AURANTII FRUCTUS IMMATURUS EXTRACT ON GROWTH, DIGESTIVE ENZYME ACTIVITY, ANTIOXIDANT CAPACITY, AND INTESTINAL FLORA OF BULLFROG (LITHOBATES CATESBEIANUS)
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摘要:
实验旨在研究枳实提取物对牛蛙(Rana catesbeiana)生长及肠道的调节作用。以牛蛙基础饲料作为对照组(CON), 在牛蛙基础饲料中添加枳实提取物作为添加组, 添加量分别为500、1000和2000 mg/kg (F500、F1000和F2000), 养殖实验持续56d。结果显示: (1)与 CON组相比, F1000与F2000组牛蛙的末均重(FBW)与增重率(WGR)显著提高(P<0.05)。(2)与CON组相比, F1000组肠道胰蛋白酶(Trypsin)、脂肪酶(LPS)、α-淀粉酶(AMS)、血清碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性均显著提高(P<0.05)。(3)枳实提取物添加组牛蛙血清中谷草转氨酶(ALT)、谷丙转氨酶(AST)活性均显著低于CON组(P<0.05)。(4)与CON组相比, 枳实提取物添加组牛蛙血清和肠道丙二醛(MDA)水平显著降低, 超氧化物歧化酶(SOD)活性显著升高(P<0.05)。枳实提取物添加组牛蛙肠道过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)、总抗氧化能力(T-AOC)水平显著提高(P<0.05)。(5)与CON组相比, 添加组牛蛙肠道菌群Shannon指数、Simpson指数显著降低(P<0.05)。在门水平上, 添加组牛蛙肠道中厚壁菌门(Firmicutes)相对丰度降低, 放线菌门(Actinobacteria)、变形菌门(Proteobacteria)和梭杆菌门(Fusobacteria)相对丰度升高。在属水平上, 枳实提取物添加组牛蛙肠道分枝杆菌属(Mycobacterium)、鲸杆菌属(Cetobacterium)、乳球菌属(Lactococcus)和红球菌属(Rhodococcus)的相对丰度高于CON组, 而多尔氏菌属(Dorea)和支原体属(Mycoplasma)则降低。综上所述, 饲料中适量添加枳实提取物可以促进牛蛙生长、提高肠道消化酶活性和抗氧化能力, 并改善牛蛙肠道菌群结构。建议牛蛙饲料中枳实提取物的最适添加量为1000 mg/kg。
Abstract:The experiment aimed to investigate the effects of Aurantii fructus immaturus (AFI) extract on the growth and intestinal regulation of bullfrog. Bullfrog basal feed was used as the control group (CON), and the additive groups received AFI extract at concentrations of 500, 1000, and 2000 mg/kg (F500, F1000, and F2000) for a 56d feeding trial. The results showed that: (1) the final body weight (FBW) and weight gain rate (WGR) in the F1000 and F2000 groups were significantly higher (P<0.05) than those in the CON group. (2) Compared with the CON group, the activities of intestinal trypsin, lipase, α-amylase, serum alkaline phosphatase, and acid phosphatase (ACP) in the F1000 group were significantly higher (P<0.05). (3) The serum alanine aminotransferase (ALT) and alanine aminotransferase (AST) activities in the AFI extract-added group were significantly lower than those in the CON group (P<0.05). (4) The serum and intestinal malondialdehyde (MDA) levels in the AFI extract-added group were significantly lower, and the superoxide dismutase (SOD) activity was significantly higher (P<0.05) compared with those in the CON group. Additionally, the levels of intestinal catalase (CAT), reduced glutathione (GSH), and total antioxidant capacity (T-AOC) were significantly increased (P<0.05) in the AFI extract addition groups. (5) Compared with the CON group, the Shannon index and Simpson index of the intestinal flora in the addition group were significantly decreased (P<0.05). At the phylum level, the relative abundance of Firmicutes (thick-walled bacteria) decreased, while Actinobacteria, Proteobacteria, and Fusobacteria increased in the additive group. At the genus level, the relative abundance of Mycobacterium, Cetobacterium, Lactococcus, and Rhodococcus were higher, whereas the relative abundance of Dorea and Mycoplasma were reduced in the AFI extract-added group. In summary, the moderate addition of AFI extract to bullfrog feed promote the growth, increase the activity of intestinal digestive enzymes and antioxidant capacity, and improve the structure of intestinal flora. It is recommended that the optimal amount of AFI extract in bullfrogs feed should be 1000 mg/kg.
