Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 5: 3025 - 3040

DOI: https://doi.org/10.12418/CJAN2023.282

RESEARCH PAPER

Effects of Bacillus coagulans and Clostridium butyricum on Growth Performance, Slaughter Performance, Serum Biochemical Indexes and Intestinal Health of Meat Ducks

  • ZOU Hanyu , 1 ,
  • WANG Baowei , 1, * ,
  • ZHANG Ming’ai 2 ,
  • FAN Wenlei 2 ,
  • YUE Bin 3 ,
  • KONG Min 3
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  • 1 College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
  • 2 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China
  • 3 Institute of Quality Waterfowl, Qingdao Agricultural University Qingdao 266109, China
*professor, E-mail:

Received date: 2022-11-16

  Online published: 2023-05-11

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Abstract

This experiment was conducted to study the effects of Bacillus coagulans and Clostridium butyricum on growth performance, slaughter performance, serum biochemical indexes and intestinal health of meat ducks. A total of 180 healthy 7-day-old Cherry Valley ducks were selected and divided into 6 groups according to their weight, with 5 replicates in each group and 6 ducks in each replicate. The control group was fed a basal diet, the BC group was fed basal diet supplemented with Bacillus coagulans, the CB group was fed the basal diet supplemented with Clostridium butyricum, the BC+CB group (1∶1) group was fed the basal diet supplemented with Bacillus coagulans and Clostridium butyricum (mixed with 1∶1 ratio), the BC+CB group (1∶2) group was fed the basal diet supplemented with Bacillus coagulans and Clostridium butyricum (mixed with 1∶2 ratio), the BC+CB group (2∶1) group was fed the basal diet supplemented with Bacillus coagulans and Clostridium butyricum (mixed with 2∶1 ratio), and the dietary total addition of probiotics was 0.3%. The pre-experimental period lasted for 5 days, and the experimental period lasted for 42 days. The results showed as follows: 1) compared with the control group, the average daily weight gain during 12 to 35 days of age and 12 to 49 days of age of BC+CB (1∶2) group was significantly increased (P<0.05), and the feed to gain ratio during 12 to 35 days of age and 12 to 49 days of age of BC+CB (1∶2) group and BC+CB (2∶1) group was significantly decreased (P<0.05). 2) There were no significant differences in dressing percentage, semi-eviscerated rate, eviscerated rate, breast muscle rate, leg muscle rate and abdominal fat rate among all groups (P>0.05). 3) The contents of high-density lipoprotein cholesterol, total protein and albumin in serum of BC+CB (1∶2) group and BC+CB (2∶1) group were significantly higher than those of the control group (P<0.05), and the serum alanine aminotransferase activity was significantly lower than that of control group and BC group (P<0.05). There were no significant differences in serum low density lipoprotein cholesterol, triglyceride, total amino acid contents and alkaline phosphatase, glutamic-oxaloacetic transaminase activities among all groups (P>0.05). 4) The intestinal secretory immunoglobulin A content of BC+CB (2∶1) group was significantly higher than that of control group and BC group (P<0.05). There were no significant differences in contents of tumor necrosis factor-α and interleukin-1β in intestine among all groups (P>0.05). 5) Compared with the control group, the villus height and villus height to crypt depth ratio in duodenum and ileum of BC+CB (1∶1) group, BC+CB (1∶2) group and BC+CB (2∶1) group were significantly increased (P<0.05). 6) Compared with the control group, the relative abundances of Ruthenibacterium and Erysipelatoclostrichaceae-unclassified in caecum of BC+CB (2∶1) group were significantly increased (P<0.05), and the relative abundances of Halomonas, Halomonadaceae, Oceanospillales and Kocuria in caecum were significantly decreased (P<0.05). In conclusion, compared with single bacterial preparation, Bacillus coagulans and Clostridium butyricum compound bacterial preparation is more conducive to promoting the growth performance of Cherry Valley ducks, increasing the contents of total protein, albumin, high-density lipoprotein cholesterol in serum, decreasing the serum alanine aminotransferase activity, improving the small intestine morphology, improving the immunity, and maintaining the intestinal flora structure. Under the test conditions, when the proportion of Bacillus coagulans and Clostridium butyrate is 2∶1, the effect is the best.

Key words： compound bacteria preparation; growth performance; serum biochemical indexes; intestinal morphology development; high throughput sequencing

Cite this article

ZOU Hanyu , WANG Baowei , ZHANG Ming’ai , FAN Wenlei , YUE Bin , KONG Min . Effects of Bacillus coagulans and Clostridium butyricum on Growth Performance, Slaughter Performance, Serum Biochemical Indexes and Intestinal Health of Meat Ducks[J]. Chinese Journal of Animal Nutrition, 2023 , 35(5) : 3025 -3040 . DOI: 10.12418/CJAN2023.282

当前集约化养殖条件下,动物多处于高密度、饲料霉变、易感病的环境或应激状态下,益生菌作为一种绿色、安全的新型饲料添加剂,不仅能够避免使用抗生素造成的抗药性以及药物残留,还能够提高营养利用率,促进动物生长,调节肠道健康,提高免疫力[1-2]。凝结芽孢杆菌是革兰氏阳性菌,属芽孢杆菌类,能够定植于小肠上部并且在结肠处产芽孢,且抗逆性强,能够在饲料加工过程中保持活性[3]。丁酸梭菌作为常用的饲料添加剂能够提高畜禽生长性能,调节肠道菌群,维护肠道健康[4-5]。单一益生菌制剂结构功能单一,纯度高,但不能够完全满足家禽养殖的多种需求,复合益生菌制剂能够弥补这一缺点[6]。陈婷等[7]研究表明,饲粮中添加复合益生菌发酵饲料能提高雪峰乌骨鸡的养分表观消化率,改善肉品质。刘淑娇等[8]试验结果表明,复合益生菌发酵饲料可以改善肉仔鸡生长性能,提高免疫功能。Fan等[9]研究发现,丁酸梭菌和凝结芽孢杆菌能够改善虹鳟鱼的生长性能,增强抗病性。Biswas等[10]研究发现,在饲粮中加入多种益生菌能够改善肉鸡性能、免疫功能以及营养代谢。目前,凝结芽孢杆菌和丁酸梭菌在猪和肉鸡饲养方面已有研究,而在肉鸭方面未见报道,且二者配合使用的效果和最佳比例的研究还处于空白。因此,本试验在饲粮中添加按不同比例混合的凝结芽孢杆菌和丁酸梭菌,旨在探究其对肉鸭生长性能、屠宰性能、血清生化指标及肠道健康的影响,为复合益生菌饲料添加剂在肉鸭生产中的应用提供理论支持。

