• ISSN 1000-0615
  • CN 31-1283/S
Volume 45 Issue 10
Oct.  2021
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Influence of dietary calcium to phosphorus ratio on growth, minerals bioaccumulation and energy metabolism in juvenile swimming crab (Portunus trituberculatus)

  • Corresponding author: ZHOU Qicun, zhouqicun@nbu.edu.cn
  • Received Date: 2020-12-14
    Accepted Date: 2021-01-29
    Available Online: 2021-08-25
  • An 8-weeks feeding trial was conducted to investigate the effects of dietary calcium/phosphorus ratios on the growth performance, minerals bioaccumulation, and energy metabolism in juvenile swimming crab (Portunus trituberculatus). Five semi-purified experimental diets were formulated to contain different ratios of calcium to phosphorus (1.00∶2.00, 1.00∶1.50, 1.00∶1.00, 1.00∶0.75 and 1.00∶0.50, respectively). A total of 150 swimming crab juveniles [initial weight (12.82 ± 0.37) g] were randomly allocated into five groups with three replicates, each and consisted of 30 crabs. The results indicated that crabs fed the diets with calcium to phosphorus ratios of 1.00∶1.00 and 1.00∶0.75 had significantly higher specific growth rate (SGR) and feed efficiency (FE) than those fed the other diets. The highest activity of GOT in hemolymph was observed at crabs fed the diet with calcium to phosphorus ratio of 1.00∶0.50, and AKP and GPT exhibited an increasing trend with the increase of dietary calcium to phosphorus ratios. Moreover, the content of phosphorus in tissues significantly increased with dietary calcium to phosphorus ratios decreasing from 1.00∶0.50 to 1.00∶2.00. Furthermore, the highest ATP content in hepatopancreas was observed in crab fed the diets calcium/phosphorus ratios of 1.00∶1.50 and 1.00∶1.00. The activities of enzymes such as hexokinase (HK) and succinate dehydrogenase (SDH) related to energy metabolism were significantly higher in hepatopancreas of crabs fed the diet with calcium to phosphorus ratio of 1.00∶1.00. Crabs fed the diet containing calcium to phosphorus ratio 1.00∶1.00 also showed significantly higher expression of genes involved in electron transport chain complex, such as nd1, sdhc, cytb and cox Ⅱ than those fed the other diets. The genes related to mitochondrial energetic metabolisms, such as atpase6, sirt3, and nrf1 were significantly up-regulated in crabs fed the diet with calcium and phosphorus ratio of 1.00∶0.75. Overall, based on broken-line and quadratic regression analysis between PWG against dietary calcium to phosphorus ratios, the optimal dietary calcium to phosphorus ratio was estimated to be 1.06-1.26, and this range could be recommended to maintain optimal phosphorus bioaccumulation and promote energy production for juvenile swimming crab.
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  • [1] National Research Council. Nutrient requirements of fish and shrimp[M]. Washington, DC: National Academies Press, 2011: 168-170.
    [2] Lall S. The minerals[J]. Fish Nutrition, 1989, 3(2): 219-257.
    [3] Lovell T. Nutrition and feeding of fish[M]. New York: Van Nostrand Reinhold, 1989.
    [4] Zubay G L. Biochemistry[M]. Reading, MA: Addison-Wesley Publishing Company, 1983.
    [5] 刘颖. 饵料中不同钙磷水平对七彩神仙鱼(Symphysodon haraldi)幼鱼生长性能、钙磷沉积及消化生理的影响[D]. 上海: 上海海洋大学, 2018.Liu Y. Effects of dietary phosphorus and calcium levels on growth, tissue deposition and digestive physiology of juvenile discus fish (Symphysodon haraldi)[D]. Shanghai: Shanghai Ocean University, 2018 (in Chinese).
    [6] 胡鹏莉. 饲料钙磷水平对花鲈生长、钙磷消化吸收和沉积的影响[D]. 厦门: 集美大学, 2019.Hu P L. The effect of dietary calcium and phosphorus levels on growth performance, digestibility, absorption and deposition of calcium and phosphorus on Lateolabrax maculatus[D]. Xiamen: Jimei University, 2019 (in Chinese).
    [7] 吉中力. 淡水环境中饲料不同的钙和磷水平对花鲈(Lateolabrax japonicus)钙磷吸收和沉积的影响[D]. 厦门: 集美大学, 2016.Ji Z L. The effects of dietary calcium and phosphorus levels on the absorption and deposition of calcium and phosphorus of Lateolabrax japonicus reared in freshwater[D]. Xiamen: Jimei University, 2016 (in Chinese).
    [8] Lee Gallagher M, Brown W D, Conklin D E, et al. Effects of varying calcium/phosphorus ratios in diets fed to juvenile lobsters (Homarus americanus)[J]. Comparative Biochemistry and Physiology-Part A: Physiology, 1978, 60(4): 467-471. doi: 10.1016/0300-9629(78)90018-X
    [9] Kitabayashi K, Kurata H, Shudo K, et al. Studies on the formula feed for kuroma prawn. I. on the relationship among glucosamine, phosphorus, and calcium[M]. Tokyo: Bull. Tokai Regional Fish Research Laboratory, 1971: 91-108.
    [10] Jin M, Zhou Q C, Zhang W, et al. Dietary protein requirements of the juvenile swimming crab, Portunus trituberculatus[J]. Aquaculture, 2013, 414-415: 303-308. doi: 10.1016/j.aquaculture.2013.08.028
    [11] Sun P, Jin M, Ding L Y, et al. Dietary lipid levels could improve growth and intestinal microbiota of juvenile swimming crab, Portunus trituberculatus[J]. Aquaculture, 2018, 490: 208-216. doi: 10.1016/j.aquaculture.2018.02.018
    [12] Han T, Wang J T, Li X Y, et al. Effects of dietary phospholipid and cholesterol levels on growth and fatty acid composition of juvenile swimming crab, Portunus trituberculatus[J]. Aquaculture Nutrition, 2018, 24(1): 164-172. doi: 10.1111/anu.12544
    [13] Ding L Y, Jin M, Sun P, et al. Cloning, tissue expression of the fatty acid-binding protein (Pt-FABP1) gene, and effects of dietary phospholipid levels on fabp and vitellogenin gene expression in the female swimming crab Portunus trituberculatus[J]. Aquaculture, 2017, 474: 57-65. doi: 10.1016/j.aquaculture.2017.03.029
    [14] Huo Y W, Jin M, Zhou P P, et al. Effects of dietary protein and lipid levels on growth, feed utilization and body composition of juvenile swimming crab, Portunus trituberculatus[J]. Aquaculture, 2014, 434: 151-158. doi: 10.1016/j.aquaculture.2014.08.011
    [15] Sun P, Jin M, Jiao L F, et al. Effects of dietary lipid level on growth, fatty acid profiles, antioxidant capacity and expression of genes involved in lipid metabolism in juvenile swimming crab, Portunus trituberculatus[J]. British Journal of Nutrition, 2020, 123(2): 149-160. doi: 10.1017/S0007114519002563
    [16] Yuan Y, Sun P, Jin M, et al. Regulation of dietary lipid sources on tissue lipid classes and mitochondrial energy metabolism of juvenile swimming crab, Portunus trituberculatus[J]. Frontiers in Phusiology, 2019, 10: 454.
    [17] Cunniff P A. Official methods of analysis of AOAC international[M]. 16th ed. Washington, DC: AOAC International, 1995.
    [18] Pan L, Yue F, Miao J, et al. Molecular cloning and characterization of a novel-type lysozyme gene in swimming crab Portunus trituberculatus[J]. Fish and Shellfish Immunology, 2010, 29(2): 286-292.
    [19] Cheng K M, Hu C Q, Liu Y N, et al. Effects of dietary calcium, phosphorus and calcium / phosphorus ratio on the growth and tissue mineralization of Litopenaeus vannamei reared in low-salinity water[J]. Aquaculture, 2006, 251(2-4): 472-483. doi: 10.1016/j.aquaculture.2005.06.022
    [20] 高红建, 田晓燕, 袁琰, 等. 三疣梭子蟹配合饲料适宜钙磷水平及其比例的研究[J]. 饲料工业, 2009, 30(6): 18-21.Gao H J, Tian X Y, Yuan Y, et al. Study on dietary of calcium and phosphorus levels and calcium/phosphorus ratio in Portunus trituberculatus[J]. Feed Industry, 2009, 30(6): 18-21(in Chinese).
    [21] Vielma J, Lall S P. Phosphorus utilization by Atlantic salmon (Salmo salar) reared in freshwater is not influenced by higher dietary calcium intake[J]. Aquaculture, 1998, 160(1-2): 117-128. doi: 10.1016/S0044-8486(97)00300-1
    [22] Ye C X, Liu Y J, Tian L X, et al. Effect of dietary calcium and phosphorus on growth, feed efficiency, mineral content and body composition of juvenile grouper, Epinephelus coioides[J]. Aquaculture, 2006, 255(1-4): 263-271. doi: 10.1016/j.aquaculture.2005.12.028
    [23] Sprague M, Dick J R, Tocher D R. Impact of sustainable feeds on omega-3 long-chain fatty acid levels in farmed Atlantic salmon, 2006-2015[J]. Scientific Reports, 2016, 6: 21892. doi: 10.1038/srep21892
    [24] Jin M, Wang M Q, Huo Y W, et al. Dietary lysine requirement of juvenile swimming crab, Portunus trituberculatus[J]. Aquaculture, 2015, 448: 1-7. doi: 10.1016/j.aquaculture.2015.05.021
    [25] Han T, Wang J T, Hu S X, et al. Effects of different dietary lipid sources on growth performance and tissue fatty acid composition of juvenile swimming crab Portunus trituberculatus[J]. Chinese Journal of Oceanology and Limnology, 2015, 33(4): 957-965. doi: 10.1007/s00343-015-4262-z
