Effects of Chronic Ammonia Stress on Serum Metabolites of Broilers Based on GC-MS
SUN YongBo, WANG Ya, SA RENNA,, ZHANG HongFuState Key Laboratory of Animal Nutrition, Institute of Animal Science/Chinese Academy of Agricultural Science, Beijing 100193
Received:2019-04-29Accepted:2019-07-3Online:2020-04-16 作者简介 About authors 孙永波,E-mail: ybsun2014@163.com。
摘要 【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、正交偏最小二乘法判别分析、t-检验等,寻找差异代谢物,并利用生物信息学方法对差异代谢物进行通路富集分析。【结果】(1)利用 GC-MS 方法,结合质谱数据库对检测到的代谢物进行快速鉴定,在肉鸡血清中共检测到204种代谢物。将代谢组数据导入SIMCA-P软件进行多元统计分析,结合t-检验筛选出 23种差异代谢物,其中上调19个,下调4个。(2)差异代谢物主要涉及能量代谢(乳酸、α-酮戊二酸)、氨基酸代谢(L-别苏氨酸、L-高丝氨酸、烟酰甘氨酸)、脂肪酸代谢(硬脂酸、亚麻酸、亚油酸、胆固醇)以及核苷酸代谢(次黄嘌呤、尿嘧啶、胸腺嘧啶)等。(3)代谢通路富集分析表明,氨气应激主要影响了肉鸡血清的脂肪代谢通路,如亚油酸代谢、花生四烯酸代谢以及α-亚麻酸代谢。【结论】GC-MS较为全面地检测到血清的代谢物,能够准确地筛选出差异代谢物,同时慢性氨气应激显著影响肉鸡的血清代谢物含量,主要影响脂肪代谢通路,为阐明氨气应激影响营养代谢的机制提供参考。 关键词:肉鸡;代谢组学;血清;代谢物;气相色谱/质谱联用技术
Abstract 【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. Keywords:broiler;metabonomics;serum;metabolites;GC-MS
PDF (2178KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 孙永波, 王亚, 萨仁娜, 张宏福. GC-MS分析慢性氨气应激对肉鸡血清代谢物的影响[J]. 中国农业科学, 2020, 53(8): 1688-1698 doi:10.3864/j.issn.0578-1752.2020.08.017 SUN YongBo, WANG Ya, SA RENNA, ZHANG HongFu. Effects of Chronic Ammonia Stress on Serum Metabolites of Broilers Based on GC-MS[J]. Scientia Acricultura Sinica, 2020, 53(8): 1688-1698 doi:10.3864/j.issn.0578-1752.2020.08.017
0 引言
【研究意义】血液理化指标能够反映动物的健康状况,分析肉鸡血液指标的变化情况能够了解其生理状态[1]。由于粪便和垫料等含氮有机物的发酵,畜禽舍内持续性的存在低浓度的氨气。动物经呼吸作用吸入氨气,一部分直接与呼吸系统黏膜接触并溶于黏膜表面,一部分通过肺泡的气血屏障进入血液循环[2]。因此,通过测定长期氨气应激肉鸡的血清代谢物的变化,对于了解氨气的有害作用及对肉鸡代谢通路的影响,分析其对畜禽健康影响的作用机制,进而科学调控养殖环境具有重要意义。【前人研究进展】氨气是一种无色、强刺激性的有毒气体,是畜禽舍内危害较大的气体,长期氨气应激损害动物的健康、诱发各种疾病[3,4]。氨气通过呼吸作用到达肺泡,然后进入血液,与血液中的血红蛋白结合,使血红素变为正铁血红素,进而降低血红蛋白的携氧能力,降低畜禽的抵抗力[5]。随着鸡舍氨气浓度的升高,血清GSH-Px活性呈二次曲线降低,CAT活性呈线性降低[6]。氨气应激显著降低肉鸡的抗氧化能力,使血液中抗氧化酶活性降低,丙二醛含量升高[7]。BORELL等[8]研究报道,50 mg·kg-1浓度氨气导致保育猪血液中巨噬细胞数量、淋巴细胞数量及皮质酮含量显著增加。WEI等[9]研究表明,70 mg·kg-1浓度氨气导致肉鸡血清球蛋白含量和溶菌酶活性降低。宋弋等[10]发现,高浓度氨气显著降低肉鸡血液中溶菌酶活性以及NK细胞杀伤活性。氨气应激还影响血液中细胞因子的含量,损害肉鸡的免疫功能[11]。李聪等[12]研究舍内不同浓度氨气对肉鸡免疫反应的作用,发现50 mg·kg-1处理组显著降低42日龄肉鸡血清中IL-6以及IL-1β浓度。【本研究切入点】目前国内外研究大多数集中在氨气应激对肉鸡血液中少量的理化指标的影响,缺乏系统性的研究。代谢组学能够反应机体出现的动态复杂的代谢应答和变化,具备其他技术所不具备的优势,在近些年越来越得到广泛的应用[13,14,15]。目前,有关氨气应激对肉鸡血清代谢物影响的研究未见报道。【拟解决的关键问题】本试验使用气相色谱–质谱联用(gas chromatography- mass spectrum,GC-MS)技术,研究慢性氨气应激条件下肉鸡血清中小分子代谢产物浓度的变化,探究氨气对肉鸡代谢的影响及可能的作用机理,从代谢方面揭示氨气的危害性,为畜禽舍舒适环境参数的制定以及合理调控舍内环境提供依据。
每个点代表一个代谢物,红色代表上调2倍及以上,蓝色代表下调2倍及以上 Each point represents a metabolite, the red representative is up 2 times and above, and the blue represents 2 times and more Fig. 6Volcano plot of serum metabolite of broilers
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