Effects of dietary supplementation with bile acid on ileal epithelial morphology， microflora composition， and relative transcript levels of IFN-γ in the ileal mucosa of goats with subacute ruminal acidosis

doi:10.11686/cyxb2024024

Abstract

Abstract:

The aim of this study was to investigate the effect of dietary supplementation with bile acid on intestinal mucosal structure， intestinal microflora composition， and the relative transcript levels of IFN-γ （encoding interferon γ）and IfnGR1 （encoding the IRN-γ receptor）in the ileal mucosa of goats with subacute ruminal acidosis （SARA） induced by a high-concentrate diet. Fifteen 3-month-old male goats （mean weight， 18.19±2.83 kg） were selected and implanted with permanent rumen fistulas by surgery. After a 2-week recovery period， the goats were randomly divided into three groups： a control group （CON）， a subacute ruminal acidosis group （SARA）， and a SARA plus bile acids treatment group （SARA+BA）， with five goats in each group. Goats in the CON group were fed with a low-concentrate diet （concentrate∶forage=3∶7）， those in the SARA and SARA+BA groups were fed with a high-concentrate diet （concentrate∶forage=7∶3）， and those in the SARA+BA group were fed with an additional 3 g of bile acid through the rumen fistula every day for 6 weeks. At the end of the experiment， the goats were slaughtered and the ileal epithelial tissues were sampled to analyze their morphology， the characteristics of goblet cells and paneth cells， and the transcript levels of IFN-γ and IfnGR1 in the mucosa. In addition， the ileal digesta were collected to analyze the composition of the microbial community. The results showed that， compared with the CON group， the SARA group showed an irregularly shaped brush border of the ileal epithelium， indicative of a degree of injury， but no significant difference in the number of cup cells and paneth cells in the villi and crypt （P>0.05）. Compared with the CON group， in the SARA group， the transcript levels of IFN-γ and IfnGR1 in the ileum mucosa were decreased （0.05<P<0.1）， the diversity of the microbial community （Ace and Shannon’s indexes） in the ileum contents was decreased （P<0.05）， and the relative abundance of the phylum Firmicutes in the microbial community in the ileum was significantly increased （P<0.05）. Compared with the CON group， the SARA group showed significantly lower relative abundance of Unclassified_f_Lachnospiraceae， Ruminococcus and Christensenellaceae_R-7_group （P<0.05） and significantly higher relative abundance of Erysipelotrichaceae_UCG-006 and Clostridium_sensu_stricto_1 （P<0.05） in the microbial community. Compared with the SARA group， the SARA+BA group had a smoother brush border of the ileal epithelium， the epithelial structure was complete， the villi height was significantly increased （P<0.05）， and the number of paneth cells was significantly increased （P<0.05）， but the number of cup cells in the villi and crypt was not significantly different （P>0.05）. Also， compared with the SARA group， the SARA+BA group showed increased transcript levels of IFN-γ in the mucosa （0.05<P<0.1）， increased transcript levels of IfnGR1 （P<0.05）， significantly increased bacterial diversity （Ace index） in the ileum microbial community （P<0.05）， and a lower relative abundance of the phylum Firmicutes （P<0.05）. At the genus level， the beneficial bacteria Unclassified_f_Lachnospiraceae， Ruminococcus and Christensenellaceae_R-7_group were more abundant in the SARA+BA group than in the SARA group （P<0.05）， and the harmful bacteria Erysipelotrichaceae_UCG-006 and Clostridium_sensu_stricto_1 were significantly less abundant （P<0.05）. In conclusion， feeding bile acid to goats with subacute ruminal acidosis improved the morphology and structure of the ileum of SARA goats， alleviated the disturbance of the microflora in the ileum， and alleviated damage to the ileum mucosa of SARA goats by up-regulating the relative mRNA expression levels of anti-inflammatory factor IFN-γ and receptor IfnGR1 in the mucosa.

Key words: bile acid, subacute ruminal acidosis, ileal mucosa, microflora composition, goat

Yue CHEN, Pin SONG, Man-man HOU, Xiao-ran YANG, Li-ping LIU, Ying-dong NI. Effects of dietary supplementation with bile acid on ileal epithelial morphology， microflora composition， and relative transcript levels of IFN-γ in the ileal mucosa of goats with subacute ruminal acidosis[J]. Acta Prataculturae Sinica, 2024, 33(12): 188-200.

