Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 7: 4333 - 4348

DOI: https://doi.org/10.12418/CJAN2025.355

RESEARCH PAPER

Effects of Compound Preparation of Clostridium butyricum Metabolites and Live Bacteria on Growth Performance and Intestinal Health of Weaned Piglets

  • WU Yifan , 1 ,
  • YU Xiaoyi 1 ,
  • WU Yujun 1 ,
  • LIU Jingjing 2 ,
  • ZANG Jianjun 1, 3 ,
  • HE Chunlan 2 ,
  • LI Xueping 2 ,
  • ZHAO Jinbiao 1, 3 ,
  • HAN Dandan 1, 3 ,
  • WANG Junjun , 1, 3, *
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  • 1 State Key Laboratory of Animal Nutrition and Feeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
  • 2 Jiangxi Heswof Biotechnology Co., Ltd., Yichun 336000, China
  • 3 The Professor Workstation of China Agricultural University, Yichun 336000, China
* professor, E-mail:

Received date: 2024-12-13

  Online published: 2025-07-12

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Abstract

The purpose of this study was to investigate the effects of dietary supplementation of compound preparation of Clostridium butyricum metabolites and live bacteria on growth performance and intestinal health of weaned piglets. A total of 192 healthy “Duroc×Landrace×Large White” weaned piglets with similar body weight at 28 days of age were selected and randomly divided into 4 group, with 6 replicates in each group and 8 piglets in each replicate (half male and half female). Piglets in the negative control group (NC group) were fed a basal diet, those in the positive control group (PC group) were fed the basal diet supplemented with 4 kg/t benzoic acid, and those in the experimental groups were fed the basal diets supplemented with 1 (MZ1 group) and 2 kg/t compound preparation (MZ2 group), respectively. The experiment lasted for 42 days and was divided into two stages as the early stage (days 1 to 21) and the late stage (days 22 to 42). The results showed as follows: 1) compared with NC group and PC group, the body weight of weaned piglets on day 42 in MZ2 group was significantly increased (P<0.05), and the average daily weight gain during days 22 to 42 and days 1 to 42 was significantly increased (P<0.05); compared with NC group, the diarrhea rate at each stage in MZ2 group was significantly decreased (P<0.05). 2) Compared with NC group, the serum immunoglobulin M content in MZ2 group was significantly increased (P<0.05), and the serum contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly decreased (P<0.05). 3) Compared with NC group, the villus height and villus height to crypt depth ratio (V/C) in jejunum and ileum in MZ2 group were significantly increased (P<0.05), the mRNA relative expression levels of claudin-1 and mucin-2 in jejunum, as well as occludin, claudin-1, mucin-1 and mucin-2 in ileum were significantly increased (P<0.05), and the mRNA relative expression levels of interleukin-6 (IL-6) and IL-1β mRNA in jejunum and ileum, as well as TNF-α in ileum were significantly decreased (P<0.05). 4) Compared with NC group, the contents of acetic acid, propionic acid and butyric acid in ileum, as well as butyric acid in colon in MZ2 group were significantly increased (P<0.05). 5) Compared with NC group and PC group, the relative abundances of Clostridium_sensu_stricto_1, Terrisporobacter and Turicibacter in ileum, as well as Clostridium_sensu_stricto_1, Terrisporobacter and Lachnospiraceae_ND3007_group in colon in MZ2 group were significantly increased (P<0.05), while the Streptococcus relative abundance in ileum and colon was significantly decreased (P<0.05). In conclusion, dietary supplementation of 2 kg/t compound preparation of Clostridium butyricum metabolites and live bacteria can improve the growth performance of weaned piglets, reduce the diarrhea rate, enhance the immune and anti-inflammatory capabilities, and promote intestinal health by improving intestinal morphology, strengthening barrier function and regulating short chain fatty acids and microbiota composition.

Key words： Clostridium butyricum metabolites; probiotics; compound preparation; weaned piglets; growth performance; intestinal health

Cite this article

WU Yifan , YU Xiaoyi , WU Yujun , LIU Jingjing , ZANG Jianjun , HE Chunlan , LI Xueping , ZHAO Jinbiao , HAN Dandan , WANG Junjun . Effects of Compound Preparation of Clostridium butyricum Metabolites and Live Bacteria on Growth Performance and Intestinal Health of Weaned Piglets[J]. Chinese Journal of Animal Nutrition, 2025 , 37(7) : 4333 -4348 . DOI: 10.12418/CJAN2025.355

