• ISSN 1000-0615
  • CN 31-1283/S
Volume 45 Issue 10
Oct.  2021
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Effects of dietary zinc on growth, serum non-specific immune indexes, disease resistance and intestinal flora structure in juvenile Litopenaeus vannamei

  • Corresponding author: YANG Qihui, qihuiyang03@163.com
  • Received Date: 2020-07-30
    Accepted Date: 2021-01-12
    Available Online: 2021-06-16
  • The trial was conducted to study the effects of dietary zinc (Zn) on growth, non-specific immune indexes, disease resistance and intestinal flora structure in juvenile Litopenaeus vannamei. Zinc sulfate heptahydrate (ZnSO4·7H2O) was used as the Zn source in this experiment. The healthy prawns [initial body weight (0.45±0.01) g] were fed diets (isonitrogenous and isolipid) containing available Zn levels (i.e., 0, 20, 40, 60, 80, 100, and 150 mg/kg) for 8 weeks, respectively. The results showed that: ① The weight gain rate (WGR) and specific growth rate (SGR) in the 60 mg/kg group were significantly higher than those in the control group, and the best feed conversion rate (FCR), the optimal protein efficiency ratio (PER) and survival rate (SR) were found in 80 mg/kg group.② The crude lipid (CL) contents in 80-150 mg/kg groups were significantly higher than that in control group, and the contents of crude ash (CA) in 40-80 mg/kg groups were significantly higher than that in control group.③ The contents of serum total protein (TP), total cholesterol (TC) and triglyceride (TG) in Zn supplemented groups were significantly higher than those in control group. ④ Dietary Zn supplement increased the activities of serum superoxide dismutase (SOD), phenol oxidase (PO), alkaline phosphatase (AKP) and acid phosphatase (ACP) and total antioxidant capacity (T-AOC), and significantly decreased the content of malondialdehyde (MDA) in serum, indicating that Zn could improve the non-specific immunity of L. vannamei.⑤ In the experiment of artificial acute infection with Vibrio harveyi, the survival rate of shrimp increased firstly and then decreased with the increase of Zn levels. The survival rate in Zn supplemented group was significantly higher than that in control group, and the highest survival rate and disease resistance were found in 80 mg/kg group. ⑥ In the analysis of intestinal flora, the numbers of effective OTUs in 40 mg/kg and 100 mg/kg groups were significantly higher than that in control group. With the increase of dietary Zn, the Ace index and Chao1 index increased significantly, while the Shannon index and Simpson index had no significant changes. The results showed that an optimal level of Zn could promote the growth and improve the disease resistance of juvenile L. vannamei. The growth performance and disease resistance of L. vannamei were poor when Zn was deficient in diets, while excessive Zn level inhibited the growth of L. vannamei to a certain extent. Overall, based on the WGR, the growth performance of L. vannamei was significantly improved by supplementing 94.46 mg/kg Zn to the diets. Based on the evaluation of disease resistance, the optimal dietary Zn supplement of L. vannamei was 80 mg/kg.
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  • [1] 王彩理, 刘丛力, 滕瑜. 南美白对虾的营养需求及饲料配制[J]. 天津水产, 2008(3-4): 7-12.Wang C L, Liu C L, Teng Y. The nutrition need and development of Penaeus vannamei[J]. Tianjin Fisheries, 2008(3-4): 7-12(in Chinese).
    [2] 农业农村部渔业渔政管理局. 中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2020.Bureau of fisheries, ministry of agriculture and rural Affairs. China Fishery Statistical Yearbook[M]. Beijing: China Agriculture Press, 2020 (in Chinese).
    [3] 安文强, 黎文伟, 谭北平, 等. 凡纳滨对虾对饲料中钙、磷的营养需求[J]. 水产科学, 2020, 39(1): 1-11.An W Q, Li W W, Tan B P, et al. Effects of dietary calcium and phosphorus levels on growth, body composition, tissue calcium and phosphorus deposition and serum biochemical indices in pacific white shrimp Litopenaeus vannamei[J]. Fisheries Science, 2020, 39(1): 1-11(in Chinese).
    [4] Baltaci A K, Yuce K, Mogulkoc R. Zinc metabolism and metallothioneins[J]. Biological Trace Element Research, 2018, 183(1): 22-31. doi: 10.1007/s12011-017-1119-7
    [5] Vallee B L, Galdes A. The metallobiochemistry of zinc enzymes[J]. Advances in Enzymology and Related Areas of Molecular Biology, 1984, 56: 283-430.
    [6] Muralisankar T, Bhavan P S, Radhakrishnan S, et al. Effects of dietary zinc on the growth, digestive enzyme activities, muscle biochemical compositions, and antioxidant status of the giant freshwater prawn Macrobrachium rosenbergii[J]. Aquaculture, 2015, 448: 98-104. doi: 10.1016/j.aquaculture.2015.05.045
    [7] Brandão-Neto J, Stefan V, Mendonça B B, et al. The essential role of zinc in growth[J]. Nutrition Research, 1995, 15(3): 335-358. doi: 10.1016/0271-5317(95)00003-8
    [8] Luo Z, Tan X Y, Zheng J L, et al. Quantitative dietary zinc requirement of juvenile yellow catfish Pelteobagrus fulvidraco, and effects on hepatic intermediary metabolism and antioxidant responses[J]. Aquaculture, 2011, 319(1-2): 150-155. doi: 10.1016/j.aquaculture.2011.06.047
    [9] Maage A, Julshamn K. Assessment of zinc status in juvenile Atlantic salmon (Salmo salar) by measurement of whole body and tissue levels of zinc[J]. Aquaculture, 1993, 117(1-2): 179-191. doi: 10.1016/0044-8486(93)90134-K
    [10] Kuz'mina V V. The influence of zinc and copper on the latency period for feeding and the food uptake in common carp, Cyprinus carpio L.[J]. Aquatic Toxicology, 2011, 102(1-2): 73-78. doi: 10.1016/j.aquatox.2010.12.018
