Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 8: 5036 - 5048

DOI: https://doi.org/10.12418/CJAN2023.467

RESEARCH PAPER

Effects of Rosemary Extract on Growth Performance, Serum Antioxidant Indices, Intestinal Morphology and Intestinal Flora of Broilers

  • WU Lifei , 1, 2 ,
  • GAO Wei , 1, 2, * ,
  • ZHAO Pengyu 1, 2 ,
  • SONG Weiguang 1, 2 ,
  • ZHAO Jian 1, 2
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  • 1 Hebei Plant Extraction Innovation Center Co.,Ltd., Handan 057250, China
  • 2 Hebei Province Plant Source Animal Health Products Technology Innovation Center, Handan 057250, China
*senior engineer, E-mail:

Received date: 2023-03-02

  Online published: 2023-08-10

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Abstract

This study was aimed to investigate the effects of different supplemental levels of rosemary extract in broiler diets on the growth performance, serum antioxidant indices, intestinal morphology and intestinal flora. A total of 576 healthy and similar weight 1-day-old male Arbor Acres (AA) broilers were randomly divided into 4 groups with 6 replicates in each group and 24 broilers per replicate. The control group (CON group) was fed a basal diet, and the experimental groups were fed adding 250 (UA250 group), 500 (UA500 group) and 750 mg/kg (UA750 group) rosemary extract experimental diets,respectively. The experiment lasted for 42 d. The results showed as follows: 1) compared with the CON group, the body weight (BW) of 21 and 42 days of age was significantly increased in the UA250 group, UA500 group and UA750 group (P<0.05). The average daily feed intake (ADFI) and average daily gain (ADG) of 22 to 42 days of age were significantly increased in the UA250 group (P<0.05). ADFI and ADG of 1 to 21 days and 22 to 42 days of age were significantly increased in the UA500 group (P<0.05). In the UA750 group, ADG of 22 to 42 days of age was increased significantly (P<0.05), the rate of death of 1 to 42 days of age was decreased significantly (P<0.05), and the rate of death of 1 to 21 days of age and 22 to 42 days of age decreased (P=0.090, P=0.085). There was no significant difference in feed/gain (F/G) between the groups (P>0.05). 2) Compared with the CON group, serum superoxide dismutase (SOD) activity was significantly increased in the UA750 group (P<0.05), and malondialdehyde (MDA) content in the UA250 group, UA500 group and UA750 group was decreased (P=0.096). There was no significant difference in serum glutathione peroxidase (GSH-Px) and glutathione S transferase (GST) activities between the groups (P>0.05). 3) Compared with the CON group, the ileal villus length (VH), duodenum, jejunum and ileal chorionic ratio (VH/CD) were significantly increased (P<0.05), and the duodenal, jejunal crypt depth (CD) was significantly reduced in the UA250 group (P<0.05). The ileal VH, duodenal and jejunal VH/CD were significantly increased (P<0.05).The duodenal and jejunal CD were significantly reduced in the UA500 group (P<0.05). The duodenal and jejunal VH and duodenal VH/CD were significantly increased (P<0.05) and duodenal CD was significantly reduced in the UA750 group (P<0.05). 4) Compared with the CON group, the Simpson and Shannon indexes in the UA750 group were significantly reduced (P<0.05), and the differences in ACE and Chao1 indexes among the groups were not significant (P>0.05). 5) Compared with the CON group, the relative abundance of Bacteroides in the UA250 group, UA500 group and UA750 group was significantly increased (P<0.05), the relative abundance of [Ruminococcus] torques group in the UA250 group and UA500 group was significantly increased (P<0.05), and the relative abundance of unclassified Clostridia_UCG_014 in UA250 group and UA750 group was significantly increased (P<0.05), the UA750 group had significantly higher relative abundances in unclassified Lachnospiraceae, uncultured rumen bacterium, and unclassified Oscillospiraceae (P<0.05).The relative abundances of Lachnoclostridium and Faecalibacterium in cecum of broilers in UA250 group, UA500 group and UA750 group were significantly reduced (P<0.05), and the relative abundance of unclassified Oscillospiraceae in UA250 group and UA500 group was significantly reduced (P<0.05). In conclusion, the addition of rosemary extract to feed can improve the growth performance, reduce the rate of death, enhance antioxidant capacity, improve intestinal morphology, optimize intestinal microbial environment, and ensure intestinal health. The optimal suitable addition level of rosemary extract in AA broiler feed is 500 mg/kg.