Figures/Tables 11

Fig.1 Test flow chart （n=5）

Table 1 Dietary formulation and nutritional composition

日粮组成Diet ingredient	对照组CON （%）	高精料组SARA （%）	营养水平Nutrient level^2）	对照组CON	高精料组SARA
苜蓿草 Alfalfa	67.45	28.91	代谢能 ME （MJ·kg^-1）	1.87	1.90
玉米 Corn	20.41	57.03	粗蛋白 Crude protein （%）	16.79	16.73
豆粕 Soybean meal	8.50	10.42	中性洗涤纤维NDF （%）	33.71	28.79
磷酸氢钙 Calcium hydrophosphate	1.10	1.10	酸性洗涤纤维 ADF （%）	18.22	14.32
石粉 Limestone	1.20	1.20	粗灰分Ash （%）	4.74	4.83
食盐 Salt	0.40	0.40	粗脂肪 Ether extract （%）	3.76	3.61
预混料 Premix^1）	0.94	0.94	淀粉Starch （%）	24.25	31.21
总计 Total	100.00	100.00	钙Calcium （%）	0.95	0.96
			磷Phosphorus （%）	0.42	0.44

Table 2 Primers used for PCR

基因Gene	引物对序列Primer pairs sequence （5′→3′）	产物大小Product size （bp）	基因库编号GeneBank ID
IFN-γ	CAGAAGTTCTTGAACGGCAGC/CTGCAGATCATCCACCGGAAT	134	NM_001285682.1
IfnGR1	AACACAGAAGATCCTGTTTGGA/CACCACAGACAGCAAGGATT	147	XM_005684807.3
IfnGR2	GGCTCGCTTATCATCAGGCT/AAGGGTCTGTCACCTGCTTG	132	XM_018046479.1
IfnAR1	GCGCATAAGAGCAGAAGAAGG/GGGGGACCAATCTGAGCTTC	105	XM_018046479.1
GAPDH	GGGTCATCATCTCTGCACCT/GGTCATAAGTCCCTCCACGA	126	XM_005680968.3

Fig.2 Effect of dietary supplementation with bile acid on pH of rumen fluid of SARA goats （n=5）

Fig.3 Effect of dietary supplementation with bile acids on ileal mucosa morphology （A）， villus height and crypt depth （B） of goats with subacute ruminal acidosis（n=5）

Fig.4 Effect of dietary supplementation with bile acid on the number of goblet cells in ileal mucosa of goats （n=5）

Fig. 5 Effect of dietary supplementation with bile acids on number of paneth cells in ileal mucosa of goats （n=5）

Fig.6 Effects of dietary supplementation with bile acids on the relative expression levels of interferon γ and its receptor mRNA in ileal mucosa of SARA goats （n=5）

Fig. 7 Effect of dietary supplementation with bile acids on α （A） and β diversity （B） of bacteria in the ileal digesta in goats

Fig.8 Effect of dietary supplementation with bile acids on the relative abundance at phylum level （A） and genus level （B） of ileal bacteria in SARA goats

Fig.9 Different bacteria at phylum level （A） and different bacteria （B） in the top 20 relative abundances at genu level of ileal bacteria

