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Alterations of growth performance, hematological parameters, and plasma constituents in the sablefish, Anoplopoma fimbria depending on ammonia concentrations

Alterations of growth performance, hematological parameters, and plasma constituents in the... Juvenile Anoplopoma fimbria (mean length 16.8 ± 2.2 cm, and mean weight 72.8 ± 5.4 g) were exposed for 2 months with different levels of ammonia (0, 0.25, 0.50, 0.75, 1.00, and 1.25 mg/L). Growth performances such as daily length gain, daily weight gain, condition factor, and hepatosomatic index were significantly decreased by ammonia exposure. Hematological parameters such as red blood cell (RBC) count, hematocrit, and hemoglobin were also significantly decreased. In plasma inorganic components, calcium and magnesium were significantly decreased by ammonia exposure. In plasma organic components, there was no alteration in cholesterol and total protein. In enzyme plasma components, glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly increased. The results of this study indicated that ammonia exposure can induce significant growth reduction and blood biochemistry alterations of A. fimbria. Keywords: Sablefish, Ammonia, Growth performance, Hematological parameters, Plasma components Background Growth factor in aquaculture is one of the most basic In Korea, aquaculture is a major industry in food secur- and critical parameters to assess toxic effects by harmful ity dimension because it can supply high-quality protein substances in aquaculture environment. In fish exposed to the public by stably breeding aquatic organisms. How- to toxic substances, growth performance is generally de- ever, ammonia hypergenesis by high density breeding in creased by energy transition from use for growth and de- aquaculture environment is a critical environmental velopment to use for tissue damage recovery (Wendelaar toxic factor to induce death. Exposure to excessive am- Bonga, 1997). In aquatic environment, excessive ammo- monia in aquatic animals induces depolarization in nia concentrations can be accumulated in body fluids in + + neuron because increased NH displaces K , which re- fish, which results in growth inhibition, tissue erosion sults in cell death in central nervous system. Therefore, and degeneration, immune suppression, and high mor- it induces convulsions, coma, and death by the cell death tality (Liang et al., 2015). (Thangam et al. 2014). In addition, acute ammonia ex- Hematological and biochemical parameters in fish can posure induces gill ventilation increase, equilibrium loss, be a critical indicator to assess alterations in circulatory convulsions, ionic balance failure, and hyper-excitability system by toxic substances in external environment in aquatic animals (Kim et al. 2015). (Vinodhini and Narayanan, 2009). Ammonia especially affects hematological parameters in fish by blocking oxy- * Correspondence: jckang@pknu.ac.kr gen transfer from gill to blood (Thangam et al. 2014). Department of Aquatic Life Medicine, Pukyong National University, Busan, Sablefish, Anoplopoma fimbria used in this study is Korea recognized as a high value fish species around the globe. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 2 of 6 In liberalization trend of the world market, aquatic prod- Table 2 Analyzed waterborne ammonia concentration from each source ucts are also involved in the trend. Therefore, develop- ment in aquaculture of a high value fish species is urgent. Waterborne ammonia concentration (mg/L) The purpose of this study was to assess toxic effects of A. Waterborne ammonia 0 0.25 0.50 0.75 1.00 1.25 fimbria exposed to ammonia a part of complete sablefish concentrations culture, and to build standard breeding guidelines of Actual ammonia 0.02 ± 0.28 ± 0.57 ± 0.81 ± 1.04 ± 1.32 ± sablefish aquaculture. concentrations 0.01 0.07 0.14 0.26 0.32 0.27 Methods Daily growth gain ¼ W −W =day f i Experimental fish and conditions Juvenile sablefish were obtained from Troutlodge Inc. inðÞ W ¼ Final length or weight; W ¼ Initial length or weight f i USA. During the acclimation period, the fish were fed diet Condition factorðÞ % ¼ W =L x100 twice daily and maintained on a 24-h dark cycle and con- stant condition at all times (Table 1). After ðÞ W ¼ weightðÞ g ; L ¼ lengthðÞ cm acclimatization, 72 fish (body length, 16.8 ± 2.2 cm; body weight, 72.8 ± 5.4 g) were randomly selected for the study. HSI ¼ðÞ liver weight=total fish weight 100 The acclimation period commenced once the final temperature had been sustained for 24 h and animals were feeding, while showing no sign of stress. Ammonia expos- Blood samples and hematological assay ure took place in tanks containing six fish per treatment Blood samples were collected within 35–40 s through group. Ammonia chloride (NH Cl) (Sigma, St. Louis, MO, the caudal vein of the fish in 1-ml disposable heparin- USA) solution was dissolved in the respective tanks. The ized syringes at the end of 1 and 2 months. The blood ammonia concentrations in the tanks were 0, 0.25, 0.50, samples were kept at 4 °C until the blood parameters 0.75, 1.00, and 1.25 mg/L, and actual ammonia concentra- were completely studied. The total red blood cell (RBC) tions are demonstrated in Table 2. Diluted 100 mg/L