    [26] Izquierdo M S, Montero D, Robaina L, et al. Alterations in fillet fatty acid profile and flesh quality in gilthead seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of fatty acid profiles by fish oil feeding[J]. Aquaculture, 2005, 250(1-2): 431-444. doi: 10.1016/j.aquaculture.2004.12.001
    [27] Koko G K D, Sarker P K, Proulx É, et al. Effects of alternating feeding regimes with varying dietary phosphorus levels on growth, mineralization, phosphorus retention and loading of large rainbow trout (Oncorhynchus mykiss)[J]. Aquaculture Living Resources, 2010, 23(3): 277-284. doi: 10.1051/alr/2010032
    [28] 张树威. 饲料磷水平对淡水养殖花鲈生长、体组成及脂代谢的影响[D]. 厦门: 集美大学, 2018.Zhang S W. Effects of dietary phosphorus levels on growth, body composition and lipid metabolism of Lateolabrax japonicus reared in freshwater[D]. Xiamen: Jimei University, 2018 (in Chinese).
    [29] Skonberg D I, Yogev L, Hardy R W, et al. Metabolic response to dietary phosphorus intake in rainbow trout (Oncorhynchus mykiss)[J]. Aquaculture, 1997, 157(1-2): 11-24. doi: 10.1016/S0044-8486(97)00141-5
    [30] Onishi T, Suzuki M, Takeuchi M. Change in carp hepatopancreatic enzyme activities with dietary phosphorus levels[J]. Bulletin of the Japanese Society of Scientific Fisheries, 1981, 47(3): 353-357. doi: 10.2331/suisan.47.353
    [31] Ossani G, Dalghi M, Repetto M. Oxidative damage lipid peroxidation in the kidney of choline-deficient rats[J]. Frontiers in Bioscience, 2007, 12: 1174-1183. doi: 10.2741/2135
    [32] 惠天朝, 施明华, 朱荫湄. 硒对罗非鱼慢性镉中毒肝抗氧化酶及转氨酶的影响[J]. 中国兽医学报, 2000, 20(3): 264-266. doi: 10.3969/j.issn.1005-4545.2000.03.018Hui T C, Shi M H, Zhu Y M. Effects of selenium on antioxidant Enzymes and transaminases of liver in cadmium chronic toxic Tilapia nilotica[J]. Chinese Journal of Veterinary Science, 2000, 20(3): 264-266(in Chinese). doi: 10.3969/j.issn.1005-4545.2000.03.018
    [33] 张树威, 鲁康乐, 宋凯, 等. 饲料羟基蛋氨酸钙、DL-蛋氨酸对花鲈生长、抗氧化能力及肠道蛋白酶活性的影响[J]. 水产学报, 2017, 41(12): 1908-1918.Zhang S W, Lu K L, Song K, et al. Effects of crystalline methionine and calcium 2-hydroxy-4-(methylthio) butyrate on growth, antioxidant ability and intestinal protease activities of Japanese seabass (Lateolabrax japonicus)[J]. Journal of Fisheries of China, 2017, 41(12): 1908-1918(in Chinese).
    [34] Zhang C X, Mai K S, Ai Q H, et al. Dietary phosphorus requirement of juvenile Japanese seabass, Lateolabrax japonicus[J]. Aquaculture, 2006, 255(1-4): 201-209. doi: 10.1016/j.aquaculture.2005.11.040
    [35] Yang S D, Lin T S, Liu F G, et al. Influence of dietary phosphorus levels on growth, metabolic response and body composition of juvenile silver perch (Bidyanus bidyanus)[J]. Aquaculture, 2006, 253(1-4): 592-601. doi: 10.1016/j.aquaculture.2005.09.002
    [36] Hurwitz S, Griminger P. The response of plasma alkaline phosphatase, parathyroids and blood and bone minerals to calcium intake in the fowl[J]. The Journal of Nutrition, 1961, 73(2): 177-185. doi: 10.1093/jn/73.2.177
    [37] Scott D, Robins S P, Nicol P, et al. Effects of low phosphate intake on bone and mineral metabolism and microbial protein synthesis in lambs[J]. Experimental Physiology, 1994, 79(2): 183-187. doi: 10.1113/expphysiol.1994.sp003751
    [38] Ladue J S, Wroblewski F. Serum glutamic pyruvic transaminase SGP-T in hepatic disease: a preliminary report[J]. Annals of Internal Medicine, 1956, 45(5): 801-811. doi: 10.7326/0003-4819-45-5-801
    [39] 姚鹰飞. 吉富罗非鱼幼鱼和成鱼饲料有效磷及钙磷比的研究[D]. 武汉: 华中农业大学, 2012.Yao Y F. Dietary available phosphorus and Ca/P requirements for juvenile and adult GIFT strain Nile tilapia (Oreochromis niloticus)[D]. Wuhan: Huazhong Agricultural University, 2012.
    [40] Wang L, Yan B, Liu N, et al. Effects of cadmium on glutathione synthesis in hepatopancreas of freshwater crab, Sinopotamon yangtsekiense[J]. Chemosphere, 2008, 74(1): 51-56. doi: 10.1016/j.chemosphere.2008.09.025
    [41] Wang W, Wu X G, Liu Z J, et al. Insights into hepatopancreatic functions for nutrition metabolism and ovarian development in the crab Portunus trituberculatus: gene discovery in the comparative transcriptome of different hepatopancreas stages[J]. PLoS One, 2014, 9(1): e84921. doi: 10.1371/journal.pone.0084921
    [42] Phillips D J H, Rainbow P S. Strategies of trace metal sequestration in aquatic organisms[J]. Marine Environmental Research, 1989, 28(1-4): 207-210. doi: 10.1016/0141-1136(89)90226-2
    [43] Conklin D E. The role of micronutrients in the biosynthesis of the crustacean exoskeleton[C]//Proceedings of the Second International Conference Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Baton Rouge: Louisiana State University, 1982: 146-165.
    [44] Tan B P, Mai K S, Liufu Z. Response of juvenile abalone, Haliotis discus hannai, to dietary calcium, phosphorus and calcium/phosphorus ratio[J]. Aquaculture, 2001, 198(1-2): 141-158. doi: 10.1016/S0044-8486(00)00595-0
    [45] 黄波, 程开敏, 刘艳妮, 等. 对虾矿物元素营养需求的研究进展[J]. 广东饲料, 2006, 15(6): 30-33. doi: 10.3969/j.issn.1005-8613.2006.06.013Huang B, Cheng K M, Liu Y N, et al. Research progress on mineral nutrient requirements of prawn[J]. Guangdong Fees, 2006, 15(6): 30-33(in Chinese). doi: 10.3969/j.issn.1005-8613.2006.06.013
    [46] 孙大业, 郭艳林, 马力耕. 细胞信号转导[M]. 第2版. 北京: 科学出版社, 1998.Sun D Y, Guo Y L, Ma L G. Cellular signal transduction[M]. 2nd ed. Beijing: Science Press, 1998.
    [47] 杨振鹏, 邓丽, 饶本强, 等. 分子通信与线粒体代谢研究[J]. 肿瘤代谢与营养电子杂志, 2019, 6(3): 277-282.Yang Z P, Deng L, Rao B Q, et al. Molecular communication and mitochondrial metabolism[J]. Electronic Journal of Metabolism and Nutrition of Cancer, 2019, 6(3): 277-282(in Chinese).
    [48] Bremer K, Monk C T, Gurd B J, et al. Transcriptional regulation of temperature-induced remodeling of muscle bioenergetics in goldfish[J]. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 2012, 303(2): R150-R158. doi: 10.1152/ajpregu.00603.2011
    [49] Rose I A, Warms J V B. Mitochondrial hexokinase: Release, rebinding, and location[J]. The Journal of Biological Chemistry, 1967, 242(7): 1635-1645. doi: 10.1016/S0021-9258(18)96139-9
    [50] 郭彪, 王芳, 侯纯强, 等. 温度突变对凡纳滨对虾己糖激酶和丙酮酸激酶活力以及热休克蛋白表达的影响[J]. 中国水产科学, 2008, 15(5): 885-889.Guo B, Wang F, Hou C Q, et al. Effects of acute temperature fluctuation on HK and PK activity, HSP70 relative content in Litopenaeus vannamei[J]. Journal of Fishery Sciences of China, 2008, 15(5): 885-889(in Chinese).
    [51] 王镜岩, 朱圣庚, 徐长法. 生物化学[M]. 第3版. 北京: 高等教育出版社, 2002: 104-105.Wang J Y, Zhu S G, Xu C F. Biochemistry[M]. 3nd ed. Beijing: Higher Education Press, 2002: 104-105.
    [52] 王茂叶. 间歇性低氧训练对小鼠机体细胞色素氧化酶和琥珀酸脱氢酶的影响[J]. 天津体育学院学报, 2005, 20(6): 26-28, 75.Wang M Y. The influence of interval hypoxic training on CCO and SDH of mice[J]. Journal of Tianjin University of Sport, 2005, 20(6): 26-28, 75(in Chinese).
    [53] Mootha V K, Lindgren C M, Eriksson K F, et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[J]. Nature Genetics, 2003, 34(3): 267-273. doi: 10.1038/ng1180
    [54] Burri L, Berge K, Wibrand K, et al. Differential effects of krill oil and fish oil on the hepatic transcriptome in mice[J]. Frontiers in Genetics, 2011, 2: 45.
    [55] Li X L. SIRT1 and energy metabolism[J]. Acta Biochimica et Biophysica Sinica, 2013, 45(1): 51-60. doi: 10.1093/abbs/gms108
    [56] Morash A J, Kajimura M, McClelland G B. Intertissue regulation of carnitine palmitoyltransferase I (CPTI): Mitochondrial membrane properties and gene expression in rainbow trout (Oncorhynchus mykiss)[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2008, 1778(6): 1382-1389. doi: 10.1016/j.bbamem.2008.02.013
    [57] Gleyzer N, Vercauteren K, Scarpulla R C. Control of mitochondrial transcription specificity factors (TFB1M and TFB2M) by nuclear respiratory factors (NRF-1 and NRF-2) and PGC-1 family coactivators[J]. Molecular and Cellular Biology, 2005, 25(4): 1354-1366. doi: 10.1128/MCB.25.4.1354-1366.2005
    [58] Wang C G, Li Z P, Lu Y N, et al. Cyclin D1 repression of nuclear respiratory factor 1 integrates nuclear DNA synthesis and mitochondrial function[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(31): 11567-11572. doi: 10.1073/pnas.0603363103
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    [20] XIANG Xiao, ZENG Benhe, ZHOU Xinghua, CHEN Jian, WANG Wenjuan. Effect of taurine supplemention at different dietary lipid levels on growth performance, body composition, hepatopancreas biochemical index and antioxidant capacity of juvenile common carp (Cyprinus carpio). Journal of fisheries of china, 2018, 42(12): 1927-1939.  doi: 10.11964/jfc.20170910961
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Influence of dietary calcium to phosphorus ratio on growth, minerals bioaccumulation and energy metabolism in juvenile swimming crab (Portunus trituberculatus)

