断奶前后非特异病原性腹泻羔羊生长生理及肠道菌群差异性比较

doi:10.3864/j.issn.0578-1752.2021.02.017

摘要/Abstract

摘要：

【目的】羔羊腹泻是由病原微生物、营养改变、环境应激等多因素互作所致的新生和断奶时期危害羔羊生长的临床常见病之一。对断奶前后腹泻羔羊的生长生理与肠道菌群变化与其同期健康羔羊进行比较。为不同生长阶段羔羊腹泻的针对性防治研究奠定科学基础。【方法】在对羔羊腹泻进行临床调查的基础上,随机选择断奶前（31—45日龄）健康和腹泻羔羊各10只、断奶后（60—75日龄）健康和腹泻羔羊各10只分成4组进行生长生理、血液生理生化指标及相关炎性因子指标测定。采集直肠粪样,用16S rRNA测序进行肠道菌群组成结构比较分析。【结果】（1）断奶前健康与腹泻羔羊体重差异不显著。而断奶后腹泻羔羊体重极显著低于健康羔羊（P<0.01）,且腹泻羔羊的体温、脉搏和呼吸频率也均显著低于健康羔羊（P<0.05）。（2）断奶前腹泻羔羊总蛋白、球蛋白、胆固醇等生化指标显著低于健康羔羊（P<0.05）。断奶后腹泻羔羊白细胞总数极显著高于健康羔羊（P<0.01）,尿素氮、肌酐、磷、血糖和总胆红素显著低于健康羔羊（P<0.05）,炎性因子IL-4、IL-6、IL-8显著高于健康羔羊。（3）断奶后健康羔羊与腹泻羔羊肠道菌群共有OTUs占比较断奶前上升近一倍。断奶前腹泻羔羊肠道菌群Alpha多样性显著降低（P<0.05）。健康与腹泻羔羊的肠道菌群PCoA分布差异显著（Weighted UniFrac Adonis和Anosim检验水平,P<0.05）,而断奶后组间差异不显著。门水平：与其同期健康羔羊相比,断奶前腹泻羔羊梭杆菌门相对丰度显著升高（P<0.05）,疣微菌门和放线菌门相对丰度显著下降（P<0.05）。断奶后,腹泻羔羊黏胶球形菌门和放线菌门相对丰度显著下降（P<0.05）。属水平：与其同期健康羔羊相比,断奶前腹泻羔羊梭杆菌属和梭菌属显著升高（P<0.05）,而艾克曼菌属、脱硫弧菌属和丁酸弧菌属显著下降（P<0.05）。断奶后,腹泻羔羊瘤胃球菌属显著下降（P<0.05）,而脱硫弧菌属显著升高（P<0.05）。【结论】本研究结果表明断奶前腹泻羔羊主要发生蛋白质和血脂降低;而断奶后腹泻羔羊的生理变化以尿素氮、肌酐和血糖等指标的下降为特点。断奶前腹泻羔羊肠道菌群失调变化明显;而断奶后腹泻羔羊炎症反应等变化明显。这些差异可能与断奶所引起的日粮组成结构和环境因素改变密切相关。

关键词: 羔羊, 断奶, 腹泻, 生理生化与炎症因子, 肠道菌群

Abstract:

【Objective】Lamb diarrhea is one of the common clinical diseases caused by the certain pathogens or the interaction with malnutrition, abnormal management, and environmental stress during newborn and weaning periods, which hazards the lamb production. There is lack of enough investigation about the differences of lambs' physio-biochemistry with their gut microbiota caused by diarrhea between suckling and weaned periods. Therefore, this study was intended to compare the growth physiology with gut microbiota of diarrheic lambs in pre-and post-weaning period, so as to lay a scientific foundation for prevention and treatment of lamb diarrhea occurred at different growth stages precisely and effectively. 【Method】10 suckling healthy lambs and 10 diarrheic lambs at age of 31-45 day, and another 10 weaned healthy lambs and 10 diarrheic lambs at age of 60-75 day were selected and paired to check the growth status, blood physio-biochemistry and inflammatory factors. The rectal fecal samples were used to analyze gut microbiota composition and structure using 16S rRNA. 【Result】1. There was no significant difference in body weight between healthy and diarrhea lambs before weaning, but after weaning, the body weight of diarrheic lambs was extremely lower than that of healthy lambs (P<0.01), and the body temperature, pulse and respiratory rate of diarrheic lambs were also significantly lower than those of healthy lambs (P<0.05). 2. Before weaning, the total protein, globulin, and cholesterol in diarrheic lambs were significantly lower than those in healthy lambs (P<0.05). After weaning, the total number of leukocytes in diarrheic lambs was extremely higher than that of healthy lambs (P<0.01). And the blood urea, nitrogen, creatinine, phosphorus, glucose and total bilirubin in diarrheic lambs were significantly lower than those in healthy lambs (P<0.05). After weaning, IL-4, IL-6 and IL-8 in diarrheic lambs were significantly higher than those in healthy lambs (P<0.05). 3. The percentage of common OTUs in the gut of healthy and diarrheic lambs after weaning was almost as doubled as that before weaning. Alpha diversity of the gut microbiota in diarrheic lambs before weaning was significantly reduced as compared to their healthy counterpart (P<0.05). And Beta diversity of the gut microbiota weighted PCoA in diarrheic lambs before weaning was significantly different from that in the healthy lambs (Weighted UniFrac Adonis and Anosim tests, P<0.05). At phylum level: before weaning, the relative abundance of Fusobacteria was significantly increased (P<0.05), whereas that of Verrucomicrobia and Actinobacteria was significantly decreased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). After weaning, the relative abundance of Lentisphaerae and Actinobacteria was significantly reduced as compared to their healthy counterpart (P<0.05). At genus level: before weaning, the relative abundance of Fusobacterium and Clostridium were significantly increased, while Akkermansia, Desulfovibrio and Butyricimonas were significantly decreased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). After weaning, the relative abundance of Ruminococcus was significantly decreased, while Desulfovibrio significantly was increased in the gut of diarrheic lambs as compared to their healthy counterpart (P<0.05). 【Conclusion】It was concluded that before weaning the content of blood proteins and the blood fat decreased significantly, whereas after weaning, the elevation of white blood cells and the decrease of CREA, UREA and the blood glucose was significant in diarrheic lambs. Before weaning, the more obvious gut microbiota dysbiosis was observed, whereas after weaning more significant gut inflammatory activity was observed in diarrheic lambs. The ration structure and the environment during weaning time could be the crucial factors for shifting aforementioned differences.

Key words: lamb, weaning, diarrhea, physio-biochemical and inflammatory factor, gut microbiota

杨柠芝,李婷,王燕,陈卓,马义诚,任强林,刘佳佳,杨会国,姚刚. 断奶前后非特异病原性腹泻羔羊生长生理及肠道菌群差异性比较[J]. 中国农业科学, 2021, 54(2): 422-434.

YANG NingZhi,LI Ting,WANG Yan,CHEN Zhuo,MA YiCheng,REN QiangLin,LIU JiaJia,YANG HuiGuo,YAO Gang. Comparison of Growth Physiology and Gut Microbiota Between Healthy and Diarrheic Lambs in Pre- and Post-Weaning Period[J]. Scientia Agricultura Sinica, 2021, 54(2): 422-434.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.02.017

https://www.chinaagrisci.com/CN/Y2021/V54/I2/422

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分组 Grouping	日龄（天） Age(d)	健康组 Health group	腹泻组 Diarrhea group
断奶前（Suckling）	31-45	H组（Health）	D组（Diarrhea）
断奶后（Weaned）	60-75	h组（health）	d组（diarrhea）

