Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 11: 7780 - 7795

DOI: https://doi.org/10.12418/CJAN2025.632

RESEARCH PAPER

Effects of Plant Protein Replacing Fish Meal and Adding Fermented Hermetia illucens L. in Diets on Serum Biochemical Indices, Antioxidant Capacity and Immune Function of Litopenaeus vannamei

  • WANG Menghua , 1, 2 ,
  • ZHOU Meng 1 ,
  • HUANG Wenqing 2 ,
  • XU Fengmeng 2 ,
  • HUANG Yanhua , 1, *
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  • 1 Innovative Institute of Animal Healthy Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
  • 2 Guangzhou Fishtech Fisheries Science and Technology Co., Ltd., Guangzhou 510640, China
*professor, E-mail:

Received date: 2025-04-18

  Online published: 2025-11-14

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Abstract

This experiment was conducted to study the effects of plant protein replacing fish meal and adding fermented Hermetia illucens L. in diets on serum biochemical indices, antioxidant capacity and immune function of Litopenaeus vannamei. The soybean meal and peanut meal were used to replace 0, 16.67%, 33.33%and 66.67% fish meal in the basal diets, and the basal diets were supplemented with 0, 4%, 8% and 16% fermented Hermetia illucens L., respectively, to make 4 kinds of iso-nitrogen and iso-lipid experimental diets, which were labeled N0, N4, N8 and N16, respectively. A total of 560 Litopenaeus vannamei with body weight of (2.86±0.01) g were randomly divided into 4 groups (N0, N4, N8 and N16 groups) with 4 replicates per group and 35 shrimps per replicate. The experimental period lasted for 8 weeks. The results showed as follows: 1) the contents of low-density lipoprotein cholesterol and cholesterol and activities of alanine aminotransferase and aspartate aminotransferase in serum of N4, N8 and N16 groups were significantly lower than those of N0 group (P<0.05), the contents of total protein and globulin in serum of N4, N8 and N16 groups were significantly higher than those of N0 group (P<0.05), the serum albumin content of N4 and N8 groups was significantly higher than that of N0 group (P<0.05), and the serum triglyceride content of N4 and N8 groups was significantly lower than that of N0 group (P<0.05). 2) The activities of peroxidase, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver of N4, N8 and N16 groups were significantly higher than those of N0 group (P<0.05), the activities of alkaline phosphatase and lysozyme in liver of N4 and N8 groups were significantly higher than those of N0 group (P<0.05), and the liver acid phosphatase activity of N4 and N8 groups was significantly lower than that of N0 group (P<0.05). 3) The serum malondialdehyde content of N8 and N16 groups was significantly lower than that of N0 group (P<0.05), and the serum SOD activity of N8 group was significantly higher than that of N0 group (P<0.05). 4) The gene relative expression levels of Dorsal-1, Relish, Cru, Pen3, SOD and GSH-Px in liver of N4 and N8 groups were significantly higher than those of N0 group (P<0.05). 5) After challenged with Vibrio parahaemolyticus Ag01, the mortalities of Litopenaeus vannamei in N0, N4, N8 and N16 groups after 7 days were 53.85%, 25.64%, 23.08% and 51.28%, respectively. In conclusion, adding 4% and 8% fermented fermented Hermetia illucens L. in diets can not only alleviate the negative effects of plant protein replacing fish meal on the health of Litopenaeus vannamei, but also enhance its antioxidant capacity and immune function. Under the conditions of this experiment, the level of plant protein replacing fish meal should not exceed 33.33%, and the appropriate supplemental level of fermented Hermetia illucens L. is 4% to 8%。

Key words： Litopenaeus vannamei; fermented Hermetia illucens L.; plant protein; fish meal; immune function

Cite this article

WANG Menghua , ZHOU Meng , HUANG Wenqing , XU Fengmeng , HUANG Yanhua . Effects of Plant Protein Replacing Fish Meal and Adding Fermented Hermetia illucens L. in Diets on Serum Biochemical Indices, Antioxidant Capacity and Immune Function of Litopenaeus vannamei[J]. Chinese Journal of Animal Nutrition, 2025 , 37(11) : 7780 -7795 . DOI: 10.12418/CJAN2025.632