    Corresponding author: ZHOU Qicun, zhouqicun@nbu.edu.cn
  • Laboratory of Fish and Shellfish Nutrition, School of Marine Sciences, Ningbo University, Ningbo    315211, China

Abstract: An 8-weeks feeding trial was conducted to investigate the effects of dietary calcium/phosphorus ratios on the growth performance, minerals bioaccumulation, and energy metabolism in juvenile swimming crab (Portunus trituberculatus). Five semi-purified experimental diets were formulated to contain different ratios of calcium to phosphorus (1.00∶2.00, 1.00∶1.50, 1.00∶1.00, 1.00∶0.75 and 1.00∶0.50, respectively). A total of 150 swimming crab juveniles [initial weight (12.82 ± 0.37) g] were randomly allocated into five groups with three replicates, each and consisted of 30 crabs. The results indicated that crabs fed the diets with calcium to phosphorus ratios of 1.00∶1.00 and 1.00∶0.75 had significantly higher specific growth rate (SGR) and feed efficiency (FE) than those fed the other diets. The highest activity of GOT in hemolymph was observed at crabs fed the diet with calcium to phosphorus ratio of 1.00∶0.50, and AKP and GPT exhibited an increasing trend with the increase of dietary calcium to phosphorus ratios. Moreover, the content of phosphorus in tissues significantly increased with dietary calcium to phosphorus ratios decreasing from 1.00∶0.50 to 1.00∶2.00. Furthermore, the highest ATP content in hepatopancreas was observed in crab fed the diets calcium/phosphorus ratios of 1.00∶1.50 and 1.00∶1.00. The activities of enzymes such as hexokinase (HK) and succinate dehydrogenase (SDH) related to energy metabolism were significantly higher in hepatopancreas of crabs fed the diet with calcium to phosphorus ratio of 1.00∶1.00. Crabs fed the diet containing calcium to phosphorus ratio 1.00∶1.00 also showed significantly higher expression of genes involved in electron transport chain complex, such as nd1, sdhc, cytb and cox Ⅱ than those fed the other diets. The genes related to mitochondrial energetic metabolisms, such as atpase6, sirt3, and nrf1 were significantly up-regulated in crabs fed the diet with calcium and phosphorus ratio of 1.00∶0.75. Overall, based on broken-line and quadratic regression analysis between PWG against dietary calcium to phosphorus ratios, the optimal dietary calcium to phosphorus ratio was estimated to be 1.06-1.26, and this range could be recommended to maintain optimal phosphorus bioaccumulation and promote energy production for juvenile swimming crab.

