复合维生素B对生长期山羊后段肠道菌群组成及肠黏膜的影响

doi:10.11686/cyxb2020562

摘要/Abstract

摘要：

本试验旨在研究日粮中添加复合维生素B对生长期山羊消化道微生物组成和肠黏膜结构的影响。试验选取10只4月龄波杂雌山羊（12.60±1.28） kg，随机分为对照组（control，CON）和复合维生素B处理组（vitamin B，VB），CON组饲喂基础日粮（n=5），VB组在基础日粮中添加复合维生素B（n=5），预饲期2周，试验期11周，试验期间自由饮水。试验结束后屠宰采样，16S rRNA测序分析盲肠和结肠内容物微生物组成，检测肠道上皮组织形态结构和相关基因与蛋白的表达。通过菌群分类学分析发现，与CON相比，VB组山羊盲肠和结肠内容物菌群的α多样性指数无显著变化（P>0.05）；在门水平上，盲肠和结肠内容物菌群中厚壁菌门（Firmicutes）和拟杆菌门（Bacteroidetes）为优势菌门，VB组盲肠拟杆菌门丰度有升高趋势（0.05<P<0.1），而螺旋菌门（Spirochaetes）丰度有降低趋势（0.05<P<0.1）；在目水平上，VB组盲肠Solirubrobacterales和Streptomycetales目菌群丰度显著下降（P<0.05）；在科水平上，VB组盲肠Christensenellaceae、Streptomycetaceae、Solirubrobacteraceae及结肠Christensenellaceae科菌群丰度显著下降（P<0.05）；在属水平上，VB组盲肠Lachnoclostridium属菌群丰度显著升高（P<0.05），盲肠Bacillus、Nocardioides及结肠unidentified_Christensenellaceae和unidentified_Gammaproteobacteria属菌群丰度显著下降（P<0.05）。与CON相比，VB组山羊空肠绒毛高度（P<0.01）、隐窝深度（P<0.05）和绒毛高度/隐窝深度（V/C，P<0.05）均显著增加；回肠隐窝深度显著降低（P<0.05），绒毛高度和V/C无显著变化（P>0.05）。实时荧光定量PCR结果显示，与CON相比，VB 组山羊盲肠上皮紧密连接和细胞增殖相关基因的表达均无显著变化（P>0.05）；而结肠黏膜上皮Occludin（P<0.05）、EGFR（P<0.01）、MIK67（P<0.05）和CCND（P<0.05）基因表达均显著上调；且VB组ZO-1蛋白表达显著上调（P<0.05）。综上表明，日粮中添加复合维生素B对生长期山羊肠道菌群多样性无显著影响，但可提高肠道中有益菌的丰度且降低有害菌的丰度，从而促进肠道绒毛的生长及紧密连接蛋白的表达，对山羊肠道健康和生长具有积极作用。

关键词: 复合维生素B, 肠道微生物区系, 肠道上皮结构, 紧密连接蛋白, 山羊

Abstract:

The aim of this study was to explore the effects of vitamin B complex on intestinal microflora composition and intestinal morphology and structure in growing goats. Ten four-month-old female goats with similar body weight （12.60±1.28 kg） were randomly divided into a control group （CON） and a group treated with vitamin B complex （VB）. Goats in the CON group were fed a basal diet （n=5）， and goats in the VB group were fed with the basal diet supplemented with vitamin B complex （n=5）. The experiment comprised an adaptive feeding period of two weeks and a treatment period of 11 weeks. Throughout the experiment， all goats were provided free access to water. At the end of the experiment， the microflora composition of cecal and colonic contents was analyzed by 16S rRNA sequencing， and the intestinal epithelial morphology structure and expression levels of related genes and proteins expression were detected. Compared to CON， VB supplementation did not alter the α diversity index obtained by bacterial taxonomic analysis （P>0.05）. At the phylum level， Firmicutes and Bacteroidetes were the dominant bacteria in the intestines of the goats. Compared to CON， there was a tendency for the abundance of Bacteroidetes in the cecum of the VB group to increase （0.05<P<0.10） and for the abundance of Spirochaetes to decrease （0.05<P<0.10）. At the taxonomic order level， the abundance of Solirubrobacterales and Streptomycetales in the cecum of goats in the VB group was significantly decreased （P<0.05）. At the taxonomic family level， the abundance of Christensenellaceae， Streptomycetaceae， Solirubrobacteraceae in the cecum and Christensenellaceae in the colon of goats in the VB group was significantly decreased （P<0.05）. At the genus level， the abundance of Lachnoclostridium in the cecum of goats in the VB group was significantly increased （P<0.05）， while the abundance of Bacillus， Nocardioides in the cecum and unidentified_Christensenellaceae and unidentified_Gammaproteobacteria in the colon of goats in the VB group was significantly decreased （P<0.05）. Compared to CON， intestinal villus height （P<0.01）， crypt depth （P<0.05） and the ratio of villus height/crypt depth （V/C， P<0.05） of the jejunum in the VB group were significantly increased； and the intestinal crypt depth of the ileum was significantly decreased （P<0.05）. Real-time PCR results showed that Occludin （P<0.05）， EGFR （P<0.01）， MKI67 （P<0.05） and CCND （P<0.05） mRNA expression was significantly up-regulated in colonic epithelium of the VB group， and ZO-1 protein expression was also significantly up-regulated （P<0.05）. It is concluded that dietary supplemention with vitamin B complex increased the abundance of certain probiotics and reduce the abundance of harmful bacteria in the cecum and colon of growing goats. Dietary supplementation with VB also promoted the growth of intestinal villi and improved intestinal structure. The observed changes from VB supplementation would benefit animal welfare and growth.