    [11] Clearwater S J, Farag A M, Meyer J S. Bioavailability and toxicity of dietborne copper and zinc to fish[J]. Comparative Biochemistry and Physiology-Part C: Toxicology & Pharmacology, 2002, 132(3): 269-313.
    [12] 郭腾飞, 黄旭雄, 苏明, 等. 饲料锌添加水平对凡纳滨对虾免疫抗菌机能和溶菌酶mRNA及Toll受体mRNA表达的影响[J]. 水产学报, 2011, 35(7): 1081-1089.Guo T F, Huang X X, Su M, et al. Effects of zinc supplementation in diet on the immunity, Vibrio-resistant ability, lysozyme mRNA and Toll receptor mRNA expressions in the white shrimp (Litopenaeus vannamei)[J]. Journal of Fisheries of China, 2011, 35(7): 1081-1089(in Chinese).
    [13] 张海涛, 陈效儒, 董晓慧, 等. 5种锌源对凡纳滨对虾生长、生化和免疫指标的影响[J]. 水产科学, 2017, 36(1): 15-21.Zhang H T, Chen X R, Dong X H, et al. Effects of Zn derived from five compounds on growth performance, biochemical and immunity indices in pacific white leg shrimp Litopenaeus vannamei[J]. Fisheries Science, 2017, 36(1): 15-21(in Chinese).
    [14] 田娟, 郜卫华, 文华. 水产动物肠道健康与饲料添加剂[J]. 动物营养学报, 2018, 30(1): 7-13. doi: 10.3969/j.issn.1006-267x.2018.01.002Tian J, Gao W H, Wen H. Research advances: intestinal health and feed additives in aquatic animals[J]. Chinese Journal of Animal Nutrition, 2018, 30(1): 7-13(in Chinese). doi: 10.3969/j.issn.1006-267x.2018.01.002
    [15] 吴金凤, 熊金波, 王欣, 等. 肠道菌群对凡纳滨对虾健康的指示作用[J]. 应用生态学报, 2016, 27(2): 611-621.Wu J F, Xiong J B, Wang X, et al. Intestinal bacterial community is indicative for the healthy status of Litopenaeus vannamei[J]. Chinese Journal of Applied Ecology, 2016, 27(2): 611-621(in Chinese).
    [16] 谭凤霞, 裴梦婷, 柴毅, 等. 抗菌肽与鱼类肠道健康研究进展[J]. 水产科学, 2020, 39(1): 135-142.Tan F X, Pei M T, Chai Y, et al. A review: Relationship between antimicrobial peptides and intestinal health in fish[J]. Fisheries Science, 2020, 39(1): 135-142(in Chinese).
    [17] 何远法, 迟淑艳, 谭北平, 等. 酵母培养物对凡纳滨对虾肠道菌群结构的影响[J]. 广东海洋大学学报, 2017, 37(4): 21-27. doi: 10.3969/j.issn.1673-9159.2017.04.004He Y F, Chi S Y, Tan B P, et al. Effect of yeast culture on intestinal microbiota of Litopenaeus vannamei[J]. Journal of Guangdong Ocean University, 2017, 37(4): 21-27(in Chinese). doi: 10.3969/j.issn.1673-9159.2017.04.004
    [18] 殷彬, 林仪彤, 迟淑艳, 等. 四种锌源对珍珠龙胆幼鱼生长性能、抗氧化能力以及矿物元素沉积的影响[J]. 水产学报, 2018, 42(7): 1111-1123.Yin B, Lin Y T, Chi S Y, et al. Effects of four forms of zinc on growth performance, antioxidant capacity and vertebrae mineral accumulation of juvenile hybrid grouper (♀Epinephelus fuscoguttatus×♂E. lanceolatu)[J]. Journal of Fisheries of China, 2018, 42(7): 1111-1123(in Chinese).
    [19] 谭丽娜. 锌对幼建鲤消化吸收能力、免疫能力和抗氧化功能的影响[D]. 雅安: 四川农业大学, 2009.Tan L N. Effect of zine on functions of digestion, immune and antioxidative of juvenile Jian carp (Cyprinus carpio var. Jian)[D]. Yaan: Sichuan Agricultural University, 2009 (in Chinese).
    [20] AOAC. Official methods of analysis of AOAC International[M]. 16th ed. Arlington, Virginia: AOAC International, 1995.
    [21] 王秀华, 宋晓玲, 黄倢. 肽聚糖制剂对南美白对虾体液免疫因子的影响[J]. 中国水产科学, 2004, 11(1): 26-30. doi: 10.3321/j.issn:1005-8737.2004.01.005Wang X H, Song X L, Huang J. Effects of peptidoglycan (PG) preparation on humoral immune factors of Litopenaeus vannamei[J]. Journal of Fishery Sciences of China, 2004, 11(1): 26-30(in Chinese). doi: 10.3321/j.issn:1005-8737.2004.01.005
    [22] 曹谨玲, 陈剑杰. 微量元素锌的生理功能及其应用研究[J]. 家畜生态, 2003, 24(4): 62-65.Cao J L, Chen J J. The biochemical function of zinc and its application research[J]. Ecology of Domestic Animal, 2003, 24(4): 62-65(in Chinese).
    [23] Senyushkina E S, Ekaterina А. Troshina. The role of zinc in the synthesis and metabolism of thyroid hormones[J]. Clinical and Experimental Thyroidology, 2021, 16(3): 25-30. doi: 10.14341/ket12697
    [24] Thirunavukkarasu M, Periyakali S B, Subramanian R, et al. Influence of two different dietary zinc sources in freshwater prawn Macrobrachium rosenbergii post larvae[J]. Journal of Oceanology and Limnology, 2018, 37(1): 290-299.
    [25] Li W W, Gong Y N, Jin X K, et al. The effect of dietary zinc supplementation on the growth, hepatopancreas fatty acid composition and gene expression in the Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae)[J]. Aquaculture Research, 2010, 41(11): 828-837. doi: 10.1111/j.1365-2109.2010.02603.x
    [26] 郭建林, 陈建明, 孙丽慧, 等. 日本沼虾幼虾对饲料中锌的需求量[J]. 动物营养学报, 2013, 25(3): 661-668. doi: 10.3969/j.issn.1006-267x.2013.03.026Guo J L, Chen J M, Sun L H, et al. Dietary zinc requirement of juvenile oriental river prawn (Macrobrachium nipponense)[J]. Chinese Journal of Animal Nutrition, 2013, 25(3): 661-668(in Chinese). doi: 10.3969/j.issn.1006-267x.2013.03.026
    [27] Shiau S Y, Jiang L C. Dietary zinc requirements of grass shrimp, Penaeus monodon, and effects on immune responses[J]. Aquaculture, 2006, 254(1-4): 476-482. doi: 10.1016/j.aquaculture.2005.10.033
    [28] 崔立娇, 张利民, 王际英, 等. 饲料中添加锌对星斑川鲽幼鱼生长、生理生化指标和机体抗氧化的影响[J]. 渔业科学进展, 2011, 32(1): 114-121. doi: 10.3969/j.issn.1000-7075.2011.01.018Cui L J, Zhang L M, Wang J Y, et al. Effects of dietary zinc on growth, blood physiological and biochemical indices and antioxidant ability of juvenile starry flounder Platichthys stellatus[J]. Progress in Fishery Sciences, 2011, 32(1): 114-121(in Chinese). doi: 10.3969/j.issn.1000-7075.2011.01.018
    [29] 王道尊, 赵亮, 俞清, 等. 锌对草鱼鱼种生长的影响[J]. 上海水产大学学报, 1995(1): 62-65.Wang D Z, Zhao L, Yu Q, et al. Effect of dietary Zn on the growth of grass carp fingerling[J]. Journal of Shanghai Fisheries University, 1995(1): 62-65(in Chinese).
    [30] 乔永刚, 谭北平, 麦康森, 等. 军曹鱼幼鱼对饲料中锌需要量的研究[J]. 中国海洋大学学报, 2007, 37(1): 105-110.Qiao Y G, Tan B P, Mai K S, et al. Study on the requirement of dietary zinc for juvenile cobia[J]. Periodical of Ocean University of China, 2007, 37(1): 105-110(in Chinese).