1 材料与方法

1.1 试验材料

凝结芽孢杆菌(经过亚铁离子驯化的富铁凝结芽孢杆菌CG.24223,活菌数1×109 CFU/mL)和丁酸梭菌(活菌数1×109 CFU/mL)均由国家水禽产业技术体系营养与饲料功能研究室提供。试验肉鸭购自新希望六和股份有限公司,所用品种为樱桃谷鸭。

1.2 试验设计

选取7日龄雄性樱桃谷肉鸭180只,根据体重均匀分为6组,每组5个重复,每个重复6只。对照组饲喂基础饲粮,BC组饲喂添加凝结芽孢杆菌的试验饲粮,CB组饲喂添加丁酸梭菌的试验饲粮,BC+CB(1∶1)组饲喂添加凝结芽孢杆菌和丁酸梭菌复合菌制剂(以1∶1比例混合)的试验饲粮,BC+CB(1∶2)组饲喂添加凝结芽孢杆菌和丁酸梭菌复合菌制剂(以1∶2比例混合)的试验饲粮,BC+CB组(2∶1)组饲喂添加凝结芽孢杆菌和丁酸梭菌复合菌制剂(以2∶1比例混合)的试验饲粮,试验饲粮中益生菌制剂总添加量均为0.3%。基础饲粮组成及营养水平见表1。
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 63.55
豆粕Soybean meal 23.85
大豆油Soybean oil 5.00
菊花梗Chrysanthemum stem 3.24
磷酸氢钙CaHPO4 1.59
石粉Limestone 1.23
食盐NaCl 0.30
DL-蛋氨酸DL-Met 0.17
L-赖氨酸盐酸盐L-Lys·HCl 0.06
色氨酸Tryptophan 0.01
预混料Premix1) 1.00
合计Total 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 13.11
粗蛋白质CP 17.01
粗纤维CF 4.31
粗脂肪EE 8.17

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 12 000 IU,VD3 2 500 IU,VE 20 mg,VK3 3 mg,VB1 3 mg,VB2 8 mg,VB6 7 mg,VB12 0.03 mg,D-泛酸 D-pantothenic acid 20 mg,烟酸 nicotinic acid 50 mg,叶酸 folic acid 1.5 mg,生物素 biotin 0.1 mg,胆碱 choline 500 mg,Cu (as copper sulfate) 9 mg,Zn (as zinc sulfate) 110 mg,Fe (as ferrous sulfate) 100 mg,Mn (as manganese sulfate) 100 mg,Se (as sodium selenite) 0.16 mg,I (as potassium iodide) 0.6 mg。

2)营养水平均为计算值。Nutrient levels were calculated values.

1.3 饲养管理

对饲养环境及器具进行严格的冲洗和消毒,通风净化后开始试验。试验鸭喂料采取少喂勤添的方式,自由采食和饮水。育雏前期保持舍内温度维持在27~31 ℃;1~7日龄24 h光照,8日龄后23 h光照;环境相对湿度为55%~65%。预试期5 d,正试期42 d。

1.4 样品采集与指标测定

1.4.1 生长性能

分别在正式试验开始时(12日龄)、试验中期(35日龄)以及试验结束(49日龄)时以重复为单位对肉鸭进行称重,计算平均日增重(ADG);称量和记录各重复肉鸭在各个阶段的饲料消耗量,计算平均日采食量(ADFI)以及料重比(F/G)。

1.4.2 屠宰性能

屠宰性能参照《家禽生产性能名词术语和度量计算方法》(NY/T 823—2020)进行测定。试验结束时,从每个重复中随机取2只肉鸭进行解剖取样,称量并记录活体重、屠体重、半净膛重、全净膛重、胸肌重、腿肌重和腹脂重,计算屠宰率、半净膛率、全净膛率、胸肌率、腹脂率和腿肌率。

1.4.3 血清生化指标

试验结束时,从每重复中随机取2只肉鸭,颈静脉血10 mL于一次性真空采血管(内含分离胶和促凝剂)中,3 000 r/min离心20 min取上清液,制得血清样品,分装,-40 ℃冷冻保存。采用试剂盒测定血清总胆固醇(T-CHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、总氨基酸(TAA)、总蛋白(TP)、白蛋白(ALB)含量及碱性磷酸酶(AKP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)活性,以上试剂盒均购自南京建成生物工程研究所。

1.4.4 肠道炎症因子以及免疫球蛋白含量测定

分别取十二指肠、空肠、回肠各10~15 cm,剪开翻转,4 ℃生理盐水冲洗内容物,刮取肠内膜组织。肠黏膜取样0.1 g,加入0.9 mL生理盐水破碎匀浆,3 000 r/min离心20 min,收集上清液。使用酶联免疫吸附测定(ELISA)试剂盒检测肠道肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、分泌型免疫球蛋白A(sIgA)含量,以上试剂盒均购自南京建成生物工程研究所。

