Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 8: 5297 - 5308

DOI: https://doi.org/10.12418/CJAN2023.489

RESEARCH PAPER

Effects of Black Soldier Fly Larvae Pulp on Serum Immune and Antioxidant Indices, Intestinal Morphology, and Disease Resistance of Hybrid Snakehead

  • XIE Yutong , 1, 2 ,
  • ZHU Xifeng 1, 3 ,
  • LI Shiyang 1, 4 ,
  • HUANG Yanhua 3, 4 ,
  • LIU Chun 4 ,
  • LU Huijie 1 ,
  • WANG Suitao 3 ,
  • WANG Guoxia , 1, **
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  • 1 Collaborative Innovation Center of Aquatic Sciences, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • 2 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
  • 3 Guangzhou Fishtech Biotechnology Co., Ltd., Guangzhou 510640, China
  • 4 Innovative Institute of Animal Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
**professor, E-mail:

*Contributed equally

Received date: 2023-02-23

  Online published: 2023-08-10

Fold

Abstract

This experiment was to investigate the effects of black soldier fly larvae pulp (BSFLP) on serum immune, and antioxidant indexes, intestinal morphology, intestinal flora, and disease resistance of hybrid snakehead. Four isonitrogen and isolipid diets were prepared by adding 0 (FM), 2% (BSFLP2), 4% (BSFLP4) and 6% (BSFLP6) of BSFLP wet matter (corresponding to the addition of 0, 9, 18, 27 g/kg BSFLP dry matter, respectively). A total of 420 snakeheads with an average body weight of (10.84±0.01) g were randomly divided into 4 groups with 3 replicates per group and 35 hybrid snakehead per replicate. The feeding period was 56 days. The results showed as follows: there were no significant differences in serum malondialdehyde content, lysozyme, catalase, alkaline phosphatase, superoxide dismutase, glutathione peroxidase activities and total antioxidant capacity among all groups (P>0.05). Compared with the FM group, the intestinal villus width and villus height in experimental groups had no significant differences (P>0.05), and the musclwe layer thickness and goblet cell number in BSFLP4 group were significantly increased (P<0.05). The Shannon index of intestinal contents in BSFLP4 group was significantly higher than that in FM group (P<0.05), and the species diversity was increased. The intestinal flora Sobs index, Chao index and Ace index in BSFLP6 group were significantly higher than those in BSFLP2 group (P<0.05). The mortality in BSFLP4 group was significantly lower than that in FM and BSFLP6 groups after Aeromonas schubertii infection (P<0.05). In conclusion, dietary addition of BSFLP can improve immune antioxidant capacity, regulate intestinal morphology and microbial structure of intestinal contents, increase microbial diversity, improve intestinal function, and enhance the ability to resist Aeromonas schubertii infection. It is recommended to add 18 g/kg BSFLP dry matter for the health of hybrid snakehead.

Key words： larvae pulp; hybrid snakehead; immune; antioxidant; intestinal structure; intestinal microbes; Aeromonas schubertii

Cite this article

XIE Yutong , ZHU Xifeng , LI Shiyang , HUANG Yanhua , LIU Chun , LU Huijie , WANG Suitao , WANG Guoxia . Effects of Black Soldier Fly Larvae Pulp on Serum Immune and Antioxidant Indices, Intestinal Morphology, and Disease Resistance of Hybrid Snakehead[J]. Chinese Journal of Animal Nutrition, 2023 , 35(8) : 5297 -5308 . DOI: 10.12418/CJAN2023.489

