瘤胃酸中毒对山羊胃肠道功能、形态和菌群的影响

doi:10.11843/j.issn.0366-6964.2024.10.045

摘要/Abstract

摘要：

旨在通过诱导瘤胃酸中毒模型，探究瘤胃酸中毒对山羊胃肠道组织和菌群结构的影响。本试验选取8只健康波尔山羊为试验对象，对照组4只，造模后的酸中毒组4只。通过瘤胃插管灌服玉米面的方法构建山羊瘤胃酸中毒模型。取胃肠道组织进行HE染色、RT-qPCR和Western blot检测，探究瘤胃酸中毒对山羊消化道组织形态、功能蛋白的影响。另外分别取瘤胃液和盲肠内容物，应用Illumina MiSeq高通量测序技术测定样本中细菌的16S rRNA，分析酸中毒对山羊胃肠道细菌群落组成的影响。结果显示：与对照组相比，酸中毒组山羊的瘤胃和小肠的组织形态出现损伤，其结构完整性被破坏；瘤胃和十二指肠中的MCT1 mRNA相对表达量和蛋白质表达量均极显著升高(P < 0.01)，而SLC5A8 mRNA相对表达量和蛋白表达量均极显著降低(P < 0.01)。对瘤胃内容物进行高通量测序后，相较于对照组，酸中毒组Alpha多样性分析中Shannon指数显著低(P < 0.05)，拟杆菌门(Bacteroidetes)、互养菌门(Synergistetes)的相对丰富度极显著降低(P < 0.01)，纤维杆菌门(Fibrobacteres)、软壁菌门(Tenericutes)的相对丰度显著升高(P < 0.05)，瘤球菌属(Ruminococcus)、普雷沃氏菌属(Prevotella)的相对丰富度显著降低(P < 0.05)。对盲肠内容物高通量测序后，与对照组相比，酸中毒组的瘤胃球菌属(Ruminococcus)、颤螺旋菌属(Oscillospira)的相对丰富度显著降低(P < 0.05)，梭菌属(Clostridium)相对丰富度显著升高(P < 0.05)。综上可知，瘤胃酸中毒可使山羊消化道组织形态完整性破坏，相关蛋白表达受影响，导致消化道菌群结构发生紊乱。

关键词: 山羊, 瘤胃酸中毒, 胃肠道形态, 高通量测序, 菌群分析

Abstract:

This study utilized a model of rumen acidosis induced by the administration of corn flour to investigate its impact on the gastrointestinal tract flora structure of goats. In this experiment, eight healthy Boer goats were selected as test subjects, 4 in the control group and 4 in the acidosis group after modeling. Gastrointestinal(GI) tissues were collected for HE staining, RT-qPCR, and Western blot assay to investigate the effects of rumen acidosis on the morphology and functional proteins of goat GI tissues. Illumina MiSeq high-throughput sequencing was used to determine the 16S rRNA of the bacteria in rumen fluid and cecum content, aimed to sutdy the effect of acidosis on the bacterial communities in the gastrointestinal tracts. The results showed that, compared with the control group, the tissue morphology and structural integrity of the rumen and small intestine were damaged in the acidosis group. The relative expression levels of MCT1 mRNA and protein in rumen and duodenum were extremely significantly increased (P < 0.01), while the relative expression levels of SLC5A8 mRNA and protein were extremely significantly decreased (P < 0.01). The results of high-throughput sequencing in rumen contents showed that, the Shannon index of Alpha diversity analysis in the acidosis group was significantly lower than that in the control group (P < 0.01), and the relative richness of Bacteroidetes and Synergistetes was significantly decreased (P < 0.01). The relative abundance of Fibrobacteres and Tenericutes was significantly increased (P < 0.05), while the relative abundance of Ruminococcus and Prevotella was significantly decreased (P < 0.05). After high-throughput sequencing of cecum contents, compared with the control group, the relative richness of Ruminococcus and Oscillospira in acidosis group was significantly decreased (P < 0.05), while the relative richness of Clostridium was significantly increased (P < 0.05). In conclusion, rumen acidosis can destroy the integrity of digestive tract morphology, affect the expression of related proteins, and disturb digestive tract flora structure.

Key words: goat, rumen acidosis, gastrointestinal morphology, high-throughput sequencing, flora analysis

中图分类号:

S858.26

张道亮, 丁红研, 王留幸, 邰文俊, 孔昊, 赵畅, 冯士彬, 王希春, 薛艳锋, 吴金节, 李玉. 瘤胃酸中毒对山羊胃肠道功能、形态和菌群的影响[J]. 畜牧兽医学报, 2024, 55(10): 4760-4772.

