Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 6: 3617 - 3630

DOI: https://doi.org/10.12418/CJAN2023.337

RESEARCH PAPER

Effects of Clostridium autoethanogenum Protein on Growth Performance, Immune Function and Intestinal Health of Yellow-Feathered Broilers

  • LIN Xiajing , 1 ,
  • LI Chongyang 2 ,
  • MAO Shenli 1 ,
  • YE Jinling 1 ,
  • FAN Qiuli 1 ,
  • CHAO Wei 2 ,
  • ZOU Fangqi 2 ,
  • JIANG Shouqun , 1, **
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  • 1 Guangdong Key Laboratory of Animal Breeding and Nutrition, Guangdong Public 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 Livestock and Poultry Breeding, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • 2 Beijing Shoulang Bio-Technology Co., Ltd., Beijing 100041,China
**professor, E-mail:

*Contributed equally

Received date: 2022-12-05

  Online published: 2023-06-08

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Abstract

This experiment was conducted to investigate the effects of Clostridium autoethanogenum protein (CAP) on growth performance, immune function and intestinal health of yellow-feathered broilers by monitoring growth performance, plasma and intestinal immune indexes, liver biochemical indexes and intestinal mucosal barrier gene expression, and to determine the appropriate supplemental level of CAP in diets of yellow-feathered broilers. The experiment used a single factor randomized design. A total of 1 280 one-day-old yellow-feathered broilers were randomly divided into 7 groups with 6 replicates in each group and 30 chickens (half male and half female) in each replicate. Broilers in 7 groups were fed the basal diets added 0 (control), 0.8%, 1.6%, 2.4%, 3.2%, 4.0%, 8.0% CAP, respectively. The experiment lasted for 56 days. Results showed as follows: 1) body weight and average daily gain at 1 to 21 days of age in 3.2% group were significantly higher than those in control group and 0.8% group (P<0.05); the feed/gain in 3.2% and 8.0% groups was significantly lower than that in control group (P<0.05). The average daily gain at 1 to 56 days of age in 4.0% and 8.0% groups was significantly higher than that in control group and 0.8% group (P<0.05), and the feed/gain in 4.0% and 8.0% groups was significantly lower than that in control group and 0.8% group (P<0.05). 2) CAP had no significant effects on organ indexes of yellow-feathered broilers at 21 and 56 days of age (P>0.05). 3) At 21 days of age, the activity of liver total superoxide dismutase (T-SOD) in 4.0% group was significantly higher than that in the control group, 0.8% and 2.4% groups (P<0.05). 4) The plasma interleukin-1β (IL-1β) content in control and 4.0% groups was significantly higher than that in 0.8% group (P<0.05); the content of plasma tumor necrosis factor-α (TNF-α) in the 1.6%, 4.0% and 8.0% groups was significantly lower than that in the control and 2.4% groups (P<0.05). Dietary CAP significantly affected plasma interleukin-6 (IL-6) content at 56 days of age (P<0.05), and the IL-6 content in 3.2%, 4.0% and 8.0% groups was significantly lower than that in control and 0.8% groups (P<0.05). 5) The relative expression level of zonula occludens protein-1 (ZO-1) mRNA in 1.6%, 2.4%, 3.2%, 4.0% and 8.0% groups was significantly higher than that in control and 0.8% groups (P<0.05). The relative expression level of Claudin1 mRNA in 1.6% group was significantly higher than that in control group and 0.8% group (P<0.05). The relative expression level of nuclear factor E2 related factor 2 (Nrf2) mRNA in 2.4%, 3.2% and 8.0% groups was significantly higher than that in control and 0.8% groups (P<0.05). 6) Dietary CAP significantly affected the crude protein metabolic rate of yellow-feathered broilers (P<0.05), and the crude protein metabolic rate in 4.0% and 8.0% groups was significantly higher than that in the control group (P<0.05). These results indicate that CAP can improve the growth performance, immune function and intestinal health of yellow-feathered broilers. The optimal dietary supplemental level of CAP for yellow-feathered broilers is 1.6% to 4.0%.

Key words： yellow-feathered broilers; Clostridium autoethanogenum protein; intestinal health; immune function; crude protein metabolic rate

Cite this article

LIN Xiajing , LI Chongyang , MAO Shenli , YE Jinling , FAN Qiuli , CHAO Wei , ZOU Fangqi , JIANG Shouqun . Effects of Clostridium autoethanogenum Protein on Growth Performance, Immune Function and Intestinal Health of Yellow-Feathered Broilers[J]. Chinese Journal of Animal Nutrition, 2023 , 35(6) : 3617 -3630 . DOI: 10.12418/CJAN2023.337

我国长久以来豆粕过于依赖进口,随着国际局势的动荡,豆粕价格的飞涨,蛋白质资源的限制严重威胁养殖业的稳定发展,寻找国产、优质蛋白质原料是目前畜牧业亟待解决的问题。乙醇梭菌蛋白(Clostridium autoethanogenum protein,CAP)是利用乙醇梭菌和特定培养基获得的菌体蛋白[1],其粗蛋白质含量高达80%以上,氨基酸组成齐全,并且富含多种维生素和微量元素[2]。乙醇梭菌蛋白具有蛋白溶解度和吸水性高的特点,所以其制粒性优于传统蛋白质原料[3]。研究发现,乙醇梭菌蛋白可以提高动物的生长性能[4]、屠宰性能[5]、抗氧化能力[6]以及改善肠道健康[7]等。目前商品化成熟的乙醇梭菌蛋白在水产养殖中已有全面的评估数据,但对于畜禽养殖方面研究非常有限,缺乏可供实际生产参考的有力依据。因此,本试验通过研究乙醇梭菌蛋白对黄羽肉鸡生长性能、免疫功能和肠道健康的影响,探讨乙醇梭菌蛋白在黄羽肉鸡应用的可能性,以期为乙醇梭菌蛋白在肉鸡生产中的合理应用提供参考依据。

