• ISSN 1000-0615
  • CN 31-1283/S
Volume 45 Issue 10
Oct.  2021
Article Contents
Turn off MathJax

Citation:

Effects of aflatoxin B1 on growth performance and liver function of juvenile Pelteobagrus fulvidraco

  • An experiment was conducted to study the effect of aflatoxin B1 on growth performance, digestive enzyme activities in intestinal tract and liver function of juvenile Pelteobagrus fulvidraco. Four experimental diets were prepared to contain 0 (control), 50, 100, 200 μg/kg aflatoxin B1. Each diet was randomly assigned to triplicate cages of 50 fish with an initial average weight of (6.00±0.10) g for 8 weeks. The results showed that: ① There were no significant differences in survival rate, feed coefficient ratio and specific growth rates of juvenile Pelteobagrus fulvidraco among all groups. The activity of trypsin in the AFB1 group significantly increased compared with the control group. When the AFB1 concentration reached 100 and 200 μg/kg, the amylase and lipase activities significantly decreased; ② With increasing AFB1 concentration, the levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), glucose, triglyceride, total bile acid and total cholesterol contents in serum significantly increased, and the AST, ALT activities in the liver significantly decreased; ③With increasing AFB1 concentration, the catalase activity and malondiadehyde content in the liver significantly increased. The superoxide dismutase activity significantly increased when the AFB1 concentration reached 100 and 200 μg/kg; ④ With increasing AFB1 concentration, the expression levels of sod and il-1β in the liver were significantly up-regulated, the expression levels of cat, il-10 and il-8 in the liver were significantly up-regulated when the AFB1 concentration reached 200 μg/kg; ⑤ Histological observations showed that an increase in the concentration of AFB1 caused some liver cells to present slight atrophy, hepatocyte nucleus shifted, cell boundary blurred, liver cell vacuolation degre. The results showed that under the experimental conditions, there was no significant effect on the growth of juvenile Pelteobagrus fulvidraco with the concentration of AFB1≤200 μg/kg, but it could affect the digestion and absorption function of the intestines, cause oxidative stress and inflammatory reactions in the liver, and result in liver function damage with the concentration of AFB1≥50 μg/kg.
  • 加载中
  • [1] 伍皓茗, 苗玉涛. 黄颡鱼营养免疫研究进展[J]. 广东饲料, 2017, 26(2): 31-33. doi: 10.3969/j.issn.1005-8613.2017.02.010Wu H M, Miao Y T. Advances in nutritional immunity of Pelteobagrus fulvidraco[J]. Guangdong Feed, 2017, 26(2): 31-33(in Chinese). doi: 10.3969/j.issn.1005-8613.2017.02.010
    [2] Santacroce M P, Conversano M C, Casalino E, et al. Aflatoxins in aquatic species: metabolism, toxicity and perspectives[J]. Reviews in Fish Biology and Fisheries, 2008, 18(1): 99-130.
    [3] Mahato D K, Lee K E, Kamle M, et al. Aflatoxins in food and feed: An overview on prevalence, detection and control strategies[J]. Frontiers in Microbiology, 2019, 10: 02266.
    [4] Nunes E M C G, Pereira M M G, Costa A P R, et al. Effects of aflatoxin B1 on performance and health of tambaqui fingerlings (Colossoma macropomum)[J]. International Aquatic Research, 2019, 11(1): 73-83.
    [5] Deng S X, Tian L X, Liu F J, et al. Toxic effects and residue of aflatoxin B1 in tilapia (Oreochromis niloticus×O. aureus) during long-term dietary exposure[J]. Aquaculture, 2010, 307(3-4): 233-240.
    [6] Ayyat M S, Ayyat A M N, Al-Sagheer A A, et al. Effect of some safe feed additives on growth performance, blood biochemistry, and bioaccumulation of aflatoxin residues of Nile tilapia fed aflatoxin-B1 contaminated diet[J]. Aquaculture, 2018, 495: 27-34.
    [7] Huang Y, Han D, Xiao X C, et al. Effect of dietary aflatoxin B1 on growth, fecundity and tissue accumulation in gibel carp during the stage of gonad development[J]. Aquaculture, 2014, 428-429: 236-242.
    [8] Arana S, Alves V A F, Sabino M, et al. Immunohistochemical evidence for myofibroblast-like cells associated with liver injury induced by Aflatoxin B1 in rainbow trout (Oncorhynchus mykiss)[J]. Journal of Comparative Pathology, 2014, 150(2-3): 258-265.
    [9] Kowalska A, Walkiewicz K, Kozieł P, et al. Aflatoxins: characteristics and impact on human health[J]. Postepy Higieny I Medycyny Doswiadczalnej, 2017, 71: 315-327.
    [10] 黄莹, 朱晓鸣, 韩冬, 等. 饲喂不同浓度黄曲霉毒素B1饲料对异育银鲫成鱼的生长和毒素积累的影响[J]. 水生生物学报, 2012, 36(5): 817-825.Huang Y, Zhu X M, Han D, et al. Growth and aflatoxin B1 accumulation of gibel carp adult fed with diets of different levels of aflatoxin B1[J]. Acta Hydrobiologica Sinica, 2012, 36(5): 817-825(in Chinese).
    [11] 黄莹, 韩金高, 朱晓鸣, 等. 饲喂不同浓度黄曲霉毒素B1饲料对花鳗鲡幼鱼生长、抗氧化能力和毒素积累的影响[J]. 水生生物学报, 2021, 45(3): 566-572.Huang Y, Han J G, Zhu X M, et al. Effects of dietary aflatoxin B1 on growth, antioxidant capacity and tissue accumulation of juvenile marbled eel (Anguilla marmorata)[J]. Acta Hydrobiologica Sinica, 2021, 45(3): 566-572(in Chinese).
    [12] Raghavan P R, Zhu X, Lei W, et al. Low levels of aflatoxin B1 could cause mortalities in juvenile hybrid sturgeon, Acipenser ruthenus ♂×A. baeri♀[J]. Aquaculture Nutrition, 2011, 17(2): e39-e47.
    [13] Zeng S L, Long W Q, Tian L X, et al. Effects of dietary aflatoxin B1 on growth performance, body composition, haematological parameters and histopathology of juvenile Pacific white shrimp (Litopenaeus vannamei)[J]. Aquaculture Nutrition, 2016, 22(5): 1152-1159.