1.4.5 肠道形态观测

分离十二指肠、空肠、回肠,取各区中部约2 cm,用生理盐水冲洗内容物,放入4%甲醛溶液中固定48 h,将固定后的十二指肠、空肠、回肠制成石蜡切片进行苏木精-伊红(HE)染色,在显微镜下测量小肠绒毛高度、隐窝深度,计算绒隐比(绒毛高度/隐窝深度)。

1.4.6 16S rDNA高变区/内源转录间隔区(ITS)的PCR扩增产物的高通量测序

将盲肠微生物进行基因组DNA提取,并通过PCR扩增纯化,引物序列及参数见表2,二次扩增后用2%琼脂糖凝胶进行电泳,然后对目的片段进行切胶回收。文库质检:使用Qubit Fluorometer检测文库DNA质量浓度,大于1.0 ng/μL为合格;使用Qseq100 DNA Analyzer检测文库DNA长度分布,符合目的片段长度、单一峰、无接头峰和大片段峰则认为合格;使用KAPA Library Quantification Kit对文库DNA的摩尔浓度进行定量,以此作为文库混合的标准。将文库混合、变性后,加入到Illumina MiSeq测序平台进行高通量并行测序。以97%的相似性对序列进行操作分类单元(OTU)聚类后,去除嵌合体序列,生成OTU代表序列与数据库(16S,RDP数据库;18S,SILVA数据库;ITS,UNITE数据库)对比完成OTU分类学注释。肠道菌群多样性分析包括样本在界(domain)、门(phylum)、纲(class)、目(order)、科(family)、属(genus)各分类学水平的物种比例和分布的群落组成分析;基于Chao1指数、ACE指数、Shannon指数、Simpson指数、Invsimpson指数的α多样性分析;通过多变量统计学方法主成分分析(PCA)、多元方差分析(ADONIS)、相似性分析(ANOSIM)、多重反应排列分析(MRPP)、分子方差分析(AMOVA)、线性判别分析效应大小(LEfSe)分析等方法,从中发现不同组之间的差异,即β多样性分析。
表2 引物序列及参数

Table 2 Primer sequences and parameters

扩增子名称
Amplifier names		 引物名称
Primer names		 引物序列
Primer sequences (5'—3')		 片段大小
Fragment size/bp
细菌16S
Bacteria 16S		 B341F
B785R		 CCTACGGGNGGCWGCAG
GACTACHVGGGTATCTAATCC		 450
古菌16S
Archaea 16S		 A349F
A806R		 CAGCCGCCGCGGTAA
GTGCTCCCCCGCCAATTCCT		 420
真菌ITS
Fungus ITS		 ITS-3
ITS-4		 GCATCGATGAAGAACGCAGC
TCCTCCGCTTATTGATATGC		 350
真菌18S
Fungus 18S		 EF4
NS2		 GGAAGGGRTGTATTTATTAG
GGCTGCTGGCACCAGACTTGC		 380

1.5 数据统计与分析

试验数据采用SPSS 25.0软件进行单因素方差统计分析(one-way ANOVA),采用Duncan氏多重比较法对分析结果进行显著性检验,P<0.05时即判定差异显著,试验数据用平均值±标准差表示。

2 结果

2.1 凝结芽孢杆菌和丁酸梭菌对肉鸭生长性能的影响

由表3可知,BC+CB(1∶2)组肉鸭12~35日龄、12~49日龄的ADG显著高于对照组和BC组(P<0.05)。各组之间各个阶段的ADFI差异不显著(P>0.05)。BC+CB(1∶2)组肉鸭12~35日龄、12~49日龄的F/G显著低于对照组(P<0.05),BC+CB(2∶1)组肉鸭12~35日龄、12~49日龄的F/G显著低于对照组和BC组(P<0.05),其中BC+CB(2∶1)组F/G最低。
表3 凝结芽孢杆菌和丁酸梭菌对肉鸭生长性能的影响

Table 3 Effects of Bacillus coagulans and Clostridium butyricum on growth performance of meat ducks

项目
Items		 日龄
Days of
age		 组别Groups P值
P-value
对照
Control		 BC CB BC+CB
(1∶1)		 BC+CB
(1∶2)		 BC+CB
(2∶1)
平均日增重
ADG/(g/d)		 12~35 72.88±2.19b 71.62±7.03c 74.02±5.19abc 76.50±0.75abc 80.43±2.45a 78.91±2.98ab 0.032
36~49 68.77±5.65 70.35±4.15 71.58±8.39 72.00±5.29 73.78±2.05 75.93±3.82 0.403
12~49 71.64±0.42b 71.31±3.50b 71.73±4.21b 73.84±4.20ab 79.12±3.16a 78.01±2.87ab 0.005
平均日
采食量
ADFI/(g/d)		 12~35 173.85±6.02 163.14±5.86 163.25±10.98 159.24±5.33 171.97±7.54 159.57±8.88 0.129
36~49 184.58±3.78 174.31±8.77 188.75±9.45 180.19±10.50 179.04±12.07 182.85±11.87 0.683
12~49 177.10±5.90 166.53±6.30 170.98±9.85 165.59±13.57 174.11±10.54 166.63±4.48 0.297
料重比
F/G		 12~35 2.37±0.03a 2.32±0.16ab 2.21±0.07abc 2.16±0.11bc 2.14±0.10bc 2.07±0.09c 0.008
36~49 2.63±0.15 2.40±0.34 2.58±0.33 2.44±0.17 2.36±0.28 2.37±0.09 0.569
12~49 2.44±0.05a 2.34±0.16ab 2.32±0.14abc 2.24±0.11abc 2.20±0.14bc 2.12±0.09c 0.023

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。表4、表5、表6和表10同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as Table 4, Table 5, Table 6 and Table 10.

