| The objective of the current study was to use the liquid helium to cryopreservethe GV oocytes of bovine instead of liquid nitrogen, and analyzed the the effect ofliquid helium vitrification on meiotic maturation of bovine GV stage oocytes, theeffect of liquid helium vitrification on expression of GDF9, BAX, ZAR1in bovine GVstage oocytes. Then try to explore the relationship between the developmentalcompetence and expression levels of genes for the vitrified oocytes.Experiment1: Bovine oocytes were randomly allocated into three groups:(1)control (fresh)(2) liquid nitrogen OPS vitrification (3) liquid helium OPS vitrification.Oocytes of both treat groups were vitrified in the same solution containing20%ethylene glycol (EG),20%dimethyl sulfoxide (DMSO) and0.5M sucrose. Aftervitrified and thawed, the oocytes were cultured in vitro(IVM, IVF and IVC). Then thedata was statistically analyzed. The results indicated that there was a significantdifference between liquid nitrogen group and liquid helium group in percentage ofmorphologically normal oocytes (81.1%vs.89.0%; P <0.05). All COCs werecultured24h,1st polor body were examined, maturation rates in liquid nitrogenvitrification and liquid helium vitrification yielded lower than control group (42.6%and50.6%vs.69.5%; P <0.05), but the maturation rate in liquid helium group wasmuch higher than liquid nitrogen group(50.6%vs.42.6%; P <0.05). Following IVFand IVC, the cleavage rate (41.1%) and blastocyst rate (10.0%) in liquid heliumgroup were also lower (P <0.05) than the controls (64.2%,40.6%) but much higher(P <0.05) than liquid nitrogen group (33.0%,4.5%). Consequently, it is feasible forhelium vitrification in bovine GV oocyte. these results imply the percentage ofmorphologically normal oocytes and the rate of maturation in liquid helium groupwere better than liquid nitrogen group. It may attribute the success to the temperatureof liquid helium is lower than liquid nitrogen. Although the practical value in thefuture may be affect because of high price of liquid helium, and evaporate quickly, the liquid helium vitrification as a device which is worth further explore to crack theimmature oocyte cryopreservation.Experiment2: The developmental competence and expression level of the genesrelated the development of oocytes including GDF9(growth/differentiation factor-9),BAX (apoptosis factor) and ZAR1(zygote arrest1) were examined. Total RNAextraction and QRT-PCR studies were carried out after24h IVM on sixty COCs (threereplicates) from each group (control, liquid nitrogen group and liquid helium group).Real-time quantitative PCR analysis indicated that the expression of GDF9was muchhigher (P <0.05) in liquid nitrogen group than control and liquid helium group, butthere was no significant different between liquid helium group and control (P>0.05).The expression of BAX in liquid nitrogen was also up-regulated (P <0.05) comparedto controls; the expression of BAX was comparatively higher in the liquid heliumgroup than that of the control group, but the regulation was not statistically significant;Although there was no significant different both the liquid nitrogen group and liquidhelium group, the latter is lower than the former. In addition, the expression of ZAR1in the liquid nitrogen group was not significant difference compared to controls (P>0.05); In liquid helium group, the expression of genes only ZAR1was down-regulated(P <0.05) than control and liquid nitrogen group. Consequently, the expression levelof genes can be alterd after the immature oocytes vitrification. the results of thepresent study indicated a relationship between the compromised developmentalcompetence and altered expression levels of genes which related the development inthe treated oocytes. Liquid helium vitrification for developmental competence inbovine GV oocyte was better than liquid nitrogen vitrification, there was probably aconnection with the expression of genes which related development in vitrified-thawedoocyte. |
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