1 材料与方法

1.1 试验动物与分组

试验采用单因子随机分组设计,选用1 260只1日龄健康、发育良好的快速型麻黄鸡,按照体重一致原则(公、母分别称重)随机分为7组,每组6个重复,每个重复30只鸡(公母各占1/2)。7组肉鸡分别饲喂在基础饲粮中加入0(对照)、0.8%、1.6%、2.4%、3.2%、4.0%、8.0%的乙醇梭菌蛋白的试验饲粮。

1.2 试验饲粮

试验采用玉米-豆粕型基础饲粮(颗粒料),参考《中国饲料成分及营养价值表》(2021年第32版)[8]和《黄羽肉鸡营养需要量》(NY/T 3645—2020)[9]配制。乙醇梭菌蛋白由北京某生物科技有限公司生产提供,乙醇梭菌蛋白营养成分见表1。具体饲粮组成及营养水平见表2至表4。各组试验鸡饲喂含不同水平乙醇梭菌蛋白的试验饲粮,各试验组营养水平保持一致。
表1 乙醇梭菌蛋白营养成分

Table 1 Nutrient composition of CAP%

项目 Items 含量 Content
表观代谢能 AME/(MJ/kg) 17.80
粗蛋白质 CP 85.00
粗纤维 CF 0.20
粗脂肪 EE 0.10
赖氨酸 Lys 9.90
蛋氨酸 Met 2.03
苏氨酸 Thr 4.85
色氨酸 Trp 0.36
异亮氨酸 Ile 6.94
精氨酸 Arg 3.90
钙 Ca 0.29
磷 P 0.31
表2 1~21日龄饲粮组成及营养水平(风干基础)

Table 2 Composition and nutrient levels of diets at 1 to 21 days of age (air-dry basis)%

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/%
0 0.8 1.6 2.4 3.2 4 8
原料 Ingredients
玉米 Corn (CP 8.7%) 54.5 55.88 56.65 57.48 58.16 59.03 63.54
豆粕 Soybean meal (CP 44.2%) 32 29.7 28.2 26.6 24.8 23 14.3
乙醇梭菌蛋白 0.8 1.6 2.4 3.2 4 8
Clostridium autoethanogenum
protein (CP 85%)
菜籽粕 Rapeseed meal (CP 35.7%) 2.4 2.4 2.4 2.4 2.4 2.4 2.4
棉籽蛋白 Cottonseed protein (CP 51.1%) 1 1 1 1 1 1 1
麦麸 Wheat bran 2.4 2.5 2.5 2.5 2.8 2.8 3
大豆油 Soybean oil 2.29 2.29 2.29 2.29 2.29 2.29 2.29
L-赖氨酸盐酸盐 L-lysine·HCl (78%) 0.44 0.41 0.36 0.31 0.25 0.24
DL-蛋氨酸 DL-Met (99%) 0.25 0.25 0.24 0.23 0.21 0.22 0.18
L-苏氨酸 L-Thr (99%) 0.17 0.16 0.15 0.14 0.12 0.11 0.06
L-精氨酸 L-Arg (99%) 0.14 0.14 0.18 0.22 0.25 0.41
石粉 Limestone 1.25 1.2 1.2 1.21 1.21 1.28 1.32
磷酸氢钙 CaHPO4 2 1.97 1.97 1.96 2.04 2.08 2.2
氯化钠 NaCl 0.3 0.3 0.3 0.3 0.3 0.3 0.3
预混料 Premix1) 1 1 1 1 1 1 1
合计 Total 100 100 100 100 100 100 100
营养水平 Nutrient levels2)
代谢能 ME/(MJ/kg) 12.12 12.12 12.12 12.12 12.12 12.12 12.12
粗蛋白质 CP 21.31 21.59 21.52 21.5 21.35 21.35 21.28
赖氨酸 Lys 1.4 1.4 1.4 1.4 1.4 1.4 1.4
蛋氨酸 Met 0.56 0.56 0.56 0.56 0.56 0.56 0.56
苏氨酸 Thr 0.93 0.93 0.93 0.93 0.93 0.93 0.93
精氨酸 Arg 1.49 1.49 1.49 1.49 1.49 1.49 1.49
钙 Ca 0.98 0.96 0.98 0.99 0.97 0.97 0.98
非植酸磷 NPP 0.47 0.47 0.47 0.47 0.47 0.47 0.47

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 600 IU,VE 45 mg,VK3 2.5 mg,VB1 2.4 mg,VB2 5.0 mg,VB6 6.0 mg,VB12 16 μg,烟酸 nicotinic acid 42 mg,D-泛酸 D-pantothenic acid 12 mg,叶酸 folic acid 1.0 mg,生物素 folic acid 0.12 mg,氯化胆碱 choline chloride 1 300 mg,抗氧化剂 antioxidant 150 mg,防霉剂 antimold 1 400 mg,Fe (as ferric sulfate) 80 mg,Cu (as copper sulfate)7 mg,Zn (as zinc sulfate)75 mg,Mn (as manganese sulfate)100 mg,Se (as sodium selenite)0.15 mg,I (as calcium iodide) 0.35 mg。

2)粗蛋白质和钙是实测值,其他为计算值。表3、表4同。CP and Ca were measured values, while the others were calculated values. The same as Table 3 and Table 4.