    [14] Yu Y Y, Niu J, Yin P, et al. Detoxification and immunoprotection of Zn(II)-curcumin in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxin B1[J]. Fish & Shellfish Immunology, 2018, 80: 480-486.
    [15] Gonçalves R A, Cam T D, Tri N N, et al. Aflatoxin B1 (AFB1) reduces growth performance, physiological response, and disease resistance in Tra catfish (Pangasius hypophthalmus)[J]. Aquaculture International, 2018, 26(3): 921-936.
    [16] 黄莹, 姚远, 朱晓鸣, 等. 饲喂不同浓度黄曲霉毒素B1饲料对草鱼幼鱼生长和毒素积累的影响[J]. 水生生物学报, 2019, 43(4): 723-730. doi: 10.7541/2019.085Huang Y, Yao Y, Zhu X M, et al. Effects of aflatoxin B1 on growth and tissue accumulation of juvenile grass carp (Ctenopharyngodon idellus)[J]. Acta Hydrobiologica Sinica, 2019, 43(4): 723-730(in Chinese). doi: 10.7541/2019.085
    [17] El-Sayed Y S, Khalil R H. Toxicity, biochemical effects and residue of aflatoxin B1 in marine water-reared sea bass (Dicentrarchus labrax L.)[J]. Food and Chemical Toxicology, 2009, 47(7): 1606-1609.
    [18] Fan Y, Liu L T, Zhao L H, et al. Influence of Bacillus subtilis ANSB060 on growth, digestive enzyme and aflatoxin residue in Yellow River carp fed diets contaminated with aflatoxin B1[J]. Food and Chemical Toxicology, 2018, 113: 108-114.
    [19] 齐灿灿, 王宝杰, 刘梅, 等. 黄曲霉毒素B1(AFB1)的短期投喂对凡纳滨对虾肠道黏膜屏障的影响[J]. 水产学报, 2017, 41(12): 1936-1945.Qi C C, Wang B J, Liu M, et al. Effects of short term addition of aflatoxin B1(AFB1) on the intestinal mucosal barrier of Litopenaeus vannamei[J]. Journal of Fisheries of China, 2017, 41(12): 1936-1945(in Chinese).
    [20] Andleeb S, Ashraf M, Hafeez-Ur-Rehman M, et al. Effect of aflatoxin B1-contaminated feed on growth and vital organs of advance fry of Catla catla[J]. Journal of Animal and Plant Sciences, 2015, 25(3): 816-825.
    [21] 毕小娟, 陈代文, 余冰, 等. 黄曲霉毒素B1对断奶仔猪生长性能、肝脏组织及肠道健康的影响[J]. 动物营养学报, 2018, 30(8): 3276-3284. doi: 10.3969/j.issn.1006-267x.2018.08.047Bi X J, Chen D W, Yu B, et al. Effects of aflatoxin B1 on growth performance, liver tissue and intestine health of weaned piglets[J]. Chinese Journal of Animal Nutrition, 2018, 30(8): 3276-3284(in Chinese). doi: 10.3969/j.issn.1006-267x.2018.08.047
    [22] 冯光德. 自然霉变玉米对肉鸭生产性能和消化生理的影响及机制研究[D]. 雅安: 四川农业大学, 2011.Feng G D. Effects of corn naturally contaminated with mycotoxins on performance and digestive physiology of ducks and its mechanism[D]. Yaan: Sichuan Agricultural University, 2011 (in Chinese).
    [23] Han X Y, Huang Q C, Li W F, et al. Changes in growth performance, digestive enzyme activities and nutrient digestibility of cherry valley ducks in response to aflatoxin B1 levels[J]. Livestock Science, 2008, 119(1-3): 216-220.
    [24] 旦增曲珍, 廖雪莲, 侯晨姝, 等. 胰蛋白酶在脓毒症大鼠模型血清及组织中的表达及意义[J]. 中华内科杂志, 2018, 57(7): 505-510. doi: 10.3760/cma.j.issn.0578-1426.2018.07.007Dan Z Q Z, Liao X L, Hou C Z, et al. The expression of trypsin in serum and vital organs of septic rats[J]. Chinese Journal of Internal Medicine, 2018, 57(7): 505-510(in Chinese). doi: 10.3760/cma.j.issn.0578-1426.2018.07.007
    [25] 何杰, 吴代武, 叶元土, 等. 饲料组胺水平对黄颡鱼生长性能、血清生化指标和胃肠道黏膜结构的影响[J]. 动物营养学报, 2018, 30(7): 2581-2593. doi: 10.3969/j.issn.1006-267x.2018.07.018He J, Wu D W, Ye Y T, et al. Effects of dietary histamine level on growth performance, serum biochemical indexes and gastrointestinal mucosa structure of Yellow Catfish (Pelteobagrus fulvidraco)[J]. Chinese Journal of Animal Nutrition, 2018, 30(7): 2581-2593(in Chinese). doi: 10.3969/j.issn.1006-267x.2018.07.018
    [26] Yan Q, Xie S, Zhu X, et al. Dietary methionine requirement for juvenile rockfish, Sebastes schlegeli[J]. Aquaculture Nutrition, 2007, 13(3): 163-169.
    [27] Han D, Xie S, Zhu X, et al. Growth and hepatopancreas performances of gibel carp fed diets containing low levels of aflatoxin B1[J]. Aquaculture Nutrition, 2010, 16(4): 335-342.
    [28] 王静. 饲料中黄曲霉毒素B1对凡纳滨对虾生长、生化指标及肝胰腺显微结构的影响[D]. 保定: 河北农业大学, 2014.Wang J. Toxic effects of aflatoxin B1 on growth performance, biochemical and hepatopancreas microstructure of Litopenaeus vannamei[D]. Baoding: Agricultural University of Hebei, 2014 (in Chinese).
    [29] 石勇, 胡毅, 刘艳莉, 等. 血根碱对LPS诱导后黄鳝免疫应激及肠道炎症相关基因表达的影响[J]. 中国水产科学, 2020, 27(1): 125-136.Shi Y, Hu Y, Liu Y L, et al. Effects of sanguinarine on immune and intestinal inflammation related to gene expression in rice field eels (Monopterus albus) induced by LPS[J]. Journal of Fishery Sciences of China, 2020, 27(1): 125-136(in Chinese).
    [30] 张媛媛, 宋理平, 胡斌, 等. 饲料中添加姜黄素对尼罗罗非鱼幼鱼生长和四氯化碳诱导肝损伤的影响[J]. 中国水产科学, 2018, 25(6): 1271-1280.Zhang Y Y, Song L P, Hu B, et al. Effect of curcumin on growth performance and protective effect of liver injury induced by carbon tetrachloride in Oreochromis niloticus[J]. Journal of Fishery Sciences of China, 2018, 25(6): 1271-1280(in Chinese).
    [31] Zhao W, Wang L, Liu M, et al. Transcriptome, antioxidant enzyme activity and histopathology analysis of hepatopancreas from the white shrimp Litopenaeus vannamei fed with aflatoxin B1(AFB1)[J]. Developmental & Comparative Immunology, 2017, 74: 69-81.
    [32] 周小新. 牛磺酸对黄曲霉毒素B1中毒大鼠脾脏功能影响的研究[D]. 沈阳: 沈阳农业大学, 2019.Zhou X X. Effect of taurine on spleen function of aflatoxin B1 poisoned rats[D]. Shenyang: Shenyang Agricultural University, 2019 (in Chinese).
    [33] Lu C, Ling F, Ji J, et al. Expression of immune-related genes in goldfish gills induced by Dactylogyrus intermedius infections[J]. Fish & Shellfish Immunology, 2013, 34(1): 372-377.
    [34] Moore K W, de Waal Malefy R, Coffman R L, et al. Interleukin-10 and the interleukin-10 receptor[J]. Annual Review of Immunology, 2001, 19(1): 683-765.
  • Relative Articles