    [31] 于万峰, 林黑着, 黄忠, 等. 卵形鲳鲹(Trachinotus ovatus)对饲料中锌的需要量[J]. 动物营养学报, 2019, 31(10): 4602-4611.Yu W F, Lin H Z, Huang Z, et al. Dietary zinc requirement of juvenile golden pompano (Trachinotus ovatus)[J]. Chinese Journal of Animal Nutrition, 2019, 31(10): 4602-4611(in Chinese).
    [32] 杨原志, 吴业阳, 董晓慧, 等. 方斑东风螺饲料中锌需要量的研究[J]. 动物营养学报, 2013, 25(3): 643-650. doi: 10.3969/j.issn.1006-267x.2013.03.024Yang Y Z, Wu Y Y, Dong X H, et al. Dietary zinc requirement of spotted Babylon, Babylonia areolate[J]. Chinese Journal of Animal Nutrition, 2013, 25(3): 643-650(in Chinese). doi: 10.3969/j.issn.1006-267x.2013.03.024
    [33] Tan B P, Mai K S. Zinc methionine and zinc sulfate as sources of dietary zinc for juvenile abalone, Haliotis discus hannai Ino[J]. Aquaculture, 2001, 192(1): 67-84. doi: 10.1016/S0044-8486(00)00435-X
    [34] 尚含乐, 郭贝贝, 彭涛, 等. 饲粮中添加益生菌制剂对舍饲山羊生长性能及血清生化指标的影响[J]. 动物营养学报, 2019, 31(2): 699-704. doi: 10.3969/j.issn.1006-267x.2019.02.026Shang H L, Guo B B, Peng T, et al. Effects of dietary probiotic preparations on growth performance and serum biochemical indexes in housed lambs[J]. Chinese Journal of Animal Nutrition, 2019, 31(2): 699-704(in Chinese). doi: 10.3969/j.issn.1006-267x.2019.02.026
    [35] 蒋明, 黄凤, 文华, 等. 饲料锌对团头鲂幼鱼生长性能、血清生化指标和抗氧化功能的影响[J]. 中国水产科学, 2015, 22(6): 1167-1176.Jiang M, Huang F, Wen H, et al. Effects of dietary zinc on growth, serum biochemical indices, and antioxidant responses in juvenile blunt snout bream, Megalobrama amblycephala[J]. Journal of Fishery Sciences of China, 2015, 22(6): 1167-1176(in Chinese).
    [36] 付志欢, 林雪, 舒绪刚, 等. 不同锌源对吉富罗非鱼生长性能、血清生化指标、血清和肝胰脏中微量元素含量的影响[J]. 动物营养学报, 2019, 31(8): 3690-3698.Fu Z H, Lin X, Shu X G, et al. Effects of different zinc sources on growth performance, serum biochemical indices and trace element contents in serum and hepatopancreas of genetic improvement of farmed tilapia[J]. Chinese Journal of Animal Nutrition, 2019, 31(8): 3690-3698(in Chinese).
    [37] Ruas C B G, dos Santos Carvalho C, de Araujo H S S, et al. Oxidative stress biomarkers of exposure in the blood of cichlid species from a metal-contaminated river[J]. Ecotoxicology and Environmental Safety, 2008, 71(1): 86-93. doi: 10.1016/j.ecoenv.2007.08.018
    [38] Tsikas D. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges[J]. Analytical Biochemistry, 2017, 524: 13-30. doi: 10.1016/j.ab.2016.10.021
    [39] Feng L, Tan L N, Liu Y, et al. Influence of dietary zinc on lipid peroxidation, protein oxidation and antioxidant defence of juvenile Jian carp (Cyprinus carpio var. Jian)[J]. Aquaculture Nutrition, 2011, 17(4): 875-882. doi: 10.1111/j.1365-2095.2011.00858.x
    [40] 王铵静, 杨奇慧, 谭北平, 等. 大豆酶解蛋白对凡纳滨对虾幼虾生长性能、血清生化指标、非特异性免疫力和抗病力的影响[J]. 广东海洋大学学报, 2018, 38(1): 14-21. doi: 10.3969/j.issn.1673-9159.2018.01.003Wang A J, Yang Q H, Tan B P, et al. Effects of enzymolytic soybean meal on growth performance, serum biochemical indices, non-specific immunity and disease resistance of juvenile Litopenaeus vannamei[J]. Journal of Guangdong Ocean University, 2018, 38(1): 14-21(in Chinese). doi: 10.3969/j.issn.1673-9159.2018.01.003
    [41] 黄辉洋, 李少菁, 王桂忠. 甲壳动物酚氧化酶活力及其在养殖中的应用[J]. 海洋通报, 2000, 19(3): 79-84. doi: 10.3969/j.issn.1001-6392.2000.03.014Huang H Y, Li S J, Wang G Z. Studies on the crustacean phenoloxidase activity and its application[J]. Marine Science Bulletin, 2000, 19(3): 79-84(in Chinese). doi: 10.3969/j.issn.1001-6392.2000.03.014
    [42] Liuxy P C, Lee K K, Chen S N. Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon[J]. Letters in Applied Microbiology, 1996, 22(6): 413-416. doi: 10.1111/j.1472-765X.1996.tb01192.x
    [43] 郁维娜, 戴文芳, 陶震, 等. 健康与患病凡纳滨对虾肠道菌群结构及功能差异研究[J]. 水产学报, 2018, 42(3): 399-409.Yu W N, Dai W F, Tao Z, et al. Characterizing the compositional and functional structures of intestinal micro-flora between healthy and diseased Litopenaeus vannamei[J]. Journal of Fisheries of China, 2018, 42(3): 399-409(in Chinese).
    [44] 郑金秀, 胡菊香, 池仕运, 等. 大宁河与香溪河细菌群落分布[J]. 环境科学与技术, 2016, 39(8): 171-177.Zheng J X, Hu J X, Chi S Y, et al. Distribution of bacterial communities in Daning River and Xiangxi River[J]. Environmental Science & Technology, 2016, 39(8): 171-177(in Chinese).
    [45] 韩少锋. 罗非鱼腐败过程菌群结构分析及腐败菌的分离、鉴定与调控[D]. 北京: 中国农业科学院, 2010.Han S F. Analysis of the bacterial community structure change in tilapia during spoilage and isolation, identification and control of spoilage organisms[D]. Beijing: Chinese Academy of Agricultural Sciences, 2010 (in Chinese).
    [46] 华蔚颖. 应用454测序技术分析菌群结构的方法学研究[D]. 上海: 上海交通大学, 2010.Hua W Y. The method study of the application of 454 pyrosequencing on microbial community analysis[D]. Shanghai: Shanghai Jiao Tong University, 2010 (in Chinese).
    [47] Turnbaugh P J, Ley R E, Mahowald M A, et al. An obesity-associated gut microbiome with increased capacity for energy harvest[J]. Nature, 2006, 444(7122): 1027-1031. doi: 10.1038/nature05414
    [48] Abid A, Davies S J, Waines P, et al. Dietary synbiotic application modulates Atlantic salmon (Salmo salar) intestinal microbial communities and intestinal immunity[J]. Fish & Shellfish Immunology, 2013, 35(6): 1948-1956.
    [49] Cahenzli J, Köller Y, Wyss M, et al. Intestinal microbial diversity during early-life colonization shapes long-term IgE levels[J]. Cell Host & Microbe, 2013, 14(5): 559-570.
    [50] Ramirez R F, Dixon B A. Enzyme production by obligate intestinal anaerobic bacteria isolated from oscars (Astronotus ocellatus), angelfish (Pterophyllum scalare) and southern flounder (Paralichthys lethostigma)[J]. Aquaculture, 2003, 227(1-4): 417-426. doi: 10.1016/S0044-8486(03)00520-9
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Effects of dietary zinc on growth, serum non-specific immune indexes, disease resistance and intestinal flora structure in juvenile Litopenaeus vannamei