表3 22~42日龄饲粮组成及营养水平(风干基础)

Table 3 Composition and nutrient levels of diets at 22 to 42 days of age (air-dry basis)%

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/%
0 0.8 1.6 2.4 3.2 4 8
原料 Ingredients
玉米 Corn (CP 8.7%) 62.58 63.53 64.21 65.16 65.96 67.04 69.89
豆粕 Soybean meal (CP 44.2%) 24.5 22.7 21.27 19.5 17.9 16 7.3
乙醇梭菌蛋白 0.8 1.6 2.4 3.2 4 8
Clostridium autoethanogenum
protein (CP 85%)
菜籽粕 Rapeseed meal (CP 35.7%) 3 3 3 3 3 3 3
棉籽蛋白 Cottonseed protein (CP 51.1%) 1.5 1.5 1.5 1.5 1.5 1.5 1.5
麦麸 Wheat bran 1 1 1 1 1 1 3
大豆油 Soybean oil 2.3 2.3 2.3 2.3 2.3 2.3 2.3
L-赖氨酸盐酸盐 L-lysine·HCl (78%) 0.48 0.44 0.39 0.34 0.29 0.25
DL-蛋氨酸 DL-Met (99%) 0.23 0.23 0.22 0.21 0.2 0.19 0.16
L-苏氨酸 L-Thr (99%) 0.2 0.19 0.18 0.17 0.16 0.08 0.08
L-精氨酸 L-Arg (99%) 0.13 0.18 0.2 0.23 0.26 0.34 0.45
石粉 Limestone 1.15 1.13 1.13 1.12 1.08 1.13 1.1
磷酸氢钙 CaHPO4 1.63 1.7 1.7 1.77 1.85 1.87 1.92
氯化钠 NaCl 0.3 0.3 0.3 0.3 0.3 0.3 0.3
预混料 Premix 1 1 1 1 1 1 1
合计Total 100 100 100 100 100 100 100
营养水平 Nutrient levels
代谢能 ME/(MJ/kg) 12.54 12.54 12.54 12.54 12.54 12.54 12.54
粗蛋白质 CP 19.29 19.25 19.3 19.21 19.25 19.2 19.32
赖氨酸 Lys 1.26 1.26 1.26 1.26 1.26 1.26 1.26
蛋氨酸 Met 0.52 0.52 0.52 0.52 0.52 0.52 0.52
苏氨酸Thr 0.88 0.88 0.88 0.88 0.88 0.88 0.88
精氨酸 Arg 1.36 1.36 1.36 1.36 1.36 1.36 1.36
钙 Ca 0.89 0.88 0.89 0.88 0.89 0.88 0.9
非植酸磷 NPP 0.41 0.41 0.41 0.41 0.41 0.41 0.41

预混料为每千克饲粮提供The premix provided the following per kg of diets: VA 9 000 IU,VD3 500 IU,VE 35 mg,VK3 2.2 mg,VB1 2.3 mg,VB2 5.0 mg,VB6 5.0 mg,VB12 15 μg,烟酸 nicotinic acid 35 mg,D-泛酸 D-pantothenic acid 10 mg,叶酸 folic acid 0.7 mg,生物素 folic acid 0.10 mg,氯化胆碱 choline chloride 1 000 mg,抗氧化剂 antioxidant 150 mg,防霉剂 antimold 1 400 mg,Fe (as ferric sulfate) 70 mg,Cu (as copper sulfate) 7 mg,Zn (as zinc sulfate) 70 mg,Mn(as manganese sulfate) 80 mg,Se (as sodium selenite) 0.15 mg,I (as calcium iodide)0.35 mg。

表4 43~56日龄饲粮组成与营养水平(风干基础)

Table 4 Composition and nutrient levels of diets at 43 to 56 days of age (air-dry basis)%

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/%
0 0.8 1.6 2.4 3.2 4 8
原料 Ingredients
玉米 Corn (CP 8.7%) 64.2 65.14 66.08 67 67.6 68.94 72.35
豆粕 Soybean meal (CP 44.2%) 19.7 18 16.2 14.54 13.2 11 3.2
乙醇梭菌蛋白 0.8 1.6 2.4 3.2 4 8
Clostridium autoethanogenum
protein (CP 85%)
菜籽粕 Rapeseed meal (CP 35.7%) 3 3 3 3 3 3 3
棉籽蛋白 Cottonseed protein (CP 51.1%) 1.8 1.8 1.8 1.8 1.8 1.8 1.8
麦麸 Wheat bran 2.5 2.5 2.5 2.5 2.5 2.5 3
大豆油 Soybean oil 4 4 4 4 4 4 4
L-赖氨酸盐酸盐 L-lysine·HCl (78%) 0.47 0.42 0.38 0.33 0.28 0.25
DL-蛋氨酸 DL-Met (99%) 0.21 0.2 0.2 0.19 0.18 0.18 0.13
L-苏氨酸 L-Thr (99%) 0.19 0.18 0.18 0.15 0.14 0.14 0.06
L-精氨酸 L-Arg (99%) 0.15 0.18 0.22 0.24 0.26 0.31 0.44
石粉 Limestone 1.06 1.06 1.06 1.07 1.06 1.03 1.06
磷酸氢钙 CaHPO4 1.42 1.42 1.48 1.48 1.48 1.55 1.66
氯化钠 NaCl 0.3 0.3 0.3 0.3 0.3 0.3 0.3
预混料 Premix 1 1 1 1 1 1 1
合计 Total 100 100 100 100 100 100 100
营养水平 Nutrient levels
代谢能 ME/(MJ/kg) 12.96 12.96 12.96 12.96 12.96 12.96 12.96
粗蛋白质 CP 18.08 17.82 18 18.07 17.81 17.81 18.06
赖氨酸 Lys 1.14 1.14 1.14 1.14 1.14 1.14 1.14
Items 0 0.8 1.6 2.4 3.2 4 8
蛋氨酸 Met 0.48 0.48 0.48 0.48 0.48 0.48 0.48
苏氨酸 Thr 0.8 0.8 0.8 0.8 0.8 0.8 0.8
精氨酸 Arg 1.23 1.23 1.23 1.23 1.23 1.23 1.23
钙 Ca 0.81 0.81 0.8 0.81 0.8 0.81 0.82
非植酸磷 NPP 0.36 0.36 0.36 0.36 0.36 0.36 0.36