  • 钙、磷参与动物体内多种生理代谢功能,在物质和能量代谢中起着十分重要的作用,是维持动物正常生长和骨骼发育的重要矿物质元素[1]。钙是构成硬质组织的结构性成分(如骨骼、外骨骼、鳞片和牙齿)的主要元素,除了结构功能以外,钙还参与了血液凝固(脊椎动物)、视力发育、肌肉收缩、卵黄形成、神经冲动传导、渗透压调节,以及作为酶促反应的辅助因子[2]。磷是细胞膜的结构物质,同时也是核酸及能量代谢过程中三磷酸腺苷的重要组成元素,直接参与动物体内每个细胞的产能反应,同时无机磷酸盐也是维持细胞内液和外液正常pH的重要缓冲剂,在细胞功能中扮演着不可或缺的角色[3-4]。几乎所有的能量转换过程都与高能磷酸键的形成和破坏有关,因此钙、磷与生物体的能量代谢和生命活动密切相关。

    钙和磷是饲料中2种重要的矿物元素[1]。饲料中适宜的钙磷比例可促进机体对钙磷及其他矿物元素的吸收和利用,加速骨骼中矿物元素的沉积;而钙磷比例过高或过低则会对动物生长产生抑制作用,甚至对动物的存活构成严重威胁[5-6]。养殖过程中最为高效便捷的补给方式就是在饲料中添加相应的矿物元素,这样可以很好地改善生长性能并提高养殖效益[7]。Gallagher等[8]评估了美洲螯龙虾(Homarus americanus)幼体饲料中适宜的钙磷比,以生长为评价指标,饲料中钙磷比为0.51时美洲鳌龙虾幼体可以获得最佳的生长性能,当钙磷比为1.55或更高时则会导致生理代谢异常。还有研究表明,饲料中钙磷比为1.0时,日本囊对虾(Penaeus japonicus)会获得较好的生长[9]

    三疣梭子蟹(Portunus trituberculatus)隶属于节肢动物门(Arthropoda)甲壳纲(Crustacea)十足目(Decapoda)梭子蟹科(Portinidae)梭子蟹属(Portunus),由于其较高的营养价值和日益增长的市场需求,自20世纪90年代以来,池塘养殖的三疣梭子蟹在我国沿海地区迅速发展[10]。随着三疣梭子蟹养殖规模的扩大,对冰鲜野杂鱼和贝类的需求日益增加,然而在生产中投喂冰鲜饵料存在许多弊端,如容易引起养殖水体污染、携带病原菌导致三疣梭子蟹病害频发、营养不均衡而致使饵料利用率低等问题日益突出,这已成为制约三疣梭子蟹健康养殖的关键因素[11]。近年来,我国已开展三疣梭子蟹营养学的研究,对推动三疣梭子蟹养殖业的健康和可持续发展具有重要意义[10-16]。然而,有关三疣梭子蟹饲料中矿物元素尤其是常量元素的研究还相对较少。因此,本实验拟通过8周投喂实验,探究不同钙磷比对三疣梭子蟹生长性能、血清生化指标、矿物质沉积和线粒体能量代谢的影响,评估三疣梭子蟹幼蟹适宜的钙磷比例。研究结果将为三疣梭子蟹商业化养殖过程中饲料钙磷的添加提供科学的指导,为三疣梭子蟹配合饲料的开发提供理论依据。

1.   材料与方法
  • 实验以秘鲁鱼粉、酪蛋白、磷虾粉和豆粕为蛋白源,鱼油和大豆卵磷脂为脂肪源,分别以NaH2PO4·2H2O、Ca(HPO4)2和CaCl2作为钙源及磷源,添加不同剂量Ca(HPO4)2(磷源利用率为92.4%)[6],用以平衡饲料中的钙磷比(过高会降低磷酸盐的生物利用率),并以海藻酸钠为饲料粘合剂,配制成5组等氮、等脂的实验饲料(粗蛋白质约为46.0%,粗脂肪约为7.5%),其钙磷比例分别为1.00∶2.00、1.00∶1.50、1.00∶1.00、1.00∶0.75和1.00∶0.50,实验饲料配方及营养成分见表1,其中药品购自中国上海国药集团化学试剂有限公司。鱼粉、豆粕和磷虾粉等干性原料用不锈钢万能粉碎机(HK-180,广东旭郎)粉碎后过80目筛分,以逐级扩大法添加矿物质和维生素预混料以及海藻酸钠等微量成分到大量原料中,混合搅拌10 min,将大豆卵磷脂、鱼油、水加入到混合机搅拌10 min,采用双螺杆挤压机(F-26,华南理工大学机械工程研究所)挤成长条状放入制粒机(G-250,华南理工大学机械工程研究所)制成2种粒径的饲料(粒径分别为3和5 mm),置于90 °C的烘箱中熟化30 min,自然风干直至水分含量检测在10%左右即可,真空包装, −20 °C冰柜中保存待用。

    项目 items饲料 (钙/磷) diet No.(Ca/P)
    Diet 1 (1.00∶2.00)Diet 2 (1.00∶1.50)Diet 3 (1.00∶1.00)Diet 4 (1.00∶0.75)Diet 5 (1.00∶0.50)
    原料 ingredients
    鱼粉 fish meal 150.00 150.00 150.00 150.00 150.00
    酪蛋白 casein 100.00 100.00 100.00 100.00 100.00
    磷虾粉 krill meal 50.00 50.00 50.00 50.00 50.00
    豆粕 soybean meal 300.00 300.00 300.00 300.00 300.00
    面粉 wheat flour 145.80 145.80 145.80 145.80 145.80
    鱼油 fish oil 16.00 16.00 16.00 16.00 16.00
    大豆卵磷脂 soybean lecithin 40.00 40.00 40.00 40.00 40.00
    维生素预混料 vitamin premix 1 10.00 10.00 10.00 10.00 10.00
    矿物质预混料 mineral premix 2 20.00 20.00 20.00 20.00 20.00
    磷酸二氢钠 NaH2PO4 ·2H2O 3 0.00 16.43 32.87 58.18 79.05
    磷酸氢二钙 ${\rm{Ca}}\left( {{\rm{HP}}{{\rm{O}}_4}} \right)_2^3$ 0.00 10.00 20.00 20.00 25.00
    氯化钙 ${\rm{CaCl}}_2^3$ 28.85 22.30 15.76 15.76 12.48
    氯化胆碱 choline chloride 3.00 3.00 3.00 3.00 3.00
    海藻酸钠 sodium alginate 20.00 20.00 20.00 20.00 20.00
    糊精 dextrin 4 116.35 96.47 76.57 51.26 28.67
    常规成分 proximate composition
    水分 moisture 117.40 119.90 116.40 117.20 112.70
    粗蛋白质 crude protein 468.10 469.50 472.80 461.70 462.70
    粗脂肪 crude lipid 76.10 74.80 76.40 75.20 74.80
    灰分 ash 93.40 90.70 91.60 92.50 91.20
    磷含量 analyzed phosphorus 32.40 22.10 17.20 12.30 7.40
    钙含量 analyzed calcium 16.50 15.70 15.70 16.10 15.30
    注:1. 维生素预混料参照Jin等[10]实验配制;2. 矿物质预混料(g/kg 预混料):柠檬酸铁 4.57,硫酸锌 9.43,硫酸锰(99%) 4.14,硫酸铜(99%) 6.61,硫酸镁 (99%) 238.97,氯化钴 (99%) 1.36;3. 磷酸二氢钠、磷酸氢二钙及氯化钙由中国上海国药化学试剂有限公司提供;4. 糊精用于平衡饲料中钙、磷的添加量
    Notes: 1. Vitamin premix was based on Jin et al [10];2. Mineral premix (g/kg premix): FeC6H5O7 4.57, ZnSO4·7H2O 9.43, MnSO4·H2O (99%) 4.14, CuSO4·5H2O (99%) 6.61, MgSO4·7H2O (99%) 238.97, CoCl2·6H2O (99%) 1.36; 3. NaH2PO4·2H2O, Ca(HPO4)2, CaCl2 were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); 4. dextrin was used to balauce the supplemental amount of calcium and phosphorus