am- count, hemoglobin (Hb), concentration, and hematocrit monia chloride (NH Cl) in 20 L glass tank to make (Ht) value were determined immediately. Total RBC respective concentrations (50, 0.25 mg/L, 100 ml; counts were counted using optical microscope with 0.50 mg/L, 150 ml; 0.75 mg/L, 200 ml; 1.00 mg/L, 250 ml; hemo-cytometer (Improved Neubauer, Germany) after 1.25 mg/L). After the exposure experiment, feed was given diluted by Hendrick’s diluting solution. The Hb concen- at a rate of 2% body weight daily (as two 1% meals per tration was determined using Cyan-methemoglobin day). There was no water flow rate. The tank water was technique (Asan Pharm. Co., Ltd.). The Ht value was de- thoroughly exchanged once per 2 days and made the same termined by the microhematocrit centrifugation tech- concentration in the respective 500 L circular tank. At the nique. The blood samples were centrifuged to separate end of each period (at 1 and 2 months), animals were plasma from blood samples at 3000 g for 5 min at 4 °C. anesthetized in buffered 3-aminobenzoic acid ethyl ester The plasma samples were analyzed for inorganic sub- methanesulfonate (Sigma Chemical, St. Louis, MO). stances, organic substances, and enzyme activity using clinical kit (Asan Pharm. Co.,Ltd.). In inorganic substances assay, calcium and magnesium were analyzed by the o- Growth cresolphthalein-complexon technique and xylidyl blue The weight and length of sablefish was measured just technique. In organic substances assay, cholesterol and before exposure, at 1 and 2 months. Daily length gain, total protein were analyzed by enzyme method and by daily weight gain, condition factor, and hepatosomatic biuret technique. In enzyme activity assay, glutamic oxal- index (HSI) were calculated by the following method. ate transaminase (GOT) and glutamic pyruvate trans- aminase (GPT) were analyzed by Kind-king technique. Table 1 The chemical components of seawater and experimental condition used in the experiments Statistical analysis Item Value The experiment was conducted in exposure periods for Temperature (°C) 13.0 ± 1.0 2 months and performed triplicate. Statistical analyses were performed using the SPSS/PC+ statistical package pH 8.2 ± 0.5 (SPSS Inc, Chicago, IL, USA). Significant differences be- Salinity (‰) 33.5 ± 0.6 tween groups were identified using one-way ANOVA Dissolved oxygen (mg/L) 7.8 ± 0.5 and Tukey’s test for multiple comparisons. The signifi- Chemical oxygen demand (mg/L) 1.21 ± 0.14 cance level was set at P < 0.05. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 3 of 6 0.4 0.6 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 0.5 1.00 mg/L 1.00 mg/L 0.3 1.25 mg/L 1.25 mg/L a a a 0.4 ab a a a a a ab ab ab b b b b 0.2 0.3 b b 0.2 0.1 0.1 0.0 0.0 1 2 1 2 Months Months 1.6 5 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 1.4 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 1.00 mg/L 1.00 mg/L 1.2 1.25 mg/L 1.25 mg/L a a a a a 1.0 ab 3 a a a a a a a b b ab 0.8 b b 0.6 0.4 0.2 0.0 0 1 2 1 2 Months Months Fig. 1 Daily length gain, daily weight gain, condition factor, and hepatosomatic index of sablefish, Anoplopoma fimbria exposed to ammonia for 2 months. Vertical bar denotes a standard error. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test Results hepatosomatic index from 0 to 0.75 mg/L ammonia ex- Growth posure after 1 and 2 months. No mortality was observed for the exposure periods. The growth performance, condition factor, and hepato- somatic index of A. fimbria is demonstrated in Fig. 1. Hematological parameters Significant decreases in daily length gain and daily RBC count, hematocrit value, and hemoglobin concen- weight gain were observed at ammonia exposure tration of A. fimbria exposed to different concentrations greater than 1.00 mg/L both in 1 and 2 months. Condi- of waterborne ammonia are demonstrated in Fig. 2. RBC tion factor was significantly decreased at ammonia ex- count was significantly decreased at ammonia exposure posure greater than 1.00 mg/L both in 1 and 2 months. greater than 1.00 mg/L in 1 month and greater than Hepatosomatic index was also significantly decreased at 0.75 mg/L in 2 months. Hematocrit value was signifi- ammonia exposure greater than 1.00 mg/L both in 1 cantly decreased at ammonia exposure greater than and 2months.However,there wasnochangeindaily 1.00 mg/L in 1 month and greater than 0.75 mg/L in length, daily weight gain, and condition factor and 2 months. Hemoglobin concentration was significantly 80 25 600 0 mg/L 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 0.25 mg/L 0.50 mg/L 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 500 0.75 mg/L 1.00 mg/L 1.00 mg/L 1.00 mg/L 1.25 mg/L 1.25 mg/L 1.25 mg/L a a a a a a a a 15 a a a a a a a ab a b b b 40 b b 300 b b b c bc 0 0 1 2 1 2 1 2 Months Months Months Fig. 2 RBC count, hematocrit, and hemoglobin of sablefish, Anoplopoma fimbria exposed to ammonia for 2 months. Vertical bar denotes a standard error. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test RBC count (x10 mm ) Condition factor (%) Daily length gain (mm/day) Hematocrit (%) Daily weight gain (g/day) Hepatosomatic index (%) Hemoglobin (g/dL) Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 4 of 6 Table 3 Changes of inorganic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a ab b b Calcium 1 1.84 ± 0.23 1.81 ± 0.26 1.75 ± 0.31 1.71 ± 0.30 1.59 ± 0.25 1.55 ± 0.21 (mg/dL) a a ab ab b b 2 1.81 ± 0.27 1.78 ± 0.23 1.62 ± 0.25 1.64 ± 0.21 1.49 ± 0.27 1.42 ± 0.24 a a a ab b b Magnesium 1 3.46 ± 0.42 3.51 ± 0.32 3.41 ± 0.27 3.29 ± 0.35 2.89 ± 0.22 2.95 ± 0.32 (mg/dL) a a ab b b b 2 3.41 ± 0.36 3.46 ± 0.38 3.28 ± 0.41 3.06 ± 0.25 2.74 ± 0.30 2.71 ± 0.24 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0 .05) as determined by Tukey’s multiple range test decreased at ammonia exposure greater than 0.75 mg/L health status by toxic substance exposure (Datta et al., in 1 and 2 months. 2007), and HSI of sablefish, A. fimbria was significantly decreased by ammonia exposure. Plasma components Blood cells in fish are generated from hematopoietic Plasma inorganic components of A. fimbria are demon- tissues of kidney and spleen, and changes in strated in Table 3. Calcium was significantly decreased hematological parameters indicate physiological effects at ammonia exposure greater than 1.00 mg/L in 1 and by stress responses (Das et al., 2004). Jeney et al. 2 months. Magnesium was also significantly decreased (1992) suggest that high levels of ammonia exposure at ammonia exposure greater than 1.00 mg/L in 1 month induce oxygen-free condition by increasing affinity of and greater than 0.75 in 2 months. Plasma organic com- hemoglobin to combine with ammonia molecules, ponents are demonstrated in Table 4. No alterations in thereby elevating ammonia concentration in blood. cholesterol and total protein were observed by water- Knoph and Thorud (1996) reported a significant borne ammonia exposure. Plasma enzyme components decrease in RBC count and hematocrit of Atlantic sal- are demonstrated in Table 5. GOT was significantly in- mon, Salmo salar exposed to ammonia. Das et al. (2004) creased at ammonia exposure greater than 1.00 mg/L in also reported a significant decrease in hemoglobin of 1 month and greater than 0.75 in 2 months. GPT was Mrigal carp, Cirrhinus cirrhosus exposed to ammonia. In also significantly increased at ammonia exposure greater this study, ammonia exposure caused a significant decrease than 1.00 mg/L in 1 month and greater than 0.75 in in RBC count, hematocrit, and hemoglobin of sablefish, A. 2 months. fimbria, which may be due to hematopoietic cell damage according to hypoxicstatusbyammoniaexposure. Discussion Calcium and magnesium in plasma inorganic com- Ammonia exposure to fish is a critical environmental ponents are critical indicators of osmotic pressure al- limited factor to inhibit growth performance by decreas- terations, and these can be increased or decreased by ing feed intake and feed utilization (Foss et al., 2003). environmental changes (Hur et al., 2001). Person-Le Many authors reported that high concentrations of am- Ruyet et al., (2003) reported that ammonia exposure monia exposure induced growth inhibition of spotted to turbot, Scophthalmus maximus induced changes in + − + 2+ wolfish, Anarhichas minor Olafsen (Foss et al., 2003), osmoticpressurebyalteringNa ,Cl ,K ,Ca con- turbot, Scophthalmus maximus (Foss et al., 2009), Atlan- centrations in plasma. In this study, calcium and tic halibut, Hippoglossus hippoglossus (Paust et al., 2011). magnesium in sablefish, A. fimbria were significantly In this study, high concentrations of ammonia induced a decreased by ammonia exposure, which indicate that significant decrease in growth of sablefish, A. fimbria, ammonia exposure affected the osmotic ion regulation which may be due to energy transition from growth and of sablefish. Cholesterol and total protein of plasma development to detoxification. Hepatosomatic index organic components in fish have been considered as a (HSI) is considered as a critical indicator to evaluate major component to assess fish health. However, there Table 4 Changes of organic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a a a a Cholesterol 1 131.5 ± 20.5 139.1 ± 18.2 140.5 ± 21.3 137.2 ± 16.8 142.2 ± 21.1 146.2 ± 18.3 (mg/dL) a a a a a a 2 135.1 ± 17.1 131.5 ± 17.1 138.2 ± 17.1 143.2 ± 21.3 148.3 ± 18.3 142.6 ± 20.2 a a a a a a Total protein 1 4.42 ± 0.53 4.35 ± 0.39 4.26 ± 0.45 4.51 ± 0.48 4.33 ± 0.37 4.59 ± 0.41 (g/dL) a a a a a a 2 4.36 ± 0.47 4.56 ± 0.35 4.37 ± 0.31 4.62 ± 0.52 4.24 ± 0.45 4.39 ± 0.51 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0 .05) as determined by Tukey’s multiple range test Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 5 of 6 Table 5 Changes of enzymatic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a a b b GOT 1 2.67 ± 0.33 2.72 ± 0.29 2.75 ± 0.35 2.81 ± 0.28 3.36 ± 0.41 3.45 ± 0.37 (karmen unit) a a ab b bc c 2 2.72 ± 0.25 2.61 ± 0.32 2.91 ± 0.21 3.24 ± 0.32 3.46 ± 0.40 3.62 ± 0.41 a a a ab b b GPT 1 1.72 ± 0.24 1.79 ± 0.19 1.84 ± 0.18 1.93 ± 0.26 2.19 ± 0.31 2.28 ± 0.27 (karmen unit) a a a b c c 2 1.75 ± 0.28 1.82 ± 0.23 1.99 ± 0.28 2.23 ± 0.31 2.41 ± 0.35 2.40 ± 0.35 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test was no significant alteration in sablefish, A. fimbria.GOT Disclosure The dataset(s) supporting the conclusions of this article is not included in and GPT in enzymatic plasma components can be easily the article. increased by hepatic tissue damage, and these are used to evaluate hepatic tissue damage (Agrahari et al., 2007). Le