Daoliang ZHANG, Hongyan DING, Liuxing WANG, Wenjun TAI, Hao KONG, Chang ZHAO, Shibin FENG, Xichun WANG, Yanfeng XUE, Jinjie WU, Yu LI. Effect of Rumen Acidosis on Gastrointestinal Function, Morphology, and Microflora in Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4760-4772.

图/表 12

表 1

图 1

表 2

图 2

图 3

图 4

图 5

图 6

图 7

图 8

表 3

表 4

参考文献 30

1	RIVERA-CHACON R , CASTILLO-LOPEZ E , RICCI S , et al. Supplementing a phytogenic feed additive modulates the risk of subacute rumen acidosis, rumen fermentation and systemic inflammation in cattle fed acidogenic diets[J]. Animals, 2022, 12 (9): 1201. doi: 10.3390/ani12091201
2	张璐, 陈爱华, 吴清明. 肠道菌群与代谢相关脂肪性肝病[J]. 临床内科杂志, 2023, 40 (1): 6- 9.
	ZHANG L , CHEN A H , WU Q M . Gut flora and metabolic associated fatty liver disease[J]. Journal of Clinical Internal Medicine, 2023, 40 (1): 6- 9.
3	STEELE M A , CROOM J , KAHLER M , et al. Bovine rumen epithelium undergoes rapid structural adaptations during grain-induced subacute ruminal acidosis[J]. Am J Physiol Regul Integr Comp Physiol, 2011, 300 (6): R1515- R1523. doi: 10.1152/ajpregu.00120.2010
4	程萌. 亚急性瘤胃酸中毒对奶山羊瘤胃上皮通透性及细胞连接蛋白表达的影响[D]. 呼和浩特: 内蒙古农业大学, 2016.
	CHENG M. Effect of subacute ruminal acidosis on rumen epithelium permeability and intercellula junction protein expression in dairy goats[D]. Hohhot: Inner Mongolia Agricultural University, 2016. (in Chinese)
5	魏子维, 邓铭, 孙宝丽, 等. 高精料饲粮对反刍动物胃肠道健康的影响及调控措施[J]. 动物营养学报, 2021, 33 (3): 1277- 1285. doi: 10.3969/j.issn.1006-267x.2021.03.010
	WEI Z W , DENG M , SUN B L , et al. Effects of high concentrate diet on gastrointestinal Health of ruminants and its regulation measures[J]. Chinese Journal of Animal Nutrition, 2021, 33 (3): 1277- 1285. doi: 10.3969/j.issn.1006-267x.2021.03.010
6	KOH A , DE VADDER F , KOVATCHEVA-DATCHARY P , et al. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites[J]. Cell, 2016, 165 (6): 1332- 1345. doi: 10.1016/j.cell.2016.05.041
7	王倩. 灰葡萄孢单羧酸转运蛋白基因BcMCT1的功能解析[D]. 杭州: 浙江工业大学, 2013.
	WANG Q. Cloning and functional analisis of monocarboxylate transporter gene (BcMCT1) in Botryils cinerea[D]. Hangzhou: Zhejiang University of Technology, 2013. (in Chinese)
8	COADY M J , WALLENDORFF B , BOURGEOIS F , et al. Establishing a definitive stoichiometry for the Na⁺/monocarboxylate cotransporter SMCT1[J]. Biophys J, 2007, 93 (7): 2325- 2331. doi: 10.1529/biophysj.107.108555
9	LI L P , PENG K L , XUE M Y , et al. An age effect of rumen microbiome in dairy buffaloes revealed by metagenomics[J]. Microorganisms, 2022, 10 (8): 1491. doi: 10.3390/microorganisms10081491
10	WANG K J , ZHENG M L , REN A , et al. Effects of high rice diet on growth performance, nutrients apparent digestibility, nitrogen metabolism, blood parameters and rumen digestibility, nitrogen metabolism, blood parameters and rumen[J]. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 2019, 25 (6): 749- 755.
11	ZHANG Y , CHOI S H , NOGOY K M , et al. Review: the development of the gastrointestinal tract microbiota and intervention in neonatal ruminants[J]. Animal, 2021, 15 (8): 100316. doi: 10.1016/j.animal.2021.100316
12	FANIYI T O , ADEGBEYE M J , ELGHANDOUR M M M Y , et al. Role of diverse fermentative factors towards microbial community shift in ruminants[J]. J Appl Microbiol, 2019, 127 (1): 2- 11. doi: 10.1111/jam.14212
13	LI Y , DING H Y , LIU L H , et al. Non-esterified fatty acid induce dairy cow hepatocytes apoptosis via the mitochondria-mediated ROS-JNK/ERK signaling pathway[J]. Front Cell Dev Biol, 2020, 8, 245. doi: 10.3389/fcell.2020.00245
14	MA J , SHAH A M , WANG Z S , et al. Potential protective effects of thiamine supplementation on the ruminal epithelium damage during subacute ruminal acidosis[J]. Anim Sci J, 2021, 92 (1): e13579. doi: 10.1111/asj.13579