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牛蛙(Rana catesbeiana)属于脊索动物门, 两栖纲, 蛙科, 蛙属, 因其优良的营养品质和良好的适应性, 被认为是国际市场上最具商业和经济价值的两栖动物之一[1, 2]。然而, 在高密度集约化养殖下, 牛蛙出现生长速度慢、饲料利用率低及对细菌病毒抵抗能力弱的情况。给行业造成了重大经济损失。中草药历史悠久, 来源丰富, 常用于预防和治疗疾病。又因其绿色无污染、易降解、无毒副作用等特性, 被作为添加剂应用于水产饲料中[3—5]。
枳实(Aurantii fructus immaturus, AFI)是芸香科柑橘属植物酸橙及其栽培变种或甜橙的干燥幼果。主产于湖南、江西、四川等地, 以湖南产量最大。枳实作为传统中药, 其生物活性成分主要包括黄酮类、挥发油类和生物碱类化合物[6]。其中最能体现枳实特征的活性成分是黄酮类化合物[7]。黄酮类化合物是药用植物中的重要成分, 具有刺激胃肠蠕动、抗氧化、保肝、抗炎等作用[8]。郑洋滨等[9]研究发现, 枳实黄酮可通过促进功能性消化不良大鼠分泌胃动素, 来改善其胃排空和小肠蠕动功能。而植物源黄酮类化合物作为饲料添加剂, 已经广泛应用于畜禽行业[10, 11]。研究表明, 在饲料中添加含黄酮类植物提取物, 提高了肉鸡[12]、肉牛[13]抗氧化能力。在水产行业, 在尼罗罗非鱼(Oreochromis niloticus)和非洲鲶(Clarias gariepinus)饲料中加入柠檬皮可以提高其抗氧化能力和免疫力[14] 。适量的黄酮类化合物可以显著提高石首鱼(Totoaba macdonaldi)的生长性能[15]。此外, 有研究表明[16], 柑橘类植物及其生物活性物质可以通过保护肠黏液层、刺激免疫系统、缓解肠道氧化应激、调节肠道菌群等多种途径保护肠道健康。鉴于枳实提取物具有的抗氧化、抗炎和免疫方面的作用, 以及牛蛙高密度集约化养殖中存在的生长缓慢和肠道病疾问题, 本实验以牛蛙为实验对象, 研究饲料中添加枳实提取物对牛蛙生长、抗氧化能力及肠道健康的影响, 以期为牛蛙健康高效养殖提供理论依据, 以及枳实提取物的开发和利用提供参考。
1. 材料与方法
1.1 实验材料及饲料
枳实提取物由张家界铭源生物科技有限公司提供, 主要生物活性成分为生物黄酮24.7%、多糖7.96%、多酚7.66%。实验牛蛙由湖南雪晖生物科技有限公司提供。以牛蛙基础饲料作为对照组(CON), 分别添加500、1000和2000 mg/kg (F500 、F1000和F2000)枳实提取物, 配置4组实验饲料。具体组成与营养水平见表 1。根据配方, 将所有原料粉碎过60目筛后, 混匀, 利用膨化机制成直径4.0 mm的膨化料, 烘干后, –20℃冰箱保存。
表 1 试验饲料组成及营养水平(干物质基础, %)Table 1. Formulation and proximate composition of the experimental diets (DM basis, %)项目Item 组别Group CON F500 F1000 F2000 鸡肉粉Chicken meal 30.00 30.00 30.00 30.00 豆粕Soybean meal 40.00 40.00 40.00 40.00 面粉Wheat flour 24.95 24.90 24.85 24.75 枳实提取物AFI extract 0.00 0.05 0.10 0.20 豆油Soybean oil 2.00 2.00 2.00 2.00 预混料Premixa 1.00 1.00 1.00 1.00 磷酸二氢钙Ca(H2PO4)2 1.50 1.50 1.50 1.50 氯化胆碱 Choline chloride 0.50 0.50 0.50 0.50 抗氧化剂Antioxidants 0.02 0.02 0.02 0.02 防霉剂Mould inhibitor 0.03 0.03 0.03 0.03 合计Total 100.00 100.00 100.00 100.00 营养水平Nutrient levelb 粗蛋白 Crude protein 40.42 40.62 40.58 40.67 粗脂肪 Crude lipid 6.69 6.56 6.62 6.66 注: a预混料组成及含量参照实验室前期研究[17]; b营养组成为测定值Note: aThe composition and content of the premix are based on the previous research conducted by our laboratory[17]; bProximate composition are measured values 1.2 实验设计与管理