    [1] PI Kun, ZHANG Min, LI Baomin, LI Gengchen. Diffusion fluxes of nitrogen and phosphorus across sediment-water interface in different aquaculture model ponds. Journal of fisheries of china, 2018, 42(2): 246-256.  doi: 10.11964/jfc.20161210622
    [2] ZHANG Lihan, LUO Zhi, YOU Wenjing, LI Dandan, WU Kun, PAN Yaxiong. Molecular characterization and tissue distribution of Frizzled (FZD) in yellow catfish (Pelteobagrus fulvidraco) by copper exposure. Journal of fisheries of china, 2018, 42(5): 625-632.  doi: 10.11964/jfc.20170410801
    [3] LU You, JIN Min, YUAN Ye, XIONG Jia, MA Hongna, ZHOU Qicun. Effects of different lipid sources on growth performance, body composition, the serum biochemical indices, fatty acids composition and antioxidant capacity in juvenile yellow catfish (Pelteobagrus fulvidraco). Journal of fisheries of china, 2018, 42(7): 1094-1110.  doi: 10.11964/jfc.20170310739
    [4] ZHANG Lihan, LUO Zhi, YOU Wenjing, LI Dandan, XU Yihuan, PAN Yaxiong. Identification of five genes from Wnt/β-catenin pathway inyellow catfish (Pelteobagrus fulvidraco) and their mRNA expression inthe ovary to waterborne copper exposure. Journal of fisheries of china, 2019, 43(4): 742-750.  doi: 10.11964/jfc.20171011012
    [5] LI Bing, ZHANG Muzi, LI Ming, YUAN Lixia, WANG Rixin. Effect of acute ammonia toxicity on genes involved in antioxidant and inflammation in head kidney macrophage of Pelteobagrus fulvidraco. Journal of fisheries of china, 2018, 42(12): 1889-1895.  doi: 10.11964/jfc.20170910969
    [6] ZHUO Meiqin, YANG Shuibo, LING Shicheng, LUO Zhi. Effects of dietary lipid on lipid metabolism, methylation and expression of PI3KCa in the ovary of yellow catfish (Pelteobagrus fulvidraco). Journal of fisheries of china, 2019, 43(10): 2186-2196.  doi: 10.11964/jfc.20190911964
    [7] CHENG Xin, PAN Tingting, JIN Min, MA Hongna, LIANG Chao, REN Zelin, ZHOU Wenhao, ZHOU Zhigang, ZHOU Qicun. Effects of dietary yeast culture supplementation on growth performance, nonspecific immunity and intestinal health of Pelteobagrus fulvidraco. Journal of fisheries of china, 2019, 43(4): 1080-1091.  doi: 10.11964/jfc.20180511291
    [8] YE Hanmei, WEI Xiaolei, TAN Xiaoying. Prokaryotic protein expression of suppressor of cytokine signaling 1 of yellow catfish (Pelteobagrus fulvidraco) and preparation of their polyclonal antibodies. Journal of fisheries of china, 2020, 44(4): 523-527.  doi: 10.11964/jfc.20190611851
    [9] ZHUO Lixin, ZHAO Hongxia, HUANG Yanhua, CAO Junming, WANG Guoxia, CHEN Bing, SUN Yuping. Influence of oxidized fish oil on the intestinal health of juvenile yellow catfish (Pelteobagrus fulvidraco) and the use of arginine as an intervention measure. Journal of fisheries of china, 2018, 42(1): 100-111.  doi: 10.11964/jfc.20160810507
    [10] HU Junru, WANG Guoxia, SUN Yuping, ZHAO Hongxia, HUANG Yanhua, CAO Junming. Effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish (Pelteobagrus fulvidraco). Journal of fisheries of china, 2019, 43(11): 2394-2404.  doi: 10.11964/jfc.20181011479
    [11] WANG Guoxia, CHEN Bing, SUN Yuping, HU Junru, PENG Kai, WU Haomin, HUANG Yanhua. Effects of replacing fish meal with defatted black soldier fly (Hermetia illucens) larvae meal on growth performance, nutrient retention, serum biochemical parameters and digestive enzymes activity of juvenile Pelteobagrus fulvidraco. Journal of fisheries of china, 2020, 44(6): 987-998.  doi: 10.11964/jfc.20190411736
    [12] SUN Liying, ZHANG Muzi, LI Ming, YUAN Lixia, WANG Rixin. Effects of acute ammonia stress on antioxidant enzyme activity and mRNA expression levels of HSP70 and HSP90 genes in tissues of yellow catfish (Pelteobagrus fulvidraco). Journal of fisheries of china, 2020, 44(5): 707-714.  doi: 10.11964/jfc.20190511815
    [13] XU Xiaoying, LI Xiaoqin, SUN Wentong, JIANG Weibo, PAN Wenqian, WANG Junpeng, TU Zhihan, LENG Xiangjun. Effects of dietary Eucommia ulmoides on growth, flesh quality, and collagen gene expression of grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2018, 42(5): 787-796.  doi: 10.11964/jfc.20170410810
    [14] LI Huifeng, LI Erchao, XU Chang, ZHOU Li, CHEN Liqiao. Effects of silymarin on growth, activities of immune-related enzymes, hepatopancreas histology and intestinal microbiota of the Pacific white shrimp (Litopenaeus vannamei) at low salinity. Journal of fisheries of china, 2021, 45(1): 98-114.  doi: 10.11964/jfc.20200612291
    [15] CHEN Gang, HUANG Jiansheng, ZHANG Jiandong, WANG Zhongliang, TANG Baogui, PAN Chuanhao. Feeding habits and growth characteristics of larvae and juvenile hybrid grouper (Epinephelus fuscoguttatus♀×E. polyphekadion♂). Journal of fisheries of china, 2018, 42(11): 1766-1777.  doi: 10.11964/jfc.20170610856
    [16] LIU Yangyang, YU Haibo, WU Wenyi, ZHONG Mingzhi, XING Junxia, ZHOU Jishu, JI Hong, XUE Min. Effects of dietary lipid levels on growth, body composition, digestive enzyme activities, serum biochemical indexes and antioxidant performance of Polyodon spathula. Journal of fisheries of china, 2018, 42(12): 1940-1956.  doi: 10.11964/jfc.20180111138
    [17] CHEN Siwang, XU Kai, WANG Wenlei, XU Yan, CHEN Changsheng, XIE Chaotian, JI Dehua. Growth and contents of C, N, and P of kelp (Saccharina japonica) cultured in Nanri Island, China and its effects on particulate and dissolved organic matter of seawater. Journal of fisheries of china, 2020, 44(8): 1306-1316.  doi: 10.11964/jfc.20190811917
    [18] LUO Yunhui, WU Bo, XU Shanliang, XU Jilin, WANG Danli. Establishment of a dynamic energy budget (DEB) growth model for Sinonovacula constricta. Journal of fisheries of china, 2021, 45(4): 578-587.  doi: 10.11964/jfc.20190911941
    [19] LI Yanhong, ZHANG Feifei, SHI Yanping, LIAO Maowen, LIU Han, LI Lin, WANG Yongjia, WU Yinglong. Effects of two kinds of polysaccharides on growth, serum antioxidant indices and tissue cadmium accumulation of Schizothorax prenanti. Journal of fisheries of china, 2021, 45(4): 588-599.  doi: 10.11964/jfc.20191012013
    [20] KONG Chun, HUA Xueming, YANG Lu, LIU Tao, YANG Jingfeng, WANG Tan, WANG Gang, WU Zhao, SHI Yonghai, SHUI Chun, SU Meiying. Nutritional physiological effects of soybean meal substituting for fish meal in the feed of obscure puffer (Takifugu fasciatus) and its relationship with soybean antigenic proteins. Journal of fisheries of china, 2017, 41(5): 734-745.  doi: 10.11964/jfc.20160810517
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(4) / Tables(6)

