Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 9: 5942 - 5953

DOI: https://doi.org/10.12418/CJAN2023.545

RESEARCH PAPER

Effects of Clostridium butyricum, Glucose Oxidase and Rosemary Extract Formulas on Growth Performance, Slaughter Performance, Immune Function and Cecal Microflora of Meat Rabbits

  • LIU Gongyan , 1 ,
  • YAN Xianfeng 2 ,
  • WU Zhiqiang 3 ,
  • ZHANG Yin 1 ,
  • ZHANG Yunhai 1 ,
  • ZHANG Yajia 1 ,
  • ZHOU Beibei 1 ,
  • REN Qifeng 1 ,
  • SUN Haitao , 1, *
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  • 1 Key Laboratory of Livestock and Poultry Multiomics of Ministry of Agriculture and Rural Affairs, Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Husbandry and Veterinary, Shandong Academy of Agricultural Sciences, Ji’nan 250100, China
  • 2 Shandong Animal Husbandry Station, Ji’nan 250100, China
  • 3 Pingyi County Animal Husbandry Development Promotion Center, Pingyi 273300, China
* associate professor, E-mail:

Received date: 2023-03-14

  Online published: 2023-09-13

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Abstract

This study aimed to study the effects of dietary Clostridium butyricum, glucose oxidase and rosemary extract formulas on growth performance, slaughter performance, immune function and cecal microflora of meat rabbits, and to provide a theoretical reference for the research and development of anti-feed additives in rabbits. A total 500 commercial Hyla rabbits (half male and female) weaned at 28-day-old were randomly divided into 5 groups with 10 replicates in each group and 10 rabbits in each replicate. Rabbits in the control group (group Ⅰ) were fed a basal diet, and others in experimental groups were fed basal diets supplemented with 100 g/t Clostridium butyrinate+1 kg/t rosemary extract (group Ⅱ), 100 g/t Clostridium butyrinate+100 g/t glucose oxidase (group Ⅲ), 1 kg/t rosemary extract+100 g/t glucose oxidase (group Ⅳ) and 100 g/t Clostridium butyrate+1 kg/t rosemary extract+100 g/t glucose oxidase (group Ⅴ), respectively. The pre-experimental period lasted for 7 days, and the experimental period lasted for 30 days. The results showed as follows: 1) the final body weight, average daily gain and survival rate of group Ⅴ were significantly higher than those of the control group (P<0.05), and the diarrhea rate was significantly lower than that of the control group (P<0.05). The pre-slaughter body weight of group Ⅴ was significantly higher than that of the control group (P<0.05). 2) The duodenal glutathione peroxidase (GSH-Px) activity of groups Ⅱ, Ⅳ and Ⅴ was significantly lower than that of the control group (P<0.05), the duodenal malondialdehyde (MDA) content of groups Ⅳ and Ⅴ was significantly higher than that of the control group (P<0.05), and the duodenal total antioxidant capacity (T-AOC) of groups Ⅱ, Ⅲ, Ⅳ and Ⅴ was significantly higher than that of the control group (P<0.05). 3) The jejunal villus height of group Ⅴ was significantly higher than that of the control group (P<0.05). 4) The ileal mucosal secretory immunoglobulin A (sIgA) content of groups Ⅱ, Ⅲ, Ⅳ and Ⅴ was significantly higher than that of the control group (P<0.05), and the ileal mucosal interferon-γ (IFN-γ) content was significantly lower than that of the control group (P<0.05); the ileal mucosal tumor necrosis factor-α (TNF-α) of group Ⅴ was significantly higher than that of other groups (P<0.05). 5) The cecal Verrucomicrobiota relative abundance of groups Ⅲ and Ⅴ was significantly higher than that of the control group (P<0.05), and the cecal Acidobacteriota relative abundance of groups Ⅲ, Ⅳ and Ⅴ was significantly lower than that of the control group (P<0.05). The relative abundances of Akkermansia, Monoglobus and Verrucomicrobium_UCG-010 in cecum of group Ⅴ were significantly higher than those of the control group (P<0.05), and the cecal Massilia relative abundance of groups Ⅱ, Ⅲ, Ⅳ and Ⅴ was significantly lower than that of the control group (P<0.05). In conclusion, dietary Clostridium butyricum, glucose oxidase and rosemary extract formulas can improve the growth performance, intestinal morphology, cecal microflora, intestinal mucosal immune function and antioxidant capacity of Hyla meat rabbits, which is beneficial to maintain intestinal health and improve the survival rate.