实验设4个处理组, 包括1个对照组(CON)和3个添加组(F500、F1000、F2000), 每个处理组3个重复, 每个重复75只蛙, 初重为(22.99±0.01) g。在0.9 m×0.9 m×0.9 m规格的帆布池进行实验, 正式试验前用基础饲料驯食2周。在试验正式开始后, 每天以体重的3%—5%饲喂2次(7:00和17:00)。根据日气温和饲喂后10min剩余饲料量, 每4d调整饲喂量, 确保牛蛙饱腹。每10d停止饲喂1d, 并用虹吸法清洁池子。在为期56d的实验中, 每天记录牛蛙的死亡数和个体重。
1.3 样品采集
在取样前, 用间氨基苯甲酸乙酯甲磺酸盐(MS-222)麻醉牛蛙。从每个帆布池中随机取3只牛蛙收集肠道粪便样本, 于1.5 mL无酶管中, –80℃保存, 以备进行16S rRNA测序分析肠道菌群结构。
在禁食24h后, 对每个池的牛蛙进行计数和体重测量, 结合养殖周期的投喂量和死亡数据, 计算出末均重(FBW)、增重率(WGR)、成活率(SR)、饲料系数(FCR)及摄食量(FI)等各种生长性能指标。
从每个池中随机取3只牛蛙采用双毁髓法处理, 用注射器抽取血于1.5 mL的无酶管中, 4℃冰箱静置24h, 离心10min (3000 r/min, 4℃), 分离出血清, –80℃保存, 用于生化指标测定。采血后进行解剖, 获取肠组织样本, 去除粪便和其他残留物后, 将清洁的组织置于1.5 mL无酶管中, 并立即在液氮中冷冻, 用于后续检测肠道消化酶和抗氧化相关指标。
1.4 指标测定
饲料营养成分的测定 采用凯氏定氮法(GB/T 6432-2018)测定粗蛋白含量, 乙醚抽提法(GB/T 6433-2006)测定粗脂肪含量。
生长指标的测定
增重率(WGR, %)=[(Wt–W0)/W0]×100;
存活率(SR, %)=(Nt/N0)×100;
饲料系数(FCR)=F/(Wt–W0);
摄食量(FI, g/只)=F/[(Nt+N0)/2]。
式中, W0为牛蛙初始重; Wt为牛蛙终末重; N0为初始牛蛙数; Nt为总存活牛蛙数; F为牛蛙摄食饲料总量。
肠道消化酶、肠道抗氧化及血清生化指标的测定 肠道胰蛋白酶(Trypsin)、α-淀粉酶(AMS)、脂肪酶(LPS)、还原型谷胱甘肽(GSH)、过氧化氢酶(CAT)活性和总抗氧化能力(T-AOC), 血清和肠道超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量, 血清谷丙转氨酶(AST)、谷草转氨酶(ALT)、碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性均使用南京建成生物工程研究所的试剂盒测定, 测定步骤按说明书进行。
肠道菌群的测定 对肠道菌群样品进行总DNA的提取, 采用16S rRNA 扩增子测序法, 采用Illumina平台对PCR纯化样品中的DNA片段进行双端(Paired-end)测序, 后续分析使用microchat (“https://mineraltsai.shinyapps.io/shinymicrochat”)进行数据分析。
1.5 数据统计
试验数据在进行正态性及方差齐质性检验后, 采用SPSS 26.0软件进行单因素方差分析和LSD多重比较, 数据以“平均值±标准误”表示, P<0.05表示差异显著。
2. 结果
2.1 饲料中添加枳实提取物对牛蛙生长性能的影响
由表 2可知, 与CON组相比, F1000与F2000组的末均重和增重率显著提高(P<0.05), F500组无显著差异(P>0.05)。F500组成活率显著高于CON组(P<0.05), 且其余添加组均有所升高。与CON组相比, 在饵料系数上, 各添加组均有所下降, 但无显著差异(P>0.05)。采食量上各组间均无显著差异(P>0.05), 但随枳实提取物添加量的增加而上升。
表 2 枳实提取物对牛蛙生长性能的影响Table 2. Effects of dietary AFI extract on the growth performance of bullfrogs项目Item 组别Group CON F500 F1000 F2000 初均重
IBW (g)22.99±0.01 22.98±0.03 23±0.01 23±0.03 末均重
FBW (g)102.3±0.49a 100.55±0.68a 113.08±2.12c 106.38±0.26b 增重率
WGR (%)345.03±2.11a 337.5±3.35a 391.53±9.29c 362.24±0.82b 存活率
SR (%)71.56±1.54a 83.11±5.55b 76.00±2.03a 75.11±2.77a 饲料系数
FCR0.96±0.05 0.86±0.04 0.89±0.03 0.91±0.03 摄食量