Ruyet et al. (1998) reported that a significant increase in Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in GOT and GPT of turbot, Scophthalmus maximus and published maps and institutional affiliations. seabream, Sparus aurata exposed to ammonia. In this study, GOP and GPT in sablefish, A. fimbria were signifi- Author details West Sea Fisheries Research Institute, National Institute of Fisheries Science, cantly increased by ammonia exposure, which may be due Incheon, Korea. Department of Aquatic Life Medicine, Pukyong National to hepatic tissue damage by ammonia. University, Busan, Korea. Department of Marine Biology, Pukyong National University, Busan, Korea. Department of Aqualife Medicine, Chonnam National University, Yeosu, Korea. Conclusion The results of this study indicate that ammonia exposure Received: 5 November 2016 Accepted: 17 March 2017 at the higher than proper concentrations affected growth performance and hematological parameters of sablefish, A. fimbria, and these changes should influence the References Agrahari S, Pandey KC, Gopal K. Biochemical alteration induced by health of sablefish, A. fimbria. In conclusion, ammonia monocrotophos in the blood plasma of fish, Channa punctatus (Bloch). Pestic concentrations at the higher than 0.75 mg/L can affect Biochem Physiol. 2007;88:268–72. various physiological effects of sablefish, A. fimbria, and Das PC, Ayyappan S, Jena JK, Das BK. Acute toxicity of ammonia and its sub- lethal effects on selected haematological and enzymatic parameters of the high concentrations of ammonia exposure require mrigal, Cirrhinus mrigala (Hamilton). Aquac Res. 2004;35:134–43. special attention in sablefish aquaculture. In addition to Datta S, Saha DR, Ghosh D, Majumdar T, Bhattacharya S, Mazumder S. Sub-lethal this environmental study, various environmental stan- concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L. Comp Biochem Physiol C dards should be established for stable sablefish Toxicol Pharmacol. 2007;145:339–49. aquaculture. Foss A, Evensen TH, Vollen T, Øiestad V. Effects of chronic ammonia exposure on growth and food conversion efficiency in juvenile spotted wolffish. Abbreviations Aquaculture. 2003;228:215–24. GOT: Glutamic oxalate transaminase; GPT: Glutamic pyruvate transaminase; Foss A, Imsland AK, Roth B, Schram E, Stefansson SO. Effects of chronic and HIS: Hepatosomatic index; RBC: Red blood cell periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus). Aquaculture. 2009;296:45–50. Acknowledgements Hur JW, Chang YJ, Lim HK, Lee BK. Stress responses of cultured fishes elicited by This research was a part of the project titled ‘Development of practical water level reduction in rearing tank and fish transference during selection techniques for the artificial seeding production of sablefish’, funded by the process. J Korean Fish Soc. 2001;34:465–72. MOF, Korea. Jeney G, Nemcsok J, Zs J, Olah J. Acute effect of sublethal ammonia concentrations on common carp (Cyprinus carpio L.). II Effect of ammonia on Authors’ contributions blood plasma transminases (GOT, GPT), G1DH enzyme activity, and ATP HJ, IK, and JM carried out the environmental toxicity studies and manuscript value. Aquaculture. 1992;104:149–56. writing. JH, DH, CW, and JS participated in the design of the study and data Kim SH, Kim JH, Park MA, Hwang SD, Kang JC. The toxic effects of ammonia analysis. JC participated in its design and coordination and helped to draft exposure on antioxidant and immune responses in Rockfish, Sebastes the manuscript. All authors read and approved the final manuscript. schlegelii during thermal stress. Environ Toxicol Pharmacol. 2015;40:954–9. Knoph MB, Thorud K. Toxicity of ammonia to Atlantic salmon (Salmo salar L.) in Competing interests seawater—Effects on plasma osmolality, ion, ammonia, urea and glucose The authors declare that they have no competing interests. levels and hematologic parameters. Comp Biochem Physiol A Physiol. 1996; 113:375–81. Consent for publication Le Ruyet JP, Boeuf G, Infante JZ, Helgason S, Le Roux A. Short-term physiological Not applicable changes in turbot and seabream juveniles exposed to exogenous ammonia. Comp Biochem Physiol A Mol Integr Physiol. 1998;119:511–8. Ethics approval and consent to participate Liang Z, Liu R, Zhao D, Wang L, Sun M, Wang M, Song L. Ammonia exposure All experimental animals used in this study were maintained under a induces oxidative stress, endoplasmic reticulum stress and apoptosis in protocol approved by the Institutional Animal Care and Use Committee of epatopancreas of pacific white shrimp (Litopenaeus vannamei). Fish Shellfish the Pukyong National University. Immunol. 2015;54:523–8. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 6 of 6 Paust LO, Foss A, Imsland AK. Effects of chronic and periodic exposure to ammonia on growth, food conversion efficiency and blood physiology in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture. 2011;315:400–6. Person-Le Ruyet J, Lamers A, Roux AL, Severe A, Boeuf G, Mayer‐Gostan N. Long‐ term ammonia exposure of turbot: effects on plasma parameters. J Fish Biol. 2003;62:879–94. Thangam Y, Perumayee M, Jayaprakash S, Umavathi S, Basheer SK. Studies of ammonia toxicity on haematological parameters to freshwater fish Cyprinus carpio (common carp). Int J Curr Microbiol App Sci. 2014;3:535–42. Vinodhini R, Narayanan M. The impact of toxic heavy metal on the hematological parameters in common carp (Cyprinus carpio L.). Iran J Environ Health Sci Eng. 2009;6:23–8. Wendelaar Bonga SE. The stress response in fish. Physiol Rev. 1997;77:591–625. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Alterations of growth performance, hematological parameters, and plasma constituents in the sablefish, Anoplopoma fimbria depending on ammonia concentrations