2.2 凝结芽孢杆菌和丁酸梭菌对肉鸭屠宰性能的影响

由表4可知,各组之间屠宰率、半净膛率、全净膛率、胸肌率、腿肌率、腹脂率差异不显著(P>0.05)。
表4 凝结芽孢杆菌和丁酸梭菌对肉鸭屠宰性能的影响

Table 4 Effects of Bacillus coagulans and Clostridium butyricum on slaughter performance of meat ducks %

项目
Items		 组别Groups P值
P-value
对照组
Control		 BC CB BC+CB
(1∶1)		 BC+CB
(1∶2)		 BC+CB
(2∶1)
屠宰率
Dressed percentage		 84.84±0.88 85.38±0.10 85.19±0.85 85.24±0.72 85.12±1.51 85.78±0.59 0.780
半净膛率
Semi-eviscerated rate		 79.31±0.40 79.37±1.27 79.96±0.73 79.82±1.50 80.29±0.78 79.68±0.82 0.704
全净膛率
Eviscerated rate		 73.68±0.61 73.68±1.33 74.02±0.83 74.30±1.53 74.43±0.99 74.18±0.58 0.857
胸肌率
Breast muscle rate		 12.20±1.18 12.48±1.81 12.81±1.44 12.74±1.26 13.03±1.60 13.24±1.00 0.895
腿肌率
Leg muscle rate		 0.56±0.05 0.59±0.04 0.59±0.04 0.59±0.03 0.58±0.03 0.60±0.05 0.791
腹脂率
Abdominal fat rate		 1.16±0.03 1.03±0.19 1.19±0.16 1.14±0.09 1.11±0.14 1.08±0.26 0.868

2.3 凝结芽孢杆菌和丁酸梭菌对肉鸭血清生化指标的影响

由表5可知,BC+CB(1∶2)组和BC+CB(2∶1)组血清HDL-C含量显著高于对照组(P<0.05),BC+CB(1∶2)组和BC+CB(2∶1)组血清TP含量显著高于对照组和BC组(P<0.05),BC+CB(1∶1)组、BC+CB(1∶2)组和BC+CB(2∶1)组血清ALB含量显著高于对照组(P<0.05),BC+CB(1∶1)组、BC+CB(1∶2)组和BC+CB(2∶1)组血清ALT活性显著低于对照组和BC组(P<0.05)。各组之间血清T-CHO、LDL-C、TG、TAA含量以及AKP、AST活性差异不显著(P>0.05)。
表5 凝结芽孢杆菌和丁酸梭菌对肉鸭血清生化指标的影响

Table 5 Effects of Bacillus coagulans and Clostridium butyricum on serum biochemical indexes of meat ducks

项目
Items		 组别Groups P值
P-value
对照
Control		 BC CB BC+CB
(1∶1)		 BC+CB
(1∶2)		 BC+CB
(2∶1)
总胆固醇
T-CHO/(mmol/L)		 0.83±0.12 0.88±0.15 0.89±0.07 0.84±0.08 0.86±0.12 0.89±0.09 0.713
高密度脂蛋白胆固醇
HDL-C/(mmol/L)		 6.05±0.90b 7.35±0.68a 6.92±0.70ab 7.12±1.00ab 7.72±1.07a 7.57±0.81a 0.023
低密度脂蛋白胆固醇
LDL-C/(mmol/L)		 2.77±0.40 2.31±1.07 2.95±0.69 2.62±0.64 3.00±0.54 3.04±0.97 0.736
甘油三酯
TG/(mmol/L)		 0.70±0.15 0.70±0.17 0.66±0.10 0.59±0.13 0.67±0.07 0.70±0.17 0.192
总氨基酸
TAA/(μmol/mL)		 14.14±3.30 13.10±1.43 13.09±0.67 11.92±1.15 16.07±3.42 16.69±1.10 0.770
总蛋白
TP/(g/L)		 26.81±3.85c 28.66±1.87bc 30.26±1.67abc 31.85±1.81abc 34.35±2.38a 35.28±3.35a 0.027
白蛋白
ALB/(g/L)		 14.97±0.87b 16.73±1.18ab 17.93±1.45a 17.32±1.57a 17.43±1.37a 18.39±0.37a 0.008
碱性磷酸酶
AKP/(U/L)		 0.15±0.00 0.15±0.00 0.15±0.00 0.15±0.00 0.15±0.00 0.15±0.00 0.691
对照
Control		 BC CB BC+CB
(1∶1)		 BC+CB
(1∶2)		 BC+CB
(2∶1)
谷丙转氨酶
ALT/(U/L)		 6.19±0.46a 6.20±0.37a 5.51±0.26ab 5.31±0.89b 5.29±0.12b 5.18±0.75b 0.029
谷草转氨酶
AST/(U/L)		 8.51±1.47 8.10±1.66 7.99±1.71 7.54±1.47 7.80±1.08 7.09±1.09 0.795

2.4 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道炎症因子及sIgA含量的影响

由表6可知,BC+CB(2∶1)组的肠道sIgA含量显著高于对照组和CB组(P<0.05)。各组之间肠道TNF-α和IL-1β含量差异不显著(P>0.05)。
表6 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道炎症因子及sIgA含量的影响

Table 6 Effects of Bacillus coagulans and Clostridium butyricum on intestinal inflammatory factors and sIgA contents of meat ducks

项目
Items		 组别Groups P值
P-value
对照
Control		 BC CB BC+CB
(1∶1)		 BC+CB
(1∶2)		 BC+CB
(2∶1)
肿瘤坏死因子-α
TNF-α/(pg/g)		 12.35±0.67 12.41±0.65 12.74±1.27 12.36±0.59 11.98±1.15 12.28±0.45 0.875
白细胞介素-1β
IL-1β/(ng/g)		 0.86±0.04 0.80±0.06 0.87±0.02 0.80±0.07 0.86±0.08 0.78±0.02 0.101
分泌型免疫球蛋白A
sIgA/(μg/g)		 2.34±0.04b 2.50±0.06ab 2.35±0.23b 2.52±0.22ab 2.57±0.18ab 2.71±0.18a 0.037