Key words： rosemary extract; broilers; growth performance; serum antioxidation; intestinal morphology; intestinal flora

Cite this article

WU Lifei , GAO Wei , ZHAO Pengyu , SONG Weiguang , ZHAO Jian . Effects of Rosemary Extract on Growth Performance, Serum Antioxidant Indices, Intestinal Morphology and Intestinal Flora of Broilers[J]. Chinese Journal of Animal Nutrition, 2023 , 35(8) : 5036 -5048 . DOI: 10.12418/CJAN2023.467

迷迭香(Rosmarinus officinalis L.),唇形科,迷迭香属,是一种天然香料植物[1]。迷迭香中含有丰富的迷迭香酸、鼠尾草酸、鼠尾草酚、精油、熊果酸和黄酮类等活性物质[2],具有抑菌、抗氧化、消炎、抗肿瘤等多种生物学功能,在动物生产应用上备受关注。有关迷迭香酸、鼠尾草酸、迷迭香精油在动物生产中的应用已有相关文献支撑,然而关于迷迭香中的熊果酸(UA)在畜牧生产上的研究鲜见报道,熊果酸是一种五环三萜类活性物质,可以从沙棘等多种常见植物中获得。黄酮类化合物存在于黄芩、木樨草等多种植物中,可分为黄酮类、黄酮醇类、异黄酮类和花色素类等[3]。研究发现,熊果酸具有抑菌、抗炎、抗氧化、保护心脏及肝脏、抗病毒等重要功效[4],而黄酮类化合物同样具有抗氧化、抗病毒以及抗肿瘤等功能,根据功效推测,以熊果酸和黄酮为主要成分的迷迭香提取物有可能是一种值得开发应用的饲料添加剂产品。因此,本试验以熊果酸和黄酮为主要成分的迷迭香提取物为研究对象,通过在基础饲粮中添加不同水平的上述迷迭香提取物,探究其对爱拔益加(AA)肉鸡的生长性能、血清抗氧化指标及肠道菌群的影响,期望研究结果能够为以熊果酸和黄酮为主要成分的迷迭香提取物在肉鸡生产中的应用提供理论依据。

1 材料与方法

1.1 试验动物和试验材料

试验动物:AA肉鸡,由河南汤阴全达家禽育种公司提供。
试验材料:试验用迷迭香提取物由河北某生物科技股份有限公司提供,主要活性物质含量为熊果酸≥50%,总黄酮≥15%,以粉剂形式拌料添加。

1.2 试验设计

试验采用单因子完全随机设计,选取1日龄健康且体重接近的AA雄性肉仔鸡576只,初重45.67~45.83 g,随机分为4组,每组6个重复,每个重复24只鸡。
对照组(CON组)饲喂玉米-豆粕型基础饲粮,试验组分别饲喂在基础饲粮中添加250(UA250组)、500(UA500组)、750 mg/kg迷迭香提取物(UA750组)的饲粮。试验期为42 d。基础饲粮为参照《鸡饲养标准》(NY/T 33—2004)和NRC(1994)进行配制的粉状配合饲料,其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 basal diets (air-dry basis) %

项目
Items		 含量Contents
1~21日龄
1 to 21 days
of age		 22~42日龄
22 to 42
days of age
原料Ingredients
玉米Corn 55.20 60.49
豆粕Soybean meal 32.70 25.80
玉米蛋白粉Corn gluten meal 5.00 5.00
豆油Soybean oil 3.00 5.30
磷酸氢钙CaHPO4 1.40 1.00
食盐NaCl 0.30 0.30
石粉Limestone 1.00 1.00
L-苏氨酸L-Thr(99%) 0.13 0.05
L-赖氨酸L-Lys(98%) 0.46 0.30
DL-蛋氨酸DL-Met(98%) 0.21 0.16
预混料Premix1) 0.60 0.60
合计Total 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.55 13.40
粗蛋白质CP 23.20 19.78
钙Ca 0.82 0.67
有效磷AP 0.56 0.50
赖氨酸Lys 1.44 1.15
苏氨酸Thr 0.97 0.78
蛋氨酸Met 0.56 0.47

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:植酸酶 phytase 750 U,VA 12 500 IU,VD3 2 600 IU,VE 50 IU,VK3 4 mg,核黄素 riboflavin 5 mg,泛酸 pantothenic acid 10 mg,烟酸 nicotinic acid 35 mg,VB12 0.03 mg,VB1 2.4 mg,VB2 5 mg,VB6 3 mg,胆碱 choline 110 mg,Mn 55 mg,Fe 75 mg,Zn 36 mg,Cu 8 mg,I 1.2 mg,Se 0.15 mg。