    Table 1.  Formulation and proximate composition of experimental diets g/kg

  • 从蟹苗场购买同一批次健康无断肢的三疣梭子蟹幼蟹,养殖实验采用蟹公寓养殖模式,在宁波市海洋与渔业科技创新基地开展。正式投喂实验饲料之前,先在蟹公寓(SCU, 33.0 cm × 22.5 cm × 25.0 cm)中暂养2周以适应养殖环境,每个蟹公寓相互独立,并提供连续的流水。期间投喂商业饲料(购自天邦食品股份有限公司,45%粗蛋白和8%粗脂质)。暂养结束后,挑选大小相近的三疣梭子蟹幼蟹150只,初始体质量为(12.82±0.37) g,随机分为5个饲料处理组,每个处理组3个平行,每个平行10只幼蟹,每只幼蟹均被放置在一个蟹公寓框中。投喂实验期间,每天18:00投喂饲料一次,次日早晨6:00检查残饵和死亡情况,做好记录及标注,并将死蟹称重并取出,投喂率为6%~8%,每2周调整1次投喂量。养殖期间,对整个养殖系统的环境因子持续监测,用 YSI Proplus 水质监测仪(YSI,Yellow Springs,Ohio, USA)测定蟹公寓中海水的温度、盐度、pH、氨氮和溶解氧水平。其中蟹公寓的海水水温为26.5~29.0 °C,盐度为24.1~28.4 g/L, pH为7.3~8.0,氨氮含量低于0.05 mg/L,溶解氧含量不低于6.0 mg/L, 磷含量为0.62 × 10-3~1.40 × 10−3 mg/L。

  • 8周养殖实验结束后,对每个蟹公寓中的三疣梭子蟹进行称重,用于计算成活率、增重率、特定生长率、饲料效率和蜕壳率。每个平行取6只幼蟹,将蟹置于冰袋上保持低温,用1 mL无菌注射器在蟹的围心窦处抽取血淋巴至1.5 mL离心管中,每2只蟹的血淋巴混为1管,共3管血淋巴样品,4 °C冰箱中静置过夜,第二天用离心机(Eppendorf 5810R,德国)在4 °C、3 500 r/min条件下离心10 min,取上清液至新的1.5 mL离心管中,储存于−80 °C超低温冰箱(Eppendorf CryoCube F570),用于血清生化指标分析。然后从取血后的三疣梭子蟹中取出肝胰腺,将大量的肝胰腺迅速收集至1.5 mL离心管中,液氮冷冻后带回实验室转移至−80 °C超低温冰箱保存,用于肝胰腺成分组成、矿物质含量和酶活性分析。再从每只梭子蟹中取绿豆大小的肝胰腺样品分装至6只1.5 mL离心管中,保存于−80 °C超低温冰箱中,用于基因表达分析。每个平行从取血和肝胰腺后的三疣梭子蟹取约10 g肌肉组织,收集至1个10 mL的离心管中,−20 °C冰箱中储存,用于测定肌肉成分组成和矿物质含量,按照上述方法,收集全部剩余甲壳至−20 °C冰箱中储存,用于矿物质含量分析。

  • 血淋巴样品按“样品采集”中所述方法进行处理。采用全自动生化分析仪(VITALAB SELECTRA Junior Pros,Netherlands)直接测定碱性磷酸酶(AKP)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、总蛋白(TP)和葡萄糖(GLU)含量。

  • 根据 AOAC[17]的方法进行饲料和组织(肝胰腺和肌肉)的常规成分分析。其中水分含量采用105 °C常压干燥法测定;粗蛋白质含量采用杜马斯燃烧法(N×6.25),通过蛋白质自动分析仪(FP-528,Leco,USA)进行测定;粗脂肪含量采用索氏抽提法,使用全自动脂肪仪(Soxtec System HT6, Tecator, Swede)进行测定,抽提所用有机试剂为石油醚(分析纯,中国国药集团化学试剂有限公司);灰分含量采用马弗炉中于550 °C高温下灼烧8 h测定。

  • 实验饲料、肌肉、肝胰腺及甲壳样品经冻干粉碎并混合均匀后,在80 °C水浴,样品中滴加70% HNO3溶液至有机物完全消解。消解后的溶液经水相注射器过滤器(SCAA -102, ANPEL Laboratory Technologies Inc, China)过滤,使用ICPOES电感耦合等离子体发射光谱仪(ICP-OES, PE 2100DV, Perkin Elmer, USA)测定样品钙、磷含量。

  • 使用南京建成生物工程研究所试剂盒,根据生产厂家说明书分析肝胰腺中ATP的含量。肌酸激酶催化三磷酸腺苷和肌酸生成磷酸肌酸,在636 nm处被吸收;通过测量吸光度值可以计算出组织中ATP含量。

  • 快速称取肝胰腺组织样品,以1∶9倍体积加入预冷的生理盐水(8.9 g/mL),用匀浆机(IKAT25, Germany)进行匀浆操作,并用离心机(Eppendorf 5810R,Germany)于4 °C、4 000 r/min条件下离心10 min。小心吸取上清液至新的1.5 mL离心管中,保存于−80 °C超低温冰箱,用于酶活性分析,主要包括丙酮酸激酶(PK)、己糖激酶(HK)和琥珀酸脱氢酶(SDH),上述所测酶采用南京建成检测试剂盒进行测定。

  • 采用总RNA提取试剂盒(R401-01,Vazyme),根据Trizol法提取三疣梭子蟹肝胰腺组织的总RNA。每个平行取6只不同梭子蟹的肝胰腺样品,提取完的总RNA均稀释到500 ng/μL,两两混样,每个平行3份RNA混合样品,采用分光光度法(Nanodrop 2000,Thermo Fisher Scientific,USA)测定总RNA 浓度。后使用反转录试剂盒Prime Script RT Reagent Kit with g DNA Eraser将RNA反转录成cDNA。荧光定量PCR前使用DNase-free水将cDNA稀释4倍,使用实时荧光定量PCR仪(Eppendorf MGOTercycler nexus GSX1,Germany)进行荧光定量PCR,反应体系为20 μL,包括上下游模板各1.0 μL,2×ChamQ Universal SYBR qPCR MGOTer Mix(Roche,Switzerland),2 μL 4倍稀释后cDNA和6 μL DEPC水。荧光定量PCR的反应程序设置:95 °C (2 min)预变形;95 °C(10 s)、58 °C (10 s)、72 °C (20 s),循环反应45次;95 °C(10 s)、60 °C (60 s)、95 °C (15 s),熔解曲线采集。在本实验中,以β-actin为内参基因,以Diet 1处理组作为相对表达中的对照组,采用2−ΔΔCt 法计算基因的相对表达量。引物设计通过参照美国国立生物技术研究所中心(NCBI)网站上的cDNA序列,使用Prime Premier 5.0引物设计软件设计荧光定量PCR扩增特异性引物,所有引物均由华大基因生物技术有限公司合成,具体的引物序列及相关信息见表2

    基因
    gene
    引物序列 (5′~3′)
    nucleotide sequence (5′-3′)
    产物长度/bp
    product size
    基因序列号或参考文献
    GenBank or references
    nd1 F: CGAAGCCGAGGTAGTGTA 228 AB093006
    R: CGATTTTGCTGAAGGAGA
    sdhc F: CGGCTCCTACCCACACTACT 179 KY406169
    R: CCCAAATCCCACACCAAG
    cytb F: GAACTACGGTTGACTTCTACG 223 KY406169
    R: AGTAATAACTGTTGCCCCTC
    cox F: TATTGTAAGTCAAGAGTCCG 254 AB093006
    R: CTCACAGCATAGAAGGTC
    cox F: GTGAATAACCCGTCTGTAACTT 145 AB093006
    R: CATCTAATAATCGGAACCCTG
    cox F: CAAAGGTTTACGGTGAGGT 132 AB093006
    R: CCATAGGGAGGTCAGTTCAT
    Atpase6 F: TAGCACTCTCTCTACCTTT 167 AB093006
    R: AGCAAGTGTTCCTGGTC
    sirt1 F: CTCCACCACTTCCAACCTTAG 318 PRJNA432636
    R: CCAGCACCAGTCAATACGATG
    sirt3 F: CAACACTGCTCACCACTTCC 259 PRJNA432636
    R: TGGCTTCACTTTGCCCTTA
    nrf1 F: CATTGACGGCATCCCCA 207 AB093006
    R: CACGACACCTTATGTTTCTGG
    β-actin F: GAAGTAGCCGCCCTGGTTGTG [18]
    R: GGGTCAGAATACCTCGCTTGCTC
    注:nd. 还原型烟酰胺腺嘌呤二核苷酸脱氢酶;sdhc. 琥珀酸脱氢酶复合体亚基C;cytb. 细胞色素b;cox. 细胞色素c氧化酶;Atpase. 腺苷三磷酸(ATP)酶;sirt. 沉默信息调节因子;nrf1. 核呼吸因子1
    Notes: nd. NADH dehydrogenase; sdhc. succinate dehydrogenase complex, subunit C; cytb. cytochrome b; cox. cytochrome c oxidase; Atpase. adenosine triphosphatase; sirt. silent information regulator; nrf1. nuclear respiratory factor 1

