Multi-Omics Analysis Reveals The Effect Of Severe Feed Restriction On The Nutrition Metabolism Of Hu Sheep In Late Pregnancy And Its Nutritional Regulation

Hu sheep is a sheep breed with excellent fecundity originating in Jiangsu and Zhejiang regions in China.The survey found that the proportion of pregnant Hu sheep with double or more lambs is as high as 80%.However,Hu sheep pregnant with multiple lambs are also prone to negative energy balance,and even pregnancy toxemia in late pregnancy.In breeding ewes,the incidence of pregnancy toxemia induced by severe negative energy balance is 20%,and the mortality rate can reach 70%-80%,causing significant economic losses to Hu sheep breeding industry in China.A model of negative energy balance in late gestation in Hu sheep was established by feed restriction in this study.The effect of severe feed restriction on the composition of blood and urine metabolites in Hu sheep,and the molecular mechanism of liver and kidney metabolism were explored in this study.Furthermore,we explored the nutritional regulation of glycerol and rumen protected choline chloride supplementation on abnormal metabolism in sheep with negative energy balance.The thesis consists of five chapters.1 Effects of severe feed restriction on blood metabolome of Hu sheep in late pregnancyThis chapter aims to explore the effects of severe feed restriction on the blood metabolome of Hu sheep in late pregnancy.20 Hu sheep(weight 60.6±4.9 kg)pregnant with 2-3 lambs were selected in this experiment and the gestation period of the sheep was 108 days.After a 7-day adaptation period,Hu sheep were randomly assigned into a control group(CON group)or severe feed restriction group(FR group)for a formal experimental period lasting 15 days.The Hu sheep in the CON group were fed to meet 100% of their nutritional requirements,while the Hu sheep in the FR group were fed to meet only 30% of their nutritional requirements.The jugular vein blood was collected before morning feeding on days 0,5,10 and 15 of the experimental periods for metabolomic analysis.Previous studies in this project showed that feed restriction significantly reduced blood glucose concentration(P < 0.05)and significantly increased blood β-hydroxybutyric acid concentration(P < 0.05),and successfully established pregnancy toxemia model induced by negative energy balance in Hu sheep.The results showed that compared with the CON group,FR significantly reduced the blood insulin concentration(P < 0.001),and significantly increased the concentrations of blood glucagon(P < 0.001),epinephrine(P < 0.001)and norepinephrine(P < 0.001)of Hu sheep.The HPLC-MS-based plasma metabolomic analysis revealed that FR significantly changed the composition of plasma metabolites in Hu sheep in late pregnancy(P = 0.001).The numbers of significantly different metabolites in the Hu sheep plasma on trial day 5,10,and 15 are 120,129,and 114,respectively.Interestingly,the most differential metabolites were related to lipid metabolism.The KEGG pathway enrichment analysis showed on experiment day 5,the NEFA and carnitine contents in the plasma of FR group increased rapidly,and feed restriction had the most significant effect on the linoleic acid metabolic pathway(P = 0.013)compared with the CON group,indicating that severe feed restriction rapidly induced lipid mobilization and fatty acid β-oxidation in Hu sheep.Compared with CON group,the choline content in plasma of Hu sheep in FR group was significantly lower(P < 0.05),suggesting fat deposition in the liver.On the experiment day 10,feed restriction led to significant changes in the tricarboxylic acid cycle(P = 0.047).Specifically,the citric acid and α-ketoglutarate in this pathway continued to decrease in the FR group,indicating that feed restriction further aggravated the changes in body metabolism.On day 15,feed restriction resulted changes in aminoacyl-t RNA biosynthesis(P = 0.004),alanine,aspartate,and glutamate metabolism(P = 0.036),valine,leucine,and isoleucine biosynthesis(P = 0.047),and arginine and proline metabolism(P = 0.050)pathways.Specifically,the content of amino acids such as arginine,valine,and aspartic acid,which can quickly enter the tricarboxylic acid cycle pathway,decreased sharply in the FR group,while the levels of ketogenic amino acids(leucine and lysine)were significantly elevated(P < 0.01),which promoted ketone body production.In addition,the study also found that feed restriction resulted in a significant increase of a number of toxic metabolites in plasma(P < 0.01),which are associated with fatty liver,brain injury and tissue damage.The results show that feed restriction can programmatically induce metabolic changes in the body,initiating with changes in lipid metabolism,further affecting amino acid metabolism through the tricarboxylic acid cycle pathway.2 Effects of severe feed restriction on urine metabolome of Hu sheep in late pregnancyThis