Study On Expression Of Genes Involved In Growth And Nutrition Metabolism，and Genetic Variation Of Insr Gene In Holstein Cattle

In present study, two parts were included:1The effects of level of nutrition on expression of genes involved in pathways of mTOR,IGF and insulin signaling in skeletal muscle of Holstein calfIn present study, we explored the molecular mechanism,by which level of nutritionaffected growth of skeletal muscle and nutrition metabolism of calf by measuring calfperformance, blood metabolites and the expression of15genes in volved inpathways ofmTOR, IGF and insulin signaling in semitendinosus tissue of Holstein calves via quantitativePCR. Twenty calves were assigned randomly to two diets based on block-design by bodyweight:(1) CON: a control diet, constituted by conventional milk replacer (20%CP,20%fat,4.90Mcal/kg ME;1.25%BW; DM basis) and conventional starter (20%CP,3.16Mcal/kg ME,DM basis);(2)HIPRO: a high-protein diet, constituted by high-protein milk replacer (28%CP,16%fat,4.84Mcal/kg ME;2%BW; DM basis) and high-protein starter (25%CP,3.20Mcal/kgME, DM basis). The final results were focused to compare the dietary differences duringpreweaning (0-5wk) and postweaning (5-10wk), respectively. The results were in thefollowings:1.1The effects of level of nutrition on calf performanceInteraction of diet and time did not significantly affect calf live BW, carcass weight,carcass composition (including protein, fat, and ash), total viscera weight, and liver weight.Except for fat weight, significant increases (P=0.01) were observed for final live BW, carcassweight, carcass composition (including protein and ash), total viscera weight and liver weightin calves fed high-protein diet vs. control diet. Greater live BW, carcass weight, carcasscomposition (including protein, fat and ash), and total viscera weight and liver weight wereobserved for calves at10wk vs.5wk.1.2The effects of level of nutrition on nutrition metabolism of calfInteraction of diet and time (P=0.01) significantly affect blood glucose concentration ofcalves (P=0.01). Rise in blood glucose concentration were observed as nutrition levelincreased for calves at5wk (P=0.04). But no alteration was found for blood glucoseconcentration as nutrition level increased for calves at10wk. In addition, decreasedconcentration of blood glucose was demonstrated for calves fed high-protein diet at10wk vs. 5wk (P=0.01).Blood urea concentration was significantly higher for calves fed high-proteindiet compared to that of calves fed control diet (P=0.01). Urea concentration (P=0.01) andBHBA (β-hydroxybutyric acid, P=0.01) concentration for calves at10wk were significantlyhigher than those for calves at5wk. Total protein concentrationfor calves at10wk weresignificantly lower than that for calves at5wk (P=0.01). Interaction of diet and time (P=0.01)had significant effects on blood NEFA (nonesterified fatty acid) concentration of calves(P=0.01).NEFA concentration increased markedly as nutrition level elevated (P=0.01). Calvesat10wk possessed lower NEFA concentration compared calves at5wk (P=0.01). There wasan overall increase between wk5and wk10for blood insulin concentration (P <0.01), andthere was an increased tendency of insulin by elevated nutrion level (P=0.09).1.3The effects of nutrion level on expression of genes involved in pathways of mTOR,IGF and insulin signaling in skeletal muscle of calfThe interaction of diet and time had significant effects on expression of IGF1R(P=0.04)in semitendinosus tissue of calf. For calves at5wk, expression of IGF1R decreased withelevated nutrition level. The interaction of diet and time also affected expression of TSC2(P=0.02). No change of expression of TSC2by nutrition level was observed for calves at5wk.But for calves at10wk, expression of TSC2was greater for calves fed high-protein dietcompared to that for calves fed control diet. Moreover, higher expression of TSC2for calvesat10wk than that for calves at5wk only occurred for calves fed high-protein diet. Nosignificant changes were found for expression of TSC1, which displayed opposite tendency tothe changes of expression of TSC2. Compared to the gene expression of calves fed controldiet, expression of RPS6KB1for calves fed high-protein diet was markedly downregulatedboth at5wk and10wk (P=0.03), that of IRS1was upregulated (P=0.02), that of INSR tendedto be downregulated (P=0.10), and that of PDPK1tended to be upregulated (P=0.10).Compared to the gene expression of calves at5wk, expression of FOXO1(P=0.04) and INSR(P=0.05) significantly downregulated, that of TSC2was significantly upregulated (P=0.01),and expression of RICTOR (P=0.07), RPTOR (P=0.10) and TSC1(P=0.06) tended to bedownregulated.2Analysis of genetic variation of bovine INSR gene in China Holstein dairy cattleTaken into account of the importance of nutritional physiology and genetics, INSR genewas selected as a candidate to find potential molecular markers, which controlled dairy cattleperformance by modulating feeding signals reception and transduction. A population of205China Holstein dairy cattle was investigated to study the genetic variation of bovine INSR viaDNA sequencing, DNA sequence analysis and PCR-RFLP techniques. We also conductedassociation analysis between the polymorphism of bovine INSR gene and milk performance (i.e., milk protein content, milk fat content, milk protein yield, milk fat yield and milk yield)of China Holstein dairy cattle, expecting to improve the future productionof dairy cattle in thecondition of intensive production and the application of MAS (marker-assist-selection). Theresults were in the followings:2.1Detection of SNPs within bovine INSR geneSequencing from two direction and SNP scanning were conducted for CDS (codingsequence) region (including22exons) in the bovine INSR gene using27pairs of primers.Thirteen mutations were revealed within bovine INSR gene via DNA sequencing, DNAsequence analysis and PCR-RFLP techniques, including7SNPs, which occurred in codingregion and resulted alterations of amino acids, and6SNPs, which occurred in non-codingregion. The mutations were observed at intron8, exon11, intron16, intron20and exon21.