仔猪良好的机体状况是生猪养殖产业生产力的基础,同时直接影响规模化猪场的经济效益。仔猪在断奶早期肠道发育和免疫系统尚未发育成熟,易遭受病原微生物的侵害,导致仔猪肠道菌群紊乱、屏障功能受损等[1],常表现出生长缓慢和腹泻等问题[2]。研究人员长期致力于探索营养调控策略,以此来缓解仔猪的断奶应激。
研究表明,断奶应激导致的肠道损伤往往会伴随着肠道菌群紊乱,进而导致肠道中短链脂肪酸(short chain fatty acids,SCFAs)含量的降低,引发机体免疫失调和对疾病的易感性[3]。丁酸作为一类SCFAs,在维持肠道正常功能中发挥着关键作用。此外,丁酸是结肠上皮重要的能量来源,断奶后补充丁酸钠可提高哺乳仔猪断奶窝重[4],同时可以通过激活G蛋白偶联受体109A(G protein-coupled receptor 109A,GPR109A)促进断奶仔猪结肠密封蛋白-1(claudin-1)的表达,从而改善肠道健康[5]。益生菌等活菌制剂已被证明可以通过维持动物肠道微生态平衡及促进SCFAs的生成来提高动物机体免疫力和生长性能。多种益生菌如屎肠球菌(Enterococcus faecium,E. faecium)、植物乳杆菌(Lactobacillus plantarum,L. plantarum)和枯草芽孢杆菌(Bacillus subtilis,B. subtilis)等已被列入饲料添加剂品种目录(2013)。研究表明,在饲粮中添加Lactiplantibacillus argentoratensis AGMB00912可以提高断奶仔猪肠道中丁酸含量,提高有益菌乳杆菌属(Lactobacillus)、Anaerobutyricum、罗斯氏菌属(Roseburia)、毛螺菌科(Lachnospiraceae)和布劳特氏菌属(Blautia)相对丰度,降低致病菌螺杆菌属(Helicobacter)和弯曲菌属(Campylobacter)相对丰度[6]。丁酸梭菌(Clostridium butyricum,C. butyricum)是一种以丁酸为主要代谢产物的严格厌氧菌,已被证明可以通过恢复肠道微生物群稳态、诱导效应T细胞分泌以及提高肠道黏蛋白-2(mucin-2)表达,以此调节肠道功能,降低腹泻率并提高生长性能[7-10]。饲粮中预先补充E. faecium NCIMB 10415能够恢复大肠杆菌感染的仔猪空肠组织的屏障和运输功能[11]。L. plantarum L47和菊粉混合物通过上调脂磷壁酸的表达,减轻产肠毒素大肠杆菌诱导的断奶仔猪回肠炎症[12]。B. subtilis也可以用于缓解葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的兔结肠炎等炎症性肠病的症状,并提高双歧杆菌等有益菌的相对丰度,促进肠道健康[13]。这些益生菌能够通过刺激肠道屏障基因表达、降低促炎细胞因子释放以及提高菌群多样性等,阻止病原微生物入侵。
SCFAs和益生菌在肠道中相互促进,共同维护肠道健康[14]。目前,丁酸、益生菌或益生元单独用于仔猪生长发育的益生作用已经被揭示。自1995年合生元概念提出以来,大量研究表明益生菌与益生元的组合效果优于单独使用。因此,本研究假设相较于直接添加丁酸盐,在仔猪饲粮中添加丁酸梭菌代谢产物加益生菌的模式在提高仔猪生长性能和改善肠道发育方面具有更好的效果。基于此,本试验使用抗腹泻效果较好的苯甲酸作为阳性对照,旨在探究在饲粮中添加不同剂量的丁酸梭菌代谢产物及3种活菌制剂(E. faecium、L. plantarum和B. subtilis)对断奶仔猪生长性能、肠道屏障功能及肠道菌群等的作用,为缓解断奶应激造成的仔猪腹泻及生长发育缓慢问题提供参考依据。

1 材料与方法

1.1 试验材料

本研究中使用的复合制剂(MZ)由丁酸梭菌代谢产物和3种益生菌复合而成。丁酸梭菌代谢产物是以定向选育的内源性丁酸梭菌菌株和天然底物(葡萄糖和玉米浆干粉等),采用“高产SCFAs”技术(流加补料等)天然发酵制成的混合物,富含丁酸、乙酸等SCFAs,产品中的丁酸以丁酸钙的形式存在,丁酸钙盐含量≥32%;3种益生菌分别为E. faecium(有效菌数≥1.0×108 CFU/g)、L. plantarum(有效菌数≥1.0×108 CFU/g)和B. subtilis(有效菌数≥2.0×108 CFU/g)。

1.2 试验设计及饲粮

本试验经中国农业大学实验动物福利与动物实验伦理审查委员会批准(批准编号:AW50214202-1-04),在农业农村部饲料工业中心丰宁动物试验基地进行。
选取28日龄初始体重为7.63 kg的健康断奶仔猪(杜×长×大)192头,随机分为4组,每组6个重复,每个重复8头猪(公母各占1/2)。阴性对照组(NC组)饲喂基础饲粮,阳性对照组(PC组)饲喂在基础饲粮的基础上添加4 kg/t苯甲酸的饲粮,试验组分别饲喂在基础饲粮的基础上添加1(MZ1组)和2 kg/t复合制剂(MZ2组)的饲粮。试验期42 d,分为前期(第1~21天)和后期(第22~42天)2个阶段。
基础饲粮参照NRC(2012)断奶仔猪营养标准配制,其组成及营养水平见表1。饲粮粗蛋白质(CP)含量参照GB/T 6432—2018中方法测定,钙和总磷含量分别参照GB/T 6436—2018和GB/T 6437—2018中方法测定;消化能=摄入总能-粪便总能,总能参照ISO 9831:1998中方法测定。
表1 基础饲粮组成及营养水平(饲喂基础)