黑水虻(Hermetia illucens L.)可将食物垃圾、变质食物、牲畜粪便等有机废物降解转化为自身的蛋白质和脂肪[1]。其幼虫蛋白质含量高,氨基酸组成平衡,优于普通豆粉和骨粉[1],且富含月桂酸、棕榈酸、微量元素和抗菌肽[2-3]。水产饲料中添加黑水虻制剂如干幼虫粉、鲜幼虫、脱脂幼虫粉和酶解幼虫浆等[4],其能提高水生动物的增重率、饲料转化率和蛋白质效率,提高血清抗氧化能力、免疫力以及抗病力[5],改善肠道微生物结构,是具有良好发展前景的功能性饲料原料。
杂交鳢又称生鱼,是乌鳢(Channa maculate♀)和斑鳢(Channa argus )的杂交后代,耐低氧,抗病能力强,生长速度比乌鳢快20%,比斑鳢快50%[6],且个体大、肉质多,是我国特色淡水养殖鱼类之一,目前年产量超过50万t[7]。舒伯特气单胞菌(Aeromonas schubertii)为气单胞菌科气单胞菌属,革兰氏阴性短杆菌,广泛存在于海水、淡水和土壤中,是一种人-畜-鱼共患的条件性致病菌,可以引起动物和人类肠炎和败血症等多种疾病[8],同时也是引起鳢科鱼类内脏类结节病的主要病原菌之一,给养殖户带来严重经济损失[9]。
已有试验证明,黑水虻虫浆(black soldier fly larvae pulp,BSFLP)可以替代乌贼膏在杂交鳢饲料中起到诱食作用,还可以提高饲料利用率,促进生长[10]。本团队试验证明,饲料中添加BSFLP(9~27 g/kg)可以提高其增重率[11],但当前BSFLP对杂交鳢血清免疫、抗氧化指标和肠道形态、菌群和抗病力等的影响鲜见报道。因此,本试验在饲料中添加不同剂量的BSFLP饲养杂交鳢,研究BSFLP对杂交鳢血清免疫、抗氧化指标和肠道形态、菌群及抗病力的影响,为BSFLP在杂交鳢配合饲料中的应用提供理论依据和参考。

1 材料与方法

1.1 试验饲料

黑水虻鲜虫浆由广州某生物科技有限公司提供,由喂食餐厨垃圾的8日龄幼虫经粉碎磨浆而得,幼虫化学成分及含量为:干物质40%,粗蛋白质14%,粗脂肪15%,粗灰分5%,甲壳素2%[12]。
用鱼粉、豆粕和菜籽粕等蛋白质源和鱼油、豆油及大豆磷脂油为脂肪源配制了4种等蛋白质(45%)和等脂肪(10%)试验饲料,其组成及营养水平如表1所示,试验饲料氨基酸和脂肪酸组成分别见表2和表3,检测方法参考胡俊茹等[3]的报道。对照组基础饲料(FM)添加35%鱼粉,试验组分别添加2%(BSFLP2)、4%(BSFLP4)和6%(BSFLP6)BSFLP湿物质(分别对应9、18、27 g/kg BSFLP干物质)。微调鱼粉和大豆磷脂油等配平营养水平。试验饲料使用实验室双螺杆挤出机(SLX-80,华南理工大学机械制造厂)挤出,切成3 mm的颗粒,55 ℃干燥,-20 ℃保存至使用。
表1 试验饲料组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of experiment diets (DM basis) %

项目
Items		 饲料Diets
FM BSFLP2 BSFLP4 BSFLP6
原料Ingredients
鱼粉Fish meal 35.00 34.50 34.00 33.50
黑水虻虫浆Black soldier fly larvae pulp 0.90 1.80 2.70
豆粕Soybean meal 16.00 16.00 16.00 16.00
菜籽粕Rapeseed meal 8.00 8.00 8.00 8.00
花生麸Peanut meal 5.00 5.00 5.00 5.00
鸡肉粉Chicken powder 5.00 5.00 5.00 5.00
高精面粉High-precision flour 19.38 19.38 19.38 19.38
鱼油Fish oil 1.00 1.00 1.00 1.00
豆油Soybean oil 1.50 1.50 1.50 1.50
大豆磷脂油Soybean lecithin 3.50 3.30 3.00 2.70
磷酸二氢钙Ca(H2PO4)2 1.00 1.00 1.00 1.00
氯化胆碱Choline chloride 0.50 0.50 0.50 0.50
维生素C磷酸酯Vitamin C phosphate 0.10 0.10 0.10 0.10
维生素预混料Vitamin premix1) 0.20 0.20 0.20 0.20
矿物质预混料Mineral premix2) 0.50 0.50 0.50 0.50
微晶纤维素Microcrystalline cellulose 3.32 3.12 3.02 2.92
合计Total 100.00 100.00 100.00 100.00
营养水平Nutrient levels3)
粗蛋白质Crude protein 44.73 45.06 45.28 44.50
粗脂肪Crude lipid 10.96 9.82 10.03 9.87
粗灰分Ash 9.40 9.70 9.63 9.61
总能Gross energy/(kJ/g) 18.91 18.72 18.63 18.58