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

1.3 饲养管理

试验肉鸡采用3层笼养,每笼体积均为60 cm(长)×60 cm(宽)×45 cm(高),每笼8只鸡。试验前对鸡舍及器具进行充分的冲洗,并严格进行消毒处理。鸡舍采用电动智能加热器进行控温,采用连续光照、自然通风结合纵向负压通风,试验期舍内光照、相对湿度和温度根据AA肉鸡饲养管理要求进行控制,即入雏前24 h将鸡舍升温至33~36 ℃,此后温度每周降低2~3 ℃,直至保持在22~24 ℃,相对湿度控制在50%~60%。
定期打扫鸡舍,并按照正常免疫程序进行免疫。整个试验期鸡只自由采食和饮水。每天08:00、12:00和18:00记录鸡舍内的温度、湿度。试验期间每天观察记录试验鸡采食、饮水及健康状况,及时记录试验鸡死淘情况及耗料量。

1.4 测定指标及方法

1.4.1 生长性能指标

分别在1、21和42日龄以重复为单位对肉鸡进行称重,统计肉仔鸡1~21日龄、22~42日龄和1~42日龄采食量以及死淘数(包括死亡鸡只和淘汰的弱雏、个体差异大的鸡只),并计算平均日采食量(ADFI)、平均日增重(ADG)料重比(F/G)和死淘率。

1.4.2 血清抗氧化指标

在试验第43天时,每个重复随机选择1只与平均体重相近的肉鸡,翅下静脉采血10 mL,3 000 r/min离心10 min,收集血清于-20 ℃保存,待测。采用酶联免疫吸附试验(ELISA)法测定血清中超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽S转移酶(GST)活性以及丙二醛(MDA)含量,具体操作严格按照试剂盒说明书进行,以上试剂盒均购自南京建成生物工程研究所。

1.4.3 肠道形态

将采血后的24只肉鸡屠宰,分离十二指肠、空肠和回肠,取肠段中部约1.5 cm固定于4%多聚甲醛溶液。将固定好的肠段用乙醇脱水,二甲苯透明,石蜡包埋进行切片、苏木精-伊红(HE)染色。使用Image Pro Plus软件测量肠段绒毛高度(VH)、隐窝深度(CD),计算绒毛高度/隐窝深度比值(绒隐比,VH/CD)。

1.4.4 盲肠微生物测定

在无菌条件下收集盲肠内容物送至百迈客生物科技有限公司,基于PacBio测序平台,利用单分子实时测序(SMRT Cell)的方法对盲肠内容物菌群基因组进行测序以及生物信息学分析。

1.5 数据统计分析

采用SPSS 21.0统计软件进行单因素方差分析(one-way ANOVA),并用Duncan氏法进行多重比较,并采用回归分析模型中曲线估计程序,对迷迭香提取物添加水平与各检测指标进行线性和二次曲线分析,结果以平均值和均值标准误表示,P<0.05表示差异显著。

2 结果与分析

2.1 饲粮中添加迷迭香提取物对1~42日龄AA肉鸡生长性能的影响

由表2可知,与CON组相比,UA250组、UA500组、UA750组肉鸡在21和42日龄体重(BW)显著增加(P<0.05);1~21日龄,UA500组ADFI和ADG显著升高(P<0.05);22~42日龄,UA250组和UA500组ADFI显著升高(P<0.05),UA250组、UA500组和UA750组ADG显著升高(P<0.05);1~42日龄,UA250组、UA500组和UA750组ADFI和ADG显著升高(P<0.05);UA250组、UA500组、UA750组3个阶段1~21日龄、22~42日龄和1~42日龄F/G均无显著差异(P>0.05)。
表2 饲粮中添加迷迭香提取物对1~42日龄AA肉鸡生长性能的影响

Table 2 Effects of dietary rosemary extract on growth performance of 1 to 42-day-old AA broilers

项目
Items		 日龄
Days of age		 对照组
Control
group		 迷迭香提取物添加水平
Rosemary extract supplemental
level/(mg/kg)		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
250 500 750
1 45.83 45.70 45.67 45.76 0.101 0.957 0.654 0.886
体重BW/g 21 753.28b 772.59a 791.64a 777.77a 5.478 0.013 0.049 0.004
42 2 120.40b 2 246.11a 2 300.63a 2 250.26a 19.480 0.002 0.011 0.001

平均日采食量		 1~21 39.75b 41.63ab 42.44a 41.52ab 0.365 0.008 0.042 0.020

ADFI/g		 22~42 115.67c 121.94ab 126.42a 120.30bc 1.208 0.008 0.017 0.006
1~42 77.39b 81.54a 83.90a 80.80a 0.709 0.005 0.011 0.002

