GC-MS分析慢性氨气应激对肉鸡血清代谢物的影响

【目的】随着养殖业集约化和规模化的发展,舍内环境对畜禽生长的影响日益突出。在密闭舍饲条件下,不断产生有毒有害气体,其中氨气的危害最大,降低畜禽生产性能,威胁动物健康。为此开展慢性氨气应激对肉鸡血清代谢物影响的研究,从小分子物质和代谢途径方面探究氨气导致机体代谢发生的变化,为舍内氨气的合理调控提供数据支持。【方法】试验采用单因素完全随机设计,将96只21日龄健康AA肉鸡随机分成对照组和试验组,每组设4个重复,每个重复12只鸡。对照组氨气浓度为0 mg·kg^-1,试验组氨气浓度为45 mg·kg^-1。试验在人工模拟呼吸舱内进行,呼吸舱采用全自动化控制温湿度等养殖条件,采用网上平养,自由采食和饮水,24h光照,试验期为21d。在肉鸡42日龄时,从每个重复中随机抽取2只,翅静脉采血,分离血清。将血清样品预处理后,采用气相色谱/质谱联用技术(gas chromatograph tandem mass spectrometer technology, GC-MS)检测代谢产物,利用质谱数据库对其进行鉴定。通过主成分分析、偏最小二乘法判别分析、正交偏最小二...

Effects of Chronic Ammonia Stress on Serum Metabolites of Broilers Based on GC-MS

【Objective】With the development of intensive and large-scale broiler breeding, the impact of environmental conditions on the growth of livestock and poultry has become increasingly prominent. Under high-density feeding conditions, a large amount of toxic and harmful gases is generated constantly, among which ammonia is the most harmful gas which reduces the performance of livestock and poultry and threatens animal health. The purpose of this experiment was to study the effects of chronic ammonia stress on serum metabolites in broiler chickens, and to explore the changes of metabolism caused by ammonia stress in terms of small molecules and metabolic pathways, so as to provide data support for reasonable regulation of ammonia in the poultry house. 【Method】Ninety-six 21-day-old healthy AA broilers were randomly divided into control group and experimental group by a single factor completely random design, with 4 replicates in each group and 12 chickens per replicate. The control group ammonia concentration was 0 mg·kg ^-1, the test group ammonia concentration was 45 mg·kg ^-1. The experiment was carried out in an artificial simulated respiratory chamber, which was fully automated to control the temperature and humidity and the other environmental conditions. Broilers were raised in the nets and the feed and water were provided ad libitum during the entire experimental period. The trial lasted 21 days with 24 h lighting program. They were routinely managed according to the AA broiler feeding manual and immunized according to the routine procedures. Two broiler chickens pre replicate from each treatment were chosen randomly and blood samples were collected from the wing vein at 42 day-old. After pretreatment of serum samples, the metabolites were detected by GC-MS technology, and identified by mass spectral database. Principal component analysis (PCA), partial least-squares discriminant analysis (PLSDA), orthogonal partial least squares discriminant analysis (OPLSDA) and t-test were used to find the different metabolites, and then the bioinformatics methods was used to analyze the differential metabolic pathways. 【Result】Results showed that: (1) A total of 204 metabolites were detected in serum of broiler chickens by GC-MS combining with rapid identification of the detected metabolites performed with a mass spectrometry database. Metabolomics data were imported into SIMCA-P software for multivariate statistical analysis, and then t-test. Twenty-three different metabolites were identified, of which 19 were up-regulated and 4 were down-regulated. (2) Differential metabolites mainly involved energy metabolism (lactic acid, α-ketoglutaric acid), amino acid metabolism (L-Allothreonine acid, L-Homoserine, Nicotinylglycine), fatty acid metabolism (stearic acid, linolenic acid, linoleic acid, cholesterol) and nucleotide metabolism (hypoxanthine, uracil, thymine) and so on. (3) Metabolic pathway enrichment analysis showed that ammonia stress mainly affected the fat metabolism pathway in serum of broiler chickens, including linoleic acid metabolism, arachidonic acid metabolism and α-linolenic acid metabolism. 【Conclusion】 It can be seen that GC-MS can detect serum metabolites comprehensively, and screen differential metabolites accurately. Chronic ammonia stress significantly affected serum metabolites content of broilers, mainly affected fat metabolism pathway.