Table 1 Composition and nutrient levels of basal diets (as-fed basis)%

项目
Items		 含量 Content
前期(第1~21天)
Early stage (days 1 to 21)		 后期(第22~42天)
Later stage (days 22 to 42)
原料 Ingredients
玉米 Corn 61.65 67.06
豆粕 Soybean meal 15.00 17.00
鱼粉 Fish meal 5.00 3.00
膨化大豆 Extruded soybean 6.00 6.00
乳清粉 Whey powder 8.00 3.00
大豆油 Soybean oil 0.35 0.40
磷酸氢钙 CaHPO4 1.15 1.10
石粉 Limestone 0.70 0.70
食盐 NaCl 0.20 0.20
L-赖氨酸盐酸盐 L-lysine·HCl 0.61 0.64
DL-蛋氨酸 DL-methionine 0.12 0.12
L-苏氨酸 L-threonine 0.22 0.23
L-色氨酸 L-tryptophan 0.50 0.05
预混料 Premix1) 0.50 0.50
合计 Total 100.00 100.00
营养水平 Nutrient levels2)
消化能 DE/(MJ/kg) 14.44 14.45
粗蛋白质 Crude protein 18.98 18.28
钙 Calcium 0.91 0.75
总磷 Total phosphorus 0.71 0.63

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:VA 12 000 IU,VD3 2 500 IU,VE 30 IU,VK3 3 mg,VB1 2.5 mg,VB2 4.0 mg,VB6 3.0 mg,VB12 12 μg,烟酸 niacin 40 mg,硫胺素 thiamine 3 mg,核黄素riboflavin 6 mg,泛酸 pantothenic acid 5 mg,叶酸 folic acid 0.7 mg,生物素 biotin 50 μg,Fe 90.0 mg,Cu 25 mg,Zn 75 mg,Mn 40 mg,I 0.35 mg,Se 0.3 mg。
2)营养水平均为实测值。Nutrient levels were measured values.

1.3 饲养管理和样品采集

试验期间仔猪自由采食和饮水,严格遵守猪场饲养管理制度,按照农场的常规程序对仔猪进行消毒、免疫和驱虫。
分别于试验第21天和第42天,每组采集6头(公母各3头,每个重复1头)仔猪新鲜粪便和前腔静脉血液样本。粪便样本在液氮中快速冷冻,保存于-80 ℃冰箱,用于微生物分析;血液样本离心后获得血清,保存于-20 ℃冰箱,用于血清指标分析。试验第42天,每个重复随机选择1头(每组6头,公母各3头)接近各组平均体重的仔猪进行屠宰和取样。屠宰后采集回肠和空肠组织固定在4%多聚甲醛中,用于形态学分析;采集回肠和空肠肠段,保存于-80 ℃冰箱,用于基因表达的分析;采集回肠和结肠食糜,保存于-80 ℃冰箱,用于SCFAs含量和微生物分析。

1.4 测定指标及方法

1.4.1 生长性能和腹泻率

试验期间,每天记录饲粮投喂量和剩余量;于试验第21天和第42天,分别称量仔猪体重,并计算仔猪各阶段平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。
采用视觉评估法观察仔猪腹泻情况,每天07:30—21:30连续观测仔猪排粪情况(粪便颜色、形状、排粪次数),对每只猪每次排出的粪便进行记载和感观评分,统计腹泻个体数并计算腹泻率。腹泻评分标准:条形或粒形粪便(0分);软粪、成形(1分);稠状、成形、粪水无分离现象(2分);液状、不成形、粪水有分离现象(3分)。将得分为2分和3分者确定为腹泻。腹泻率计算公式如下:
腹泻率(%)=100×仔猪腹泻头数总和/(仔猪头数×试验天数)。

1.4.2 血清生化和免疫指标

血清生化指标采用全自动生化分析仪(CLS880,江苏泽成生物技术有限公司)测定,包括血清总蛋白(TP)、白蛋白(ALB)、球蛋白(GLB)、葡萄糖(GLU)、尿素氮(UN)含量及谷草转氨酶(AST)、谷丙转氨酶(ALT)活性;血清免疫球蛋白A(IgA)、免疫球蛋白G(IgG)、免疫球蛋白M(IgM)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)和肿瘤坏死因子-α(TNF-α)含量采用猪酶联免疫吸附试验(ELISA)试剂盒(武汉伊莱瑞特生物科技股份有限公司)进行测定。

1.4.3 肠道组织形态学分析

取出4%多聚甲醛固定的肠道样品,经脱水、透明、包埋、冷却凝固和修正后制成包埋蜡块,再依次经过切片、漂浮和烤片制成切片,进行苏木精-伊红(HE)染色后,用显微镜观察和测定绒毛高度(villus height,VH)和隐窝深度(crypt depth,CD),计算绒毛高度/隐窝深度(V/C)值。

1.4.4 肠道食糜SCFAs含量

称取500 mg回肠和结肠食糜制备原液,移入10 mL离心管中,用8 mL超纯水充分混合并振荡1~2 min;吸取160 μL原液到新的10 mL离心管中,补超纯水7.84 mL至8 mL,振荡混匀;用2 mL注射器取稀释液,过滤膜(0.22 μm)装入进样瓶。采用离子色谱仪(戴安ICS-3000)测定SCFAs(乙酸、丙酸和丁酸)含量。

1.4.5 实时荧光定量PCR(RT-qPCR)

