Key Proteins Screening And Its Effect Mechanism Of Dairy Cows With Postpartum Anestrus Caused By Negative Energy Balance

The negative postpartum energy balance(NEB)of dairy cows inhibits the development of follicles and increases the rate of anestrus.Although intensive cattle farms use the Timed Artificial Insemination(TAI)to improve the estrus rate of dairy cows,there are still some NEB dairy cows that have no diviation follicles for the body to ovulate after they are treated with TAI.At present,the increased lack of estrus caused by NEB in high-production dairy cows has become a major problen restricting the reproductive efficiency of dairy cows.To this end,this study aimed at this problem and carried out the proteomic analysis of NEB hypothermic cow serum,follicular fluid and ovarian cortex.The relationship between differential protein(adiponectin,ADPN)and postpartum hypoestrus in dairy cows and its role as an early warning system for the risk of postpartum hypoestrus in dairy cows.The characteristics of the effects of ADPN on the growth and development and the synthesis of steroid hormones in the in vitro granular cells(GCs)of dairy cows cultured with low glucose.To clarify the proteomic changes characteristics of NEB dairy cows postpartum hypoestrus.ADPN affects the molecular mechanism of postpartum fatigue in dairy cows and the growth and development of GCs in vitro.1.In vivo experiments.In order to clarify the proteomic profile of postpartum hypoxia in dairy cows and the role of differential proteins.In this test,dairy cows were divided into negative energy balance group(NEB,BHBA)concentration of serum?-hydroxybutyric acid(BHBA)higher than 1.20 mmol/L and glucose(Glu)concentration lower than 2.8 mmol/L in the 14-21 d postpartum.N=30)and energy balance group(PEB,n=30).Follow the two groups of cows to 55-60 days postpartum.In the 50-55 d postpartum,select NEB cows in anestrus(NEB-A,n=6,follicle diameter<8 mm)and PEB cows in estrus(PEB-E,n=6,the diameter of the diviation follicle is 15-20 mm)according to whether they are in estrus and the diameter of follicles.(15-20 mm)slaughter and collect samples.Then,carry out the experiment,the results are as follows:(1)TMT/LC-MS/MS proteomics technology is applied to screen the differentially expressed protein(DEP)of NEB-A and PEB-E cow serum,follicular fluid and ovarian cortex.Analyze DEP function and enrichment pathways.Compared with the PEB-E group,the DEP in the cow serum of the NEB-A group has 82down-regulations and 78 up-regulations.The main enrichment pathways include:complement and coagulation system,P13K-AKT pathway,glucagon signaling pathway,and MAPK pathway;There are 135 down-regulations and 37 up-regulations of DEP in the follicular fluid of cows in the NEB-A group.The main enrichment pathways include:lipid metabolism,IGF regulation system,and immune system;There are 88 down-regulations and 70 up-regulations of DEP in the follicular cavity cortical tissue of cows in the NEB-A group.The main enrichment pathways include:steroid hormone metabolism,fatty acid metabolism,and immune system.(2)Western blot was used to verify the selected 5 DEPs,RBP4(retinol binding protein 4),ADPN,SOD(superoxide dismutase),IGF2(insulin-like growth factor 2)and CRP(C-reactive protein)The expression level in the organization,and the results are consistent with the omics results.The phosphorylation levels of AKT and ERK1/2in the ovarian tissue were verified.(3)Immunohistochemistry were used to locate ADPN in ovarian tissue.Compared with the PEB-E group,the ADPN of the cows in the NEB-A group was down-regulated in the ovarian medulla,which was consistent with the down-regulation in blood,follicular fluid and adipose tissue(p<0.05),confirming that ADPN was mainly distributed in the ovarian medulla.(4)NEB and PEB dairy cow serum liver function indexes,insulin regulation-related indexes,and ADPN and Glu in serum and follicular fluid,after biochemical analysis,cluster analysis,risk index analysis,binary logistic regression analysis and subject work characteristics Curve(ROC)analysis found.ADPN in serum was strongly correlated with insulin regulation and liver function indexes,and Glu in follicular fluid was positively correlated with ADPN.In addition,the ADPN<2.365?g/m L in the serum of dairy cows 14-21 days postpartum is an early warning indicator and threshold value for the occurrence of postpartum anestrus in dairy cows.The results showed that the protein components in the serum,follicular fluid and follicular cavity cortex tissue of dairy cows after 14-21 d postpartum affect the estrus at 50-55 d postpartum,and ADPN in serum,follicular fluid and ovarian tissue at 50-55 d postpartum.Significant down-regulation changes occurred and ADPN is a risk factor for the occurrence of anestrus in dairy cows.2.In vitro test.In order to clarify the role of low glucose/ADPN in the follicular development and GCs growth and development of dairy cows,this experiment is based on the establishment of a low glucose cultured in vitro bovine GCs model.A series of experiments were carried out,and the results are as follows:(1)Western blot is used to detect the levels of bovine GCs under low glucose culture and cell cycle proteins(CCNA1,CCND1,CCNE2,CDK1),steroid hormone synthesis proteins(St AR,HSD3B1),and ADPN protein receptors(Adipo R2)under normal culture.The expression level.Compared with the normal group,the expression levels of cyclin,steroid hormone synthesis protein,and Adipo R2 were significantly down-regulated under low glucose culture(p<0.05).(2)Under low Glu culture,add low-dose(0.1?g/m L),medium-dose(0.5?g/m L)and high-dose(1?g/m L)recombinant bovine ADPN protein.Only the protein coating solvent was added as the control group.After treatment for 12 h,24 h,48 h,CCK8and Western blot were used to detect cell viability,ADPN receptor,cyclin,and steroid hormone synthesis protein protein expression levels,and radioimmunoassay was used to detect E₂and P₄content in the medium.Compared with other groups.the ability of GCs to secrete E₂in the low-dose group did not change significantly,and the high concentration of ADPN significantly increased the ability to synthesize E₂.Compared with the control group,different doses of ADPN had no significant effect on the secretion of P₄by GCs.(3)Cultivate GCs with low Glu.add LY294002(PI3K/AKT inhibitor)and high-dose ADPN under low-sugar culture.Treatment for 12 h,24 h,48 h.Western blot was used to detect the phosphorylation level of AKT and the protein levels of DCK2,St AR,HSD3B1,and Adipo R2.q-PCR was used to detect the gene expression level of CYP19A1 and 3?-HSD.Radioimmunoassay is used to detect the content of E₂and P₄.Compared with the control group,the cell viability of the ADPN+PI3K inhibitor group was significantly decreased(p<0.01),the CDK2 protein level was significantly decreased(p<0.01),and the St AR and HSD3B1 protein levels were significantly decreased(p<0.01).The content of P₄in the base did not change significantly,but the content of E₂decreased significantly(p<0.05).The results show that ADPN affects the secretion and growth of GCs steroid hormones in a low Glu environment through the Adipo R2-PI3K-AKT pathway.In summary,this study obtained the proteome profile of postpartum anestrus in dairy cows,and found that NEB through the complement and coagulation system,P13K-AKT pathway,glucagon signaling pathway,MAPK pathway,fatty acid metabolism,immune system regulation,and IGF system pathways affect postpartum follicular development in dairy cows,and establish the early warning effect of ADPN on the risk of postpartum anestrus in NEB dairy cows,confirming that ADPN can inhibit follicular development by affecting the proliferation of low Glu dairy cow GCs and the synthesis of steroid hormones through PI3K-AKT pathway.