Study On Key Factors Of In Vitro Culture Medium That Supporting Porcine Blastocysts Continuous Development

In vitro culture of embryos is of great significance to explore the mechanism of embryonic development.In vitro culture mediums for mouse,human and non-human primate post hatching embryos have been established,and post-hatch embryo development events have been observed in vitro.However,an effective culture medium that can support the in vitro development of post hatching embryos in livestock has not yet been obtained.The purpose of this study was to explore the key factors affecting the continuous development of Porcine blastocysts in vitro,and to lay the foundation for the construction of a culture medium that supports the development of Porcine embryos in vitro to the differentiation stage of three germ layers.Therefore,in this study,Porcine blastocysts that developed to the day 6 after parthenogenetic in vitro were used as the research object,and were continuously cultured in vitro.Morphological characteristics and gene expression of porcine parthenogenetic blastocysts developed to day 8 were analyzed by detection,systematic evaluation of the effects of PZM-3,Advanced DMEM/F12 and Knock Out ^TM DMEM/F12 basal medium,and the addition of fetal bovine serum（FBS）,Knock Out serum replacement（KSR）,L-Glutamine and ITS-X on the sustained development of Porcine blastocysts in vitro.The main results are as follows.（1）To explore the effect of basal medium on embryonic development,we replaced Porcine blastocysts that developed to day 6 after parthenogenesis to PZM-3,Advanced DMEN/F12 and Knock Out ^TM DMEM/F12 basal medium.The results showed that 48h after the replacement of the basal medium,there was no significant difference in embryo diameter,embryo cell number and blastocyst hatching rate between the Knock Out ^TM DMEM/F12 treatment group and the control group（PZM-3）（254.22±82.01μm and 218.90±54.17μm;75.88±32.03 and 58.00±16.40;16.61±4.88%and 11.24±2.98%）;Compared with the control group,Advanced DMEN/F12 treatment group can significantly increase embryo diameter,embryo cell number and blastocyst hatching rate（262.19±59.60μm vs.218.90±54.17μm;101.92±28.15 vs.58.00±16.40;25.29±3.39%vs.11.24±2.98%,P<0.05）.（2）In order to explore the effect of FBS on embryonic development,based on Experiment 1,we added different concentrations（0%,5%,10%,20%）of FBS to the Advanced DMEM/F12 basal medium,and counted and observed Embryos diameter,embryo cell number and blastocyst hatch rate.The results showed that after changing the culture medium for 48 hours,there was no significant difference in embryo diameter,embryo cell number and blastocyst hatching rate between10%FBS and 20%FBS and the control group（0%FBS）（278.34±66.04μm,264.40±47.70μm and 245.83±66.70μm;95.56±28.97,70.88±20.62 and 98.21±30.40;21.72±6.85%,22.59±9.48%and 17.51±5.59%）;There was no significant difference in the number of embryonic cells with the addition of 5%FBS compared with the control group（131.00±41.64 and 98.21±30.40）,but significantly improved embryo diameter and blastocyst hatch rate（318.57±63.30μm vs.245.83±66.70μm;22.59±9.48%vs.17.51±5.59%,P<0.05）.（3）In order to explore the effect of L-Glutamine on embryonic development,we based on Experiment 2,different concentrations（0m M,1m M,2m M,4m M）of L-Glutamine were added to detect the embryo diameter,embryo cell number and blastocyst hatching rate.It was found that after48 hours of changing the culture medium,there was no significant difference in embryo diameter,embryo cell number and blastocyst hatching rate between 1 m M L-Glutamine,2 m M L-Glutamine,4 m M L-Glutamine group and control group（0 m M L-Glutamine）（293.22±50.32μm,327.96±50.49μm,338.99±58.20μm and 308.71±58.32μm;84.27±19.81,116.58±24.73,131.25±18.64 and 117.50±37.21;38.89±9.62%,34.12±1.37%,58.31±18.92%and 30.05±4.93%）.The mean diameter,mean hatching rate and mean cell number of embryos treated with 4 m M L-Glutamine were larger than those of the other three groups.（4）In order to explore the effect of ITS-X on embryonic development,based on experiment 3,we added 1×ITS-X and counted the embryo diameter,embryo cell number and blastocyst hatching rate.It was found that after 48 hours of changing the culture medium,compared with the control group,there was no significant difference in embryo diameter and blastocyst hatching rate between the experimental group added with 1×ITS-X（347.37±74.53μm and 313.64±56.10μm;54.60±13.51%and 63.49±17.55%）,and the number of embryonic cells increased significantly（146.92±22.88 vs.116.25±28.38,P<0.05）.（5）In order to explore the effect of KSR on embryonic development,we based on Experiment4,experimental group Advanced DMEN/F12 added 5%FBS,4 m M L-Glutamine and 1×ITS-X for AFGI,experimental group Advanced DMEN/F12 added 5%KSR,4 m M L-Glutamine and 1×ITS-X for AKGI,Embryos diameter,embryo cell number and blastocyst hatch rate were measured.It was found that after 48 hours of changing the culture medium,Compared with the control group（PZM-3）,the embryo diameter and blastocyst hatching rate in the AFGI group and AKGI group were significantly increased（343.17±45.48μm,385.71±43.45μm vs.218.71±52.65μm;57.19±3.89%,52.01±11.45%vs.12.38±4.84%,P<0.05）;embryo cells in the AFGI and AKGI groups compared with the control group Numbers increased significantly（97.21±19.49 vs.70.51±12.87,P<0.05;203.00±14.75 vs.70.51±12.87,P<0.01）.（6）In order to explore the effect of porcine blastocysts continuous development in vitro culture medium AKGI on embryonic development,we based on Experiment 5,the biological characteristics and gene expression of Porcine blastocysts developed to the 8th day after parthenogenetic activation were detected by Real-Time PCR,TUNEL and immunofluorescence.Real-Time PCR results showed that compared with the control group（PZM-3）,in the AKGI culture medium,cell proliferation-related genes（CDKL2 and GDF）,anti-apoptosis-related genes BCL-xl,ICM pluripotency-related genes（SOX2,NANOG and OCT4）and metabolism-related genes（LDHA,GAPDH,G6PD,CS,HSL,and PPARγ）the expression level was significantly increased（P<0.05）;TUNEL results showed that compared with the control group,the proportion of apoptotic cells in Porcine blastocysts to total cells in the AKGI culture medium was significantly lower（P<0.01）.Immunofluorescence results showed that ICM cells expressed both NANOG and SOX2 in the AKGI culture medium,while the control group ICM only expressed SOX2;although there was no significant difference in the ratio of ICM cells to total cells between the two groups,the number of ICM cells in blastocysts in the AKGI culture medium was significantly higher than that in the control group（13.80±2.59 vs.5.50±2.20,P<0.05）.In summary,this study preliminarily explored the main factors affecting the continuous development of Porcine blastocysts in vitro,and found that AKGI（Advanced DMEN/F12supplemented with 5%KSR,4 m M L-Glutamine and 1×ITS-X）is the most suitable training medium.The Porcine blastocysts were cultured in vitro to the 8th day after parthenogenetic activation,and the diameter of embryos,the number of embryo cells and the hatching rate of blastocysts were significantly increased.In addition,it was found that the AKGI culture medium can increase the expression levels of factors related to cell proliferation,reduce the proportion of apoptotic cells in the embryo,promote NANOG expression in ICMs.It laid the foundation for the establishment of an in vitro culture medium for Porcine embryos after hatching.