Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 10: 6637 - 6650

DOI: https://doi.org/10.12418/CJAN2025.540

RESEARCH PAPER

Effects of Exopolysaccharides from Lactobacillus reuteri on Growth Performance, Serum Biochemical Indexes, and Intestinal Health of Weaned Piglets

  • RUAN Shihui , 1, 2 ,
  • XU Wentuo 1, 2 ,
  • LIU Huiliang 1, 2 ,
  • FENG Zhangjian 2 ,
  • WU Qiwen 2 ,
  • XIONG Yunxia 2 ,
  • WANG Li 2 ,
  • LIU Wenchao 1 ,
  • YI Hongbo , 2, *
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  • 1 College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524000, China
  • 2 Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, State Key Laboratory of Swine and Poultry Breeding Industry, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
*professor, E-mail:

Received date: 2025-03-06

  Online published: 2025-10-15

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Abstract

This experiment aimed to investigate the effects of exopolysaccharides from Lactobacillus reuteri (LR-EPS) on growth performance, serum biochemical indexes, and intestinal health of weaned piglets. A total of 48 twenty-one-day-old “Duroc×Landrace×Yorkshire” crossbred weaned piglets with an initial body weight of (5.13±0.10) kg were randomly divided into 2 groups: the control group (CON group, fed a basal diet) and the polysaccharide group (EPS group, fed the basal diet supplemented with 300 mg/kg LR-EPS). Each group consisted of 6 pens with 4 piglets per pen (half male and half female), and the experimental period lasted for 21 days. The results showed as follows: 1) compared with the CON group, the average daily gain (ADG) of weaned piglets in the EPS group was extremely significantly increased (P<0.01), and the feed-to-gain ratio (F/G) was extremely significantly decreased (P<0.01). 2) Compared with the CON group,the serum albumin (ALB) content in the EPS group was extremely significantly increased (P<0.01), and the contents of total protein (TP), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) activity were significantly higher than those in the CON group (P<0.05). 3) Compared with the CON group, the activities of T-AOC and GSH-Px in the jejunum were significantly increased (P<0.05), and the GSH-Px activity in the ileum was significantly increased (P<0.05); LR-EPS extremely significantly increased the villus height of the jejunum and ileum in weaned piglets (P<0.01). 4) Compared with the CON group, the relative mRNA expression levels of Claudin-1 and Occludin in the jejunum of piglets in the EPS group were significantly increased (P<0.05); the relative mRNA expression level of Claudin-1 in the ileum was significantly increased (P<0.05). 5) The Chao1 index and Dominance index indicated that LR-EPS could significantly improve the evenness and richness of the intestinal flora in piglets (P<0.05). At the phylum level, the relative abundance of Bacteroidetes in the EPS group was extremely significantly increased (P<0.01); at the genus level, the relative abundance of Prevotellaceae_NK3B31_group in the EPS group was significantly increased (P<0.05). 6) Spearman correlation analysis showed that the relative abundances of Bacteroidetes and Prevotellaceae_NK3B31_group in the intestine were extremely significantly positively correlated with the relative mRNA expression levels of intestinal barrier-related genes such as jejunal Claudin-1, ileal Claudin-1, and Occludin (P<0.01). In conclusion, dietary supplementation with 300 mg/kg LR-EPS can effectively improve the growth performance of weaned piglets, enhance the antioxidant capacity of the body, and improve intestinal barrier function.

Key words： exopolysaccharides from Lactobacillus reuteri; weaned piglets; growth performance; intestinal barrier; gut microbiota

Cite this article

RUAN Shihui , XU Wentuo , LIU Huiliang , FENG Zhangjian , WU Qiwen , XIONG Yunxia , WANG Li , LIU Wenchao , YI Hongbo . Effects of Exopolysaccharides from Lactobacillus reuteri on Growth Performance, Serum Biochemical Indexes, and Intestinal Health of Weaned Piglets[J]. Chinese Journal of Animal Nutrition, 2025 , 37(10) : 6637 -6650 . DOI: 10.12418/CJAN2025.540

