| Establishment of yak in vitro produced embryo system was essential for the basicstudy of yak zygote and embryo as well as the commercial application of assistedreproduction technology in yak. In order to improve the efficiency of yak in vitroembryo production, clarify the effect of FSH and FBS on apoptosis in cumulus cellsand oocyte developmental competence, and reveal the chronological onset ofapoptosis and ultrastructural characteristics during pre-implantation embryodevelopment in yak, several experiments were conducted in this study. The resultswere showed as following.(1) In the present study, apoptosis in both oocytes and cumulus cells wasdetected by TUNEL labelling and the expression of Fas and Fasl protein was studiedby indirect immunofluorescence, meanwhile, the effect of FSH and FBS on apoptosisand protein expression of cumulus cells as well as the developmental competence ofoocytes was analyzed. The results indicated that COCs with more than five layeredcumulus showed a low apoptotic or nonapoptotic cumulus investment at the onset ofIVM, while the increase in apoptosis in cumulus cells was observed in COCs withless than five layered cumulus, TUNEL reaction was not detected in oocytes; At theonset of IVM, Fas and Fasl protein was found to be present in cumulus cells, Fasprotein expression was localized at the cellular membrane of oocytes, but Fasl proteincould not be detected in oocytes; After IVM culture, the distribution of apoptoticcumulus cells showed an irregular pattern with a higher incidence at the middle andperipheral layers of the complex, and fewer or no signs of apoptosis in the inner layers,the distribution of Fas and Fasl protein was similar; Addition of FSH or FBS to M199medium significantly enhanced the developmental competence of oocytes, alsosignificantly decreased the incidence of apoptosis and the expression of Fas and Faslprotein in cumulus cells, noticeably, when FSH and FBS was added into M199medium together, the blastocyst rate and hatching rate was highest, while theincidence of apoptosis and the expression of Fas and Fasl protein in cumulus cells waslowest; In conclusion, the inhibition of apoptosis in yak cumulus cells by FSH and FBS during IVM could be partly via the Fas-Fasl signaling pathway, finally thedevelopmental competence of oocytes was improved.(2) This study was designed to determine the effect of swim-up or percollgradient methods on yak sperm quality and in vitro embryo development, the effect ofvarious sperm-oocyte co-incubation times, different oocyte donor ages, differentculture medium and culture density on yak in vitro embryo development. The resultsindicated that swim-up separated sperm showed a higher motility, while theconcentration of spermatozoa recovered and percent recovery of motile sperm werehigher with percoll separation, according to the optical and electron microscopy,swim-up produced higher percentage of sperm with intact plasma membrane andacrosome than percoll separation, however, there was no difference in the percentageof sperm with intact chromatin between two treatment groups, the blastocyst rate wassimilar in both groups, whereas cleavage rate was higher when swim-up was used;The lower cleavage and blastocyst rates were obtained when sperm-oocyteco-incubated for6and12h, both values significantly increased at18h, but nodifferences were observed between18and24h; Oocytes from prepubertal heifers andadult yaks were in vitro matured, fertilized and cultured, oocytes from prepubertalheifers had lower blastocyst rate when compared to those from adult yaks, whereasthe cleavage rates were similar; No differences were observed in terms of cleavagerate, blastocyst rate and blastocyst cell number between SOFaa and CR1aa medium,however, significant differences were observed in terms of blastocyst rate andblastocyst cell number among different culture density, the blastocyst rate andblastocyst cell number at a density of1:2(25zygotes/50μL) were significantly higherthan these at a density of1:10(40zygotes/400μL); Overall, oocytes from adult yakswere in vitro matured, then co-incubated with swim-up separated sperm for18h,when25zygotes were cultured in50μL SOFaa medium, the day8blastocystformation rate was more than20%.(3) During pre-implantation development, DNA degradation and apoptoticnuclear morphology of yak IVP embryos was detected using TUNEL reaction andDAPI staining, respectively, ultrastructural characteristics of yak embryos was observed by transmission electron microscopy. It was found that apoptoticcharacteristics were not observed in any two-cell embryos, the earliest observation ofthe TUNEL reaction was in condensed/fragmented nucleus of three-cell embryo, afew early cleavage stage embryos displayed less nuclei with general apoptotic nuclearmorphology and TUNEL reaction, while most morula and all blastocysts displayedmore nuclei with these apoptotic features; The average cell number in morulae andblastocyst was42.6and97.3, the incidence of apoptosis in morulae and blastocystwas2.9%and5.6%, respectively, in morulae and blastocysts,26.6%of theTUNEL-positive nuclei did not display apoptotic nuclear morphology, and47.4%ofthe nuclei displaying apoptotic morphology were not concurrently TUNEL-labelled,according to our criteria these cells were not regarded as apoptotic; Ultrastructuralfeatures of apoptosis were found in yak blastocysts, but not present in other stageembryos, apoptotic ICM and trophoblast cells were usually shrinking withcytoplasmic and organelle condensation and contained multiple small vesicles,vacuoles and lipid droplets, and also contained a few secondary lysosomes, andsecondary lysosomes contained phagocytized material including degeneratedorganelles and nuclear material, chromatin condensation and marginalization alongthe nuclear envelope and highly condensed nuclei were frequently seen, budding andblebbing of the plasma membrane was occasionally observed; In addition, theultrastructural features of cellular organelles, cytoplasmic components, the surfaceand nucleus of blastomeres possessed obvious changes as development progressed. |
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