-semen from bull Awere higher than those of the other bulls. After the correlation anova, we found that the percentage ofAN–/PI–spermatozoa was positively correlated with cleavage rate and blastocyst rate of IVF (0.8119,0.8898)(P<0.01), and the percentage of AN+/PI+spermatozoa was negatively correlated with thecleavage rate and blastocyst rate (-0.8250,-0.9008)(P<0.01).Experiment4: In vitro production of sexed embryos with clear pedigree informationThe effects of the sex-sorted X-sperm/oocyte ratio and the size of fertilization drop on IVF werestudied in the experiment. The results showed that the cleavage rate and blastocyst rate were relativelyhigher when the ratio of sperm/oocyte was2500:1and the volume of fertilization drop was50μL[(23.75±9.43%),(22.50±2.55)%]. And using the optimal system established above, we havested18unsexed embryos from3Piedmont beef cattle and3sexed embryos from1dairy cattle with clearpedigree.