采用TRIzol法(北京艾德莱生物科技有限公司)提取肠道组织总RNA,测定浓度和纯度。使用RNA反转录试剂盒(北京擎科生物科技股份有限公司)SynScriptTM Ⅲ cDNA Synthesis Mix将1 μg总RNA逆转录成cDNA。使用2×TSINGKE® Master qPCR混合物(SYBR Green Ⅰ)(北京擎科生物科技股份有限公司)在Light Cycler System(Roche,美国)上进行RT-qPCR测定。以β-肌动蛋白(β-actin)作为内参基因,测定肠道屏障功能相关基因包括claudin-1、闭合蛋白(occludin)、闭锁小带蛋白-1(ZO-1)、黏蛋白-1(mucin-1)、mucin-2以及炎症细胞因子相关基因包括IL-1β、IL-6、IL-10、TNF-α,RT-qPCR引物(引物序列见表2)由北京擎科生物科技股份有限公司合成。测定数据按照2-ΔΔCt方法计算目的基因mRNA相对表达量。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')
密封蛋白-1
Claudin-1		 F:AAGGACAAAACCGTGTGGGA
R:CTCTCCCCACATTCGAGATGATT
闭合蛋白
Occludin		 F:CAGTGGTAACTTGGAGGCGT
R:CCGTCGTGTAGTCTGTCTCG
闭锁小带蛋白-1
ZO-1		 F:GCCATCCACTCCTGCCTAT
R:CGGGACCTGCTCATAACTTC
黏蛋白-1
Mucin-1		 F:GTGCCGACGAAAGAACTG
R:TGCCAGGTTCGAGTAAGAG
黏蛋白-2
Mucin-2		 F:CTGTGTGGGGCCTGACAA
R:AGTGCTTGCAGTCGAACTCA
白细胞介素-1β
IL-1β		 F:CGTCTGCGATGATGACCCAA
R:AGCACCAGGGCTTTTTCATCT
白细胞介素-6
IL-6		 F:ACCGGTCTTGTGGAGTTTCA
R:GGTGGTGGCTTTGTCTGGAT
白细胞介素-10
IL-10		 F:CGGCGCTGTCATCAATTTCT
R:GGCTTTGTAGACACCCCTCTC
肿瘤坏死因子-α
TNF-α		 F:TTATCGGCCCCCAGAAGGAA
R:CGACGGGCTTATCTGAGGTT
β-肌动蛋白
β-actin		 F:TGGAACGGTGAAGGTGACAG
R:CTTTTGGGAAGGCAGGGACT

1.4.6 微生物16S rRNA测序

使用QIAamp R Fast DNA Stool Mini Kit(Qiagen,德国)提取肠道食糜总基因组DNA。使用2%琼脂糖凝胶电泳检测扩增产物,使用AxyPrep DNA凝胶提取试剂盒(Axygen Bio,美国)纯化,并通过Qubit 2.0荧光计(Thermo Fisher Scientific,美国)定量。将纯化的PCR产物合并为等摩尔量,并在Illumina HiSeq 2500平台上测序,产生300 bp的配对末端读数。基于Illumina HiSeq高通量测序平台对16S rRNA基因V3~V4区(338F(5'-ACTCCTACGGGAGGCAGCAG-3')和806R(5'-GGACTACHVGGGTWTCTAAT-3')进行PCR扩增、文库构建和测序,获得具有代表性的扩增序列变异(amplicon sequence variants,ASVs)序列。相关分析过程在美吉生物云平台上进行。

1.5 数据统计分析

试验数据经过Excel 2019处理后,采用SPSS 26.0软件进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行多重比较,结果数据采用平均值和均值标准误(SEM)表示;采用GraphPad Prism 9.4软件进行图片制作,P<0.05表示差异显著,0.05≤P<0.10表示差异有显著趋势。

2 结果与分析

2.1 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪生长性能和腹泻率的影响

由表3可知,与NC组和PC组相比,MZ2组断奶仔猪第42天体重显著提高(P<0.05);各组间第21天体重无显著差异(P>0.05)。第1~21天,各组间ADFI、ADG和F/G均无显著差异(P>0.05);与NC组相比,MZ1组、MZ2组和PC组腹泻率均显著降低(P<0.05);与PC组相比,MZ2组腹泻率显著降低(P<0.05)。第22~42天,与NC组、PC组和MZ1组相比,MZ2组ADG显著提高(P<0.05);与NC组和MZ1组相比,MZ2组和PC组腹泻率显著降低(P<0.05),且MZ2组与PC组间无显著差异(P>0.05);各组间ADFI和F/G无显著差异(P>0.05)。第1~42天,与NC组相比,MZ1组和MZ2组ADG显著提高(P<0.05);与PC组和MZ1组相比,MZ2组ADG显著提高(P<0.05);与NC组和MZ1组相比,MZ2组和PC组腹泻率显著降低(P<0.05),且MZ2组与PC组间无显著差异(P>0.05);各组间ADFI和F/G无显著差异(P>0.05)。
表3 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪生长性能和腹泻率的影响

Table 3 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on growth performance and diarrhea rate of weaned piglets

项目
Items		 时间
Time		 组别 Groups 均值标准误
SEM		 P值
P-value
NC MZ1 MZ2 PC


体重
BW/kg		 第1天 Day 1 7.61 7.66 7.68 7.51 0.253 0.895
第21天 Day 21 16.95 17.33 17.36 16.85 0.405 0.499
第42天 Day 42 29.51b 30.38ab 31.40a 29.93b 0.611 0.017


平均日采食量
ADFI/g		 第1~21天 Days 1 to 21 672.22 678.77 680.67 675.47 12.210 0.906
第22~42天 Days 22 to 42 1 079.25 1 093.49 1 111.24 1 094.93 26.353 0.687
第1~42天 Days 1 to 42 875.74 886.13 895.95 885.20 18.367 0.750


平均日增重
ADG/g		 第1~21天 Days 1 to 21 445.19 460.12 460.71 445.21 9.099 0.137
第22~42天 Days 22 to 42 598.12b 621.33b 668.80a 622.50b 11.884 <0.001
第1~42天 Days 1 to 42 521.65c 540.72b 564.76a 533.85bc 9.048 0.001