原料 Ingredient	日粮组成 Diet composition (%)		营养成分 Nutritional components	营养水平 Nutrient levels
原料 Ingredient	断奶前Suckling	断奶后Weaned	营养成分 Nutritional components	断奶前Suckling	断奶后Weaned
玉米Corn	43.12	25.20	干物质 DM (%)	82.29	86.65
麸皮Wheat bran	3.75	4.40	消化能DE (MJ·kg^-1)	10.41	6.43
豆粕Sybean meal	16.50	4.40	粗蛋白 CP (%)	16.76	10.76
棉粕Cottonseed meal	3.00	1.20	粗纤维 CF (%)	11.01	24.48
葵粕Sunflower meal	2.63	2.00	粗灰分Ash (%)	4.43	6.27
酵母Brewers dried yeast	0.70	1.60	钙 Ca (%)	0.57	0.51
食盐NaCl	0.80	0.40	总磷 P (%)	0.34	0.23
石粉Limestone	0.80	0.40
碳酸氢钠Sodium bicarbonate	3.70	0.40
麦秸Wheat straw	10.00	40.00
苜蓿草粉Alfalfa meal	14.00	19.00
复合预混料Premix	1.00	1.00
合计Total	100.00	100.00

日龄 Age (d)	总头数 N. lambs	腹泻只（次）数 N. diarrhea	腹泻率 Diarrhea rate	死亡数 N. death	病死率 Mortality
1-15	80	34	2.83	18	52.90
16-30	122	55	3.01	12	21.80
31-45	59	22	2.49	9	40.90
60-75	200	68	2.27	14	20.60
总计Total	461	179	2.59	53	29.60

指标 Index	断奶前Suckling		断奶后Weaned
指标 Index	健康Health	腹泻Diarrhea	健康Health	腹泻Diarrhea
白细胞WBC	8.69±0.39	9.12±0.45	8.96±0.36A	11.20±0.47B
淋巴细胞LY	4.42±0.32	5.00±0.26	5.84±0.34	6.23±0.42
中性粒细胞Gran	4.14±0.46	4.33±0.85	5.36±0.87	5.29±0.72
血红蛋白HGB	107.00±3.78	108.00±5.16	127.00±4.44	141.00±5.06
红细胞RBC	9.43±0.24	8.97±0.43	8.69±0.24	9.30±0.34
血小板PLT	507.00±38.9	487.00±38.40	750.00±40.00	768.00±55.00

指标 Index	断奶前Suckling		断奶后Weaned
指标 Index	健康Health	腹泻Diarrhea	健康health	腹泻diarrhea
碱性磷酸酶ALKP(U·L^-1)	355.00±90.30	169.20±73.13	38.80±9.20	69.80±38.27
丙氨酸转移酶ALT(U·L^-1)	186.40±152.42	228.00±258.80	37.20±16.77	37.40±18.77
胰淀粉酶AMYL(U·L^-1)	64.60±12.88	59.20±34.78	32.00±30.54	54.80±30.26
白蛋白ALB(g·L^-1)	19.00±2.35	19.60±1.82	15.20±5.72	19.80±6.61
球蛋白GLOB(g·L^-1)	48.80±3.97a	35.60±5.35b	56.80±12.48	47.40±14.08
总蛋白TP(g·L^-1)	66.40±15.79a	55.00±5.24b	72.00±9.05	67.20±9.41
胆固醇CHOL(mmol·L^-1)	3.70±1.87a	2.52±0.86b	0.58±0.34	0.77±0.22
肌酐CREA(mmol·L^-1)	39.00±14.07	27.20±5.72	35.80±7.60A	22.90±5.60B
尿素氮UREA(mmol·L^-1)	4.18±1.45	3.64±1.28	9.15±2.50A	6.48±0.94B
总胆红素BIL(mmol·L^-1)	2.80±1.64	2.00±2.23	4.00±2.94a	1.20±0.44b
血糖GLU(mmol·L^-1)	5.36±0.71	5.42±1.72	4.85±1.01a	3.93±0.64b
钙离子Ca(mmol·L^-1)	2.50±0.08	2.35±0.20	2.14±0.27	2.11±0.10
磷PHOS(mmol·L^-1)	2.89±0.82a	2.18±0.40b	2.52±0.58A	1.64±0.31B