    Table 2.  Real-time quantitative PCR primers for electron transport chain and mitochondrial energy metabolism related genes

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

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

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

    饲料效率(feed efficiency rate,FER,%)=(WtW0)/Wf×100%

    蜕壳率(molting ratio,MR,%)=2×Nm/(Nt+N0)×100%

    式中,Nt为养殖实验结束时梭子蟹的数量,N0为养殖实验开始时梭子蟹的数量,Wt为终末体质量(g),W0为初始体质量(g),t为实验天数(d),Wf为梭子蟹摄入饲料量(g),Nm为养殖期间梭子蟹蜕壳的次数。

  • 所有的实验数据均以平均值±标准误(n=3)表示,用Levene氏法确定是否符合正态分布和方差齐性,之后再采用SPSS 22.0软件(Chicago,USA)对所有数据进行单因素方差分析(One-Way ANOVA),若存在显著差异,则用Tukey氏检验对数据进行多重比较分析,当P<0.05时,则认为组间在统计学上具有显著性差异。使用GraphPad Prism 8.0.2软件(San Diego,CA,United States)进行图形处理。

2.   结果
  • 三疣梭子蟹幼蟹的成活率为76.67%~83.33%,各饲料组幼蟹的成活率在统计学上无显著性差异(P>0.05)。终末均重(FBW)随着钙磷比例的升高呈现先上升后下降的趋势,投喂钙磷比例1.00∶1.00饲料组三疣梭子蟹增重率、特定生长率显著高于投喂钙磷比例1.00∶2.00饲料组(P<0.05)。以增重率(WGR)为指标,根据折线模型和二次回归曲线模型分析得出三疣梭子蟹幼蟹饲料中适宜钙磷比例分别为1.06和1.26 (图1)。投喂钙磷比为1.00∶1.00和1.00∶0.75饲料组的三疣梭子蟹特定生长率(SGR)和饲料效率(FER)均显著高于投喂其他饲料组,其中投喂钙磷比例1.00∶0.50饲料组的三疣梭子蟹的SGR和FER最低(P<0.05)。蜕壳率(MR)随着钙磷比例的升高呈现先上升后下降的趋势,投喂钙磷比例1.00∶1.00和1.00∶0.75饲料的三疣梭子蟹显著高于投喂钙磷比例1.00∶2.00和1.00∶0.50饲料组(P<0.05)(表3)。

    Figure 1.  Relationship between the weight gain rate and dietary calcium to phosphorus ratios

    参数
    parameters 
    饲料 (钙/磷) diet No. (Ca/P)
    diet 1(1.00∶2.00)diet 2(1.00∶1.50)diet 3(1.00∶1.00)diet 4(1.00∶0.75)diet 5(1.00∶0.50)
    成活率/% survival 83.33 ± 3.33 76.67 ± 3.33 76.67 ± 3.33 80.00 ± 5.77 76.67 ± 3.33
    初始均重/g IBW 11.43 ± 0.51 12.36 ± 0.54 12.47 ± 1.03 12.03 ± 0.32 12.93 ± 0.94
    终末均重/g FBW 28.24 ± 0.95a 31.24 ± 1.40ab 36.52 ± 4.12b 33.54 ± 0.35ab 31.78 ± 2.50ab
    增重率/% WGR 148.07 ± 19.29a 153.34 ± 2.38ab 192.81 ± 6.55c 178.45 ± 1.88bc 145.67 ± 3.41a
    特定生长率/(%/d) SGR 1.62 ± 0.07a 1.66 ± 0.02a 1.92 ± 0.04b 1.83 ± 0.01b 1.60 ± 0.03a
    饲料效率/% FER 0.51 ± 0.03a 0.51 ± 0.02a 0.66 ± 0.05b 0.61 ± 0.02b 0.49 ± 0.01a
    蜕壳率/% MR 1.40 ± 0.03a 1.48 ± 0.05ab 1.78 ± 0.05c 1.65 ± 0.04bc 1.37 ± 0.07a
    注:同行数据上标不同小写字母表示差异显著(P<0.05),下同
    Notes: values in the same row with different superscript letters are significantly different (P < 0.05),the same below

    Table 3.  Effects of dietary calcium phosphorus ratios on growth performance and feed utilization in juvenile P. trituberculatus

  • 不同钙磷比显著影响血淋巴碱性磷酸酶、谷丙转氨酶和谷草转氨酶活性,并随着钙磷比水平的增加呈上升趋势,钙磷比1.00∶0.50饲料组谷草转氨酶活力显著高于其他各组(P<0.05),钙磷比1.00∶0.50饲料组的碱性磷酸酶和谷丙转氨酶酶活力显著高于(1.00∶2.00)、(1.00∶1.50)和(1.00∶1.00)饲料组(P<0.05)。饲料不同钙磷比对血淋巴总蛋白和葡萄糖含量无显著影响(P>0.05)(表4)。

    参数
    parameters 
    饲料(钙/磷) diet No. (Ca/P)
    diet 1(1.00∶2.00)diet 2(1.00∶1.50)diet 3(1.00∶1.00)diet 4(1.00∶0.75)diet 5(1.00∶0.50)
    碱性磷酸酶/(U/L) AKP 15.75 ± 0.49a 17.04 ± 0.21a 18.09 ± 0.15ab 20.48 ± 0.63bc 21.98 ± 0.90c
    总蛋白/(g/L) TP 38.37 ± 1.98 39.20 ± 1.24 36.10 ± 0.46 37.55 ± 0.40 38.19 ± 1.54
    谷丙转氨酶/(U/L) GPT 124.44 ± 8.74a 135.08 ± 8.55ab 140.06 ± 5.27ab 155.74 ± 4.79bc 175.41 ± 1.81c
    谷草转氨酶/(U/L) GOT 141.61 ± 2.80a 152.54 ± 2.50a 150.04 ± 5.68a 184.15 ± 9.64b 219.54 ± 4.64c
    葡萄糖/(mmol/L) GLU 0.87 ± 0.10 0.85 ± 0.05 0.86 ± 0.03 1.01 ± 0.06 1.00 ± 0.09

    Table 4.  Effects of dietary calcium to phosphorus ratios on hemolymph biochemical parameters in juvenile P. trituberculatus

  • 饲料中添加不同钙磷比对幼蟹肝胰腺以及肌肉中的水分和蛋白质含量均无显著性影响(P>0.05)。不同钙磷比显著影响三疣梭子蟹肝胰腺和肌肉中的粗脂肪和灰分含量,幼蟹组织中的灰分含量均随饲料添加钙磷比水平的升高而降低,而脂肪含量显著增加(P<0.05)(表5)。