Key words： Clostridium butyrate; glucose oxidase; rosemary extract; meat rabbits; growth performance; immune function; microflora

Cite this article

LIU Gongyan , YAN Xianfeng , WU Zhiqiang , ZHANG Yin , ZHANG Yunhai , ZHANG Yajia , ZHOU Beibei , REN Qifeng , SUN Haitao . Effects of Clostridium butyricum, Glucose Oxidase and Rosemary Extract Formulas on Growth Performance, Slaughter Performance, Immune Function and Cecal Microflora of Meat Rabbits[J]. Chinese Journal of Animal Nutrition, 2023 , 35(9) : 5942 -5953 . DOI: 10.12418/CJAN2023.545

消化道疾病是家兔最为多发的疾病之一,仔兔腹泻是困扰家兔生产的一个重大问题,尤其是在断奶阶段[1-2]。抗生素曾被广泛用于预防和治疗动物消化道疾病,这不仅导致养殖成本增加,而且会影响产品质量。随着人们对环境保护及食品安全的关注,抗生素作为生长促进剂在畜禽养殖中所造成的副作用日益增加。我国也于2020年全面禁止在饲料中添加抗生素,因此,抗生素替代物的开发利用成为当务之急[3]。益生菌、酶制剂和植物提取物具有绿色、安全、生态等突出优点,成为新型饲料添加剂的首选。益生菌能够调节动物肠道菌群平衡,发挥预防及治疗疾病的功效。饲粮中添加丁酸梭菌(Clostridium butyricum)等益生菌能够改善猪、家兔、鲈鱼等动物肠道菌群组成,提高机体抗氧化能力和免疫功能,对维持肠道健康具有重要意义[4⇓-6]。葡萄糖氧化酶(glucose oxidase)是一种有氧脱氢酶,能够催化β-D-葡萄糖氧化为葡萄糖酸和过氧化氢。Wang等[7]研究表明,饲粮中单独添加葡萄糖氧化酶及分解淀粉芽孢杆菌SC06与葡萄糖氧化酶配合使用可提高肉鸡抗氧化能力和免疫功能,改善肠道菌群组成,且分解淀粉芽孢杆菌SC06与葡萄糖氧化酶配合使用的效果优于单独添加葡萄糖氧化酶。迷迭香(Rosmarinus officinalis L.)提取物作为一种重要的植物提取物,具有多种药理和生物活性。饲粮中添加迷迭香提取物可以改善断奶仔猪的生长性能、营养物质消化率、抗氧化能力、肠道形态和微生物区系,适宜添加剂量为200 mg/kg[8]。此外,迷迭香提取物作为肉兔饲料添加剂能够降低兔肉中胆固醇含量,增加肌肉营养价值,提高肌肉抗氧化能力[9]。Yadollahi等[10]研究了饲料中添加维生素E、益生菌、虾青素或迷迭香提取物对虹鳟鱼生长性能、血液学指标、抗氧化和肝脏酶活性的影响,结果表明饲料中添加维生素E(50 μg/kg)复配迷迭香提取物(50 mg/kg)或杆菌益生菌(500 mg/kg)可以有效地维持虹鳟鱼的健康状况。本试验在前期开展的丁酸梭菌、迷迭香提取物等单品的试验结果[5,9]基础上,探讨饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔生长性能、屠宰性能、免疫功能和盲肠微生物区系的影响,为家兔新型、绿色、安全的替抗产品研发提供理论参考。

