动物营养学报 >

2023 , Vol. 35 >Issue 11: 7099 - 7109

DOI: https://doi.org/10.12418/CJAN2023.646

研究论文

包被植物乳杆菌和低聚果糖对蛋鸡生产性能、蛋品质、免疫能力、粪便臭味及盲肠微生物的影响

  • 宋丹 , 1, 2 ,
  • 段涛 2 ,
  • 陈炳旭 1 ,
  • 陈丽仙 2 ,
  • 王薇薇 , 2, * ,
  • 闵育娜 , 1, *
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  • 1 西北农林科技大学动物科技学院,杨陵 712100
  • 2 国家粮食和物资储备局科学研究院粮食品质营养研究所,北京 100037
* 王薇薇,副研究员,硕士生导师,E-mail: ;
闵育娜,教授,博士生导师,E-mail:

宋 丹(1988—),女,陕西渭南人,博士研究生,动物营养与饲料科学专业。E-mail:

Copy editor: 田艳明

收稿日期: 2023-05-31

  网络出版日期: 2023-11-12

基金资助

中央级公益性科研院所基本科研业务费专项(ZX2215)

国家现代农业产业技术体系(CARS-40)

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Effects of Coated Lactobacillus plantarum and Fructooligosaccharide on Performance, Egg Quality, Immune Capacity, Fecal Odor and Cecum Microbiota of Laying Hens

  • SONG Dan , 1, 2 ,
  • DUAN Tao 2 ,
  • CHEN Bingxu 1 ,
  • CHEN Lixian 2 ,
  • WANG Weiwei , 2, * ,
  • MIN Yu'na , 1, *
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  • 1 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
  • 2 Institute of Grain Quality and Nutrition Research, Academy of National Food and Strategic Reserves Administration, Beijing 100037, China
* WANG Weiwei, associate professor, E-mail: ;
MIN Yu'na, professor, E-mail:

Received date: 2023-05-31

  Online published: 2023-11-12

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摘要

本试验旨在研究包被植物乳杆菌和低聚果糖对蛋鸡生产性能、蛋品质、免疫能力、粪便臭味及盲肠微生物的影响。选取360只健康的34周龄海兰褐蛋鸡,随机分为4组,每组6个重复,每个重复15只。对照组(CON组)饲喂基础饲粮,试验组分别饲喂在基础饲粮的基础上添加1 g/kg包被植物乳杆菌(MLP组,活菌数为1.0×1010 CFU/g)、1 g/kg低聚果糖(FOS组)以及1 g/kg包被植物乳杆菌+1 g/kg低聚果糖(SYN组)的饲粮。试验期24周。结果表明:1)饲粮中添加包被植物乳杆菌和低聚果糖对蛋鸡生产性能无显著影响(P>0.05)。2)与对照组相比,FOS组和SYN组蛋白高度和哈氏单位显著提高(P<0.05)。3)与对照组相比,MLP组和FOS组蛋鸡血浆干扰素-γ(INF-γ)、白细胞介素-6(IL-6)和白细胞介素-2(IL-2)含量显著降低(P<0.05),SYN组血浆白细胞介素-10(IL-10)和IL-6含量显著提高(P<0.05),且SYN组血浆IL-10、IL-6和IL-2含量显著高于MLP组和FOS组(P<0.05)。4)与对照组相比,试验组蛋鸡粪便中吲哚和粪臭素含量均显著降低(P<0.05)。5)在门水平上,与对照组相比,MLP组蛋鸡盲肠厚壁菌门(Firmicutes)和FOS组盲肠拟杆菌门(Bacteroidota)相对丰度提高,试验组盲肠螺旋菌门(Spirochaetota)和脱硫菌门(Desulfobacterota)相对丰度降低。在属水平上,与对照组相比,MLP组盲肠乳杆菌属(Lactobacillus)相对丰度提高,盲肠拟杆菌属(Bacteroides)和理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)相对丰度降低;FOS组盲肠拟杆菌属和罗姆布茨菌属(Romboutsia)相对丰度提高;SYN组盲肠瘤胃球菌属扭链群(Ruminococcus_torques_group)和考拉杆菌属(Phascolarctobacterium)相对丰度有所提高。线性判别分析效应大小(LEfSe)分析表明,对照组蛋鸡盲肠显著富集有害菌脱硫弧菌属(Desulfovibrio)[线性判别分析(LDA)评分>2,P<0.05],SYN组盲肠显著富集有益菌霍氏真杆菌属(Eubacterium_hallii_group)和韦荣氏球菌属(Veillonella)(LDA评分>2,P<0.05)。综上可知,饲粮中添加包被植物乳杆菌和低聚果糖能够提高蛋鸡蛋品质和免疫能力,改善盲肠微生物组成,降低粪便臭味物质含量;其中,低聚果糖在改善蛋品质和降解臭味物质方面效果较佳,包被植物乳杆菌和低聚果糖联合添加在提高机体免疫能力和改善盲肠微生物组成方面效果较佳。

