| Previous studies have shown that the supplementation of 1% arginine can enhance the reproductive performance in pregnant sows.However,due to the high price of arginine in the market,the cost of dietary arginine supplementation at 1% is too expensive,which limits the wide application of arginine in practical production.Therefore,this study was carried out to investigate the effects of adding 0.1% L-arginine-HCl in wheat-soybean meal basal diets on the farrowing performance of the gilts,and determine the effects and possible mechanism of arginine and its metabolites on the regulation of porcine placental trophectoderm cells(p Tr2).The results of our study may provide scientific guidance for the future application of arginine in sow production.Experiment 1,a total of 120 gilts(Landrace× Large White)with similar age and body weight,and consistent genetic background and estrus date were selected,and were.randomly allocated into either Control group or Arginine group.Each group had 60 replicates,with one gilt per replicate.The gilts in the Arginine group were fed the basal wheat-soybean meal diets supplemented with0.1% L-arginine-HCl,while those in the Control group were fed 0.17% L-alanine-supplemented diets as isonitrogenous balance during 30 to 110 days of gestation.During the experiment,the litter size,born piglets alive,body weight of piglets born as well as placental weights were recorded,and plasma free amino acid concentrations and other biomedical paramenters(estrogen,polyamine,nitric oxide,ammonia and insulin-like growth factor-I in plasma)at 30,70 and 90 days of gestation were also determined..Experiment 2,The effect of different concentrations of arginine on the proliferation of p Tr2 cells was carried out in vitro.The p Tr2 cells were incubated in the arginine-free specialized DMEM medium for 6 h-arginine starvation.Then different concentrations of arginine were supplemented with arginine(0.2,0.4,0.8,1.6,and 3.2 m M)to the medium for incubation of 2,4,and 6 days.The proliferation of the placenta trophectoderm cells was detected by MTT assay.Apoptosis was detected by mitochondrial membrane potential kits.The expression of genes involved in cell proliferation was detected by quantitative RT-PCR(q RT-PCR).The growth factor and estrogen synthesis were detected by ELISA kits,while expression of proteins involved in the mTOR signaling pathway were determined by western blotting analyses.Experiment 3,the optimal arginine concentration for enhancing the proliferation of p Tr2 cells was selected based on the results of Experiment 2.Then the total nitric oxide synthase inhibitor(L-NMMA),amine synthesis inhibitor(DFMO),nitric oxide donor sodium nitroprusside,and putrescine were used to dertermine the effects of NO and putrescine on the regulation of cell proliferation of p Tr2 cells.The proliferation of placenta trophectoderm cells was detected by MTT assay.The cell cycle was detected by flow cytometry.The expression of genes involoved cell proliferation was detected by qRT-PCR.The expression of proteins in the m TOR and β-catenin signaling pathways were detected by western blotting analyses.The results of this study showed that:1)Compared with the Control group,the supplementation of 0.1% L-arginine-HCl in the wheat-soybean meal diet for gilts between 30-110 days of gestation significantly increased the litter size by 1.1(11.28%)and increased the numver of live births by 1.1(12.64%)(P <0.05).The plasma concentrations of arginine,ornithine,and NO in the gilts were significantly increased by0.1% L-arginine-HCl supplementation on days 70 and 90 of gestation(P<0.05).The concentrations of IGF-I,proline and spermine in plasma of gilts were significantly increased on day 90 of gestation(P<0.05),while the plasma NH3 concentration was decreased on day 90 of gestation(P<0.01).2)The proliferation of p Tr2 cells was significantly higher in different arginine-supplemented groups than that in the control group at days 2,4,and 6(P<0.05),with 0.4 mM most profound,while at the same time 0.4 mM arginine inhibited the cell apotosis at days 2 and 4.The addition with either 0.4 mM or 0.8 m M arginine significantly increased the relative expression of PCNA mRNA in pTr2 cells at day 4(P<0.05).The expression of arginine decarboxylase(ADC)m RNA increased significantly at day 4(P>0.05).The addition of 0.4 mM or 0.8 m M arginine significantly incrased the m RNA expression of arginase in pTr2 cells at day 4(P<0.05).The levels of NO,EGF,estradiol and progesterone in the culture medium of pTr2 cells on days 2 and 4 were significantly higher than those in the control group(P<0.05).The expression of m TOR,4EBP-1,RPS6,p-4EBP-1 and p-RPS6 proteins in p Tr2 cells was all increased by 0.4 mM arginine(P<0.05).3)The cell proliferation of pTr2 cells on days 2,4 and 6 was increased by the addition of either sodium nitroprusside and putrescine compared to the NO or putrescine inhibitors groups(P<0.05).The percentage of G2+S phase in p Tr2 cells was increased on day 2 and 4 based on the cell cycle assay(P<0.05).The expression of PCNA,IGF-I,EGF,mTOR and β-catenin mRNA in pTr2 cells was all increased on 2 day by sodium nitroprusside and putrescine(P<0.05).The protein expression of m TOR,β-catenin and p-β-catenin in p Tr2 cells was significantly increased on day 2after addition of putrescine(P<0.05).The expression of m TOR,β-catenin and p-β-catenin in p Tr2 cells at day 4 were significantly increased by putrescine treatment(P<0.05).In summary,this study found that the supplementation of low-dose of arginine-HCl(0.1%)to wheat-soybean meal improved the farrowing performance of gilts.The addition of 0.4 mM arginine in the arginine-free culture medium promoted the cell proliferation and inhibited the cell apoptosis,increased the synthesis of estrogen,and upregulated the mRNA expression of arginine metabolic pathway associated enzymes in p Tr2 cells.The metabolites of arginine,mainly NO andputrescine,enhanced the cell proliferation of pTr2 cells,at least by the activations of both theβ-catenin and mToR signal pathways. |
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