预混料为每千克饲粮提供The premix provided the following per kg of diets: VA 6 000 IU,VD3 500 IU,VE 25 mg,VK3 1.7 mg,VB1 1.0 mg,VB2 4.0 mg,VB6 5.0 mg,VB12 8 μg,烟酸 nicotinic acid 20 mg,D-泛酸 D-pantothenic acid 8 mg,叶酸 folic acid 0.3 mg,生物素 folic acid 0.02 mg,氯化胆碱 choline chloride 750 mg,抗氧化剂 antioxidant 150 mg,防霉剂 antimold 1 400 mg,Fe (as ferric sulfate)64 mg,Cu (as copper sulfate)7 mg,Zn (as zinc sulfate) 68 mg,Mn (as manganese sulfate) 78 mg,Se (as sodium selenite)0.15 mg,I (as calcium iodide)0.35 mg。

1.3 饲养管理

试验期56 d,分3个阶段(1~21日龄、22~42日龄和43~56日龄)饲养。试验期间,安排专人负责饲养,试验鸡自由采食和饮水,每天自然光照和通风,保持禽舍清洁卫生。试验鸡饲养方式为地面平养(垫料为谷壳),按照常规的饲养操作规程。

1.4 测定指标及方法

1.4.1 生长性能

21、42、56日龄前1 d 20:00断料不断水,于次日08:00以重复为单位称体重、剩余饲粮重,计算试验期肉鸡的平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。每天观察试验鸡健康和粪便状况,统计每个饲养阶段试验鸡的死亡率。

1.4.2 粗蛋白质代谢率

42日龄时,从7个组的每个重复中选取4只鸡(公母各占1/2),一共选用168只鸡,采用笼养,每2只试验鸡放1个鸡笼,整个试验期自由采食(试验饲粮与43~56日龄饲粮相同)。采用全收粪法进行代谢试验,代谢试验开始前,鸡舍和鸡笼彻底清洗干净,待干燥后,用高锰酸钾和甲醛进行消毒。每个代谢试验期共6 d,前3 d为适应期,不收集排泄物,第3天开始正式试验。试验鸡自由采食,同时以每重复为单位准确收集排泄物,喷洒少量10%盐酸用来固定挥发性氮。仔细剔除羽毛、皮屑等其他杂物,并捡出遗洒的饲粮,将排泄物刮入培养皿中,然后放入65~70 ℃的烘箱中烘干至发脆,取出在空气中回潮24 h后再进行称重。然后用粉碎机粉碎并过40目筛,装袋,于-20 ℃冰箱保存,供分析备用。粗蛋白质的代谢率计算公式如下:
DC(%)= F × N F - f × N F F × N F×100。
式中:DC为饲粮中粗蛋白质代谢率(%);F为饲粮采食量(g);f为粪便排泄量(g);NF为饲粮中粗蛋白质含量(%);Nf为排泄物中粗蛋白质含量(%)。

1.4.3 器官指数

21日龄时,从每个重复中选取2只试验鸡(公母各1只)进行称重,然后完整剖摘肝脏、法氏囊、脾脏,并称重,计算各器官指数。试验结束时(56日龄),从每个重复中选取2只试验鸡(公母各1只)进行称重,然后完整剖摘心脏、肝脏、脾脏、肺脏、肾脏,并称重,计算各器官指数。

1.4.4 血浆和肠道免疫指标

21、56日龄时,从每个重复中选取2只试验鸡(公母各1只),使用抗凝管翅静脉采血2 mL,离心分离血浆,剪取空肠肠段并刮取黏膜,测定血浆和空肠黏膜免疫炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)含量,测定采用酶联免疫吸附测定(ELISA)法使用多功能酶标仪(spectra max M-5,Molecular Devices公司,美国)测定,试剂盒采购于北京厚泽鸿业科技有限公司。

1.4.5 肝脏生化指标

21、56日龄时,从每个重复中选取2只试验鸡(公母各1只),剖取肝脏,测定肝脏中总超氧化物歧化酶(T-SOD)、碱性磷酸酶(AKP)、琥珀酸脱氢酶(SDH)、胆碱酯酶(CHE)活性和谷胱甘肽(GSH)含量,使用多功能酶标仪(spectra max M-5,Molecular Devices公司,美国)检测,试剂盒采购于南京建成生物工程研究所。

1.4.6 空肠黏膜屏障相关基因表达

56日龄时,从每个重复中选取1只试验鸡(每组公母各6只),剪取空肠肠段,刮取适量的空肠黏膜,按RNAsio试剂说明提取总RNA,用NanoDrop ND2000进行定量后,用反转录试剂盒Prime ScriptTM RT Reagent Kit (Perfect Real Time)对RNA样品进行RT反应合成cDNA;然后以β-肌动蛋白(β-actin)为内参,用荧光定量PCR试剂盒iTaq Universal SYBR® GreenSupremix (BIO-RAD)对目标基因[碱性氨基酸转运载体(rBAT)、封闭蛋白1(Occludin1)、闭合蛋白1(Claudin1)、核因子E2相关因子2(Nrf2)、闭锁小带蛋白-1(ZO-1)]进行定量检测,引物序列见表5,引物由生工生物工程(上海)股份有限公司合成,反应体系为:10 μL iTaq Universal SYBR® GreenSupremix,0.4 μL上游引物(10 μmol/L),0.4 μL下游引物(10 μmol/L),2.0 μL上述RT产物稀释液,H2O补足至20 μL;反应条件为:95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34 s,40个循环,PCR反应结束后进行熔解曲线分析,收集数据进行统计分析,以β-actin为内参基因,按照2-ΔΔCt法计算各目的基因的mRNA相对表达量。
表5 实时荧光定量PCR引物序列