平均日增重		 1~21 33.69b 34.61ab 35.52a 34.86ab 0.244 0.007 0.118 0.022

ADG/g		 22~42 65.47b 70.17a 71.85a 70.12a 0.711 0.004 0.009 0.001
1~42 49.39b 52.39a 53.69a 52.49a 0.464 0.002 0.011 0.001
1~21 1.18 1.20 1.19 1.19 0.007 0.338 0.269 0.547
料重比F/G 22~42 1.77 1.73 1.76 1.72 0.009 0.094 0.648 0.707
1~42 1.57 1.56 1.56 1.54 0.005 0.121 0.921 0.681

死淘率		 1~21 4.39 4.16 4.16 1.36 0.580 0.090 0.693 0.409

Mortality rate/%		 22~42 3.80 2.14 3.59 0.69 0.584 0.085 0.698 0.734
1~42 8.21a 6.24a 7.63a 2.05b 0.767 0.010 0.967 0.312

同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同小写字母表示差异显著(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 small letter superscripts mean significant difference (P<0.05). The same as below.

与CON组相比,1~21日龄和22~42日龄,UA750组有降低死淘率的趋势(P=0.090、P=0.085);1~42日龄,UA750组死淘率显著降低(P<0.05)。
随饲粮中迷迭香提取物添加水平的增加,21和42日龄BW、3个阶段(1~21日龄、22~42日龄和1~42日龄)ADFI、2个阶段(22~42日龄和1~42日龄)ADG呈显著线性和二次曲线变化(P<0.05),1~21日龄ADG呈显著的二次曲线变化(P<0.05)。

2.2 饲粮中添加迷迭香提取物对42日龄AA肉鸡血清抗氧化指标的影响

由表3可知,与CON组相比,UA750组血清中SOD的活性显著提高(P<0.05);UA250组、UA500组和UA750组血清MDA含量有降低的趋势(P=0.096),分别降低了31.84%、24.41%和30.86%;UA250组、UA500组和UA750组血清中GSH-Px和GST活性均无显著差异(P>0.05)。
表3 饲粮中添加迷迭香提取物对42日龄AA肉鸡血清抗氧化指标的影响

Table 3 Effects of dietary rosemary extract on serum antioxidant indices of 42-day-old AA broilers

项目
Items		 对照组
Control
group		 迷迭香提取物添加水平
Rosemary extract supplemental
level/(mg/kg)		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
250 500 750
超氧化物歧化酶SOD/(U/mL) 121.94b 149.68ab 161.67ab 189.68a 9.650 0.015 0.476 0.121
谷胱甘肽过氧化物酶
GSH-Px/(U/mL)		 1 240.26 1 245.45 1 294.81 1 298.70 21.270 0.400 0.953 0.467
谷胱甘肽S转移酶GST/(U/L) 6.56 6.64 6.23 6.85 0.279 0.505 0.881 0.983
丙二醛MDA/(nmol/L) 5.12 3.49 3.87 3.54 0.315 0.096 0.107 0.175

2.3 饲粮中添加迷迭香提取物对42日龄AA肉鸡肠道形态的影响

由表4可知,与CON组相比,UA750组十二指肠VH显著增加(P<0.05);UA250组、UA500组和UA750组十二指肠CD均显著降低(P<0.05),十二指肠VH/CD显著增加(P<0.05),且CD和VH/CD随饲粮迷迭香提取物添加水平的增加呈显著线性和二次曲线变化(P<0.05)。
表4 饲粮中添加迷迭香提取物对42日龄AA肉鸡肠道形态的影响

Table 4 Effects of dietary rosemary extract on intestinal morphology of 42-day-old AA broilers

