Neuroendocrine Regulation Mechanism On Gluconeogenesis And Fat Mobilization Of Ketosis And Fatty Liver In Dairy Cows

In this study, healthy multiparous dairy cows, the diseased dairy cows withketosis and fatty liver in field, primary cell culture in vitro, and quantitive RT-PCRwere applied to probe the neuroendocrine regulation mechanism on gluconeogenesisand fat mobilization in dairy cows with ketosis and fatty liver.In the animal trial, twenty-four peripartum cows , 5-8 yrs and 3-5 parities, wererandomly assigned into three treatments, each of 10, fed on basal diets with1.79NND/kg from antepartum 28d to parturition, and fed on hay freedom frompostpartum 1d to 56d. The body weight (BW), dry matter intake (DMI), milk yield(MY), blood biochemical parameters, serum hormones, and expression level ofPEPCK-C mRNA in liver, HSL mRNA and LP mRNA in tail fat tissue weredetermined from antepartum 28d and 14d to postpartum 1d, 14d, 28d and 56d. Theresults as follows: peripartum dairy cows was prone to develop hypoglycemia , dairycows with hypoglycemia at antepartum tended to relapse at postpartum, but usuallyrecovery after postpartum 28d. There was the highest level of serum NEFA, TBI atpostpartum 1d, and of serum BHBA, AST at postpartum 14d in two groups. Dairycows with hypoglycemia developed subclinical ketosis at postpartum 14d and had anincreasing concentration of serum NEFA. There was higher level of serum NPY andLP/NPY at postpartum 1d, 14d, 28d, of HSL mRNA expression in tail fat atpostpartum 1d and 14d, and of DMI at postpartum 56d in dairy cows withhypoglycemia than those in control(P<0.05). There was lower level of serum P4 atantepartum 28d and at postpartum 1d, 28d, 56d, of liver PEPCK-C mRNA expressionat antepartum 28d and 14d, and of LP mRNA expression in tail fat at postpartum 56din dairy cows with hypoglycemia than those in control (P<0.05). There was nodifference in other blood parameters such as serum AST, TBI, TG, INS, GN, LP, E2,GH, and INS/GN in two groups。 In the experiment of dairy cows with ketosis and fatty liver, ketosis and fatty liveroccur in early lactating dairy cows, which had obviously low blood sugar and milkyield(P<0.05), and 18.8% and 41.98% of TG content in liver, respectively . There wassignificant increase in serum AST, γ-GT, TBI, CHE and Cr of dairy cows with fattyliver, which indicated a risk of hepatosis and renal disfunction, but no obvious increasein dairy cows with ketosis. One reason of low conception rate is possibly due to lowconcentration of serum E2 and P4 in dairy cows with ketosis and fatty liver. There wasmarkedly decrease in serum INS, LP and INS/GN, fat HSL mRNA and LP mRNAexpression of dairy cows with ketosis, accompanied by increase in liver PEPCK-CmRNA expression, which facilitated to enhance DMI, gluconeogenesis and fatmobilization. There was a higher value in serum INS, INS/GN in dairy cows with fattyliver, accompanied by low level of serum LP, and low expression of liver PEPCK-CmRNA, fat HSL mRNA and LP mRNA , which facilitated to decrease gluconeogenesisand fat mobilization and increase DMI. But, The reason that causes low appetite,hypoglycemia, and high serum NEFA, BHBA in dairy cows with ketosis and fattyliver is worthy of research more. According to the publishing sequence, two pairs of primers were designed andRT-PCR was employed to amplify the partial cDNA of PEPCK-C and β-actin, twotarget sequences were recombinated into cloning vector respectively, then sequencinghomology of the two plasmids was 98.8% and 98% for PEPCK-C and β-actin ,respectively. Using the 909bp fragment of β-actin as inner marker, grading-up halfquantitive RT-PCR with 57℃anneal, 2.40mmol/L Mg2+, 30 cycle, 2.6ng/ml cDNA,2:1 primer ratio of PEPCK-C toβ-actin was employed to determine the expressinglevel of 416bp fragment of PEPCK-C in primary culture liver cell which was treatedwith different concentrations of INS (0 mmol/L, 5 mmol/L, 10 mmol/L, 20 mmol/L,50 mmol/L, 100 mmol/L), GN(0 pg/ml, 25 pg/ml, 100 pg/ml, 200 pg/ml, 500pg/ml,1000 pg/ml), and LP(0 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100ng/ml) for 12h. The result showed that the expression level of PEPCK-C mRNA inliver cell was gradually decreased with increasing concentration of INS in culturemedia, on the contrary the expression level of PEPCK-C mRNA in liver cell wasgradually increased with increasing concentration of GN in culture media. But, leptinhad no obvious effect on expression of PEPCK-C mRNA in liver cell. According to the publishing sequence, four pairs of primers were designed andRT-PCR was employed to amplify the partial cDNA of HSL and LP, four targetsequences were recombinated into cloning vector respectively, then homology of thetwo long sequence plasmids was 98.3% and 99.6% for 299bp fragment of HSL and480bp fragment of LP respectively, the two short sequences were 100% for 91bpfragment of LP and 99bp fragment of HSL. According to previous reports, a model ofcalf preadipocyte culture in vitro was successfully established, which was proved bymorphology, growth curve, oil red O fat staining. A fluorescent Q-PCR was adopted toprobe the expressing level of HSL mRNA and LP mRNA in primary adipocytes whichwere treated with different concentrations of INS (0mmol/L,5 mmol/L,10 mmol/L,20mmol/L,50 mmol/L,100 mmol/L), GN(0 pg/ml,25 pg/ml,100 pg/ml,200 pg/ml,500pg/ml,1000 pg/ml), and LP(0 ng/ml,2.5 ng/ml,5 ng/ml,10 ng/ml,50 ng/ml,100 ng/ml)for 12h.. The result showed that the expression level of HSL mRNA in adipocyte cellgradually decreased as increasing concentration of INS in culture media, but on thecontrary the expression level of LP mRNA in adipocyte cell gradually increased.Furthermore, when the concentration of GN increased in culture media, the expression...