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Juvenile Anoplopoma fimbria (mean length 16.8 ± 2.2 cm, and mean weight 72.8 ± 5.4 g) were exposed for 2 months with different levels of ammonia (0, 0.25, 0.50, 0.75, 1.00, and 1.25 mg/L). Growth performances such as daily length gain, daily weight gain, condition factor, and hepatosomatic index were significantly decreased by ammonia exposure. Hematological parameters such as red blood cell (RBC) count, hematocrit, and hemoglobin were also significantly decreased. In plasma inorganic components, calcium and magnesium were significantly decreased by ammonia exposure. In plasma organic components, there was no alteration in cholesterol and total protein. In enzyme plasma components, glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly increased. The results of this study indicated that ammonia exposure can induce significant growth reduction and blood biochemistry alterations of A. fimbria. Keywords: Sablefish, Ammonia, Growth performance, Hematological parameters, Plasma components Background Growth factor in aquaculture is one of the most basic In Korea, aquaculture is a major industry in food secur- and critical parameters to assess toxic effects by harmful ity dimension because it can supply high-quality protein substances in aquaculture environment. In fish exposed to the public by stably breeding aquatic organisms. How- to toxic substances, growth performance is generally de- ever, ammonia hypergenesis by high density breeding in creased by energy transition from use for growth and de- aquaculture environment is a critical environmental velopment to use for tissue damage recovery (Wendelaar toxic factor to induce death. Exposure to excessive am- Bonga, 1997). In aquatic environment, excessive ammo- monia in aquatic animals induces depolarization in nia concentrations can be accumulated in body fluids in + + neuron because increased NH displaces K , which re- fish, which results in growth inhibition, tissue erosion sults in cell death in central nervous system. Therefore, and degeneration, immune suppression, and high mor- it induces convulsions, coma, and death by the cell death tality (Liang et al., 2015). (Thangam et al. 2014). In addition, acute ammonia ex- Hematological and biochemical parameters in fish can posure induces gill ventilation increase, equilibrium loss, be a critical indicator to assess alterations in circulatory convulsions, ionic balance failure, and hyper-excitability system by toxic substances in external environment in aquatic animals (Kim et al. 2015). (Vinodhini and Narayanan, 2009). Ammonia especially affects hematological parameters in fish by blocking oxy- * Correspondence: jckang@pknu.ac.kr gen transfer from gill to blood (Thangam et al. 2014). Department of Aquatic Life Medicine, Pukyong National University, Busan, Sablefish, Anoplopoma fimbria used in this study is Korea recognized as a high value fish species around the globe. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 2 of 6 In liberalization trend of the world market, aquatic prod- Table 2 Analyzed waterborne ammonia concentration from each source ucts are also involved in the trend. Therefore, develop- ment in aquaculture of a high value fish species is urgent. Waterborne ammonia concentration (mg/L) The purpose of this study was to assess toxic effects of A. Waterborne ammonia 0 0.25 0.50 0.75 1.00 1.25 fimbria exposed to ammonia a part of complete sablefish concentrations culture, and to build standard breeding guidelines of Actual ammonia 0.02 ± 0.28 ± 0.57 ± 0.81 ± 1.04 ± 1.32 ± sablefish aquaculture. concentrations 0.01 0.07 0.14 0.26 0.32 0.27 Methods Daily growth gain ¼ W −W =day f i Experimental fish and conditions Juvenile sablefish were obtained from Troutlodge Inc. inðÞ W ¼ Final length or weight; W ¼ Initial length or weight f i USA. During the acclimation period, the fish were fed diet Condition factorðÞ % ¼ W =L x100 twice daily and maintained on a 24-h dark cycle and con- stant condition at all times (Table 1). After ðÞ W ¼ weightðÞ g ; L ¼ lengthðÞ cm acclimatization, 72 fish (body length, 16.8 ± 2.2 cm; body weight, 72.8 ± 5.4 g) were randomly selected for the study. HSI ¼ðÞ liver weight=total fish weight 100 The acclimation period commenced once the final temperature had been sustained for 24 h and animals were feeding, while showing no sign of stress. Ammonia expos- Blood samples and hematological assay ure took place in tanks containing six fish per treatment Blood samples were collected within 35–40 s through group. Ammonia chloride (NH Cl) (Sigma, St. Louis, MO, the caudal vein of the fish in 1-ml disposable heparin- USA) solution was dissolved in the respective tanks. The ized syringes at the end of 1 and 2 months. The blood ammonia concentrations in the tanks