References 39

1	Ba L J D M， Ao R G L， Wang C J， et al. Pathogenesis and research progress of subacute rumen acidosis in ruminants. Feed Research， 2023， 46（21）： 145-149.
	巴拉吉德玛，敖日格乐，王纯洁，等. 反刍动物亚急性瘤胃酸中毒的发病机理及研究进展. 饲料研究， 2023， 46（21）： 145-149.
2	Dong G， Liu S， Wu Y， et al. Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows： Impacts on immunity and metabolism. Acta Veterinaria Scandinavica， 2011， 53（1）： 48-55.
3	Acciarino A， Diwakarla S， Handreck J， et al. The role of the gastrointestinal barrier in obesity-associated systemic inflammation. Obesity Reviews， 2024， 25： e13673.
4	Elmhadi M E， Ali D K， Khogali M K， et al. Subacute ruminal acidosis in dairy herds： Microbiological and nutritional causes， consequences， and prevention strategies. Animal Nutrition， 2022， 10： 148-155.
5	Shi W R， Zhou F， Meng Q H， et al. Function and application in animal production of bile acids. Feed Research， 2023，46（15）： 167-172.
	施文瑞，周凡，孟庆辉，等. 胆汁酸的功能及其在动物生产中的应用. 饲料研究， 2023， 46（15）： 167-172.
6	He S Q， Li L X， Yao Y N， et al. Bile acid and its bidirectional interactions with gut microbiota： A review. Critical Reviews in Microbiology， 2023， doi： 10.1080/1040841X.2023.2262020.
7	Hegyi P， Maléth J， Walters J R， et al. Guts and gall： Bile acids in regulation of intestinal epithelial function in health and disease. Psychological Review， 2018， 98（4）： 1983-2023.
8	Cai J， Rimal B， Jiang C， et al. Bile acid metabolism and signaling， the microbiota， and metabolic disease. Clinical Pharmacology & Therapeutics， 2022， doi： 10.1016/j.pharmthera.2022.108238.
9	Tao S Y. Effects of high-concentrate diet on hindgut epithelial barrier in lactating dairy goats and the mechanisms involved. Nanjing： Nanjing Agricultural University， 2018.
	陶诗煜. 高精料日粮对泌乳期奶山羊后段肠道上皮屏障的影响及其机制. 南京：南京农业大学， 2018.
10	Deng L Q. Make regular paraffin sections quickly. Journal of Hubei University for Nationalities （Medical Edition）， 2001（4）： 59.
	邓利群. 快速制作常规石蜡切片. 湖北民族学院学报（医学版）， 2001（4）： 59.
11	Caporaso J G， Lauber C L， Walters W A， et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America， 2011， 108（S1）： 4516-4522.
12	Plaizier J C， Danesh M M， Derakhshani H， et al. Review： Enhancing gastrointestinal health in dairy cows. Animal， 2018， 12（S2）： 399-418.
13	Camilleri M， Madsen K， Spiller R， et al. Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterology and Motility， 2012， 24（6）： 503-512.
14	Chelakkot C， Ghim J， Ryu S H. Mechanisms regulating intestinal barrier integrity and its pathological implications. Experimental and Molecular Medicine， 2018， 50（8）： 1-9.
15	Tao S Y， Duan M L， Dong H B， et al. High concentrate diet induced mucosal injuries by enhancing epithelial apoptosis and inflammatory response in the hindgut of goats. PLoS One， 2014， 9（10）： e111596.
16	Lai Z， Lin L， Zhang J， et al. Effects of high-grain diet feeding on mucosa-associated bacterial community and gene expression of tight junction proteins and inflammatory cytokines in the small intestine of dairy cattle. Journal of Dairy Science， 2022， 105（8）： 6601-6615.
17	Huai Y Y， Liang Z S， Ji S L， et al. Effects of recombinant epidermal growth factor on the proliferation of the intestinal epithelial cells in early-weaned pigs. Animal Husbandry and Veterinary Medicine， 2016， 48（4）： 70-75 .
	槐玉英，梁梓森，纪少丽，等. 重组表皮生长因子对仔猪小肠上皮细胞增殖活性的影响. 畜牧与兽医， 2016， 48（4）： 70-75.
18	Guo Y S， Yan S M， Shi B L， et al. Effects of Lactobacillus fermentum supplementation on the small intestinal villus structure of broilers. Chinese Journal of Animal Nutrition， 2011， 23（7）： 1194-1200.
	郭元晟，闫素梅，史彬林，等. 发酵乳酸杆菌对肉鸡小肠绒毛形态的影响. 动物营养学报， 2011， 23（7）： 1194-1200.
19	Allaire J M， Crowley S M， Law H T， et al. The intestinal epithelium： Central coordinator of mucosal immunity. Trends in Immunology， 2018， 39（9）： 677-696.