关键词： 包被植物乳杆菌; 低聚果糖; 蛋鸡; 生产性能; 粪便臭味物质; 盲肠微生物

本文引用格式

宋丹 , 段涛 , 陈炳旭 , 陈丽仙 , 王薇薇 , 闵育娜 . 包被植物乳杆菌和低聚果糖对蛋鸡生产性能、蛋品质、免疫能力、粪便臭味及盲肠微生物的影响[J]. 动物营养学报, 2023 , 35(11) : 7099 -7109 . DOI: 10.12418/CJAN2023.646

Abstract

This experiment was conducted to investigate the effects of coated Lactobacillus plantarum and fructooligosaccharide on performance, egg quality, immune capacity, fecal odor and cecum microbiota of laying hens. A total of 360 healthy Hy-Line brown laying hens of 34-week-old were randomly divided into 4 groups with 6 replicates per group and 15 hens per replicate. Hens in the control group (CON group) were fed a basal diet, and those in experimental groups were fed the basal diet supplemented with 1 g/kg coated Lactobacillus plantarum (MLP group, the viable bacteria count was 1.0×1010 CFU/g), 1 g/kg fructooligosaccharide (FOS group) and 1 g/kg coated Lactobacillus plantarum+1 g/kg fructooligosaccharide (SYN group) diets, respectively. The experiment lasted for 24 weeks. The results showed as follows: 1) dietary Lactobacillus plantarum and fructooligosaccharide had no significant effects on performance of laying hens (P>0.05). 2) Compared with the control group, the albumen height and Haugh unit in FOS and SYN groups were significantly increased (P<0.05). 3) Compared with the control group, the contents of interferon-γ (INF-γ), interleukin-6 (IL-6) and interleukin-2 (IL-2) in plasma of laying hens in MLP and FOS groups were significantly decreased (P<0.05), and the contents of interleukin-10 (IL-10) and IL-6 in plasma in SYN group were significantly increased (P<0.05), meanwhile the contents of IL-10, IL-6 and IL-2 in plasma in SYN group were significantly higher than those in MLP and FOS groups (P<0.05). 4) Compared with control group, the contents of indole and skatole in feces of laying hens in experimental groups were significantly decreased (P<0.05). 5) At the phylum level, compared with the control group, the Firmicutes relative abundance in cecum of laying hens in MLP group and the Bacteroidota relative abundance in cecum in FOS group were increased, and the relative abundances of Spirochaetota and Desulfobacterota in cecum in experimental groups were decreased. At the genus level, compared with the control group, the Lactobacillus relative abundance in cecum in MLP group was increased, and the relative abundances of Bacteroides and Rikenellaceae_RC9_gut_group were decreased; the relative abundances of Bacteroides and Romboutsia in cecum in FOS group were increased; the relative abundances of Ruminococcus_torques_group and Phascolarctobacterium in cecum in SYN group were increased. Linear discriminant analysis effect size (LEFSe) analysis showed that the harmful bacterium as Desulfovibrio was significantly enriched in cecum of laying hens in the control group [linear discriminant analysis (LDA) score>2, P<0.05], and the beneficial bacteria as Eubacterium_hallii_group and Veillonella were significantly enriched in cecum in SYN group (LDA score>2, P<0.05). In conclusion, dietary coated Lactobacillus plantarum and fructooligosaccharide can improve egg quality and immune capacity, improve cecum microbial composition, and reduce fecal odorous substance contents. Among them, fructooligosaccharide has better effects on improving egg quality and degrading odors, and the combination of Lactobacillus plantarum and fructooligosaccharide has better effects on improving immune capacity and cecum microbial composition.

Key words： coated Lactobacillus plantarum; fructooligosaccharide; laying hens; performance; fecal odorous substance; cecal microbiota