料重比
F/G		 第1~21天 Days 1 to 21 1.51 1.47 1.47 1.52 0.082 0.256
第22~42天 Days 22 to 42 1.81 1.75 1.66 1.75 0.074 0.252
第1~42天 Days 1 to 42 1.67 1.63 1.58 1.65 0.077 0.250


腹泻率
Diarrhea rate/%		 第1~21天 Days 1 to 21 7.04a 5.95b 4.76d 5.26c 0.232 <0.001
第22~42天 Days 22 to 42 4.66a 4.76a 3.87b 3.97b 0.308 0.001
第1~42天 Days 1 to 42 5.85a 5.36a 4.32b 4.61b 0.261 0.001

同行数据肩标相同字母或无字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下表同。

In the same row, values with the same letter or no letter superscripts indicated no significant difference (P>0.05), while with different letter superscripts indicated significant difference (P<0.05). The same as below.

综合生长性能和腹泻率结果,MZ2组比MZ1组提升和改善效果更佳,因此后续指标选择MZ2组与NC组和PC组进行比较。

2.2 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪血清生化和免疫指标的影响

由表4可知,各组间断奶仔猪血清TP、ALB、GLB、GLU、UN含量及AST、ALT活性均无显著差异(P>0.05)。与NC组相比,MZ2组和PC组血清IgM含量显著提高(P<0.05),且MZ2组与PC组间无显著差异(P>0.05);MZ2组血清IgA含量有提高趋势,但差异不显著(P>0.05);各组间血清IgG含量无显著差异(P>0.05)。与NC组相比,MZ2组和PC组血清TNF-α和IL-1β含量显著降低(P<0.05),且MZ2组与PC组间无显著差异(P>0.05);各组间血清IL-6和IL-10含量无显著差异(P>0.05)。
表4 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪血清生化和免疫指标的影响

Table 4 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on serum biochemical and immune indices of weaned piglets

项目
Items		 组别 Groups 均值标准误
SEM		 P值
P-value
NC MZ2 PC
总蛋白 TP/(g/L) 57.90 57.27 57.84 2.987 0.974
白蛋白 ALB/(g/L) 31.78 31.94 31.70 1.338 0.984
球蛋白 GLB/(g/L) 26.12 25.34 26.14 3.033 0.956
葡萄糖 GLU/(mmol/L) 5.41 5.62 5.34 0.374 0.752
尿素氮 UN/(g/L) 2.32 2.09 1.99 0.427 0.744
谷草转氨酶 AST/(U/L) 52.09 49.02 49.33 9.067 0.936
谷丙转氨酶 ALT/(U/L) 50.08 50.83 48.86 5.230 0.937
免疫球蛋白A IgA/(g/L) 1.12 1.29 1.25 0.070 0.071
免疫球蛋白M IgM/(g/L) 0.88b 1.03a 1.02a 0.052 0.006
免疫球蛋白G IgG/(g/L) 9.48 9.62 9.54 0.159 0.225
白细胞介素-1β IL-1β/(ng/L) 308.60a 264.05b 272.07b 13.944 0.014
白细胞介素-6 IL-6/(ng/L) 56.82 50.72 53.31 3.902 0.319
白细胞介素-10 IL-10/(ng/L) 26.13 25.04 23.52 1.783 0.364
肿瘤坏死因子-α TNF-α/(ng/L) 62.73a 54.36b 55.98b 2.990 0.031

2.3 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道形态以及屏障功能和炎症细胞因子相关基因表达的影响

如图1所示,NC组断奶仔猪空肠和回肠绒毛完整性较差,排列相对松散;MZ2组和PC组空肠和回肠绒毛结构完整,排列整齐,无明显组织学损伤。由表5可知,与NC组相比,MZ2组断奶仔猪空肠VH和V/C值显著提高(P<0.05),PC组空肠V/C值显著提高(P<0.05),MZ2组和PC组空肠CD无显著差异(P>0.05);MZ2组回肠VH和V/C值显著提高(P<0.05),PC组回肠VH显著提高(P<0.05),MZ2组和PC组回肠CD无显著差异(P>0.05)。
图1 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道形态的影响

Fig.1 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on intestinal morphology of weaned piglets

表5 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道形态的影响

Table 5 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on intestinal morphology of weaned piglets

项目
Items		 组别 Groups 均值标准误
SEM		 P值
P-value
NC MZ2 PC


空肠 Jejunum		 绒毛高度 Villus height/μm 579.93b 626.89a 603.21ab 15.649 0.029
隐窝深度 Crypt depth/μm 197.78 190.08 193.77 5.831 0.439
绒毛高度/隐窝深度 V/C 2.94b 3.30a 3.12a 0.085 0.003


回肠 Ileum		 绒毛高度 Villus height/μm 565.10b 646.07a 612.23a 16.437 0.001
隐窝深度 Crypt depth/μm 191.70 181.14 187.51 7.091 0.351
绒毛高度/隐窝深度 V/C 2.97b 3.58a 3.27ab 0.157 0.005
如图2所示,与NC组相比,MZ2组断奶仔猪空肠claudin-1和mucin-2 mRNA相对表达量显著提高(P<0.05),空肠occludin、ZO-1和mucin-1 mRNA相对表达量无显著差异(P>0.05);与NC组相比,MZ2组回肠occludin、claudin-1、mucin-1和mucin-2 mRNA相对表达量显著提高(P<0.05),回肠ZO-1 mRNA相对表达量无显著差异(P>0.05)。此外,与PC组相比,MZ2组回肠occludin、claudin-1和mucin-2 mRNA相对表达量显著提高(P<0.05) 。
图2 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道屏障功能相关基因表达的影响

数据柱标记不同字母表示差异显著(P<0.05)。图3和图4同。

Fig.2 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on intestinal barrier function related gene expression of weaned piglets

Value columns marked with different letters indicated significant difference (P<0.05). The same as Fig.3 and Fig.4.

