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Effects of butaphosphan and cyanocobalamin mixture on immunity and stress in olive flounder, Paralichthys olivaceus

Effects of butaphosphan and cyanocobalamin mixture on immunity and stress in olive flounder,... Background: The study evaluated the effects of a butaphosphan and cyanocobalamin mixture on the immune system and stress in olive flounders, Paralichthys olivaceus. Methods: The mixture was intramuscularly injected into olive flounders at the current recommended dose. Furthermore, to determine the toxicity of overdose, a histological examination was performed after injection of 1-, 2-, and 4-fold higher than the recommended dose. Results: Immunity parameters were altered during the first 2 weeks after a single intramuscular injection of the mixture in olive flounders (average weight 20.5 ± 1.1 g). The levels of all tested items, except glutathione and anti- protease, were higher in the treated group than in the control group in the first week; the levels of all tested items were even higher in the second week in the treated group than in the control group. The level of nitro-blue tetrazolium, myeloperoxidase, and superoxide dismutase between the two groups differed significantly. Changes in the stress response to different seawater temperatures (increase or decrease in seawater temperature by 3–5°C using 50 L heated or cooled seawater tanks) were studied by determining the changes in cortisol and glucose levels on days 1 and 7. Both cortisol and glucose levels were significantly lower in the treated group than in the control group. Histological analysis did not reveal any abnormalities after intramuscular injection of the mixture at doses that were 1-, 2-, and 4-fold higher than the recommended dose. Conclusions: Intramuscular injection of a butaphosphan and cyanocobalamin mixture is safe and effective in reducing stress and improving immunity in olive flounders. Keywords: Butaphosphan, Cyanocobalamin, Immunity, Olive flounder, Stress Background rate, stunted growth, and decline in the capability to Olive flounder is a popular seafood and a commercially maintain homeostasis and necessary immunity are important fish in Korea; it accounted for about 46.2% of reasons why the commercial production of this fish has the total cultured fish domestic production of 37,238 resulted in economic losses. These problems are associ- tons in 2018 (KOSIS 2018). However, increased infection ated with poor water quality and stress caused by high- density culture to meet the excessive demand for this fish (Maule et al. 1989; Kang et al. 2007). * Correspondence: leesj73@korea.kr Most domestic fish farms of olive flounder are set up Seaweed Research Center, National Institute of Fisheries Science, Mokpo by using running water at the natural water temperature 58746, South Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 2 of 9 (Kim et al. 2015). However, large water temperature Immunity and stress markers were quantified in the fluctuations during summer and winter are unsuitable blood and serum. Each analysis was performed in for the optimum growth of olive flounder; consequently, triplicate. this puts the fish under stress (Barton and Iwama 1991). Stress increases the level of cortisol and glucose in fish Test material (Davis and Parker 1990; Demers and Bayne 1997) and BPC mixture, approved by the National Institute of weakens the immune system by reducing the activities Fisheries Science’s as an abdominal and intramuscular of complement, lysozyme, etc. (Sunyer and Tort 1995). injection aquaculture drug (butaphosphan 100 mg and The butaphosphan and cyanocobalamin aquaculture cyanocobalamin 0.05 mg/mL), was used at the recom- drug mixture (BPC) is being used in domestic fish farms mended dose (0.5 mL/kg of fish body weight). to reduce stress and improve growth and immunity and, thus, to reduce the negative effects of the environment. BPC has been used widely in the livestock industry (e.g., Biochemical measurements and safety assessment for the production of cattle and pigs) and reported to (recommended BPC dose) improve liver function, tissue regeneration, and absorp- Olive flounders (n = 180; average weight 20.5 ± 1.1 g) tion of nutrients by increasing hematogenesis (Flasshoff were placed into six (30 fish each/three petitions per 1974) and reduce stress by lowering the cortisol concen- group) 120-L water tanks containing seawater and tration (Deniz et al. 2009). Reduced cortisol levels in fish grouped as follows: control group intramuscularly ad- help to maintain normal immune parameters (Seo et al. ministered 0.01 mL of phosphate-buffered saline and test 2020), and even under high-density culture stress, a groups intramuscularly administered BPC. reduction in the levels of IL-B, AST, and ALT helps to The feeding, behavior, branchial movement, internal prevent loss of immunity function and tissue damage and external features, and mortality were evaluated daily (Mechesso et al. 2019). However, research