1) 维生素预混料为每千克饲料提供 Vitamin premix provided the following per kilogram of diets: VD 2 000 IU,VE 50 IU,VK 1 mg,VB1 1 mg,VB2 6 mg,胆碱 choline 1 000 mg,VB6 5 mg,烟酸 nicotinic acid 10 mg,VA 2 500 IU,生物素 biotin 0.14 mg,D-泛酸钙 D-calcium pantothenate 20 mg,叶酸 folic acid 1 mg,VC 50 mg。

2) 矿物质预混料为每千克饲料提供 Mineral premix provided the following per kilogram of diets: FeSO4·H2O 13 mg,ZnSO4·H2O 60 mg,NaCl 1 200 mg,MnSO4·H2O 32 mg,CuSO4·H2O 7 mg,KI 8 mg。

3) 营养水平为实测值。Nutrient levels were measured values.

表2 试验饲料氨基酸组成

Table 2 Amino acid composition of experimental diets g/100 g 饲料

项目
Items		 饲料Diets
FM BSFLP2 BSFLP4 BSFLP6
天冬氨酸Asp 4.07 3.64 3.51 3.53
谷氨酸Glu 8.11 7.53 7.42 7.31
丝氨酸Ser 1.90 2.13 2.07 1.98
组氨酸His 1.05 1.02 1.01 0.95
甘氨酸Gly 2.33 2.64 2.65 2.49
苏氨酸Thr 1.66 1.85 1.83 1.73
精氨酸Arg 2.54 3.02 2.97 2.83
丙氨酸Ala 1.96 2.49 2.49 2.25
酪氨酸Tyr 0.94 1.27 1.24 1.21
缬氨酸Val 2.54 2.71 2.78 2.51
蛋氨酸Met 0.78 0.84 0.81 0.76
苯丙氨酸Phe 1.86 2.11 2.14 2.01
异亮氨酸Ile 1.91 1.76 1.76 1.67
亮氨酸Leu 2.27 2.62 2.61 2.46
赖氨酸Lys 3.45 3.00 3.03 2.82
表3 试验饲料脂肪酸组成

Table 3 Fatty acid composition of experimental diets g/100 g总脂肪酸

项目
Items		 饲料Diets
FM BSFLP2 BSFLP4 BSFLP6
C12∶0 0.02 0.07 0.07 0.10
C14∶0 0.23 0.20 0.21 0.21
C16∶0 1.53 1.47 1.47 1.46
C16∶1n-9 0.23 0.20 0.21 0.20
C18∶0 0.44 0.43 0.43 0.43
C18∶1n-9 1.84 1.80 1.81 1.83
C18∶2n-6 2.57 2.51 2.42 2.37
C18∶3n-3 0.30 0.29 0.28 0.28
C20∶5 0.43 0.36 0.36 0.34
C22∶6n-6 0.32 0.36 0.37 0.35
饱和脂肪酸总量∑SFA 2.38 2.33 2.34 2.34
单不饱和脂肪酸总量∑MUFA 2.16 2.09 2.11 2.13
多不饱和脂肪酸总量∑PUFA 3.70 3.59 3.50 3.41
n-3多不饱和脂肪酸总量∑n-3 PUFA 0.30 0.29 0.28 0.28
n-6多不饱和脂肪酸总量∑n-6 PUFA 2.90 2.87 2.79 2.72

1.2 试验设计与饲养管理

养殖试验在广东省农业科学院动物科学研究所白云实验基地循环系统玻璃缸中进行,杂交鳢鱼苗购于广州市锦龙渔业有限公司。经过1周暂养后,选取初始体重为(10.84±0.01) g的健康杂交黑鱼420尾,随机分为4组,每组3个重复(缸),每缸35尾,圆柱形玻璃纤维缸大小为直径80 cm×高70 cm,容积为350 L。每天09:00和16:00各投喂1次,饲养56 d。养殖期间水质条件为:溶解氧含量>5 mg/L,水温24.0~29.0 ℃,pH 7.6~7.9,氨氮含量≤0.1 mg/L,亚硝酸盐含量≤0.02 mg/L。

1.3 舒伯特气单胞菌感染试验

饲养试验结束后,从每组随机挑选30尾杂交鳢,用舒伯特气单胞菌(采用稀释涂布法确定浓度后用生理盐水配制成菌悬液)于玻璃鱼缸(20 L)中进行感染试验,每次10尾鱼,重复做3次。感染方式为30 ℃条件下菌液浸泡2 h,菌液浓度为2×106 CFU/mL,浸泡2 h后鱼转移至另一个同样大小的鱼缸中养殖,用加热棒加热保持30 ℃水温,气石充氧保证溶氧充足,不投喂试验饲料,观察并记录杂交鳢0、24、48、96 和168 h内各组死亡情况,计算168 h(7 d)内的累积死亡率。

