Effects Of RP-Arg And NCG Supplementation On Fetal And Placental Development In Underfed Hu Sheep During Pregnancy

Hu sheep are a local breed in China characterized by precociousness and hyper-prolificacy.At present,the feeding and management of Hu sheep is expanded,but there is some improper feed management.Moreover,because of Hu sheep prolificacy,pregnancy ewes may be malnourished,which will be lead to intrauterine fetal growth restriction(IUGR).Arginine(Arg)is one of the most abundant amino acid nitrogen carrier in tissue protein in animals and humans,which serves as a precursor for synthesis of urea,nitric oxide and polyamines.Arg is rapidly degraded in rumen,and the price of rumen-protected arginine(RP-Arg)is uneconomical.N-carbamylglutamate(NCG)is low in rumen degradation,can enhance endogenous Arg synthesis.However,the research of effects of dietary RP-Arg and NCG supplementation on development of fetus and placenta in nutrient restriction Hu sheep during pregnancy is insufficient.In this paper,we focused on effects of RP-Arg and NCG supplementation on fetal and palcental development in underfed Hu sheep during pregnancy.First,we applied the metabolomic technique to explore the changes of blood metabolism at different time points of Hu sheep in pregnancy.Secondly,this part aimed to evaluate how dietary supplementation of RP-Arg and NCG in underfed pregnant Hu ewes would affect(1)metabolomic profiling in umbilical venous plasma of fetus;(2)palcental development;(3)fetal amino acids metabolism and somatotropic axis genes expression;(4)the function of fetal liver.The experiment is divided into five main parts as follows:1.Metabolic profiling of stages of healthy pregnancy in Hu sheep using nuclear magnetic resonance(NMR)To understand the differences in ovine metabolic changes that occur during normal pregnancy,pregnant ewes carrying twin fetuses(n = 8)were selected at 35 d of pregnancy.All ewes received 100%of National Research Council(NRC)requirements of all nutrients and energy during this experiment.At 50,70,90,and 110 d of pregnancy,maternal plasma samples were collected and designated as one of four corresponding time points(T1,T2,T3,and T4,respectively).Maternal plasma samples were analyzed using 1H-nuclear magnetic resonance(1H-NMR)spectroscopy to compare their c.We used multivariate pattern recognition to screen for different metabolites in the plasma of the ewes.The body weight and food intake of the ewes were significantly(P<0.05)different at the four time points,and increased with the passage of pregnancy time.The principal component analysis model results showed that the metabolic states at time points T2 and T3 moved gradually further away from that at T1 and were furthest away from that at T1 at time point T4.Among the different time points,there were thirteen significantly differential metabolites in the maternal plasma(P<0.05).These metabolites were closely related to amino acid metabolism and lipid metabolism,which might occur at different time points in pregnant ewes.In particular,newly observed changes in 1-methylhistidine and malonate were the first such changes found in maternal plasma.These results demonstrate that the metabolomics approach has value for evaluating metabolism in pregnancy with advancing pregnancy.In conclusion,during normal pregnancy in Hu sheep,related metabolites play an important role in amino acid and lipid metabolism for meeting the nutritional demands of pregnant ewes.2.Metabolomic profiling in umbilical venous plasma reveals effects of dietary RP-Arg and NCG supplementation in nutrient restriction hu sheep during pregnancyIt is a major health problem worldwide,which lead to IUGR and fetal undernutrition accompanies placental insufficiency.Arginine is critical to health,growth,and reproduction.NCG,a key enzyme in arginine synthesis,is not extensively degraded in rumen.The aim of this study was to investigate ameliorating effects of RP-Arg and NCG supplementation on dietary in undernourished Hu sheep during pregnancy.From d 35 to 110 of pregnancy,32 Hu ewes carrying twin fetues were randomly divided into 4 groups,a control(CG)group(n = 8;100%NRC requirements for pregnant sheep),a underfed(RG)group(n = 8;fed 50%NRC requirements,which included 50%mineral-vitamin mixture),and 2 treatment(ARG and NCG)groups(n = 8;fed 50%NRC requirements supplemented with 20 g/d RP-Arg or 5 g/d NCG,which included 50%mineral-vitamin mixture.The umbilical venous plasma samples of fetuses were tested by 1H-NMR.Thirty-two differential metabolites were identified,indicating altered metabolic pathways of amino acid,carbohydrate and energy,lipids,oxidative stress metabolism among the 4 groups.Our results demonstrate that the beneficial effect of dietary RP-Arg and NCG supplementation on mammalian reproduction is associated with complex metabolic networks.3.Effects of dietary RP-Arg and NCG supplementation on development of maternal and fetal placenta in nutrient restriction Hu sheep during pregnancyThis study aimed to evaluate the effects of RP-Arg and NCG supplementation on fdevelopment of maternal and fetal placenta in nutrient restriction Hu sheep during pregnancy.Thirty-two Hu ewes carrying twin fetues were randomly divided into 4 groups(CG,RG,ARG,and NCG groups)from d 35 to 110 of pregnancy.At d 110 of pregnancy,fetal umbilical venous plasma,maternal placenta(caruncle)and fetal placenta(cotyledons)was collected.The present study evaluated the concentration of metabolites in umbilical venous plasma,the expression of this growth factor family