Article views(998) PDF downloads(11) Cited by()

Related
Proportional views

Effects of aflatoxin B1 on growth performance and liver function of juvenile Pelteobagrus fulvidraco

    Corresponding author: WANG Jinlong, 124532076@qq.com
    Corresponding author: HU Yi, huyi740322@163.com
  • 1. Hunan Engineering Research Center for Utilization of Characteristics Aquatic Resources, Hunan Agricultural University, Changsha 410128, China
  • 2. Collage of Life and Environmental Sciences, Hunan University of Arts and Science, Changde    415000, China
  • 3. Hunan Fisheries Science Institute, Changsha    410153, China
  • 4. College of Life Sciences, Hunan Normal University, Changsha    410081, China
  • 5. Jiangsu Aomai Bio-Technology Co.LTD, Nanjing    210000, China
  • 6. Wuhan DBN Aquaculture Technology Co. LTD, Wuhan    430090, China

Abstract: An experiment was conducted to study the effect of aflatoxin B1 on growth performance, digestive enzyme activities in intestinal tract and liver function of juvenile Pelteobagrus fulvidraco. Four experimental diets were prepared to contain 0 (control), 50, 100, 200 μg/kg aflatoxin B1. Each diet was randomly assigned to triplicate cages of 50 fish with an initial average weight of (6.00±0.10) g for 8 weeks. The results showed that: ① There were no significant differences in survival rate, feed coefficient ratio and specific growth rates of juvenile Pelteobagrus fulvidraco among all groups. The activity of trypsin in the AFB1 group significantly increased compared with the control group. When the AFB1 concentration reached 100 and 200 μg/kg, the amylase and lipase activities significantly decreased; ② With increasing AFB1 concentration, the levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), glucose, triglyceride, total bile acid and total cholesterol contents in serum significantly increased, and the AST, ALT activities in the liver significantly decreased; ③With increasing AFB1 concentration, the catalase activity and malondiadehyde content in the liver significantly increased. The superoxide dismutase activity significantly increased when the AFB1 concentration reached 100 and 200 μg/kg; ④ With increasing AFB1 concentration, the expression levels of sod and il-1β in the liver were significantly up-regulated, the expression levels of cat, il-10 and il-8 in the liver were significantly up-regulated when the AFB1 concentration reached 200 μg/kg; ⑤ Histological observations showed that an increase in the concentration of AFB1 caused some liver cells to present slight atrophy, hepatocyte nucleus shifted, cell boundary blurred, liver cell vacuolation degre. The results showed that under the experimental conditions, there was no significant effect on the growth of juvenile Pelteobagrus fulvidraco with the concentration of AFB1≤200 μg/kg, but it could affect the digestion and absorption function of the intestines, cause oxidative stress and inflammatory reactions in the liver, and result in liver function damage with the concentration of AFB1≥50 μg/kg.

  • 黄颡鱼(Pelteobagrus fulvidraco)养殖产量高,是我国重要的水产养殖品种之一,其肉质细嫩、少细刺、味道鲜美,深受人们喜爱[1],集约化养殖需大量的配合饲料。在高温潮湿的环境中,饲料原料或成品会因加工、运输和存储等管理不当等而滋生霉菌,产生黄曲霉毒素(Aflatoxin, AFT)污染[2],可能会降低黄颡鱼配合饲料的品质,进而影响黄颡鱼的健康养殖。AFT是黄曲霉和寄生曲霉的次级代谢产物,其衍生物约有20多种[3],其中黄曲霉毒素B1 (Aflatoxin B1, AFB1)毒性最大,致癌性最强。已有研究发现AFB1在降低其生长[4-6]及繁殖性能[7]、造成机体抗氧化损伤及组织病变的同时[4-6,8],还能在水产动物组织中蓄积[4],进而影响水产动物品质。

    肝脏是水产动物重要的解毒器官,其结构的完整是机体行使正常生理功能的关键。AFB1被水产动物摄入后,能很快被胃肠道吸收并进入循环系统,在动物各组织中蓄积,还会被转化为AFB1-8,9-环氧化物(AFBO),以共价键形式与DNA、RNA及蛋白质结合形成加合物,进而损伤肝细胞[2,9]。有研究表明AFB1被水产动物摄食后,不仅会沉积到肝脏组织中,还会损伤水产动物的肝脏结构,造成肝细胞空泡、坏死及细胞核萎缩[4]。黄莹等[10]在异育银鲫(Carassius auratus gibelio)及花鳗鲡(Anguilla marmorata)[11]的研究中发现,水产动物肝脏中AFB1的积累量随AFB1水平的升高而升高。Raghavan等[12]研究发现在杂交鲟(Acipenser ruthenus ♂×A. baeri♀)饲料中添加40 μg/kg的AFB1会造成局部性肝细胞坏死,产生炎症细胞。Zeng等[13]研究发现AFB1会影响凡纳滨对虾(Litopenaeus vannamei)肝脏储存细胞及分泌细胞的数量,造成肝胰腺细胞萎缩等。此外,AFB1还能造成水产动物肝功能损伤,影响机体抗氧化能力及免疫能力,并降低水产动物的抗病能力,研究发现杂交罗非鱼(Oreochromis niloticus×O. aureus)在摄食AFB1饲料后机体免疫能力降低[5];凡纳滨对虾在摄食AFB1饲料后会引发肝脏氧化应激,造成肝功能受损[14];越南巴沙鱼(Pangasius hypophthalmus)[15]在摄食AFB1后不仅会使机体肝功能受损,还会降低其抗病能力。但也有研究发现,饲料中添加2 000 μg/kg的AFB1对水产动物肝功能及肝脏组织结构均无显著影响[7,16]