    Corresponding author: YANG Qihui, qihuiyang03@163.com
  • 1. Laboratory of Aquatic Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang    524088, China
  • 2. Aquatic Animals Precision Nutrition and High-Efficiency Feed Engineering Research Centre of Guangdong Province, Zhanjiang 524088, China

Abstract: The trial was conducted to study the effects of dietary zinc (Zn) on growth, non-specific immune indexes, disease resistance and intestinal flora structure in juvenile Litopenaeus vannamei. Zinc sulfate heptahydrate (ZnSO4·7H2O) was used as the Zn source in this experiment. The healthy prawns [initial body weight (0.45±0.01) g] were fed diets (isonitrogenous and isolipid) containing available Zn levels (i.e., 0, 20, 40, 60, 80, 100, and 150 mg/kg) for 8 weeks, respectively. The results showed that: ① The weight gain rate (WGR) and specific growth rate (SGR) in the 60 mg/kg group were significantly higher than those in the control group, and the best feed conversion rate (FCR), the optimal protein efficiency ratio (PER) and survival rate (SR) were found in 80 mg/kg group.② The crude lipid (CL) contents in 80-150 mg/kg groups were significantly higher than that in control group, and the contents of crude ash (CA) in 40-80 mg/kg groups were significantly higher than that in control group.③ The contents of serum total protein (TP), total cholesterol (TC) and triglyceride (TG) in Zn supplemented groups were significantly higher than those in control group. ④ Dietary Zn supplement increased the activities of serum superoxide dismutase (SOD), phenol oxidase (PO), alkaline phosphatase (AKP) and acid phosphatase (ACP) and total antioxidant capacity (T-AOC), and significantly decreased the content of malondialdehyde (MDA) in serum, indicating that Zn could improve the non-specific immunity of L. vannamei.⑤ In the experiment of artificial acute infection with Vibrio harveyi, the survival rate of shrimp increased firstly and then decreased with the increase of Zn levels. The survival rate in Zn supplemented group was significantly higher than that in control group, and the highest survival rate and disease resistance were found in 80 mg/kg group. ⑥ In the analysis of intestinal flora, the numbers of effective OTUs in 40 mg/kg and 100 mg/kg groups were significantly higher than that in control group. With the increase of dietary Zn, the Ace index and Chao1 index increased significantly, while the Shannon index and Simpson index had no significant changes. The results showed that an optimal level of Zn could promote the growth and improve the disease resistance of juvenile L. vannamei. The growth performance and disease resistance of L. vannamei were poor when Zn was deficient in diets, while excessive Zn level inhibited the growth of L. vannamei to a certain extent. Overall, based on the WGR, the growth performance of L. vannamei was significantly improved by supplementing 94.46 mg/kg Zn to the diets. Based on the evaluation of disease resistance, the optimal dietary Zn supplement of L. vannamei was 80 mg/kg.

  • 凡纳滨对虾(Litopenaeus vannamei),主要分布在南美太平洋沿岸水域[1],是当今世界上三大养殖虾类中单位产量最高的品种。据统计,2019年我国凡纳滨对虾养殖总产量高达181.5万t[2]。凡纳滨对虾生长快、环境适应力强[3],因其肉质鲜嫩、养殖效益高而在水产养殖上得到广泛的认可,前景可观。锌是维持生物机体生理活性所必需的微量元素,只需极少量即可发挥其重要的生物作用。锌几乎存在于动物的所有细胞中,在维持生物膜的稳定性和完整性方面起着重要作用[4]。锌参与动物体内许多酶的组成或激活,从而调节机体的免疫和能量代谢[5-7]

    在水产养殖上,锌可以维持鱼类的正常生长和发育、提高免疫力。尽管鱼类可以从水体中吸收锌,但大多数养殖水体中锌的含量无法满足鱼类的生长需求[8]。缺乏锌元素会影响鱼的生理状况,比如生长迟缓、发育畸形等,甚至会导致免疫功能受损;相反,饲料中锌添加量过高,不仅增加饲料成本,而且会影响水产动物对钙、镁、铁等矿物元素的吸收和利用,严重时会导致锌中毒[9-11]。因此,饲料中添加适量锌是非常有必要的。适量的锌有利于鱼类的生长,提高饲料转化率,节约养殖成本,并提高抗病力。凡纳滨对虾是较低等的水生动物,不具备特异性免疫机能,仅依靠非特异性免疫机能维持自身免疫。已有研究表明,饲料中添加锌无机盐或锌螯合盐,可以促进凡纳滨对虾幼虾的生长及改善机体的免疫抗菌机能[12-13]。目前,肠道健康作为水产领域研究的一个热点[14-17],其往往取决于肠道菌群的结构和分布,肠道微生态的平衡可促进肠道免疫系统的完善。有研究表明在水产动物饲料中添加锌有利于水产动物肠道健康[18-19],而有关饲料中锌水平对水产动物肠道菌群的研究相对较少。

    本实验通过在饲料中添加不同水平的锌,研究其对凡纳滨对虾非特异性免疫力、抗病力及肠道菌群结构的影响,从而评估锌在凡纳滨对虾幼虾饲料中的适宜添加量,为锌在凡纳滨对虾饲料中的合理使用提供理论依据。

1.   材料与方法
  • 实验以脱维酪蛋白(粗蛋白质含量为85%)、明胶、白鱼粉为主要蛋白质源,鱼油和玉米油为主要脂肪源配制基础饲料,配方具体比例与营养水平见表1。以ZnSO4·7H2O作为本实验的锌源,在每千克基础饲料中分别添加0 (对照组)、20、40、60、80、100、150 mg/kg的有效锌,制成7组等氮等脂的实验饲料。各原料经粉碎机粉碎后过80目筛网,按配方准确称取各饲料组分并做相应标记。各组原料按配方比例采用逐级扩大法混合均匀后,加入鱼油、玉米油以及适量的纯净水充分混匀,使用双螺杆挤条机制成粒径为1.0和1.5 mm 2种规格的实验饲料,经后熟化(60 °C,30 min)在阴凉干燥处风干后,分装密封,置−20 °C保存待用。