    参数
    parameters
    饲料 (钙/磷) diet No. (Ca/P)
    diet 1(1.00∶2.00)diet 2(1.00∶1.50)diet 3(1.00∶1.00)diet 4(1.00∶0.75)diet 5(1.00∶0.50)
    肌肉 muscle
    水分 moisture 81.59 ± 0.83 80.59 ± 0.83 81.69 ± 0.53 80.02 ± 0.66 80.11 ± 1.42
    粗蛋白质 crude protein 16.35 ± 0.40 16.03 ± 0.89 16.51 ± 0.50 16.18 ± 0.18 16.32 ± 0.29
    粗脂肪 crude lipid 6.35 ± 0.08a 6.39 ± 0.06a 6.70 ± 0.01b 7.01 ± 0.06c 7.06 ± 0.03c
    灰分 ash 1.91 ± 0.08c 1.85 ± 0.04bc 1.60 ± 0.06ab 1.44 ± 0.09a 1.49 ± 0.13a
    肝胰腺 hepatopancreas
    水分 moisture 78.63 ± 1.54 79.37 ± 1.86 80.65 ± 1.31 79.58 ± 1.49 81.29 ± 2.31
    粗蛋白质 crude protein 6.79 ± 0.46 6.72 ± 1.35 7.14 ± 0.67 7.35 ± 0.62 6.88 ± 0.94
    粗脂肪 crude lipid 10.53 ± 0.29a 10.48 ± 0.31a 12.76 ± 0.41b 13.60 ± 0.19b 13.48 ± 0.31b
    灰分 ash 1.23 ± 0.06b 1.20 ± 0.06b 0.99 ± 0.03a 0.97 ± 0.05a 0.92 ± 0.02a

    Table 5.  Effects of dietary calcium to phosphorus ratios on proximate composition in hepatopancreas and muscle in juvenile P. trituberculatus (wet weight) %

  • 饲料中添加不同比例钙磷对幼蟹的肝胰腺、肌肉和甲壳中的钙含量均无显著差异(P>0.05)。随着饲料中钙磷比水平从1.00∶2.00增加至1.00∶0.50,肝胰腺、肌肉和甲壳中的磷含量显著降低,且投喂钙磷比为1.00∶2.00饲料组幼蟹磷含量显著高于投喂其他饲料组(P<0.05)(图2)。

    Figure 2.  Minerals content in hepatopancreas, muscle and carapace of juvenile P.trituberculatus fed diets containing different calcium to phosphorus ratios

  • 在饲料中添加不同钙磷比显著影响梭子蟹肝胰腺中的ATP含量,投喂钙磷比为1.00∶1.50和1.00∶1.00饲料组幼蟹显著高于投喂其他饲料组(P<0.05)(图3)。

    Figure 3.  ATP content of hepatopancreas of juvenile P.trituberculatus fed diets containing different calcium to phosphorus ratios

  • 投喂钙磷比为1.00∶1.00饲料组幼蟹肝胰腺中HK酶活性显著高于投喂钙磷比为1.00∶2.00组,而随着饲料中钙磷比的进一步增加,肝胰腺HK酶活性逐步降低(P<0.05)。幼蟹肝胰腺中SDH酶活性呈现出于HK相同趋势,投喂钙磷比为1.00∶1.00饲料组SDH活性显著高于钙磷比1.00∶2.00和1.00∶0.50饲料组(P<0.05)。饲料中钙磷比对三疣梭子蟹肝胰腺PK酶活性无显著影响(P>0.05)(图4)。

    Figure 4.  Enzyme activity related to energetic metabolism in the hepatopancreas of juvenile P.trituberculatus fed diets containing different calcium to phosphorus ratios

  • 投喂钙磷比1.00∶2.00饲料组幼蟹肝胰腺nd1和sdhc的相对表达量显著低于其他饲料组(P<0.05)。投喂钙磷比1.00∶1.00和1.00∶0.75饲料组三疣梭子蟹肝胰腺cytb的表达水平显著上调(P<0.05)。投喂钙磷比1.00∶1.50和1.00∶1.00饲料组能显著上调肝胰腺cox Ⅱ的表达水平(P<0.05),然而除钙磷比为1.00∶2.00饲料组的其余4组相比则无显著性差异(P>0.05)。梭子蟹肝胰腺cox Ⅰ和cox Ⅲ的相对表达水平不受饲料中钙磷比的影响(P>0.05)(图5)。

    Figure 5.  Relative mRNA expression levels of genes related to of electron transport chain (a) and mitochondrial energetic metabolism (b) in hepatopancreas of juvenile P. trituberculatus fed diets containing different calcium to phosphorus ratios

    投喂钙磷比为1.00∶0.75和1.00∶0.50饲料组能显著上调肝胰腺ATPase6的表达水平(P<0.05)。而投喂钙磷比1.00∶1.50和1.00∶1.00饲料组幼蟹肝胰腺sirt3的相对表达量显著高于其他饲料组(P<0.05)。此外,投喂钙磷比1.00∶1.50、1.00∶1.00和1.00∶0.75饲料组能显著上调梭子蟹肝胰腺nrf1的表达水平(P<0.05),而投喂钙磷比为1.00∶1.00和1.00∶0.75饲料组相比则无显著性差异(P>0.05)。饲料中钙磷比对幼蟹肝胰腺sirt1的相对表达水平无显著影响(P>0.05)(图5)。

3.   讨论
  • 生长性能是用于评价饲料中营养物质引起机体变化最常用的指标[1]。已有大量的实验评估了饲料中不同钙磷比对动物生长性能的影响,结果表明适宜的钙磷比能够显著提高凡纳滨对虾[19](Litopenaeus vannamei)和七彩神仙鱼[5](Symphysodon haraldi)的增重率和特定生长率,本实验也得出了类似的结果。三疣梭子蟹摄食钙磷比为1.00∶1.00的饲料比摄食其余组饲料表现出更好的生长性能。本实验中,三疣梭子蟹的增重率为145.67%~192.81%,高于已有的对同一研究品种获得的结果,其增重率仅仅为31.25%~39.56%[20]。两项研究中增重率的差异主要是由于幼蟹初始体质量不同造成的,与之前研究中幼蟹的初重(31.04±0.81) g相比,本实验的幼蟹初重(12.82±0.37) g明显较小。在本实验中,通过对增重率与钙磷比例的折线和二次回归方程分析,得到三疣梭子蟹幼蟹最适钙磷比为1.06~1.258。该值与已有甲壳类动物的结果相似,如日本囊对虾[9]饲料中推荐的钙磷比为1,同样在鱼类的研究中也表明,商业配方中钙磷比小于2可以使得大西洋鲑(Salmo salar)[21]和石斑鱼(Epinephelus coioides)[22]获得较好的生长。在本实验条件下,投喂过高或过低钙磷比的饲料三疣梭子蟹增重率、特定生长率和饲料效率显著低于最适组,结果表明饲料中过高或过低的钙磷比会导致三疣梭子蟹生长迟缓和饲料利用率降低,饲料中适宜的钙磷比是维持三疣梭子蟹正常生长的必要条件。此外,蜕壳是甲壳动物生长发育所必经的生理活动,三疣梭子蟹的生长受蜕壳和蜕壳频率的极大影响,能量供应不足以及蜕壳后能量稳态的紊乱都会造成甲壳动物蜕壳失败,最终死亡[23]。本实验中,钙磷比为1.00∶0.50饲料组三疣梭子蟹蜕壳率最低,存活率也相对较低,揭示了钙磷比可能在三疣梭子蟹生存及蜕壳方面有一定关联。

  • 常规成分是水生动物反应营养摄入和健康状况的指标[24]。本研究中的三疣梭子蟹幼蟹肌肉的常规成分与以往研究结果值相近[25]。有研究表明,肌肉中脂质与水分的含量之间呈负相关关系[26]。本实验中肌肉水分与脂质含量虽无显著差异,但呈现相同的反比趋势。有研究发现当饲料磷含量不足时,会造成动物肝脏脂肪的沉积[27-28],本研究结果表明,三疣梭子蟹摄食高钙磷比的饲料一定程度上促进了组织中脂质的积累,其原因可能是饲料钙含量过高抑制了磷的吸收,降低丙酮酸激酶的活力,导致氧化磷酸化过程受损,使得三羧酸循环被抑制,最终导致肌肉和肝胰腺组织中的粗脂肪含量显著升高[29-30]。另外,本实验的结果也发现,随着钙磷比的降低,灰分和磷的沉积呈现升高的趋势,这表明钙磷比是影响组织矿化程度的主要因素。