1 材料与方法

1.1 试验设计

试验选用28日龄断奶的体重相近、健康状况良好的商品伊拉肉兔500只(公母各占1/2),随机分为5组,每组10个重复,每个重复10只。对照组(Ⅰ组)饲喂基础饲粮,根据前期丁酸梭菌、迷迭香提取物等单品的试验结果[5,9],试验组分别饲喂添加100 g/t丁酸梭菌+1 kg/t迷迭香提取物(Ⅱ组)、100 g/t丁酸梭菌+100 g/t葡萄糖氧化酶(Ⅲ组)、1 kg/t迷迭香提取物+100 g/t葡萄糖氧化酶(Ⅳ组)及100 g/t丁酸梭菌+1 kg/t迷迭香提取物+100 g/t葡萄糖氧化酶(Ⅴ组)的试验饲粮。饲粮营养水平参考《肉兔营养需要量》(NY/T 4049—2021)[11],基础饲粮组成及营养水平见表1。经7 d预试期后进入30 d正试期。
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)%

原料
Ingredients		 含量
Content		 营养水平
Nutrient levels2)		 含量
Content
玉米 Corn 5.0 消化能 DE/(MJ/kg) 10.23
豆粕 Soybean meal 8.0 干物质 DM 89.82
大麦 Barley 6.0 粗蛋白质 CP 16.12
麸皮 Wheat bran 15.0 粗脂肪 EE 2.80
玉米胚芽粕 Corn germ meal 16.0 粗纤维 CF 17.38
玉米皮 Corn husk 17.0 中性洗涤纤维 NDF 38.74
苜蓿草粉 Alfalfa meal 15.0 酸性洗涤纤维 ADF 23.08
豆秸粉 Soybean straw powder 7.0 酸性洗涤木质素 ADL 6.29
稻壳粉 Rice hull powder 8.0 粗灰分 Ash 9.03
磷酸氢钙 CaHPO4 1.5 钙 Ca 0.95
氯化钠 NaCl 0.5 总磷 TP 0.45
预混料 Premix1) 1.0 赖氨酸 Lys 0.60
合计 Total 100.0 蛋氨酸+半胱氨酸 Met+Cys 0.65

1)预混料为每千克饲粮提供 Premix provided the following per kg of the diet: VA 8 000 IU,VD3 1 500 IU,VE 45 mg,VK3 2.0 mg,VB1 1.0 mg,VB2 3.0 mg,VB6 1.5 mg,烟酸 nicotinic acid 30 mg,泛酸 pantothenic acid 50 mg,叶酸 folic acid 0.5 mg,氯化胆碱 choline chloride 100 mg,Fe 50 mg,Cu 10 mg,Zn 50 mg,Mn 10 mg,I 0.5 mg,Se 0.05 mg,赖氨酸 Lys 1.5 g,蛋氨酸 Met 0.5 g,其余由麦饭石载体补足 the rest was Maifan stone carrier complement.

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

1.2 样品采集与制备

试验结束当天,每个重复选取1只与该重复平均体重相近的家兔屠宰,用于统计屠宰性能;同时采集十二指肠、空肠、回肠黏膜和盲肠内容物样品,用于十二指肠抗氧化指标、空肠组织形态结构、回肠黏膜免疫指标和盲肠微生物区系测定。

1.3 测定指标和方法

1.3.1 生长性能

在试验正式开始(35日龄)和结束当日(65日龄),称量每个重复家兔重量,统计试验期间每个重复采食量,计算平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)比。以重复为单位,计算试验期间家兔腹泻率和成活率[6]。

1.3.2 屠宰性能

试验兔屠宰前称重,记录宰前活体重,屠宰后依次对欧洲胴体重、半净膛胴体重和全净膛胴体重以及心脏、肝脏和肾脏进行称重,计算欧洲屠宰率、半净膛屠宰率、全净膛屠宰率以及心脏、肝脏和肾脏相对重量[12]。

1.3.3 十二指肠抗氧化指标

十二指肠谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(malondialdehyde,MDA)含量及总抗氧化能力(total antioxidant capacity,T-AOC)使用南京建成生物工程研究所的试剂盒测定(货号分别为:A005、A001-1、A003-1和A015)。