    项目   
    items   
    含量
    content
    原料/% ingredient
     脱维酪蛋白 vitamin-free casein 33.00
     明胶 gelatin 8.00
     白鱼粉 white fish meal 8.00
     玉米淀粉 corn starch 25.00
     鱼油 fish oil 2.00
     玉米油 corn oil 2.00
     维生素预混料1 vitamin premix 1.00
     矿物质预混料2 mineral premix 2.00
     氯化胆碱 choline chloride 0.10
     抗坏血酸 ascorbic acid 0.05
     磷酸二氢钙 monocalcium phosphate 1.50
     羧甲基纤维素钠 sodium carboxymethylcellulose 2.00
     微晶纤维素 microcrystalline cellulose 15.35
     合计 total 100.00
    营养水平 nutrient levels
     粗蛋白质/% crude protein 39.55
     粗脂肪/% crude lipid 7.12
     锌/(mg/kg) zinc 2.88
    注:1. 维生素预混料为每千克饲料提供:维生素A 10.00 mg,维生素D 120.00 mg,维生素E 99.00 mg,维生素K 10.00 mg,纤维素 589.65 mg,硫胺素 25.50 mg,核黄素 25.00 mg,吡哆醇 50.00 mg,氰钴胺素 0.10 mg,叶酸 6.25 mg,泛酸钙 61.00 mg,肌醇 800.00 mg,烟酸 201.00 mg,生物素 2.50 mg;2. 矿物质预混料为每千克饲料提供:高碘酸钾 0.06 mg,六水合氯化钴 8.14 m,五水硫酸铜 39.68 mg,柠檬酸铁 27.42 mg,七水合硫酸锰 0.24 mg,磷酸钙 160.00 mg,一水硫酸镁 24.86 mg,氯化钾 30.66 mg,亚硒酸钠 4.00 mg,沸石粉 1 648.38 mg
    Notes: 1. Vitamin premix provided the following ingredients per kg of feed: vitamin A 10.00 mg, vitamin D 120.00 mg, vitamin E 99.00 mg, vitamin K 10.00 mg, cellulose 589.65 mg, thiamin 25.50 mg, riboflavin 25.00 mg, pyridoxine 50.00 mg, cyanocobalamine 0.10 mg, folic acid 6.25 mg, calcium pantothenate 61.00 mg, inositol 800.00 mg, niacin 201.00 mg, biotin 2.50 mg;2. mineral premix provided the following ingredients per kg of feed: KIO4 0.06 mg, CoCl2·6H2O 8.14 mg, CuSO4·5H20 39.68 mg, Ferric citrate 27.42 mg, MnSO4·7H2O 0.24 mg, Ca3(PO4)2 160.00 mg, MgSO4·H2O 24.86 mg, KCl 30.66 mg, NaSeO3 4.00 mg, Zeolite powder 1 648.38 mg

    Table 1.  Formula and nutrients levels of the basic diets (dry matter)

  • 本实验于广东海洋大学海洋生物研究基地室内养殖系统进行。实验虾苗购于湛江雷州兴海农虾苗厂,暂养于室外池中至规格为0.30~0.70 g。24 h禁食后,挑选健康、规格均匀的幼虾[初始体质量为(0.45±0.01) g]进行随机分组至28个0.30 m3的玻璃纤维钢桶。实验设计为7个处理,4个重复,每个重复40尾幼虾。初次日投喂量为幼虾体质量的10%,每日7:00、11:00、16:00和21:00定时投喂。根据对虾生长状况、摄食量、水温等因素的变化,调整投喂量。养殖期间换水量为30%~50%,前期隔天换水,后期每天换水。定期测量水温[(29.5±0.8) °C]、盐度(29~31)、溶解氧(5~6 mg/L)、氨氮含量(<0.03 mg/L)、pH(7.8~8.2)等,并观察虾的生长活动,及时记录实验期间虾的死亡情况。养殖周期为8周。

  • 养殖周期结束后停喂24 h,捞取凡纳滨对虾计数并称重记录。每个重复随机取10尾对虾进行围心腔抽血,将血液合并于1.5 mL的离心管,4 °C静置12 h后进行离心(离心参数为4 °C,4 000 r/min,10 min),然后取上清液保存于−80 °C超低温冰箱中,用于血清生化指标及非特异性免疫指标的测定。用经过灭菌的镊子及剪刀解剖对虾,获得肝胰腺和全肠分别置于液氮中速冻,随后转移至−80 °C冻存,肝胰腺用于测定非特异性免疫酶活性,全肠用于肠道菌群分析。另外,每个重复再随机取5尾对虾置于密封袋中保存于−20 °C,用于后期全虾体成分的分析。

  • 根据实验开始到结束的记录,包括幼虾初始体质量、日投喂量、各重复死亡情况、实际养殖天数等,计算增重率、饲料系数、特定生长率、蛋白质效率及存活率。

    式中,NtN0分别为实验终末存活对虾的数量和实验初始幼虾的数量(尾);WtW0分别为实验终末和实验初始对虾的总重(g);Wz为实验期间死亡对虾的总重(g);t为养殖天数(d);F为对虾摄食饲料的干重(g);P为饲料粗蛋白质含量(%)。

  • 实验饲料及全虾的常规分析方法[20]如下:水分(moisture)含量的测定采用105 °C常压干燥恒重法;粗蛋白质(CP)含量的测定采用凯氏定氮仪检测法;粗脂肪(CL)含量的测定采用索氏抽提法;粗灰分(CA)含量的测定采用550 °C马弗炉灼烧法。

  • 血清中总蛋白(TP)、总胆固醇(TC)、甘油三酯(TG)的含量,以及血清中超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)的活性以及丙二醛(MDA)含量、总抗氧化能力(T-AOC)的测定均严格按照试剂盒(南京建成生物工程研究所)说明书进行操作。另外,血清中酚氧化物酶(PO)活性的测定参照王秀华等[21]的方法。

  • 肠道微生物群结构由北京百迈客生物科技有限公司进行高通量测序分析。通过对虾肠道有效操作分类单元(operational taxonomic units,OTUs)统计、物种多样性分析及肠道菌群结构3个方面来分析饲料中添加锌对凡纳滨对虾肠道菌群的影响。

  • 本实验所用哈维氏弧菌(Vibrio harveyi) 菌株来自广东省水产经济动物病原生物学及流行病学重点实验室。菌种经活化、扩大培养后连续稀释,获得不同浓度的菌液用于攻毒实验。经预实验,获得哈维氏弧菌对凡纳滨对虾的半数致死量(LD50,7 d)为1.0×107 cfu/mL。养殖结束后,每重复随机取10尾虾进行攻毒实验,取50 μL此浓度的哈维氏弧菌菌液注射到实验对虾背部肌肉中,以注射等量的生理盐水作为对照。统计凡纳滨对虾被攻毒后7 d的死亡数量,计算存活率。