on the effects until day 14 after BPC administration. Immunity status of BPC in fish is scant. was evaluated on day 7 and 14 after BPC administration BPC is regarded as safe and is free of any residual and and was based on the levels of lysozyme (LZM), nitro- toxic components (MFDS 2013; EPMAR 2014). blue tetrazolium (NBT), superoxide dismutase (SOD), However, there have been reports drawing a correlation myeloperoxidase (MPO), glutathione peroxidase (GPx), between BPC administration and tissue damage and anti-protease (AP), and the total immunoglobulin (TIg) mortality in fish. It is possible that the dose used at some analyses. fish-farming sites is higher than that recommended. However, to get more clarity, we conducted this study to LZM analysis obtain relevant safety and efficacy data of BPC adminis- Serum LZM activity was determined by a turbidometric tration in the cultured olive flounder. assay (Yeh et al. 2008) utilizing lyophilized Micrococcus lysodeikticus cells (Sigma, USA). Briefly, each well of an Methods ELISA plate was filled with 10 μL of serum from individ- Fish ual fish, mixed with 200 μLof an M. lysodeikticus sus- Olive flounders (Paralichthys olivaceus) with no history pension (0.2 mg/mL in 0.05 M sodium phosphate buffer of disease or infection were purchased from a local fish [pH 6.2]), maintained at 25 °C, and the absorbance mea- farm in Jeju Island. Fish were moved to the Marine sured at 530 nm after 1 and 6 min using a plate reader. Environment Research Institute of Jeju National Univer- Each unit of LZM activity corresponded to a 0.001/min sity and were acclimatized for 1 week to the experimen- decrease in sample absorbance. tal environment (Fig. 1). The photoperiod was maintained at 12 h using fluores- cent lamps. The temperature of seawater used for cul- NBT analysis turing was maintained at 20–23 °C. Fish were fed The activation of serum macrophages was determined standard food (approximately 3% of the body weight) by measuring the neutrophil oxidative-radical generation twice daily at 9 am and 6 pm. using the method of Kumari and Sahoo (2005). Samples of whole blood (50 μL) and NBT solution (0.2%) were Sample collection placed in a glass test tube, to which 1 mL of dimethyl Five fish were selected at random from the control and formamide was added to reduce the generated formazan. BPC groups. The fish were anesthetized using 2- After centrifugation at 2000Íg for 5 min, the extent of phenoxyethanol solution (100 ppm). Blood samples were NBT reduction was determined by measuring the ab- taken from the tail vein using a heparin-treated syringe sorbance of the supernatant at 540 nm (Genesys 10UA, and separated by centrifugation (5000 rpm for 10 min). USA). Dimethyl formamide was used as the blank. Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 3 of 9 Fig. 1 Map showing the location of the study area. a Location of Jeju Island and b map of Jeju Island showing topography and location of Hamdeok SOD analysis GPx analysis Serum SOD activity was quantified using the SOD assay Serum GPx activity was measured following the instruc- kit (Sigma, 19160). Samples (20 μL) and 20 μLof WST tions of the GPx activity kit (Biovision, Inc., Milpitase, working solution were placed in a 96-well microplate, CA, USA). Serum samples were placed into a 96-well 15 μL of the enzyme solution was added, the mixture plate; solutions of NADH, glutathione, and glutathione was diluted with 2.5 mL of the dilution buffer, and the reductase were added; and absorbance was measured at content was mixed. After incubation for 20 min at 37 °C, 340 nm using a microplate reader. the absorbance was measured at 450 nm with a micro- plate reader (Thermo, USA). The results were calculated AP analysis using the formula Serum AP activity was determined using the method of Ellis (1990). Serum samples (20 μL) were mixed with ½ Ablank 1 - Ablank 3 -ðÞ Sample - Blank 2 =Ablank 1 20 μL of trypsin solution and reacted at 22 °C for 10 min. - Ablank 3 100 Then, 200 μL of phosphate buffer (0.1 M, pH 7.0) and 250 μL of azocasein (2%) were added to each sample, and the mixtures were reacted at 22 °C for 30 min. The samples were centrifuged at 6000Íg, and 100 μL aliquots MPO analysis of supernatants were placed in the wells of a 96-well Serum MPO activity was determined using the method plate together with 100 μL of NaOH (1 N). The absorb- of Kumari and Sahoo (2005). Each well of a 96-well plate ance was determined at 430 nm. was filled with 80 μL of Hanks balanced salt solution and 20 μL of serum, 20 mM of 3,3′,5,5′-tetramethyben- zidine, and 5 mM H O were added. After a reaction TIg analysis 2 2 time of 2 min, then 15 μLof 4M H SO solution was TIg was determined in the serum using the method of 2 4 added before the absorbance was measured at 450 nm Siwicki and Anderson (1993). Immunoglobulins were using a microplate reader (Thermo, USA). precipitated with 12% polyethylene glycol (Sigma, USA) Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 4 of 9 and quantified using the