1.4 样品采集与指标测定

1.4.1 血清生化指标测定

投喂试验结束后,禁食24 h取样。从每个鱼缸中随机选取6条鱼,用1 mL无菌注射器鱼尾静脉取血,3 500 r/min离心(80-2台式电动离心机)10 min,取上清液保存于-80 ℃冰箱,分析血清抗氧化酶活性。血清样本采用商业试剂盒(南京建成生物工程研究所)测定,测定指标包括:丙二醛(malondialdehyde,MDA)含量、总抗氧化能力(total antioxidant capacity,T-AOC)及过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、溶菌酶(lysozyme,LZM)、碱性磷酸酶(alkaline phosphatase,AKP)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性。

1.4.2 肠道组织切片制作及测量

从每个鱼缸中随机取1尾鱼,取中肠1 cm于10%福尔马林溶液中固定,制备石蜡组织切片,经PAS染色后用PANNORAMIC全景切片扫描仪扫描,观察肠绒毛形态与结构。将处理好的肠切片用CaseViewer2.2软件在10倍光镜下观察测量黏膜厚度、绒毛高度,40倍光镜下测量肌层厚度。每张切片分别测定10个肠绒毛高度、绒毛宽度和肌层厚度,得到平均值作为测定数据进行分析。绒毛高度:肠绒毛基部至顶端的垂直距离;绒毛宽度:绒毛宽度共分3个部分,从肠绒毛基部、中部、尖端部分别测量再求其平均值;肌层厚度:肠的环肌层和纵肌层的厚度,即黏膜下层基部至浆膜层的垂直距离。用Image-Pro Plus 6.0软件计数杯状细胞数量。

1.4.3 肠道菌群

每个鱼缸随机取2尾鱼,取腹腔后肠样本和肠道内容物样本,分别装入2.0 mL的冷冻管中,立即置于液氮中,后保存在-80 ℃备用。提取肠道和肠道内容物总DNA并对其数量和质量进行检测和评估,正向引物:5'-ACTCCTACGGGAGGCAGCA-3';反向引物:5'-GGACTACHVGGGTWTCTAAT-3',测序区域为V3+V4,PCR扩增后用Illumina HiSeq 2500(北京百迈客生物科技有限公司)进行测序。在7%的相似度水平下进行操作分类单元(OTU)聚类分析和物种分类学分析。

1.5 数据统计与分析

试验数据均用平均值±标准误表示,采用SPSS 22.0软件中的one-way ANOVA程序进行单因素方差分析,采用Duncan氏法进行组间多重比较。先对数据进行方差齐性检验,若不满足方差齐性则采用Dunnett-T3检验法进行多重比较。P<0.05表示显著差异。

2 结果

2.1 血清免疫、抗氧化指标

由表4可知,与FM组相比,各试验组血清MDA含量无显著变化(P>0.05),但BSFLP4组显著高于BSFLP6组(P<0.05);各组血清AKP、CAT、LZM、SOD、GSH-Px活性和T-AOC无显著差异(P>0.05),但试验组AKP、CAT活性均高于对照组。
表4 黑水虻虫浆对杂交鳢血清免疫、抗氧化指标的影响

Table 4 Effects of BSFLP on serum immune and antioxidation indices of hybrid snakehead

项目
Items		 组别Groups P值
P-value
FM BSFLP2 BSFLP4 BSFLP6
碱性磷酸酶AKP/(U/L) 9.92±1.93 13.78±0.28 11.92±1.43 12.00±0.14 0.441
溶菌酶LZM/(mg/mL) 6.00±1.17 5.21±1.44 6.46±1.37 5.55±1.47 0.923
过氧化氢酶CAT/(U/mL) 28.15±3.99 38.99±2.56 42.01±5.13 34.18±5.35 0.210
超氧化歧物酶SOD/(U/mL) 12.82±2.48 13.24±1.28 12.89±0.78 14.55±0.82 0.835
总抗氧化能力T-AOC/(mmol/mL) 0.38±0.02 0.33±0.02 0.40±0.05 0.45±0.08 0.330
谷胱甘肽过氧化物酶GSH-Px/(U/mL) 214.67±13.36 193.33±4.81 178.67±21.35 201.78±5.24 0.291
丙二醛MDA/(mmol/mL) 7.69±0.81ab 6.48±0.53ab 8.61±1.05b 5.60±0.89a 0.035