鱼粉因蛋白质含量高、含有丰富且均衡的必需氨基酸和多不饱和脂肪酸、抗营养因子水平低、适口性好等优点,一直是水产饲料中重要的优质蛋白质源[1]。随着水产养殖行业的发展,鱼粉需求量越来越大,然而由于海洋动物资源有限、环保要求及可持续发展需要,鱼粉产量急剧下降,且价格居高不下[2]。为保护海洋渔业资源、促进水产行业的绿色可持续发展,以及应对鱼粉供不应求的市场局面,寻找替代鱼粉的其他蛋白质源,开发低鱼粉、无鱼粉饲料已成为国内外饲料界的一个重要课题[1]。植物蛋白质原料来源广、价格低,是理想的鱼粉替代品[3]。但植物蛋白质存在抗营养因子水平高、氨基酸不平衡和纤维素含量高等问题,其替代鱼粉在水产养殖上仍存在一些争议[4-6]。
黑水虻幼虫在水产饲料及养殖中的应用研究已较多,如大黄鱼(Larimichthys crocea)[7]、乌鳢(Channa argus)[8]、罗氏虾(Macrobrachium rosenbergii)[9]、黄颡鱼(Pelteobagrus fulvidraco)[10]、罗非鱼(Oreochromis niloticus )[11-12]、中华鳖(Pelodiscus sinensis)[13]、凡纳滨对虾(Litopenaeus vannamei)[14]、黑斑蛙(Pelophylax nigromaculatus)[15]、大西洋鲑鱼(Salmo salar)[16]和鹦鹉鱼(Amphilophus)[17]等。然而,无论是黑水虻鲜虫、干虫、虫油还是脱脂黑水虻,主要研究还是集中在粗蛋白质和粗脂肪等常规营养成分方面,对黑水虻幼虫功能性物质的挖掘和深加工产品的应用研究还很缺乏。黑水虻幼虫含有甲壳素[18]、抗菌肽[19]和月桂酸[20]等多种生物活性物质,虫浆在乌鳢[21]和大口黑鲈(Micropterus salmoides)[22]饲料中应用可以提高机体的抗氧化能力和免疫力,同时黑水虻虫粉和脱脂虫粉在凡纳滨对虾[23-24]和大口黑鲈[25]饲料中应用均可提高其抗氧化能力和免疫力,这与其含有的抗菌肽[26]和壳聚糖[27]等活性物质密切相关。黑水虻的发酵和酶解等加工处理可进一步提升饲料原料品质,不仅可以延长饲料原料保质期[28],而且可将大分子营养物质转化为更易吸收的小分子活性物质,大大提高了其营养价值。同时,深加工处理可增加饲料原料的功能作用,但目前对于黑水虻深加工产品的应用研究较为缺乏。因此,本试验针对加工处理的黑水虻——发酵黑水虻,研究植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾血清生化指标、抗氧化能力和免疫功能的影响,评估用植物蛋白质替代鱼粉的饲料中添加发酵黑水虻进而缓解植物蛋白质过多对水产动物机体健康带来的副作用,为其在甲壳类动物上的应用提供理论依据。

1 材料与方法

1.1 试验设计

分别用豆粕和花生粕替代基础饲料中0、16.67%、33.33%和66.67%的鱼粉,并在基础饲料中添加0、4%、8%、16%的发酵黑水虻,制成4种等氮、等脂试验饲料,分记为N0、N4、N8和N16。饲料的制作参见王梦华等[29]。发酵黑水虻主要营养成分见表1,制作方法参见发明专利[30]中实施案例2。试验饲料组成及营养水平见表2,试验饲料氨基酸组成见表3。选取体重为(2.86±0.01) g的凡纳滨对虾560尾,随机分为4组(N0、N4、N8和N16组),每组4个重复,每个重复35尾虾。试验期8周。
表1 发酵黑水虻主要营养成分(湿重基础)

Table 1 Main nutritional components of fermented Hermetia illucens L. (wet weight basis)%

项目
Items		 发酵黑水虻
Fermented
Hermetia illucens L.
粗蛋白质CP 17.42
粗脂肪EE 6.91
水分Moisture 49.40
粗灰分Ash 5.11
钙Ca 1.19
磷P 0.39
酸溶蛋白Acid-soluble protein 6.62
食盐NaCl 0.63
总酸Total acid 3.67
天门冬氨酸Asp 0.95
谷氨酸Glu 3.09
丝氨酸Ser 0.42
组氨酸His 0.24
甘氨酸Gly 0.76
苏氨酸Thr 0.44
精氨酸Arg 0.54
丙氨酸Ala 1.61
酪氨酸Tyr 0.48
缬氨酸Val 0.74
蛋氨酸Met 0.10
苯丙氨酸Phe 0.74
异亮氨酸Ile 0.60
亮氨酸Leu 1.84
赖氨酸Lys 0.64
多肽Polypeptide 11.02
壳聚糖Chitosan 0.61
胆固醇Cholesterol 1.52
表2 试验饲料组成及营养水平(干物质基础)