如图3所示,与NC组相比,MZ2组和PC组断奶仔猪空肠和回肠IL-6 mRNA相对表达量均显著降低(P<0.05),MZ2组空肠和回肠IL-1β mRNA相对表达量显著降低(P<0.05);MZ2组回肠TNF-α mRNA相对表达量显著降低(P<0.05),空肠TNF-α mRNA相对表达量无显著差异(P>0.05);MZ2组和PC组空肠和回肠IL-10mRNA相对表达量均无显著差异(P>0.05)。此外,与PC组相比,MZ2组回肠IL-1β mRNA相对表达量显著降低(P<0.05)。
图3 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道炎症细胞因子相关基因表达的影响

Fig.3 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on intestinal immune cytokine related gene expression of weaned piglets

2.4 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道SCFAs含量和菌群的影响

如图4所示,与NC组相比,MZ2组断奶仔猪回肠乙酸、丙酸和丁酸含量均显著提高(P<0.05),PC组回肠乙酸和丙酸含量显著提高(P<0.05);MZ2组和PC组结肠丁酸含量显著提高(P<0.05),结肠乙酸和丙酸含量无显著差异(P>0.05)。
图4 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道SCFAs含量的影响

Fig.4 Effects of compound preparation of Clostridium butyricum metabolites and live bacteria on intestinal SCFAs contents of weaned piglets

基于物种的主坐标分析(PCoA)结果(图5)表明,各组间断奶仔猪回肠菌群多样性存在显著差异(P<0.05),结肠菌群多样性存在显著差异趋势(P=0.051)。如图6所示,各组间肠道微生物在属水平上的组成上存在明显差异,与NC组相比,MZ2组和PC组回肠狭义梭菌属1(Clostridium_sensu_stricto_1)相对丰度明显提高,回肠链球菌属(Streptococcus)和乳杆菌属(Lactobacillus)相对丰度有所降低;MZ2组和PC组结肠乳杆菌属相对丰度有所降低,结肠埃希氏-志贺氏菌属(Escherichia-Shigella)相对丰度有所提高。同时,与PC组相比,MZ2组回肠和结肠狭义梭菌属1相对丰度明显提高,回肠和结肠链球菌属相对丰度明显降低。
图5 断奶仔猪肠道菌群多样性

Fig.5 Intestinal microbiota diversity of weaned piglets

图6 断奶仔猪肠道菌群在属水平上组成

Clostridium_sensu_stricto_1:狭义梭菌属1;Streptococcus:链球菌属;Lactobacillus:乳杆菌属;Escherichia-Shigella:埃希氏-志贺氏菌属;Terrisporobacter:特里斯孢菌属;Actinobacillus:放线杆菌属;Turicibacter:苏黎世杆菌属;Prevotella:普雷沃氏菌属;Faecalibacterium:粪杆菌属;Mycoplasma:支原体属;Dialister:戴阿利斯特杆菌属;Agathobacter:琼脂杆菌属;Romboutsia:罗姆布茨菌属;Unclassified_o_Lactobacillales:未分类乳杆菌目;Enterococcus:肠球菌属;Unclassified_k_norank_d_Bacteria:未分类细菌;Bacillus:芽孢杆菌属;Blautia:布劳特氏菌属;Subdoligranulum:罕见小球菌属;Veillonella:韦荣氏球菌属;Norank_f_norank_o_Clostridia_UCG-014:未定级梭菌纲UCG-014;Christensenellaceae_R-7_group:克里斯滕森菌科R-7群;Norank_f_Muribaculaceae:未定级鼠杆状菌科;Norank_f_Eubacterium_coprostanoligenes_group:未定级产粪甾醇真杆菌群;Coprococcus:粪球菌属;Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;Lachnospira:毛螺菌属;Unclassified_f_Lachnospiraceae:未分类毛螺菌科;Others:其他。

Fig.6 Intestinal microbiota composition of weaned piglets at genus level

多级物种线性判别分析(LDA)效应大小(LEfSe)分析结果(图7)和属水平上Kruskal-Wallis秩和检验多重比较分析结果(图8)表明,与NC组和PC组相比,MZ2组断奶仔猪回肠狭义梭菌属1、特里斯孢菌属(Terrisporobacter)和苏黎世杆菌属(Turicibacter)相对丰度以及结肠狭义梭菌属1、特里斯孢菌属和毛螺菌科ND3007群(Lachnospiraceae_ND3007_group)相对丰度显著提高(P<0.05),回肠和结肠链球菌属相对丰度显著降低(P<0.05)。
图7 断奶仔猪肠道菌群LEfSe分析

Fig.7 LEfSe analysis of intestinal microbiota of weaned piglets

图8 断奶仔猪肠道菌群Kruskal-Wallis秩和检验多重比较分析

Fig.8 Multiple comparison analysis of Kruskal-Wallis rank sum test on intestinal microbiota of weaned piglets