standard Bradford assay kit solution for 24 h, and dehydrated using 70% EtOH. (Sigma C-690). Tissues were embedded in paraffin (Leica EG 1150HC, Germany) using an embedder (Leica Jung 820, Statistical analysis Germany), sliced using a microtome, attached to glass SPSS (Ver. 21) statistical analysis program was used to slides, dried, and stained with hematoxylin and eosin. perform the T test. Statistical significance was set at P < Microscopic examination of these tissues was carried 0.05. out using an optical microscope (Zeiss LT60, Germany) to observe any abnormal histological changes that may Analysis of stress caused by changes in seawater have been caused by BPC. temperature Olive flounders (n = 120; average weight 20.5 ± 1.1 g) Results were placed in six (20 fish each/three petitions per Biochemical measurements and safety assessment group) 50-L water tanks that could be heated or cooled (recommended BPC dose) and grouped as follows: control group intramuscularly The safety of BPC was tested in comparison with a con- injected with 0.01 mL of phosphate-buffered saline and trol group. No increased mortality or abnormal findings test groups intramuscularly injected with BPC. Water in food intake, internal/external features, and branchial temperature was cooled and heated (by 3–5 °C) alter- movement were seen (data not shown). LZM, NBT, nately every 24 h by using cooling (17–19 °C) thermo- SOD, MPO, and TIg values, except for GPx and AP ac- stats and heating (20–22 °C) rods, respectively. Cortisol tivity, were higher in the BPC group than in the control and glucose levels were determined on day 1 and day 7. group in the first week, but no significant differences The study was conducted for 7 days only since the possi- were observed except for the LZM activity (Table 1). bility of olive flounders dying because of the changes in The values of all these immunity markers were higher in water temperature needed to be considered; hence, the the second week than in the first week, with higher fish mortality rate was also observed. values being observed in the BPC group than in the con- trol group for all markers. Significant differences were Cortisol analysis observed in NBT, MPO, and SOD activities between the The serum cortisol level was measured by an enzyme two groups (Table 1). immunoassay kit (Bio vision, USA). Reduction of stress caused by changes in seawater Glucose analysis temperature Serum glucose was determined using an automatic Significantly lower levels of cortisol and glucose were biochemical analyzer (IDDXX, USA). found in the BPC group than in the control group (Fig. 2). Fish deaths associated with changes in seawater Statistical analysis temperature occurred from day 5 in the control group Statistical analysis was conducted in the same way as for and reached 20% mortality on the last day of the test, the immunological method using SPSS (Ver. 21) statis- whereas no mortality was observed during the same tical analysis program to perform the T test and to period in the BPC group (Fig. 3). determine the data statistical significance at P < 0.05. Safety assessment (BPC overdose) Safety assessment (BPC overdose) During the 14 days of the study, no abnormality or mor- Olive flounders (n = 240; average weight 20.5 ± 1.1 g) tality was observed in any of the groups (data not were placed in 12 (20 fish each/three petitions per shown). Histological analysis showed similar results for group) 100-L water tanks containing seawater and all the BPC groups. The gill plates and gill filaments grouped as follows: control group intramuscularly showed a constant configuration, and no signs of tox- injected with 0.01 mL of phosphate-buffered saline and icity, such as bleeding, congestion on gills, or necrosis of test groups intramuscularly injected with 0.01 mL, 0.02 epithelial cells, were observed in any of the test groups. mL, and 0.04 mL of BPC. Histological analysis was No fish showed abnormalities in the muscle injected conducted to evaluate the effect of an overdose of BPC with BPC, and while minor degeneration was seen in the on day 7 and day 14 after administration using five muscle fiber, similar minor degeneration was observed randomly collected fish from each group. even in the control group. No bleeding or infiltration of inflammatory cells was observed. No toxic effects such Histological analysis as misalignment of liver cells due to damage to the hep- The liver, kidneys, spleen, gill, intestines, and muscles atic cord or degeneration of the tubular epithelial cells (injected area) were extracted from fish, fixed in Bouin’s in the kidneys caused by the administration of BPC were Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 5 of 9 Table 1 The effect of BPC on immune responses for 1 week and 2 weeks. BPC was injected intramuscularly at a dose of 0.01 mL Groups LZM NBT SOD MPO GPx AP TIg (units/mL) (absorbance) (%, inhibition) (absorbance) (mU/mL) (%, inhibition) (mg/mL) 1 week Control 16 ± 1 0.87 ± 0.01 49 ± 7.46 1.64 ± 0.28 15.1 ± 2.02 60.3 ± 3.51 21.2 ± 2.26 BPC 18.3 ± 4.04* 0.88 ± 0.005 53 ± 0.73 1.73 ± 0.32 13.3 ± 3.27 59.3 ± 1.53 21.3 ± 0.66 2 weeks Control 22.3 ± 3.78 0.93 ± 0.002 51.2 ± 3.52 1.81 ± 0.11 28.2 ± 2.89 75 ± 3.61 25.6 ± 2.64 BPC 25 ± 4.58 0.95 ± 0.007* 56.1 ± 5.32* 2.01 ± 0.09* 30.1 ± 3.08 77 ± 2 27.6 ± 1.93 Values are mean ± SE (n =3) LZM lysozyme, NBT nitro-blue tetrazolium, SOD superoxide dismutase, MPO myeloperoxidase, GPx glutathione peroxidase, AP anti-protease, Tig total immunoglobulin *Significant difference between the control group and the BPC group based on the t test (P < 0.05) observed. Red blood cells were observed in the spleen An increase in non-specific immune response reduces tissue of olive flounders in both the treated and control mortality and improves fish health (Ji et al. 2007; Yuan groups, but no increase in white blood cells was ob- et al. 2007; Citarasu 2010). LZM is a widely distributed served. BPC injection did not cause any inflammation or enzyme that plays a major role in the defense mechan- other toxic effects. There were no changes over time to ism of higher animals as it functions as an opsonin and the normal histology after BPC administration (Fig. 4). protects against microbes (e.g., viruses and bacteria) and disease conditions (e.g., cancer) (Jollès and Jollès 1984). Discussion NBT increases reactive oxygen species (ROS) formation BPC is used in domestic fish farms of olive flounder as a by hydrolyzing invading organisms such as viruses and registered aquaculture drug to improve immunity and bacteria. MPO plays a role in the inflammatory response; reduce stress in fish (Mechesso et al. 2019; National In- it induces toxicity in the deceased host when destroying stitute of Fisheries Sciences 2018; Seo et al. 2020). Al- the invading organisms (Palić et al. 2005). Activation of though BPC is shown to be effective based on its SOD and GPx antioxidant enzymes is known to prevent application in the livestock industry (Pereira et al. 2013a, ROS-mediated cell damage (Lopes et al. 2001). TIg im- 2013b; Tabeleao et al. 2017), its effects on fish remain proves immunity as it increases acidophilic granulocytes unclear. In this study, BPC was intramuscularly injected (Picchietti et al. 2007). Our study showed higher im- into olive flounders at the current recommended dose, munity in fish treated with BPC than in control fish; this and tests were conducted to determine the effects of was inferred as all measured parameters improved in the BPC on immunity and stress. Furthermore, the toxic ef- BPC groups whereas only one the control group that im- fects of BPC overdose (1-, 2-, and 4-fold higher than the proved in the second week. Hasi et al. 2005a, 2005b recommended dose) were studied based on histological reported that butaphosphan improved humoral and cell- examination. mediated immune responses in mice; we observed the Fig. 2 Effect of BPC on stressed responses. BPC was injected intramuscularly at a dose of 0.01 mL. a Cortisol and b glucose Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 6 of 9 Fig. 3 Cumulative mortality in the control group and the BPC group in 1 week Fig. 4 Histological results of the gills (a), muscles (b), kidneys (c), liver (d), spleen (e), and intestine (f) after injecting different concentrations of BPC for 1 week. Hematoxylin and eosin stating (scale bar = 20 μm). Ch, chondrocyte; Gc, goblet cell; Gf, gill filament; Gl, gill lamella; Glo, glomerulus; He, hepatocyte; Mb, muscle bundle; Mc, mucous cell; Ml, mucosal layer; MMC, melanomacrophage; Pc, pillar cell; RBC, red blood cell; Rt, renal tubule; Si, sinusoid; Sl, submucosal layer; WBC, white blood cell-enriched region Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 7 of 9 same in the present study in fish. We observed that fish energy metabolism dynamics that occur when fish are treated with BPC showed excellent feeding behaviors under stress (Barton and Iwama 1991). We determined than control fish when similar feed amounts were whether the reduced glucose concentration in the BPC supplied. This result may also have some effect on group was related to the energy consumed under stress. immunity. However, we could not assess the possible ac- Although our results match those by Chang et al. (2001) tion mechanisms of BPC that would promote appetite and show a close correlation with the decreased cortisol and immunity; additional tests will be needed to identify levels, further studies might be beneficial to elucidate such mechanisms. the mechanism connecting stress, glucose, cortisol, and Factors that could activate stress in fish farms might BPC. include physical (selection or transport), chemical The permitted residual quantities of veterinary drugs (changes in seawater temperature, salinity, or dissolved used in domestic animals are determined by combining oxygen), and mental and social stress (high-density cul- toxicological evaluation and permitted residual quantity ture or sound) (Donaldson 1981). Unlike fish grown in evaluation considering international