项目
Items		 指标
Indexes		 对照组
Control
group		 迷迭香提取物添加水平
Rosemary extract supplemental
level/(mg/kg)		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
250 500 750
绒毛高度
VH/μm		 845.30
±120.16b		 997.50
±113.38ab		 931.79
±51.57ab		 1 059.21
±65.00a		 31.845 0.022 0.266 0.303
十二指肠
Duodenum		 隐窝深度
CD/μm		 128.77
±25.11a		 77.02
±7.41b		 79.85
±7.63b		 99.66
±10.92b		 6.817 0.004 0.001 0.001
绒隐比
VH/CD		 6.61
±0.44c		 12.95
±0.70a		 11.71
±0.74ab		 10.68
±0.95b		 0.708 <0.001 <0.001 <0.001
绒毛高度
VH/μm		 692.07
±52.59b		 808.30
±123.63ab		 839.60
±23.38ab		 894.91
±135.59a		 29.511 0.041 0.396 0.151
空肠
Jejunum		 隐窝深度
CD/μm		 153.29
±31.27a		 88.35
±28.97b		 97.29
±27.73b		 113.37
±16.13ab		 8.577 0.036 0.005 0.012
绒隐比
VH/CD		 4.59
±0.57b		 9.58
±2.24a		 9.26
±2.82a		 7.98
±1.45ab		 0.681 0.039 0.008 0.013
绒毛高度
VH/μm		 407.01
±24.67b		 513.65
±77.16a		 513.91
±90.69a		 408.03
±35.16b		 19.974 0.047 0.011 0.046
回肠
Ileum		 隐窝深度
CD/μm		 74.13
±10.32		 60.79
±8.17		 74.95
±28.36		 58.48
±6.39		 4.061 0.357 0.529 0.793
绒隐比
VH/CD		 5.57
±0.83b		 8.61
±2.10a		 7.36
±2.13ab		 7.01
±0.61ab		 0.454 0.021 0.013 0.051
空肠形态学统计结果显示,与CON组相比,UA750组空肠的VH显著增加(P<0.05);UA250组、UA500组空肠的CD显著降低(P<0.05),同时空肠的VH/CD显著增加(P<0.05),且CD和VH/CD随饲粮迷迭香提取物添加水平的增加呈显著线性和二次曲线变化(P<0.05)。
与CON组相比,UA250组、UA500组回肠的VH显著增加(P<0.05),其中UA250组回肠的VH/CD显著增加(P<0.05),且VH随饲粮迷迭香提取物添加水平的增加呈显著线性和二次曲线变化(P<0.05),VH/CD呈显著二次曲线变化(P<0.05)。

2.4 饲粮中添加迷迭香提取物对42日龄AA肉鸡盲肠微生物的影响

2.4.1 盲肠内容物有效操作分类单元(OTU)数统计

由图1-A的Shannon指数稀释曲线可知,Shannon指数曲线趋于平缓,说明测序量足够大,表明OTU数不会再随着测序量的增加而增长,可真实反映群落各物种间的相对比例关系,可以进行数据分析。由图1-B可知,试验组与对照组盲肠内容物中共有的OTU数为430个。
图1 盲肠内容物有效操作分类单元数统计

A:Shannon指数的稀释曲线;B:不同添加水平迷迭香提取物的肉鸡肠道微生物菌群韦恩图。

Fig.1 Statistics of effective operational taxonomic unit of cecal contents

A:dilution curve of Shannon index;B:Venn diagram of intestinal microflora of broilers added with different supplemental levels of rosemary extract.

2.4.2 饲粮中添加迷迭香提取物对42日龄AA肉鸡盲肠微生物α多样性的影响

由表5可知,与CON组相比,UA750组Simpson和Shannon指数显著降低(P<0.05),且Shannon指数随饲粮迷迭香提取物添加水平的增加呈显著线性和二次曲线变化(P<0.05);UA250组和UA500组的Shannon指数最高,同时更接近于CON组水平。各组间ACE、Chao1指数无显著差异(P>0.05)。
表5 饲粮中添加迷迭香提取物对42日龄AA肉鸡盲肠微生物α多样性的影响

Table 5 Effects of dietary rosemary extract on cecal microbial alpha diversity of 42-day-old AA broilers

项目
Items		 对照组
Control
group		 迷迭香提取物添加水平
Rosemary extract supplemental
level/(mg/kg)		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
250 500 750
ACE指数ACE index 236.36 237.80 223.84 243.55 7.328 0.417 0.911 0.831
Chao1指数Chao1 index 242.28 235.49 216.76 248.69 6.646 0.132 0.993 0.362
Simpson指数Simpson index 0.960a 0.964a 0.964a 0.941b 0.001 <0.001 0.348 0.639
Shannon指数Shannon index 5.48a 5.56a 5.55a 5.08b 0.043 <0.001 0.001 <0.001

2.4.3 饲粮中添加迷迭香提取物对42日龄AA肉鸡盲肠微生物β多样性的影响

β多样性分析可以用于比较不同样品间在物种多样性方面存在的差异,在主坐标分析(PCoA)中,样本距离越接近,则表示物种组成结构越相似。由图2可知,饲粮中添加不同水平迷迭香提取物组和CON组分界清晰,即各试验组和CON组在β多样性方面有明显的区分,微生物群落组成有较大差异,而各组间菌群结构分布相对紧凑,相似度较高,群落构成差异较小。
图2 饲粮中添加迷迭香提取物对42日龄AA肉鸡盲肠微生物β多样性的影响

Fig.2 Effects of dietary rosemary extract on cecal microflora beta diversity of 42-day-old AA broilers