    目前,国内外在AFB1对水产动物毒性机理的研究甚少,已有的研究表明不同水产动物对AFB1敏感程度存在差异[8,15,17-18],温水性鱼类对AFB1的耐受性较冷水性鱼类更强[2,8,10,12],且关于AFB1对黄颡鱼影响的研究未见详细报道。因此本实验以黄颡鱼幼鱼为研究对象,研究黄颡鱼配合饲料中添加AFB1对其生长、肠道消化酶及肝脏功能的影响,旨在探明AFB1引发黄颡鱼幼鱼肝脏功能受损的浓度,为黄颡鱼健康养殖提供依据。

1.   材料与方法
  • 参照黄颡鱼商品饲料配方,配制一种以鱼粉和豆粕为主要蛋白源,鱼油为脂肪源的基础饲料,在基础饲料中分别添加0、50、100、200 μg/kg的AFB1,配制成含不同AFB1梯度的4种等氮等脂(40.17%蛋白质,6.95%脂肪)实验饲料(表1)。毒素浓度设计梯度参考已有AFB1报道[5,10,18],添加的AFB1购自美国Sigma公司。饲料原料粉碎后过80目筛,按照配方要求准确称量各种原料,混匀后用饲料制粒机挤压出2.0 mm粒径的颗粒饲料,在阴凉处风干后置于−20 °C冷存备用。其中,AFB1的添加方法按照等比例扩大法,首先将AFB1与少量面粉等比例混合稀释,再按照设计浓度添加到相应饲料中。饲料制作完成后,利用高效液相色谱法检测各组饲料中AFB1的实际值分别为2.13、65.93、102.93、193.98 μg/kg。

    项目
    item
    含量
    content
    饲料原料 ingredients
    秘鲁蒸汽鱼粉 Peru steam fish meal 360
    豆粕 soybean meal 284
    面粉 wheat flour 279.6
    鱼油 fish oil 34
    预混料 premix 25
    氯化胆碱 choline chloride 2
    磷酸二氢钙 Ca(H2PO4) 2 15
    防霉剂 anti-mildew agent 0.3
    抗氧化剂 antioxidant 0.1
    合计 total 1 000
    营养成分 nutritional composition
    粗蛋白质 crude protein 401.7
    粗脂肪 crude lipid 69.5
    粗灰分 crude ash 77.1
    注:预混料为每千克饲料提供碘化钾 (1%) 60 mg、氯化钾 200 mg、六水氯化钴(l%) 50 mg、一水硫酸亚铁400 mg、五水亚硒酸钠(1%) 65 mg、一水硫酸锌 400 mg、五水硫酸铜 30 mg、一水硫酸锰 150 mg、一水硫酸镁 2 000 mg、维生素B1 12 mg、沸石粉 3 645.85 mg、核黄素 12 mg、维生素B6 8 mg、维生素K3 8 mg、维生素B12 0.05 mg、肌醇 100 mg、烟酸 50 mg、泛酸 40 mg、叶酸5 mg、维生素A 25 mg、生物素 0.8 mg、维生素D 35 mg、维生素C 100 mg、维生素E 50 mg、乙氧基喹啉 150 mg、小麦粉 2 434.15 mg
    Notes: The premix provided the following per kg diets: KI(1%) 60 mg, KCl 200 mg, CoCl2·6H2O(1%) 50 mg, FeSO4·H2O 400 mg, Na2Se O3·5H2O(1%) 65 mg, ZnSO4·H2O 400 mg, CuSO4·5H2O 30 mg, MnSO4·H2O 150 mg, MgSO4·H2O 2 000 mg, VB1 12 mg, zeolite power 3 645.85 mg, riboflavin 12 mg, VB6 8 mg, VK3 8 mg, VB12 0.05 mg, inositol 100 mg, niacin acid 50 mg, pantothenic acid 40 mg, folic acid 5 mg, VA 25 mg, biotin 0.8 mg, VD 35 mg, VC 100 mg, VE 50 mg, ethoxyquin 150 mg, wheat flour 2 434.15 mg

    Table 1.  Diet formulation and chemical composition of experiment diets g/kg

  • 实验用鱼购自湖南某省级良种场,养殖实验在湖南省水产科学研究所进行。实验前期,挑选规格一致的黄颡鱼,通过养殖池(3.0 m×1.5 m×1.0 m)暂养驯化,用对照组饲料驯化2周,饥饿24 h后,挑选初始体质量为(6.00±0.10) g的黄颡鱼600尾,随机分成4组,每组3个重复,共12个养殖池(3.0 m×1.5 m×1.0 m),每个养殖池放养黄颡鱼50尾。投喂量以黄颡鱼体质量的3%~5%为标准,日投喂 3 次(8:00、12:00、18:00),每3天调整投喂量。整个养殖实验持续 8周,养殖期间24 h不间断充氧,保持微流水,水温26.0~32.0 °C,溶解氧含量≥6.0 mg/L,氨氮含量≤0.20 mg/L。

  • 实验开始及结束时记录养殖池黄颡鱼数量和重量,投喂饲料总量等用以计算黄颡鱼的各项生长指标。养殖实验结束后,从每个网箱中随机取5尾黄颡鱼,测定体长、体质量以及内脏、肝脏重量,用以计算黄颡鱼肝体比、脏体比、肥满度。

    成活率(survival rate, SR, %)= Nt /N0×100%

    增重率(weight gain rate, WGR, %)=(WtW0)/W0 ×100%

    饲料系数(feed conversion ratio, FCR)=Wf /(WtW0)

    特定生长率(specific growth rate, SGR, %/d)=(lnWt−lnW0) /t ×100%

    肥满度(condition factor, CF, g/cm3)=W/L3×100

    肝体比(hepatosomatic index, HSI, %)= Wh/W×100%

    脏体比(viscerosomatic index, VSI, %)= Wv/W×100%

    式中,Nt为终末尾数;N0为初始尾数;Wt为终末体质量(g);W0为初始体质量(g);Wf为摄入饲料量(g);t为饲喂天数(d);Wh为鱼肝脏重(g);Wv为鱼内脏重(g);W为鱼体质量(g);L为鱼体长(cm)。