Table 2 Composition and nutrient levels of experimental diets (DM basis)%

项目
Items		 组别Groups
N0 N4 N8 N16
原料Ingredients
鱼粉Fish meal 24.00 20.00 16.00 8.00
花生粕Peanut meal 15.00 17.07 19.14 23.29
豆粕Soybean meal 15.00 17.07 19.14 23.29
虾粉Shrimp powder 5.00 5.00 5.00 5.00
发酵黑水虻Fermented Hermetia illucens L. 0.00 4.00 8.00 16.00
高筋面粉Bread flour 12.00 12.00 12.00 12.00
微晶纤维素Microcrystalline cellulose 18.00 13.86 9.72 1.42
豆油Soybean oil 2.00 1.96 1.92 1.85
大豆卵磷脂Soybean lecithin 1.50 1.50 1.50 1.50
鱼油Fish oil 1.50 1.50 1.50 1.50
沸石粉Zeolite 2.39 2.11 1.87 1.33
磷酸二氢钙Ca(H2PO4)2 1.50 1.50 1.50 1.50
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
维生素C磷酸酯Vitamin C phosphate ester 0.20 0.20 0.20 0.20
黏合剂Adhesive 0.50 0.50 0.50 0.50
氧化钇Y2O3 0.04 0.04 0.04 0.04
食盐NaCl 0.30 0.30 0.30 0.30
赖氨酸Lys3) 0.23 0.43 0.61 1.00
蛋氨酸Met4) 0.14 0.26 0.36 0.58
合计Total 100.00 100.00 100.00 100.00
营养水平Nutrients levels5)
粗蛋白质CP 35.21 35.31 35.26 35.39
粗脂肪EE 8.24 8.26 8.38 8.24
粗灰分Ash 10.60 10.88 10.69 10.65
钙Ca 1.52 1.53 1.47 1.37
磷P 1.21 1.23 1.19 1.12

1)每千克维生素预混料含有 One kilogram of vitamin premix contained the following:VA 4 000 000 IU,VD3 2 000 000 IU,VE 30 g,VK3 10 g,VB1 5 g,VB2 15 g,VB6 8 g,泛酸钙 calcium pantothenate 25 g,叶酸 folic acid 2.5 g,生物素 biotin 0.08 g,烟酸 nicotinic acid 40 g,VB12 0.02 g,肌醇 inositol 150 g。

3)50%包膜结构的赖氨酸。50% encapsulated structure Lys.

4)50%胞膜结构的蛋氨酸。50% encapsulated structure Met.

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

2)每千克矿物质预混料含有 One kilogram of mineral premix contained the following:MgSO4·H2O 12 g,KCl 90 g,Met-Cu 3 g,FeSO4·H2O 1 g,ZnSO4·H2O 10 g,Ca(IO3)2 0.06 g,Met-Co 0.16 g,NaSeO3 0.003 6 g。

表3 试验饲料氨基酸组成(干物质基础)

Table 3 Amino acid composition of experimental diets (DM basis)%

项目
Items		 组别Groups
N0 N4 N8 N16
天门冬氨酸Asp 3.53 3.56 3.53 3.55
谷氨酸Glu 6.04 6.05 6.07 6.10
丝氨酸Ser 1.52 1.54 1.53 1.54
组氨酸His 0.80 0.82 0.81 0.80
甘氨酸Gly 1.79 1.81 1.80 1.81
苏氨酸Thr 1.31 1.31 1.32 1.29
精氨酸Arg 2.32 2.35 2.33 2.36
丙氨酸Ala 1.95 1.96 1.94 1.95
酪氨酸Tyr 0.92 0.93 0.92 0.94
缬氨酸Val 1.54 1.60 1.57 1.56
蛋氨酸Met 0.61 0.59 0.59 0.59
苯丙氨酸Phe 1.71 1.71 1.72 1.73
异亮氨酸Ile 1.47 1.52 1.47 1.48
亮氨酸Leu 2.51 2.52 2.51 2.54
赖氨酸Lys 2.07 2.07 2.07 2.09
总氨基酸TAA 30.09 30.34 30.17 30.34

1.2 饲养管理

凡纳滨对虾苗购买于阳江市某公司[水产苗种生产许可证:粤渔种(529823009)],购回后暂养1周。在规格为200 L的16个循环系统缸中进行养殖试验,饲养8周,养殖过程管理参见王梦华等[29]。养殖试验期间为自然光照,水温27~31 ℃,水体盐度5‰,氨氮浓度<0.20 mg/L,亚硝酸盐浓度<0.01 mg/L,溶氧浓度>5.00 mg/L,pH 7.80~8.20。

1.3 样品采集

养殖试验结束后停食24 h,每个缸随机选取12尾虾,快速放入40 mg/L的间氨基苯甲酸乙酯甲磺酸盐(MS-222)溶液中麻醉,用于血液采集,将血液室温条件下静置2 h,2 200×g离心10 min,取上清液置于0.5 mL离心管中,保存在-80 ℃冰箱,用于血清生化、免疫和抗氧化指标测定。采血后的虾放于冰上解剖,分离肝胰腺,用于检测肝脏免疫、抗氧化指标以及肝脏免疫、抗氧化相关基因的表达。