  • 利用SPSS Version 17.0软件对实验数据进行分析,采用单因素方差分析方法(One-Way ANOVA)。若存在显著性差异(P<0.05),则进行Duncan氏多重比较检验,以平均值±标准差(mean ± SD)的形式表示实验结果。

2.   结果
  • 终末体质量 (FBW)、WGR、SGR、SR和PER随着饲料中锌添加量的增加呈先升后降的趋势,其中FBW、WGR和SGR均在60 mg/kg组达到最大值,SR和PER在80 mg/kg组达到最高值,均显著高于对照组 (P<0.05);FCR则呈先降后升的趋势,在80 mg/kg组达到最低值。锌添加组FBW、WGR、SGR、FCR和PER组间均无显著差异(P>0.05),而SR在最高添加组(150 mg/kg组)显著低于80 mg/kg组(P<0.05)(表2)。以WGR作为判断依据,经多项式模型分析得出:凡纳滨对虾幼虾饲料中锌的最适添加量为94.46 mg/kg (图1)。

    锌添加量/(mg/kg)
    Zn supplement
    初始体质量/g
    IBW
    终末体质量/g
    FBW
    增重率/%
    WGR
    特定生长率/(%/d)
    SGR
    饲料系数
    FCR
    存活率/%
    SR
    蛋白质效率
    PER
    00.45±0.018.88±0.22a1872.99±49.75a5.32±0.04a1.94±0.05b74.17±3.82a1.35±0.04a
    200.45±0.019.36±0.35ab1980.10±78.21ab5.42±0.07ab1.74±0.10a77.50±2.50ab1.50±0.08ab
    400.45±0.019.60±0.13ab2033.96±28.00ab5.47±0.02ab1.69±0.12a77.50±4.33ab1.55±0.11b
    600.45±0.0110.03±1.04b2128.34±230.97b5.54±0.18b1.67±0.16a76.67±5.20ab1.57±0.15b
    800.45±0.019.74±1.06ab2065.52±234.97ab5.48±0.20ab1.60±0.11a82.50±4.33b1.64±0.10b
    1000.45±0.019.82±0.40ab2081.31±87.84ab5.50±0.07ab1.72±0.04a75.83±1.44ab1.52±0.04b
    1500.45±0.019.64±0.18ab2042.22±40.18ab5.47±0.03ab1.76±0.04a75.00±2.50a1.49±0.03ab
    注:同一列上标不同字母表示差异显著(P<0.05),下同
    Notes: Different letters in the same column show significant difference (P<0.05), the same below

    Table 2.  Effects of Zn supplement in dietary on growth performance of L. vannamei n=4

    Figure 1.  Relationship between Zn supplement and WGR of L. vannamei

  • 饲料中添加锌对凡纳滨对虾体水分含量的影响较小;锌添加组对虾粗蛋白含量与对照组无显著差异(P>0.05),而20 mg/kg组和100 mg/kg组粗蛋白含量显著低于80 mg/kg组(P<0.05);对照组粗脂肪含量显著低于80~150 mg/kg添加组(P<0.05);40~80 mg/kg添加组对虾粗灰分含量显著高于对照组(P<0.05)(表3)。

    锌添加量/(mg/kg)
    Zn supplement
    水分/%
    moisture
    粗蛋白质/%
    CP
    粗脂肪/%
    CL
    粗灰分/%
    CA
    075.70±0.6372.65±0.53ab7.70±0.64a13.12±0.07a
    2075.56±0.2072.03±0.70a8.27±0.50ab13.12±0.08a
    4075.83±0.1672.97±0.58ab8.45±0.31ab13.37±0.05b
    6075.79±0.7372.51±0.62ab8.51±0.72ab13.37±0.16b
    8075.69±0.8773.54±0.82b9.14±0.27b13.33±0.06b
    10075.69±0.2871.91±0.59a8.70±0.48b13.22±0.08ab
    15075.55±0.0972.54±0.49ab9.19±0.63b13.13±0.07a

    Table 3.  Effects of Zn supplement on body composition of L. vannamei (dry matter basis) n=4

  • 饲料中添加锌显著影响凡纳滨对虾的各项血清生化指标(P<0.05),随着饲料中锌添加水平的提高,各指标均呈先上升后下降的趋势。锌添加组对虾血清中TP、TC均显著高于对照组(P<0.05),均在80 mg/kg组达到最大值;对照组TG含量显著低于40~150 mg/kg组(P<0.05),在60 mg/kg组达到最大值(表4)。

    锌添加量/(mg/kg)
    Zn supplement
    总蛋白/(g/L)
    TP
    总胆固醇/(mmol/L)
    TC
    甘油三酯/(mmol/L)
    TG
    0 46.960±0.270a 0.073±0.001a 0.067±0.001a
    20 57.140±1.080b 0.082±0. 000b 0.069±0.003ab
    40 57.450±1.840b 0.084±0.001c 0.071±0.001bc
    60 56.330±0.940b 0.085±0.001c 0.072±0.001c
    80 57.480±1.130b 0.085±0.001c 0.071±0.002bc
    100 55.990±1.040b 0.084±0.001c 0.070±0.002bc
    150 55.460±2.130b 0.084±0.001c 0.069±0.001bc

    Table 4.  Effects of Zn supplement on serum biochemical index of L. vannamei n=4

  • 随着饲料中锌水平的提高,对虾血清中SOD、PO、T-AOC、AKP、ACP的活性均呈现先上升后下降的趋势,其中SOD、PO、T-AOC和AKP的活性均在80 mg/kg组达到最大值,而ACP活性在100 mg/kg组达到最大值,均显著高于对照组(P<0.05);血清中MDA的含量随着锌添加量的增加呈先降后升的趋势,80 mg/kg组MDA含量最低,显著低于对照组(P<0.05)(表5)。

    锌添加量/
    (mg/kg)
    Zn supplement
    超氧化物歧化酶/
    (mmol/L)
    SOD
    酚氧化酶/
    (mmol/L)
    PO
    丙二醛/
    (mmol/L)
    MDA
    总抗氧化能力/
    (mM)
    T-AOC
    碱性磷酸酶/
    (金氏单位/100 mL)
    AKP
    酸性磷酸酶/
    (金氏单位/100 mL)
    ACP
    0170.88±5.47a441.67±6.80a20.62±0.36e8.79±0.37a2.63±0.29a5.95±0.12a
    20177.48±4.16abcd445.83±10.76ab19.57±0.37d9.33±0.31ab3.09±0.04b6.07±0.09a
    40186.37±3.84bcd460.42±14.23bc18.99±0.20bc9.63±0.34bc3.52±0.24cd6.27±0.07b
    60187.17±4.02cd472.92±12.50c18.74±0.20ab9.93±0.14bc3.53±0.08cd6.95±0.09c
    80188.55±9.46d493.75±7.98d18.50±0.45a10.23±0.24c3.69±0.14d7.07±0.09c
    100176.07±8.82abc464.58±7.98c19.20±0.10cd9.84±0.33bc3.31±0.05bc7.11±0.17c
    150175.30±11.59ab458.33±6.80bc19.28±0.14cd9.80±0.18bc3.14±0.13b6.98±0.16c