15	HU H L , YANG S Q , CHENG M , et al. Long-term effect of subacute ruminal acidosis on the morphology and function of rumen epithelial barrier in lactating goats[J]. J Integr Agric, 2022, 21 (11): 3302- 3313. doi: 10.1016/j.jia.2022.08.087
16	薛春旭. 高精料日粮对山羊小肠微生物发酵、微生物区系及上皮形态结构的影响[D]. 南京: 南京农业大学, 2017.
	XUE C X. The impact of high concentrate diet on small intestinal fermentation, microbial community and epithelial morphology of goats[D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)
17	DALILE B , VAN OUDENHOVE L , VERVLIET B , et al. The role of short-chain fatty acids in microbiota-gut-brain communication[J]. Nat Rev Gastroenterol Hepatol, 2019, 16 (8): 461- 478. doi: 10.1038/s41575-019-0157-3
18	BIONAZ M , VARGAS-BELLO-PÉREZ E , BUSATO S . Advances in fatty acids nutrition in dairy cows: from gut to cells and effects on performance[J]. J Anim Sci Biotechnol, 2020, 11 (1): 110. doi: 10.1186/s40104-020-00512-8
19	YAN L , ZHANG B , SHEN Z M . Dietary modulation of the expression of genes involved in short-chain fatty acid absorption in the rumen epithelium is related to short-chain fatty acid concentration and pH in the rumen of goats[J]. J Dairy Sci, 2014, 97 (9): 5668- 5675. doi: 10.3168/jds.2013-7807
20	刘军花, 朱伟云, 毛胜勇. 高谷物日粮促进山羊瘤胃上皮单羧酸转运蛋白1及钠钾ATP酶mRNA的表达[J]. 草业学报, 2017, 26 (2): 95- 101.
	LIU J H , ZHU W Y , MAO S Y . A high-grain diet promotes expression of MCT1 and Na⁺/K⁺-ATPase mRNAs in the ruminal epithelium of goats[J]. Acta Prataculturae Sinica, 2017, 26 (2): 95- 101.
21	苏效双, 张春刚, 刘光磊, 等. 单羧酸转运蛋白: 在挥发性脂肪酸转运中的作用及影响基因表达的因素[J]. 动物营养学报, 2016, 28 (9): 2709- 2714.
	SU X S , ZHANG C G , LIU G L , et al. Monocarboxylate transporters: function in volatile fatty acid transport and gene expression influencing factors[J]. Chinese Journal of Animal Nutrition, 2016, 28 (9): 2709- 2714.
22	MAO S Y , ZHANG R Y , WANG D S , et al. Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing[J]. Anaerobe, 2013, 24, 12- 19. doi: 10.1016/j.anaerobe.2013.08.003
23	KHAFIPOUR E , LI S C , PLAIZIER J C , et al. Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis[J]. Appl Environ Microbiol, 2009, 75 (22): 7115- 7124. doi: 10.1128/AEM.00739-09
24	KIM Y H , NAGATA R , OHKUBO A , et al. Changes in ruminal and reticular pH and bacterial communities in Holstein cattle fed a high-grain diet[J]. BMC Vet Res, 2018, 14 (1): 310. doi: 10.1186/s12917-018-1637-3
25	LIU J H , XU T T , ZHU W Y , et al. High-grain feeding alters caecal bacterial microbiota composition and fermentation and results in caecal mucosal injury in goats[J]. Br J Nutr, 2014, 112 (3): 416- 427. doi: 10.1017/S0007114514000993
26	金磊, 王立志, 王之盛, 等. 基于高通量测序技术对山羊盲肠细菌多样性的分析[J]. 微生物学通报, 2019, 46 (6): 1423- 1433.
	JIN L , WANG L Z , WANG Z S , et al. Analysis of cecum bacterial diversity of goat based on Illumina MiSeq sequencing[J]. Microbiology China, 2019, 46 (6): 1423- 1433.
27	TIGCHELAAR E F , BONDER M J , JANKIPERSADSING S A , et al. Gut microbiota composition associated with stool consistency[J]. Gut, 2016, 65 (3): 540- 542.
28	王悦. 湖羊结肠微生物区系及上皮形态和功能对高精料日粮的适应性应答研究[D]. 南京: 南京农业大学, 2018.
	WANG Y. The adaptive response of the microbiota and epithelial morphology and function to high grain diets in the colon of Hu sheep[D]. Nanjing: Nanjing Agricultural University, 2018. (in Chinese)
29	HOSSAIN M E . Sub-acute ruminal acidosis in dairy cows: its causes, consequences and preventive measures[J]. Online J Anim Feed Res, 2020, 10 (6): 302- 312.
30	SCHIRMER M , GARNER A , VLAMAKIS H , et al. Microbial genes and pathways in inflammatory bowel disease[J]. Nat Rev Microbiol, 2019, 17 (8): 497- 511.