FI (g/只)56.26±2.96 56.57±0.37 60.21±1.15 59.96±0.33 注: 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); 下同Note: In the same row, values with no letter or the same letter superscripts indicate no significant difference (P>0.05), while different small letter superscripts indicate significant differences (P<0.05); The same applies below 2.2 饲料中添加枳实提取物对牛蛙肠道消化酶活性的影响
由表 3可知, 与CON组相比, F1000与F2000组胰蛋白酶和脂肪酶活性显著提高(P<0.05), F500组无显著差异(P>0.05)。各添加组的α-淀粉酶活性均显著高于CON组(P<0.05), 且F1000组活性最高。
表 3 枳实提取物对牛蛙肠道消化酶的影响Table 3. Effects of dietary AFI extract on digestive enzyme activity of bullfrogs项目Item 组别Group CON F500 F1000 F2000 胰蛋白酶
Trypsin
(U/mg prot)101.09±3.07a 106.99±1.78a 164.99±2.72b 163.66±2.26b 脂肪酶LPS
(U/g prot)26.62±0.27b 24.63±0.32ab 41.59±1.41c 42.78±0.91c α-淀粉酶
AMS
(U/g prot)64.5±3.31a 77.1±1.04b 116.25±0.71c 73.02±0.36b 2.3 饲料中添加枳实提取物对牛蛙血清生化指标的影响
由表 4可知, 除F500组外, 其余添加组MDA含量均显著低于CON组(P<0.05)。与CON组相比, 各添加组牛蛙血清中SOD活性均显著升高(P<0.05), 其中F1000组活性最高。添加组ALT和AST活性均显著低于CON组(P<0.05), 最低值分别在F2000与F1000组。牛蛙血清中AKP、ACP活性均呈现先上升后下降趋势, F1000组显著高于其他各组(P<0.05)。
表 4 枳实提取物对牛蛙血清生化指标的影响Table 4. Effects of dietary AFI extract on serum biochemical indexes of bullfrogs项目Item 组别 Group CON F500 F1000 F2000 丙二醛MDA
(nmol/mL)2.9±0.42b 2.52±0.17b 1.22±0.01a 1.77±0.04a 超氧化物
歧化酶SOD
(U/mL)9.12±0.31a 10.14±0.04b 11.16±0.02c 10.05±0.35b 谷草转氨酶
ALT(U/L)34.03±1.04c 29.77±2.81b 23.71±2.15a 20.81±1.04a 谷丙转氨酶
AST(U/L)28.64±0.78d 19.79±0.28b 18.24±0.23a 24.09±0.45c 碱性磷酸酶AKP
(金氏单位/
100 mL)1.69±0.1b 1.79±0.09b 4.85±0.11c 1.37±0.01a 酸性磷酸酶ACP
(金氏单位/
100 mL )1.98±0.14a 2.49±0.12b 4.46±0.34d 3.06±0.18c 2.4 饲料中添加枳实提取物对牛蛙肠道抗氧化指标的影响
由表 5可知, 与CON组相比, 各添加组MDA含量均显著低于CON组(P<0.05)。F500和F1000 组牛蛙肠道SOD活性显著升高(P<0.05), F2000组无显著差(P>0.05)。添加组牛蛙肠道中GSH含量随枳实提取物添加量的增加而升高, 且均显著高于CON组(P<0.05)。添加组CAT、T-AOC水平均显著高于CON组(P<0.05), 分别在F1000组和F2000组达到最高。
表 5 枳实提取物对牛蛙肠道MDA含量及抗氧化指标的影响Table 5. Effects of AFI extract on intestinal MDA level and antioxidant indexes of bullfrogs项目 Item 组别 Group CON F500 F1000 F2000 丙二醛MDA
(nmol/mg prot)0.40±
0.01c0.23±
0.01b0.19±
0.01a0.19±
0.00a超氧化物歧化酶
SOD (U/mg prot)34.42±
0.59a41.83±
0.08b46.31±
0.61c33.89±
0.49a还原型谷胱甘肽
GSH (μmol/g prot)60.69±
