饲粮纤维水平对猪肠道屏障功能、结肠微生物及代谢产物的影响

doi:10.3864/j.issn.0578-1752.2023.22.014

Abstract

Abstract:

【Objective】 Adding fiber raw materials is one of the effective methods to reduce the feed costs. The aim of this study was to investigate the effects of different fiber level diets on the intestinal health of Mashen (MS) pig and Duroc × Landrace × Large (DLY) pig, so as to provide the basis for the rational use of fiber. 【Method】 In this study, 80 MS pigs and 80 DLY pigs with an initial body weight of (20 ± 0.5) kg were used as test objects (half of the sows and half of the boars). MS and DLY pigs were divided into four groups each assigned to different diets, with five replicates per treatment and four pigs per replicate. When 0%, 9.35%, 18.64% and 28.03% soybean hulls were added to the corn soybean meal basal diet, the NDF content was 9% (9N), 13.5% (13.5N), 18% (18N) and 22.5% (22.5N), respectively. The test lasted for 30 days. 【Result】 For MS pigs, the content of IL-10 in ileum of 18N group and TNF-α in cecum of 13.5N and 22.5N groups was significantly decreased (P<0.05). The number of goblet cells in jejunum, ileum and caecum was increased with the increase of fiber level. In colon, the number of goblet cells and the expression level of MUC2 were significantly increased in 13.5N and 18N groups (P<0.05). The expressions of Claudin-2, Occludin, E-cadherin, ZO-1, and ZO-2 of 18N and 22.5N groups were increased significantly (P<0.05). The abundance of Escherichia-Shigella in 13.5N, 18N and 22.5N groups were decreased significantly (P<0.05), while the abundance of Lactobacillus in 13.5N group, Prevoteaceae_NK3B31_group in 18N group and Methanobrevibacter in 22.5N group were increased significantly (P<0.05). Palmitic acid, stearic acid, lauric acid and capric acid in 13.5N, 18N and 22.5N groups were significantly increased (P<0.05), while lithocholic acid and cholic acid were significantly decreased (P<0.05). Differential metabolites were mainly concentrated in the pathways related to lipid metabolism and carbohydrate digestion and absorption. For DLY pigs, the TNF-α content in cecum of 13.5N group was significantly increased (P<0.05). The number of goblet cells in each intestinal segment of 13.5N, 18N and 22.5N groups was significantly higher than those of 9N group (P<0.05). The expression level of colonic MUC2 was increased significantly in 13.5N and 18N groups (P<0.05) and decreased significantly in 22.5N group (P<0.05). The expression levels of Claudin-2, Occludin, E-cadherin and ZO-1 in 18N and 22.5N groups were decreased significantly (P<0.05). The abundance of Methanobrevibacter in 13.5N group, Streptococcus in 18N group and Lachnospira in 22.5N group was significantly increased (P<0.05). L-tyrosine, L-glutamic acid, L-pyroglutamic acid, phenylalanine and other derivatives in 13.5N, 18N group were significantly increased (P<0.05), while significantly decreased (P<0.05) in 22.5N group. Differential metabolites were mainly concentrated in amino acid metabolism pathway. In MS pigs and DLY pigs, there was a strong correlation between microflora and intestinal barrier related genes, as well as tryptophan and bile acid metabolites. 【Conclusion】 Improving the dietary fiber level could strengthen the intestinal barrier function of pigs, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, regulate the metabolism of lipid and amino acids in the colon, affect the metabolism of short-chain fatty acids, tryptophan and bile acids through the microbial pathway, and promote intestinal health.

Key words: dietary fiber level, intestinal barrier, microbiota, metabolites, pig

LIU Chang, CUI ZiXu, ZUO Zhou, YUN HongMei, NIU Jin, YANG Yang, GUO XiaoHong, LI BuGao, GAO PengFei, ZHAO Yan, CAO GuoQing. Effects of Dietary Fiber Level on Intestinal Barrier Function, Colonic Microbiota and Metabolites in Pigs[J].Scientia Agricultura Sinica, 2023, 56(22): 4532-4551.