3 讨论

在传统养殖生产过程中,饲粮中预防性添加抗生素可有效减少断奶仔猪腹泻的发生,降低死亡率[15]。随着我国禁止在畜禽饲粮中添加抗生素的政策实施,微生态制剂、SCFAs和酶制剂等替抗产品在促进幼畜生长和减轻断奶应激方面发挥出巨大潜力[16]。本研究对丁酸梭菌代谢产物及活菌复合制剂在断奶仔猪上应用的有效性进行了评价。

3.1 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪生长性能和腹泻率的影响

丁酸或丁酸盐作为饲料添加剂能够提高家禽和猪的营养物质消化率和吸收利用率。饲粮中添加丁酸盐能够显著提高断奶仔猪ADG和饲料转化效率,这可能与丁酸能够诱导内源性分泌物有关[17]。ADFI、ADG、F/G和腹泻率是衡量仔猪生长性能最直接的指标。本课题组前期研究发现,丁酸梭菌代谢产物及活菌复合制剂能够改善母猪繁殖性能,提高出生窝重、仔猪个体重以及哺乳仔猪的生长性能。然而,部分研究结果显示,单独添加1 000 mg/kg(1 kg/t)丁酸钠能降低断奶仔猪腹泻率,但对ADG和F/G均无显著影响[18]。饲粮中添加150和300 mg/kg丁酸钠补充剂对肉鸡生长性能也没有显著影响[19]。本试验中,MZ1组和MZ2组实际所添加丁酸盐含量小于1 kg/t(约为0.32和0.64 kg/t),但对断奶仔猪生长性能均有促进作用,且MZ2组试验后期(第22~42天)和全期(第1~42天)在降低断奶仔猪腹泻率以及提高ADG和末重方面均表现出更好的效果。这可能是因为丁酸梭菌代谢产物除丁酸盐外,还含有其他SCFAs(如乙酸盐、丙酸盐),多种SCFAs共同作用有效地刺激了肠道蠕动和食糜排空速率[14]。另外,复合制剂中含有的3种活菌成分E. faecium、L. plantarum和B. subtilis均能够通过改善动物肠道内环境,刺激肠道内源厌氧菌增殖,促进双歧杆菌、乳杆菌等有益菌定植;B. subtilis还能够广谱抑制致病性大肠杆菌、产气荚膜梭菌等致病菌,提高对营养物质的消化吸收能力,以此降低腹泻率,提高生长性能[20-23]。这3种活菌与丁酸梭菌代谢产物协同作用,在促进断奶仔猪生长性能方面发挥出更大优势。
综上所述,饲粮中添加1和2 kg/t丁酸梭菌代谢产物及活菌复合制剂均对断奶仔猪生长性能有促进作用,且MZ2组的效果更优,MZ1组与PC组效果基本一致。因此,后续指标分析以MZ2组断奶仔猪为试验组研究对象,探究其效果优于NC组和PC组的潜在机制。

3.2 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪血清生化指标的影响

血清生化指标可以反映动物机体的健康状况。本研究中,各组间断奶仔猪血清TP、ALB和GLB等指标没有显著差异,表明丁酸梭菌代谢产物及活菌复合制剂在一定程度上维持了宿主稳态。针对与肝脏损伤密切相关的指标血清AST活性,MZ2组低于NC组和PC组,而血清AST活性增高表明肝功能受损,这说明饲喂MZ具有保护断奶仔猪肝脏功能的作用。IgA、IgG和IgM是体液免疫的主要成分,能够特异性结合相应的抗原并调节免疫功能。研究表明,有机酸能够通过提高血清免疫球蛋白含量来增强仔猪的免疫系统功能[24]。口服灌喂C. butyricum和丁酸能够激活蛋白激酶B(Akt)-哺乳动物雷帕霉素靶蛋白(mTOR)通路提高小鼠血浆IgG、IgM和IgA含量[25]。这与本研究结果一致,说明丁酸梭菌代谢产物及活菌复合制剂能够促进断奶仔猪的免疫系统发育,在提高体液免疫能力方面具有潜在优势。

3.3 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道形态和屏障功能的影响

肠道绒毛和隐窝发育在维持肠道正常功能方面起到重要作用。断奶应激常导致仔猪肠道绒毛高度降低,隐窝深度提高,小肠结构和功能发生不可逆的损伤,进而导致生长迟缓。黏蛋白和紧密连接蛋白作为肠道屏障的主要蛋白,广泛存在于肠道黏膜中,能够调节肠道通透性,二者共同反映肠道屏障功能。本研究结果显示,MZ2组断奶仔猪回肠mucin-1和mucin-2 mRNA相对表达量显著高于NC组。研究表明,丁酸能够刺激黏蛋白-3、黏蛋白-4和黏蛋白-12的分泌,进而改变微生物群落在肠道中的黏附作用[26]。mucin-2还可与丁酸盐共同作用促进抗菌肽生成,抵抗DSS诱导的小鼠结肠炎发生[27]。这表明MZ2组断奶仔猪肠道中的丁酸含量能够促进黏蛋白的表达,提高肠道黏液屏障功能。
PC组添加的苯甲酸是一种良好的抗腹泻有机酸,具有促进肠道屏障发育的作用[28]。但在本研究中,苯甲酸未能表现出良好的改善肠道屏障功能的作用;而MZ2组断奶仔猪空肠和回肠VH和V/C以及claudin-1、occludin、mucin-1和mucin-2 mRNA相对表达量均表现出优于PC组的效果。研究报道,苯甲酸多以包被的形式用作饲料添加剂,这可能是因为包被后能起到缓释效果,使苯甲酸被微生物降解的速率更慢,增加其在小肠内的停留时间[29]。而本研究中使用的苯甲酸未经包被,MZ2组能起到更好效果的原因可能是活菌成分改变了断奶仔猪的肠道环境,其中L. plantarum和B. subtilis在繁殖和定植过程中消耗大量氧气,降低了肠道内pH,促进了优势微生物的生长[30-31]。同时,有研究表明,饲喂L. plantarum能够促进仔猪空肠和回肠肠道绒毛发育,B. subtilis和E. faecium通过上调回肠紧密连接蛋白的表达增强肠道屏障功能[32-33]。肠道内环境的改变能够提高丁酸盐到达肠道的稳定性,使得丁酸盐能够更加直接在肠道中被肠上皮的刷状缘吸收和代谢,为机体提供营养,促进mucin-1和mucin-2 mRNA相对表达量的提高[34]。