随着人们对肉蛋奶需求的增加,集约化养殖成为畜禽生产的主要养殖模式,但该养殖模式会产生大量的动物粪便,而动物粪便处理不当会严重污染环境,同时传播病原菌[1]。在需求增加的同时,畜禽产品的品质也日益受到关注,抗生素的限制使用加剧了动物感染细菌性炎症的风险,进而会影响畜禽产品的品质。家禽粪便中的臭味物质排放和抗生素替代品的选择已成为家禽业面临的主要挑战,因此寻找既能减少臭味物质排放又能增强机体免疫能力的高效环保策略具有巨大的生态和科学价值。饲粮中添加益生菌和益生元是目前应用最为广泛的策略之一,植物乳杆菌(Lactobacillus plantarum)和低聚果糖是常见的益生菌和益生元。Qiao等[2]研究表明,饲粮中添加植物乳杆菌能够显著提高蛋鸡产蛋率和粪便乳酸菌数量,产蛋率比对照组提高了2.8%。大量研究指出,饲粮中添加植物乳杆菌能够增强机体免疫和抗氧化能力,改善肠道屏障功能和肠道微生物组成[3⇓-5],降低氨气的排放[6]。Shang等[7]研究表明,低聚果糖能够显著提高肉鸡回肠绒毛高度、隐窝深度和黏膜总厚度,提高回肠中白细胞介素-10(interleukin-10,IL-10)和干扰素-γ(interferon-γ,IFN-γ)mRNA表达量,提高肉鸡的免疫能力。除此之外,饲粮中添加低聚果糖可以改善肠道菌群[8-9]。但是,低聚果糖对蛋鸡的影响研究很少,同时植物乳杆菌和低聚果糖对臭味物质粪臭素、吲哚的影响尚未研究。同时,植物乳杆菌的抗逆性差是目前限制其使用的关键因素之一,微囊化包被和保护剂可以有效增强其抗逆性[10],但微囊化和保护剂双层包被植物乳杆菌对蛋鸡的影响尚未可知。低聚果糖为植物乳杆菌提供能量,促进其定植,二者联合使用效果是否优于单独使用也未有报道。因此,本试验旨在研究双层包被植物乳杆菌和低聚果糖对蛋鸡生产性能、蛋品质、免疫能力、粪便臭味及盲肠微生物的影响,为植物乳杆菌和低聚果糖在蛋鸡养殖中的应用提供科学依据。

1 材料与方法

1.1 试验材料

本试验所用植物乳杆菌LP15-1为本课题组保藏菌种,包被植物乳杆菌采用国家粮食和物资储备局科学研究院专利技术(ZL202011208280.7)生产,该技术利用多层包被技术提高了植物乳杆菌的抗逆性,其中活菌数为1.0×1010 CFU/g;低聚果糖来自山东某生物股份有限公司,其中主要成分为35%蔗果三糖、50%蔗果四糖和10%蔗果五糖。

1.2 试验设计和饲粮

试验选取360只健康的34周龄海兰褐蛋鸡,随机分为4组(基础产蛋率分别为96.86%、96.47%、96.86%和96.86%),每组6个重复,每个重复15只。对照组(CON组)饲喂基础饲粮,试验组分别饲喂在基础饲粮的基础上添加1 g/kg包被植物乳杆菌(MLP组)、1 g/kg低聚果糖(FOS组)以及1 g/kg包被植物乳杆菌+1 g/kg低聚果糖(SYN组)的饲粮。基础饲粮为参照《鸡饲养标准》(NY/T 33—2004)配制的玉米-豆粕型饲粮,其组成及营养水平见表1。试验期24周。
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 61.00
豆粕Soybean meal 25.58
大豆油Soybean oil 1.80
磷酸氢钙CaHPO4 1.90
石粉Limestone 9.00
氯化钠NaCl 0.15
氯化胆碱Choline chloride (50%) 0.20
预混料Premix1) 0.22
DL-蛋氨酸DL-Met 0.10
L-赖氨酸盐酸盐L-Lys·HCl 0.05
合计Total 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 11.41
粗蛋白质CP 16.50
钙Ca 3.64
总磷TP 0.63
赖氨酸Lys 0.81
蛋氨酸Met 0.35

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 9 500 IU,VB2 6 mg,VB12 0.025 mg,VD3 62.5 μg,VE 30 IU,VK3 2.65 mg,生物素 biotin 0.032 5 mg,叶酸 folic acid 1.25 mg,D-泛酸 D-pantothenic acid 12 mg,烟酸 nicotinic acid 50 mg,Cu 8 mg,Fe 80 mg,Mn 100 mg,Zn 75 mg,I 0.35 mg,Se 0.15 mg。

2)代谢能为计算值,其余为实测值。ME was a calculated value, while the others were measured values.

1.3 饲养管理和样品采集

本试验在天津畜牧兽医研究所武清基地进行,试验采用3层阶梯笼养,每笼3只鸡,鸡舍环境温度维持在21~24 ℃,采用16 h人工光照,按正常免疫程序进行免疫接种。试验期间,鸡只自由采食和饮水,每天07:00、12:00和17:00各投料1次,全程监测鸡群健康状况并记录死淘鸡数。试验结束当天,每重复随机选取1只鸡,翅静脉采血于抗凝管中,2 000×g、4 ℃离心15 min,制备血浆;同时,收集新鲜粪便于离心管中,存于-20 ℃待测。鸡只采血后,采用二氧化碳(CO2)窒息后颈静脉放血致死,取盲肠食糜于离心管中,于-80 ℃保存待测。