1.4 指标测定

1.4.1 原料和试验饲料营养成分

粗脂肪含量采用GB/T 6433—2006的方法测定,粗灰分含量采用GB/T 6438—2007的方法测定,水分含量采用GB/T 6435—2014的方法测定,粗蛋白质含量采用GB/T 6432—2018的方法测定,钙含量采用GB/T 6436—2002的方法测定,磷含量采用GB/T 6437—2018的方法测定,总酸含量采用GB/T 12456—2008的方法测定,酸溶蛋白含量采用NY/T 3801—2020的方法测定,食盐含量采用GB/T 6439—2007的方法测定,胆固醇含量采用GB 5009.128—2016的方法测定,多肽和氨基酸含量采用高效液相色谱(HPLC)法测定。壳聚糖以盐酸氨基葡萄糖计,样品经酸水解后采用高效液相色谱法测定。

1.4.2 血清生化指标

血清总蛋白(TP)、白蛋白(ALB)、球蛋白(GLOB)、尿素氮(UN)、胆固醇(CHOL)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、葡萄糖(GLU)含量及谷丙转氨酶(ALT)、谷草转氨酶(AST)活性采用全自动生化分析仪进行检测分析。

1.4.3 血清和肝脏免疫、抗氧化指标

超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化氢酶(GSH-Px)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、过氧化物酶(POD)、溶菌酶(LZM)活性和丙二醛(MDA)含量采用试剂盒(南京建成生物工程研究所),参照试剂盒说明书上的测定步骤和计算公式进行测定。

1.4.4 肝脏免疫、抗氧化相关基因表达

根据Chen等[31]的研究设计凡纳滨对虾免疫、抗氧化相关基因的特异性引物,引物序列见表4。参照Trizol试剂盒(Invitrogen,美国)说明书的操作步骤及试剂提取凡纳滨对虾肝脏的总RNA,再参照Prime ScriptTM反转录试剂盒(TaKaRa,日本)说明书的操作步骤除去提取总RNA中的DNA,随后利用反转录试剂将RNA反转录为cDNA,获得的cDNA利用双蒸水稀释2倍,-20 ℃条件下保存备用。
表4 引物序列

Table 4 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')		 GenBank登录号
GenBank accession number
Dorsal-1 F:TGGGGAAGGAAGGATGC
R:CGTAACTTGAGGGCATCTTC		 FJ998202.1
Relish F:CTACATTCTGCCCTTGACTCTGG
R:GGCTGGCAAGTCGTTCTCG		 EF432734
抗脂多糖因子
ALF		 F:CGCTTCACCGTCAAACCTTAC
R:GCCACCGCTTAGCATCTTGTT		 GQ227486.1
Cru F:GGTGTTGGTGGTGGTTTCCC
R:CAGTGGCTTGTGCCAGTTCC		 AY486426.1
Pen3 F:ATACCCAGGCCACCACCCTT
R:TGACAGCAACGCCCTAACC		 DQ206403.1
超氧化物歧化酶
SOD		 F:GCAATGAATGCCCTTCTACC
R:CAGAGCCTTTCACTCCAACG		 AB108065.1
谷胱甘肽过氧化氢酶
GSH-Px		 F:GGCACCAGGAGAACACTAC
R:CGACTTTGCCGAACATAAC		 AY973252.2
β-肌动蛋白
β-actin		 F:GAGCAACACGGAGTTCGTTGT
R:CATCACCAACTGGGACGACATGGA		 AF300705.2
实时定量PCR(qPCR)体系由0.5 μL上游引物(10 μmol/L)、0.5 μL下游引物(10 μmol/L)、1 μL第1链cDNA、12.5 μL 2×SYBR® Premix Ex Taq(TaKaRa,日本)及10.5 μL双蒸水组成。PCR程序为:95 ℃ 2 min,1个循环;95 ℃ 10 s,55 ℃ 10 s,72 ℃ 20 s,40个循环后,利用熔解曲线检验PCR反应产物的单一性。参考Livak等[32]的方法,以β-肌动蛋白为参比基因,采用2-ΔΔCt方法得到目的基因的相对表达量。

1.4.5 抗病原菌感染试验

正式抗病原菌感染试验开始前开展预试验,探索副溶血弧菌Ag01(菌株由南海海洋研究所提供)半致死浓度,累计用暂养虾300余尾,最终确定半致死浓度为5×104 CFU/尾,即每尾虾肌肉注射50 μL浓度为1×106 CFU/mL的无菌生理盐水稀释的副溶血弧菌Ag01菌液。试验分为4组,每组3个重复,每个重复13尾虾。N0组对虾注射50 μL无菌生理盐水,N4、N8、N16组对虾注射50 μL浓度为1×106 CFU/mL的无菌生理盐水稀释的副溶血弧菌Ag01菌液。每隔2 h进行一次死亡数据统计,连续统计52 h;后续改为每天观察统计1次,观察7 d后,统计各组对虾死亡情况。