2.5 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道形态的影响

由表7、表8和表9可知,与对照组相比,BC+CB(1∶1)组、BC+CB(1∶2)组和BC+CB(2∶1)组的十二指肠绒毛高度分别显著提高了6.41%、8.56%、13.62%(P<0.05),隐窝深度分别显著降低了34.46%、33.26%、29.60%(P<0.05),绒隐比分别显著提高了38.46%、50.17%、50.00%(P<0.05)。各组之间空肠绒毛高度、隐窝深度和绒隐比差异不显著(P>0.05)。与对照组相比,BC+CB(1∶1)组、BC+CB(1∶2)组和BC+CB(2∶1)组的回肠绒毛高度分别显著提高了3.91%、3.92%、3.91%(P<0.05),绒隐比分别显著提高了24.22%、18.84%、29.18%(P<0.05),隐窝深度差异不显著(P>0.05)。
表7 凝结芽孢杆菌和丁酸梭菌对肉鸭十二指肠形态的影响

Table 7 Effects of Bacillus coagulans and Clostridium butyricum on duodenum morphology of meat ducks

项目
Items		 绒毛高度
Villous height/μm		 隐窝深度
Crypt depth/μm		 绒隐比
V/C
组别Groups
对照Control 898.87±49.53b 169.71±24.99a 5.72±0.89c
BC 956.67±89.49ab 156.19±9.92a 6.13±0.48bc
CB 899.10±25.33b 124.01±22.24b 6.95±1.10b
BC+CB(1∶1) 965.56±32.77a 111.23±22.52b 7.92±0.80a
BC+CB(1∶2) 975.85±30.90a 113.27±9.65b 8.59±0.40a
BC+CB(2∶1) 1 021.28±19.36a 119.48±6.44b 8.58±0.25a
P值P-value <0.001 <0.001 <0.001

同列数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。表8和表9同。

In the same column, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as Table 8 and Table 9.

表8 凝结芽孢杆菌和丁酸梭菌对肉鸭空肠形态的影响

Table 8 Effects of Bacillus coagulans and Clostridium butyricum on jejunum morphology of meat ducks

项目
Items		 绒毛高度
Villous height/μm		 隐窝深度
Crypt depth/μm		 绒隐比
V/C
组别Groups
对照Control 914.93±11.77 108.93±5.94 8.04±0.47
BC 902.16±28.30 106.23±3.83 8.45±0.49
CB 921.55±36.33 106.12±5.63 8.80±0.97
BC+CB(1∶1) 915.21±22.36 104.60±10.61 8.52±1.00
BC+CB(1∶2) 911.88±16.32 108.26±7.30 8.12±0.58
BC+CB(2∶1) 933.88±11.23 108.73±6.34 8.77±0.27
P值P-value 0.310 0.797 0.339
表9 凝结芽孢杆菌和丁酸梭菌对肉鸭回肠形态的影响

Table 9 Effects of Bacillus coagulans and Clostridium butyricum on ileum morphology of meat ducks

项目
Items		 绒毛高度
Villous height/μm		 隐窝深度
Crypt depth/μm		 绒隐比
V/C
组别Groups
对照Control 901.06±14.55b 116.92±4.17 7.06±0.56b
BC 903.87±4.97b 116.95±9.26 7.36±0.75b
CB 902.34±34.89b 111.63±7.83 7.22±0.52b
BC+CB(1∶1) 931.12±22.88a 107.46±11.84 8.77±1.16a
BC+CB(1∶2) 936.34±10.32a 114.24±8.67 8.39±0.66a
BC+CB(2∶1) 936.29±13.01a 104.71±10.95 9.12±0.42a
P值P-value 0.004 0.140 <0.001

2.6 凝结芽孢杆菌和丁酸梭菌对肉鸭盲肠微生物组成的影响

根据上述试验结果综合考虑,选取BC+CB(2∶1)组与对照组进行对比,分析凝结芽孢杆菌和丁酸梭菌对肉鸭盲肠微生物组成的影响。

2.6.1 门水平

由图1可知,在门水平上,对照组与BC+CB(2∶1)组的微生物组成基本相同,主要由厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)组成,其中厚壁菌门和拟杆菌门为优势菌门,约占微生物菌落总比例的80%。
Fig.1 肉鸭盲肠微生物门水平菌落组成

A:对照组 control group;B:BC+CB(2∶1)组 BC+CB(2∶1) group。下图同 the same as below.

Caecum microbial colony composition at phylum level of meat ducks

Firmicutes:厚壁菌门;Bacteroidetes:拟杆菌门;Proteobacteria:变形菌门;Actinobacteria:放线菌门;Fusobacteria:梭杆菌门;Bacteria-unclassified:未分类细菌;Others:其他。

2.6.2 属水平

由图2可知,在属水平上,对照组与BC+CB(2∶1)组的盲肠菌落主要由拟杆菌属(Bacteroides)、另枝菌属(Alistipes)、普雷沃氏菌科(Prevotellaceae)、梭杆菌属(Fusobacterium)、未分类毛螺菌科(Lachnospiraceae-underclassified)、脱硫弧菌属(Desulfovibro)、未分类瘤胃球菌科(Ruminococcaceae-underclassified)、罗姆布茨菌(Romboutsia)、未分类迟缓埃菌科(Eggerthellaceae-underclassified)、粪杆菌属(Faecalibacterium)、巨单胞菌属(Megamonas)、罕见小球菌属(Subdoligranulum)等组成,其中BC+CB(2∶1)组中属于肠道有益菌属的拟杆菌属、普雷沃氏菌科、梭杆菌属等的相对丰度所占的比例高于对照组,而属于肠道有害菌属的脱硫弧菌属、另枝菌属、未分类毛螺菌科等的相对丰度所占的比例低于对照组。
图2 肉鸭盲肠微生物属水平菌落组成