3.4 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪机体免疫和抗炎能力的影响

断奶会导致免疫功能障碍,使得促炎细胞因子过度分泌,引发肠道炎症,阻碍断奶仔猪的生长。丁酸梭菌和丁酸盐能够通过增强机体免疫反应及降低促炎反应发挥积极作用。Zhang等[35]探究了口服丁酸梭菌和丁酸盐对烧伤小鼠肠道损伤的影响,结果发现口服丁酸梭菌能提高肠道丁酸盐含量,降低TNF-α和IL-6含量,重塑肠道微生物群落结构,抑制烧伤小鼠的肠道损伤。本试验中,饲粮中添加丁酸梭菌代谢产物及活菌复合制剂降低了断奶仔猪空肠和回肠中炎性细胞因子IL-6、IL-1β和TNF-α mRNA相对表达量,表明仔猪具有更好的抗炎能力。这与Liu等[36]在肉鸡饲粮中添加丁酸梭菌和丁酸钠的试验结果一致。此外,免疫球蛋白也是免疫系统的重要组成部分。血清生化指标结果显示,饲粮中添加2 kg/t丁酸梭菌代谢产物及活菌复合制剂提高了断奶仔猪血清IgM和IgA含量,降低了血清中促炎细胞因子IL-1β和TNF-α含量,表明复合制剂更倾向于通过降低血清和肠道组织中促炎细胞因子水平的作用机制发挥作用。由此说明,丁酸梭菌代谢产物及活菌复合制剂在改善断奶仔猪抗炎能力和调节机体免疫力方面具有潜在益生作用。

3.5 丁酸梭菌代谢产物及活菌复合制剂对断奶仔猪肠道SCFAs含量和菌群的影响

SCFAs对宿主肠道健康、免疫调节和对病原体的易感性有积极影响[37]。本研究中,MZ2组饲粮中含有较高含量的丁酸盐,对提高回肠和结肠食糜中丁酸含量的结果是显而易见的。除此之外,L. plantarum也能够在肠道中利用纤维底物,代谢产生丁酸、丙酸等SCFAs[38]。高水平的丁酸盐促进了肠道发育,有助于维持肠道屏障正常功能。
SCFAs还是肠道微生物群与宿主之间相互作用的关键介质[37]。本试验中,与NC组和PC组相比,MZ2组断奶仔猪回肠和结肠狭义梭菌属1和特里斯孢菌属相对丰度显著提高。狭义梭菌属1在肠道中具有降解多糖产生乙酸和丁酸的功能;此外,在MZ2组结肠中富集的毛螺菌科ND3007群也能够利用淀粉、菊粉和阿拉伯木聚糖等多糖产生丁酸和其他SCFAs[39],这可能也是回肠乙酸和丁酸含量升高的原因之一。根据上述结果,笔者认为肠道中维持较高含量的丁酸是MZ2组断奶仔猪回肠mucin-1和mucin-2 mRNA相对表达量远高于NC组和PC组的主要原因。丁酸含量的维持一方面与MZ2组较高添加量有关,另一方面复合制剂刺激了狭义梭菌属1和、毛螺菌科ND3007等产丁酸菌的生成,使丁酸含量保持在适宜仔猪生长的范围。此外,复合制剂中的活菌成分L. plantarum能够提高菌群多样性,如提高厚壁菌门(Firmicutes)相对丰度,阻碍致病菌黏附[21];同时,E. faecium和B. subtilis也能够降低肠道pH,促进肠道厌氧菌生成[20]。特里斯孢菌属作为一类严格厌氧菌,能够产生具有抗菌能力的次级代谢产物,并增强机体消化酶活性和碳水化合物代谢[40]。苏黎世杆菌属也是一类具有抗炎特性的潜在有益菌,能够改变宿主胆汁酸谱和脂质代谢能力,经苏黎世杆菌属发酵后的多糖还能够通过影响血清素系统抑制结直肠肿瘤的发生[41-43]。这些潜在益生菌相对丰度的提高,能够帮助仔猪更好地消化和利用饲粮中的纤维和碳水化合物,调节肠道微生态,提高菌群多样性,并提高对营养物质的消化吸收能力。综上所述,本研究中的丁酸梭菌代谢产物及活菌复合制剂有利于提高肠道潜在有益菌的相对丰度,改善肠道健康,从而促进断奶仔猪生长发育。

4 结论

饲粮中添加2 kg/t丁酸梭菌代谢产物及活菌复合制剂能够提高断奶仔猪生长性能,降低腹泻率,提高机体免疫和抗炎能力,并通过改善肠道形态、增强屏障功能以及调节SCFAs和菌群组成促进肠道健康。

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