Table 5 Real-time fluorescent quantitative PCR primer sequences

基因 Genes 引物序列 Primer sequences (5'—3')
闭锁小带蛋白-1 ZO-1 F:CCAAAGACAGCAGGAGGAGA
R:TGGCTAGTTTCTCTCGTGCA
闭合蛋白1 Claudin1 F:GAGGATGACCAGGTCAAGAAG
R:TGCCCAGCCAATGAAGAG
封闭蛋白1 Occludin1 F:TCATCCTGCTCTGCCTCATCT
R:CATCCGCCACGTTCTTCAC
核因子E2相关因子2 Nrf2 F:ATCACCTCTTCTGCACCGAA
R:GCTTTCTCCCGCTCTTTCTG
碱性氨基酸转运载体 rBAT F:CCCGCCGTTCAACAAGAG
R:CAGAAAACCTCAGAGCTCCCTTT

1.5 数据处理与统计分析

试验数据采用SAS 8.0软件的一般线性模型(GLM)程序进行单因素方差分析,并对试验数据进行Duncan氏法多重比较,统计显著水平为P<0.05,试验数据均以平均值和均值标准误(SEM)表示。

2 结果

2.1 乙醇梭菌蛋白对黄羽肉鸡生长性能的影响

由表6可知,饲粮中乙醇梭菌蛋白显著影响黄羽肉鸡1~21日龄和整个试验期的生长性能,其中3.2%组1~21日龄体重和平均日增重显著高于对照组和0.8%组;3.2%和8.0%组料重比显著低于对照组(P<0.05);4.0%和8.0%组1~56日龄的平均日增重显著高于对照组和0.8%组(P<0.05),4.0%和8.0%组料重比显著低于对照组和0.8%组(P<0.05)。
表6 乙醇梭菌蛋白对黄羽肉鸡生长性能的影响

Table 6 Effects of Clostridium autoethanogenum protein on growth performance of yellow-feathered broilers

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
1~21日龄 1 to 21 days of age
平均日增重 ADG/g 14.17b 14.21b 14.85ab 14.46ab 15.10a 14.66ab 15.00ab 0.12 0.046
平均日采食量 ADFI/g 26.32 25.99 26.92 26.16 26.89 26.53 26.43 0.17 0.776
料重比 F/G 1.86a 1.83ab 1.81ab 1.81ab 1.78b 1.81ab 1.76c 0.01 0.045
死亡率 Mortality/% 0.00 1.19 0.57 0.57 1.15 0.57 1.11 0.27 0.864
22~42日龄 22 to 42 days of age
平均日增重 ADG/g 34.47 33.27 34.29 33.96 33.37 35.34 36.66 2.40 0.458
平均日采食量 ADFI/g 81.35 79.67 79.67 79.79 79.17 80.55 81.65 0.47 0.415
料重比F/G 2.37 2.40 2.33 2.36 2.39 2.29 2.23 0.03 0.812
死亡率 Mortality/% 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.635
43~56日龄 43 to 56 days of age
平均日增重 ADG/g 46.32 45.38 47.13 47.24 47.75 49.69 47.67 0.66 0.185
平均日采食量 ADFI/g 121.26 121.20 121.29 120.29 120.42 121.59 123.33 0.65 0.350
料重比 F/G 2.63 2.67 2.59 2.55 2.53 2.46 2.60 0.03 0.552
死亡率 Mortality/% 0.00 0.00 0.69 0.69 0.69 0.00 0.00 0.20 0.121
1~56日龄 1 to 56 days of age
平均日增重 ADG/g 29.82bc 28.78c 30.21ab 29.97abc 30.12ab 31.17a 31.29a 0.21 0.033
平均日采食量 ADFI/g 76.31 75.83 76.03 75.41 75.49 76.22 77.13 0.37 0.648
料重比 F/G 2.56ab 2.64a 2.52bc 2.52bc 2.51bc 2.45c 2.47c 0.02 0.017
死亡率 Mortality/% 0.56 1.11 1.11 1.67 1.67 0.56 0.56 0.30 0.326

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。各组具体死亡情况:对照组(公鸡1只)、0.8%组(公鸡2只)、1.6%组(公鸡1只)、2.4%组(公鸡1只,母鸡2只)、3.2%组(公鸡2只,母鸡1只)、4.0%组(母鸡1只)、8.0%组(母鸡1只)。

In the same row, values with the same letter or no letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below. Death in each group: control group (1 male broiler), 0.8% group (2 male broilers), 1.6% group (1 male broiler), 2.4% group (1 male broiler, 2 female broilers), 3.2% group (2 male broilers, 1 female broiler), 4.0% group(1 female broiler), 8.0% group (1 female broiler).