2.4.4 门水平和属水平相对丰度分析

由图3所示,在门水平中,厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)和变形菌门(Proteobacteria)相对丰度达90%以上;在属水平中,除未知菌属和其他菌属,4个组在属水平上相对丰度超过1%的有拟杆菌属(Bacteroides)、未分类的瘤胃细菌(uncultured rumen bacterium)、未分类梭状芽孢杆菌属UCG_014(unclassified Clostridia_UCG_014)、未分类毛螺菌科(unclassified Lachnospiraceae)、未分类瘤胃球菌科(unspecified Ruminococcaceae)、巴恩斯氏菌属(Barnesiella)、瘤胃球菌属torques群([Ruminococcus] torques group)、副拟杆菌属(Parabacteroides)、未分类颤螺旋菌科(unclassified Oscillospiraceae)、Lachnoclostridium、Alistipes、Negativibacillus、瘤胃菌属UCG_005、未分类梭状芽胞杆菌目vadinBB60群(unclassified Clostridia vadinBB60 group)、Incertae_Sedis、粪杆菌属(Faecalibacterium)、埃希氏杆菌属_志贺氏杆菌属(Escherichia_Shigella)、未分类柔膜菌纲RF39菌属(unclassified Mollicutes_RF39)、Butyricicoccus、理研菌属(Rikenella)。
图3 42日龄AA肉鸡盲肠微生物门水平和属水平上的组成

Fig.3 Composition of cecal microflora of 42-day-old AA broilers at phylum and genus levels

由表6可知,与CON组相比,UA250组、UA500组、UA750组肉鸡盲肠拟杆菌属的相对丰度显著提高 (P<0.05),UA250组、UA500组瘤胃球菌属torques群的相对丰度显著提高(P<0.05),UA250组、UA750组未分类梭状芽孢杆菌属UCG_014的相对丰度显著提高(P<0.05),UA750组未分类毛螺菌科、未分类的瘤胃细菌和未分类颤螺旋菌科的相对丰度显著提高 (P<0.05)。
表6 42日龄AA肉鸡盲肠微生物优势门和优势属相对丰度的影响

Table 6 Effects of relative abundance of dominant phylum and dominant genus in cecal microflora of 42-day-old AA broilers

项目
Items		 对照组
Control
group		 迷迭香提取物添加水平
Rosemary extract supplemental
level/(mg/kg)		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
250 500 750
门水平Phylum level
厚壁菌门Firmicutes 0.691b 0.773a 0.608c 0.701b 0.015 <0.001 0.247 0.008
拟杆菌门Bacteroidota 0.298ab 0.190c 0.342a 0.288b 0.014 <0.001 0.028 0.001
属水平Genus level
拟杆菌属Bacteroides 0.125c 0.143b 0.159ab 0.185a 0.007 0.006 0.646 0.011
未分类梭状芽孢杆菌属UCG_014
Unclassified Clostridia_UCG_014		 0.049c 0.109a 0.053bc 0.058b 0.005 <0.001 <0.001 <0.001
瘤胃球菌属torques群
[Ruminococcus] torques group		 0.013 2b 0.054 5a 0.053 5a 0.012 7b 0.004 <0.001 <0.001 <0.001
未分类毛螺菌科
Unclassified Lachnospiraceae		 0.065 3b 0.051 7b 0.052 5b 0.088 8a 0.005 0.008 0.065 0.079
未分类的瘤胃细菌
Uncultured rumen bacterium		 0.065 0b 0.048 1c 0.043 1c 0.128 0a 0.007 <0.001 0.032 0.010
Lachnoclostridium 0.037 4a 0.026 7b 0.025 3b 0.022 8b 0.001 <0.001 0.011 <0.001
粪杆菌属Faecalibacterium 0.024 0a 0.018 6b 0.013 8c 0.013 5c 0.001 <0.001 0.078 <0.001
未分类颤螺旋菌科
Unclassified Oscillospiraceae		 0.030 8b 0.017 0c 0.018 1c 0.050 1a 0.003 <0.001 0.004 0.002
UA250组、UA500组、UA750组肉鸡盲肠Lachnoclostridium、粪杆菌属相对丰度显著降低(P<0.05),UA250组、UA500组肉鸡盲肠未分类颤螺旋菌科相对丰度显著降低(P<0.05)。

