Effects Of Glycine/Melatonin On The Cryopreservation In Mink Oocytes At Germinal Vesicle Stage

Mink is a kind of fur animal with high economic value.The application of advanced reproductive biotechnology in mink breeding lags behind livestock seriously.Cryopreservation of oocyte is the most important and basic biotechnology.At present,it has been widely used in mammalian oocyte with low fat content.However,for oocytes of animals with high fat content,vitrification efficiency is low,How to improve the efficiency of cryopreservation is one of the hotspots in biological science.Glycine（Gly）and Melatonin（MT）have been fully verified in improving the vitrification efficiency of low-fat oocytes such as mice.However,whether they can improve the vitrification efficiency of mink oocytes with high fat content still needs further study.In this study,Germinal vesicle（GV）oocytes are used to vitrify as the research object,and immunofluorescence,qRT-PCR and other methods are used to investigate the influence of adding 1mM Gly or 10^-9M MT in vitrified,thawed and matured on the quality of mink oocytes.Including recovery rate of oocyte,nuclear maturation rate,distribution of mitochondria and cortical granules,and the changes of glycolysis-related gene（Ldh1,Pkm2 and Pfkp）expression in granulosa cells which surround oocyte.Furthermore,the effects of Gly and MT addition on the cryopreservation efficiency of leech oocytes were studied from multiple levels of genes,organelles and cells.The contents of this study are as follows:1.The addition of 1mM Gly（73±1.8%vs 55±4.1%）or 10^-9M MT（70±2.7%vs 55±4.1%）during the vitrification,thawing and in vitro maturation of mink GV stage oocytes could significantly improve the GVBD rate of oocytes（P<0.05）.2.The addition of 1mM Gly（54±3.2%vs 30±2.3%）during the vitrification and thawing of mink GV stage oocytes could significantly reduce the mitochondrial damage which induced by vitrification（P<0.05）,and improve the correct distribution of active mitochondria.3.The addition of 1mM Gly（42±4.2%vs 34±1.8%）during the vitrification,thawing and in vitro maturation of mink GV stage oocytes could significantly reduce the loss of cortical granules which induced by vitrification（P<0.05）,and improve the correct distribution of cortical granules.4.The addition of 1mM Gly or 10^-9M MT during vitrification and thawing of mink GV stage oocytes could significantly increase the expression of glycolysis-related genes（Ldh1,Pkm2,Pfkp）in granulosa cells surrounding oocytes（P<0.05）.Thus,the bidirectional signal transduction between oocyte and its surrounding granular cells is protected,and the oocyte quality is indirectly improved.5.The addition of 1mM Gly or 10^-9M MT during the vitrification,thawing and in vitro maturation of mink GV stage oocytes could improve the efficiency of oocyte cryopreservation.The addition of1mM Gly was significantly better than 10^-9M MT in improving mitochondrial distribution and cortical granule distribution（P<0.05）,but there was no significant difference in nuclear maturation rate（P>0.05）.