  • 养殖实验结束后,停食24 h,每个养殖池随机取5尾黄颡鱼,解剖取其整个肠道,去除肠道内容物及其附着物,用冰双蒸水清洗肠道,滤纸吸干,于−80 °C冰箱内保存。测定前,取肠道组织捣碎后加生理盐水1∶9稀释,并放入匀浆仪内进行冰水浴匀浆。匀浆完毕,在4 °C下3 000 r/min 离心15 min,取上清液备用,于24 h内分析完毕。胰蛋白酶(trypsin)、肠道淀粉酶(amylase)和脂肪酶(lipase)活性均采用南京建成生物工程研究所的试剂盒测定。

  • 养殖实验结束后,停食24 h,从每个养殖池中随机取5尾黄颡鱼,经过丁香酚麻醉后,采取尾静脉抽血。采血后,解剖取整个肝脏,滤纸吸干,于−80 °C冰箱内保存。血液在4 °C下静置过夜后,3500 r/min离心15 min,取上清置于−80 °C冰箱备用。黄颡鱼血清葡萄糖(glucose, GLU)、总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)、总胆汁酸(total bile acid, TBA)、谷草转氨酶(aspartate aminotransferase, AST)、谷丙转氨酶(alanine aminotransferase, ALT)以及肝脏超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、丙二醛(malondiadehyde, MDA)、AST、ALT等均利用南京建成生物工程研究所的试剂盒进行测定。

  • 养殖实验结束后,禁食24 h,在每个养殖池中随机选取3尾黄颡鱼,解剖进行肝脏采样,用4%多聚甲醛固定肝组织24 h。常规酒精脱水、二甲苯透明、浸蜡、包埋、切片、H.E染色后封片观察,染色剂购自南京建成科技有限公司。观察时每组选取3张非连续性切片,送至武汉赛维尔生物科技有限公司进行切片扫描,扫描结束后采用Case Viewer图像分析系统分析,每张切片选取同一视野,分别在100倍与200倍下观察黄颡鱼肝脏组织结构,并进行拍照。

  • 在1.5 mL无酶管中加入1.0 mL Trizol试剂,取30~50 mg在‒80 °C冻存的肝脏组织于无酶管中,用低温匀浆仪进行匀浆处理;匀浆完毕后,室温放置5 min,使肝脏样品充分裂解;在裂解好的样品中加入200 μL氯仿,充分振荡15 s,室温放置2~3 min;将样品12000 r/min,4 °C离心15 min;吸取400 μL上层水相到新的1.5 mL无酶管中,加入400 μL异丙醇,颠倒混匀,室温放置10 min后,样品12 000 r/min,4 °C离心10 min;弃上清,加入1.0 mL 75%乙醇,小心振荡,洗涤沉淀,然后12 000 r/min,4 °C离心3 min;弃75%乙醇,并重复上步骤;弃75%乙醇,干燥,使管内酒精充分挥发;用30 µL DEPC·H2O溶解沉淀,于‒80 °C存备用。

  • cDNA的合成应用PrimeScript®RT reagent Kit with gDNA Eraser试剂盒。cDNA的合成分两步:①基因组DNA的除去反应,反应体系共10 μL(5×gDNA Eraser Buffer 2 μL,gDNA Eraser 1 μL,模板RNA 1 μL,RNase Freed H2O 6 μL),于42 °C孵育2 min;②反转录反应,反应体系共20 μL(5×PrimeScript®Buffer2 4 μL,PrimeSycript®RT Enzyme Mix1 1 μL,RT Primer Mix 1 μL,反应液10 μL,RNase Freed H2O 4 μL),于37 °C孵育15 min,并在85 °C下加热5 s合成cDNA模板。

  • 黄颡鱼肝脏内参基因(β-actin)、超氧化物歧化酶(superoxide dismutase, sod)、过氧化氢酶(catalase, cat)、白介素-1β (interleukin-1βil-1β)、白介素-8(interleukin-8,il-8)、白介素-6(interleukin-6,il-6)、白介素-10 (interleukin-10,il-10)等引物序列见表2。引物设计过程:通过NCBI查找黄颡鱼的基因序列;Primer Premier 5软件设计挑选引物;上海生工有限公司合成引物。Real Time PCR反应体系共12.5 μL (SYBR 6 μL,Forward primer 0.5 μL,Reverse primer 0.5 μL,cDNA 0.5 μL,RNase Freed H2O 5 μL),实时荧光定量反应程序为95 °C预变性30 s,95 °C变性5 s,55 °C退火25 s,72 °C延伸15 s,共40个循环,最后于70~95 °C下收集数据60 s,荧光定量的结果运用表达量E=2−ΔΔCt方法进行计算,其中ΔΔCt=(Ct目的基因‒Ct内参基因)实验组‒(Ct目的基因‒Ct内参基因)对照组

    目的基因
    target gene
    序列号
    accession no.
    上游引物
    forward primer(5′ to 3′)
    下游引物
    reverse primer(5′ to 3′)
    产物长度/bP
    product length
    β-actin EU161066.1 CTGCTGCCTCTTCCTCCTCT CCGCAGGACTCCATACCC 133
    sod KX455916.1 TTGGAGACAATACAAATGGGTG CATCGGAATCGGCAGTCA 129
    cat KX455919.1 GAAGTTCTACACCGATGAGGGA ACAGGGTTGCCTTCAGAGTTTA 288
    il-1β MF770571.1 AGCGACTGTGGATTTGACTCA CCTGGATACGGATTCCTTTGT 109
    il-8 KY218792.1 TGAAGGTCTGCCTGAATCCA GCTTGTTGTTATGTTGACCCTTG 96
    il-10 KY218793.1 AGGTTCCTCCTGCTTTCTTTG CTGTTGAATAGTGCGGTGTCC 194
    il-6 XM_027176013.1 CTGCACTATCTTGCCCTGTTG TCTGGTCTGGAGCGTGAGC 121

    Table 2.  Primer sequences used in the study

  • 实验结果先利用WPS表格进行数据整理,并采用SPSS 19.0对实验数据进行统计分析,实验结果用平均值±标准误(mean±SE)表示。首先对实验数据进行单因素方差分析(One-Way ANOVA),如果组间差异显著(P<0.05),采用Duncan氏进行多重比较。

2.   结果
  • 各处理组黄颡鱼幼鱼特定生长率、增重率、饲料系数、成活率、脏体比和肥满度均无显著差异(P>0.05),但随着AFB1添加量升高,黄颡鱼幼鱼肝体比呈上升趋势,当饲料中AFB1添加量为200 μg/kg时肝体比达到最高且显著高于对照组(P<0.05)(表3)。