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原料 Ingredient	组别 Group				营养水平 Nutrient levels	组别 Group
原料 Ingredient	9N	13.5N	18N	22.5N	营养水平 Nutrient levels	9N	13.5N	18N	22.5N
玉米 Corn	75.09	67.19	59.30	52.75	干物质 DM (%)	87.12	87.44	87.77	88.08
大豆皮 Soybean hull		9.35	18.64	28.03	净能 NE (MJ·kg^-1)	10.61	10.02	9.44	8.85
豆粕 Soybean meal	20.43	19.19	17.97	15.24	消化能 DE (MJ·kg^-1)	14.40	13.85	13.31	12.75
赖氨酸 L-Lys·HCl	0.29	0.23	0.17	0.15	粗蛋白质 CP (%)	15.50	15.50	15.50	15.00
蛋氨酸 L-Met	0.11	0.09	0.08	0.07	粗纤维 CF (%)	2.00	5.00	8.00	11.00
苏氨酸 L-Thr	0.13	0.10	0.08	0.07	中性洗涤纤维 NDF (%)	9.00	13.50	18.00	22.50
色氨酸 L-Trp	0.02	0.01	0.00	0.00	酸性洗涤纤维 ADF (%)	5.30	8.10	11.67	14.79
石粉 Limestone	0.85	0.71	0.58	0.44	标准回肠可消化赖氨酸 SID Lys (%)	0.90	0.85	0.80	0.75
磷酸氢钙 CaHPO₄	0.83	0.88	0.92	0.99	标准回肠可消化赖氨酸/净能 SID Lys/NE (%)	0.085	0.085	0.085	0.085
氯化钠 NaCl	0.25	0.25	0.26	0.26	钙 Ca (%)	0.58	0.58	0.58	0.58
预混料 Premix ^1）	2.00	2.00	2.00	2.00	磷 P (%)	0.46	0.46	0.46	0.46
合计 Total	100.00	100.00	100.00	100.00

基因 Gene	引物序列 Primer sequence (5′→3′)	登录号 Accession No.	产物长度 Products size (bp)
MUC2	F: CTGTGCGACTACAACTTCGC R: AGATGGTGTCGTCCTTGACC	XM_021082584.1	139
Claudin-2	F: ACTGCAAGGAAATCGCTCCA R: TCTTGGCTTTGGGTGGTTGA	NM_001161638.1	98
Occludin	F: CCTCCTCCCCTTTCGGACTA R: TCACTTTCCCGTTGGACGAG	NM_001163647.2	70
E-cadherin	F: GTGGTTCCGAAGCTGCTAGT R: CCCCACTCGTTCAGGTAGTC	NM_001163060.1	85
ZO-1	F: GAGAAAGGTGAAACCCTGCTG R: GTCAGGAGTCATGGACGCAC	XM_047432990.1	111
ZO-2	F: ATTCGGACCCATAGCAGACATAG R: GCGTCTCTTGGTTCTGTTTTAGC	NM_001206404.1	90
18S	F: ATAAACGATGCCGACTGGCGAT R: CAATCTGTCAATCCTGTCCGTGT	NR_046261.1	219

项目 Item	MS				DLY				SEM	P-value
项目 Item	9N	13.5N	18N	22.5N	9N	13.5N	18N	22.5N	SEM	Diet	Breed	Diet×Breed
回肠 Ileum
白介素10 IL-10（pg·mL^-1）	181.86a	158.02ab	148.60b	157.57ab	168.05ab	151.94b	173.40ab	160.70ab	3.539	0.095	0.699	0.110
肿瘤坏死因子α TNF-α（pg·mL^-1）	185.56	180.72	164.58	164.20	175.47	176.17	170.86	171.61	4.648	0.416	0.972	0.745
分泌型免疫球蛋白A sIgA（µg·mL^-1）	41.51	37.17	36.19	41.19	42.93	34.09	32.41	35.53	1.585	0.139	0.251	0.726
盲肠Cecum
白介素10 IL-10（pg·mL^-1）	160.26	153.09	150.62	154.66	152.17	148.81	165.62	164.28	2.857	0.502	0.470	0.215
肿瘤坏死因子α TNF-α（pg·mL^-1）	171.95bc	147.62d	169.50bc	150.44d	184.57b	162.93cd	168.10bc	203.01a	2.558	0.007	0.001	0.004
分泌型免疫球蛋白A sIgA（µg·mL^-1）	41.72	35.61	39.53	42.39	39.53	37.21	36.43	38.42	1.586	0.521	0.417	0.826
结肠Colon
白介素10 IL-10（pg·mL^-1）	158.92	163.16	152.42	146.80	153.32	155.33	163.35	151.74	3.148	0.446	0.894	0.446
肿瘤坏死因子α TNF-α（pg·mL^-1）	171.94	153.25	153.98	158.20	181.41	167.39	163.51	167.38	4.684	0.279	0.149	0.992
分泌型免疫球蛋白A sIgA（µg·mL^-1）	36.08	38.86	43.60	42.40	37.75	35.65	36.87	37.42	1.220	0.436	0.091	0.404