断奶应激易引起肠道炎症、肠道屏障受损,导致仔猪腹泻、生长迟缓,甚至死亡[1],给养猪业带来巨大的经济损失。为响应国家“禁抗限锌”的号召,研发绿色高效的替抗产品是大势所趋。罗伊氏乳杆菌(Lactobacillus reuteri,LR)具有缓解氧化应激和炎症反应的作用[2],在断奶仔猪上具有重要应用价值。前期本课题组从健康仔猪粪便中分离出新型猪源罗伊氏乳杆菌LR1,并发现其可以提升断奶仔猪平均日增重(ADG)[3],增强肠道屏障功能[4],促进仔猪氨基酸吸收和脂肪酸合成[5]。然而,LR1发挥益生作用的组分及机制仍有待深入解析。近年研究表明,益生菌的胞外多糖(exopolysaccharides,EPS)具有调节机体免疫[6]、红细胞结合能力[7]、肠道菌群[8]、细胞衰老与凋亡[9]等作用。基于此,本课题组分离纯化出LR1胞外多糖(LR-EPS)进行试验,旨在探讨饲粮中添加LR-EPS对断奶仔猪生长性能、血清生化指标以及肠道健康的影响,为LR-EPS在断奶仔猪上的合理应用提供基础数据。

1 材料与方法

1.1 试验材料

LR-EPS分离提纯自猪源罗伊氏乳杆菌LR1(活菌数为5×1012 CFU/L),由广东省农业科学院动物科学研究所提供。

1.2 试验设计

本试验通过广东省农业科学院动物科学研究所实验动物福利伦理委员会的审查(批准号GDIAS2023116)。
试验选用48头初始体重为(5.13±0.10) kg的21日龄“杜×长×大”三元杂交断奶仔猪,根据性别和体重随机分为2组,每组分为6栏,每栏4头(公母各占1/2)。对照组(CON组)饲喂基础饲粮,多糖组(EPS组)在基础饲粮中添加300 mg/kg LR-EPS,添加量参考路则庆等[10]的研究。试验期为21 d。试验结束后,每栏中随机选取接近平均体重的1头仔猪屠宰取样(n=6,公母各占1/2)。除钙、磷水平根据前期研究结果[11]确定外,基础饲粮营养水平的设计参照NRC(2012)猪营养需要,其组成及营养水平见表1。基础饲粮中粗蛋白质、粗纤维、粗脂肪、钙、总磷含量分别按照GB/T 6432—2018、GB/T 6434—2022、GB/T 6433—2006、GB/T 6436—2018和GB/T 6437—2018中方法进行测定。
表1 基础饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis) %

项目Items 含量Content
原料Ingredients
玉米Corn 58.06
豆粕Soybean meal 16.07
低蛋白乳清粉Low protein whey powder 8.00
浓缩乳清蛋白Concentrated whey protein 10.00
鱼粉Fish meal 3.00
大豆油Soybean oil 1.00
蔗糖Sucrose 1.00
50%氯化胆碱50% choline chloride 0.20
氯化钠NaCl 0.40
L-赖氨酸L-lysine 0.22
DL-蛋氨酸DL-methionine 0.08
L-苏氨酸L-threonine 0.01
氧化锌ZnO 0.20
石粉Limestone 0.44
磷酸氢钙CaHPO4 0.30
植酸酶Phytase 0.02
预混料Premix1) 1.00
合计Total 100.00
营养水平Nutrient levels2)
净能NE/(MJ/kg) 10.87
粗蛋白质CP 22.49
粗纤维CF 1.92
粗脂肪EE 3.56
标准回肠可消化赖氨酸SID Lys 1.46
标准回肠可消化苏氨酸SID Thr 0.86
标准回肠可消化蛋氨酸SID Met 0.47
标准回肠可消化色氨酸SID Trp 0.29
标准回肠可消化缬氨酸SID Val 1.00
钙Ca 0.49
标准全消化道可消化钙STTD Ca 0.33
总磷TP 0.41
标准全消化道可消化磷STTD P 0.26

1)预混料为每千克饲粮提供 Premix provided the following per kg of the diet: VA 9 920 IU, VD3 2 240 IU, VB1 2.4 mg, VB2 8 mg, VB6 6.4 mg, VB12 32 mg, 烟酸 nicotinic acid 32 mg,泛酸 pantothenic acid 12 mg, 叶酸 folic acid 0.8 mg, 生物素 biotin 64 mg, Fe(as ferrous sulfate) 90 mg, Cu (as copper sulfate) 12 mg, Mn (as manganese sulfate) 52.5 mg, I (as potassium iodide) 0.525 mg, Se (as sodium selenite) 0.36 mg。2)粗蛋白质、粗纤维、粗脂肪、钙、总磷为实测值,其余为参照NRC(2012)计算值。CP, CF, EE, Ca and TP were measured values, while the others were calculated values according to NRC (2012).

