Effect Of Non-Invasive Lipid Lowering On Freezing Resistance For Porcine Oocytes And Parthenogenetic Embryos

China is the country with the largest resource of pig breeds,but the population size of most local pig breeds has been significantly reduced,even endangered.Therefore,how to efficiently and effectively protect pig germplasm resources is a key scientific issue at present.Among them,the cryopreservation technology based on the gene bank of germplasm resources is an effective method to enhance the population sizes and restore rare breeds,of which is a necessary supplement to the conservation farm and the last-ditch in the protection of pig germplasm resources.However,porcine oocyte and embryo undergo severe cryodamage,as low oocyte survival rate and embryonic developmental potential after cryopreservation,of which the reason is ascribed to high lipid content.Those lipids hinder the penetration and replacement of cryoprotectants,resulting in cellular cryodamage.Degreasing or reducing lipid is considered to be one of the most effective methods to improve cryotolerance.Traditional methods of degreasing require high-speed centrifugation and micromanipulation,which may cause mechanical damage to oocytes.Therefore,exploring non-invasive lipid lowering has practical significance in preventing porcine oocytes from cryodamage.In this study,oocytes and embryos were coincubated with10 μM Forskolin,a chemical lipid-lowering agent,during in vitro maturation and embryonic development,vitrified and thawed using Cryotop,followed with a series of determination to reveal the effects and mechanism of non-invasive lipid-lowering on the cryotolerance of porcine oocytes and parthenogenetic embryos.This study provided theoretical and technical support for solving the problem of lipid-lowering in the cryopreservation of porcine oocytes and embryos.The main methods and results of this study are as follows:1.Effect of non-invasive lipid lowering on freezing damage of porcine oocytes and its mechanismThe purpose of this study was to investigate the effect of non invasive lipidlowering on the cryotolerance of porcine oocytes.4 groups were set: fresh oocytes(Fresh),oocytes coincubated with 10 μM Forskolin during maturation(Forskolin),vitrified oocytes(Vitrified)and 10 μM Forskolin pretreated vitrified oocytes(Vitrified+ Forskolin).Nile red staining was used to determine the lipid content in each group.The ultrastructure of lipid droplets were observed by transmission electron microscopy.Oocyte metabolites were measured by metabonomics.Mitochondrial membrane potential was detected by JC-1.DCFH-DA was used to detect intracellular reactive oxygen species generation.Annexin V was used to detect the level of early apoptosis.The oocyte survival rate after cryopreservation was detected using FDA.Embryonic developmental potential was determined after parthenogenetic activation.As the results,forskolin-addition to in vitro maturation medium not showed significant effect on maturation rate compared to fresh oocytes(P > 0.05).The concentration of forskolin was safe and feasible to be used in subsequent experiments.The number(192.34 vs 459.86,P < 0.05)and relative area(0.62 vs 1.00,P < 0.05)of lipid droplets in forskolin treated oocytes before and after freezing both significantly decreased.The area and quantity of intracellular lipid droplets decreased.After freezing,lipid droplets mainly existed as heterogeneous lipid droplets,with uneven distribution and increased cavitation levels.Metabolomic studies have also shown that treatment of oocytes with Forskolin alters the level and pathway of lipid metabolism of intracellular metabolites.The treatment with hirsutin did not significantly affect the mitochondrial membrane potential,reactive oxygen species levels,early apoptosis rate,survival rate,and developmental potential of fresh oocytes(P > 0.05).Compared with frozen oocytes,Forskolin pretreatment significantly increased the mitochondrial membrane potential(1.04 vs 0.51)of frozen oocytes,alleviated oxidative stress caused by freezing(11.57 vs 22.77),significantly reduced the occurrence of early apoptosis(68.30% vs 86.03%),and significantly improved the survival rate(71.17% vs 51.47%)and developmental potential of frozen oocytes.The cleavage rate increased from 8.23% to 16.63%(P<0.05),the morula rate increased from 53.19% to 62.21%(P<0.05).This study confirmed that the non-invasive lipidlowering agent Forskolin reduces the lipid content of oocytes,improves mitochondrial depolarization,oxidative stress,and early apoptosis after freezing,and improves the survival rate and developmental potential of porcine oocytes after freezing.2.The effect and mechanism of non-invasive lipid lowering on freezing damage of porcine parthenogenetic embryosBased on the above research results,this experiment aims to explore the effect of non-invasive lipid-lowering agent Forskolin on freezing injury of porcine embryos.Set up 4 groups: fresh embryo group(Fresh),10 μM Forskolin treated embryos(Foskolin),frozen embryos(Vitrified)and the frozen embryo group pretreated with10μM Forskolin(Vitrified+Forskolin).Detection of the effects of embryo lipid content,lipid ultrastructure,mitochondrial membrane potential levels,oxidative stress indicators,post freezing survival rate,and embryo developmentThe results showed that the lipid content of fresh embryos was significantly reduced by the addition of Foskolin during the entire process of oocyte maturation and embryo development.Compared with frozen embryos,the number of lipid droplets(140.70 vs 197.30,P<0.05)and relative area(0.44 vs 0.65,P<0.05)of frozen embryos pretreated with Foskolin significantly decreased;Ultrastructural observation showed that the bright stripes of heterogeneous lipid droplets in frozen embryos pretreated with Foskolin increased and widened,some of them gradually dissolved or even disappeared,and the number of heterogeneous lipid droplets decreased.Compared with frozen embryos,frozen embryos pretreated with Foskolin significantly increased the mitochondrial membrane potential of frozen embryos(1.24 vs 0.81),significantly reduced the release of reactive oxygen species(17.27 vs 32.06),significantly decreased the level of early apoptosis of frozen embryos(56.09% vs80.13%),and significantly increased the survival rate of frozen embryos(83.06% vs66.61%)(P<0.05).This study confirmed that the non-invasive lipid-lowering agent Foskolin can effectively reduce the lipid content of embryos,alleviate embryo damage,oxidative stress,and early apoptosis caused by freezing,and improve the quality of frozen embryos.This study supported the application of non-invasive lipid lowering agents in the cryopreservation of porcine oocytes and embryos,and provided a theoretical basis for promoting the resolution of technical bottlenecks in porcine oocyte vitrification.