Effects Of High Concentration Of Blood NEFA On The Synthesis Of Milk Protein And Fat In Dairy Cows With Clinical Ketosis

Ketosis is a majormetabolic disease of dairy cows in early lactation.Cows with clinical ketosis always show relatively high plasma concentration of non-esterified fatty acids(NEFA)and ketone bodies,and associate with decreased milk protein percentage and increased milk fat percentage.The high levels of NEFA in the blood can be absorbed by the bovine mammary gland and used to synthesize milk fat.Milk protein and fat synthesis are affected by several factors.The JAK2/STAT5 and mTOR signaling pathways are central to the regulation of milk protein synthesis.Cell death-inducing DFFA-like effector a(CIDEA)is a lipid droplet coat protein with closely relating to lipid metabolism.Previous studies have shown that CIDEA plays an important role in the regulation of milk fat synthesis and secretion in mice,and highly expressed in the mammary gland of lactating dairy cows.However,whether and how the surge in uptake of NEFA by bovine mammary gland during clinical ketosis plays a role in regulating milk protein and milk fat synthesis is unknown.Therefore,in this study,mammary gland of healthy and clinically ketotic dairy cows were collected in vivo,bovine mammary epithelial cells(BMEC)were isolated and cultured with different concentrations of NEFA in vitro to study the effect and mechanism of high concentration of NEFA on the synthesis of milk protein and milk fat.Blood and milk samples from healthy and clinically ketotic cows were collected to detect blood metabolic indexes and milk composition parameters.The results showed that serum concentrations of NEFA and the content of milk fat were greater in cows with clinical ketosis,and milk protein was lower in cows with clinical ketosis.Subsequent experiments study the effect of high levels of NEFA on the synthesis of milk protein and milk fat respectively.Mammary tissue from clinically-ketotic and healthy dairy cows were collected,BMEC were cultured in vitro and divided into 3 groups: treated with 0,0.3,0.6,1.2 mM NEFA,methionine(Met)co-cultured with 1.2 mM NEFA,prolactin(PRL)co-cultured with 1.2 mM NEFA.In vivo results showed that the JAK2/STAT5 and mTOR signaling pathways were inhibited and the abundance of ?-casein was lower in mammary tissue of cows with clinical ketosis.In vitro,NEFA decreased the abundance of ?-casein in BMEC.In addition,the phosphorylation level of JAK2,STAT5,mTOR,S6K1 and the mRNA abundance of STAT5,S6K1,and 4EBP1 was downregulated with NEFA.Met and PRL can significantly alleviate the inhibitory effects of high concentration NEFA on ?-caseinexpression,JAK2/STAT5 and mTOR signaling pathways.The results indicated that high levels of NEFA suppressed JAK2/STAT5 and mTOR signaling pathways and further inhibited ?-casein synthesis in BMEC.Mammary tissue from clinically-ketotic and healthy dairy cows were collected,BMEC were cultured in vitro and divided into 3 groups: infected with CIDEA overexpression adenovirus group,treated with 0,0.3,0.6,1.2 mM NEFA,infected with CIDEA-silencing adenovirus and 1.2 mM NEFA.In vivo,compared with healthy cows,the abundance of CIDEA,fatty acid synthesis-related enzymes(FASN,ACACA),lactolipid drop secretion related enzymes(BTN1A1,XDH)was greater in mammary tissue of cows with clinical ketosis.In vitro,overexpression of CIDEA and treated with NEFA in BMEC increased the abundance of FASN,ACACA,BTN1A1,and XDH,and increased triacylglycerol(TAG)content.Importantly,knockdown of CIDEA reversed the upregulation of FASN,ACACA,BTN1A1,and XDH abundance and TAG content induced by NEFA treatment.The results demonstrate that high levels of NEFA stimulate the expression of CIDEA and enhance milk fat synthesis and secretion.Our results indicated that the low milk protein percentage and high milk fat percentage were related to the high concentration of NEFA.These results suggest that high blood concentration NEFA is an important starting factor leading to low milk protein rate and high milk fat rate,and its effect on milk protein or milk fat rate is mediated by JAK2/STAT5 and mTOR signaling pathways or CIDEA,respectively.