项目 Item	MS				DLY				SEM	P-value
项目 Item	9N	13.5N	18N	22.5N	9N	13.5N	18N	22.5N	SEM	Diet	Breed	Diet×Breed
十二指肠Duodenum	23.20a	24.87a	25.07a	25.40a	18.13b	22.40a	22.47a	22.80a	0.397	0.002	<0.001	0.502
空肠Jejunum	20.73b	25.47a	25.40a	24.33a	16.80c	20.67b	20.53b	20.07b	0.393	<0.001	<0.001	0.939
回肠Ileum	20.40c	24.33b	25.00b	31.93a	13.93e	16.13d	20.60c	21.13c	0.534	<0.001	<0.001	<0.001
盲肠Cecum	28.07de	33.20c	35.80b	39.13a	22.73f	26.60e	28.80de	30.13d	0.529	<0.001	<0.001	0.154
结肠Colon	58.47d	70.07a	72.47a	61.00cd	49.27f	53.20e	66.33b	62.80bc	0.818	<0.001	<0.001	<0.001

属名Genus	MS_9N	MS_13.5N	MS_18N	MS_22.5N	P-value
乳酸杆菌属 Lactobacillus	10.05b	23.15a	8.42b	4.87c	<0.001
甲烷短杆菌属 Methanobrevibacter	1.78b	4.53b	0.78b	29.21a	<0.001
普雷沃氏菌科_NK3B31属 Prevotellaceae_NK3B31_group	3.24b	3.73b	18.53a	1.68b	<0.001
瘤胃菌科UCG-005属 Ruminococcaceae_UCG-005	3.29b	9.28a	0.87c	4.01b	<0.001
土孢杆菌属 Terrisporobacter	4.44ab	1.83c	2.84bc	6.25a	0.014
大肠埃希菌-志贺氏菌属 Escherichia-Shigella	10.45a	0.18c	0.19c	1.86b	<0.001
梭状芽孢杆菌科_sensu_stricto_1属Clostridium_sensu_stricto_1	6.23a	1.09b	1.40b	1.93b	<0.001
真杆菌属 Eubacterium_coprostanoligenes_group	5.03a	2.42b	1.34c	1.27c	<0.001
理研氏菌科_RC9属 Rikenellaceae_RC9_gut_group	0.83c	3.42a	2.15b	2.90a	<0.001
瘤胃球菌_1属 Ruminococcus_1	1.50bc	4.24a	0.89c	2.32b	<0.001
毛螺菌属 Lachnospira	0.42c	1.39b	4.91a	0.84bc	<0.001
克里斯滕森菌科_R-7属 Christensenellaceae_R-7_group	2.62a	1.86ab	0.95c	1.58bc	0.009
粪杆菌属 Faecalibacterium	0.15b	0.58b	5.76a	0.17b	0.001
考拉杆菌属 Phascolarctobacterium	1.65b	0.78c	2.03a	1.56b	<0.001
瘤胃菌科UCG-014属 Ruminococcaceae_UCG-014	0.29c	2.10a	2.01a	1.50b	<0.001
链球菌属 Streptococcus	1.26b	1.05b	2.82a	0.37b	0.033
密螺旋体_2属 Treponema_2	2.04a	1.57ab	0.36c	1.43b	<0.001
罕见小球菌属 Subdoligranulum	1.01b	0.87b	2.96a	0.21c	<0.001
Agathobacter	2.21a	0.55c	1.80b	0.14d	<0.001
毛螺菌科_XPB1014属 Lachnospiraceae_XPB1014_group	0.35c	2.58a	0.11c	1.30b	<0.001

Effects of Dietary Fiber Level on Intestinal Barrier Function, Colonic Microbiota and Metabolites in Pigs

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属名Genus	DLY_9N	DLY_13.5N	DLY_18N	DLY_22.5N	P-value
乳酸杆菌属 Lactobacillus	40.04a	21.08bc	19.11c	26.55b	0.001
甲烷杆菌属 Methanobrevibacter	6.85ab	10.43a	2.62b	4.05b	0.041
链球菌属 Streptococcus	2.86c	4.83b	8.37a	3.68bc	<0.001
瘤胃菌科UCG-005属 Ruminococcaceae_UCG-005	6.47a	3.09b	2.74b	4.87a	0.002
密螺旋体_2属 Treponema_2	0.41b	7.00a	4.77ab	0.77b	0.060
巨型球菌 Megasphaera	3.06a	0.08b	2.89a	5.59a	0.013
毛螺菌属 Lachnospira	0.96b	0.90b	1.12b	7.68a	<0.001
瘤胃菌科UCG-014属 Ruminococcaceae_UCG-014	1.25b	1.21b	4.61a	2.45ab	0.117
梭状芽孢杆菌科_sensu_stricto_1属 Clostridium_sensu_stricto_1	3.35	3.49	2.06	0.56	0.168
拟普雷沃菌属 Alloprevotella	0.78b	0.10b	5.76a	1.83ab	0.075
克里斯滕森菌科_R-7属 Christensenellaceae_R-7_group	0.77b	5.73a	1.17b	0.60b	<0.001
土孢杆菌属 Terrisporobacter	1.83	2.70	2.26	1.22	0.346
毛螺菌科_XPB1014属 Lachnospiraceae_XPB1014_group	1.00b	5.37a	0.86b	0.70b	<0.001
理研氏菌科_RC9属 Rikenellaceae_RC9_gut_group	0.78b	1.90a	1.88a	1.71a	0.001
真杆菌属 Eubacterium_coprostanoligenes_group	1.10b	1.63a	1.91a	1.18b	<0.001
瘤胃球菌_1属 Ruminococcus_1	0.49	0.83	1.48	1.57	0.140
瘤胃菌科UCG-002属 Ruminococcaceae_UCG-002	1.22ab	1.79a	0.88bc	0.46c	0.008
艾克曼菌属 Akkermansia	3.69a	0.27b	0.12b	0.21b	0.022
罕见小球菌属 Subdoligranulum	0.81b	0.50b	1.69a	1.22ab	0.029
粪杆菌属 Faecalibacterium	0.77bc	0.10c	1.74a	1.48ab	0.004