Others:其他;Bacteroides:拟杆菌属;Alistipes:另枝菌属;Lachnospiraceae-underclassified:未分类毛螺菌科;Desulfovibro:脱硫弧菌属;Ruminococcaceae-UGG-014:瘤胃球菌科-UGG-014;Ruminococcaceae-underclassified:未分类瘤胃球菌科;Romboutsia:罗姆布茨菌;Eggerthellaceae-underclassified:未分类迟缓埃菌科;Ruminococcaceae-ge:瘤胃球菌科-ge;Mucispirillum:多胞菌属;Bacteroides-unclassified:未分类拟杆菌属;Faecalibacterium:粪杆菌属;Subdoligranulum:罕见小球菌属;Fournierella:福尼尔菌属;Parabacteroides:副拟杆菌属;Synergistes:互养菌属;Prevotellaceae:普雷沃氏菌科;Fusobacterium:梭杆菌属;Prevotellaceae-UGG-001:普雷沃氏菌科-UGG-001;Megamonas:巨单胞菌属;Phascolarctobacterium:考拉杆菌属;Aeromonadales:假单胞菌;Olsenella:寡源杆菌属;uncultured:未培养的。

Fig.2 Caecum microbial colony composition at genus level of meat ducks

2.7 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道菌群多样性与丰富度的影响

2.7.1 α多样性分析

由表10可知,BC+CB(2∶1)组与对照组之间Chao1指数、ACE指数、Shannon指数、Simpson指数和Invimpson指数差异不显著(P>0.05)。
表10 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道菌群α多样性指数的影响

Table 10 Effects of Bacillus coagulans and Clostridium butyricum on intestinal flora α diversity index of meat ducks

项目
Items		 组别Groups P值
P-value
对照Control BC+CB(2∶1)
Chao1指数Chao1 index 2 406.29±551.40 2 793.00±749.08 0.380
ACE指数ACE index 2 667.89±731.33 3 391.85±522.95 0.141
Shannon指数Shannon index 4.86±0.44 4.94±0.53 0.821
Simpson指数Simpson index 0.03±0.01 0.02±0.01 0.418
Invimpson指数Invimpson index 40.43±21.03 46.43±11.33 0.633
覆盖率Coverage 0.99±0.00 0.98±0.00 0.340

2.7.2 β多样性分析

β多样性分析是对各样本的微生物群落组成进行对比分析,即分析样品间的微生物菌落多样性。PCA基于OTU丰度表,运用方差分解,将多维数据的差异反映在二维坐标图上,坐标轴为能够最大程度解释方差的2个特征根,样本组成越相似,在PCA图中越聚集,而不同组的样本可能表现出分散分布。图3是肉鸭盲肠微生物PCA聚类分析图,从图中可以看出,对照组与BC+CB(2∶1)组微生物群落差异性小,物种组成基本相似。
图3 肉鸭盲肠微生物PCA聚类分析图

Fig.3 PCA cluster analysis map of cecal microorganisms of meat ducks

由表11可知,β多样性指数包括ADONIS、ANOSIM、MRPP、AMOVA,为数据间差异显著性评价提供依据。BC+CB(2∶1)组与对照组之间β多样性指数差异均不显著(P>0.05)。
表11 肉鸭盲肠微生物β多样性指数

Table 11 β diversity indexes of cecum microorganisms of meat ducks

项目
Items		 P值
P-value
多元方差分析ADONIS 0.237
相似性分析ANOSIM 0.243
多重反应排列分析MRPP 0.189
分子方差分析AMOVA 0.178

2.7.3 LEfSe分析

LEfSe分析则体现了组与组之间寻找具有统计学差异的生物标识。由图5可知,BC+CB(2∶1)组与对照组相比仍有相对丰度差异物种,盲肠鲁森杆菌属(Ruthenibacterium)、未分类的丹毒丝菌科(Erysipelatoclostrichaceae-unclassified)的相对丰度显著提高(P<0.05),盲肠盐单胞菌属(Halomonas)、盐单胞菌科(Halomonadaceae)、海洋螺杆菌目(Oceanosprillales)和考克斯菌属(Kocuria)的相对丰度显著降低(P<0.05)。

3 讨论

3.1 凝结芽孢杆菌和丁酸梭菌对肉鸭生长性能的影响

大量研究表明,饲粮中添加益生菌能够有效提高动物的生长性能与饲料利用率,从而提高养殖利润[11⇓-13]。多项研究证明,凝结芽孢杆菌可以提高动物ADG、降低F/G,改善生长性能[14-15]。Li等[16]研究发现,饲粮中添加丁酸梭菌膳食补充剂可以改善肠道盲肠生物菌落,从而提高生长性能以及饲料利用率。武丽达等[17]研究证明,饲粮中添加丁酸梭菌有利于提高白羽肉鸡的生长性能,提高机体抗氧化能力。本试验发现,凝结芽孢杆菌和丁酸梭菌混合饲喂提高了肉鸭饲养前期ADG,降低了饲养前期与整个饲养期的F/G,与前人研究结论一致。这可能是因为凝结芽孢杆菌能够发酵产大量淀粉酶、蛋白酶、脂肪酶等消化酶,促进营养物质的充分消化吸收[18]。丁酸梭菌能够分解有机物转化为丁酸,代谢产生多种维生素与短链脂肪酸,为细胞生长和机体发育提供能量[19]。
图4 肉鸭盲肠微生物LEfSe分析柱状图