1.3.4 空肠组织形态结构

采集3 cm空肠样品置于4%多聚甲醛固定液固定,固定良好后进行修剪、脱水、包埋、切片、染色、封片。使用正置白光拍照显微镜(Nikon,Eclipse Ci-L)拍照,选取小肠组织的目的区域进行40倍成像,成像完成后使用Image-Pro Plus 6.0分析软件,分别测量每张切片中5处完整肠绒毛高度、绒毛宽度、隐窝宽度、黏膜层厚度和肌层厚度,取平均值,同时计算绒毛高度/隐窝深度。

1.3.5 回肠黏膜免疫指标

回肠黏膜分泌型免疫球蛋白A(secretory immunoglobulin A,sIgA)、肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-2(interleukin-2,IL-2)和白细胞介素-6(interleukin-6,IL-6)含量使用江苏晶美生物科技有限公司酶联联免疫试剂盒测定(货号分别为:JM-00776R1、JM-00660R1、JM-00710R1、JM-00621R1和JM-00722R1)。

1.3.6 盲肠微生物区系

采集盲肠内容物样品,置于液氮保存,委托北京诺禾致源科技股份有限公司提取DNA,基于Illumina Nova测序平台测序,构建PCR-free文库,然后进行双末端(paired-end)测序,检测微生物区系多样性。

1.4 数据处理

采用SAS 9.4.0统计软件中的GLM程序进行数据的方差分析,采用Duncan氏法进行数据的多重比较,结果以平均值和均方根误差(root mean square error,RMSE)表示,P<0.05为差异显著。

2 结果

2.1 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔生长性能的影响

由表2可知,在初始体重无显著差异(P>0.05)的情况下,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔终末体重、平均日增重、料重比、成活率和腹泻率有显著影响(P<0.05)。其中,Ⅴ组的终末体重、平均日增重和成活率显著高于对照组(P<0.05),腹泻率显著低于对照组(P<0.05);Ⅳ组的料重比显著高于Ⅴ组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔平均日采食量无显著影响(P>0.05)。
表2 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔生长性能的影响

Table 2 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on growth performance of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
初始体重 IBW/g 883.1 833.6 846.0 887.9 869.9 51.813 4 0.500 8
终末体重 FBW/g 2 161.3b 2 122.5b 2 163.8b 2 155.0b 2 244.2a 86.777 4 0.043 1
平均日增重 ADG/(g/d) 42.60b 42.96b 43.93ab 42.24b 45.81a 1.695 8 0.026 9
平均日采食量 ADFI/(g/d) 167.08 168.92 168.33 170.50 169.00 6.320 4 0.820 4
料重比 F/G 3.94ab 3.94ab 3.85ab 4.05a 3.70b 0.274 1 0.049 8
成活率 Survival rate/% 86.00b 92.00ab 93.00ab 92.00ab 97.00a 7.272 5 0.030 9
腹泻率 Diarrhea rate/% 17.00a 8.00b 9.00b 7.00b 4.00b 8.576 5 0.021 6

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)。下表同。

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 below.

2.2 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔屠宰性能的影响

由表3可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔宰前活体重有显著影响差异(P<0.05)。其中,Ⅴ组的宰前活体重显著高于对照组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔欧洲屠宰率、半净膛屠宰率和全净膛屠宰率以及心脏、肝脏和肾脏相对重量无显著影响(P>0.05)。
表3 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔屠宰性能的影响

Table 3 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on slaughter performance of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
宰前活体重
Pre-slaughter body weight/g		 2 141.0b 2 142.4b 2 191.3b 2 160.9b 2 282.5a 165.981 5 0.042 5
欧洲屠宰率
European slaughter ratio/%		 55.19 55.91 55.49 57.48 56.80 2.616 1 0.276 7
半净膛屠宰率
Semi clean slaughter ratio/%		 49.99 49.95 49.84 51.26 50.42 2.070 6 0.528 0
全净膛屠宰率
Full clean slaughter ratio/%		 44.86 44.83 44.68 46.04 45.36 2.169 8 0.621 8
心脏相对重量
Heart relative weight/(g/kg)		 3.53 3.00 3.56 3.41 3.55 0.563 8 0.153 3
肝脏相对重量
Liver relative weight/(g/kg)		 42.21 40.82 41.19 40.31 39.59 4.221 2 0.707 6
肾脏相对重量
Kidney relative weight/(g/kg)		 8.04 7.81 8.43 7.95 8.29 0.765 6 0.358 3