chapter aims to investigate the effect of severe feed restriction on the urine metabolome of Hu sheep in late pregnancy.The experimental design was the same as described in chapter 1.On the day 15,Hu sheep were slaughtered 4 hours after morning feeding,and bladder urine was collected for metabolomic analysis.The samples of Hu sheep in the CON group and the FR group could be clearly distinguished by principal component analysis,indicating significant differences in urine metabolic compositions of the two groups(P < 0.05).135 differential urinary metabolites identified between CON group and FR group were mainly involved in glucose metabolism,fatty acid metabolism,amino acid metabolism,and others.Compared with the CON group,feed restriction significantly reduced gluconeogenesis-related metabolites(D-glucose,lactate,levoglucan,α-ketoglutarate,phosphohydroxypyruvate,and glucose 6-phosphate),while significantly increased the content of small amounts of fatty acids,carnitine and ketogenic amino acids(leucine and lysine)in urine(P < 0.01).Consistent with the metabolic results,the enrichment analysis based on KEGG metabolic pathways showed significant changes in the urinary metabolites related to amino acid metabolism pathways in the FR group(P < 0.05).Among the related metabolic pathways,feed restriction had the most significant effect on D-glutamine and D-glutamate metabolic pathway(impact value = 1).Our results showed that ketogenic amino acids could be excreted by kidney,even though the large amount of fatty acids produced by lipid mobilization could not be excreted.3 The mechanism study of severe feed restriction affecting liver metabolism of Hu sheep in late pregnancy based on lncRNA technologyThis chapter aims to explore the mechanism of severe feed restriction affecting liver metabolism of Hu sheep in late pregnancy.The experimental design was the same as described in chapter 1.On the experiment day 15,Hu sheep were slaughtered 4 hours after morning feeding,and liver samples were collected.The results showed that compared with the CON group,a total of 296 differentially expressed(DE)lncRNAs(189 up-regulated and 107 down-regulated)and 972 DE mRNAs(514 up-regulated and 458 down-regulated)were identified in the liver of Hu sheep in the FR group.The DE lncRNA target genes and DE mRNAs were enriched in the lipid metabolism,glucose metabolism,amino acid metabolism,and inflammatory response in liver under the condition of negative energy balance.Compared with the CON group,feed restriction significantly reduced the mRNA expressions of genes related to hepatic gluconeogenesis and glucose catabolism(P < 0.05),and significantly increased the absorption and transport of fatty acids to the Hu sheep liver in the FR group.Feed restriction increased expressions of genes related to β-oxidation,triglyceride synthesis,and catabolism(P < 0.05),as well as mRNA expression of genes related to ketogenic amino acid catabolism(P < 0.05),but significantly reduced mRNA expressions of genes related to choline synthesis(P < 0.05).Compared with the CON group,feed restriction significantly up-regulated the mRNA expression of NF-kappa B signaling pathway genes(IRAK1,DDX58,MALT1 and IKBKB)in the liver(P < 0.05),and significantly down-regulated the gene expression of IL-10(P < 0.05).Correlation analysis showed that the expression of choline synthesis genes in the liver was negatively correlated with the expression of NF-kappa B signaling pathway genes(P < 0.05),while it was significantly positively correlated with the mRNA expression of IL-10(P < 0.05).In addition,this study found that lncRNAs MSTRG.28837.1 and MSTRG.139925.2 have regulatory effects on the choline synthesis genes PLA2G16 and PEMT,respectively.The results showed that feed restriction decreased the activities of glucose catabolism in the liver of Hu sheep,while enhanced the activities of lipid metabolism and amino acid catabolism in the liver.During this process,the choline synthesis capacity of Hu sheep liver cells was significantly reduced by feed restriction,which may be closely related to the up-regulation of inflammation-related gene expression and it’s a key target for metabolic disorders and dysfunction.4 The mechanism study of severe feed restriction affecting kidney metabolism of Hu sheep in late pregnancy based on lncRNA technologyThis chapter aims to explore the mechanism of severe feed restriction affecting kidney metabolism of Hu sheep in late pregnancy.The experimental design was the same as mentioned above.On the 15 th day of the experimental period,Hu sheep were culled 4 hours after morning feeding and kidney samples were collected.The kidney morphology analysis showed no significant difference in kidney lipid deposition between the two groups.Compared with the CON group,a total of 156 differentially expressed(DE)lncRNAs(70 up-regulated and 86 down-regulated)and 1251 DE mRNAs(694 up-regulated and 