and mTOR signaling pathway’s intermediates(mammalian target of rapamycin,mTOR;p70 ribosomal protein S6 kinase,p70S6K;factor 4E-binding protein 1,4EBP1)in caruncle and cotyledons.The results showed that the ratio of fetal body weights to placental weight was decreased in RG group compared with CG group;however,the placental weights were no differences among CG,ARG and NCG groups.The results showed that the ratio of fetal body weights to placental weight was decreased in RG group compared with CG group;however,the placental weight,weight of cotyledons and caruncula were no differences among CG,ARG and NCG groups.The mRNA expressions of vascular endothelial grovwth factor A(VEGFA),vascular endothelial growth factor receptor-1(VEGFR-1),placenta growth factor-1(PLGF1),angiopoietin-1(Ang-1),and angiopoietin-2(Ang-2)were no significant in cotyledons and caruncula of four groups.Significantly increased mRNA expression of vascular endothelial growth factor receptor-2(VEGFR-2)was detected in RG group compared to CG,ARG,and NCG groups,but there was decreased tyrosine kinase receptors-2(Tie-2)mRNA expression in RG group.In RG group,mTOR signaling was inhibited,as deter:mined by increased p70S6K and 4EBP1 mRNA expressions,and increased p70S6K,4EBP1,and p-p70S6K proteins expression.The data provide important insight into the maternal nutrition levels alter developmental programming of cotyledons and caruncula.Dietary RP-Arg and NCG supplementation to underfed ewes during pregnancy could promote the growth and development of cotyledons and caruncula.4.Effects of dietary RP-Arg and NCG supplementation on fetal amino acids metabolism and somatotropic axis genes expression in nutrient restriction Hu sheep during pregnancyThis study was focused on the hypothesis that dietary RP-Arg and NCG supplementation in underfed ewes is effective in enhancing fetal growth.Between d 35 and 110 of pregnancy,32 multiparous ewes carried two fetuses were randomly assigned to four groups(CG,RG,ARG,and NCG groups).At d 110 of pregnancy,all ewes were slaughtered and fetal weight and fetal organ weight recorded.Metabolites and hormones in fetal plasma,amino acids concentrations in fetal liver and longissimus dorsi muscle,and somatotropic axis genes mRNA expressions were were determined and analyzed.Most fetal organs weights expressed as a percentage of BW was increased in ARG and NCG groups compared with RG group(P<0.05).In the RG group,fetal plasma concentrations of insulin(INS),insulinlike growth factor-I(IGF-I),total amino acid(TAA),lactate(LD),thyroxine(T3),and thyroxine/triiodothyronine(T4/T3)were significantly decreased compared with the other groups(P<0.05),but the concentrations of growth hormon e(GH),non-esterified free fatty acid(NEFA),and total cholesterol(TC)were significantly increased(P<0.05).Maternal RP-Arg or NCG supplementation markedly improved concentrations of amino acids in fetal tissues,and influenced somatotropic axis genes mRNA abundance within nutrient restricted ewes.Collectively,these novel findings show that maternal RP-Arg and NCG supplementation to underfed ewes prevented fetal growth restriction,at least in part,by improving metabolic homeostasis of fetal endocrinology,increasing the availability of amino acids in fetal liver and longissimus dorsi muscle,and influence the somatotropic axis genes expression levels.While these alterations in fetal growth and development may be beneficial to fetuses in the face of maternal nutrient restriction,their effects later in postnatal life need further investigation.5.Effects of dietalry RP-Arg and NCG supplementation on development of fetal liver in nutrient restriction Hu sheep during pregnancyThis study aimed to determine how dietary supplementation of RP-Arg and NCG in underfed pregnant Hu ewes would affect(1)growth and development of fetal liver;(2)anti-oxidation activies in fetal liver;and(3)anti-apotosi activies in fetal liver.Thirty-two Hu ewes(n=32)carrying twin fetuses were allocated to 4 groups from d 35 to 110 of pregnancy:CG,RG,ARG,and NCG groups.The results showed that the decreased dry matter,water,fat,protein and ash components of fetal livers in RG group were found compared to CG group,these components were significantly increased in NCG group compared to RG group(P<0.05).The DNA,RNA and protein concentrations,the RNA and protein contents,and protein/DNA was decreased in RG group compared with CG group(P<0.05).The DNA,RNA and protein concentrations,protein contents,and protein/DNA was increased in NCG group compared with RG group(P<0.05).The concentrations of cholinesterase(CHE),nitric oxide(NO),nitric oxide synthase(NOS),total antioxidant capacity(TAC),superoxide dismutase(SOD),alanine aminotransferase(ALT)and total protein(TP)in RG group were lower than those of CG group(P<0.05),and there was increased glutathione peroxidase(GSH-Px),maleic dialdehyde(MDA),and aspartate aminotransferase(AST)in RG group relative to CG group(P<0.05).In RG group,the mRNA and proteins expression of p53 and Bax was significantly increased(P<0.05)compared with CG group,and the genes expression of FasL and Bcl-2,the ratio of Bcl-2 to Bax,and the protein expression of Bcl-2 in RG group was significantly decreased(P<0.05)compared with CG group.These results indicate that maternal alters developmental programming of fetal liver.Dietary supplementation of RP-Arg and NCG to underfed ewes during pregnancy could influence the growth and development of fetal liver,ameliorate anti-oxidant imbalance and dysfunction,and inhibits apoptosis cell death.