4.45a81.82±
1.86b86.09±
1.12bc93.58±
1.91d过氧化氢酶
CAT (U/mg prot)55.10±
0.58a89.71±
0.93c101.20±
0.94d67.32±
0.19b总抗氧化能力
T-AOC (mmol/g prot)0.16±
0.01a0.23±
0.01b0.28±
0.01c0.29±
0.00c2.5 饲料中添加枳实提取物对牛蛙肠道菌群的影响
如图 1所示, PCoA分析结果表明, 2维图像可以充分解释枳实提取物引起的牛蛙肠道微生物群落差异(轴1解释度: 53.25%, 轴2解释度: 26.27%)。各添加组与CON组均无明显聚集, 表明在饲料中添加枳实提取物改变了牛蛙肠道菌群的结构。
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图 1 牛蛙肠道菌群Beta多样性(PcoA)分析Figure 1. Beta diversity principal coordinates analysis (PCoA) of intestinal microflora in bullfrogs牛蛙肠道菌群Alpha多样性分析结果如图 2 所示, F1000与F2000组Simpson指数显著低于CON组(P<0.05), F500组无显著变化(P>0.05)。与CON组相比, 添加组Shannon指数随添加量的增加而下降, 其中F2000组Shannon指数显著降低(P<0.05)。
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图 2 牛蛙肠道菌群Alpha多样性分析Figure 2. Alpha diversity analysis of intestinal microflora in bullfrogs如图 3所示在门水平上, 在牛蛙肠道中优势菌门主要为软壁菌门(Tenericute)、变形菌门(Proteobacteria)、梭杆菌门(Fusobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria)。与CON组相比, 添加组软壁菌门与厚壁菌门相对丰度降低, 放线菌门、变形菌门和梭杆菌门相对丰度升高。
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图 3 牛蛙肠道菌群在门和属水平上的相对丰度Figure 3. Relative abundance of intestinal microflora of bullfrogs at phylum and genus levels在属水平上, 分枝杆菌属(Mycobacterium)、鲸杆菌属(Cetobacterium)、多尔氏菌属(Dorea)、乳球菌属(Lactococcus)、支原体属(Mycoplasma)和红球菌属(Rhodococcus)是牛蛙肠道的主要菌属。与CON组相比, F500组鲸杆菌属相对丰度升高。F1000和F2000组占主导地位的菌属为分枝杆菌属与红球菌属, 此外, F2000组的优势菌属还包括乳球菌属, 且其相对丰度均高于CON组。各添加组中多尔氏菌属、支原体属的相对丰度均低于CON组。
3. 讨论
3.1 饲料中添加枳实提取物对牛蛙生长及肠道消化酶活性的影响
黄酮类化合物被认为是枳实的主要生物活性成分之一[18]。有研究表明, 来自植物的黄酮类化合物用作人类和动物的免疫刺激剂, 可增强抗氧化和抗炎特性, 并刺激免疫反应, 进而改善生长[19]。Lopes等[20]研究发现喂食柑橘精油的银鲶(Rhamdia quelen)较对照组表现出更好的生长性能。此外, Tejaswini等[21]研究表明饲料中添加富含黄酮类的柠檬皮提取物对南亚野鲮(Labeo rohita)的采食量和生长性能产生积极影响。在本研究中, F1000和F2000组牛蛙的末均重、增重率显著升高, 表明枳实提取物能够改善牛蛙的生长性能。枳实提取物的促生长作用可能与消化酶活性及肠道有益菌相对丰度有关。枳实中富含的黄酮类化合物可以促进肠胃运动, 消除积食[22], 并提高消化酶活性[23], 促进牛蛙对饲料的消化吸收, 从而提高其生长性能。在本研究中, 饲料中添加枳实提取物显著提高了牛蛙肠道消化酶活性, 其中, 胰蛋白酶和脂肪酶活性在F1000 和F2000组中显著提高。Shabana等[24]进行的一项类似研究表明, 饲喂柑橘皮提取物的卡特拉鲃(Catla catla)蛋白酶活性显著升高。在肥育猪[25]与仔猪[26]上也有类似研究。有研究推测, 黄酮类化合物可能促进肠道上皮细胞的生长, 从而提高肠道对营养物质的消化吸收, 间接刺激肠道消化酶的分泌[27]。而脂肪酶活性的升高可能是枳实提取物通过激活脂肪酶活性来刺激脂质的利用[28]。然而, Koochi等[29] 发现, 高剂量的柠檬皮提取物具有抗淀粉酶活性和抗葡萄糖苷酶活性。柠檬皮中的黄酮类化合物与淀粉酶和葡萄糖苷酶竞争性结合, 从而减少碳水化合物的消化。这可能是本研究中, 当枳实提取物添加量到达2000 mg/kg时淀粉酶活性降低的原因。综上, 在饲料中适量添加枳实提取物可以促进牛蛙胃肠蠕动, 并提高肠道消化酶活性, 来改善其生长性能。