1.5 数据统计分析

利用SPSS 26.0进行统计分析,用单因素方差分析(one-way ANOVA)和Duncan氏法多重比较进行组间差异分析。结果采用平均值±标准误(mean±SE)形式表示,差异显著性水平为P<0.05。

2 结果

2.1 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾血清生化指标的影响

由表5可知,各组之间凡纳滨对虾血清GLU和HDL-C含量无显著差异(P>0.05)。N0组血清LDL-C含量显著高于N4、N8和N16组(P<0.05),且N16组显著高于N4组(P<0.05)。N4、N8和N16组血清TP含量显著高于N0组(P<0.05),且N8组显著高于N4组(P<0.05)。N0和N16组血清ALB含量显著低于N4和N8组(P<0.05)。N4、N8和N16组血清GLOB含量显著高于N0组(P<0.05),且N8和N16组显著高于N4组(P<0.05)。N0和N16组血清ALB/GLOB显著低于N4和N8组(P<0.05)。N4和N8组血清TG含量显著低于N0组(P<0.05),且N0组显著低于N16组(P<0.05)。N0组血清CHOL含量显著高于其他各组(P<0.05),且N16组显著高于N4组(P<0.05)。N0组血清ALT和AST活性显著高于其他各组(P<0.05),且N4和N8组显著高于N16组(P<0.05)。
表5 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾血清生化指标的影响

Table 5 Effects of plant protein replacing fish meal and adding fermented Hermetia illucens L. in diets on serum biochemical indices of Litopenaeus vannamei

项目
Items		 组别Groups
N0 N4 N8 N16
葡萄糖GLU/(mmol/L) 1.50±0.03 1.47±0.02 1.53±0.02 1.50±0.06
低密度脂蛋白胆固醇LDL-C/(mmol/L) 0.68±0.01c 0.47±0.03a 0.51±0.00ab 0.54±0.01b
高密度脂蛋白胆固醇HDL-C/(mmol/L) 0.28±0.00 0.28±0.00 0.27±0.00 0.28±0.00
总蛋白TP/(g/L) 57.30±0.28a 60.60±0.12b 62.38±0.27c 61.31±0.76bc
白蛋白ALB/(g/L) 5.95±0.12a 7.09±0.02b 7.08±0.10b 6.12±0.05a
球蛋白GLOB/(g/L) 51.34±0.18a 53.51±0.12b 55.30±0.33c 55.19±0.75c
白蛋白/球蛋白ALB/GLOB/% 11.59±0.22a 13.26±0.04b 12.80±0.24b 11.10±0.17a
甘油三酯TG/(mmol/L) 0.48±0.01b 0.36±0.01a 0.38±0.01a 0.58±0.02c
胆固醇CHOL/(mmol/L) 1.01±0.05c 0.73±0.01a 0.79±0.01ab 0.82±0.02b
谷丙转氨酶ALT/(U/L) 370.11±3.26c 326.34±2.94b 325.42±2.22b 232.20±0.90a
谷草转氨酶AST/(U/L) 213.05±2.11c 193.01±2.09b 197.28±2.06b 159.74±2.35a

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 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏和血清免疫、抗氧化指标的影响

由表6可知,各组之间凡纳滨对虾肝脏TP含量无显著差异(P>0.05)。N0和N16组肝脏ACP活性显著高于N4和N8组(P<0.05)。N4和N8组肝脏AKP活性显著高于N0组和N16组(P<0.05),且N8组显著高于N4组(P<0.05)。N0组肝脏POD活性显著低于N4、N8和N16组(P<0.05),且N16组显著低于N8组(P<0.05),N8组显著低于N4组(P<0.05)。N4和N8组肝脏LZM活性显著高于N0组(P<0.05),且N0组显著高于N16组(P<0.05)。N0组肝脏SOD活性显著低于其他各组(P<0.05),且N16组显著低于N8组(P<0.05),N8组显著低于N4组(P<0.05)。N0组肝脏GSH-Px活性显著低于其他各组(P<0.05),且N16组显著低于N4组(P<0.05),N4组显著低于N8组(P<0.05)。
表6 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏免疫、抗氧化指标的影响

Table 6 Effects of plant protein replacing fish meal and adding fermented Hermetia illucens L. in diets on liver immune and antioxidant indices of Litopenaeus vannamei