2.3 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔十二指肠抗氧化指标的影响

由表4可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔十二指肠GSH-Px活性、MDA含量和T-AOC有显著影响(P<0.05)。其中,Ⅱ组、Ⅳ组和Ⅴ组的十二指肠GSH-Px活性显著低于对照组(P<0.05),Ⅳ组和Ⅴ组的十二指肠MDA含量显著高于对照组(P<0.05),Ⅱ组、Ⅲ组、Ⅳ组和Ⅴ组的十二指肠T-AOC显著高于对照组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔十二指肠SOD活性无显著影响(P<0.05)。
表4 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔十二指肠抗氧化指标的影响

Table 4 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on duodenal antioxidant indices of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
谷胱甘肽过氧化物酶
GSH-Px/(U/mg prot)		 13.78a 11.09bc 13.48ab 10.78c 10.94bc 2.434 4 0.032 6
超氧化物歧化酶 SOD/(U/mg prot) 272.27 305.10 302.64 299.08 320.31 52.714 1 0.489 1
丙二醛 MDA/(nmol/mg prot) 0.51c 0.47c 0.56bc 0.79a 0.72ab 0.172 4 0.002 1
总抗氧化能力
T-AOC/(mmol/g prot)		 0.56c 1.93b 3.03ab 2.73ab 3.37a 1.182 5 0.000 3

2.4 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔空肠组织形态结构的影响

由表5可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔空肠绒毛高度有显著影响(P<0.05)。其中,Ⅴ组的空肠绒毛高度显著高于对照组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔空肠绒毛宽度、隐窝深度、黏膜层厚度、肌层厚度和绒毛高度/隐窝深度无显著影响(P>0.05)。
表5 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔空肠组织形态结构的影响

Table 5 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on jejunal histological structure of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
绒毛高度 Villus height/μm 625.36b 806.71ab 825.50ab 804.57ab 914.70a 187.245 9 0.048 2
隐窝深度 Crypt depth/μm 119.65 113.98 124.95 114.91 124.12 20.184 7 0.733 4
绒毛宽度 Villus width/μm 107.48 124.14 128.89 114.47 140.45 26.117 4 0.129 3
黏膜层厚度
Mucosal layer thickness/μm		 787.01 955.70 979.06 951.78 1 069.63 196.691 3 0.094 2
肌层厚度 Muscle layer thickness/μm 169.92 142.93 174.27 169.93 140.13 41.424 6 0.302 2
绒毛高度/隐窝深度
Villus height/crypt depth		 5.23 7.29 6.68 7.06 7.50 1.652 7 0.070 4

2.5 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔回肠黏膜免疫指标的影响

由表6可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔回肠黏膜sIgA、TNF-α和IFN-γ含量有显著影响(P<0.05)。其中,Ⅱ组、Ⅲ组、Ⅳ组和Ⅴ组的回肠黏膜sIgA含量显著高于对照组(P<0.05),回肠黏膜IFN-γ含量显著低于对照组(P<0.05);Ⅴ组的回肠黏膜TNF-α含量显著高于其他各组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔回肠黏膜IL-2和IL-6含量无显著影响(P>0.05)。
表6 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔回肠黏膜免疫指标的影响

