| The experiment studied on effects of starter feeds of two different physical forms(pelleted and textured)on growth performances,rumen morphology and functions,rumen microflora,transcriptome and metabolome taking Hu lambs as the research objects,and carried out the correlation analysis among their results,which provided a systematical and comprehensive theoretical basis for regulation of rumen development of ruminant and research and development of starter feeds in production of mutton sheep.The experiment was adopted to one-way ANOVA design and divided into two dietary treatments: 1)a pelleted starter feed(PS,with a mean particle size of six mm diameter),and 2)a textured starter feed(TS,which included coarse mashed steam-flaked corn,also with a mean particle size of six mm diameter).Twenty-four eight-day-old male Hu lambs(5.04 ± 0.75 kg body weights)were chosen as experimental animals,and randomly assigned to one of two dietary treatments,12 lambs every treatment.From zero to seven days of age,the lambs were fed milk along with their mothers.At eight days of age,the lambs were separated from their mothers,reared in individual cages and began to be fed milk replacer(MR)at 2% of body weight at day eight and starter feed with ad lib.The MR was stopped to be fed at thirty-five days of age.The experiments ended at forty-two days of age,and the clean fresh water was being readily available during the whole experiments.The empty body weights of all lambs were weighted at every weekend,and the amounts of starter feed offered and refused were recorded daily for each lamb.Finally,the average daily gain(ADG),dry matter intake of starter feed(DMI)and feed efficiency(FE)were calculated.Six lambs for each treatment were euthanized at twenty-one(pre-weaning)or forty-two(post-weaning)days of ages to determine the relative weight of ruminant stomach,morphology,fermentation parameters and enzyme activities of rumen.Two samples of contents of rumen were collected.One samples was adopted to 16 S r DNA and analyzed using next-generation sequencing Mi Seq,and the function of microbial was predicted;and the other samples were analyzed the metabolites of rumen,enrichment of metabolites and pathways of metabolism of different metabolites based on GC-MS metabolome of rumen.The ventral region of each animal’s rumen was collected,and analyzed the different expression of genes using RNA-Seq between two treatments.Finally the functions of different expression of genes were annotated by GO(Gene Ontology),and the pathways of enrichment were analyzed by KEGG(Kyoto Encyclopedia Genes and Genomes).Based on data of the genomes,transcriptomes and metabolomes,we carried out to correlation analysis between metabolomes and genomes,metabolomes and transcriptomes respectively.The results were following:1.The TS lambs had a greater final body weight(P = 0.039)and higher DMI(P= 0.023)for post-weaning lambs,and which increased respectively by 25.19%,13.18%compared them of PS lambs.And post-weaning ADG of TS had trends to higher than that of PS(P = 0.072),and increased by 40.45%.The results showed compared to PS,TS were more beneficial to increase the growth performances for pre-weaning and post-weaning lambs early.2.Compared to PS,TS increased significantly the papillae length,circular muscle thickness,muscle layer thickness,sectional area,total VFA(volatile fatty acids),propionate and butyrate contents of rumen for pre-weaned and post-weaned lambs(P < 0.05),and papillae width and relative weight of rumen for post-weaned lambs(P < 0.05).At same time,TS had trends to increase the relative weight of rumen for pre-weaned lambs compared to PS(P = 0.072).The other fermentation parameters and enzymatic activities of rumen for pre-weaning or post-weaning lambs between two groups had no differences(P > 0.05).TS were more beneficial to increasing of the relative weight,development of morphology and fermentation of rumen than them of PS for pre-weaning and post-weaning lambs early.3.Similar analysis(ANOSIM)showed compared to PS,microflora of rumen of TS for pre-weaning lambs were more similar to them of TS for post-weaning lambs.The diversities of microflora of rumen showed the relative abundance of Bacteroidetes of TS has increased to 61.96%,and the relative abundance of Firmicutes and Proteobacteria of TS has decreased to 32.08% and 3.99% for pre-weaning lambs respectively.And the Firmicutes and Proteobacteria of TS for pre-weaning lambs were replaced by Bacteroidetes,which became the first dominant phylum,and achieved the similar levels to them for post-weaning lambs(65.36%).After post-weaning,the relative abundance of Bacteroidetes of TS increased further,and achieved 65.36%;the relative abundance of Firmicutes of TS decreased further,and achieved 24.03%;the relative abundance of Proteobacteria of TS increased again from 3.99% to 7.17%;but the Bacteroidetes was still the first dominant phylum.The changes of relative abundance of Bacteroidetes and Firmicutes of PS were consistent with them of TS,but the relative abundance of Proteobacteria of PS was decreased continuously for post-weaning lambs,and achieved 1.15%;the relative abundance of Bacteroidetes(57.28%)of PS for post-weaning lambs was still lower than them of TS for pre-weaning(61.96%)and post-weaning(65.36%)lambs.At levels of genus,the relative abundance of Sharpea of TS was higher significantly than that of PS for pre-weaning lambs,and it was continuous higher significantly than that of PS for post-weaning lambs(P < 0.05).Compared to pre-weaning lambs,13 genus of PS changed significantly,and only one genus of TS changed significantly for post-weaning lambs(P < 0.05).As far as functional prediction of rumen microbe,only the “Transporters” and “Fatty acid degradation” of PS were increased significantly and “Amino acid related enzymes” was decreased significantly for pre-weaning lambs compared to those of TS in KEGG level 3 pathways(P < 0.05).The results showed the physical forms of