    Table 5.  Effects of Zn supplement on serum non-specific immune indexes of L. vannamei n=4

  • 凡纳滨对虾经哈维氏弧菌攻毒7 d后,锌添加组对虾存活率均显著高于对照组(P<0.05)(图2)。随着饲料中锌水平的提高其存活率显著升高后下降,在80 mg/kg组达到峰值。

    Figure 2.  The survival rate of L. vannamei challenged with V. harveyi

  • 随着饲料中锌添加量的增加,对虾肠道有效OTU数目先升后降,以对照组OTU最低,添加组中40和100 mg/kg组OTU显著高于对照组(P<0.05),其余各组间无显著差异(P>0.05)(图3)。

    Figure 3.  Effective OTUs in intestine of L. vannamei

  • 微生物多样性是基于Illumina HiSeq测序平台,利用双末端测序(Paired-End)的方法,构建小片段文库进行测序。肠道微生物群落的丰富度和多样性可以用Alpha多样性分析反映。覆盖率指数(Coverage)在各组中均大于0.99,说明各组样品均达到测序要求。100 mg/kg组丰富度Ace指数显著高于对照组(P<0.05),其他添加组与对照组差异不显著(P>0.05);对照组丰富度Chaol指数显著低于40、60和100 mg/kg组(P<0.05)。其余物种多样性指数,香农指数(Shannon)和辛普森指数(Simpson)在各组之间无显著差异(P>0.05)(表6)。

    锌添加量/(mg/kg)
    Zn supplement
    Ace指数
    Ace index
    Chao1指数
    Chao1 index
    香农指数
    Shannon index
    辛普森指数
    Simpson index
    覆盖率指数
    Coverage
    0251.64±74.48a248.95±76.06a3.82±0.820.86±0.060.99±0.00
    20300.72±7.48ab296.47±4.24abc4.47±0.550.88±0.030.99±0.00
    40321.57±50.62ab325.04±53.54bc4.49±0.300.87±0.020.99±0.00
    60321.77±19.81ab328.60±16.08bc4.00±0.260.83±0.030.99±0.00
    80273.77±20.89a273.24±15.80ab4.07±0.660.86±0.020.99±0.00
    100347.75±14.63b354.03±11.58c4.19±0.300.86±0.020.99±0.00
    150281.56±17.36ab279.11±23.45ab3.94±0.450.86±0.010.99±0.00

    Table 6.  Effects of Zn supplement on gut species diversity of L. vannamei n=4

  • 本实验凡纳滨对虾肠道菌群主要由变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、软壁菌门(Tenericutes)、放线菌门(Actinobacteria)等门类组成。各组对虾肠道中的菌群组成门类相似,但在各组所占比例有一定的差异。80和150 mg/kg组变形菌门的相对丰度明显低于对照组,其余各组则明显高于对照组。80 mg/kg组厚壁菌门的相对丰度明显高于对照组。除40 mg/kg组外,其余各组软壁菌门的相对丰度均低于对照组。与对照组相比,锌添加组放线菌门的相对丰度均有所下降(图4)。

    Figure 4.  Communities and relative content of bacteria at the level of Phylum in intestine of L. vannamei

    凡纳滨对虾肠道菌群在属水平上除了部分无法鉴别的菌属(unclassified)外,主要有鲁杰氏菌属(Ruegeria)、Motilimonas、弧菌属(Vibrio)和Hopperia等。锌添加组对虾肠道中鲁杰氏菌属和Motilimonas的相对丰度均高于对照组;而Hopperia相对丰度均低于对照组。另外,弧菌属在80和150 mg/kg组相对丰度明显高于对照组,其余各组差异不大(图5)。

    Figure 5.  Communities and relative content of bacteria at the level of Genus in intestine of L. vannamei

3.   讨论
  • 锌是动物细胞内含量最多的微量元素,参与动物体内蛋白质、脂类、糖类、维生素和矿物质等的代谢过程[22]。动物对饲料中微量元素的需求均有一个适宜的范围,锌也一样。锌具有促生长的作用,其原因可能是锌参与碳酸酐酶、DNA聚合酶、RNA聚合酶、碱性磷酸酶、乳酸脱氢酶等酶的合成或激活,从而促进动物机体细胞的分裂和增殖,可加快动物的生长和发育[22-23]。本实验以七水合硫酸锌作为锌的来源添加到饲料中,可减少锌与其他影响因子结合的几率,从而提高凡纳滨对虾对锌的吸收。本实验得出,饲料中添加适量的锌可显著提高凡纳滨对虾幼虾的增重率、特定生长率及蛋白质效率,这与张海涛等[13]的研究结果相似。同样,有研究表明锌有利于提高罗氏沼虾(Macrobrachium rosenbergii)[24]和中华绒螯蟹(Eriocheir sinensis)[25]的采食量,从而促进生长并提高养殖成活率。另外,缺锌会导致罗氏沼虾[6]、日本沼虾(Macrobrachium nipponense)[26]、斑节对虾(Penaeus monodon)[27]、星斑川鲽(Platichthys stellatus)[28]、草鱼(Ctenopharyngodon idella)[29]、军曹鱼(Rachycentron canadum)[30]、卵形鲳鲹(Trachinotus ovatus)[31]等甲壳动物和鱼类生长受阻、体格消瘦畸形、成活率下降。

    在对虾体成分分析中,适宜的锌添加量可显著影响粗脂肪和粗灰分含量,但不影响水分和粗蛋白含量。而张海涛等[13]研究表明,饲料中添加锌同时影响凡纳滨对虾体粗脂肪、粗灰分和粗蛋白的含量。实验对象同样是凡纳滨对虾幼虾,存在研究结果差异的原因可能是饲料配方粗蛋白的含量不同。在其他甲壳动物和软体动物的相关研究中,饲料中添加锌对日本沼虾[27]、方斑东风螺(Babylonia areolata)[32]和皱纹盘鲍(Haliotis discus hannai)[33]体粗蛋白、粗脂肪及粗灰分含量均无显著影响。因此,关于锌水平影响对虾等无脊椎水产动物体组成的机制有待进一步探究。

    据上述研究可说明锌是水产动物生长中所必需的微量元素,对大多数水产动物具有促生长作用,且需通过在饲料中添加锌的方式来满足水产动物的生长需要。本实验以增重率作为评价指标,通过二项式模型拟合,得出凡纳滨对虾幼虾饲料中锌的最适添加量为94.46 mg/kg。

  • 血液对维持动物机体内环境的稳态有着重要的作用,因此通过测定血液生化指标,可反映动物机体营养物质代谢、病理变化以及健康免疫状况[34],是衡量动物健康和生理状态的重要依据。血清中的TP含量在一定程度上能反映动物对蛋白质的代谢能力[35]。本实验中锌添加组凡纳滨对虾血清TP含量显著升高,此结果表明锌在一定程度上提高了凡纳滨对虾对饲料中蛋白质的消化和吸收能力,从而促进幼虾生长。血清中的TC和TG是衡量动物血清中脂质含量的重要指标。本实验结果显示,锌可显著提高凡纳滨对虾血清中TC和TG的含量,这与张海涛等[13]的研究结果一致,且在团头鲂(Megalobrama amblycephala)[35]和吉富罗非鱼(GIFT Oreochromis niloticus)[36]上得到的结论相似,这说明锌对水产动物脂质代谢具有一定的促进作用。