    项目   
    items   
    AFB1添加量/(μg/kg diet) supplemented AFB1
    050100200
    初始均重/g IBW 6.00±0.01 6.01±0.00 5.99±0.00 6.01±0.01
    终末均重/g FBW 18.17±0.39 19.82±1.61 18.34±1.33 18.37±1.47
    增重率/% WGR 202.70±6.25 229.98±26.92 206.18±22.27 205.66±24.07
    特定生长率/(%/d) SGR 1.88±0.04 2.01±0.14 1.89±0.12 1.88±0.14
    饲料系数 FCR 2.06±0.10 1.97±0.36 1.97±0.19 1.96±0.24
    成活率/% SR 86.00±9.02 85.00±0.58 86.00±3.46 94.00±2.31
    肝体比/% HSI 2.06±0.15a 2.38±0.11ab 2.41±0.08ab 2.74±0.13b
    脏体比/% VSI 13.13±0.60 11.76±0.53 12.07±0.36 12.01±0.41
    肥满度/(g/cm3) CF 0.97±0.01 1.04±0.03 0.97±0.02 1.01±0.02
    注:表中同一行数据右上标字母不同表示差异显著,P<0.05;下同
    Notes: Data in the same lime with different superscript letters mean significant difference (P<0.05),the same below

    Table 3.  Effect of aflatoxin B1 on the growth of juvenile P. fulvidraco n=3

  • 饲料中添加AFB1显著提高了黄颡鱼幼鱼胰蛋白酶活性(P<0.05),50 μg/kg AFB1添加组胰蛋白酶活性最高。与对照组相比,100 μg/kg和200 μg/kg AFB1添加组肠道淀粉酶和脂肪酶活性显著降低(P<0.05)(表4)。

    项目   
    items   
    AFB1添加量/(μg/kg diet) supplemented AFB1
    050100200
    胰蛋白酶/(U/mg prot) trypsin 1656.17±48.48a 1906.71±56.02b 1847.09±42.88b 1834.37±29.45b
    淀粉酶/(U/mg prot) amylase 58.80±1.51c 54.63±4.70bc 41.82±2.21a 48.02±1.13ab
    脂肪酶/(U/g prot) lipase 23.48±1.28b 43.64±1.25c 14.30±1.30a 18.31±2.00a

    Table 4.  Effect of aflatoxin B1 on the digestive enzyme activities in the intestinal tract of juvenile P. fulvidraco n=3

  • 随着饲料中AFB1添加量升高,黄颡鱼幼鱼血清GLU和TG含量呈显著上升趋势(P<0.05),TC含量在200 μg/kg AFB1添加组显著高于对照组(P<0.05),其余各处理组间无显著差异。与对照组相比,AFB1添加组血清AST与ALT活性显著升高(P<0.05),100 μg/kg AFB1添加组血清TBA含量显著高于其他各组(P<0.05)(表5)。

    项目   
    items   
    AFB1添加量/(μg/kg diet) supplemented AFB1
    050100200
    葡萄糖/(mg/dL) GLU 108.72±0.96a 120.72±2.41b 157.16±3.15c 226.84±2.49d
    总胆固醇/(mmol/L) T-CHO 5.50±0.15a 5.62±0.14ab 5.69±0.05ab 5.93±0.05b
    甘油三酯/(mmol/L) TG 2.62±0.04a 2.77±0.02b 4.07±0.05d 3.29±0.08c
    总胆汁酸/(μmol/L) TBA 11.33±0.55a 11.50±0.54ab 13.42±0.55c 13.12±0.67bc
    谷丙转氨酶/(U/L) ALT 4.88±0.33a 7.95±0.25b 7.36±0.86b 18.70±1.26c
    谷草转氨酶/(U/L) AST 23.93±1.68a 34.38±2.35b 33.24±0.63b 43.08±2.06c

    Table 5.  Effect of aflatoxin B1 on serum biochemical indexes of juvenile P. fulvidraco n=3

  • 饲料中AFB1含量低于50 μg/kg对黄颡鱼幼鱼肝脏AST、ALT活性无影响,当AFB1浓度达100 μg/kg时,黄颡鱼幼鱼肝脏AST活性(图1-a)显著下降(P<0.05),而肝脏ALT活性(图1-b)则在AFB1浓度达200 μg/kg时显著下降(P<0.05)。

    Figure 1.  Effect of aflatoxin B1 on activities of AST, ALT in liver of juvenile P. fulvidraco

  • 经含AFB1的饲料投喂56 d后,各实验组黄颡鱼幼鱼出现绿肝现象。肝脏组织学(图版)观察可知,50 μg/kg组与对照组相比无明显影响,而当AFB1浓度达200 μg/kg,黄颡鱼幼鱼部分肝细胞出现轻微萎缩、肝细胞核移位、细胞界限模糊、肝细胞内空泡化程度增加。

    Figure 图版.  Effect of aflatoxin B1 on liver tissue structure of juvenile P.fulvidraco

  • 随着AFB1添加量的升高,黄颡鱼幼鱼肝脏CAT活力和MDA含量显著升高(P<0.05)。当AFB1添加量达100 μg/kg时,黄颡鱼幼鱼肝脏SOD活性显著升高(P<0.05),其余各组间无显著差异(P>0.05)(表6)。黄颡鱼幼鱼肝脏sod (图2-a)基因表达量会着随AFB1浓度上升显著上调(P<0.05),cat(图2-b)基因表达量则在200 μg/kg AFB1组显著上调(P<0.05)。

    项目   
    items   
    AFB1添加量/(μg/kg diet) supplemented AFB1
    050100200
    过氧化氢酶/(U/mg) CAT9.46±1.67a13.65±0.38b15.51±1.95b19.42±0.56c
    超氧化物歧化酶/(U/mg) SOD151.62±14.51ab137.18±10.11a167.51±7.57bc193.20±4.59c
    丙二醛/(nmol/mg) MDA8.31±0.45a10.97±0.61b18.06±0.99c26.03±1.31d

    Table 6.  Effect of aflatoxin B1 on the antioxidant indexes in the liver of juvenile P. fulvidraco n=3

    Figure 2.  Effect of aflatoxin B1 on liver antioxidant related gene expression of juvenile P. fulvidraco

  • 随着AFB1浓度的升高,il-1β (图3-a)基因表达量显著上调(P<0.05),当AFB1水平达200 μg/kg时,il-8 (图3-b)基因及il-10 (图3-c)表达量出现显著上调(P<0.05),而各毒素组il-6 (图3-d)基因表达量与对照组无显著差异(P>0.05)。

    Figure 3.  Effect of aflatoxin B1 on liver inflammatory related gene expression of juvenile P. fulvidraco

3.   讨论
  • AFB1是一种剧毒物质,高剂量AFB1易造成机体死亡,低剂量AFB1会因累积效应导致机体慢性中毒,阻碍生长发育及其对营养物质的吸收[19],造成水产动物体表黄化、生长下降、行为异常等[2]。研究发现,杂交鲟[12]摄食80 μg/kg AFB1饲料25 d后存活率显著下降,巴沙鱼[15]、喀拉鲃(Catla catla)[20]摄食50 μg/kg AFB1饲料后增重率显著降低。然而,在本实验研究发现黄颡鱼幼鱼摄食200 μg/kg AFB1饲料56 d后其生长性能与对照组无明显差异,这与黄莹等[10]研究结果类似,造成这些不同结果的可能原因有:养殖时间短,低剂量累积效应不足以阻碍动物的生长发育;添加AFB1的浓度和方法不同;不同品种或不同阶段水产动物对AFB1生物转化能力有差异等。与此同时,本实验中的饲料系数相对偏高,推测其原因是养殖用水为池塘水,能见度较低,实验所用饲料为沉性料,在投喂时易造成浪费。