starter feeds affected microflora of rumen.TS was better adaptabilities compared to PS,and could induce rumen to establish the dominant Bacteroidetes and Sharpea for lambs in pre-weaning,which were beneficial to growth of lambs and development of rumen.However,functional predictions of rumen microbe of two groups for pre-weaning and post-weaning lambs were similar.4.The sequencing of transcriptome of rumen showed,compared to PS,5 genes of TS up-regulated significantly,and 10 genes of TS down-regulated significantly for pre-weaning lambs;284 genes of TS up-regulated significantly,and 462 genes of TS down-regulated significantly for post-weaning lambs(|log2(Fold Change)| > 1 and FDR < 0.05);TS up-regulated significantly the genes related to VFA(for pre-weaning lambs,BDH2;for post-weaning lambs,BDH1、HMGCL、HMGCS2、PPARD、ACSS2)in fatty acid metabolisms、protein and protein kinase of cell cycles(for pre-weaning lambs,CDK1;for post-weaning lambs,CDK18、CDK9、CDK2AP2、CCNK、CCND3、SART3)、growth(for post-weaning lambs,IGFBP5 and IGFBP4)in cell proliferation(|log2(Fold Change)| > 1 and FDR < 0.05),and down-regulated significantly the genes related to apoptosis of rumen epithelium in cell apoptosis(for pre-weaning lambs,CARD14;for post-weaning lambs,CARD14、CASP8AP2、CASP8)(|log2(Fold Change)| > 1 and FDR < 0.05).The genes of different expression between two treatments for post-weaning lambs were more than them for pre-weaning lambs obviously.Seven genes were chosen randomly and verified by PCR,and the results were consistent with the sequencing of transcriptome.The above results showed the TS prompted the growth and development of rumen for pre-weaning and post-weaning lambs,and the effects for post-weaning lambs were more beneficial than them for pre-weaning lambs from a transcriptome point of view.5.Compared to PS,the relative contents of 398 metabolites of TS increased significantly,and the relative contents of 97 metabolites of TS reduced significantly for pre-weaning lambs(VIP > 1.0,|log2(Fold Change)| > 1 and P < 0.05),which key metabolic pathways were metabolism ofα-linolenic acid,arginine,proline,alanine,aspartic acid,Glutamic acid,histidine(P<0.05),synthesis of arginine(P = 0.053),metabolism of 2-oxycarboxylic acid(P = 0.098).The relative contents of 60 metabolites of TS increased significantly,and the relative contents of 307 metabolites of TS reduced significantly for post-weaning lambs(VIP > 1.0,|log2(Fold Change)| > 1 and P < 0.05),which key metabolic pathways were metabolism of arachidonic acid(P = 0.067).TS increased significantly the contents of amino acids(for pre-weaning lambs,L-aspartic acid,L-Glutamic acid and Lhistidine;for post-weaning lambs,L-histidine),arachidonic acid and glyceraldehyde 3-phosphate(for post-weaning lambs)significantly(P < 0.05),and decreased the contents of sucrose、citric acid and D-Gluconic acid(for pre-weaning lambs),L-Phenylalanine(for post-weaning lambs)significantly(P < 0.05).The results showed TS changed the composition of metabolites of rumen,which were related to promote the development of rumen,metabolism of amino acids and fatty acids for lambs.6.TS increased the relative abundance of Blautia for pre-weaning lambs,and the relative abundance of Bacteroides,Porphyromonas and Intestinimonas for post-weaning lambs compared to PS,which promoted the metabolism of fatty acids(for pre-weaning lambs,methyl jasmonate and glyceryl 1-5(hydroxydodecanoate);for post-weaning,Ip A),improved the supplying of energy(for post-weaning lambs,decreasing of contents of pregnanediol-3-glucuronide and cinnamic acid),strengthened the amino acids synthesis(for pre-weaning lambs,anthranoyllycoctonine and N-(tert-Butoxycarbonyl)-L-leucine).These results were beneficial to growth and development of rumen for pre-weaning and post-weaning lambs.Additionally,the decreasing of the relative abundance of Lachnospira and Howardella strengthened the utilization of Bacteroides,Porphyromonas and Intestinimonas to starch and sugar further,which promoted the production of propionate and butyrate,and growth and development of rumen epithelium.Compared to PS,TS up-regulated the expression of CXCL14 and DHCR24,down-regulated the expression of COX1、COX2 and COX3,which were related to metabolism of fatty acids,and increased the contents of essential fatty acids(arachidonic acid);up-regulated the expression of ACAT1、BDH2 and HMGCL,which were related to metabolism of ketone bodies,and decreased the contents of citric acid,pregnanediol-3-glucuronide,cinnamic acid,improved the supplying of energy,strengthened the synthesis of amino acids(anthranoyllycoctonine,N-Glycosyl-L-asparagine,L-Histidine,L-Aspartic acid and Indoleacetyl Glutamic Acid).Additionally,TS up-regulated the expression of TGM2、CDK1、CSRP2,which prompted the proliferation of rumen epithelium,inhibited the apoptosis of rumen epithelium.Hence,TS were better beneficial to growth and development of rumen for pre-weaning and post-weaning lambs.All in all,the physical forms of starter feeds affected significantly the growth,phenotype of rumen development,microflora of rumen,transcriptomes and metabolomes for pre-weaning and post-weaning lambs.Compared to PS,TS increased the relative abundance of Bacteroidetes at levels of phylum and the relative abundance of sharpea at levels of genus;up-regulated the expression of genes related to VFA,protein and protein kinase of cell cycles,growth,and down-regulated the expression of genes related to apoptosis of rumen epithelium;improved the contents of many amino acids,arachidonic acid and glyceraldehyde 3-phosphate,reduced the contents of sucrose,citric acid,D-Gluconic acid and L-Phenylalanine.These results prompted the increasing of relative weight of rumen,development of rumen morphology and fermentation of rumen.Hence TS were more beneficial to growth and development of rumen,and the effects for post-weaning lambs were better than them for pre-weaning lambs. |
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