VTs和CPAs对玻璃化冷冻后牛MⅡ期卵母细胞代谢指纹图谱和发育能力的影响OA

Vitrfication is commonly used for oocyte cryopreservation,however,cryoinjury and cryoprotectant toxicity results in damage to the structure and poor developmental capacity of oocytes after freezing.Liquid helium(LHe,-269℃)as cryogen,can improve cooling rate of vitrification.And high cooling rate is the key factor ensuring the success of vitrification of oocytes.In this study,laser tweezers Raman spectroscopy(LTRS)was used to non-invasively investigate the effects of varying vitrification temperatures(VTs)and cryoprotective agent concentrations(CPAs)(17.5%ethylene glycol(EG)+17.5%dimethyl sulfoxide(DMSO),20%EG+20%DMSO)on the metabolic fingerprint profiles of bovine(Bos taurus)MⅡ stage oocytes after vitrification-thawing,and through in vitro developmental competence tests,the feasibility of using Raman spectroscopy to detect the quality of oocytes was explored.Bovine MⅡ oocytes were collected and randomized into 5 groups:Fresh oocytes(Fresh),oocytes vitrified in liquid helium including LHe-EDS35 and LHe-EDS40,oocytes vitrified in liquid nitrogen(LN,-196℃)including LN-EDS35 and LN-EDS40.The fresh oocytes and 4 experimental groups oocytes after vitrification-thawing were cultured in vitro for 4 h,then the culture media for single oocyte in different experimental groups were collected and separately analyzed using LTRS.Subsequently,they were evaluated embryo developmental competence after in vitro fertilization.These results showed that the significant changes were observed in the Raman spectra of culture media for oocyte after vitrification-thawing(P＜0.05).The intensities of the characteristic peaks at 862 cm-1(tyrosine),1 631 cm-1(Amide I:β-sheet),and 1 451 cm-1(lipids)for culture media of oocytes in LHe-EDS35,LHe-EDS40 were significantly lower than those in LN-EDS35,LN-EDS40 groups(P＜0.05).Furthermore,vitrification induced a transformation of the protein secondary structure from the α-helices to the β-sheet form in oocytes of LN-EDS35 group.The results of in vitro development of oocytes from 5 experimental groups showed that cleavage rate,blastocyst rate of oocytes in LHe-EDS35 were significantly higher than those in LN-EDS35,LN-EDS40,LHe-EDS40 groups(P＜0.05).In summary,LHe as cryogen,the supplementation of 17.5%EG and 17.5%DMSO cryoprotectant in the vitrification solution could reduce cryoinjury caused by vitrification at some extent,and significantly improved developmental capacity in vitro of MⅡoocytes after vitrification-thawing,which helped to optimize the vitrification procedures.This study provides a theoretical basis for the efficient vitrification of bovine MⅡoocytes using LHe as cryogen.