Ruthenibacterium:鲁森杆菌属;Erysipelatoclostrichaceae-unclassified:未分类丹毒梭菌科;Halomonas:盐单胞菌属;Oceanosprillales:海洋螺杆菌目;Halomonadaceae:盐单胞菌科;Kocuria:考克斯菌属;LDA score:线性判别分析得分。

Fig.4 LEfSe analysis histogram of cecal microorganisms of meat ducks

3.2 凝结芽孢杆菌和丁酸梭菌对肉鸭屠宰性能的影响

屠宰性能反映了动物机体对营养物质的吸收利用情况,是衡量肉禽经济效益的重要指标。有研究表明,添加不同益生菌可提高动物屠宰性能,还可以降低腹脂率[20⇓-22]。而Rehman等[23]研究发现,饲粮中添加益生菌和益生元对俄罗斯肉鸡的胴体重、腿肌重、胸肌重无显著影响。也有研究结果表明,复合益菌制剂对家禽的屠宰性能及器官发育指数均无显著影响[24⇓-26]。本试验结果也表明,不同比例的凝结芽孢杆菌和丁酸梭菌混合饲喂对肉鸭的屠宰率、半净膛率、全净膛率、胸肌率、腿肌率、腹脂率有一定改善作用,但影响不显著,与前人研究结果一致。

3.3 凝结芽孢杆菌和丁酸梭菌对肉鸭血清生化指标的影响

血清生化指标可反映动物体内营养水平、代谢情况以及生理健康。Mohammadi等[27]研究发现,投喂植物乳杆菌的罗非鱼血清中TP、ALB、球蛋白、总免疫球蛋白含量显著提高。张伟等[28]研究表明,饲粮中添加丁酸梭菌和屎肠球菌组成的复合微生态制剂对蛋鸡血清TG、T-CHO含量无显著影响,但是可以显著提高血清TP与ALB含量。本试验结果与前人研究结果一致。HDL-C可以运载组织中的胆固醇到肝脏中代谢,促进胆固醇的代谢排出,与患心血管疾病的风险呈负相关。血清TP、ALB含量则与肉鸭对饲料蛋白质的摄入与代谢利用相关。ALT和AST是检验肝功能是否正常的重要指标,其活性升高则表明肝脏受损。这说明凝结芽孢杆菌和丁酸梭菌复合菌制剂能够提高肉鸭对饲料中脂类、蛋白质的吸收利用,维护调节肝脏功能,保证机体正常生理活动。

3.4 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道炎症因子和sIgA含量的影响

炎症因子水平体现了机体炎症的发生与炎症程度,而免疫球蛋白是一种非常重要的抗体因子,尤其是sIgA有4个抗原结合位点,比普通抗体分子具有更高的亲和力,同时也具有很高的稳定性。sIgA主要存在于分泌液中,如唾液、泪液、鼻和支气管黏膜、肠胃液及黏膜,是机体黏膜局部抗感染免疫的主要抗体。胡婷等[29]研究发现,不同剂量的凝结芽孢杆菌BC-HYI均能显著降低蛋雏鸡血清TNF-α含量,同时显著提高空肠黏膜sIgA含量。Zhang等[30]研究表明,将凝结芽孢杆菌代替抗生素饲喂肉鸡,显著降低了血清促炎因子含量,显著提高了血清免疫球蛋白含量。Zhang等[31]通过口服丁酸梭菌修复小鼠肠道,增加了肠道丁酸含量,降低了TNF-α和白细胞介素-6(IL-6)含量,抑制了烧伤小鼠的肠道损伤。本试验结果显示,凝结芽孢杆菌和丁酸梭菌复合菌制剂能够调节肠道炎症因子的水平,肠道TNF-α和IL-1β含量有一定程度的降低,同时提高了肠道sIgA含量,减少肠道炎症的发生,提高了肠道免疫力。

3.5 凝结芽孢杆菌和丁酸梭菌对肉鸭肠道形态的影响

小肠作为吸收和消化的主要场所,小肠黏膜向内弯曲凸出形成大量环形褶皱与绒毛,使表面积增加,小肠的绒毛高度、隐窝深度和绒隐比反映了机体对营养物质消化吸收的效率,是衡量小肠吸收功能的重要指标。大量研究表明,益生菌能够改善动物肠道形态,从而提高饲料利用率[32⇓⇓-35]。Zhang等[36]研究表明,在雄性樱桃谷鸭饲粮中添加枯草芽孢杆菌,提高了空肠、回肠的绒毛高度和绒隐比。李修宇等[37]研究发现,饲粮中添加丁酸梭菌或复合益生菌均能提高黎村黄鸡的小肠绒毛高度。本试验结果与前人研究结果相似,凝结芽孢杆菌和丁酸梭菌混合添加能够改善肠道形态,提高吸收效率,尤其是显著提高了十二指肠和回肠绒毛高度和绒隐比。凝结芽孢杆菌为兼性厌氧菌,进入肠道后吸氧繁殖,为丁酸梭菌创造无氧条件,二者联合产生的氨基酸、短链脂肪酸等物质可以加快肠蠕动,从而改善肠道的形态与消化功能。

