| Vitrification is of great significance for the conservation of genetic resources.It has been successfully applied to the preservation of various animal oocytes and embryos.However,the survival rate of pig oocytes and embryos after freezing is relatively low.The fundamental reason mainly is from the lipid droplets(LDs)of pig oocytes,that LDs content is high and their freezing tolerance is poor.The developmental stage of pig embryos also affects the effect of vitrification.Therefore,L-carnitine was used for pig oocytes during in vitro maturation(IVM)to explore its possible effect on the vitrification of pig oocytes.The date of parthenogenetic activation of porcine oocytes was recorded as D0 to explore the optimal freezing period of D4,D5 and D6 embryos,so as to improve the application efficiency of cryopreservation of embryos in pig breeding and germplasm resources conservation.1.The effect of L-carnitine on vitrification of oocytesOocytes was divided into four groups according to whether L-carnitine(L-IVM)was added in IVM and whether vitrification was performed: The nonvitrification(NV)group was oocytes only from IVM,while the vitrification(V)group was oocytes from IVM.The NVL(nonvitrification with L-carnitine addition in IVM)group was oocytes from L-IVM,and the VL(vitrification with L-carnitine addition in IVM)group was oocytes from L-IVM.After culturing in vitro for 16 h,Cryotop vitrification was performed.The oocytes had fully adapted to the in vitro culture.After thawing,they were put back into the original culture well for further culture for 26-30 h.The results showed that vitrification reduced the survival rate of oocytes and the content of LDs.Although the addition of L-carnitine did not improve the survival rate of oocytes,it had a positive effect on the metabolism of LDs,triacylglycerol and oxidative stress.The addition of L-carnitine improved the metabolism of triacylglycerol in NVL,the expression levels of CPT-1 and HSL genes were significantly increased(p<0.05),the content of LDs and ROS level in the cytoplasm were reduced(p<0.01),and the expression of SOD1 was increased(p<0.01),which enhanced its antioxidant capacity.The addition of L-carnitine reduced the metabolism of triacylglycerol in VL,the expression of HSL gene was significantly reduced(p<0.01),the content of LDs was increased(p<0.01),and the level of ROS in the cytoplasm decreased(p<0.01),which reduced oxidative stress.Therefore,the addition of L-carnitine had a positive effect on vitrified oocytes.2.Selection of the best freezing period for pig embryosPig embryos from three developmental stages(morula D4,early blastocyst D5,and expanded blastocyst D6)were subjected to Cryotop vitrification.After freezing-thawing,embryos were further in vitro cultured for 24 h.Subsequently,the recovery rate of embryo was counted,while lipid droplets,mitochondria,and cytoskeleton were detected.The results show that vitrification reduced the number of blastomeres in embryos(p<0.01),but also that DNA and cytoskeleton were damaged,and uneven distribution of mitochondria was detected.In vitrificated-thawing pig embryos,LDs were affected obviously that evenly distributed and small LDs are aggregated into large LDs.However,the survival rate was relatively higher when D5 embryos were vitrification than those embryos from D4 or D6(p<0.05),which indicating that the optimal development stage for pig embryos vitrification might be on D5 of early blastocyst.Therefore,D5 embryos were selected for further gene expression research,while results showed that the m RNA expression levels of perilipin PLIN3 and triacylglycerol synthesis related genes AGPAT1 and DGAT were significantly reduced(p<0.05),which might indicate that vitrification might further affect the synthesis of Lipid,which may have an irreversible impact on embryonic development. |
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