同行数据肩标不同字母表示显著差异(P<0.05)。下表同。

In the same row, values with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 杂交鳢肠道形态

由表5可知,与FM组相比,各试验组绒毛宽度、绒毛高度无显著变化(P>0.05),但BSFLP2组绒毛宽度显著高于BSFLP4组(P<0.05);肌层厚度随BSFLP添加量增加,呈先增加后下降的趋势,BSFLP4组显著高于FM组(P<0.05);BSFLP4组杯状细胞数显著高于FM组和BSFLP2组(P<0.05)。
表5 黑水虻虫浆对杂交鳢肠道形态的影响

Table 5 Effects of BSFLP on intestinal morphology of hybrid snakehead

项目
Items		 组别Groups P值
P-value
FM BSFLP2 BSFLP4 BSFLP6
绒毛高度Villus height/μm 580.95±63.97 598.87±42.83 468.21±17.37 453.91±17.16 0.166
绒毛宽度Villous width/μm 104.72±8.90ab 114.18±5.24b 90.54±0.77a 105.09±3.93ab 0.042
肌层厚度Muscular thickness/μm 57.95±6.56a 86.91±1.29ab 141.89±40.38b 88.24±2.65ab 0.030
杯状细胞数
Number of goblet cells/个		 4 402.00±222.01ab 3 262.33±325.94a 5 655.50±562.50c 4 435.58±200.52bc 0.012
由图1可知,与FM组相比,BSFLP2~BSFLP6组肠道组织没有损伤,肌层厚度先增加后下降,BSFLP4组最高;随着BSFLP添加量增加,紫色小圆点数量呈先增加后降低的趋势,即杯状细胞数先增加后降低。
图1 各组杂交鳢的肠道形态结构(PAS染色)

Fig.1 Intestinal morphology and structure in hybrid snakehead of each group (PAS stain,100×)

2.3 杂交鳢肠道微生物

由表6可知,肠道内容物菌群试验组Sobs指数均增加,BSFLP4组Shannon指数显著高于FM组(P<0.05),物种多样性增加。后肠菌群Sobs指数、Chao指数和Ace指数呈上升趋势,物种丰度增加,BSFLP6组显著高于BSFLP2组(P<0.05)。各组肠道内容物/后肠微生物在门和属水平中的物种分布如图2所示,试验组肠道变形菌门相对丰度下降,厚壁菌门、假单胞菌属和乳球菌属相对丰度增加。
表6 黑水虻虫浆对杂交鳢肠道微生物的影响

Table 6 Effects of BSFLP on intestinal microbes of hybrid snakehead

项目
Items		 组别Groups P值
P-value
FM BSFLP2 BSFLP4 BSFLP6
肠道内容物Intestinal contents
Shannon指数Shannon index 1.94±0.17b 1.17±0.14a 2.86±0.16c 1.35±0.31ab 0.006
Simpson指数Simpson index 0.60±0.05 0.40±0.06 0.67±0.06 0.41±0.12 0.133
覆盖度Coverage/% 100.00±0.00 99.99±0.01 100.00±0.01 100.00±0.00 0.596
Chao指数Chao index 84.85±10.65 84.97±2.26 117.51±46.58 138.83±34.84 0.363
Ace指数Ace index 86.36±9.89 85.13±2.97 116.78±42.29 139.22±35.76 0.376
Sobs指数Sobs index 79.00±6.00 82.67±2.96 110.33±43.97 134.67±34.68 0.340
后肠Hindgut
Shannon指数Shannon index 1.48±0.28 1.76±0.21 1.44±0.04 1.18±0.34 0.361
Simpson指数Simpson index 0.51±0.12 0.60±0.05 0.39±0.14 0.29±0.07 0.124
覆盖度Coverage/% 99.99±0.01 100.00±0.01 100.00±0.01 99.99±0.01 0.648
Chao指数Chao index 59.14±5.47ab 29.75±6.75a 63.79±22.58ab 155.81±39.22b 0.048
Ace指数Ace index 61.06±5.26ab 31.37±8.05a 60.09±19.69b 156.84±37.75b 0.040
Sobs指数Sobs index 58.00±6.00ab 28.50±5.50a 48.50±25.50ab 144.67±31.47b 0.038
图2 肠道内容物/后肠微生物在门和属水平中的物种分布图

