Relation Of Ketone Body Level To Antioxidative,Leptin In Cows And In Vitro Effect Of Leptin On Antioxidative Enzyme Expression In Calf’s Hepatocyte

It is important to investigate relationship of whey milk and blood plasma level of ketone body to that of anti oxidative indexes, leptin for understanding pathogenesis of ketosis. In this study, whey milk level of ketone body (KET), Total anti oxidative capacity(TAC), malondialdehyde(MDA), catase(CAT), glutathione perioxidase (GPX), non-esterified fatty acid (NEFA) whey milk level and blood plasma level of leptin, insulin, IGF-I were determined, correlation between them were analysed according to different level of ketone body. By radioimmuno assay, real time fluorescence quantitative PCR and primary culture of calf’s hypatocyte, adding different level of leptin, insulin and IGF-I, the mRNA expression level of CAT, GPX, MDA in calf s hypatocyte and TAC, MDA concentration in vitro were analysed.Test 1:To understand the correlation between ketone body and some oxidative/antioxidative index,98 Holstein cows in eary lactation were chosen from a dairy farm in Guangxi, whey milk analytic data indicated that all cows were divided into groups according to whey milk level of ketone body, the correlation between them was analysed. Results:the whey milk level of KET, TAC, MDA, GPX, and CAT was 13.8±6.7 mg/dl,5.0±3.1 U/ml,2.2±1.8 nmol/ml,56.1±47.1 U/ml and 3.0±2.0 U/ml, respectively. High KET group (milk KET>20 mg/dl, n=18) had higher whey milk level of all indexes except CAT than middle KET group (milk KET:10-19.9 mg/dl, n=47) or low KET group (milk KET<10 mg/dl, n=33). There was significant differrence in KET level among three groups. There was significant differrence in TAC level between middle and low KET groups. In all cows, a significant correlation existed between KET and TAC, (r=0.237), between TAC and MDA (r=-0.233). In high KET group, a significant negative correlation existed between MDA and TAC (r=-0.586), MDA and KET (r=-0.482). In group with milk KET<20 mg/dl, a significant positive correlation existed between TAC and KET (r=0.298, n=80, p<0.01) Conclusion, whey milk level of KET and some oxidative/antioxidative indexes was obtained. High milk KET level had a significant effect on correlation between milk oxidative/antioxidative indexes, It made the correlation weak between TAC and KET, made the negative correlation strong between MDA and TAC, It changed correlation from significant positive correlation to significant negative correlation between KET and MDA.Test 2:To investigate whey milk level and blood plasma level of leptin, insulin, NEFA and their correlation between them,7 healthy cows and 7 cows with ketosis were chosen, blood and milk samples were collected once a week from second week to seventh week postpartum, the whey milk level and blood plasma level of leptin, insulin, NEFA were determined. Results showen that both whey milk level and blood plasma level of leptin and insulin were lower, those of NEFA higher in ketostic cows compred with the healthy cows. The whey milk level of leptin, insulin, NEFA was significant higher than the blood plasma level of them. A significant correlation existed betwee whey milk and blood plasma in leptin (n=84, r=0.64, P=0.025), insulin (n=84, r=-0.627, P=0.029), and NEFA (n=84, r=0.810, P=0.001). Conclusion, the characterestics was found about whey milk level and blood plasma level of leptin, insulin, and NEFA, It could be possible to detect NEFA by whey milk instead of blood plasma.Test3:in this test, natal calf’s haptocyte was cultivated, effect of leptin, insulin and IGF-I with different level on mRNA expression level of antioxidative enzyme. In Leptin (0,2.5,5,10,50,100 ng/ml), insulin (0, 5,10,20,50,100 nM), and IGF-I (0,10,30,50,100,150 ng/ml) was added into the culture medium of primary haptocyte, after 24 hours cultivating. At same time, level of MDA, TAC, GPX, CAT in the medium was determined before and after leptin, insulin, and IGF-I added. Results shown that amplification efficiency of CAT, GPx, β-actin by SYBRGreen I fluorescence quantitative PCR was 100%. Standard curve equation and R2 was y=-3.315x+14.72, R2=0.9992; y=-3.228x+8.78, R2=0.9998; y=-0.301 x+8.9344, R2=0.9997, respectively. Solubility curve of fluorescence quantitative PCR was a single peak, that means no dimer and good specificity. Conclusion, high level of insulin and IGF-I could increased actavity and mRNA level of CAT, GPX of bovine hepatocte.