1.4 检测指标及方法

1.4.1 生产性能和蛋品质

试验期间,每天按重复记录产蛋数、蛋重、喂料量和破蛋数,在试验结束后计算各组的产蛋率、平均日采食量、平均蛋重、料蛋比和破蛋率。试验结束当天,每重复收集5枚鸡蛋进行蛋品质分析,其中蛋壳强度采用蛋壳强度测定仪EFG-0503测定,蛋壳厚度采用蛋壳厚度测定测量仪ETG-1061测定,蛋黄颜色、哈氏单位和蛋白高度采用蛋品质分析仪EMT-5200测定,蛋形指数采用游标卡尺测定。

1.4.2 血浆免疫指标和粪便臭味物质含量

采用鸡特异性酶联免疫吸附测定(ELISA)试剂盒(北京中科凯泽科技有限公司)检测血浆INF-γ、白细胞介素-2(interleukin-2,IL-2)、白细胞介素-6(interleukin-6,IL-6)和IL-10含量。根据Zhu等[11]描述的方法,采用高效液相色谱法测定粪便中臭味物质吲哚和粪臭素含量。

1.4.3 盲肠微生物

盲肠微生物采用16S rRNA高通量测序方法进行分析,包括DNA提取、PCR扩增、荧光定量以及Illumina文库构建和测序[12]。其中,测序采用Illumina公司的MiSeq PE300平台(上海美吉生物医药科技有限公司)进行,随后在美吉生信云平台(https://cloud.majorbio.com)上进行数据分析。利用操作分类单元(operational taxonomic unit,OTU)信息,采用Mothur 1.30.1软件计算盲肠微生物α多样性指数,包括Shannon指数、Simpson指数、Chao1指数和ACE指数。通过线性判别分析(linear discriminant analysis,LDA)和线性判别分析效应大小(linear discriminant analysis effect size,LEfSe)分析,确定不同类群间细菌从门到属显著富集的类群(LDA评分>2,P<0.05)[13]。

1.5 数据统计分析

试验数据先用Excel 2016进行初步处理,然后采用SPSS 22.0软件中的单因素方差分析(one-way ANOVA)进行统计分析,随后采用Duncan氏法进行多重比较检验,试验结果以平均值和均值标准误(SEM)表示,P<0.05表示差异显著。

2 结果与分析

2.1 包被植物乳杆菌和低聚果糖对蛋鸡生产性能的影响

由表2可知,饲粮中添加包被植物乳杆菌和低聚果糖对蛋鸡产蛋率、平均蛋重、平均日采食量、料蛋比和破蛋率均无显著影响(P>0.05)。不过,与对照组(CON组)相比,试验组产蛋率和破蛋率均有改善,其中MLP组改善最为明显,其次是SYN组;MLP组产蛋率较对照组提高了2.68%,破蛋率降低了43.40%。
表2 包被植物乳杆菌和低聚果糖对蛋鸡生产性能的影响

Table 2 Effects of coated Lactobacillus plantarum and fructooligosaccharide on performance of laying hens

项目
Items		 组别Groups SEM P值
P-value
CON MLP FOS SYN
产蛋率Laying rate/% 93.77 96.28 95.30 95.57 0.427 0.208
平均蛋重Average egg weight/g 65.66 64.24 63.84 64.49 0.471 0.596
平均日采食量ADFI/g 113.51 110.62 108.23 112.15 1.323 0.568
料蛋比F/G 1.85 1.79 1.78 1.82 0.025 0.805
破蛋率Broken egg rate/% 0.53 0.30 0.40 0.33 0.077 0.734

同行数据肩标无字母或相同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 no letter or the same letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 包被植物乳杆菌和低聚果糖对蛋鸡蛋品质的影响

由表3可知,饲粮中添加包被植物乳杆菌和低聚果糖对蛋白高度和哈氏单位有显著影响(P<0.05),对蛋形指数、蛋黄颜色、蛋壳强度和蛋壳厚度均无显著影响(P>0.05)。与对照组相比,FOS组和SYN组蛋白高度和哈氏单位显著提高(P<0.05);MLP组蛋品质各项指标均无显著差异(P>0.05),但蛋壳强度、蛋白高度、蛋黄颜色和哈氏单位均有所提高(P>0.05)。
表3 包被植物乳杆菌和低聚果糖对蛋鸡蛋品质的影响

Table 3 Effects of coated Lactobacillus plantarum and fructooligosaccharide on egg quality of laying hens

项目
Items		 组别Groups SEM P值
P-value
CON MLP FOS SYN
蛋形指数Egg shape index 1.30 1.30 1.28 1.30 0.005 0.645
蛋壳厚度Eggshell thickness/mm 0.41 0.41 0.41 0.40 0.003 0.796
蛋壳强度Eggshell strength/N 36.06 37.78 35.32 39.16 0.947 0.508
蛋白高度Albumen height/mm 5.69b 6.38ab 7.11a 7.17a 0.188 0.005
蛋黄颜色Yolk color 7.00 7.26 6.80 6.93 0.067 0.089
哈氏单位Haugh unit 71.48b 77.17ab 82.79a 82.47a 1.374 0.002