2.2 乙醇梭菌蛋白对黄羽肉鸡器官指数的影响

由表7可知,乙醇梭菌蛋白对黄羽肉鸡21和56日龄各器官指数均无显著影响(P>0.05)。
表7 乙醇梭菌蛋白对黄羽肉鸡器官指数的影响

Table 7 Effects of Clostridium autoethanogenum protein on organ indexes of yellow-feathered broilers%

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照Control) 0.8 1.6 2.4 3.2 4 8
21日龄 21 days of age
脾脏 Spleen 0.16 0.15 0.18 0.16 0.17 0.19 0.15 0.007 0.938
法氏囊 Bursa of Fabricius 0.39 0.38 0.35 0.36 0.35 0.4 0.41 0.009 0.329
胸腺 Thymus 0.49 0.52 0.52 0.46 0.44 0.52 0.46 0.018 0.774
肝脏 Liver 3.12 3.21 3.29 3.14 3.3 3.4 3.23 0.043 0.841
56日龄 56 days of age
心脏 Heart 0.53 0.59 0.55 0.53 0.52 0.5 0.52 0.018 0.111
肝脏 Liver 2.7 2.62 2.66 2.64 2.47 2.4 2.61 0.066 0.72
脾脏 Spleen 0.21 0.21 0.23 0.2 0.2 0.21 0.21 0.005 0.498
肺脏 Lung 0.69 0.77 0.71 0.66 0.64 0.65 0.66 0.026 0.671
肾脏 Kidney 0.8 0.79 0.79 0.79 0.72 0.72 0.76 0.022 0.779

2.3 乙醇梭菌蛋白对黄羽肉鸡肝脏生化指标的影响

由表8可知,饲粮添加乙醇梭菌蛋白对21日龄试验鸡肝脏T-SOD活性有显著影响(P<0.05),其中4.0%组肝脏T-SOD活性显著高于对照组、0.8%和2.4%组(P<0.05)。乙醇梭菌蛋白对56日龄试验鸡肝脏生化指标均无显著影响(P>0.05)。
表8 乙醇梭菌蛋白对黄羽肉鸡肝脏生化指标的影响

Table 8 Effects of Clostridium autoethanogenum protein on biochemical indexes in liver of yellow-feathered broilers

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
21日龄 21 days of age
胆碱酯酶
CHE/(U/mg prot)		 0.36 0.39 0.33 0.32 0.25 0.31 0.43 0.021 0.834
碱性磷酸酶
AKP/(U/g prot)		 81.97 85.56 79.09 81.99 72.95 84.43 74.05 2.490 0.302
琥珀酸脱氢酶
SDH/(U/mg prot)		 0.81 0.68 0.88 0.65 0.53 0.53 0.64 0.055 0.434
总超氧化物歧化酶
T-SOD/(U/mg prot)		 48.22bc 45.45c 49.15abc 47.32bc 53.78ab 56.20a 54.46ab 1.097 0.028
谷胱甘肽
GSH/(μmol/g prot)		 25.36 25.92 25.36 19.14 18.88 21.44 28.75 1.055 0.588
56日龄 56 days of age
胆碱酯酶
CHE/(U/mg prot)		 0.77 0.80 0.73 0.76 0.83 0.70 0.75 0.022 0.236
碱性磷酸酶
AKP/(U/g prot)		 19.05 21.23 20.31 18.86 21.91 21.67 19.30 0.487 0.250
琥珀酸脱氢酶
SDH/(U/mg prot)		 1.10 1.27 1.19 1.18 1.05 1.05 1.23 0.032 0.731
总超氧化物歧化酶
T-SOD/(U/mg prot)		 7.49 7.62 7.47 7.21 7.92 7.21 7.04 0.244 0.103
谷胱甘肽
GSH/(μmol/g prot)		 11.69 11.22 11.79 12.67 11.70 11.29 14.00 0.358 0.140

2.4 乙醇梭菌蛋白对黄羽肉鸡血浆免疫因子含量的影响

由表9可知,饲粮添加乙醇梭菌蛋白显著影响21日龄试验鸡血浆免疫因子IL-1β和TNF-α含量(P<0.05),其中对照组和4.0%组血浆IL-1β含量显著高于0.8%组(P<0.05);1.6%、4.0%和8.0%组血浆TNF-α含量显著低于对照组和2.4%组(P<0.05)。乙醇梭菌蛋白显著影响56日龄黄羽肉鸡血浆免疫因子IL-6含量(P<0.05),3.2%、4.0%和8.0%组血浆IL-6含量显著低于对照组和0.8%组(P<0.05)。
表9 乙醇梭菌蛋白对黄羽肉鸡血浆免疫因子含量的影响

Table 9 Effects of Clostridium autoethanogenum protein on plasma immune factor contents of yellow-feathered broilersng/L

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
21日龄 21 days of age
白细胞介素-1β IL-1β 13.77a 11.68b 12.69ab 12.87ab 13.25ab 14.17a 13.62ab 0.252 0.014
γ-干扰素IFN-γ 51.47 43.00 50.18 49.54 47.94 48.95 0.41 1.589 0.683
白细胞介素-6 IL-6 13.57 14.59 10.47 12.83 13.17 0.59 13.61 0.486 0.541
肿瘤坏死因子-α TNF-α 58.03a 52.80ab 38.99bc 59.79a 49.51abc 34.90c 37.73b 4.155 0.006
56日龄 56 days of age
白细胞介素-1β IL-1β 4.35 4.73 4.49 4.36 4.46 3.88 4.00 0.096 0.900
γ-干扰素 IFN-γ 12.02 12.41 12.60 12.46 11.80 12.12 11.43 0.244 0.346
白细胞介素-6 IL-6 5.13a 5.17a 4.85ab 4.83ab 4.37b 4.35b 4.10b 0.103 0.019
肿瘤坏死因子-α TNF-α 13.75 13.72 13.13 13.62 14.25 11.72 13.78 0.347 0.123

2.5 乙醇梭菌蛋白对黄羽肉鸡空肠黏膜免疫炎症因子含量的影响

由表10可知,乙醇梭菌蛋白对试验鸡空肠黏膜免疫因子含量均无显著影响(P>0.05)。
表10 乙醇梭菌蛋白对黄羽肉鸡空肠黏膜免疫炎症因子含量的影响

Table 10 Effects of Clostridium autoethanogenum protein on jejunum mucosal immune factor contents of yellow-feathered broilersng/g prot