基因 Gene	基因序列号 GenBank accession no.	产物大小/bp Product size	引物序列(5′→3′) Primer sequence(5′→3′)
β-actin	NM_001314342.1	91	F：TCCTTCCTGGGCATGGAATC
			R：CGTAAAGGTCCTTGCGGATG
MCT1	XM_018046576.1	144	F：AAGAGGAGGAACTTTGAGGC
			R：TGAACACCTGGTCTGACTTC
SLC5A8	XM_018048162.1	87	F：CTATTGCCTTGTGCTTGCAT
			R：TTATAGGCTGGGATGCAGTG

项目 Parameter	参考范围 Reference range	对照组 Control group	酸中毒组 Acidosis group	P 值 P-value
pH	7.31~7.42	7.49±0.06	7.35±0.08	0.031
二氧化碳分压/mmHg PCO₂	32~46	35.50±5.20	32.50±5.07	0.442
碳酸氢根/(mmol·L^-1) HCO₃^-	20.0~29.0	24.10±1.83	16.45±4.77	0.024
阴离子间隙/(mmol·L^-1) AnGap	—	15.18±1.59	14.20±3.13	0.599
二氧化碳总量/(mmol·L^-1) TCO₂	21~28	25.08±1.84	17.33±4.89	0.025
剩余碱/(mmol·L^-1) BE	-3~3	3.10±2.14	-5.68±5.29	0.022
缓冲碱/(mmol·L^-1) BB	42~52	51.10±2.14	42.33±5.29	0.022
钠离子/(mmol·L^-1) Na⁺	144~160	143.50±5.32	140.00±3.56	0.316
钾离子/(mmol·L^-1) K⁺	3.5~5.8	4.00±0.37	3.10±0.22	0.005
氯离子/(mmol·L^-1) Cl^-	109~122	110.50±0.58	108.75±2.36	0.212

项目 Parameter	对照组 Control group	酸中毒组 Acidosis group	P 值 P-value
厚壁菌门Firmicutes	0.249 4±0.153 4	0.672 2±0.313 2	0.067
拟杆菌门Bacteroidetes	0.716 3±0.151 5	0.090 3±0.114 4	0.001
放线菌门Actinobacteria	0.001 1±0.000 2	0.186 0±0.252 2	0.193
互养菌门Synergistetes	0.004 1±0.001 4	0.000 1±0.000 1	0.001
螺旋菌门Spirochaetes	0.005 3±0.004 6	0.001 5±0.001 6	0.181
软壁菌门Tenericutes	0.000 3±0.000 4	0.000 9±0.000 6	0.021
纤维杆菌门Fibrobacteres	0.000 0±0.000 0	0.000 4±0.000 6	0.006

项目 Parameter	对照组 Control group	酸中毒组 Acidosis group	P 值 P-value
瘤胃球菌属Ruminococcus	0.008 60±0.003 41	0.000 18±0.000 15	0.003
梭菌属Clostridium	0.004 92±0.003 37	0.050 05±0.030 01	0.024
颤螺旋菌属Oscillospira	0.060 83±0.023 58	0.000 16±0.000 31	0.014
链球菌Streptococcus	0.000 00±0.000 00	0.064 83±0.064 01	0.136
双歧杆菌属Bifidobacterium	0.000 04±0.000 06	0.104 20±0.118 70	0.178
乳杆菌属Lactobacillusru	0.000 71±0.000 12	0.140 76±0.198 76	0.254
志贺杆菌Shigella	0.010 00±0.018 59	0.141 65±0.209 66	0.298
巨型球菌属Megasphaera	0.000 00±0.000 00	0.246 70±0.275 47	0.123