2.3 包被植物乳杆菌和低聚果糖对蛋鸡血浆免疫指标的影响

由表4可知,饲粮中添加包被植物乳杆菌和低聚果糖对蛋鸡血浆免疫指标均有显著影响(P<0.05)。与对照组相比,MLP组和FOS组蛋鸡血浆INF-γ、IL-6和IL-2含量显著降低(P<0.05),SYN组血浆IL-10和IL-6含量显著提高(P<0.05),且SYN组血浆IL-10、IL-6和IL-2含量显著高于MLP组和FOS组(P<0.05)。
表4 包被植物乳杆菌和低聚果糖对蛋鸡血浆免疫指标的影响

Table 4 Effects of coated Lactobacillus plantarum and fructooligosaccharide on plasma immune indices of laying hens pg/mL

项目
Items		 组别Groups SEM P值
P-value
CON MLP FOS SYN
干扰素-γ INF-γ 317.05a 112.31b 114.34b 108.95b 27.189 0.004
白细胞介素-10 IL-10 49.90bc 47.18c 50.32b 54.71a 0.803 0.001
白细胞介素-6 IL-6 20.77b 17.30c 17.11c 22.10a 0.532 <0.001
白细胞介素-2 IL-2 185.58a 168.62b 169.46b 191.73a 2.801 <0.001

2.4 包被植物乳杆菌和低聚果糖对蛋鸡粪便臭味物质含量的影响

由表5可知,饲粮中添加包被植物乳杆菌和低聚果糖对蛋鸡粪便臭味物质含量有显著影响。与对照组相比,试验组蛋鸡粪便中吲哚和粪臭素含量均显著降低(P<0.05),其中FOS组降低效果最好,粪便中吲哚和粪臭素含量比对照组均降低87%以上,其次是MLP组,降低79%以上。
表5 包被植物乳杆菌和低聚果糖对蛋鸡粪便臭味物质含量的影响

Table 5 Effects of coated Lactobacillus plantarum and fructooligosaccharide on fecal odorous substance contents of laying hens ng/mg

项目
Items		 组别Groups SEM P值
P-value
CON MLP FOS SYN
吲哚Indole 20.27a 4.20b 2.37b 5.28b 2.078 0.001
粪臭素Skatole 1.58a 0.32b 0.19b 0.39b 0.188 0.017

2.5 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物的影响

2.5.1 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物α多样性的影响

由表6可知,饲粮中添加包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物α多样性各项指数均无显著影响(P>0.05)。不过,与对照组相比,FOS组Shannon指数、Chao1指数和ACE指数有所降低,Simpson指数有所升高,表明饲粮中添加低聚果糖降低了蛋鸡盲肠微生物的多样性和丰富度;SYN组Chao1指数和ACE指数有所升高,表明饲粮同时添加包被植物乳杆菌和低聚果糖提高了盲肠微生物的丰富度。
表6 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物α多样性的影响

Table 6 Effects of coated Lactobacillus plantarum and fructooligosaccharide on α diversity of cecal microbiota of laying hens

项目
Items		 组别Groups SEM P值
P-value
CON MLP FOS SYN
Shannon指数Shannon index 4.75 4.74 4.51 4.73 0.040 0.093
Simpson指数Simpson index 0.019 0.019 0.028 0.023 0.001 0.088
ACE指数ACE index 746.37 725.30 688.64 787.10 15.726 0.159
Chao1指数Chao1 index 745.50 732.26 714.28 820.08 17.106 0.128

2.5.2 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物组成的影响

由图1可知,在门水平上,与对照组相比,MLP组蛋鸡盲肠厚壁菌门(Firmicutes)相对丰度有所提高,FOS组盲肠拟杆菌门(Bacteroidota)相对丰度有所提高,试验组盲肠螺旋菌门(Spirochaetota)和脱硫菌门(Desulfobacterota)相对丰度有所降低,其中MLP组盲肠螺旋菌门和FOS组盲肠脱硫菌门相对丰度降低最多,分别较对照组降低了89.18%和71.28%。在属水平上,与对照组相比,MLP组盲肠乳杆菌属(Lactobacillus)相对丰度有所提高,盲肠拟杆菌属(Bacteroides)和理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)相对丰度有所降低;FOS组盲肠拟杆菌属和罗姆布茨菌属(Romboutsia)相对丰度有所提高;SYN组盲肠瘤胃球菌属扭链群(Ruminococcus_torques_group)和考拉杆菌属(Phascolarctobacterium)相对丰度有所提高。
图1 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物在门和属水平上相对丰度的影响