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
21日龄 21 days of age
白细胞介素-1β IL-1β 1.16 1.23 1.34 1.34 1.46 1.51 1.55 0.057 0.418
γ-干扰素 IFN-γ 3.82 4.02 4.18 4.91 5.00 4.75 4.99 0.196 0.497
白细胞介素-6 IL-6 2.05 1.98 2.31 2.37 1.96 2.62 3.13 0.127 0.448
肿瘤坏死因子-α TNF-α 4.33 5.21 5.47 4.71 5.50 5.10 4.32 0.388 0.301
56日龄 56 days of age
白细胞介素-1β IL-1β 15.25 18.51 18.66 18.74 15.86 18.07 16.69 0.579 0.370
γ-干扰素 IFN-γ 55.69 69.54 60.61 62.29 58.16 59.92 61.81 2.793 0.410
白细胞介素-6 IL-6 11.69 14.57 12.12 14.17 12.21 14.63 11.08 0.494 0.209
肿瘤坏死因子-α TNF-α 63.97 74.39 81.69 67.70 64.05 75.58 75.13 2.322 0.586

2.6 乙醇梭菌蛋白对黄羽肉鸡空肠黏膜屏障基因表达的影响

由表11可知,乙醇梭菌蛋白显著影响1~21日龄黄羽肉鸡空肠黏膜屏障基因表达(P<0.05),其中1.6%、2.4%、3.2%、4.0%和8.0%组空肠ZO-1 mRNA相对表达量显著高于对照组和0.8%组(P<0.05);1.6%组空肠Claudin1 mRNA相对表达量显著高于对照组和0.8%组(P<0.05);2.4%、3.2%和8.0%组空肠Nrf2 mRNA相对表达量显著高于对照组和0.8%组(P<0.05)。饲粮添加乙醇梭菌蛋白对56日龄黄羽肉鸡空肠黏膜屏障基因mRNA相对表达量无显著影响(P>0.05)。
表11 乙醇梭菌蛋白对黄羽肉鸡空肠黏膜屏障基因表达的影响

Table 11 Effects of Clostridium autoethanogenum protein on mucosal barrier gene expression in jejunum of yellow-feathered broilers

项目
Items		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
21日龄 21 days of age
闭锁小带蛋白-1 ZO-1 1.01b 1.00b 1.46a 1.54a 1.88a 1.62a 1.91a 0.079 0.013
闭合蛋白1 Claudin1 1.06bc 0.91c 2.33a 1.76ab 1.90ab 1.98ab 1.92ab 0.153 0.021
封闭蛋白1 Occludin1 1.07 0.98 1.35 1.51 1.51 1.23 1.23 0.087 0.130
核因子E2相关因子2 Nrf2 1.09bc 0.91c 1.42abc 1.82a 1.84a 1.61ab 1.84a 0.104 0.038
碱性氨基酸转运载体
rBAT		 1.02 1.17 1.33 1.02 1.40 0.90 0.99 0.087 0.177
56日龄 56 days of age
闭锁小带蛋白-1 ZO-1 0.83 0.58 0.83 0.41 0.64 0.46 0.69 0.085 0.115
闭合蛋白1 Claudin1 0.78 0.58 0.66 0.85 0.78 0.86 0.53 0.048 0.064
封闭蛋白1 Occludin1 1.65 1.46 1.06 1.39 1.11 1.46 1.09 0.170 0.267
核因子E2相关因子2 Nrf2 1.02 0.89 0.70 0.89 0.87 1.03 0.75 0.071 0.188
碱性氨基酸转运载体
rBAT		 0.37 0.53 0.60 0.45 0.35 0.49 0.55 0.066 0.356

2.7 乙醇梭菌蛋白对黄羽肉鸡粗蛋白质代谢率的影响

由表12可知,饲粮中添加乙醇梭菌蛋白显著影响黄羽肉鸡粗蛋白质代谢率(P<0.05),其中4.0%和8.0%组粗蛋白质代谢率显著高于对照组(P<0.05)。
表12 乙醇梭菌蛋白对黄羽肉鸡粗蛋白质代谢率的影响

Table 12 Effects of Clostridium autoethanogenum protein on crude protein metabolic rate of yellow-feathered broilers%

项目
Item		 饲粮乙醇梭菌蛋白水平 Dietary CAP level/% SEM P值
P-value
0(对照
Control)		 0.8 1.6 2.4 3.2 4.0 8.0
粗蛋白质代谢率
Crude protein
metabolic rate		 50.73b 52.76b 53.39b 53.62b 50.55b 61.70a 60.25a 1.037 0.036

3 讨论

3.1 乙醇梭菌蛋白对黄羽肉鸡生长性能的影响

本试验结果显示,饲粮中添加乙醇梭菌蛋白对黄羽肉鸡生长性能有改善作用,其中1~21日龄饲粮添加3.2%乙醇梭菌蛋白时生长性能最佳,且显著高于对照组;试验全期饲粮添加4.0%和8.0%乙醇梭菌蛋白生长性能显著高于对照组。这说明乙醇梭菌蛋白在黄羽肉鸡不同生长阶段适宜添加量存在差异。吴雨珊[10]在白羽肉鸡的试验中发现,添加1%和4%乙醇梭菌蛋白替代豆粕可以显著提高饲养前期的生长性能,试验全期添加2%~5%乙醇梭菌蛋白替代豆粕可以显著降低料重比。该结果与本试验结果相似,也与Zhu等[11]和Li等[12]在水产上使用乙醇梭菌蛋白替代鱼粉的研究结果相近。乙醇梭菌蛋白能够提高动物生产性能的原因可能是由于其蛋白质消化率高,易被动物机体消化吸收[13]。Li等[12]研究显示,乙醇梭菌蛋白替代鱼粉可以显著提高饲粮中的蛋白质利用率。也有研究发现,饲粮使用乙醇梭菌蛋白替代豆粕可以有效提高肉鸡血浆中总蛋白和白蛋白的含量[10]。本试验的结果与上述研究结果相似,结果也显示出乙醇梭菌蛋白替代豆粕能够显著提高粗蛋白质代谢率。