A: 肠道内容物微生物门水平的物种分布图;B: 肠道内容物微生物属水平的物种分布图;C: 后肠微生物门水平的物种分布图;D: 后肠微生物属水平的物种分布图。

Fig.2 Map of species distribution of intestinal contents/hindgut microbes at phylum and genera levels

A: map of species distribution of intestinal contents microbes at phylum level; B: map of species distribution of intestinal contents microbes at genera level; C: map of species distribution of hindgut microbes at phylum level; D: map of species distribution of hindgut microbes at genera level.

2.4 杂交鳢抗舒伯特气单胞菌感染的结果

由表7可知,用舒伯特气单胞菌感染后,试验组杂交鳢死亡率均低于FM组,BSFLP4组死亡率显著低于FM组和BSFLP6组(P<0.05)。
表7 黑水虻虫浆对杂交鳢抗舒伯特气单胞菌感染的影响

Table 7 Effects of BSFLP on ani-infection of Aeromonas schubertii of hybrid snakehead %

项目
Item		 组别Groups P值
P-value
FM BSFLP2 BSFLP4 BSFLP6
死亡率Mortality 80.00±8.16b 63.33±30.91ab 33.33±9.43a 76.67±4.71b 0.042

3 讨论

3.1 黑水虻虫浆对杂交鳢抗病力的影响

LZM和AKP是常见的判断机体免疫能力的指标[13],LZM是非特异性免疫因子和杀菌酶,其活性在一定程度上可以反映机体的免疫防护能力和机体免疫状态[14],AKP参与物质转运和离子分泌,是衡量免疫防御和消化吸收的重要指标[15]。氧化应激是由自由基产生易引起机体衰老和疾病的一种负面状态,动物机体内CAT、SOD、GSH-Px等活性决定了动物机体抗氧化能力[16-17]。本试验中添加BSFLP,杂交鳢血清AKP、CAT活性呈上升趋势,锦鲤(Cyprinus carpio haematopterus)[18]结果与上述结果一致,也是AKP、CAT活性呈上升趋势。本试验中,BSFLP4组舒伯特气单胞菌感染后的7 d累积死亡率降低,与许丰孟[19]报道的大口黑鲈饲料中添加适量BSFLP(10 g/kg干物质)减少嗜水气单胞菌感染累计死亡率结果相似,有效提高宿主抗病抑菌能力。分析原因可能与黑水虻含有甲壳素、月桂酸和抗菌肽有关,如Gopalakannan等[20]添加10 g/kg甲壳素显著提高鲤鱼血清LZM活性和白细胞数,提高抗嗜水气单胞菌能力。任秀芳等[21]添加5 g/kg壳聚糖饲养克氏原螯虾(Procambarus clarkii),血清酸性磷酸酶(ACP)和CAT活性显著高于对照组。本试验中,随着BSFLP添加量增加,饲料C12∶0含量从0.2 g/kg上升至1 g/kg,饲料实测值也增加。张文阁[22]研究表明,饲喂C12∶0和其衍生物能够减少沙门氏菌在小鼠肠道定植,减少细菌入侵的肠道损伤。体外抑菌试验中C12∶0对大肠埃希菌、金黄色葡萄球菌、肠炎沙门氏菌和鸡白痢沙门氏菌也表现出显著的抑菌效用[23]。另外也可能与BSFLP中含有抗菌肽有关,据报道,在黄鳝(Monopterus albus)饲料中添加600 mg/kg抗菌肽,其血清中SOD、AKP、ACP、CAT活性提高,抗嗜水气单胞菌能力增加[24]。本试验结果与之相似,添加BSFLP使杂交鳢免疫能力改善、抗舒伯特气单胞菌感染能力增强。