[1]	娜梅拉, 李科南, 杜海东, 郭文亮, 娜仁花. 不同日龄内蒙古白绒山羊瘤胃及粪便真菌多样性差异研究[J]. 畜牧兽医学报, 2024, 55(8): 3526-3540.
[2]	程玉婷, 阿比克哈莫, 杨晨, 张丁中, 任云鑫, 岳华, 汤承. 山羊Aichivirus C的分离鉴定及演化分析[J]. 畜牧兽医学报, 2024, 55(8): 3612-3622.
[3]	武永杰, 徐英环, 刘腾飞, 马琳, 陈鸿, 徐永平. 阴囊热应激对山羊血睾屏障结构和功能的影响[J]. 畜牧兽医学报, 2024, 55(7): 2973-2982.
[4]	李陇平, 李托, 曹培文, 朱海鲸, 张小玲, 张宸, 肖普辉, 董书伟, 冯平, 屈雷, 毕台飞. 饲粮不同能量水平对陕北白绒山羊断奶公羔瘤胃发酵特性和微生物组成的影响[J]. 畜牧兽医学报, 2024, 55(7): 3011-3023.
[5]	郑焕琴, 姜晓敏, 岳红, 王宝岩, 刘洋, 张兴晓, 张建龙, 朱洪伟. 猫1型疱疹病毒分离鉴定及部分生物学特性分析[J]. 畜牧兽医学报, 2024, 55(7): 3040-3048.
[6]	李京宇, 陈金铭, 张明一, 赵姗姗, 陶德良, 宋军科, 杨新, 樊莹莹, 赵光辉. 犬新孢子虫miRNAs的鉴定与分析[J]. 畜牧兽医学报, 2024, 55(7): 3085-3093.
[7]	董书餐, 毛帅翔, 伍翠莹, 李耀坤, 孙宝丽, 郭勇庆, 邓铭, 刘德武, 柳广斌. 雄激素受体抑制剂恩杂鲁胺对山羊卵泡颗粒细胞增殖凋亡的影响[J]. 畜牧兽医学报, 2024, 55(5): 2022-2031.
[8]	李秋云, 田芯源, 廖文圣, 张焕容, 任玉鹏, 杨发龙, 朱江江, 向华. SOCS2对山羊鼻甲骨细胞增殖、周期及凋亡的影响[J]. 畜牧兽医学报, 2024, 55(5): 2226-2240.
[9]	李鹏飞, 高桂琴, 周广青, 吴锦艳, 颜新敏, 曹小安, 何继军, 袁莉刚, 尚佑军. 山羊地方性鼻内肿瘤病毒TaqMan荧光定量RT-PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(5): 2259-2266.
[10]	彭佩雅, 陈钰焓, 杨龙, 王铭, 赵芮葶, 何俊, 印遇龙, 刘梅. 家畜基因组拷贝数变异研究进展[J]. 畜牧兽医学报, 2024, 55(4): 1356-1369.
[11]	肖敏, 赵威, 孙武, 娜日苏, 赵乐, 刘陶禄, 张继攀, 赵永聚. 山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究[J]. 畜牧兽医学报, 2024, 55(3): 1019-1029.
[12]	康佳, 段香茹, 尹雪姣, 杨若晨, 李太春, 单新雨, 陈美静, 张英杰, 刘月琴. 半胱氨酸、蛋氨酸对体外培养绒山羊次级毛囊生长及毛乳头细胞增殖的影响[J]. 畜牧兽医学报, 2024, 55(2): 515-527.
[13]	宋艳, 袁永丰, 钱虹宇, 李鑫灿, 罗洪艳, 王芝英, 周作勇. 羊伪结核棒状杆菌的分离鉴定及部分生物学特性分析[J]. 畜牧兽医学报, 2024, 55(2): 680-687.
[14]	苟想珍, 杨军祥, 赵子惠, 冯玲霞, 陈万辉, 李玉洁, 张忠钰, 马克岩, 蒋东平, 常嵘, 文亚洲, 王珂, 马友记. 基于简化基因组测序(Super-GBS)的子午岭黑山羊保种群遗传结构评估[J]. 畜牧兽医学报, 2024, 55(10): 4334-4345.
[15]	李聪聪, 黄子珂, 黄念旎, 马诗语, 刘晗, 肖志标, 宋果, 蒋亮, 彭为波, 杨联熙, 郭云涛, 黄生强. 九疑山兔线粒体基因组组装及系统进化分析[J]. 畜牧兽医学报, 2024, 55(10): 4417-4427.