A:在门水平上的物种组成与相对丰度 species composition and relative abundance at phylum level;B:在属水平上的物种组成与相对丰度 species composition and relative abundance at genus level。

Bacteroidota:拟杆菌门;Firmicutes:厚壁菌门;Actinobacteriota:放线菌门;Spirochaetota:螺旋菌门;Unclassified_k_norank_d_Bacteria:未分类未定级细菌;Desulfobacterota:脱硫菌门;Others:其他;Bacteroides:拟杆菌属;Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;Unclassified_o_Bacteroidales:未分类的拟杆菌目;Unclassified_f_Lachnospiraceae:未分类的毛螺菌科;Lactobacillus:乳杆菌属;Ruminococcus_torques_group:瘤胃球菌属扭链群;Romboutsia:罗姆布茨菌属;Subdoligranulum:罕见小球菌属;Olsenella:欧陆森氏菌属;Phascolarctobacterium:考拉杆菌属;Faecalibacterium:粪杆菌属;Peptococcus:消化球菌属;Blautia:布劳特氏菌属;Prevotellaceae_UCG-001:普雷沃氏菌科UCG-001;Shuttleworthia:沙特尔沃思菌属;Alistipes:另枝菌属;Christensenellaceae_R-7_group:克里斯滕森菌科R-7群;Butyricicoccus:丁酸球菌属;Turicibacter:苏黎世杆菌属;Parabacteroides:副拟杆菌属;Treponema:密螺旋体属;Norank_f_norank_o_Clostridia_UCG-014:未定级梭菌纲UCG-014;Unclassified_f_Peptostreptococcaceae:未分类消化链球菌科;Bifidobacterium:双歧杆菌属。

Fig.1 Effects of coated Lactobacillus plantarum and fructooligosaccharide on relative abundance of cecum microbiota at phylum and genus levels of laying hens

2.5.3 蛋鸡盲肠差异微生物LDA和LEfSe分析

由图2可知,对照组蛋鸡盲肠显著富集的微生物是鳞球菌属(Sphaerochaeta)、毛螺菌科NK4A136群(Lachnospiraceae_NK4A136_group)和脱硫弧菌属(Desulfovibrio)(LDA评分>2,P<0.05),MLP组显著富集的微生物是未分类消化链球菌科(unclassified_f_Peptostreptococcaceae)(LDA评分>2,P<0.05),FOS组显著富集的微生物是假黄杆菌属(Pseudoflavonifractor)和双歧杆菌科(Bifidobacteriaceae),SYN组显著富集的微生物是霍氏真杆菌属(Eubacterium_hallii_group)和韦荣氏球菌属(Veillonella)(LDA评分>2,P<0.05)。综上可知,饲粮中添加包被植物乳杆菌和低聚果糖改变了蛋鸡盲肠微生物的组成,表现为提高了有益菌(如双歧杆菌科、霍氏真杆菌属等)相对丰度,降低了有害菌(如脱硫弧菌属)相对丰度。
图2 蛋鸡盲肠差异微生物LDA和LEfSe分析

A:线性判别分析效应大小分析 LEfSe analysis;B:线性判别分析 LDA。

Desulfobacterota:脱硫菌门;Actinobacteria:放线菌纲;Desulfovibrionia:脱硫弧菌纲;Bifidobacteriales:双歧杆菌目;Desulfovibrionales:脱硫弧菌目;Bifidobacteriaceae:双歧杆菌科;Desulfovibrionaceae:脱硫弧菌科;Desulfovibrio:脱硫弧菌属;Eubacterium_hallii_group:霍氏真杆菌属;Lachnospiraceae_NK4A136_group:毛螺菌科NK4A136群;Pseudoflavonifractor:假黄杆菌属;Sphaerochaeta:鳞球菌属;Veillonella:韦荣氏球菌属;unclassified_f_Peptostreptococcaceae:未分类消化链球菌科。