3.2 饲料中添加枳实提取物对牛蛙血清生化的影响
血清生化指标在一定程度反映组织健康状况和机体代谢机能。血清谷草转氨酶(ALT)和谷丙转氨酶(AST)活性可以反映鱼类肝损伤的程度。本研究表明, 饲料中添加枳实提取物显著降低了牛蛙血清中ALT和AST活性。这与Wangkahar等[30]的研究结果相似, 表明饲料中补充枳实提取物可以降低牛蛙的肝脏损伤。有研究证实[31], 富含黄酮的水果提取物可以改善酒精诱导的氧化性肝损伤。酸性磷酸酶(AKP)和碱性磷酸酶(ACP)与机体非特异免疫密切相关, 具有破坏和抑制病原体的效果。在本实验中, 饲料中添加枳实提取物显著提高了牛蛙血清中AKP和ACP的活性, 表明枳实提取物对牛蛙具有免疫刺激作用。在用脱水柠檬皮[32]和柠檬桔梗果皮精油[33]等不同柑橘属植物喂养的不同鱼类中也有类似的结果。综上, 枳实提取物对牛蛙肝脏保护及免疫刺激具有积极影响。
3.3 饲料中添加枳实提取物对牛蛙血清及肠道抗氧化指标的影响
动物组织损伤与氧化损伤密切相关[34]。枳实中富含的黄酮类化合物等活性成分都具有较强的抗氧化能力[7]。其结构多样性使其能够与不同的靶点相互作用[35]。SOD和CAT是动物体内的主要抗氧化酶。GSH是细胞内重要的调节代谢物质, 是衡量健康的标志之一。T-AOC代表并反映了抗氧化酶和非酶系统对外部刺激做出反应和维持身体功能的代偿能力。MDA是脂质氧化反应的稳定产物, 具有一定的生物毒性。Kamboh等[10]发现, 橙皮苷显著提高了肉鸡血清中SOD活性, 降低了MDA含量, 从而改善其抗氧化状态。Rahman等[14]发现, 以柠檬皮为食的非洲鲶CAT和SOD活性及GSH含量均显著提高, 缓解了氧化应激损伤。Mohamed 等[36]对罗非鱼的研究中, 添加甜橙和柠檬提取物显著提高了罗非鱼CAT和SOD活性, 并刺激免疫反应, 改善其生长性能。除此之外, 饲料中添加橙皮苷后, 克氏原螯虾(Procambarus clarkii) T-AOC和SOD活性显著提高, MDA含量降低[37]。本试验有相似的结果, 在基础饲料中添加枳实提取物不仅能显著提高牛蛙血清及肠道SOD、肠道CAT、GSH和T-AOC的水平, 并显著降低血清及肠道MDA的含量。这表明添加枳实提取物显著提高了牛蛙的抗氧化能力。究其原因, 可能是枳实中含有黄酮类活性物质能中和牛蛙体内的自由基, 减少氧化应激, 缓解脂质过氧化、从而提高机体抗氧化能力。已有研究证明[38], 柑橘皮提取物具有清除自由基, 提高细胞活力和减少细胞内 ROS 存在的能力。柑橘柠檬精油可以通过防止脂质过氧化, 表现出抗氧化作用[39]。Xi等[40] 研究证实了柠檬皮可防止氧化应激并破坏反应性自由基。此外, 有研究表明, 在动物饲料中添加柑橘黄酮可以提高动物抗氧化能力, 改善动物的生长性能[41]。黄酮类化合物被认为是一类天然免疫刺激剂和抗氧化剂。并且在修复肠道屏障及调节肠道菌群方面有积极影响[42]。
3.4 饲料中添加枳实提取物对牛蛙肠道菌群的影响
动物肠道菌群具有多种生理作用[43], 包括抵抗病原体定植、外源性和内源性化合物的代谢转化、短链脂肪酸的产生及与宿主免疫系统的相互作用。肠道微生物稳态是整个代谢系统平衡的重要组成部分, 因此肠道菌群的生态失调也被认为是代谢疾病发展的重要因素。研究发现, 肠道菌群与黄酮类化合物之间存在相互作用, 肠道菌群将黄酮类化合物转化为其代谢物, 导致黄酮类化合物生物利用度增加, 而黄酮类化合物又通过抑制病原菌和刺激有益菌来调节肠道微生物群落的组成[44]。在本实验中, 与CON组相比, F1000与F2000组Shannon指数和Simpson指数下降, 表明枳实提取物的添加降低了牛蛙肠道菌群的多样性。这可能是因为厚壁菌门相对丰度降低导致, 而厚壁菌门的下降主要是由于支原体属的相对丰度下降。支原体是导致肠道相关疾病(如肠炎)的潜在致病细菌[45]。此外, F2000组乳球菌属相对丰度上升。而乳球菌属被证明可以促进鱼类生长发育, 提高免疫力和消化酶活性, 以及维持鱼类血液蛋白质和肠道菌群的平衡[46]。这说明添加枳实提取物一定程度缓解牛蛙肠道炎症, 并有利于肠道菌群的平衡。Zhao等[47]研究证实, 柑橘类黄酮可以降低奶牛的内毒素水平和全身炎症。放线菌门在维持肠道菌群稳态方面起着关键作用[48], 其产生的次级代谢产物可以对病原菌产生抑制和致死作用[49]。在本研究中, F1000与2000组放线菌门相对丰度增加, 主要是由于红球菌属和分枝杆菌属的相对丰度提高。红球菌属可分泌大量的活性酶, 充分利用有机化合物作为能源和碳源[50]。而分枝杆菌属菌群多为条件致病菌[51], 加之有益菌的增殖, 使得F1000与F2000组中并未暴发疾病[52] 。但具体作用机理还有待研究。此外, F500组鲸杆菌属相对丰度增加, 多尔氏菌属相对丰度下降。鲸杆菌属能够产生丁酸和维生素B12, 为鱼体提供营养物质, 调节肠道健康[53]。多尔氏菌属是血脂异常、Ⅱ型糖尿病和肥胖进展的潜在生物标志物[54]。这表明枳实提取物可以缓解脂质沉积, 改善牛蛙肠道健康。总体来说, 添加枳实提取物可以通过上调牛蛙肠道有益菌的相对丰度, 同时抑制有害细菌的生长, 来改善牛蛙肠道菌群结构, 从而促进肠道健康。