    肠道是消化吸收的主要器官,摄食AFB1会引发肠道氧化应激,导致肠道屏障功能受损,从而降低动物的生长性能[19,21]。冯光德等[22]发现肉鸭摄食霉变玉米(含AFB1)后胰淀粉酶、脂肪酶活性无明显影响;Han等[23]发现,饲喂20或40 μg/kg AFB1饲粮时,肉鸭十二指肠内食糜中胰蛋白酶和淀粉酶活性增加。而在本实验中,AFB1浓度达100 μg/kg时,淀粉酶、脂肪酶活性显著下降,这表明适量的AFB1不会对黄颡鱼幼鱼脂肪、糖类等的消化造成影响,但饲料中AFB1浓度过高会加重肠道的负担,从而对淀粉酶、脂肪酶的分泌造成影响。胰蛋白酶一般是以酶原的形式通过胰腺细胞分泌,当动物肠道黏膜屏障损伤时,胰蛋白酶会侵入肠壁,导致胰蛋白酶活性升高[24],实验中AFB1浓度升高,黄颡鱼幼鱼胰腺等发生慢性炎症,胰腺细胞大量释放酶原,使得黄颡鱼幼鱼肠道内的胰蛋白酶活性异常升高,肠道对营养物质的消化吸收能力并未加强。

  • 肝脏是AFB1的主要靶器官,AFB1的毒性作用表现:动物在摄食AFB1饲料后,饲料中的AFB1在机体内通过细胞色素P450氧化酶(cytochrome P450, CYP450)代谢为AFBO,随后以共价键形式与DNA、RNA及蛋白质结合形成加合物[2,9],损伤肝细胞。而肝脏是葡萄糖、脂肪和蛋白质代谢的重要场所。血清甘油三酯、胆固醇能够反映出机体对脂类代谢状况[25],当机体脂质代谢与葡萄糖代谢出现紊乱时,血清葡萄糖、甘油三酯、胆固醇含量升高。转氨酶主要存在于肝细胞胞浆内,参与蛋白质的代谢,催化氨基酸与酮酸之间氨基的转移,ALT活性升高表明机体氨基酸代谢旺盛,合成代谢增强,蛋白质分解减弱,有利于氮的沉积,AST活性升高则表明尿素生成速度加快,氨基酸代谢产物的毒害作用削弱,两者的活性高低可反映机体蛋白质合成及分解的情况[26],当细胞受损时转氨酶可逸出细胞外,导致血清中ALT和AST活性升高,血清中两者的活性常作为反映肝细胞受损的主要敏感指标。本实验中,黄颡鱼摄食AFB1饲料后,血清中GLU、TG、TC和TBA含量以及AST和ALT活性升高,肝脏中AST和ALT活性降低,表明AFB1进入黄颡鱼幼鱼机体后,形成的加合物会造成黄颡幼鱼葡萄糖、脂肪和蛋白质代谢紊乱,肝脏功能受损。本研究结果与在凡纳滨对虾[13]、巴沙鱼[15]、异育银鲫[27]上的研究一致,而在草鱼(Ctenopharyngodon idella)[16]上的研究却发现摄食AFB1含量≤500 μg/kg饲料56 d后AST、ALT无显著变化,未造成肝功能受损,说明不同水产动物对AFB1耐受程度存在差异,但具体机制还需进一步研究。

    肝脏是鱼类最主要的解毒器官,可将体内新陈代谢产生的有毒物质化解成为无毒,毒性较轻或容易被溶解的物质,再经胆汁或尿液排出体外,肝脏结构完整是其行使正常生理功能的重要保障。本实验中,随着饲料中AFB1浓度的上升,黄颡鱼幼鱼机体内形成的加合物逐渐增多,肝脏的解毒作用不足以抵消AFB1的毒性作用,导致鱼体肝体比逐渐增大,部分肝细胞出现轻微萎缩、肝细胞核移位、细胞界限模糊、肝细胞内空泡化程度增加,肝细胞结构受损。有研究发现,罗非鱼摄食超过245 μg/kg AFB1饲料20周以上后会导致肝形态异常,肝脏空泡化严重[5];凡纳滨对虾摄入AFB1(400~2 000 μg/kg)后肝细胞坏死程度加深,肝胰腺空泡数量增加[28],与本实验结果相似,推测AFB1造成肝脏葡萄糖、脂肪和蛋白质代谢紊乱及肝功能破坏可能与肝脏形态损伤有关。

    SOD、CAT作为抗氧化系统中的重要指标,在维持机体氧化与抗氧化平衡中起关键作用,两者可在一定程度上反映机体抗氧化能力[29],MDA作为自由基引发脂质过氧化反应后的最终分解产物,其含量不仅能反映机体脂质过氧化程度,还能反映机体细胞损伤程度[30]。本研究中,随着AFB1浓度的升高,黄颡鱼幼鱼肝脏CAT、SOD活性、MDA含量均呈现升高趋势,sodcat基因表达量也出现显著上调,这与凡纳滨对虾摄食15 mg/kg AFB1后SOD、CAT、GST活性增加类似[31],推测可能是由于AFB1浓度增加,毒性增强,肝脏损伤,产生氧化应激反应,机体生理失衡。白细胞介素(il)是由淋巴细胞等产生的细胞因子,在免疫反应、造血以及炎症过程中起着调节作用。其中,il-6可通过诱导B细胞的产生和T细胞增殖和分化来参与机体的免疫应答[32]il-8能促进炎症细胞趋化和诱导细胞增殖,il-1β作为早期促炎症的关键细胞因子,不仅能调节机体免疫反应,还能在细菌感染时诱导一系列炎症反应[33],而il-10则具有抗炎特性,能减少促炎介质的产生,也能抑制抗原呈递[34]。有研究表明,AFB1能诱发机体炎症反应[32],与本实验研究结果相似,随着饲料中AFB1水平的升高,黄颡鱼幼鱼肝脏促炎症因子il-1βil-8表达量以及抗炎因子il-10表达量显著上调,说明饲料中添加AFB1诱发了黄颡鱼幼鱼肝脏炎症反应。

4.   结论
  • 在本实验条件下,饲料中AFB1含量低于200 μg/kg不影响黄颡鱼幼鱼生长性能,但饲料中AFB1≥50 μg/kg时会影响黄颡鱼幼鱼肠道的消化吸收功能,同时引起肝脏氧化应激及炎性反应,造成肝脏功能损伤。

Reference (34)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return