1.3 饲养管理

试验仔猪饲养于广东省农业科学院白云试验基地仔猪舍,试验各组猪只在同一栋猪舍不同栏饲养,饲养管理条件一致。每日根据前1天采食情况调整投放饲粮量,并于08:00、12:00、16:00、20:00投料。试验期间所有仔猪自由采食和饮水,按照养殖场常规饲养管理和免疫。

1.4 样品采集

试验第22天,用20 mL一次性注射器采集前腔静脉血,血清分离后于-80 ℃冰箱保存待测。仔猪采血后屠宰并采集样品。取中段空肠和远端回肠的组织样品,经生理盐水清洗后,一部分完全浸泡于4%多聚甲醛中,用于组织形态结构观察;一部分储存于2 mL冻存管中,液氮速冻后移至-80 ℃冰箱待测。

1.5 测定指标及方法

1.5.1 生长性能与腹泻率

试验期间每天早上称量剩余的饲粮重量,并于试验第1、22天称量仔猪空腹体重,计算仔猪ADG、平均日采食量(ADFI)、料重比(F/G)。
试验期间每日观察仔猪粪便形态、外观,记录腹泻仔猪头数与腹泻天数,计算仔猪腹泻率。
腹泻率(%)=[该组腹泻仔猪头次/(该组仔猪头数×试验天数)]×100。

1.5.2 血清生化指标

利用日立7600全自动生化分析仪检测断奶仔猪血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总蛋白(TP)、白蛋白(ALB)、肌酐(CRE)、尿素(UREA)、葡萄糖(GLU)、甘油三酯(TG)、胆固醇(CHO)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)等指标含量或活性,所用试剂购于中生北控生物科技有限公司。

1.5.3 抗氧化指标

采用南京建成生物工程研究所的试剂盒,检测血清、空肠组织和回肠组织的抗氧化指标:丙二醛(MDA)含量、谷胱甘肽过氧化酶(GSH-Px)活性和总抗氧化能力(T-AOC)。详细步骤按照试剂盒说明书进行。

1.5.4 肠道形态结构观察

将4%多聚甲醛固定24 h后的肠段进行脱水、石蜡包埋处理,取5 μm横切面制成苏木精-伊红(HE)染色切片,并进行形态学测量。利用光学显微镜(尼康,日本)捕获染色样品的图像后,用ImageJ软件测定肠道绒毛高度、隐窝深度,并计算绒隐比。

1.5.5 肠道屏障基因表达

采用实时荧光定量PCR(qRT-PCR)技术检测仔猪空肠、回肠组织中闭锁蛋白-1(Claudin-1)、闭合蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)、黏蛋白-2(MUC-2)的mRNA相对表达量。利用TRIzol(TaKaRa,日本)试剂提取组织总RNA,超微量紫外分光光度计NanoDrop-1000(Thermo Fisher Scientific,美国)评估总RNA的浓度和纯度。根据反转录试剂盒(EZBioscience)说明书将RNA反转录为cDNA,用于实时荧光定量。根据NCBI中猪的目的基因mRNA序列,利用Primer Premier 5.0软件设计引物(表2),引物由生工生物工程(上海)股份有限公司合成。10 μL的反应体系如下:5 μL SYBR Premix Ex Taq,上、下游引物各0.5 μL,4 μL cDNA。反应条件为:95 ℃预变性5 min,95 ℃变性5 s,60 ℃退火34 s,95 ℃延伸15 s,扩增循环次数为40次。以β-肌动蛋白(β-actin)为内参基因,结果用2-△△Ct法计算各目的基因的mRNA相对表达量。
表2 qRT-PCR引物序列

Table 2 Primer sequences for qRT-PCR

基因
Genes		 引物序列
Primer sequences(5'—3')		 产物长度
Product length/bp		 登录号
Accession number
闭锁蛋白-1 Claudin-1 F: ACGGCCCAGGCCATCTAC
R: TGCCGGGTCCGGTAGATG		 221 AJ318102.1
闭锁小带蛋白-1 ZO-1 F: AGCCCGAGGCGTGTTT
R: GGTGGGAGGATGCTGTTG		 147 XM_013993251
闭合蛋白Occludin F: GCACCCAGCAACGACAT
R: CATAGACAGAATCCGAATCAC		 144 XM_005672525
黏蛋白-2 MUC-2 F: CTGCTCCGGGTCCTGTGGGA
R: CCCGCTGGCTGGTGCGATAC		 100 XM_007465997.1
β-肌动蛋白β-actin F: CTGCGGCATCCACGAAACT
R: AGGGCCGTGATCTCCTTCTG		 380 XM_003124280