3.6 凝结芽孢杆菌和丁酸梭菌对肉鸭盲肠微生物组成的影响

肠道微生物组成对机体消化吸收营养有着重要作用,微生物分类水平有界、门、纲、目、科、属。本试验从门、属水平分析对比不同组的肉鸭盲肠内容物菌落组成,盲肠微生物菌群中厚壁菌门和拟杆菌门为禽类主要优势菌门,与郝永胜等[38]研究结果一致。厚壁菌门的细菌能够起到降解膳食纤维的作用,产生乙酸、丁酸等代谢产物,改善肠道通透性,拟杆菌门的细菌被认为是多糖主要降解的物,值得一提的是厚壁菌门与拟杆菌门的比值(F/B)被广泛认为对肠道稳定有重要影响,F/B过高表现为肥胖,F/B过低表现为炎症性肠病[39⇓-41]。另外有研究发现,益生菌能够通过调节肠道中厚壁菌门与拟杆菌门比例来影响机体脂质代谢,降低因肥胖导致的其他并发症的几率,从而达到调节体重与健康的目的[42-43]。本试验结果显示,凝结芽孢杆菌和丁酸梭菌混合添加能够增加一些肠道有益菌的相对丰度,普雷沃氏菌科在禽类肠道中暂时没有深度研究,但有研究表明[44],反刍动物肠道中的普雷沃氏菌科有助于分解蛋白质和碳水化合物食物,尤其是对半纤维素的消化,并参与淀粉和果胶的消化过程。拟杆菌属被证实可以参与脂质代谢,调节脂肪肝[45],尤其是脆弱拟杆菌,能够修复人体免疫缺陷和后天免疫紊乱,增加白细胞含量和增强巨噬免疫细胞功能,消除感染以及癌症风险,双向调节免疫力。梭杆菌属中有部分菌种属于肠道致病菌,但仍有研究表明,梭杆菌属相对丰度降低会导致炎症性肠病[46]。同时,益生菌能够通过发酵分泌有机酸,抑制有害菌生长。目前对毛螺菌科有争议,有研究发现毛螺菌科的不同类群与肠内外疾病有关[47]。脱硫弧菌属也被称为硫酸盐还原菌(SRB),属于变形菌门,可以吸收硫酸盐而不是氧气,产生硫化氢(H2S),因此被认为会对肠道上皮产生毒性,导致胃肠道疾病,属于肠道致病菌。另枝菌属作为一种较新的细菌属,在菌群失调与疾病中有高度相关性,Parker等[48]研究发现,另枝菌属可能对一些疾病有保护作用,包括肝纤维化、结肠炎、肿瘤免疫治疗和心血管疾病,并且会导致结直肠癌。综上所述,肉鸭的肠道菌群结构因受饲粮成分、饲养环境、遗传背景等多种因素影响[49],凝结芽孢杆菌和丁酸梭菌复合菌制剂虽然没有改变盲肠菌群主要菌群结构,但是可以提高有益菌的相对丰度,降低有害菌的相对丰度,对调节肠道健康有一定的作用。

3.7 凝结芽孢杆菌和丁酸梭菌对肉鸭盲肠微生物多样性的影响

肠道微生物菌落多样性通过α多样性与β多样性来体现,α多样性是对单个样品中物种多样性的分析,即分析样本内的微生物菌落多样性,通过单样本的多样性分析(可以反映样本内的微生物菌落的丰富度和多样性。β多样性是对不同样本的微生物菌落组成进行比较分析,即分析样本间的微生物菌落多样性。鲁森杆菌属是厚壁菌门、梭菌纲、梭菌目、瘤胃球菌科的一个属,目前只包括1个物种——乳酸鲁森杆菌,可以发酵一系列碳水化合物,产生D-乳酸和琥珀酸;丹毒丝菌科属厚壁菌门、柔膜菌纲、枝原体目;盐单胞菌科原产地为中国,需氯化钠才能生长,最适氯化钠浓度为3%,产淀粉酶、过氧化氢酶,不能水解明胶,硝酸盐还原阳性,不能发酵果糖、葡萄糖、甘露糖、半乳糖、乳糖、麦芽糖、蔗糖产酸;海洋螺杆菌目是基于16S rRNA基因系统发育关系建立的γ-变形菌纲中的重要分支,此类有机异养型细菌普遍表现出嗜盐或耐盐的特性,在海洋环境中广泛分布。多项研究显示不同益生菌对不同物种的肠道内菌群多样性的影响不同[50-51]。Vargas-Albores等[52]研究发现,益生菌制剂不会对动物肠道的微生物多样性产生显著影响;王纯[53]证实饲喂芽孢杆菌的动物肠道微生物多样性指数均无显著变化;覃振斌等[54]研究表明复合芽孢杆菌能够提高清远麻羽鸡的Chao指数、Shannon指数,而复合乳酸菌则会降低该指数。本试验中,α多样性指数的变化体现了凝结芽孢杆菌和丁酸梭菌混合饲喂的肉鸭盲肠微生物多样性有所增加,但与对照组的盲肠微生物多样性差异不显著,与很多研究结果[55-56]相似,可能由于每个物种的肠道优势菌群不同,凝结芽孢杆菌和丁酸梭菌的发酵产物能够促进有益菌定殖,抑制有害菌生长[57⇓-59],肠道菌群多样性变化非固定规律,而是动态变化的,且随时间增长,机体肠道各项功能发育完善,肠道微生态趋于平衡。

4 结论

① 饲粮中添加凝结芽孢杆菌和丁酸梭菌复合菌制剂能够提高肉鸭ADG,降低F/G,改善生长性能,提高血清HDL-C、TP、ALB含量,增强机体营养利用能力。
② 饲粮中添加凝结芽孢杆菌和丁酸梭菌复合菌制剂能够提高肉鸭肠道sIgA含量,提高小肠绒毛高度和绒隐比,降低隐窝深度,增强肠道消化吸收能力。
③ 饲粮中添加凝结芽孢杆菌和丁酸梭菌复合菌制剂不影响盲肠微生物菌群结构,菌群多样性差异不显著,盲肠鲁森杆菌属、未分类的丹毒丝菌科、盐单胞菌属、盐单胞菌科、海洋螺杆菌目、考克斯菌属的相对丰度有显著差异。
④ 饲粮中添加凝结芽孢杆菌和丁酸梭菌复合菌制剂对肉鸭生物学效应的影响优于单一菌制剂,且当二者比例为2∶1时效果最优。

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