3 讨论

3.1 饲粮中添加迷迭香提取物对1~42日龄AA肉鸡生长性能的影响

畜禽生长性能的提高会直接影响养殖场的经济效益,而生长性能常用ADFI、ADG和F/G等数据来衡量[5]。陈翔等[6]研究表明,肉鸡饲粮中添加熊果酸对肉鸡的生长性能没有显著影响,但可降低F/G。
同时有研究表明,饲粮中添加黄酮类物质可以显著改善肉鸡的生长性能[7]。郑旭等[8]研究结果显示,饲粮中添加槲皮万寿菊素(一种黄酮类物质)能够显著提高肉鸡的ADG,显著降低ADFI和F/G,从而改善其生长性能。本研究结果显示,UA250组、UA500组、UA750组均可以显著提高肉鸡21和42日龄BW以及22~42日龄肉鸡的ADG;UA500组显著提高1~21日龄肉鸡的ADFI和ADG;UA250组和UA500组显著提高22~42日龄肉鸡的ADFI。此外,本研究还发现,UA750组显著降低1~42日龄肉鸡的死淘率,同时有降低1~21日龄和22~42日龄肉鸡死淘率的趋势。本试验研究结果与陈翔等[6]、郑旭等[8]的研究结果均存在差异,初步分析原因可能为:熊果酸具有调节肠道菌群结构和数量、维持肠道菌群稳态平衡、改善肠道形态等生理功能[9-10],黄酮类化合物具有较强的体外抗氧化功能,可以有效防止饲粮的氧化,保证饲粮质量,而熊果酸与黄酮类物质之间可能具有协同增效作用,两者共同发挥作用,提高肉鸡的ADFI和ADG,进而提高肉鸡的生长性能。

3.2 饲粮中添加迷迭香提取物对42日龄肉鸡血清抗氧化指标的影响

熊果酸是一种具有强抗氧化性能的天然抗氧化剂,可以有效清除超氧阴离子和羟自由基[11-12]。张海全等[13]研究发现,苦丁茶熊果酸可以显著提高由四氯化碳(CCl4)引起的肝脏损伤小鼠肝脏组织SOD和GSH-Px活性,同时降低肝脏组织中MDA含量。陈翔等[6]研究表明,熊果酸能够有效降低肉鸡血清中的MDA的含量,提高SOD的活性。张毅峰[14]研究表明,饲粮中添加槲皮万寿菊素可以显著提高肉鸡血清SOD和GSH-Px活性以及T-AOC,同时显著降低MDA含量。本研究结果表明,UA250组、UA500组、UA750组有降低血清MDA含量的趋势,UA750组可显著提高血清中SOD活性,此结果与上述研究结果基本一致,但本研究中血清GSH-Px和GST活性无显著影响,分析原因可能由于与熊果酸及黄酮抗氧化机制有关,同时与使用剂量、肉鸡的品种以及饲养环境有关。有关熊果酸与黄酮联合使用对肉鸡抗氧化性能的机制还未见系统报道,故仍需要大量的研究证实。综上可知,迷迭香提取物有可以通过增强体内抗氧化酶的活性来提高AA肉鸡的抗氧化能力。

3.3 饲粮中添加迷迭香提取物对42日龄肉鸡肠道形态的影响

肠道绒毛是肠道的重要组成部分,其形态结构直接影响着机体对营养物质的消化和吸收,而肠道VH、CD、VH/CD是评价肠道消化吸收能力的重要指标[15]。关于熊果酸在肉鸡上的相关报道较少,而有研究表明黄酮类物质具有改善肉鸡肠道健康的功效,马绍航[16]研究表明,山银花黄芩提取物(一种黄酮类物质)能够显著提高肉鸡肠道VH和VH/CD。本研究结果表明,UA250组、UA500组、UA750组均可以不同程度提高十二指肠、空肠、回肠的VH和VH/CD,同时降低十二指肠、空肠的CD,本试验与马绍航[16]的研究结果基本一致,推测可能是由于熊果酸可以通过提高肠上皮细胞间紧密连接蛋白表达、减少上皮细胞凋亡[17],从而增加小肠绒毛高度,提高VH/CD;也可能是由于熊果酸调节了肠道菌群结构,给小肠绒毛发育提供了良好环境,进而刺激肠道组织形态发育;同时也可能与黄酮类化合物能够通过增加细胞内碱性磷酸酶活性及肠道上皮细胞DNA的合成进而促进肠道发育有关[18]。综上可知,富含熊果酸与黄酮的迷迭香提取物能够有效改善肉鸡肠道组织结构,促进肠道对营养物质消化与吸收,从而促进其生长。