1.5.6 肠道微生物16S rRNA基因测序

回肠黏膜微生物16S rRNA基因测序由北京诺禾致源科技股份有限公司进行测定。利用DNA提取试剂盒(TianGen)提取回肠黏膜微生物总基因组DNA,通过1%琼脂糖凝胶电泳检测DNA的纯度和浓度。PCR扩增选用细菌16S V4区特异性引物515F和806R(引物序列F:5'-GTGCCAGCMGCCGCGGTAA-3';R:5'-GGACTACHVGGGTWTCTAAT-3'),PCR扩增程序为:98 ℃预变性1 min,98 ℃变性10 s,50 ℃退火30 s,72 ℃延伸30 s,循环30次,最后在72 ℃下保持5 min。随后用NEB Next UltraTM Ⅱ FS DNA PCR-free Library Prep Kit建库试剂盒(New England Biolabs)进行文库构建,用NovaSeq-6000进行PE 250上机测序。菌群多样性、相对丰度在诺禾致源分析平台完成。

1.6 数据统计与分析

数据采用SPSS 26.0软件进行独立样本t检验,结果用“平均值±标准误”表示,P<0.05为差异显著,P<0.01为差异极显著。

2 结果

2.1 LR-EPS对断奶仔猪生长性能及腹泻率的影响

由表3可知,试验开始时,2组仔猪的初重无显著差异(P>0.05);试验结束后,EPS组的末重、ADG与CON组相比极显著升高(P<0.01)。与CON组相比,EPS组ADFI显著增加(P<0.05),F/G极显著下降(P<0.01)。2组仔猪腹泻率无显著差异(P>0.05)。
表3 LR-EPS对断奶仔猪生长性能及腹泻率的影响

Table 3 Effects of LR-EPS on growth performance and diarrhea rate of weaned piglets

项目
Items		 组别Groups P值
P-value
CON EPS
初重IW/kg 5.11±0.09 5.14±0.11 0.826
末重FW/kg 10.91±0.22 12.57±0.27 0.001
平均日采食量ADFI/(g/d) 397.02±5.82 442.66±15.22 0.029
平均日增重ADG/(g/d) 276.19±11.79 353.49±11.15 0.001
料重比F/G 1.44±0.05 1.25±0.02 0.003
腹泻率Diarrhea rate/% 2.38±0.84 1.98±0.98 0.761

2.2 LR-EPS对断奶仔猪血清生化指标的影响

由表4可知,与CON组相比,EPS组血清ALP活性和TP、ALB含量显著或极显著上升(P<0.05或P<0.01);其他血清生化指标均无显著差异(P>0.05)。
表4 LR-EPS对断奶仔猪血清生化指标的影响

Table 4 Effects of LR-EPS on serum biochemical indexes of weaned piglets

项目
Items		 组别Groups P值
P-value
CON EPS
谷丙转氨酶ALT/(U/L) 54.05±4.22 59.76±5.45 0.439
谷草转氨酶AST/(U/L) 68.17±2.69 81.74±4.00 0.056
碱性磷酸酶ALP/(U/L) 262.26±9.79 352.86±18.78 0.004
乳酸脱氢酶LDH/(U/L) 532.34±70.62 686.79±75.46 0.061
总蛋白TP/(g/L) 38.46±2.17 45.59±1.50 0.029
白蛋白ALB/(g/L) 16.50±0.50 24.38±0.82 <0.001
肌酐CRE/(μmol/L) 77.35±7.87 93.57±2.56 0.109
尿素UREA/(μmol/L) 2.81±0.53 3.25±0.34 0.481
葡萄糖GLU/(mmol/L) 5.12±0.55 4.59±0.58 0.563
甘油三酯TG/(mmol/L) 0.30±0.07 0.38±0.02 0.254
胆固醇CHO/(mmol/L) 1.69±0.10 1.72±0.10 0.909
高密度脂蛋白HDL/(mmol/L) 0.63±0.06 0.56±0.03 0.325
低密度脂蛋白LDL/(mmol/L) 0.83±0.09 0.80±0.05 0.764