项目
Items		 组别Groups
N0 N4 N8 N16
总蛋白TP/(g/kg prot) 6.17±0.03 6.14±0.05 6.23±0.06 6.17±0.10
酸性磷酸酶ACP/(金氏单位/g prot) 35.21±1.02b 25.92±0.45a 27.41±0.24a 37.23±0.70b
碱性磷酸酶AKP/(金氏单位/g prot) 16.80±0.12a 17.89±0.17b 19.06±0.23c 16.78±0.37a
过氧化物酶POD/(U/g prot) 3.71±0.14a 7.13±0.40d 6.42±0.09c 5.08±0.13b
溶菌酶LZM/(U/g prot) 14.68±0.14b 15.40±0.20c 16.00±0.30c 13.25±0.14a
超氧化物歧化酶SOD/(U/g prot) 2.18±0.03a 5.08±0.05d 3.16±0.13c 2.46±0.06b
谷胱甘肽过氧化氢酶GSH-Px/(U/kg prot) 168.85±2.59a 230.42±5.25c 251.24±4.20d 210.26±4.52b
由表7可知,各组之间凡纳滨对虾血清CAT活性无显著差异(P>0.05)。N8和N16组血清MDA含量显著低于N0和N4组(P<0.05)。N8组血清SOD活性显著高于N0组(P<0.05)。
表7 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾血清抗氧化指标的影响

Table 7 Effects of plant protein replacing fish meal and adding fermented Hermetia illucens L. in diets on serum antioxidant indices of Litopenaeus vannamei

项目
Items		 组别Groups
N0 N4 N8 N16
过氧化氢酶CAT/(U/mL) 268.44±4.26 277.70±4.42 273.01±6.03 266.58±5.82
丙二醛MDA/(nmol/mL) 2.94±0.07b 2.78±0.08b 2.28±0.14a 2.33±0.06a
超氧化物歧化酶SOD/(U/mL) 87.70±1.44a 98.79±1.97ab 104.27±7.61b 94.37±1.24ab

2.3 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏免疫、抗氧化相关基因表达的影响

由图1可知,随着饲料中鱼粉替代量和发酵黑水虻添加量的增加,凡纳滨对虾肝脏Dorsal-1、Relish、Cru、Pen3、SOD和GSH-Px基因相对表达量均呈先增加后降低的变化趋势。各组之间凡纳滨对虾抗脂多糖因子(ALF)基因相对表达量无显著差异(P>0.05)。N4组肝脏Dorsal-1基因相对表达量显著高于N8组(P<0.05),且N8组显著高于N0组(P<0.05),N0组显著高于N16组(P<0.05)。N8组肝脏Relish基因相对表达量显著高于N4组(P<0.05),且N4组显著高于N0组(P<0.05),N0组显著高于N16组(P<0.05)。N4和N8组肝脏Cru基因相对表达量显著高于N0和N16组(P<0.05),且N4组显著高于N8组(P<0.05),N16组显著高于N0组(P<0.05)。N4组肝脏Pen3基因相对表达量显著高于N8组(P<0.05),且N8组显著高于N16组(P<0.05),N16组显著高于N0组(P<0.05)。N4和N8组肝脏SOD基因相对表达量显著高于N0和N16组(P<0.05),且N8组显著高于N4组(P<0.05),N0组显著高于N16组(P<0.05)。N4和N8组肝脏GSH-Px基因相对表达量显著高于N0和N16组(P<0.05),且N0组显著高于N16组(P<0.05)。
图1 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏免疫、抗氧化相关基因表达的影响

数据柱标相同小写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)。

Fig.1 Effects of plant protein replacing fish meal and adding fermented Hermetia illucens L. in diets on liver immune and antioxidant related genes expression of Litopenaeus vannamei

Value columns with the same small letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05).

2.4 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾抗病原菌感染的影响

由图2可知,用副溶血弧菌Ag01攻毒后,随着时间的推移,各组试验对虾死亡率逐渐增加,48 h后基本稳定,死亡率不再增加。7 d后,N0、N4、N8和N16组对虾死亡率分别为53.85%、25.64%、23.08%和51.28%。
图2 凡纳滨对虾死亡率