4. 结论
综上所述, 在本实验条件下, 饲料中适量添加枳实提取物可以促进牛蛙的生长、提高肠道消化酶活性、抗氧化能力, 同时改善牛蛙肠道菌群结构, 从而促进肠道健康。在本实验条件下枳实提取物在牛蛙饲料中的最适添加量为1000 mg/kg。
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图 1 牛蛙肠道菌群Beta多样性(PcoA)分析
Figure 1. Beta diversity principal coordinates analysis (PCoA) of intestinal microflora in bullfrogs
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图 2 牛蛙肠道菌群Alpha多样性分析
Figure 2. Alpha diversity analysis of intestinal microflora in bullfrogs
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图 3 牛蛙肠道菌群在门和属水平上的相对丰度
Figure 3. Relative abundance of intestinal microflora of bullfrogs at phylum and genus levels
表 1 试验饲料组成及营养水平(干物质基础, %)
Table 1 Formulation and proximate composition of the experimental diets (DM basis, %)
项目Item 组别Group CON F500 F1000 F2000 鸡肉粉Chicken meal 30.00 30.00 30.00 30.00 豆粕Soybean meal 40.00 40.00 40.00 40.00 面粉Wheat flour 24.95 24.90 24.85 24.75 枳实提取物AFI extract 0.00 0.05 0.10 0.20 豆油Soybean oil 2.00 2.00 2.00 2.00 预混料Premixa 1.00 1.00 1.00 1.00 磷酸二氢钙Ca(H2PO4)2 1.50 1.50 1.50 1.50 氯化胆碱 Choline chloride 0.50 0.50 0.50 0.50 抗氧化剂Antioxidants 0.02 0.02 0.02 0.02 防霉剂Mould inhibitor 0.03 0.03 0.03 0.03 合计Total 100.00 100.00 100.00 100.00 营养水平Nutrient levelb 粗蛋白 Crude protein 40.42 40.62 40.58 40.67 粗脂肪 Crude lipid 6.69 6.56 6.62 6.66 注: a预混料组成及含量参照实验室前期研究[17]; b营养组成为测定值Note: aThe composition and content of the premix are based on the previous research conducted by our laboratory[17]; bProximate composition are measured values 
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表 2 枳实提取物对牛蛙生长性能的影响
Table 2 Effects of dietary AFI extract on the growth performance of bullfrogs
项目Item 组别Group CON F500 F1000 F2000 初均重
IBW (g)22.99±0.01 22.98±0.03 23±0.01 23±0.03 末均重
FBW (g)102.3±0.49a 100.55±0.68a 113.08±2.12c 106.38±0.26b 增重率