557 down-regulated)were identified in the kidneys of Hu sheep in the FR group.KEGG pathway enrichment analysis showed that DE lncRNA target genes and mRNAs were enriched in adrenaline signaling pathway,alanine,aspartate,and glutamate metabolism,aldosterone-regulated sodium reabsorption,arginine,and proline metabolism,c AMP signaling pathway,histidine metabolism,and less pathways were related to lipid metabolism.Compared with the CON group,feed restriction significantly increased the relative gene expressions of renal gluconeogenesis genes G6PC(P < 0.05),PC(P < 0.01),FBP(P < 0.05)and PCK2(P < 0.01),and significantly decreased the relative expression of renal glucose catabolism(P < 0.05).Feed restriction significantly upregulated the gene expressions related to fatty acid synthesis and β-oxidation,triglyceride synthesis,and catabolism(P < 0.05),significantly increased the choline synthesis genes choline cytidyltransferase(PCYT1),and APOB mRNA gene expression(P < 0.05).At the same time,compared with the CON group,the expressions of genes related to amino acid catabolism in the FR group were significantly down-regulated(P < 0.05),and the gene expressions of catecholamine receptor(GPCR)and genes in the c AMP-gluconeogenesis signaling pathway were significantly up-regulated(P < 0.05).The gene expressions of SNAT3 and GLUT1 were significantly up-regulated.In addition,this study found that the lncRNAs MSTRG.166985.16 and MSTRG.68020.9 could regulate the SNAT3 and the gluconeogenesis gene PC,respectively,indicating that they may play an important role in renal gluconeogenesis.The results showed that feed restriction increased the choline synthesis and triglyceride transport capacity in Hu sheep kidneys,but it did not lead to renal lipid deposition.Catecholamines can activate the c AMP-gluconeogenesis signaling pathway by up-regulating its receptor expression in the kidney,thereby enhancing the reabsorption of gluconeogenic substrates to maintain the body’s glucose homeostasis.5 The counteracting effects of glycerol or rumen protected choline chloride supplementation on abnormal metabolism induced by feed restriction in Hu sheepThis chapter aims to investigate the nutritional regulation effects of glycerol or rumen protected choline chloride supplementation on abnormal metabolism induced by feed restriction in Hu sheep.18 Hu sheep(weight 67.7±7.7 kg)with 100-day gestation period,similar parity(2-3)and 2-3 litters were selected in this study.The sheep were randomly divided into three groups: control group(CON)were fed to meet 30% of their nutritional requirements only,glycerol group(GLY)were fed to meet 30% of their nutritional requirements + 40 ml/d glycerol,while rumen protected choline chloride group(RPC)were fed to meet 30% of their nutritional requirements + 10 g/d of rumen protected choline chloride twice a day.The experimental period was 9 days.The body weight of the sheep was measured before morning feeding at the beginning and end of the test,and jugular vein blood was collected every 3 days before morning feeding.On the ninth day of the experiment,rumen fluid was collected orally 4 hours after morning feeding.The results showed that sheep in the GLY group had significantly less weight loss compared to the CON group(P < 0.05).With the extension of the time,the blood BHBA concentration in the CON group continued to increase,while remained stable in the GLY group and the RPC group.Compared with the CON group,glycerol significantly increased the blood glucose concentration of Hu sheep(P < 0.05),while RPC significantly increased the concentrations of NEFA,glucose,and very low density lipoprotein in the blood(P < 0.05).Glycerol significantly decreased ruminal p H(P < 0.05),acetic acid molar ratio(P < 0.01)and acetate to propionate ratio(P < 0.01),and significantly increased rumen propionic acid molar ratio(P < 0.01),butyric acid molar ratio(P < 0.05)and valeric acid molar ratio(P < 0.05).The metabolomics results showed that there were significant differences in the composition of blood metabolites among the three groups.Compared with CON group,there were 32 differential metabolites in GLY group,which were significantly enriched in aminoacyl-t RNA biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis,ketone body synthesis and degradation,phenylalanine metabolism and vitamin B6 metabolism.14 differential metabolites were identified between the CON and the RPC groups,and these differential metabolites were mainly enriched in the synthesis and degradation of ketone bodies(P = 0.025)and butyrate metabolism(P = 0.096).The results showed that glycerol and rumen protected choline chloride could ameliorate the abnormal glucose,lipid and amino acid metabolism induced by feed restriction.Most importantly,glycerol mainly alleviated the amino acid and lipid metabolism disorders of the body,while rumen protected choline chloride mainly improved lipid metabolism.