类 Class	差异代谢物 Differential metabolite	差异倍数 Fold change
类 Class	差异代谢物 Differential metabolite	13.5N/9N	18N/9N	22.5N/9N
脂肪酰类 Fatty acyls	棕榈酸 Palmitic acid	2.90	1.48	1.12
	硬脂酸Stearic acid	3.09	1.18	1.06
	花生四烯酸 Arachidonic acid	2.32	3.80	0.77
	油酸 Oleic acid	1.76	1.59	0.34
	月桂酸 Lauric acid	7.35	16.22	2.21
	癸酸 Decanoic acid	6.75	24.33	1.09
	肾上腺素酸 Adrenic acid	1.24	1.68	0.28
	二十二碳五烯酸 Docosapentaenoic acid	1.55	3.42	0.69
	11Z-二十碳烯酸 11Z-Eicosenoic acid	1.03	3.35	0.51
	二十二碳六烯酸 Docosahexaenoic acid	1.03	3.35	0.51
	20-羟基二十碳四烯酸 20-HETE	1.24	3.13	0.64
脂质和类脂分子 Lipids and lipid-like molecules	石胆酸 Lithocholic Acid	0.60	0.36	0.75
类固醇和类固醇衍生物 Steroids and steroid derivatives	熊去氧胆酸 Glycoursodeoxycholic acid	0.27	0.17	18.46
	胆酸 Cholic acid	0.66	0.77	0.33
	脱氧胆酸 Deoxycholic acid	0.38	0.61	1.60
	甘胆酸 Glycocholic acid	0.56	0.41	9.30
	胆钙化醇 Cholecalciferol	0.64	0.29	0.42
	骨化三醇 Calcitriol	1.03	2.72	0.68
吲哚和衍生物 Indoles and derivatives	色胺 Tryptamine	1.29	0.09	1.31
	5-羟基吲哚-3-乙酸5-Hydroxyindole-3-acetic acid	4.06	0.69	1.31
	褪黑素 Melatonin	1.27	1.05	5.36
	吲哚-3-乙酸 Indole-3-acetic acid	0.05	0.18	0.58
羧酸及其衍生物 Carboxylic acids and derivatives	烟酸 Nicotinic acid	0.94	1.18	0.47
	N-乙酰-L-谷氨酸 N-Acetyl-L-glutamic acid	5.75	1.04	0.39
	L-酪氨酸 L-Tyrosine	1.49	1.65	1.38
	L-苯丙氨酸 L-Phenylalanine	2.94	2.40	1.16
	L-谷氨酸 L-Glutamic acid	2.72	2.41	0.78
	N6-乙酰-L-赖氨酸 N6-Acetyl-L-lysine	5.17	2.37	0.65
	L-焦谷氨酸 L-Pyroglutamic acid	2.36	1.62	0.95
	5-氧代脯氨酸 5-oxoproline	3.97	1.70	1.01

二级分类通路 KEGG_B_class	代谢通路 Pathways	差异代谢物（MS） Differential metabolites	差异代谢物（DLY） Differential metabolites
免疫系统 Immune system	血小板活化 Platelet activation	环化GMP Cyclic GMP 花生四烯酸 Arachidonic acid	环化GMP Cyclic GMP 前列腺素H2 Prostaglandin H2
	FcγR介导的吞噬作用 Fc gamma R-mediated phagocytosis	花生四烯酸 Arachidonic acid	-
	FcεRI信号通路 Fc epsilon RI signaling pathway	花生四烯酸 Arachidonic acid	-

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