3.4 饲粮中添加迷迭香提取物对42日龄肉鸡盲肠微生物的影响

盲肠是机体水分调节、尿素循环和碳水化合物发酵的最重要场所,对肉鸡肠道营养健康至关重要[19]。研究表明,肠道菌群的多样性和丰富度与肉鸡发生疾病的概率息息相关[20-21]。一般采用Shannon和Simpson指数判定菌群多样性,通常菌群多样性指数越高,就越难破坏菌群区系的平衡。而Chao1指数和ACE指数反映菌群丰富度,该值越大,表明菌群丰富度和多样性越大。本研究发现,UA750组Simpson指数显著降低,然而该组Shannon指数显著低于对照组相应指标,可能与肉鸡个体差异有关。由此推测,剂量过大的迷迭香提取物可能会改变菌群均匀度,提高菌群丰富度。β多样性结果表明,饲粮中添加迷迭香提取物可以影响肉鸡盲肠的微生物区系,与CON组相比微生物群落组成有较大差异。田力等[10]研究表明,熊果酸能够改善肿瘤小鼠的肠道菌群结构,具有恢复肿瘤模型小鼠肠道菌群紊乱的作用。马浩然等[9]研究表明,熊果酸可能通过调节肠道菌群结构和数量,修复肠黏膜屏障,改善肠道微生态,以缓解酒精性肝脏损伤。Cuervo等[22]研究发现,黄酮类化合物可以通过与微生物互作进而影响体内微生物稳态,同时可以改善不合理饮食导致的肠道微生态失调。
厚壁菌门与拟杆菌门主要通过酵解动物体内的营养成分而参与机体新陈代谢。相比于拟杆菌门,厚壁菌门能产生更多的短链脂肪酸(SCFAs)和脂质产物,促进机体脂质的沉积,同时厚壁菌门的细菌还能参与改善宿主的健康调节[23⇓-25]。研究表明,动物盲肠中拟杆菌门相对丰度增加表明盲肠中参与SCFAs代谢途径的菌群发达,促进饲粮营养物质尤其是饲粮纤维的利用,进而为宿主提供能量和氨基酸等营养物质[26]。Polansky等[27]研究表明,SCFAs在为肠道上皮细胞提供能量和抑制病原菌产生致病因子等方面发挥重要的作用。本试验结果表明,在门水平上,迷迭香提取物能够显著提高厚壁菌门的相对丰度,一定程度上提高拟杆菌门的相对丰度。
毛螺菌科能发酵膳食纤维产生的SCFAs,具有降低肠道炎症反应、提高黏膜屏障等功能,同时研究表明,毛螺菌科的相对丰度与肉鸡生长性能之间存在显著的正相关关系[28-29]。拟杆菌属是发酵产生SCFAs的主要菌群之一,同时在含氮物质的利用以及胆汁酸和其他类固醇的生物转化中也具有重要作用[30]。Guglielmi等[31]研究发现,肠道瘤胃菌科细菌可能通过激活免疫系统来增强抗肿瘤反应。瘤胃球菌科同样是SCFAs的产生者,在降解多种多糖和纤维中发挥着关键作用[32]。梭状芽孢杆菌属是碳水化合物发酵产生SCFAs的主要菌群,同时可增强机体免疫能力,通过释放生长因子、增加肠道黏膜能量以及增强免疫力等方式在肠道健康中发挥着重要作用[33]。Oscillospira在肠道同样可产生丁酸为主的各种SCFAs,进而促进鸡肠上皮细胞增殖[34]。在本研究中,UA750组可以显著提高未分类毛螺菌科、拟杆菌属、未分类的瘤胃细菌、瘤胃球菌属torques群、未分类梭状芽孢杆菌属UCG_014以及未分类颤螺旋菌科的相对丰度。由此推测,迷迭香提取物可以通过调节盲肠中的微生物,优化肠道微生物环境,促进肉鸡的肠道健康,为肉鸡的健康生长提供了保障。
研究表明,Lachnoclostridium丰度的升高会引起炎症因子的高表达[35]。本试验结果显示,饲喂迷迭香提取物可显著降低Lachnoclostridium的相对丰度,从而减少炎症反应的发生。粪杆菌属细菌广泛存在人体肠道中,在下一代益生菌或生物治疗药物方面有潜在开发前景[36]。从试验结果可知,饲喂迷迭香提取物可显著降低粪杆菌属相对丰度,由此推断AA肉鸡肠道中粪杆菌属相对丰度对迷迭香提取物可能比较敏感。
综上所述,饲粮中添加迷迭香提取物可促进盲肠有益菌生长,抑制部分有害菌增殖,优化肉鸡肠道菌群组成。

4 结论

饲粮中添加迷迭香提取物可以提高AA肉鸡的生长性能、血清抗氧化能力,改善盲肠形态,优化盲肠菌群结构。综合分析,1~21日龄和22~42日龄AA肉鸡均以饲粮中添加500 mg/kg迷迭香提取物为宜。

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