Table 6 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on ileum mucosal immune indices of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
分泌型免疫球蛋白A sIgA/(μg/g) 168.80b 219.69a 226.34a 250.32a 218.45a 42.796 0 0.010 7
肿瘤坏死因子-α TNF-α/(ng/g) 3.40b 2.95b 3.56b 3.38b 4.76a 0.960 8 0.008 8
干扰素-γ IFN-γ/(ng/g) 6.70a 4.65b 4.82b 4.91b 5.29b 1.025 0 0.002 1
白细胞介素-2 IL-2/(ng/mg) 44.33 46.38 46.85 38.48 42.68 8.988 2 0.356 8
白细胞介素-6 IL-6/(ng/mg) 778.46 723.85 736.45 836.02 783.47 180.658 0 0.749 3

2.6 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠微生物区系的影响

2.6.1 扩增子序列变体(amplicon sequence variants,ASVs)分析

为了使信息分析的结果更加准确、可靠,首先对原始数据进行拼接、过滤,得到有效数据;然后基于有效数据通过DADA2进行降噪,并过滤掉相对丰度小于5的序列,从而获得最终的ASVs,各组ASVs分布韦恩图见图1。
图1 ASVs分布韦恩图

Fig.1 Venn diagram of ASVs

2.6.2 α多样性指数分析

由表7可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠微生物菌群ASVs数量和Chao1指数有显著影响(P<0.05)。其中,Ⅴ组的盲肠微生物菌群ASVs数量和Chao1指数显著低于Ⅰ组、Ⅱ组和Ⅳ组(P<0.05)。饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠微生物菌群Shannon指数、Simpson指数和测序深度指数无显著影响(P>0.05)。
表7 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠微生物α多样性指数的影响

Table 7 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on cecum microorganisms α diversity indexes of meat rabbits

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
ASVs数量 ASVs number 1 038.3a 1 058.9a 922.1ab 1 017.3a 758.3b 206.929 7 0.037 5
Chao1指数Chao1 index 1 041.4a 1 063.4a 927.1ab 1 022.0a 760.6b 208.391 9 0.037 9
Shannon指数Shannon index 8.302 5 8.528 1 8.263 9 8.531 6 7.983 3 0.409 9 0.250 7
Simpson指数 Simpson index 0.985 4 0.990 8 0.986 6 0.989 9 0.986 0 0.011 6 0.844 4
测序深度指数 Goods_coverage index 0.999 5 0.999 4 0.999 4 0.999 3 0.999 9 0.000 6 0.276 4

2.6.3 盲肠微生物物种丰富度分析

2.6.3.1 门水平分析

由表8可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠疣微菌门(Verrucomicrobiota)和酸杆菌门(Acidobacteriota)相对丰度有显著影响(P<0.05)。其中,Ⅲ组和Ⅴ组的盲肠疣微菌门相对丰度显著高于对照组(P<0.05),Ⅲ组、Ⅳ组和Ⅴ组的盲肠酸杆菌门相对丰度显著低于对照组(P<0.05)。
表8 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠菌群门水平的影响(前10位)

Table 8 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on cecal microflora at phylum level of meat rabbits (top 10)

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
厚壁菌门 Firmicutes 24 054 28 619 29 173 31 397 31 085 7 751.204 0 0.348 0
拟杆菌门 Bacteroidota 10 337 8 201 8 737 8 749 8 529 4 854.347 0 0.918 1
变形菌门 Proteobacteria 3 384 1 080 776 1 261 311 2 138.467 0 0.063 4
疣微菌门 Verrucomicrobiota 301.9c 1 054.0bc 1 754.9ab 795.8bc 2 590.5a 1 315.486 0 0.013 3
酸杆菌门 Acidobacteriota 714.1a 373.0ab 130.1b 90.9b 3.5b 498.656 7 0.049 1
放线菌门 Actinobacteriota 387.4 427.9 307.9 613.3 302.4 363.348 6 0.435 5
WPS-2 454.8 141.0 79.3 80.8 0.0 320.059 0 0.064 6
广古菌门 Euryarchaeota 23.8 172.9 610.3 403.5 80.3 438.692 8 0.061 7
脱硫杆菌门 Desulfobacterota 142.6 236.0 375.8 209.8 322.5 268.582 7 0.448 7
绿弯菌门 Chloroflexi 184.4 83.5 36.3 35.3 0.0 132.723 9 0.075 5

