| Ammonia（NH3）,the most aerial pollutant in animal facilities,seriously affects animal growth and health.Skeletal muscle is a very critical tissue for the animal body to utilize lipid and glucose.The disorder of lipid metabolism in skeletal muscle is not only related to the maintenance of skeletal muscle function,but also directly related to the occurrence and development of metabolic diseases.Recent studies have demonstrated that aerial ammonia exposure affected breast muscle characterization and altered body fat distribution in broiler chickens.At present,the effects of atmospheric ammonia inside the pig houses on lipid metabolism of animal skeletal muscle and the specific mechanism of action are still unclear.In this dissertation,the following results were obtained by studying the effects of different concentrations of NH3 on lipid metabolism in skeletal muscle of growing pigs and its mechanism.1.Atmospheric ammonia affects myofiber development and lipid metabolism in growing pig muscle.In order to elucidate the effects of aerial ammonia exposure on the physiological characteristics and lipid metabolism of skeletal muscle in growing pigs,a single factor completely randomized design was used in this trial,where 18 binary growing pigs were exposed to 0,10 or 25 mg/m3 ammonia（6repetitions per group,1 pig per repetition）for 25 days,respectively.The results showed that ammonia exposure increased fat content in the longissimus dorsi muscle（P=0.007）,and meat color got lighter（P=0.001）after 25 mg/m3 ammonia exposure.Analysis of My HC isoforms showed an increased My HC IIx（P=0.057）but decreased My HC I（P=0.029）after ammonia exposure.Although hyperammonemia was reported to upregulate myostatin（MSTN）and inhibit downstream mTOR pathway,no changes have been found in the m RNA expression level of MSTN and protein expression level of the mTOR signal pathway after ammonia exposure.RNA-Seq showed that 10 mg/m3 ammonia exposure altered genes related to myofiber development,whereas 25 mg/m3 ammonia affected genes associated with fatty acid synthesis andβ-oxidation.Our findings in this chapter showed aerial ammonia exposure appears to regulate myofiber development and lipid metabolism in the skeletal muscle.2.High aerial ammonia exposure regulates lipid metabolism in the pig skeletal muscle via mTOR pathway.This chapter aimed to further explore the potential pathways of lipid metabolism disturbance in skeletal muscle using a high ammonia exposure model.The single factor completely randomized design was used in this trial,where 12 growing pigs were exposed to 0 or 35 mg/m3 ammonia（6 repetitions per group,1 pig per repetition）for 25 days.The data showed that ammonia exposure also caused an increased long-chain fatty acids concentrations and fat content in the longissimus dorsi muscle,and also elevated serum total triglyceride and Apo B contents（P<0.05）.The pigs exposed to 35 mg/m3ammonia had high serum free amino acids including branched-chain amino acids（BCAAs）（P<0.05）.RNA-Seq analysis showed that genes encoding enzymes involved in lipid synthesis and uptake were up-regulated,whereas genes related to lipolysis,transport andβ-oxidation were down-regulated.Furthermore,the activation of mTOR signal pathway（P<0.05）and inhibition of AMPK signal（P<0.01）were observed in the pig exposure to 35 mg/m3 atmospheric ammonia.These findings in this chapter showed that high concentration of atmospheric ammonia exposure greatly interferes with amino acid metabolism,resulting in increased BCAAs and aromatic amino acids.The increased BCAAs production can up-regulate lipid synthesis and down-regulateβ-oxidation by activating mTOR signaling and inhibiting AMPK signaling.3.Aerial ammonia exposure induces the perturbation of the interorgan ammonia disposal and branched-chain amino acid catabolism in growing pigs.Based on the pig model exposed to different concentrations of aerial ammonia（10,25 or 35 mg/m3）in the previous two chapters,the study in this chapter further explored how different organs were coordinated to defend against aerial ammonia exposure and the effect of aerial ammonia on BCAA metabolism.The data demonstrated that after above25 mg/m3 ammonia exposure,decreased aspartate（P=0.016）and glutamate（P=0.030）,increased ornithine（P=0.002）were found in the ammonia-removing liver;and after high ammonia（35 mg/m3）exposure,glutamine synthetase expression was increased（P=0.012）.Increased glutamate（P=0.004）and decreased glutaminase expression（P=0.083）were observed in the lungs after high ammonia exposure.There was also an increasing trend of free glutamine in the kidneys after high ammonia exposure（P=0.066）.For BCAA catabolism,high ammonia exposure increased BCAA content in both the lungs and muscle,whereas below 25 mg/m3 ammonia exposure increased BCAA only in the lungs（P<0.05）.The expression of BCAA transaminase and dehydrogenase complex were inhibited to a varying degree in the liver,lungs and muscle after above 25 mg/m3 ammonia exposure,especially high ammonia exposure.These results indicated that with aerial ammonia increasing,both increased urea cycle and glutamine synthesis were ammonia defensive strategies,and high ammonia exposure impaired the BCAA catabolism.4.Exposure to high aerial ammonia causes hindgut dysbiotic microbiota and alterations of microbiota-derived metabolites in growing pigs.Recent researches have shown that gut microbes play a vital role in skeletal muscle function and metabolism.Hence,this chapter aimed to investigate how aerial ammonia exposure affected the hindgut microbiota and its metabolites based on a high ammonia（35 mg/m3）exposure model.The results demonstrated that an upward trend in hyocholic acid（HCA）,lithocholic acid（LCA）,hyodeoxycholic acid（HDCA）（P<0.10）;a downward trend in tauro-deoxycholic acid（P=0.075）;and a reduced tauro-HDCA level（P=0.005）were found in the serum bile acid（BA）profiles after ammonia exposure.Ammonia exposure notably raised microbial alpha-diversity in colonic and cecal chyme and clearly exhibited a distinct microbial cluster in hindgut indicated by principal coordinate analysis（P<0.01）,indicating that ammonia exposure induced alterations of microbial community structure and composition in the hindgut.Further analysis displayed that ammonia exposure increased the number of potentially harmful bacteria,and decreased the number of beneficial bacteria（FDR<0.05）.Analysis of microbiota-derived metabolites in the hindgut showed that ammonia exposure increased acetate and decreased isobutyrate or isovalerate in the cecum or colon（P<0.05）,respectively.Unlike the alteration of serum BA profiles,cecal BA data showed that high ammonia exposure had a downward trend in cholic acid,HCA,and LCA（P<0.10）;a downward trend in deoxycholic acid and HDCA（P<0.05）;and an upward trend in glycol-chenodeoxycholic acid（P=0.013）.The findings in this chapter illustrated that high ammonia exposure induced the dysbiotic microbiota in the hindgut,thereby affecting the production of microbiota-derived short-chain fatty acids and BAs.In conclusion,the results of this research found that environmental ammonia exposure interfered with lipid metabolism in skeletal muscle of growing pigs.The alterations of ammonia defense strategy of animal body and the imbalance of gut microbiota play an important role in mediating the activation of downstream signaling pathways induced by aerial ammonia to change the expression of lipid metabolism-related genes in growing pig muscle. |
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