| Wooden breast is a histopathology that occurs in the pectoralis major muscle of broilers with high incidence.Wooden breast reduces the meat quality and consumer desire to purchase significantly,causing huge economic losses to the poultry industry.Wooden breast is a challenge for the broiler industry,and the mechanisms underlying those defects are still poorly understood.These limit the development of diagnosis,prevention,mitigation and regulation for wooden breast myopathy.In this experiment,AA broiler chickens were raised,and single-cell transcriptome sequencing was performed on severe wooden breast and normal chicken breast meat.Combined with the metabolome of breast muscle,the potential mechanism of gut microbiota that affecting wooden breast was revealed.Subsequently,the effects of nutritional modulation of ursolic acid on the incidence of wooden breast were evaluated.Part one,Pathogenetic implications of wooden breast.Three hundred 1-d-old broilers(AA broilers)were selected and fed.On 49 d,all birds were slaughtered and sampled.Based on necropsy and histomorphological analysis,those broilers were classified into normal(NORM),mild wooden breast(MILD)and severe wooden breast(SEV).For NORM group,the whole breast fillets were soft;for MILD group,the hard area was less than 50 %;for SEV group,more than 50 % of breasts fillets were hard.In Experiment 1,phenotype index in wooden breast fillets.Twenty replicates each in the NORM,MILD and SEV groups were selected to compare the differences in meat quality,histomorphological differences,muscle fiber traits,inflammatory cytokine m RNA expression levels,antioxidant indexes,satellite cell repair capacity and fibrosis of the three chicken breasts.The results showed that broilers with wooden breast accounted for 27.02 %,including 19.76 % for mild wooden breast and 7.26 % for severe wooden breast.The L*value,a*,p H,and drip loss of severe WB were higher than normal breasts significantly(P <0.05).Myofiber cytoplasmic vacuolization,atrophy and degeneration,infiltration of connective tissue and inflammatory cells in the intima and interstitial membranes were observed in severe wooden breast.The average myofiber area and myofiber diameter decreased significantly in mild wooden breast and severe wooden breast(P < 0.05).MDA level increased significantly and antioxidant enzyme activities were also up-regulated significantly in severe wooden breast(P < 0.05).It was normal for the cellular m RNA expression positions of PAX7 and MYOD1 in severe wooden breast.In Experiment 2,single-cell sequencing combined with metabolome analysis revealed alternations in cell types and transcriptional metabolic profiles of wooden breast.Three fillets each from NORM and SEV groups were selected.Differences in cell types and in cell transcriptional profiles between the two groups were analyzed by single-cell sequencing.The metabolic profiles were also analyzed to reveal the effects of cells on the composition of breast fillets metabolites.The results showed that 10 cell types presented in breast fillets,including myoblasts,satellite cells,fibroblasts,erythroid-like cells,endothelial cells,T cells,macrophages,B cells and neutrophils.The amount of PTPRC+ immune cells was significantly higher in WB fillets than that in normal fillets.Severe wooden breast had a macrophage that neither similar to M1-nor M2-type macrophages.Myocytes in wooden breast highly expressed ADIPOQ and had oxidative stress.Collagen genes expressed in satellite cells was up-regulated in wooden breast and the differentiation of them was heterogeneous.Cellular communication relationships differed in wooden breast.The communication intensity between the macrophages and other cell types weakened.Metabolomic results showed that muscle metabolites exhibited a stepwise progression with the severity of wooden breast.Cells were predominantly involved in the pathyway of lysine degradation,protein ubiquitination modifications and muscle fiber contraction.In Experiment 3,cecal digesta metabolome and plasma metabolome mediated by gut microbiota in wooden breast broilers were analyzed.Twenty samples of each group were selected to perform 16 S r RNA sequencing and cecum chyme and plasma metabolomic analysis.The results showed that gut microbiota and diversity in birds with severe wooden breast differed from normal birds.At phylum level,Firmicutes/Bacteroidetes ratio in birds with severe wooden breast decreased compared to normal birds(P = 0.098),but an increased abundance of Verrucomicrobia(P < 0.05).At species level,the abundance of potential pathogenic bacteria such as Clostridium spiroforme increased significantly(P < 0.05).While the abundance of probiotic such as Lactobacillus agilis and Streptococcus alactolyticus decreased(P < 0.05).Gut microbiota related metabolites were mainly involved in pathways of valine,leucine and isoleucine biosynthesis and arginine biosynthesis.A total of 161 metabolites were regulated,and these metabolites were positively correlated with glutamine and glutamate metabolism,and arginine biosynthesis metabolic pathways and significantly negatively correlated with tyrosine metabolic pathways(P < 0.05).Five metabolites in plasma were positive with severity of wooden breast including Anserine,1-methylhistidine,2-oxyarginine,γ-glutamyl-β-aminopropionitrile,and choline(P < 0.05).Four plasma metabolites could be identified to microbial-related metabolites,including guanylacetic acid,3,4,5-trimethoxyphenol,2-hexadecenoylcarnitine and 3-methyladenine,correlated with meat quality and muscle fiber trait indices.Part two,the relief effect of wooden breast by ursolic acid.In Experiment 1,the selection of optimal supplementary of ursolic acid in broilers.A total of 360 1-day-old AA broilers were divided into 5 groups with 6 replicates each randomly,with diets containing 0,150,300,450 and 600 mg/kg of ursolic acid(UA),respectively.The results showed that supplement with different concentrations of UA to the diets increased the average daily feed intake and average daily gain,in which the best concentrations were 450 mg/kg and 600 mg/kg.But UA have little effect on broiler breast muscle rate.Supplement with more than 150 mg/kg UA reduced the leg muscle rate of broilers but increased the leg muscle yield(P < 0.05).450 mg/kg of UA improved the tenderness and water retention capacity of breast meat(P < 0.05).UA improved the antioxidant capacity of breast muscle.The best concentrations were 300 mg/kg and 450mg/kg.In conclusion,supplementary with 450 mg/kg UA promoted growth,altered meat quality and antioxidant index of breast muscle,which can be used in further research.In Experiment 2,the relief effect of wooden breast by ursolic acid.Two hundred 1-d broilers were selected and divided into control and UA groups,fed the basal diet or diet containing 450 mg/kg UA,respectively.The results showed that 450 mg/kg UA reduced the incidence of wooden breast(P = 0.068),increased the average diameter and area of myofibers(P < 0.05),and reduced serum AST and ALP levels.7In conclusion,various cell types in wooden breast fillets have different transcriptional profile compared to normal breast fillets.Macrophages in wooden breast had engulfed myofiber fragments in wooden breast tissues.Myocytes in wooden breast had oxidative stress.Satellite cells in wooden breast had normal MYOD1 and PAX7 m RNA expression positions,but differentiation trajectories of satellite cells were heterogeneous,and their collagen family gene expression was up-regulated.Communication between cells are weakened in wooden breast,with intercellular communication mainly involved in the lectin receptor-ligand family.Metabolites in wooden breast were different with normal breast fillets,in pathway of lysine degradation,protein ubiquitination modifications,and muscle fiber contraction.The gut microbiota were also altered in severe wooden breast broilers.Differential bacteria in birds with wooden breast were mainly involved in plasma glutamine metabolism and arginine metabolism,and microbioal-related plasma metabolites were associated with meat quality and muscle fiber traits.Supplementary with 450 mg/kg ursolic acid reduced the incidence of wooden breast and contribute to myofiber growth. |
PDF Full Text Request