2.6.3.2 属水平分析

由表9可知,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠克曼菌属(Akkermansia)、果胶单核菌属(Monoglobus)、马赛菌属(Massilia)和疣微菌属UCG-010(Verrucomicrobium_UCG-010)相对丰度有显著影响(P<0.05)。其中,Ⅲ组和Ⅴ组的盲肠克曼菌属相对丰度显著高于对照组(P<0.05),Ⅳ组和Ⅴ组的盲肠果胶单核菌属相对丰度显著高于对照组(P<0.05),Ⅲ组、Ⅳ组和Ⅴ组的盲肠疣微菌属UCG-010相对丰度显著高于对照组(P<0.05),Ⅱ组、Ⅲ组、Ⅳ组和Ⅴ组的盲肠马赛菌属相对丰度显著低于对照组(P<0.05)。
表9 丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对肉兔盲肠菌群属水平的影响(前20位)

Table 9 Effects of Clostridium butyricum, glucose oxidase and rosemary extract formulas on cecal microflora at genus level of meat rabbits (top 20)

项目
Items		 组别 Groups 均方根
误差
RMSE		 P值
P-value
Ⅰ
(对照 Control)		 Ⅱ Ⅲ Ⅳ Ⅴ
Muribaculaceae 8 300 6 476 6 280 6 763 7 539 4 995.129 0 0.922 8
梭菌属_UCG-014
Clostridium_UCG-014		 3 328.3 4 172.9 4 063.6 5 137.5 3 784.5 1 991.468 0 0.476 3
克曼菌属 Akkermansia 301.0c 1 023.3bc 1 754.9ab 795.8bc 2 590.5a 1 314.927 0 0.012 9
果胶单核菌属 Monoglobus 509.6c 753.8bc 1 273.1bc 1 761.3ab 2 695.9a 1 100.171 0 0.002 7
梭菌属vadinBB60群
Clostridium_vadinBB60_group		 1 921.1 2 254.4 2 477.3 2 505.5 820.9 1 377.491 0 0.110 9
马赛菌属Massilia 1 269.5a 111.1b 66.1b 71.9b 0.0b 794.456 3 0.012 7
V9D2013群 V9D2013_group 968.3 1 370.3 1 265.6 1 774.1 1 445.1 962.882 3 0.573 8
瘤胃球菌属 Ruminococcus 1 944.1 1 964.1 1 773.1 2 533.8 1 971.0 809.376 9 0.409 6
NK4A214群 NK4A214_group 853.0 1 106.9 1 202.0 1 296.1 1 615.5 689.683 7 0.287 9
瘤胃球菌科Ruminococcaceae 964.4 838.9 906.8 651.6 923.6 767.129 7 0.932 7
优杆菌属西雷姆群
Eubacterium_siraeum_group		 1 142.9 1 815.5 1 597.6 1 649.4 1 436.8 571.375 0 0.199 7
青枯菌属 Ralstonia 10.5 3.6 166.9 326.1 4.4 425.219 0 0.478 4
克里斯滕森菌科R-7群
Christensenellaceae_R-7_group		 649.4 692.6 691.8 760.4 632.0 475.502 2 0.985 8
疣微菌属UCG-010
Verrucomicrobium_UCG-010		 544.9b 767.0ab 1 024.0a 1 030.0a 1 011.9a 373.533 2 0.048 8
WPS-2 454.8 141.0 79.3 80.8 0.0 320.059 0 0.064 6
甲烷芽孢杆菌属
Methanobrevibacter		 23.8 168.8 607.8 398.5 78.8 434.465 3 0.059 3
芽孢杆菌属 Bacillus 443.4 107.9 192.8 262.4 0.0 413.886 3 0.287 7
考拉杆菌属
Phascolarctobacterium		 106.8 354.1 41.0 121.1 31.9 288.964 7 0.186 6
理研菌科RC9肠道群
Rikenellaceae_RC9_gut_group		 390.8 216.6 260.8 513.5 37.1 459.145 8 0.314 3
鞘氨醇单胞菌属
Sphingomonas		 359.9 191.5 68.4 46.5 0.0 262.990 2 0.065 4