3.2 乙醇梭菌蛋白对黄羽肉鸡肝脏生化指标的影响

胆碱酯酶和碱性磷酸酶均是判断肝脏功能的重要指标[14-15],本试验结果显示,使用乙醇梭菌蛋白替代豆粕对黄羽肉鸡肝脏中的胆碱酯酶和碱性磷酸酶活性均无显著影响,这个结果与本试验的器官指数结果相呼应,说明乙醇梭菌蛋白与豆粕一样是安全的蛋白质原料,不会对黄羽肉鸡的器官造成负面影响。
T-SOD是一种能够将超氧阴离子催化位过氧化氢和氧气的金属结合酶,是体内重要的氧化自由基清除剂,它的活性是反映机体抗氧化水平的关键判断指标之一[16]。在肉鸡的试验中发现,添加乙醇梭菌蛋白可以提高饲养全期超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性,但这种作用并没有与乙醇梭菌蛋白的添加量呈线性变化[10]。在草鱼的试验中发现,使用乙醇梭菌蛋白替代豆粕会显著降低血浆中MDA含量,对SOD活性无显著影响,并且当乙醇梭菌蛋白的添加量达到10%会造成肝脏的损伤[4]。本试验结果显示,使用乙醇梭菌蛋白替代豆粕可以显著提高肝脏中T-SOD的活性,说明乙醇梭菌蛋白对提高黄羽肉鸡抗氧化水平具有一定的作用。有研究发现,使用乙醇梭菌蛋白可以显著提高Nrf2信号通路基因[Nrf2、铜锌超氧化物歧化酶(Cu/Zn-SOD)、HO-1和过氧化氢酶(CAT)]的表达水平[12],本试验中也发现,乙醇梭菌蛋白可以显著提高肠道黏膜中Nrf2的表达水平,推测乙醇梭菌蛋白可能是通过调控Nrf2信号通路改善机体抗氧化水平,具体是如何调控还需进一步深入研究。

3.3 乙醇梭菌蛋白对黄羽肉鸡免疫功能的影响

机体中的免疫炎症细胞因子在对抗有害物质和病原菌等方面发挥着重要的作用,检测细胞因子的含量被认为是评价动物机体免疫状况的重要指标[17-18]。TNF-α主要由巨噬细胞、单核细胞和T细胞分泌,参与调节炎症反应和细胞免疫反应的吞噬作用[19]。IL-1β和IL-6主要由单核细胞和T细胞分泌,具有调节免疫反应、炎症和抗体诱导的功能[20]。IL-1β、IL-6、IFN-γ和TNF-α均参与促炎反应,增强炎症反应,提高机体抗感染的能力[21]。本试验的结果显示,乙醇梭菌蛋白替代豆粕可以显著降低黄羽肉鸡血浆中免疫炎症因子IL-1β、IL-6和TNF-α的含量,说明乙醇梭菌蛋白对黄羽肉鸡的免疫功能起到一定的积极改善作用。研究发现,肉鸡饲粮使用乙醇梭菌蛋白替代豆粕后可以显著提高盲肠和血液中的短链脂肪酸浓度[10],而短链脂肪酸可以通过抑制脱乙酰基酶(HDACs)作用于单核血细胞和中性粒细胞,减少促炎因子(IL-2、IL-6、IL-8)的表达[22⇓-24],这可能是乙醇梭菌蛋白改善机体免疫功能的原因,具体机理还需要进一步研究。

3.4 乙醇梭菌蛋白对黄羽肉鸡空肠黏膜屏障关键基因表达的影响

空肠作为肠道中最长的部分,是整个胃肠道表面积最大的部位,其在营养吸收过程中占有非常重要的地位[25]。肠道黏膜屏障是肠道机械屏障中最重要的一环,它主要由紧密连接组成[26],紧密连接则是由跨膜蛋白(Occludin、Claudins等)、胞质蛋白闭锁小带蛋白(ZO)家族和细胞骨架结构共同构成的复合物[27-28]。许多研究发现,提高Occludin、Claudins、ZO-1在肠道黏膜的表达水平可以促进肠道屏障功能,改善肠道健康[29-30]。本试验研究结果发现,饲粮中使用乙醇梭菌蛋白可以显著提高空肠黏膜中ZO-1和Claudin1的表达水平,说明乙醇梭菌蛋白对肠道健康有一定的促进作用,分析原因可能是由于饲粮的颗粒质量对肠道黏膜屏障有显著的影响作用[31-32],而乙醇梭菌蛋白较豆粕更有助于提高颗粒饲料的硬度和耐久性[10];此外,乙醇梭菌蛋白的胃蛋白酶消化率高达90.4%[30],极易被肠道吸收,对肠道黏膜也能起到一定的保护作用。

4 结论

在本试验条件下,饲粮中加入乙醇梭菌蛋白可促进黄羽肉鸡生长,增强机体免疫功能,提高抗氧化能力和改善空肠黏膜屏障。因此,根据多项指标综合分析,黄羽肉鸡饲粮中乙醇梭菌蛋白适宜添加量为1.6%~4.0%。

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