3.2 黑水虻虫浆对杂交鳢肠道组织形态的影响

肠道组织形态学被用来评估潜在的饲料中的负面影响,其中蛋白质来源将特别影响肠道绒毛的构建[25-26]。肠绒毛与营养物质的吸收和利用有关,绒毛高度、绒毛宽度与绒毛面积密切相关,绒毛面积减小可能导致营养吸收不良和肠道炎症[27]。Wang等[28]发现,在花鲈(Lateolabrax japonicus)饲料中用脱脂黑水虻干虫粉高达192 g/kg时,肠道的绒毛高度、绒毛宽度没有显著改变。在饲料中添加脱脂黑水虻干虫粉含量高达26.4或400 g/kg时,不会显著影响虹鳟(Oncorhynchus mykiss)的绒毛面积[29-30]。本试验中,添加BSFLP对杂交鳢肠道绒毛高度、绒毛宽度也没有显著影响,与上述报道结果一致。
肌层厚度影响肠道蠕动能力,肌层厚度有利于肠道内容物的运输和营养物质的吸收[31];杯状细胞是分泌型细胞,分泌物质起到的润滑与保护作用[32],杯状细胞数增加可增强肠黏膜的防御和屏障功能[33]。本试验中,试验组肌层厚度、绒毛杯状细胞数均高于对照组,说明添加BSFLP后能够促进杂交鳢对营养物质吸收和增强肠黏膜防御,BSFLP4组生长性能和抗舒伯特气单胞菌感染的能力提高也验证了这一点。这可能与黑水虻中甲壳素或其衍生物壳聚糖有关,本试验中添加9~27 g/kg BSFLP干物质,杂交鳢饲料中甲壳素含量为1.8~5.4 g/kg,低于报道的5 g/kg壳聚糖可显著提高草鱼(Ctenopharyngodon idella)中肠肌层厚度[34]和饲料中添加16 g/kg稀土壳聚糖螯合盐提高鲫鱼(Carassius auratus)肠道绒毛杯状细胞数[35],推测跟肉食性鱼类的甲壳素或壳聚糖用量低有关,有待更多试验验证。

3.3 黑水虻虫浆对杂交鳢肠道微生物的影响

肠道菌群在与宿主进化过程中形成共生关系,并在调节宿主的消化吸收、代谢和免疫应答等方面起着至关重要的作用,是评价肠道健康的关键指标[36]。肠道菌群物种的Alpha多样性分析可以反映微生物物种多样性和物种丰度。有研究表明,添加黑水虻虫油替代豆油后草鱼肠道菌群多样性增加[37];黑水虻虫粉显著增加鸡盲肠中挥发性脂肪酸产量,改善肠道菌群,提高盲肠微生物多样性[38-39]。本试验结果与上述报道类似,饲料添加BSFLP,杂交鳢肠道内容物菌群多样性得到改善。
变形菌门相对丰度增加是菌群失调、疾病风险的潜在标志[40]。本研究中,试验组肠道变形菌门相对丰度下降,厚壁菌门、假单胞菌属和乳球菌属相对丰度增加,说明添加BSFLP改善杂交鳢肠道菌群结构,可以减少疾病风险,增强肠道屏障功能。Xu等[41]也研究表明,饲料添加BSFLP饲养大口黑鲈,肠道抗氧化能力得到了加强和改善,致病菌(支原体属)相对丰度减少,本试验结果与之相似。据报道,黑水虻肠道具有种类丰富的酶、高活力的抗菌肽、独特的肠道菌群[42],厚壁菌门、拟杆菌门和变形菌门是黑水虻肠道的优势菌[43]。厚壁菌门可以通过乳酸发酵氧化糖,产生短链脂肪酸,抵御外界病原体入侵,增加肠道屏障功能[44],所以BSFLP改善鱼类肠道菌群可能是因为黑水虻的优势菌的作用。另外黑水虻改善动物肠道菌群结构与黑水虻富含甲壳素和抗菌肽有关,甲壳素在机体酶的作用下可降解为壳聚糖和壳寡糖[45],调节肠道微生物群组成。有试验证明,饲粮添加5 g/kg壳聚糖可以抑制马驹肠道大肠杆菌,增加双歧杆菌和乳酸杆菌相对丰度[46];饲料添加20 mg/kg壳寡糖虹鳟肠道假单胞菌属消失,气单胞菌属相对丰度均上升[47]。BSFLP中抗菌肽具抗菌谱较广,且有热稳定性[48],在饲料加工处理后保持对病原菌的杀灭作用[49]。

4 结论

本试验条件下,在饲料中添加BSFLP可以改善杂交鳢免疫、抗氧化能力,调节肠道形态和肠道内容物菌群结构,增加菌群多样性进而提高肠道功能,还可以增强抗舒伯特气单胞菌感染的能力。综合来看,杂交鳢饲料中添加18 g/kg BSFLP干物质为宜。

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