Relationship Between ADPN,LEP,IR And Inactive Ovary In Dairy Cows With NEB

Background and purpose:The perinatal period is a critical period of dairy cow production.In this period,dairy cows are prone to negative energy balance（NEB）,and NEB will increase the incidence of postpartum inactive ovary（IO）in dairy cows,resulting in a decrease in estrus mating rate of dairy cows,causing huge losses to dairy industry.Although the etiology of postpartum IO in dairy cows has been reported in many studies,the relationship between NEB,adiponectin（ADPN）and leptin（LEP）and insulin resistance（IR）in dairy cows and their effects on IO are still unclear.Therefore,this research applied blood clinicopathology,immunology and molecular biology techniques to study the relationship between ADPN,LEP,IR and NEB inactive ovary,and laid a foundation for further research on the pathogenesis and new preventive measures of postpartum IO in dairy cows.Methods:The dairy cows with a previous lactation of more than 9 tons were selected as experimental animals in a large intensive dairy farm of Heilongjiang province,which were divided into positive energy balance group（PEB）and negative energy balance group（NEB）according to plasma BHBA and Glu levels at postpartum 14d,and 30 cows in each group.Subsequently,PEB and NEB cows were assigned into IR group and non-IR group according to the occurrence of IR,and 30 cows in each group.Then,at postpartum 50 to 60 d,according to whether or not estrus and follicle development status,the cows were again divided into IO group from NEB（NEB-IO）and estrus group from PEB（PEB-E）,and 6 cows in each group.Then,biochemical methods were used to detect the levels of blood biochemical indexes（Glu,BHBA,ADPN,LEP,NEFA,E2,P4,INS,IGF-1,GH）of experimental cows.Fluorescence quantitative PCR and Western Blot were used to detect the gene and protein expression levels of ADPN and LEP in adipose tissue and ovarian tissue of NEB-IO and PEB-E dairy cows,respectively.Immunohistochemical method was used to detect the ADPN receptor levels in these two tissues.All the data were analyzed by independent sample t test,Pearson correlation analysis and ROS analysis to determine the changes and relationships of blood biochemical indicators and to evaluate the risk of disease occurrence.Results:（1）The postpartum body condition loss of NEB dairy cows increased,and the production performance decreased.At the same time,plasma levels of LEP and ADPN decreased at postpartum 21 and 50 days,plasma IR index,TG,NEFA,BHBA,INS,and GH increased,while TC,Glu,E2,IGF-1 and LEP decreased（P<0.01）.These results suggest that NEB causes abnormal energy metabolism,insulin resistance,imbalance of adipocytokines,reproductive hormones and metabolic hormones in dairy cows,leading to the occurrence of postpartum IO in dairy cows.（2）Postpartum NEB in dairy cows increases the risk of IO by 4.33 times and the risk of IR by 2.7 times.IR increases the risk of IO by 3.2 times.NEB increases the risk of abnormal plasma LEP or ADPN levels by 3.33 times or 4.5 times.These results indicate that NEB or IR is an important risk factor for the occurrence of IO in dairy cows,and NEB is also an important risk factor for the occurrence of IR or abnormal levels of LEP and ADPN in dairy cows.（3）Plasma IR index of cows in the NEB and PEB groups was positively or negatively correlated with NEFA,BHBA or Glu,and E₂,P₄,GH,ADPN,and LEP were negatively correlated（P<0.01）.In NEB or NEB-IO cows,ADPN and LEP in blood and follicular fluid decreased when IR occurred,and E₂and P₄in follicular fluid also decreased（P<0.05）.These results indicate that IR exacerbates energy metabolism disorders,adipocytokines abnormalities,and reproductive hormone imbalances in dairy cow,thereby promoting the occurrence of IO.（4）The m RNA levels of ADPN and LEP in the adipose tissue and ovarian tissue and the protein levels of ADPN and LEP in adipose tissue,ovarian tissue,follicular fluid,serum,and ADPN receptor levels in ovarian tissue were all significantly decreased in the NEB-IO group（P<0.05）.These results suggest that ADPN and LEP play an important role in molecular regulation of IO in dairy cows.（5）The single indicator for early warning of the risk of IO in dairy cows at postpartum 14days is ADPN>2.365 mmol/L,LEP>5.565 mmol/L,or IR>3.796 which has certain diagnostic significance.The combined indicator judgment index is LEP+ADPN,or LEP+ADPN+IR,which can improve the specificity and use value of diagnosis.Conclusion:NEB is an important risk factor for the occurrence of IO and IR in dairy cows,and IR is also an important risk factor for IO.The correlation between NEB,IR and adipocytokines（ADPN,LEP）plays an important role in the occurrence of postpartum IO in dairy cows.The risk early warning and evaluation indexes and thresholds for IO in postpartum dairy cows have been determined.This study laid a theoretical foundation for more effective prevention and treatment of postpartum IO anestrus in dairy cows in the future.