2.3 LR-EPS对断奶仔猪血清及肠道抗氧化指标的影响

由图1可知,与CON组相比,EPS组血清T-AOC、GSH-Px活性显著上升(P<0.05)。在空肠中,EPS组T-AOC、GSH-Px活性显著高于CON组(P<0.05);在回肠中,EPS组GSH-Px活性显著高于CON组(P<0.05)。与CON组相比,EPS组空肠、回肠MDA含量显著降低(P<0.05)。
图1 LR-EPS对断奶仔猪血清及肠道的抗氧化指标的影响

A:血清抗氧化指标;B:空肠、回肠抗氧化指标。A: serum antioxidant indexes; B: jejunum and ileum antioxidant indexes.*表示差异显著(P<0.05)。下图同。 * mean significant difference (P<0.05). The same as below.

Fig.1 Effects of LR-EPS on serum and intestinal antioxidant indexes of weaned piglets

2.4 LR-EPS对断奶仔猪肠道形态的影响

肠组织HE染色切片显示(图2),少量绒毛出现断裂,肠道组织形态相对完整。如表5所示,与CON组相比,EPS组空肠、回肠的绒毛高度均极显著上升(P<0.01);且EPS组空肠的绒隐比极显著高于CON组(P<0.01)。但与CON组相比,EPS组仔猪回肠的隐窝深度极显著增加(P<0.01)。
图2 肠道HE染色形态

Fig.2 HE staining morphology of intestine (10×)

表5 LR-EPS对断奶仔猪肠道形态的影响

Table 6 Effects of LR-EPS on intestinal morphology of weaned piglets

项目
Items		 组别Groups P值
P-value
CON EPS
空肠Jejunum
绒毛高度Villus height/μm 379.77±14.15 547.42±38.15 0.002
隐窝深度Crypt depth/μm 312.57±21.89 258.07±15.79 0.071
绒隐比V/C 1.24±0.07 2.14±0.15 0.001
回肠Ileum
绒毛高度Villus height/μm 317.42±10.55 480.56±12.61 <0.001
隐窝深度Crypt depth/μm 266.02±12.80 351.10±19.96 0.001
绒隐比V/C 1.24±0.08 1.41±0.07 0.127

2.5 LR-EPS对断奶仔猪肠道屏障相关基因mRNA表达的影响

如图3所示,与CON组相比,添加LR-EPS可以显著提高空肠Claudin-1、Occludin和回肠Claudin-1的mRNA相对表达量(P<0.05)。
图3 LR-EPS对断奶仔猪肠道屏障的影响

Fig.3 Effects of LR-EPS on intestinal barrier of weaned piglets

2.6 LR-EPS对断奶仔猪肠道微生物多样性的影响

利用16S rDNA扩增子测序技术对断奶仔猪回肠黏膜进行检测。如图4-A所示,CON组回肠微生物含有1 790个操作分类单元(OTU),特有OTU数目为1 721个;EPS组回肠微生物含有1 666个OTU,其中特有OTU数目为1 597个OTU。如图4-B显示,EPS组的Chao1指数、Observer_feature指数显著低于CON组(P<0.05),且Dominance指数极显著低于CON组(P<0.01)。
图4 LR-EPS对断奶仔猪肠道微生物的影响

A:操作分类单元(OTU)水平物种韦恩图;B:α多样性分析;C:无度量多维标定法统计;D:门和属水平上的差异微生物;E:微生物差异分析条形图(LDA>4.0)。A: Venn diagram for species at OTU; B: α-diversity analysis; C: NMDS statistics; D: differential microbes at phylum and genus levels; E: microbial difference analysis bar chat (LDA>4.0).* *表示差异极显著(P<0.01)。* * mean extremely significant difference (P<0.01).

Fig.4 Effects of LR-EPS on gut microbiota of weaned piglets

图4-C为断奶仔猪回肠微生物的无度量多维标定法(NMDS)统计,反映样本的群落组成及组间、组内差异。R值大于0且接近1,表示2组间物种组成、丰度分布等微生物群体结构差异显著大于组内差异(P<0.05)。
通过断奶仔猪回肠微生物多样性分析,结果表明,饲粮添加LR-EPS会改变断奶仔猪肠道微生物的组成结构。如图4-D所示,在门水平的前10种高丰度菌中,EPS组的拟杆菌门相对丰度极显著高于CON组(P<0.01),EPS组的热脱硫杆菌门相对丰度极显著低于CON组(P<0.01);在属水平的前10种高丰度菌中,EPS组的普雷沃氏菌科NK3B31群相对丰度显著高于CON组(P<0.05),CON组的密螺旋体属相对丰度显著高于EPS组(P<0.05)。
图4-E为线性判别分析(LEfSe),进一步从门到属水平分析2组间的差异群落,线性判别分析(LDA)评分阈值设置为4。CON组中优势菌为密螺旋体属,EPS组中优势菌为拟杆菌门、拟杆菌纲、拟杆菌目、普雷沃氏菌科、普雷沃氏菌科NK3B31群。