Fig.2 LDA and LEfSe analysis of differential cecal microbiota of laying hens

3 讨论

3.1 包被植物乳杆菌和低聚果糖对蛋鸡生产性能和蛋品质的影响

植物乳杆菌和低聚果糖是目前应用广泛的益生菌和益生元,但研究主要集中在肉鸡上,且饲粮中添加植物乳杆菌和低聚果糖可以提高肉鸡的生长性能[14⇓⇓-17]。但Ding等[18]和Al-Khalaifa等[19]分别在饲粮中添加植物乳杆菌和低聚果糖未显著改善肉鸡生长性能。Qiao等[2]在饲粮中添加植物乳杆菌显著提高蛋鸡产蛋率和采食量。Feng等[20]在饲粮中添加低聚果糖可以提高黄羽肉种鸡的产蛋率、蛋重和料蛋比。本研究发现,饲粮中添加包被植物乳杆菌和低聚果糖未显著影响蛋鸡生产性能,但是产蛋率和破蛋率均有改善。生产性能的改善可能是因为包被植物乳杆菌和低聚果糖提高了机体的免疫能力和改善了肠道微生物组成,进而促进了营养物质的消化和吸收[3]。而饲粮中添加包被植物乳杆菌和低聚果糖对生产性能的影响不同的原因可能是因为菌株类别和活性、动物品种以及饲养环境不同所致[21]。随着生活水平的提升,人们不仅仅关注生产性能,蛋品质的优劣也是重点关注之一。本研究发现,饲粮中联合添加包被植物乳杆菌和低聚果糖(SYN组)显著提高蛋白高度和哈氏单位,这与Xu等[3]研究益生菌(枯草芽孢杆菌、乳酸菌和酵母菌)提高产蛋高峰期和产蛋后期的蛋白高度和哈氏单位的结果一致,其原因有待进一步研究。

3.2 包被植物乳杆菌和低聚果糖对蛋鸡免疫能力的影响

细胞因子主要由Th1细胞和Th2细胞分泌,在调节免疫和炎症方面具有重要意义[22]。细胞因子IL-2和INF-γ是由Th1细胞在病毒或细胞内细菌感染后产生,具有促进炎症和细胞吞噬作用;而IL-10是由Th2细胞分泌产生,有助于抗体合成并抑制炎症[23-24];IL-6既是促炎细胞因子又是抗炎细胞因子,在单核细胞和巨噬细胞中产生[25]。有研究指出,植物乳杆菌可减少Th1类细胞因子(降低血清IFN-γ含量和回肠IL-6表达),上调Th2类细胞因子IL-4在回肠中表达[24]。饲粮中添加低聚果糖能提高肉鸡回肠中IL-10的mRNA表达量[26]。上述结果与本研究一致,本研究结果表明,饲粮中单独添加包被植物乳杆菌或低聚果糖显著降低了蛋鸡血浆IL-6、IL-2和INF-γ含量,联合添加则显著提高了血浆IL-10含量。植物乳杆菌和低聚果糖增强机体免疫能力是通过升高IL-10含量,调节先天性和适应性T细胞反应,有效抑制炎症反应实现的[25];同时,其能够通过减少IL-6、IL-2和INF-γ炎症细胞因子,抑制肠道结构和屏障功能受损,从而提高机体对营养物质的消化和吸收能力[27];此外,还有可能是其能够平衡促炎因子和抗炎因子,保护机体免受病原体入侵,并且维持肠道内环境的稳定[28]。

3.3 包被植物乳杆菌和低聚果糖对蛋鸡粪便臭味物质的影响

恶臭排放已成为畜牧业面临的主要挑战之一,粪臭素和吲哚是导致鸡排泄物恶臭的主要原因,二者导致多种生物体疾病,并且不易生物降解[1]。本实验室前期研究发现,植物乳杆菌能够降低粪便粪臭素和吲哚含量分别达20.0%和36.0%[10]。Zhu等[11]在饲粮中添加低聚糖显着降低了肉鸡排泄物中的吲哚和粪臭素含量。本研究中,植物乳杆菌经过新工艺包被后添加,蛋鸡粪便粪臭素和吲哚含量降低了79%以上,饲粮中添加低聚果糖使粪便粪臭素和吲哚含量降低了87%以上,但二者联合使用未出现协同效果。吲哚和粪臭素是由微生物活动对L-色氨酸进行多步降解产生的,因此,粪臭素和吲哚的产生受可用L-色氨酸水平、肠道微生物组成和饲粮组成的调节[11]。高浓度的吲哚和粪臭素可能通过解偶联,从而抑制氧化磷酸化在线粒体中形成,对蛋鸡的生产性能产生不利影响[29]。饲粮中添加包被植物乳杆菌和低聚果糖降低臭味物质含量可能是因为其能够增加肠道内有益菌数量,降低肠道pH,促进更多的蛋白质和氨基酸消化吸收,从而抑制色氨酸转化为粪臭素和吲哚,最终降低臭味物质的排放。