20	Kim J J， Khan W I. Goblet cells and mucins： Role in innate defense in enteric infections. Pathogens， 2013， 2（1）： 55-70.
21	Okumura R， Takeda K. Roles of intestinal epithelial cells in the maintenance of gut homeostasis. Experimental and Molecular Medicine， 2017， 49（5）： e338.
22	van der Flier L G， Clevers H. Stem cells， self-renewal， and differentiation in the intestinal epithelium. Annual Review of Physiology， 2009， 71： 241-260.
23	Johansson M E， Hansson G C. Immunological aspects of intestinal mucus and mucins. Nature Reviews Immunology， 2016， 16（10）： 639-649.
24	Cui C， Li L， Wu L， et al. Paneth cells in farm animals： Current status and future direction. Journal of Animal Science and Biotechnology， 2023， 14（1）： 118.
25	Hou Q， Huang J， Ayansola H， et al. Intestinal stem cells and immune cell relationships： Potential therapeutic targets for inflammatory bowel diseases. Frontiers in Immunology， 2021， doi： 10.3389/fimmu.2020.623691.
26	Günther C， Martini E， Wittkopf N， et al. Caspase-8 regulates tnf-α-induced epithelial necroptosis and terminal ileitis. Nature， 2011， 477（7364）： 335-339.
27	Ergun E， Ergun L， Asti R N， et al. Light and electron microscopic morphology of paneth cells in the sheep small intestine. Revue De Medecine Veterinaire， 2003， 154（5）： 351-355.
28	El Bougrini J， Pampin M， Chelbi-Alix M K. Arsenic enhances the apoptosis induced by interferon gamma： Key role of IRF-1. Cellular and Molecular Biology （Noisy-le-grand）， 2006， 52（1）： 9-15.
29	Gocher A M， Workman C J， Vignali D A A. Interferon-gamma： Teammate or opponent in the tumour microenvironment？ Nature Reviews Immunology， 2022， 22（3）： 158-172.
30	Richard N， Savoye G， Leboutte M， et al. Crohn’s disease： Why the ileum？ World Journal of Gastroenterolog， 2023， 29（21）： 3222-3240.
31	Thoetkiattikul H， Mhuantong W， Laothanachareon T， et al. Comparative analysis of microbial profiles in cow rumen fed with different dietary fiber by tagged 16S rRNA gene pyrosequencing. Current Microbiology， 2013， 67（2）： 130-137.
32	Plaizier J C， Li S， Danscher A M， et al. Changes in microbiota in rumen digesta and feces due to a grain-based subacute ruminal acidosis （SARA） challenge. Microbial Ecology， 2017， 74（2）： 485-495.
33	Zhong X X， Huang J， Liu Z Y， et al. Effects of mannan oligosaccharides and complex probiotics on growth performance and intestinal morphology，volatile fatty acid contents and microbial structure of weaned piglets. Chinese Journal of Animal Nutrition， 2020， 32（7）： 3099-3108.
	钟晓霞，黄健，刘志云，等. 甘露寡糖和复合益生菌对断奶仔猪生长性能及肠道形态结构、挥发性脂肪酸含量和菌群结构的影响. 动物营养学报， 2020， 32（7）： 3099-3108.
34	Crost E H， Coletto E， Bell A， et al. Ruminococcus gnavus： Friend or foe for human health. FEMS Microbiology Reviews， 2023， https：//doi.org/10.1093/femsre/fuad014.
35	Zeng Y， Gao Y H， Peng Z L， et al. Effects of yeast culture supplementation in diets on rumen fermentation parameters and microflora of house-feeding yak. Chinese Journal of Animal Nutrition， 2020， 32（4）： 1721-1733.
	曾钰，高彦华，彭忠利，等. 饲粮中添加酵母培养物对舍饲牦牛瘤胃发酵参数及微生物区系的影响. 动物营养学报， 2020， 32（4）： 1721-1733.
36	Guo J， Mu R， Li S， et al. Characterization of the bacterial community of rumen in dairy cows with laminitis. Genes， 2021， doi： 10.3390/genes12121996.
37	Wang K， Ren A， Zheng M， et al. Diet with a high proportion of rice alters profiles and potential function of digesta-associated microbiota in the ileum of goats. Animals， 2020， doi： 10.3390/ani10081261.
38	Chiang J Y. Bile acid metabolism and signaling. Comprehensive Physiology， 2013， 3（3）： 1191-1212.
39	Fogelson K A， Dorrestein P C， Zarrinpar A， et al. The gut microbial bile acid modulation and its relevance to digestive health and diseases. Gastroenterology， 2023， 164（7）： 1069-1085.

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