WGR (%)345.03±2.11a 337.5±3.35a 391.53±9.29c 362.24±0.82b 存活率
SR (%)71.56±1.54a 83.11±5.55b 76.00±2.03a 75.11±2.77a 饲料系数
FCR0.96±0.05 0.86±0.04 0.89±0.03 0.91±0.03 摄食量
FI (g/只)56.26±2.96 56.57±0.37 60.21±1.15 59.96±0.33 注: 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); 下同Note: In the same row, values with no letter or the same letter superscripts indicate no significant difference (P>0.05), while different small letter superscripts indicate significant differences (P<0.05); The same applies below
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表 3 枳实提取物对牛蛙肠道消化酶的影响
Table 3 Effects of dietary AFI extract on digestive enzyme activity of bullfrogs
项目Item 组别Group CON F500 F1000 F2000 胰蛋白酶
Trypsin
(U/mg prot)101.09±3.07a 106.99±1.78a 164.99±2.72b 163.66±2.26b 脂肪酶LPS
(U/g prot)26.62±0.27b 24.63±0.32ab 41.59±1.41c 42.78±0.91c α-淀粉酶
AMS
(U/g prot)64.5±3.31a 77.1±1.04b 116.25±0.71c 73.02±0.36b
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表 4 枳实提取物对牛蛙血清生化指标的影响
Table 4 Effects of dietary AFI extract on serum biochemical indexes of bullfrogs
项目Item 组别 Group CON F500 F1000 F2000 丙二醛MDA
(nmol/mL)2.9±0.42b 2.52±0.17b 1.22±0.01a 1.77±0.04a 超氧化物
歧化酶SOD
(U/mL)9.12±0.31a 10.14±0.04b 11.16±0.02c 10.05±0.35b 谷草转氨酶
ALT(U/L)34.03±1.04c 29.77±2.81b 23.71±2.15a 20.81±1.04a 谷丙转氨酶
AST(U/L)28.64±0.78d 19.79±0.28b 18.24±0.23a 24.09±0.45c 碱性磷酸酶AKP
(金氏单位/
100 mL)1.69±0.1b 1.79±0.09b 4.85±0.11c 1.37±0.01a 酸性磷酸酶ACP
(金氏单位/
100 mL )1.98±0.14a 2.49±0.12b 4.46±0.34d 3.06±0.18c
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表 5 枳实提取物对牛蛙肠道MDA含量及抗氧化指标的影响
Table 5 Effects of AFI extract on intestinal MDA level and antioxidant indexes of bullfrogs
项目 Item 组别 Group CON F500 F1000 F2000 丙二醛MDA
(nmol/mg prot)0.40±
0.01c0.23±
0.01b0.19±
0.01a0.19±
0.00a超氧化物歧化酶
SOD (U/mg prot)34.42±
0.59a41.83±
0.08b46.31±
0.61c33.89±
0.49a还原型谷胱甘肽
GSH (μmol/g prot)60.69±
4.45a81.82±
1.86b86.09±
1.12bc93.58±
1.91d过氧化氢酶
CAT (U/mg prot)55.10±
0.58a89.71±
0.93c101.20±
0.94d67.32±
0.19b总抗氧化能力
T-AOC (mmol/g prot)0.16±
0.01a0.23±
0.01b0.28±
0.01c0.29±
0.00c
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