2.7 相关性分析

为了确定肠道差异微生物与肠道屏障功能变化的关系,利用Spearman相关性分析对上述EPS组的优势菌群与肠道屏障指标进行评估,结果如图5所示。拟杆菌门、拟杆菌纲、拟杆菌目、普雷沃氏菌科NK3B31群相对丰度与空肠Claudin-1、回肠Claudin-1、Occludin、ZO-1、MUC-2 mRNA相对表达量呈极显著正相关(P<0.01),说明LR-EPS可能通过肠道微生物调控肠道屏障相关基因的表达,从而改善仔猪肠道健康。
图5 断奶仔猪肠道微生物群与肠道屏障指标的相关性分析热图

* *表示极显著相关(P<0.01)。

Fig.5 Heatmap of correlation between gut microbiota and intestinal barrier indexes of weaned piglets

* * indicates extremely significant correlation (P<0.01).

3 讨论

3.1 LR-EPS对断奶仔猪生长性能的影响

微生物EPS是天然存在的高分子质量碳水化合物,因具有抗炎、抗菌、抗癌等生物活性而逐渐受到重视[12]。现有研究指出,饲粮中添加植物乳杆菌EPS可以提高仔猪消化率,从而提高断奶仔猪生长性能[13]。路则庆等[10]研究发现,阴沟肠杆菌产生的富硒EPS可以显著提升断奶仔猪的ADG,而对仔猪腹泻没有改善效果。此外,有报道指出,LR能够显著增加断奶犊牛的ADG,并显著降低其F/G[14],LR-EPS作为LR的代谢产物,具有相似的作用。本试验结果表明,饲粮中添加LR-EPS显著提高仔猪ADFI,且仔猪ADG极显著增加,F/G极显著下降,与前人研究结果相似,这提示LR-EPS可能对仔猪消化吸收功能有促进作用。

3.2 LR-EPS对断奶仔猪血清生化指标的影响

血清生化指标主要提示肝脏功能、肾脏功能、血脂代谢和血糖水平等几个方面的健康状况,也是评价应激的重要参考[15]。ALT、AST、ALP属于肝脏功能的常规指标。其中,ALP参与脂质运输,其活性可以反映机体生长性能及肝脏代谢功能。有研究指出,ALP活性易受氧化应激影响[16],且与动物日增重呈正相关[17]。同时,高ALP活性可以改善营养代谢,提高机体对钙、磷的吸收利用率,促进骨骼生长[18],这与本研究中断奶仔猪ADG极显著提高的结果相符。因此,EPS组仔猪血清ALP活性极显著增加可能是仔猪快速成长的标志,也提示着LR-EPS可能缓解断奶仔猪体内氧化应激。尽管血清ALP活性上升可能表明肝脏受损[19],但本试验中仔猪血清ALP活性均在正常参考值范围内[20],对仔猪正常生长没有产生负面影响。血清TP含量越高,越有益于蛋白质在机体内的沉积和组织器官的生长发育,并对仔猪增重具有促进作用。ALB是TP的组成成分之一,参与机体内氨基酸的合成与代谢,同时有维持血液胶体渗透压、清除自由基、运输机体代谢原料及废物等功能[21]。LR-EPS的添加显著或极显著提高仔猪血清TP和ALB含量,表明LR-EPS有效改善仔猪肝脏细胞代谢功能,使更多能量和氨基酸用于ALB合成,并由此减轻仔猪体内氧化应激和肠道炎症。此外,有研究表明,血清ALB可反映仔猪的肝脏蛋白质代谢状况[22],说明LR-EPS可能影响仔猪肝脏蛋白质代谢,有利于仔猪体内蛋白质的沉积,提高仔猪增重速度。