  • 血清GOT和GPT活性在一定程度上可以作为判断肝脏损伤的指标[31-32]。血清中GOT和GPT的活性降低可能与肝脏损伤变小有关[33]。本实验GOT和GPT活性随着饲料钙磷比的增加呈现升高的趋势,且均在钙磷比为1.00∶0.50时活性达到最高值。GOT和GPT活性升高,可能是由于钙磷比的增大升高了肝胰腺脂肪的沉积,加剧了肝胰腺的损伤。GOT和GPT在钙磷比0.5~1.0的范围内没有显著差异,说明过低或适宜的钙磷比不会对造成肝胰腺的损伤。AKP是反映骨骼代谢的重要指标,经常被用于衡量动物体内的营养情况以及骨骼对钙磷的吸收程度,在水产动物的矿化过程中发挥着重要作用[34-35]。一般认为,饲料中钙磷缺乏时,机体矿化不足,成骨细胞活动增强,AKP活性升高,饲料中磷含量升高,骨磷周转代谢变慢,AKP活性降低[36-37],而当饲料中钙磷含量满足需要时,为维持机体钙磷平衡,AKP活性也增强,使钙磷向骨骼中沉积[38]。本实验结果表明,梭子蟹血淋巴AKP活性显著受饲料钙磷比的影响,且钙磷比1.00∶0.75和1.00∶0.50处理组显著高于钙磷比1.00∶2.00和1.00∶1.50处理组。原因推测是低钙磷比组饲料促进了钙磷在骨骼中的沉积,以维持钙磷的平衡。然而,不同鱼类其研究结果各异,在尼罗罗非鱼(Oreochromis niloticus)[39]中血清AKP活性随饲料钙磷比降低呈先升高后降低的趋势,造成AKP活性差异的原因可能是不同动物对钙磷的需要量不同。

  • 肝胰腺作为甲壳类动物消化吸收以及储存营养物质的中心,在物质代谢、维持营养物质水平和能量储存等方面起着不可替代的作用,尤其是作为矿物质的储存部位,已经在甲壳动物中得到证实[40-42]。甲壳是角质层的一个容易分离的组织,被确定为一个矿化的结构组织,主要有碳酸钙和少量的镁、磷、硫和微量矿物质,因此,甲壳是研究甲壳动物矿物质沉积的理想器官[43]。甲壳矿化所需的钙直接从生境中摄取,组织中的钙仅储存备用[44]。有研究发现,印度对虾(Penaeus indicus)在不同生理活动期间,钙含量在机体各组织中波动性较大,尤其蜕皮后期肝胰腺中钙的含量最高,外骨骼则最低[45]。本研究中,各个组织中钙含量无显著差异,造成以上2个物种结果不同的原因可能是不同物种对钙的敏感程度不同,三疣梭子蟹组织中的钙含量还可能受水环境和蜕壳期的影响,而与饲料中钙磷比例无关。甲壳动物中磷含量较少,在甲壳的矿化中作用稍次于钙,本研究表明,作为与甲壳相邻的致密层的主要元素,饲料中的钙磷比同样影响到其他组织中的磷沉积,为诱导与控制碳酸钙的沉积提供密切关系。

  • 动物的生长、发育,核酸、蛋白质的生物合成、机体运动包括肌肉的收缩以及生物膜的传递、运输功能等都需要消耗能量,起捕获和储存能量作用的分子腺嘌呤核苷三磷酸,是由ADP和无机磷酸合成[46]。ATP、ADP和无机磷酸广泛存在于生物体的各个细胞内,起着传递能量的作用,柠檬酸循环和氧化磷酸化作为形成ATP的主要阶段,在能量代谢中起关键作用[47]。内源性和外源性因素会改变机体能量需求和供应的平衡,为了应对平衡的变化并维持能量稳态,机体会调节线粒体的代谢状态以协调ATP的产生[48]。本研究发现,钙磷比1.00∶1.50和1.00∶1.00处理组肝胰腺中ATP含量显著高于其他组,说明添加适量钙磷比能消耗更多的能量来运输离子并建立跨膜电势,驱动线粒体产生更多的ATP,所以用于细胞各项代谢活动的能量储存远远多于其他处理组。

    能量代谢的调节划分为分子水平、细胞水平和整体水平,分子水平主要是浓度的调节和酶的调节,包括酶的数量调节以及酶活性的调节等[47]。已糖激酶是一种调节酶,作用于葡萄糖发生糖酵解的第一步,是将D-葡萄糖分子在第6位的磷酸化催化形成产物葡萄糖-6-磷酸和ADP,能使己糖激酶受到变构抑制[49]。有研究表明,无机磷酸有解除葡萄糖-6-磷酸和ADP对己糖激酶有抑制的作用,这需要保持一定的酵解速度以维持能量的需要有关,只要有少量的无机磷酸存在,就能解除葡萄糖-6-磷酸的抑制作用,使酵解中间物维持在一定水平[50]。本实验中,钙磷比1.00∶1.00组己糖激酶活力显著高于钙磷比1.00∶2.00组,表明钙磷比1.00∶1.00组酵解速度更快,钙磷比1.00∶2.00组在代谢过程中产生的无机磷酸可能抑制了ATP的形成。琥珀酸脱氢酶,是唯一一个结合于线粒体内膜上的多亚基酶,其是连接氧化磷酸化与电子传递的枢纽之一,可为真核细胞线粒体需氧和产能的呼吸链提供电子,作为参与三羧酸循环的关键酶,琥珀酸脱氢酶是反映线粒体功能的标志酶之一,其活性可作为评价三羧酸循环运行程度的指标[51-53]。三疣梭子蟹肝胰腺中钙磷比1.00∶1.00组饲料中琥珀酸脱氢酶的活性显著高于钙磷比1.00∶2.00和1.00∶0.50组,同时联系本实验中饲料钙磷比对三疣梭子蟹肝胰腺电子传递链上sdhc表达的影响,可以发现投喂升高钙磷比饲料的幼蟹显著上调了肝胰腺sdhc的表达水平,可见琥珀酸脱氢酶以FAD作为其脱下电子受体的前提,在琥珀酸脱氢酶酶活性和相关基因表达水平上都论证了饲料中适宜的钙磷比可以提高三疣梭子蟹幼蟹线粒体能量代谢的能力。

    真核生物通过细胞线粒体内膜内的一系列蛋白复合体形成ATP,氧化磷酸化很大程度上是由编码复合体I (NADH脱氢酶)的基因表达驱动[53-54]。在本实验中,电子传递链中nd1基因表达的上调,说明线粒体电子传递链的核编码基因的上调与饲料中钙磷比升高密切相关,对于维持ATP水平至关重要。除了线粒体复合物的基因表达量增加外,在钙磷比1.00∶1.50和1.00∶1.00组中还观察到sirt3基因表达也出现上调。近十几年来,研究者对Sirtuin蛋白家族开展了多角度全方位的研究[55]Sirt3去乙酰化酶能激活脂肪酸氧化中的各种目标底物,是线粒体能量代谢的调节因子,在能量代谢过程中扮演了重要的角色[56]。此外,核呼吸因子1编码一种同型二聚蛋白,直接调控呼吸链复合体亚基的核基因表达,并间接调控负责呼吸链、线粒体DNA转录和拷贝的3种线粒体基因[57-58]。钙磷比1.00∶1.00组三疣梭子蟹肝胰腺nrf1高表达,表明适宜的钙磷比能上调能量代谢通路上相关基因的表达。综上所述,线粒体产生的能量可以用来维持三疣梭子蟹蜕壳前后的能量供给以及稳态,这可以部分解释了钙磷比1.00∶1.00组中三疣梭子蟹蜕壳率最高。

4.   结论
  • 本研究表明,饲料中不同的钙磷比对三疣梭子蟹幼蟹生长性能、健康状态、矿物质沉积及能量代谢等的影响显著;适宜的钙磷比不仅能够改善三疣梭子蟹的生长性能和饲料利用,有效提高能量稳态水平。以增重率为评价指标,三疣梭子蟹幼蟹最适钙磷比为1.06~1.26。

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