Key words: vitamin B complex, intestinal microflora, gut epithelial structure, tight junction, goats

吴晨, 姚志浩, 梅文晴, 冯宇妍, 陈渠, 倪迎冬. 复合维生素B对生长期山羊后段肠道菌群组成及肠黏膜的影响[J]. 草业学报, 2021, 30(11): 170-180.

Chen WU, Zhi-hao YAO, Wen-qing MEI, Yu-yan FENG, Qu CHEN, Ying-dong NI. Effects of vitamin B complex on intestinal microflora composition and gut epithelial structure in growing goats[J]. Acta Prataculturae Sinica, 2021, 30(11): 170-180.

图/表 11

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项目 Item	原料 Ingredients （%）	项目 Item	营养水平 Nutrient levels^{2 ）}
精粗比The ratio of concentrate to forage	40∶60	消化能Digestible energy （MJ·kg^-1）	8.25
苜蓿青贮Alfalfa silage	60	粗蛋白Crude protein （%）	18.05
玉米 Corn	22.4	粗脂肪Ether extract （%）	3.00
麸皮Wheat bran	4.8	中性洗涤纤维Neutral detergent fiber （%）	35.07
豆粕 Soybean meal	10.8	酸性洗涤纤维Acid detergent fiber （%）	24.50
小苏打Baking soda	0.4	钙Calcium （%）	0.88
预混料 Premix^1）	1.6	总磷Total phosphorus （%）	0.35
总计 Total	100

项目 Item	原料 Ingredients （%）	项目 Item	营养水平 Nutrient levels^{2 ）}
精粗比The ratio of concentrate to forage	40∶60	消化能Digestible energy （MJ·kg^-1）	8.25
苜蓿青贮Alfalfa silage	60	粗蛋白Crude protein （%）	18.05
玉米 Corn	22.4	粗脂肪Ether extract （%）	3.00
麸皮Wheat bran	4.8	中性洗涤纤维Neutral detergent fiber （%）	35.07
豆粕 Soybean meal	10.8	酸性洗涤纤维Acid detergent fiber （%）	24.50
小苏打Baking soda	0.4	钙Calcium （%）	0.88
预混料 Premix^1）	1.6	总磷Total phosphorus （%）	0.35
总计 Total	100

基因 Gene	引物序列 Primer sequences （5′—3′）	基因序列号 GenBank accession No.
CCND	R：AAAGCCCTCTTCCATACA F：CTCCGCCTTCCCTAAC	XM_005680985.1
MKI67	R：TCAGTGAGCAGGAGGCAGTA F：GGAAATCCAGGTGACTTGCT	XM_004004769.1
Claudin 1	R：CACCCTTGGCATGAAGTGTA F：AGCCAATGAAGAGAGCCTGA	HM117762.1
Claudin 4	R：AAGGTGTACGACTCGCTGCT F：GACGTTGTTAGCCGTCCAG	HM117763.1
Occludin	R：GTTCGACCAATGCTCTCTCAG F：CAGCTCCCATTAAGGTTCCA	BC133617.1
EGFR	R：AACTGTGAGGTGGTCCTTGG F：CACTGTGTTGAGGGCAATGA	XM_005695500.1
GAPDH	R：GGGTCATCATCTCTGCACCT F：GGTCATAAGTCCCTCCACGA	HM043737.1

基因 Gene	引物序列 Primer sequences （5′—3′）	基因序列号 GenBank accession No.
CCND	R：AAAGCCCTCTTCCATACA F：CTCCGCCTTCCCTAAC	XM_005680985.1
MKI67	R：TCAGTGAGCAGGAGGCAGTA F：GGAAATCCAGGTGACTTGCT	XM_004004769.1
Claudin 1	R：CACCCTTGGCATGAAGTGTA F：AGCCAATGAAGAGAGCCTGA	HM117762.1
Claudin 4	R：AAGGTGTACGACTCGCTGCT F：GACGTTGTTAGCCGTCCAG	HM117763.1
Occludin	R：GTTCGACCAATGCTCTCTCAG F：CAGCTCCCATTAAGGTTCCA	BC133617.1
EGFR	R：AACTGTGAGGTGGTCCTTGG F：CACTGTGTTGAGGGCAATGA	XM_005695500.1
GAPDH	R：GGGTCATCATCTCTGCACCT F：GGTCATAAGTCCCTCCACGA	HM043737.1

项目 Items	指标 Index	对照组 CON	VB组 VB	P值P-value
盲肠内容物 Cecal digesta	ACE指数 ACE	1649.32±108.40	1525.63±105.40	0.45
	Chao 1指数 Chao 1	1672.20±130.08	1521.72±132.24	0.45
	香农指数 Shannon index	7.87±0.12	7.67±0.04	0.15
结肠内容物 Colonic digesta	ACE指数ACE	1383.60±106.60	1383.32±42.36	1.00
	Chao 1指数Chao 1	1343.51±104.90	1355.62±33.51	0.92
	香农指数Shannon index	7.70±0.15	7.42±0.18	0.27