Fig.2 Mortality of Litopenaeus vannamei

3 讨论

3.1 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾血清生化和抗氧化指标的影响

动物的血清生化指标通常反映其生理代谢情况、健康程度及对营养和环境的适应情况[33]。鱼类血清生化指标与机体的营养、代谢和健康状况密切相关[34]。虾机体内具有一系列抗氧化系统,以防止体内过氧化,保护细胞免受氧化损伤。血清TP含量可作为评价鱼类生理状态的典型指标[35]。血清ALB含量是表征脊椎动物机体营养状态的可靠参数[36],反映肝脏功能的损伤程度,机体发生炎症时血清ALB含量会下降20%左右[37-38]。血清GLOB是由B细胞转化成浆细胞后分泌的,反映机体的抵抗力,在防御系统中起着血液蛋白质转运的作用[39]。ALB/GLOB则是肝脏和肾脏疾病等低蛋白质血症的指标[40]。血清TP和GLOB含量的升高可能是鱼类免疫反应过程中保护蛋白质升高的体现[41-42]。本研究中,N4和N8组的凡纳滨对虾血清ALB含量和ALB/GLOB显著高于N0和N16组,可能与适量添加发酵黑水虻提高了凡纳滨对虾机体免疫功能有关。
ALT和AST活性能较准确反映肝脏健康状况,肝脏病变或受损时,血清ALT和AST活性提高[43]。研究发现,用植物蛋白质替代花鲈饲料中的鱼粉会出现营养物质代谢紊乱和营养性肝病,血清ALT活性显著提高[44]。本研究中,N4和N8组的凡纳滨对虾血清ALT和AST活性显著低于N0组,这与蔺玉珍等[10]用黑水虻幼虫粉替代饲料中鱼粉饲喂黄颡鱼导致其血清ALT和AST活性降低的结果一致。此外,用蛹肽蛋白替代饲料中的鱼粉也可降低养殖动物血清ALT和AST活性[45]。这可能与昆虫粉含有的甲壳低聚糖和壳聚糖等免疫多糖,具有提高机体免疫力和保护肝脏的作用有关[46]。本研究发现,各组凡纳滨对虾的血清GLU和HDL-C含量无显著差异,这与用黑水虻虫粉替代鱼粉不影响建鲤(Cyprinus carpiovar Jian)[47]和花鲈(Lateolabrax japonicus)[48]血清GLU和HDL-C含量的研究结果近似。
本研究中,N4、N8和N16组凡纳滨对虾血清LDL-C含量均显著低于N0组,可能与发酵黑水虻中含有的几丁质多糖有关。研究表明,在饲料中添加黑水虻幼虫粉可以降低建鲤[49]、欧洲鲈鱼(Dicentrarchus labrax)[50]、黄颡鱼[51]、花鲈[48]和凡纳滨对虾[52]血清CHOL含量。本研究中,N8和N16组凡纳滨对虾血清CHOL和TG含量显著低于N0组,可能与鱼粉中胆固醇含量比黑水虻中胆固醇含量高[49],以及黑水虻中含有较高的几丁质多糖抑制CHOL的吸收、增加肝脏内TG的水解和胆汁酸的排泄有关[50,53-55]。
MDA是动物机体自由基的指示物,被普遍当作各种氧化自由基损伤机体的指标[56]。研究表明,氨暴露、低温和空气暴露等应急条件下,虾体内MDA含量显著升高[57-58]。MDA含量的高低间接反映机体细胞受自由基攻击的严重程度,机体MDA含量变化反映脂质氧化程度[59]。用植物蛋白质替代鱼粉可使凡纳滨对虾血清ALT、AST活性和MDA含量显著提高[60]。本研究中,N8和N16组的凡纳滨对虾血清MDA含量显著低于N0和N4组,说明在饲料中添加8%和16%的发酵黑水虻可以提高凡纳滨对虾机体抗氧化能力,有利于凡纳滨对虾机体健康。

3.2 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏免疫、抗氧化指标的影响

磷酸酶是一种关键的溶酶体酶,可作为生物系统环境应激评估的标志物,以评估虾的免疫状态[61]。据报道,AKP参与消化和解毒过程,ACP是遇到组织或细胞损伤时由溶酶体释放[62],ACP活性受膳食营养水平的影响[63]。南美白对虾幼鱼肝脏AKP和ACP活性可能会随着饲料中鱼粉含量的降低而降低[64]。本研究结果与上述研究结果不尽相同,肝脏ACP活性呈先降低后增加的趋势,N0和N16组肝脏ACP活性显著高于N4和N8组;而AKP活性变化与ACP活性变化相反,说明在饲料中添加发酵黑水虻对凡纳滨对虾机体组织有一定的保护作用。SOD是一种重要的内源性抗氧化酶,是机体对抗活性氧物质的一线防御系统的一员[65-66]。SOD和CAT是动物机体内抗氧化防御系统的重要酶类,其可以有效清除机体内的活性氧自由基,对细胞膜及细胞内的核酸起到一定保护作用[67]。Bae等[68]研究发现,用豆粕替代凡纳滨对虾饲料中40%的鱼粉,可显著降低对虾血清SOD活性。本研究发现,在饲料中添加发酵黑水虻组肝脏SOD和POD活性显著提高,说明在饲料中添加发酵黑水虻提高了凡纳滨对虾机体抗氧化能力。这与用黑水虻干虫粉替代饲料中的50%鱼粉显著降低乌鳢幼鱼血清MDA含量、显著提高其血清SOD和LZM活性结果[69]相似。也与用黑水虻幼虫粉替代适宜水平的鱼粉能显著提高凡纳滨对虾血清抗氧化指标(总抗氧化能力和SOD活性)研究结果[14]相似。这可能与黑水虻幼虫粉和发酵黑水虻中几丁质的抗氧化作用[70-71]有关。