3.4 包被植物乳杆菌和低聚果糖对蛋鸡盲肠微生物的影响

维持肠道微生物群落的动态平衡对于减轻炎症和改善动物肠道健康具有重要意义[25]。本研究结果表明,饲粮中联合添加包被植物乳杆菌和低聚果糖能够提高蛋鸡盲肠微生物Chao1指数和ACE指数,表明包被植物乳杆菌和低聚果糖联合添加提高了盲肠菌群的丰富度,菌群丰富度提高可能是因为包被植物乳杆菌和低聚果糖降低了臭味物质含量,减少了其对肠道上皮细胞的氧化损伤,从而能够维护肠道氧化还原稳态;还有可能是因为低聚果糖促进了植物乳杆菌的增殖,增加了有益菌的数量。
本试验中,饲粮中添加包被植物乳杆菌和低聚果糖在门和属水平上均改变了蛋鸡盲肠微生物组成。在门水平,与对照组相比,MLP组盲肠厚壁菌门相对丰度提高,试验组盲肠螺旋菌门和脱硫菌门相对丰度降低。厚壁菌门在恢复肠道稳态和营养物质消化利用方面发挥着重要作用,其丰度与能量和营养物质吸收呈正相关[30]。研究指出,饲粮中添加植物乳杆菌能够提高北京鸭肠道厚壁菌门相对丰度,促进其生长性能提高并降低血清促炎因子白细胞介素-1β含量[24]。因此,本研究饲粮中添加包被植物乳杆菌可能通过提高厚壁菌门丰度而改善了蛋鸡的营养利用和抗炎状态,并最终改善了产蛋性能和降低了臭味物质的排放。螺旋菌门丰度的提高会降低饲料转化率,进而影响繁殖和生产性能[31]。脱硫菌门可能释放脂多糖(lipopolysaccharide,LPS)到肠道引起炎症反应,同时扰乱肠道能量代谢,诱导肠上皮细胞的异常增殖,损伤肠道屏障[32]。饲粮中添加包被植物乳杆菌和低聚果糖可能是通过降低螺旋菌门和脱硫菌门丰度,提高饲料转化率,降低肠道炎症和肠道屏障损伤,从而提高蛋鸡的生产性能和机体免疫能力。
已有研究报道,乳杆菌属通过产生有机酸(乳酸和乙酸)提高机体免疫能力和肠道完整性,从而抵抗沙门氏菌等病原体的感染,并促进营养吸收,进而改善生长性能[33-34];拟杆菌属会导致肠道炎症并降低营养物质消化吸收[30]。Lan等[35]指出,理研菌科RC9肠道群与高脂饮食诱导的“有害菌”呈正相关,与“有益菌”呈负相关,其丰度的提高可能会进一步诱导菌群紊乱。本研究中,与对照组相比,MLP组蛋鸡盲肠乳杆菌属相对丰度提高,盲肠拟杆菌属和理研菌科RC9肠道群相对丰度降低,这就进一步解释了饲粮中添加包被植物乳杆菌可能是通过改变微生物组成提高了营养物质的利用率,并降低了机体的炎症反应,进而提高了生产性能。有研究指出,盲肠中拟杆菌属、嗜胆菌属(Bilophila)和埃希氏杆菌属(Escherichia)相对丰度的下降导致肠道粪臭素减少[36],这也就解释了MLP组臭味物质含量降低是通过降低拟杆菌属和理研菌科RC9肠道群丰度实现的。大量研究指出,双歧杆菌科有利于树突状细胞的成熟以及T细胞和T辅助细胞发育的调节,并产生宿主产生能量所需的必需短链脂肪酸[37-38]。饲粮中添加果聚糖能够提高双歧杆菌丰度,并通过促进肉鸡盲肠的低pH来抑制病原体(大肠杆菌和沙门氏菌)增殖[38];低聚果糖能够增强肠道发酵,增加短链脂肪酸的产生,刺激双歧杆菌等有益菌的生长[8],这与本研究结果一致,本研究饲粮中添加低聚果糖显著富集了双歧杆菌科。
瘤胃球菌属扭链群通过增加脱氧胆酸的产生和通过胆汁酸-脂肪-G蛋白偶联胆汁酸受体Gpbar1(TGR5)轴改善肥胖而有益于代谢[39];考拉杆菌属通过产丙酸降低LPS结合蛋白和C反应蛋白的水平来减少炎症和保护肠道黏膜屏障[40]。本研究发现,饲粮中添加联合包被植物乳杆菌和低聚果糖提高了蛋鸡盲肠瘤胃球菌属扭链群和考拉杆菌属相对丰度,进一步进行LEfSe多级物种差异判别分析发现,SYN组显著富集霍氏真杆菌属和韦荣氏球菌属。韦荣氏球菌属是短链脂肪酸乙酸盐和丙酸盐的生产者,可以维持能量稳态,其丰度的提高可以增强肠道菌群的能量代谢[41]。霍氏真杆菌属在调节加强肠道屏障功能和调节全身炎症的生物途径中发挥有益作用[42]。因此,饲粮中联合添加包被植物乳杆菌和低聚果糖可能是通过改变微生物组成,减少炎症和改善肠道屏障功能,进而促进代谢。

4 结论

饲粮中添加包被植物乳杆菌和低聚果糖能够提高蛋鸡蛋品质和免疫能力,改善盲肠微生物组成,降低粪便臭味物质含量;其中,低聚果糖在改善蛋品质和降解臭味物质方面效果较佳,包被植物乳杆菌和低聚果糖联合添加在提高机体免疫能力和改善盲肠微生物组成方面效果较佳。

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