    SOD是一种抗氧化酶,可以将动物体内有害的超氧自由基(O2−)转化为无害的H2O2,从而降低O2−对细胞的损害[37]。若O2−作用于脂质,使其发生过氧化反应,会生成具有毒性的MDA,机体MDA含量越高,则表明细胞膜脂质过氧化程度越高,细胞受损就越严重[5]。有研究表明,在饲料中添加80 mg/kg锌可提高罗氏沼虾肝胰腺中SOD和过氧化氢酶(CAT)的活性,从而提高罗氏沼虾的抗氧化能力[38]。本实验中,随着饲料锌添加量的增加,血清SOD活性显著升高,MDA含量显著下降,对虾总抗氧化能力明显提升,此结果与郭腾飞等[12]在凡纳滨对虾上的研究一致。此外,在建鲤(Cyprinus carpio var. Jian)[39]和珍珠龙胆石斑鱼(Epinephelus fuscoguttatus ♀×E. lanceolatu ♂)[18]上也获得相似的研究结果。锌影响水生动物抗氧化能力的主要原因可能是,锌参与CuZn-超氧化物歧化酶的合成,能有效清除机体内的O2−,具有增强免疫的作用。此外,锌还可以诱导合成金属硫蛋白,从而抵抗氧自由基对细胞的损伤[19]

    凡纳滨对虾只能通过自身的细胞免疫和体液免疫两种非特异性免疫来抵御外界环境的伤害。PO、ACP和AKP三者在抵御外界病原入侵方面有着重要作用[40]。PO以酶原的形式存在于对虾等甲壳动物体内,当外来病原入侵机体和受到环境应激时PO酶原被激活从而进行免疫识别与防御[41]。AKP和ACP具有催化磷酸单酯水解的功能,是动物体内解毒体系的重要组成。其中锌参与AKP的合成,能够较真实地反映养殖动物锌的营养状况[32]。已有研究表明,饲料中添加适量的锌可显著提高凡纳滨对虾PO、ACP和AKP的活性,从而提高对虾的非特异性免疫力[13]。本实验中,锌添加组中血清PO、AKP和ACP活性均显著高于对照组,说明适量的锌对凡纳滨对虾非特异性免疫功能有积极的作用。有研究指出,动物机体内的锌含量不足时,会影响组织合成AKP[31];锌可以通过提高ACP的活性增强建鲤幼鱼白细胞的溶菌能力[19]

    哈维氏弧菌广泛存在于海洋环境中,是一种革兰氏阴性菌,会导致对虾患发光病、肝胰腺细胞坏死,最终死亡,其危害极大[42]。对养殖动物进行人工攻毒是评估动物免疫力和抗病力的有效方法。本实验攻毒结果表明,饲料中添加适量的锌可提高凡纳滨对虾的抗病力,降低死亡率,其中80 mg/kg组对虾存活率最高、抗病力最强。基于上述凡纳滨对虾非特异性免疫指标和哈维氏弧菌攻毒结果得出,饲料中添加80 mg/kg锌有助于增强凡纳滨对虾的非特异性免疫力,从而提高抗病力。

  • 肠道是消化吸收的重要器官,存在着大量群体结构复杂的微生物。在动物长期的进化过程中,肠道微生物在消化吸收中扮演着重要的角色,与宿主之间存在相互依赖、相互制约的关系[43]

    丰富度指数和多样性指数是检测微生物多样性和复杂性的重要指标,其数值的差异反映的是群落结构和物种数目的不同,其指标由划分的有效OTU数所反映。本实验中40和100 mg/kg组的有效OUT数目显著高于对照组,表明40和100 mg/kg组样本所含物种多于对照组。常用Ace指数和Chao1指数来计算菌群丰富度,二者数值越大代表样本中所含物种总数越多。Shannon指数和Simpson指数用于计算菌群多样性,二者数值越大则表示物种多样性越高[44]。本研究发现,随着饲料中锌含量的增加,Ace指数和Chao1指数值均显著提高,而Shannon指数和Simpson指数值无显著变化,表明在饲料中添加锌可改变凡纳滨对虾肠道微生物群的物种丰富度而不改变其多样性。

    肠道菌群从门水平来看,各组间的菌类组成几乎一致,而菌类所占比例有所不同。在本研究中,变形菌门、拟杆菌门以及厚壁菌门三者是凡纳滨对虾肠道中的优势菌群,而各处理组间菌门所占比例存在差异,这表明在凡纳滨对虾肠道中,菌群的变化在一定程度上受到水体环境和个体健康问题的影响。变形菌门与鱼类样品腐败有着密切的关系,并且有报道证实,患病动物肠道内的变形菌门的相对比例会显著升高[43-45]。在本实验中,80和150 mg/kg组凡纳滨对虾肠道内变形菌门的相对丰度低于对照组,这说明在一定条件下锌可抑制肠道部分有害菌的繁殖,改善肠道的健康状况。有研究表明,拟杆菌门参与糖类、胆汁酸及类固醇代谢,可能影响机体的脂质代谢[46]。本实验中随着饲料中锌添加量的增加,拟杆菌门所占比例先降低后升高,类固醇代谢能力先下降后上升,导致凡纳滨对虾血清中胆固醇和甘油三酯含量先升后降,与血清生化指标的变化趋势一致。厚壁菌门部分亚群能帮助宿主从饮食中吸收能量而促进生长[47]

    肠道菌群从属水平来看,各组间的菌类组成不变。本研究中鲁杰氏菌属、Motilimonas、弧菌属和Hopperia为凡纳滨对虾肠道中的优势菌属。部分弧菌属有助于凡纳滨对虾肠道中碳水化合物的代谢利用;而有些为致病菌,若在肠道中大量繁殖,会危害对虾健康。本实验中,80和150 mg/kg组对虾肠道中弧菌属含量高于对照组,可能是锌促进了弧菌属中有益菌在肠道的定植,从而改善肠道功能。肠道其余菌群也有所变化,其原因可能是对虾在实验过程中受养殖水体中微生物的影响。根据相关研究和本试验结果,肠道菌群结构和组成会影响宿主的营养代谢过程、生理机能,并通过形成防御屏障来保护宿主免受外源病原菌入侵,从而改善免疫系统,有助于提高抗病力[48-50]

4.   结论
  • 综上所述,饲料中添加不同水平的锌对凡纳滨对虾幼虾的生长、非特异性免疫指标、抗病力以及肠道微生物结构具有显著的影响。本实验以增重率作为评价指标时,根据其二项式模型得出,凡纳滨对虾幼虾饲料中锌的最适添加量为94.46 mg/kg;以抗病力作为评价依据时,凡纳滨对虾幼虾饲料中锌的最适添加量为80 mg/kg。

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