3.3 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾肝脏免疫、抗氧化相关基因表达的影响

对虾机体内具有一系列抗氧化系统,以防止体内过氧化,保护机体免受氧化损伤。在本试验中,以对虾肝脏免疫和抗氧化相关基因为切入点开展相关基因表达的研究。Dorsal-1和Relish基因是节肢动物中核因子-κB(NF-κB)家族成员[72],Relish基因参与免疫缺陷信号通路并介导抗革兰氏阴性菌的免疫过程,Dorsal-1基因则主要参与Toll样信号通路介导抗革兰氏阳性菌以及真菌的免疫相关过程[73]。ALF、Cru、Pen3主要参与机体抗病原菌感染,与机体免疫功能有关,SOD和GSH-Px基因与机体抗氧化作用有关。本研究结果显示,在饲料中添加适量发酵黑水虻显著提高了肝脏Dorsal-1基因相对表达量,替代过量时使Dorsal-1基因相对表达量显著降低;这与低鱼粉饲料会导致凡纳滨对虾内质网应激的结果[64]相似。GSH-Px是一种重要的细胞内酶,可将过氧化氢分解成水,从而保护细胞免受氧化应激[74]。研究发现,用植物蛋白质替代花鲈饲料中的鱼粉会出现营养性肝病,肝脏GSH-Px活性显著降低[44]。而用黑水虻幼虫粉替代花鲈饲料中7.5%~30.0%的鱼粉可显著提高血清GSH-Px活性[48],在建鲤[47,49] 、黄颡鱼[75]、凡纳滨对虾[76]和镜鲤(Cyprinus carpio)[77]上也得到了相似的结果。这可能与黑水虻幼虫粉和发酵黑水虻中几丁质的抗氧化作用[70-71]、提高鱼类非特异性免疫力、激活巨噬细胞活性[78]有关。ALF是虾中的一种抗菌肽分子,具有强大的广谱抗微生物作用,有助于虾抵抗病原入侵[79]。本研究发现,在饲料中添加4%、8%和16%发酵黑水虻组凡纳滨对虾肝脏ALF基因相对表达量有所提高,Cru和Pen3基因相对表达量显著提高,说明用发酵黑水虻适量替代鱼粉可提高凡纳滨对虾免疫功能,这可能与其中含有的几丁质和抗菌肽成分有关。研究发现,在饲料中添加适量黑水虻幼虫粉显著提高了黄颡鱼免疫力,主要归因于其中几丁质和抗菌肽的作用[75]。在鲤鱼饲料中直接添加抗菌肽可显著增强机体免疫力[80]。由此可见,黑水虻中的几丁质和抗菌肽均能提高养殖动物机体免疫功能[81-82]。

3.4 植物蛋白质替代鱼粉并在饲料中添加发酵黑水虻对凡纳滨对虾抗病原菌感染的影响

凡纳滨对虾是世界上具有重要商业价值的海洋物种。但是,由于虾的各种疾病,养虾业面临着严重的问题[83]。其中,弧菌感染造成的经济损失最为严重[84],尤其是弧菌引起的急性肝胰腺坏死病,在整个行业造成了巨大的经济损失[85]。且植物蛋白质中含有的抗营养因子会降低鱼类的免疫力[86],用豆粕替代凡纳滨对虾饲料中50%的鱼粉使其免疫力显著降低[87]。本研究用副溶血弧菌Ag01对各组凡纳滨对虾进行注射攻毒试验,用于评估虾的免疫功能,发现用植物蛋白质替代鱼粉的饲料中添加适量发酵黑水虻可明显降低凡纳滨对虾的死亡率,与未添加发酵黑水虻组相比,添加4%和8%发酵黑水虻组的死亡率可降低约50%,这与上述研究发现在饲料中添加发酵黑水虻提高了凡纳滨对虾机体抗氧化能力和免疫功能相对应,可能是发酵黑水虻中的几丁质、抗菌肽和益生菌成分综合作用的结果。

4 结论

饲料中添加4%和8%的发酵黑水虻不仅可以缓解植物蛋白质替代鱼粉对凡纳滨对虾健康带来的负面作用,而且可以提高凡纳滨对虾抗氧化能力,增强免疫功能;但当鱼粉替代过量时,即使增加发酵黑水虻的添加水平到16%时,凡纳滨对虾抗氧化能力和免疫功能仍受到了不良影响。在本试验条件下,植物蛋白质替代鱼粉水平不宜超过33.33%,且发酵黑水虻的适宜添加水平为4%~8%。

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