(1) Mutation nt12689G>A occurred at intron8, resulting Hinf I polymorphism. Threegenotypes were observed via Hinf I PCR-RFLP analysis: genotype GG(315bp+149bp+34bp+5bp), genotype GA (315bp+218bp+149bp+97bp+34bp+5bp) andgenotype AA (218bp+149bp+97bp+34bp+5bp).(2) Mutation nt26301G>A, nt26334G>C, nt26496A>G, nt26504C>T and nt26526A>Goccurred at exon11, resulting alterations of amino acids as followings: Arg509His、Arg520Pro、Gln574Arg、Arg577Trp、His584Arg. No linkage was observed among the5SNPs.(3) Mutations nt44018G>A, nt44024A>G and nt44067C>T, which were linked together,occurred at intron16, resulting Hha I polymorphism, and three genotypes were observed:genotype GG (526bp+345bp), genotype GA (871bp+526bp+345bp) and genotype AA(871bp).(4) Mutations nt121892A>G and nt121877A>G occurred at intron20; Mutationsnt122326T>C and nt122362T>C occurred at exon21，resulting alterations of amino acids asfollowings: Val1231Ala and Leu1243Pro. Linkage among mutations nt121877A>G,nt122326T>C and nt122362T>C possessed Hha I polymorphism: genotype GG (621bp),genotype GA (621bp+517bp+104bp) and genotype AA (517bp+104bp). Three genotypeswere observed via forced Pvu II PCR-RFLP for mutation nt121877A>G: genotype AA(577bp), genotype AG (577bp+542bp+35bp) and genotype GG (542bp+35bp).2.2Genetic parameters of population for bovine INSR gene(1) Three genotypes were observed for all loci of bovine INSR. Data of He(Heterozygosity) and PIC (Polymorphism Information Content) suggested that polymorphismof INSR gene was not rich in the investigated population. Consistent with values ofHe(0.4085-0.4971), values of PIC at locus GT2, GT4, GT5and GT9implied a higher genetic polymorphism.(2) All loci in bovine INSR gene were not in Hardy-Weinberg balance in presentinvestigated population (P<0.05).(3) In present population, values of D’between two loci within INSR gene were higherthan0, suggesting linkage disequilibrium between two loci (0<D’<1) with different degree.Values of D’between locus GT2and GT3, between locus GT4and GT5, GT6were1,suggesting completely linkage disequilibrium. In addition, except for the value of r2betweenlocus GT1and GT8was0，which suggested linkage equilibrium, values of r2between otherloci were0.001-0.353，suggesting linkage disequilibrium between two loci with differentdegree.2.3The effects of polymorphism of bovine INSR on milk performance in ChinaHolstein dairy cattleAssociation analysis between9loci of bovine INSR gene and milk performance (milkprotein content, milk fat content, milk protein yield, milk fat yield and milk yield) of205China Holstein dairy cattle revealed the followings:(1) There was no effect of polymorphisms of locus GT1、GT3、GT5、GT7and GT8onmilk performance, respectively (P>0.05).(2) Locus GT2might be important for milk fat content, milk protein yield and milk yield.Individuals with genotype AA had higher milk fat content than those with genotype GG(3.7%)and GA(3.9%), and no difference were observed for milk fat content between individuals withgenotype GG and GA. The interaction of lactation and GT2significantly influenced milkprotein yield (P=0.015),milk yield (P=0.008), with more milk protein yield and milk yield forindividuals with genotype GG than those with genotype AA at first lactation. The interactionof lactation and GT2tended to influenc milk fat yield (P=0.078).(3) Locus GT4might be important for milk fat content, milk fat yield, milk protein yieldand milk yield. Individuals with genotype GG had higher milk fat content than those withgenotype AG (3.7%). The interaction of season and GT4significantly influenced milk fatcontent (P=0.018), individuals with genotype AG had less milk fat content when deliveryoccurred from June to Augast than those from September to November and those fromDecember to Febrary. Interaction of lactation and GT2tended to influenc milk fat yield(P=0.078). Individuals with genotype AA had11.58%higher milk fat yield than those withgenotype GG at locus GT4. Genotype of locus GT4tended to affect milk protein yield(P=0.081) and milk yield (P=0.053).(4) Locus GT6might be important for milk fat content and milk protein content.Individuals with genotype GG had higher milk fat content than those with genotype AG (4.7%) at locus GT6. Individuals with genotype GG possessed higher milk protein contentthan those with genotype AG (0.6%).(5) Although there was no effect of polymorphism of locus GT9on milk fat content,milk fat yield, milk protein yield and milk yield (P>0.05), it showed tendency for milk proteincontent (P=0.085)，suggesting that locus GT9probably was important for milk proteincontent.