3.3 LR-EPS对断奶仔猪抗氧化指标的影响

仔猪在断奶应激的情况下,机体会产生过量的活性氧,破坏体内活性氧稳态,从而损害DNA、蛋白质和组织功能,导致仔猪生长迟缓。据研究报道,仔猪早期断奶后,GSH-Px、超氧化物歧化酶等抗氧化酶活性急剧下降,肝脏氧化自由基和MDA含量显著上升,表明断奶会加剧仔猪氧化应激[23]。T-AOC是抗氧化能力的综合指标,反映机体非酶抗氧化防御系统的能力[24]。前人研究证实,植物乳杆菌EPS可以逆转镉中毒大鼠机体T-AOC等抗氧化指标[25]。EPS组仔猪血清和肠道中T-AOC显著升高,表示仔猪系统性增强对氧化应激的抵御能力,应对氧化应激的缓冲容量提升。LR-EPS能够下调肠道因氧化应激而损伤的风险,并支持高速生长下的代谢需求。GSH-Px是清除过氧化氢和脂质过氧化物的关键酶,可以通过特异性催化谷胱甘肽还原机体内过量的氧化自由基,防止氧化自由基与细胞相互作用,是动物体内抵抗氧化自由基的最主要抗氧化酶[26]。Wang等[27]发现,德氏乳杆菌EPS通过Kelch样ECH相关蛋白1/核因子E2相关因子2(Keap1/Nrf2)途径减轻IPEC-J2细胞中过氧化氢诱导的氧化损伤应激,提高细胞中T-AOC和GSH-Px活性。EPS组断奶仔猪血清、肠道中GSH-Px活性显著增加,说明LR-EPS能够有效清除血清、肠黏膜中的过氧化物,并缓解肠道内的氧化损伤。前期研究发现,LR1可以显著提高断奶仔猪肠道中Toll样受体的表达[4]。基于此,LR-EPS或许也具有相似功能,通过上调肠道中Toll样受体的表达,激活肠道抗氧化信号。MDA是脂质过氧化的主要产物,反映体内脂质过氧化速率、强度以及细胞损伤程度[28]。研究表明,乳酸菌EPS和乳杆菌EPS具有较强的抗氧化活性,可以清除自由基,能够分解过氧化氢以及螯合铜铁离子[29]。陈晓宇等[30]发现,LR肽聚糖可以提高蛋雏鸡血浆GSH-Px活性,并显著降低血浆MDA含量。本试验中,EPS组的肠道MDA含量显著低于CON组,与前人研究结果相似,并说明LR-EPS具有与LR相似的抗氧化活性,可以通过减少断奶仔猪体内脂质过氧化、增强仔猪抗氧化酶活性,从而提高全身抗氧化能力。