3 讨论

益生菌、酶制剂和植物提取物具有绿色、安全、生态等突出优点,成为新型绿色饲料添加剂的首先。丁酸梭菌能够代性产生丁酸,具有广泛的生物学功能,饲粮中添加丁酸梭菌可改善生猪、家禽和反刍动物的生长性能[13⇓-15]。葡萄糖氧化酶作为一种需氧脱氢酶,能够氧化分解葡萄糖产生葡萄糖酸,促进动物肠道健康,提高生长性能。饲粮中添加葡萄糖氧化酶能够提高肉仔鸡生长性能、回肠表观氨基酸消化率和回肠微生物区系,且分解淀粉芽孢杆菌SC06与葡萄糖氧化酶配合使用效果优于单独添加葡萄糖氧化酶[7,16]。迷迭香提取物具有多种药理和生物活性,饲粮中添加迷迭香提取物可以改善动物的生长性能、营养物质消化率、抗氧化能力和免疫功能[8⇓-10]。本研究发现,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方能够提高肉兔生长性能,降低腹泻率,这与前人研究报道基本一致。
丁酸梭菌具有抑制肠道致病菌侵入的功能,在维持动物肠道健康方面发挥重要调节作用[17⇓⇓-20]。此外,Huang等[21]研究报道,在家兔饲粮中添加丁酸梭菌能够提高小肠肠绒毛高度和促进回肠sIgA的分泌。饲粮中添加葡萄糖氧化酶可以促进肠道屏障的完整性,对改善霉菌毒素造成肉鸡肝脏氧化应激具有一定缓解功能[22-23]。迷迭香提取物中含有较高的多酚及黄酮含量,具有较大的自由基清除活性,迷迭香提取物可能通过增加抗氧化能力减少应激对虹鳟鱼的影响[24⇓-26]。Hadree等[27]研究报道迷迭香提取物和维生素E具有降低氧化应激和激活新西兰白兔的抗氧化保护作用。本研究发现,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方对能够提高肉兔小肠绒毛高度、抗氧化能力和sIgA含量,对维持肠道健康具有重要作用。
家兔盲肠内富含大量微生物,在营养物质消化、吸收、代谢和肠黏膜免疫调节中发挥重要作用,饲粮营养物质含量和种类是影响笼养家兔盲肠微生物区系组成的重要因素[28⇓-30]。在门水平上,家兔盲肠内容物相对丰度最大是厚壁菌门,其次是拟杆菌门,厚壁菌门与碳水化合物和蛋白质的吸收有关,拟杆菌门主要是促进碳水化合物发酵、吸收和降解多糖,这两大菌门的稳定对促进肉兔肠道健康十分重要[31-32]。大量研究表明,丁酸梭菌可通过增加牛、猪、鸭等动物肠道微生物菌群多样性,增强肠道屏障功能和免疫系统[33⇓-35]。此外,饲粮中添加250 U/kg葡萄糖氧化酶也可以提高肉仔鸡第22~42天的生长性能,这与肠道形态、短链脂肪酸组成和回肠微生物组成的改变有关[16]。本研究发现,饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方能够影响肉兔盲肠微生物菌群中疣微菌门和酸杆菌门相对丰度,影响肉兔盲肠微生物菌群中克曼菌属、果胶单核菌属、马赛菌属和疣微菌属UCG-010相对丰度。然而,目前家兔盲肠微生物菌属的变化与生长性能的关系尚不清楚。因此,需要更多的研究来理解家兔生长性能和盲肠内各种微生物群之间的相互关系。

4 结论

饲粮中添加丁酸梭菌、葡萄糖氧化酶和迷迭香提取物组方能够改善肉兔小肠形态结构和盲肠微生物区系,提高肠道黏膜免疫功能和机体抗氧化能力,有益于维持肠道健康,降低腹泻率,提高生长性能。

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