were 0, 0.25, 0.50, samples were kept at 4 °C until the blood parameters 0.75, 1.00, and 1.25 mg/L, and actual ammonia concentra- were completely studied. The total red blood cell (RBC) tions are demonstrated in Table 2. Diluted 100 mg/L am- count, hemoglobin (Hb), concentration, and hematocrit monia chloride (NH Cl) in 20 L glass tank to make (Ht) value were determined immediately. Total RBC respective concentrations (50, 0.25 mg/L, 100 ml; counts were counted using optical microscope with 0.50 mg/L, 150 ml; 0.75 mg/L, 200 ml; 1.00 mg/L, 250 ml; hemo-cytometer (Improved Neubauer, Germany) after 1.25 mg/L). After the exposure experiment, feed was given diluted by Hendrick’s diluting solution. The Hb concen- at a rate of 2% body weight daily (as two 1% meals per tration was determined using Cyan-methemoglobin day). There was no water flow rate. The tank water was technique (Asan Pharm. Co., Ltd.). The Ht value was de- thoroughly exchanged once per 2 days and made the same termined by the microhematocrit centrifugation tech- concentration in the respective 500 L circular tank. At the nique. The blood samples were centrifuged to separate end of each period (at 1 and 2 months), animals were plasma from blood samples at 3000 g for 5 min at 4 °C. anesthetized in buffered 3-aminobenzoic acid ethyl ester The plasma samples were analyzed for inorganic sub- methanesulfonate (Sigma Chemical, St. Louis, MO). stances, organic substances, and enzyme activity using clinical kit (Asan Pharm. Co.,Ltd.). In inorganic substances assay, calcium and magnesium were analyzed by the o- Growth cresolphthalein-complexon technique and xylidyl blue The weight and length of sablefish was measured just technique. In organic substances assay, cholesterol and before exposure, at 1 and 2 months. Daily length gain, total protein were analyzed by enzyme method and by daily weight gain, condition factor, and hepatosomatic biuret technique. In enzyme activity assay, glutamic oxal- index (HSI) were calculated by the following method. ate transaminase (GOT) and glutamic pyruvate trans- aminase (GPT) were analyzed by Kind-king technique. Table 1 The chemical components of seawater and experimental condition used in the experiments Statistical analysis Item Value The experiment was conducted in exposure periods for Temperature (°C) 13.0 ± 1.0 2 months and performed triplicate. Statistical analyses were performed using the SPSS/PC+ statistical package pH 8.2 ± 0.5 (SPSS Inc, Chicago, IL, USA). Significant differences be- Salinity (‰) 33.5 ± 0.6 tween groups were identified using one-way ANOVA Dissolved oxygen (mg/L) 7.8 ± 0.5 and Tukey’s test for multiple comparisons. The signifi- Chemical oxygen demand (mg/L) 1.21 ± 0.14 cance level was set at P < 0.05. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 3 of 6 0.4 0.6 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 0.5 1.00 mg/L 1.00 mg/L 0.3 1.25 mg/L 1.25 mg/L a a a 0.4 ab a a a a a ab ab ab b b b b 0.2 0.3 b b 0.2 0.1 0.1 0.0 0.0 1 2 1 2 Months Months 1.6 5 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 1.4 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 1.00 mg/L 1.00 mg/L 1.2 1.25 mg/L 1.25 mg/L a a a a a 1.0 ab 3 a a a a a a a b b ab 0.8 b b 0.6 0.4 0.2 0.0 0 1 2 1 2 Months Months Fig. 1 Daily length gain, daily weight gain, condition factor, and hepatosomatic index of sablefish, Anoplopoma fimbria exposed to ammonia for 2 months. Vertical bar denotes a standard error. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test Results hepatosomatic index from 0 to 0.75 mg/L ammonia ex- Growth posure after 1 and 2 months. No mortality was observed for the exposure periods. The growth performance, condition factor, and hepato- somatic index of A. fimbria is demonstrated in Fig. 1. Hematological parameters Significant decreases in daily length gain and daily RBC count, hematocrit value, and hemoglobin concen- weight gain were observed at ammonia exposure tration of A. fimbria exposed to different concentrations greater than 1.00 mg/L both in 1 and 2 months. Condi- of waterborne ammonia are demonstrated in Fig. 2. RBC tion factor was significantly decreased at ammonia ex- count was significantly decreased at ammonia exposure posure greater than 1.00 mg/L both in 1 and 2 months. greater than 1.00 mg/L in 1 month and greater than Hepatosomatic index was also significantly decreased at 0.75 mg/L in 2 months. Hematocrit value was signifi- ammonia exposure greater than 1.00 mg/L both in 1 cantly decreased at ammonia exposure greater than and 2months.However,there wasnochangeindaily 1.00 mg/L in 1 month and greater than 0.75 mg/L in length, daily weight gain, and condition factor and 2 months. Hemoglobin concentration was significantly 80 25 600 0 mg/L 0 mg/L 0 mg/L 0.25 mg/L 0.25 mg/L 0.25 mg/L 0.50 mg/L 0.50 mg/L 0.50 mg/L 0.75 mg/L 0.75 mg/L 500 0.75 mg/L 1.00 mg/L 1.00 mg/L 1.00 mg/L 1.25 mg/L 1.25 mg/L 1.25 mg/L a a a a a a a a 15 a a a a a a a ab a b b b 40 b b 300 b b b c bc 0 0 1 2 1 2 1 2 Months Months Months Fig. 2 RBC count, hematocrit, and hemoglobin of sablefish, Anoplopoma fimbria exposed to ammonia for 2 months. Vertical bar denotes a standard error. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test RBC count (x10 mm ) Condition factor (%) Daily length gain (mm/day) Hematocrit (%) Daily weight gain (g/day) Hepatosomatic index (%) Hemoglobin (g/dL) Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 4 of 6 Table 3 Changes of inorganic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a ab b b Calcium 1 1.84 ± 0.23 1.81 ± 0.26 1.75 ± 0.31 1.71 ± 0.30 1.59 ± 0.25 1.55 ± 0.21 (mg/dL) a a ab ab b b 2 1.81 ± 0.27 1.78 ± 0.23 1.62 ± 0.25 1.64 ± 0.21 1.49 ± 0.27 1.42 ± 0.24 a a a ab b b Magnesium 1 3.46 ± 0.42 3.51 ± 0.32 3.41 ± 0.27 3.29 ± 0.35 2.89 ± 0.22 2.95 ± 0.32 (mg/dL) a a ab b b b 2 3.41 ± 0.36 3.46 ± 0.38 3.28 ± 0.41 3.06 ± 0.25 2.74 ± 0.30 2.71 ± 0.24 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0 .05) as determined by Tukey’s multiple range test decreased at ammonia exposure greater than 0.75 mg/L health status by toxic substance exposure (Datta et al., in 1 and 2 months. 2007), and HSI of sablefish, A. fimbria was significantly decreased by ammonia exposure. Plasma components Blood cells in fish are generated from hematopoietic Plasma inorganic components of A. fimbria are demon- tissues of kidney and spleen, and changes in strated in Table 3. Calcium was significantly decreased hematological parameters indicate physiological effects at ammonia exposure greater than 1.00 mg/L in 1 and by stress responses (Das et al., 2004). Jeney et al. 2 months. Magnesium was also significantly decreased (1992) suggest that high levels of ammonia exposure at ammonia exposure greater than 1.00 mg/L in 1 month induce oxygen-free condition by increasing affinity of and greater than 0.75 in 2 months. Plasma organic com- hemoglobin to combine with ammonia molecules, ponents are demonstrated in Table 4. No alterations in thereby elevating ammonia concentration in blood. cholesterol and total protein were observed by water- Knoph and Thorud (1996) reported a significant borne ammonia exposure. Plasma enzyme components decrease in RBC count and hematocrit of Atlantic sal- are demonstrated in Table 5. GOT was significantly in- mon, Salmo salar exposed to ammonia. Das et al. (2004) creased at ammonia exposure greater than 1.00 mg/L in also reported a significant decrease in hemoglobin of 1 month and greater than 0.75 in 2 months. GPT was Mrigal carp, Cirrhinus cirrhosus exposed to ammonia. In also significantly increased at ammonia exposure greater this study, ammonia exposure caused a significant decrease than 1.00 mg/L in 1 month and greater than 0.75 in in RBC count, hematocrit, and hemoglobin of sablefish, A. 2 months. fimbria, which may be due to hematopoietic cell damage according to hypoxicstatusbyammoniaexposure. Discussion Calcium and magnesium in plasma inorganic com- Ammonia exposure to fish is a critical environmental ponents are critical indicators of osmotic pressure al- limited factor to inhibit growth performance by decreas- terations, and these can be increased or decreased by ing feed intake and feed utilization (Foss et al., 2003). environmental changes (Hur et al., 2001). Person-Le Many authors reported that high concentrations of am- Ruyet et al., (2003) reported that ammonia exposure monia exposure induced growth inhibition of spotted to turbot, Scophthalmus maximus induced changes in + − + 2+ wolfish, Anarhichas minor Olafsen (Foss et al., 2003), osmoticpressurebyalteringNa ,Cl ,K ,Ca con- turbot, Scophthalmus maximus (Foss et al., 2009), Atlan- centrations in plasma. In this study, calcium and tic halibut, Hippoglossus hippoglossus (Paust et al., 2011). magnesium in sablefish, A. fimbria were significantly In this study, high concentrations of ammonia induced a decreased by ammonia exposure, which indicate that significant decrease in growth of sablefish, A. fimbria, ammonia exposure affected the osmotic ion regulation which may be due to energy transition from growth and of sablefish. Cholesterol and total protein of plasma development to detoxification. Hepatosomatic index organic components in fish have been considered as a (HSI) is considered as a critical indicator to evaluate major component to assess fish health. However, there Table 4 Changes of organic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a a a a Cholesterol 1 131.5 ± 20.5 139.1 ± 18.2 140.5 ± 21.3 137.2 ± 16.8 142.2 ± 21.1 146.2 ± 18.3 (mg/dL) a a a a a a 2 135.1 ± 17.1 131.5 ± 17.1 138.2 ± 17.1 143.2 ± 21.3 148.3 ± 18.3 142.6 ± 20.2 a a a a a a Total protein 1 4.42 ± 0.53 4.35 ± 0.39 4.26 ± 0.45 4.51 ± 0.48 4.33 ± 0.37 4.59 ± 0.41 (g/dL) a a a a a a 2 4.36 ± 0.47 4.56 ± 0.35 4.37 ± 0.31 4.62 ± 0.52 4.24 ± 0.45 4.39 ± 0.51 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0 .05) as determined by Tukey’s multiple range test Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 5 of 6 Table 5 Changes of enzymatic plasma components in sablefish, Anoplopoma fimbria exposed to ammonia for 2 months Parameters Period Ammonia (mg/L) (month) 0 0.25 0.50 0.75 1.00 1.25 a a a a b b GOT 1 2.67 ± 0.33 2.72 ± 0.29 2.75 ± 0.35 2.81 ± 0.28 3.36 ± 0.41 3.45 ± 0.37 (karmen unit) a a ab b bc c 2 2.72 ± 0.25 2.61 ± 0.32 2.91 ± 0.21 3.24 ± 0.32 3.46 ± 0.40 3.62 ± 0.41 a a a ab b b GPT 1 1.72 ± 0.24 1.79 ± 0.19 1.84 ± 0.18 1.93 ± 0.26 2.19 ± 0.31 2.28 ± 0.27 (karmen unit) a a a b c c 2 1.75 ± 0.28 1.82 ± 0.23 1.99 ± 0.28 2.23 ± 0.31 2.41 ± 0.35 2.40 ± 0.35 Values are mean ± SE. Values with different superscript are significantly