3.4 LR-EPS对肠道健康的影响

肠道是消化吸收的主要部位,肠道形态结构的完整性是断奶仔猪对营养物质消化吸收的保证[31]。肠道形态包括绒毛高度、隐窝深度和绒隐比,这些指标可以反映肠道发育情况与功能是否完善[32]。绒毛高度和绒隐比下降说明肠黏膜功能受损,肠道消化吸收能力下降[33]。研究表明,仔猪早期断奶可导致仔猪肠道形态损伤,包括肠道绒毛高度下降、隐窝深度增加及绒隐比降低。断奶后仔猪肠道绒毛高度和绒隐比远低于断奶前,直到断奶后第14天才恢复至断奶前的水平[34]。在结肠炎小鼠的研究中发现,枯草芽孢杆菌EPS可以降低小鼠结肠的隐窝深度,减少肠道炎症[35],缓解小鼠肠道黏膜和隐窝损伤。同时,从粪便中分离出的唾液乳杆菌对断奶仔猪肠道的绒毛高度和绒隐比都有显著增加效果[36]。Yi等[3]的研究表明,罗伊氏乳杆菌LR1可以显著降低断奶仔猪空肠隐窝深度,提高回肠绒毛高度和绒隐比,本试验结果与上述研究结果相似。当LR-EPS进入肠道后,可能会被肠道微生物利用再产生短链脂肪酸,驱动隐窝干细胞增殖、分化,以维持肠黏膜更新。因此,仔猪肠道隐窝深度的增加是隐窝细胞不断增殖以修复受损黏膜的结果。LR-EPS改善断奶仔猪肠道形态,绒毛高度与绒隐比的提升直接关联于生长性能优化,而隐窝深度增加反映肠黏膜修复能力增强,多途径保护仔猪不受断奶应激影响,从而促进仔猪生长发育。
肠上皮是病原体、毒素、过敏原等从消化道进入体内的第1道物理屏障,肠道紧密连接蛋白对于维持肠道物理屏障功能的完整性至关重要。肠道紧密连接蛋白主要由Claudin、Occludin、ZO等蛋白复合物组成,为肠道提供结构支撑,是构成肠道物理屏障的核心组分。紧密连接蛋白的表达水平与肠道屏障功能呈正相关,其功能异常可能造成肠道屏障损伤及肠道通透性增加,进而诱发肠道炎症反应[37]。仔猪在断奶阶段因饮食结构的变化,肠道炎症状况频发。本研究通过评估LR-EPS对肠道紧密连接蛋白的调控,探究其对肠道屏障功能的改善效果。试验结果显示,LR-EPS的添加可以显著增加空肠、回肠Claudin-1的mRNA相对表达量,表明LR-EPS可能降低断奶仔猪肠道通透性。并且,前人研究结果表示罗伊氏乳杆菌LR1可以提升断奶后42 d仔猪结肠的ZO-1和Occludin的基因表达量[4]。另有研究证实,短双歧杆菌的EPS能够增强结肠炎小鼠肠道屏障功能,减轻小鼠肠道炎症[38],与本研究结果相似。由此可见,LR-EPS与LR1具有相似的保护肠道屏障功能,可以有效上调肠道紧密连接蛋白的表达,强化肠道物理屏障。
肠道菌群稳态对维持宿主机体的生长发育、营养消化吸收有重要作用,更丰富的微生物群能够降低宿主的发病率,影响宿主的健康。为探究LR-EPS对仔猪肠道微生物群的影响,本研究对2组仔猪回肠黏膜进行了微生物组学分析。已有研究表明,EPS能够通过改变肠道微生物的数量与结构促进肠道稳态[39],本研究结果印证了这一观点。EPS组的Chao1指数、Observed_features指数显著下降,提示LR-EPS可能通过选择性抑制部分微生物或促进优势菌定植,减少稀有菌种比例,使群落结构趋向种群少而功能精的局势;Dominance指数下降说明LR-EPS的添加促使仔猪肠道微生物均匀度提升,优势菌过度增殖现象缓解,肠道微生态稳定性可能增强。NMDS统计可以量化群落结构差异,强调优势菌群的影响。NMDS统计结果中,2组间差异大于组内差异,表明LR-EPS处理对结肠菌群结构的重塑作用强于个体差异或环境波动的影响。而PCoA反映物种组成的相似性,用于识别2组间微生物的整体分异。EPS组可能富集特定功能菌,而CON组保留更多环境适应性广的杂菌。为了研究LR-EPS对断奶仔猪肠道菌群结构的影响,本试验继续分析其在不同水平上的菌落相对丰度。研究指出,仔猪肠道中厚壁菌门和拟杆菌门占比最高[40],本试验也得出了相似的结论。在门水平上,EPS组拟杆菌门的相对丰度增加,可能与拟杆菌的特性有关。拟杆菌可以降解高分子质量的碳水化合物,进而运输到菌体内代谢利用,促进自身生长[41]。这表明LR-EPS的添加通过改变仔猪肠道微生物群落结构,从而促进肠道消化吸收高分子质量的碳水化合物。在属水平上,EPS组仔猪肠道中普雷沃氏菌属的相对丰度显著增加。普雷沃氏菌属参与纤维的降解,分泌木聚糖酶、阿拉伯呋喃糖苷酶,分解植物细胞壁多糖[42],有助于仔猪对麦麸等植物性饲料的消化吸收,提高仔猪ADG。与此同时,Wang等[43]认为普雷沃氏菌属在减轻肠道应激方面起着关键作用,且能有效提高宿主的抗氧化能力。而EPS组相对丰度显著下降的密螺旋体属的部分菌种可能会引发猪痢疾,破坏仔猪肠道黏液层和Occludin中的蛋白酶并诱发炎症[44],这也是LR-EPS能够增强仔猪肠道物理屏障功能的原因。Spearman相关性分析结果显示,拟杆菌门和普雷沃氏菌属的相对丰度与肠道紧密连接蛋白的mRNA表达呈显著正相关,这表明EPS组仔猪肠道中的优势菌群可能增强肠道物理屏障,从而提升仔猪生长性能。

4 结论

综上所述,饲粮中添加300 mg/kg LR-EPS能够有效提高断奶仔猪生长性能,增强机体抗氧化能力,改善肠道屏障功能。

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