| Wuzhumuqin sheep,labeled as a "GREEN FOODS",which possess a variety of essential amino acids and minerals,is nearly without any fleshy or greasy smell in the fresh mutton.Wuzhumuqin sheep's body is solid and compact with a wide back and waist,also can endure extreme weather,including heat,cold,draught etc.Meanwhile,the sheep shares high levels in the lambs survival rate,meat yield and meat quality.Owing to the existence of multiple spines in the individuals,Wuzhumuqin sheep is known as the"NATIONAL PROTECTION RESOURCES of LIVESTOCK and POULTRY BREEDING in CHINA".Their living environment is sparsely populated within vast territory,and the sheep is used to "natural grazing" conducting extensive management.Nowadays the ecological environment(involving the grassland,soil,plant etc.)deteriorated,and this feeding manner might have a subtle impact on grassland ecosystems as well as animal development.In this paper,muscle development in the individual growth acted as the base point,starting from the myogenic regulatory factor(MRFs)gene expression and muscle fiber morphological changes to determine the effect of natural grazing conditions on muscle development.Subsequently,MRFs family full-length sequence obtained from Wuzhumuqin sheep and was accompanied by relationship between the gene polymorphisms and individual growth traits to offer the original data in individual germplasm resources of the multi-spine Wuzhumuqin sheep.Therefore,the signification of this study was to present reliable basic background for the muscular mechanism and the genetic breeding of Wuzhumuqin sheep through all above explorations.The specific results are as follows:1.Three major genes of MRFs family were cloned and sequenced by conventional PCR technology,and then performed homologous alignments with sequences in GenBank termed as Myf5(KT899092),Myf6(KT899093)and Myogenin.The full-length gene sequences of the MRFs family were composed of three exons and two introns sharing high sequence homology,with different sizes in the fragments.The corresponding predictive proteins also had higher conserved regions,referred to as the basic helix-loop-helix(bHLH)domain.Comparing the Myf5 senquence of the Wuzhumuqin sheep with the corresponding sequences of sheep in NCBI,no point mutation site was found in the full length of the Myf5 gene.The Myf6 full length sequence had two mutated sites located at g.1150 C>A and g.1197 C>T,belonging to silent mutation sites.Ten silent mutations were found in the full-length Myogenin gene,in which two of them were unique for the Wuzhumuqin sheep,but the remaining eight were as same as the corresponding sequences of goat in NCBI.2.In this section,the Wuzhumuqin sheep(adult ewes,n=100)was used as study animal with different growth traits.The polymorphisms of Myf5,Myf6 and Myogenin genes were analyzed by direct sequencing during the whole genetic sequences.The results suggested that no SNPs was found in the Myf5 gene in the tested samples,but a polymorphic locus in the Myf6 gene located at g.242 AC>CC>CT.There were significant differences in body weight and body height between different genotypes in Myf6 gene.Simultaneously,there were four polymorphic loci in Myogenin gene,located in the intron two region g.212 C>A,g.401 G>A,g.536 C>G,g.556 C>T,and no significant differences was found between the different genotypes of the same locus in myogenin gene within the growth traits.These results further indicated that the Myf6 gene might be the candidate genes or molecular markers for the growth index in Wuzhumuqin sheep.3.The different expression patterns of MRFs family(Myf5,MRP4,myogenin)were obtained by fluorescence quantitative PCR(Real time PCR)in the Wuzhurmuqin sheep during postnatal growth.The results showed that the three genes of the MRFs family had relatively stable gene expression levels after birth,and there was no significant decrease or increase between age stages(P>0.05).It is hypothesized that the muscle development under natural feeding pattern has no adverse stimuli on the mRNA expression of MRFs family in the tested animals.In other words,different patterns of exercise,exercise intensity,seasonal climate change(forage and temperature)and normal growth processes may have no significant stress or site specificity on MRFs gene expression during muscle development.4.Muscle fiber-type composition and fiber diameter were measured using histochemistry and morphological analysis in autonomous feeding Wuzhumuqin sheep during postnatal life.The results showed that fiber diameters were increased gradually in two major muscles(dorsal longissimus dorsi and biceps femoris)until 9 months,at which the value reached the highest levels simultaneously in the tested animals.Afterwards,the diameter was slightly reduced at 12 months in both muscles,and then it remained relatively stable at 18 months,without further decrease or increase.However,muscle fiber diameter was progressively increased until 18 months in the triceps muscle.Thus,muscle fiber diameters might have anatomical site specificity during muscle development.In the LD muscle,changes in the proportion of each of fiber types(?,?a,and ?b)were relatively small.A higher proportion of type I and a 6.19-fold lower proportion of type ?a fibers were observed(P<0.05)in the BF muscle.In addition,the compositions of type ? and ? a fibers continuously changed in the TB muscle(P<0.05).In summary,the increase in the fiber diameters and the percentage change in muscle fiber type may have a specific relationship in Wuzhumuqin sheep during their aging process under natural grazing.5.A mounts of significant correlations were observed between MRFs gene expression,muscle fiber composition,and fiber diameter in three major muscles,as follows:In the longissimus dorsi muscle,the expression of Myf5 gene was significantly correlated with muscle fiber type ?(r=-0.87,P<0.05),fiber rate ?/?Ib(r=-0.86,P<0.05),and accompanied by positive correlations with muscle fiber ?a(r=0.81,P<0.05),fiber rate ?a/?b(r=0.81,P<0.05)and fiber rate ?a/?(r=0.89,P<0.05).At the same time,fiber diameter was highly correlated with type ? muscle fiber(r=0.70,P>0.05),muscle fiber rate ?/?b(r=0.68,P>0.05),and Myf5 gene expression(r=-0.64,P>0.05).In biceps femoris,MyoG gene expression was intensely correlated with fiber rate ?/?b(r=0.81,P<0.05)and type ?b muscle fibers(r=-0.80,P>0.05).At the same muscle,fiber diameter and MyoG gene expression had a high negative correlation(r=-0.79,P>0.05).In the triceps muscle,fiber diameter shares amounts of significant correlations with muscle fiber ?(r=-0.93,P<0.05),fiber rate ?/?b(r=-0.88,P<0.05),muscle fiber ?a(r=0.90,P<0.01),and fiber rate ?a/?(r=0.92,P<0.01).In the meanwhile,fiber diameter remain correlated with fiber rate ?a/?b(r=0.72,P<0.05)and myogenin gene expression(r=-0.78,P>0.05).These results further indicated that muscle development must be closely linked with all three characteristics of muscle physiology during aging process with site specificity in anatomy.In summary,the diameter of muscle fibers reach the highest value at the age of 9 months in the natural grazing Wuzhumuqin sheep during postnatal life,and is relatively stable in further stages involving 12 months and 18 months.There was a hypothesis that step-grade transformations might be existed in changes of fiber-types proportion,as?>?a>?b and/or ?b>?a>? patterns.The activation/inhibition of this model may be affected by animal ages(little lamb,youth lamb,adult sheep),natural environment(temperature,season,climate,pasture etc.),and feeding behaviors(animal feeding habits,seasonal,feeding environment,regional vegetation structure etc.)and these potential factors might have a mixed effects on muscle development.There were very few researches regarding full-length sequences in MRFs family were reported in ruminant animal in GenBank database.This study would make up this vacancy and accompanied by the SNPs with growth traits,suggesting the Myf6 gene as an effective candidate gene in muscle development.At the same time,mRNA levels of the MRFs family did not change significantly after birth in the tested animals. |
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