Effect Of Changes In Hepatic Lipid Metabolism On Follicular Development In Postpartum Dairy Cows With Negative Energy Balance

Negative energy balance（NEB）in dairy cows may lead to disturbance of lipid metabolism and thus inhibit follicular development because energy intake cannot meet the energy requirement of milk synthesis.This study aims to analyze the mechanism of postpartum liver lipid metabolism disorder on follicular development through metabolomics and to verify the effects of differentially expressed non-esterified fatty acids screened in vivo on the development of granulosa cells（GCs）and steroid hormone synthesis in vitro by in vitro experiments.According to the serum levels of glucose,β-hydroxybutyric acid,and non-esterified fatty acids,the group K（n=30）and a healthy control group H（n=30）on the 14 to 21 days postpartum,and were tracked until estrus and mating on the 45-60 days postpartum.According to whether the cows are in estrus,NEB cows without estrus in group K are selected as group N（n=6,undominated follicles）;Healthy cows with normal estrus performance in group H were selected as the healthy control group C（n=6,advantageous follicles）.,and liver tissue samples were collected from two groups at 45-60 days postpartum.To verify the effects of different non-esterified fatty acids on the growth of GCs and steroid hormone synthesis.Based on the results of quantitative metabolomics analysis,based on primary cow GCs model in vitro,some differentially expressed NEFAs（2.1 m M palmitic acid,2.7 m M stearic acid,2.5 m M trans oleic acid,1.6 m M linoleic acid,1.1 m M eicosatetraenoic acid）screened by targeted metabolomics were added to verify their effects on the cycle and steroid hormones of GCs.The results showed that a total of 35 differentially expressed metabolites were identified by non-targeted metabolomics.The metabolic enrichment pathways included amino acid metabolism and synthesis,c AMP signaling pathway,MAPK signaling pathway,pyruvate metabolism,etc.Targeted metabolomics determined the content of 38 non-esterified fatty acids（NEFAs）in the liver.Compared with group C,the liver contents of eicosatrienoic acid and arachidonic acid in group N were significantly increased（P<0.05）.The highest fatty acid content in the liver was palmitic acid,followed by trans-oleic acid,stearic acid,oleic acid,linoleic acid,and eicosatrienoic acid.There are 30 types of fatty acid metabolites showing an increasing trend.In vitro test results showed that the activity of GCs and the gene and protein expression of cyclin（CCNA1,CCND1,CCNE2,CDK1）and steroid synthesis-related proteins（St AR,HSD3B1,E₂,P₄）were decreased in the NEFAs supplementation group compared with the non-supplementation group.In summary:Hepatic lipid metabolism disorders in dairy cows with NEB affect follicular development.Among them,hepatic metabolic disorders cause an increase in total NEFAs,and changes in the proportion of palmitic acid,stearic acid,trans-oleic acid,linoleic acid and eicosatrienoic acid in NEFAs affect the activity of GCs and the expression of steroid hormones,thereby inhibiting postpartum follicular development in dairy cows.