different at 1 and 2 months (P < 0.05) as determined by Tukey’s multiple range test was no significant alteration in sablefish, A. fimbria.GOT Disclosure The dataset(s) supporting the conclusions of this article is not included in and GPT in enzymatic plasma components can be easily the article. increased by hepatic tissue damage, and these are used to evaluate hepatic tissue damage (Agrahari et al., 2007). Le Ruyet et al. (1998) reported that a significant increase in Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in GOT and GPT of turbot, Scophthalmus maximus and published maps and institutional affiliations. seabream, Sparus aurata exposed to ammonia. In this study, GOP and GPT in sablefish, A. fimbria were signifi- Author details West Sea Fisheries Research Institute, National Institute of Fisheries Science, cantly increased by ammonia exposure, which may be due Incheon, Korea. Department of Aquatic Life Medicine, Pukyong National to hepatic tissue damage by ammonia. University, Busan, Korea. Department of Marine Biology, Pukyong National University, Busan, Korea. Department of Aqualife Medicine, Chonnam National University, Yeosu, Korea. Conclusion The results of this study indicate that ammonia exposure Received: 5 November 2016 Accepted: 17 March 2017 at the higher than proper concentrations affected growth performance and hematological parameters of sablefish, A. fimbria, and these changes should influence the References Agrahari S, Pandey KC, Gopal K. Biochemical alteration induced by health of sablefish, A. fimbria. In conclusion, ammonia monocrotophos in the blood plasma of fish, Channa punctatus (Bloch). Pestic concentrations at the higher than 0.75 mg/L can affect Biochem Physiol. 2007;88:268–72. various physiological effects of sablefish, A. fimbria, and Das PC, Ayyappan S, Jena JK, Das BK. Acute toxicity of ammonia and its sub- lethal effects on selected haematological and enzymatic parameters of the high concentrations of ammonia exposure require mrigal, Cirrhinus mrigala (Hamilton). Aquac Res. 2004;35:134–43. special attention in sablefish aquaculture. In addition to Datta S, Saha DR, Ghosh D, Majumdar T, Bhattacharya S, Mazumder S. Sub-lethal this environmental study, various environmental stan- concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L. Comp Biochem Physiol C dards should be established for stable sablefish Toxicol Pharmacol. 2007;145:339–49. aquaculture. Foss A, Evensen TH, Vollen T, Øiestad V. Effects of chronic ammonia exposure on growth and food conversion efficiency in juvenile spotted wolffish. Abbreviations Aquaculture. 2003;228:215–24. GOT: Glutamic oxalate transaminase; GPT: Glutamic pyruvate transaminase; Foss A, Imsland AK, Roth B, Schram E, Stefansson SO. Effects of chronic and HIS: Hepatosomatic index; RBC: Red blood cell periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus). Aquaculture. 2009;296:45–50. Acknowledgements Hur JW, Chang YJ, Lim HK, Lee BK. Stress responses of cultured fishes elicited by This research was a part of the project titled ‘Development of practical water level reduction in rearing tank and fish transference during selection techniques for the artificial seeding production of sablefish’, funded by the process. J Korean Fish Soc. 2001;34:465–72. MOF, Korea. Jeney G, Nemcsok J, Zs J, Olah J. Acute effect of sublethal ammonia concentrations on common carp (Cyprinus carpio L.). II Effect of ammonia on Authors’ contributions blood plasma transminases (GOT, GPT), G1DH enzyme activity, and ATP HJ, IK, and JM carried out the environmental toxicity studies and manuscript value. Aquaculture. 1992;104:149–56. writing. 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Ethics approval and consent to participate Liang Z, Liu R, Zhao D, Wang L, Sun M, Wang M, Song L. Ammonia exposure All experimental animals used in this study were maintained under a induces oxidative stress, endoplasmic reticulum stress and apoptosis in protocol approved by the Institutional Animal Care and Use Committee of epatopancreas of pacific white shrimp (Litopenaeus vannamei). Fish Shellfish the Pukyong National University. Immunol. 2015;54:523–8. Kim et al. Fisheries and Aquatic Sciences (2017) 20:4 Page 6 of 6 Paust LO, Foss A, Imsland AK. Effects of chronic and periodic exposure to ammonia on growth, food conversion efficiency and blood physiology in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture. 2011;315:400–6. Person-Le Ruyet J, Lamers A, Roux AL, Severe A, Boeuf G, Mayer‐Gostan N. Long‐ term ammonia exposure of turbot: effects on plasma parameters. J Fish Biol. 2003;62:879–94. Thangam Y, Perumayee M, Jayaprakash S, Umavathi S, Basheer SK. Studies of ammonia toxicity on haematological parameters to freshwater fish Cyprinus carpio (common carp). Int J Curr Microbiol App Sci. 2014;3:535–42. Vinodhini R, Narayanan M. The impact of toxic heavy metal on the hematological parameters in common carp (Cyprinus carpio L.). Iran J Environ Health Sci Eng. 2009;6:23–8. Wendelaar Bonga SE. The stress response in fish. Physiol Rev. 1997;77:591–625. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

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Fisheries and Aquatic SciencesSpringer Journals

Published: Mar 24, 2017

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