standards. However, their natural environment, cultured fish may always be butaphosphan and vitamins are classified as safe and are exposed to stress as they are cultured in a closed and exempt from the evaluation of the residual quantity and limited space (Pickering 1990). toxicity limits (MFDS 2013; EPMAR 2014). Fish cultured under stress for a long time may have in- Aquaculture drugs used in fish farms are excreted by creased disease incidence owing to their decreased abil- or disappear naturally in fish if the dose and usage ity to maintain homeostasis, lesser resistance to stress, comply with the existing standards. This can ensure the and lower immunity, all of which contribute to increased safety of sea foods. However, some fish farms tend to mortality (Heo et al. 2002; Yang et al. 2016). administer drugs in excessive amounts over the recom- This study adopted the anesthetic approach suggested mended dose. We conducted histological analysis after by Wer et al. (2009), which involved limiting the time BPC was administered at 1-, 2-, and 4-fold higher than under anesthesia to 3 min and the recovery time to 5 the recommended dose to determine its safety. The min to suppress a possible long-term physiological stress results showed that BPC did not alter the histological due to the applied anesthetic. findings and that it was safe for olive flounders even at a We observed significantly lower levels of cortisol and dose 4-times higher (0.04 mL) than the recommended glucose in the BPC groups than in the control group. dose. No side effects were observed with the high dose Seo et al. (2020) tested stress in BPC-treated olive floun- of BPC. ders kept under high-density culture conditions and re- Seo et al. (2020) reported that the AST level signifi- ported that cortisol levels decreased sharply in the BPC cantly increased within 24 h after injecting 0.3 mL/kg group compared to those in the control group (phos- BPC into olive flounders without any toxicity. Histo- phate-buffered saline-treated fish); the results of our logical examination in this study showed normal liver study are similar. BPC is an amino acid phosphonic acid tissues, which is a natural consequence of a long-term derivative; while its exact mechanism of action has not investigation as the drug is excreted from the body or been elucidated (EMEA 1999), it was reported that it disappears naturally over time with no complications can accelerate the production of ATP by supplying inor- seen histologically. ganic phosphate to support oxidative phosphorylation The results of our study indicate that intramuscular that is an important metabolic process in ATP gener- injection of BPC improves the immunity of, and reduces ation (Hasi et al. 2005a, 2005b). Seo et al. (2020) re- stress in, olive flounders. The relationships between im- ported that ATP concentration in olive flounders that munity and appetite and between immunity and stress received an intramuscular injection of BPC increased are still to be elucidated. In addition, field test data on compared with that in the control group (phosphate- BPC use will be required. buffered saline-treated fish). It is likely that the increase in ATP production contributed to the stress reduction Conclusion by compensating for the stress-induced energy loss; BPC effectively and safely reduces stress levels by redu- elucidation of the exact underlying mechanisms here cing the production of cortisol and glucose. BPC would be of interest. improves the health of olive flounder, especially when Hematological factors are used as indicators of physio- administered before or during stressful situations. logical stress in fish in response to changes in water temperature (Adam 1990; Cataldi et al. 1998). Glucose is Abbreviations BPC: Butaphosphan and cyanocobalamin mixture; LZM: Lysozyme; one of the factors that, via a secondary reaction, in- GPx: Glutathione peroxidase; AP: Anti-protease; NBT: Nitro-blue tetrazolium; creases cortisol level; increased blood glucose acts as an MPO: Myeloperoxidase; ROS: Reactive oxygen species; SOD: Superoxide energy source and compensates for the changes in dismutase; TIg: Total immunoglobulin Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 8 of 9 Acknowledgements Flasshoff FH. Clinical and chemical blood serum investigations in cattle and This work was supported by a grant from the National Institute of Fisheries treatment studies with ornithine-aspartate product HMV20 and with catosal Science (R2020060). for the reduction of fertility and health disorders. Germany: Tierarztliche Hochschule Hannover; 1974. Hasi S, Du X, Jiang J, Zhu B. Study on immune stimulating properties of Authors’ contributions compound butafosfan solution in mice. 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Effects of butaphosphan and cyanocobalamin mixture on immunity and stress in olive flounder, Paralichthys olivaceus

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Abstract

Background: The study evaluated the effects of a butaphosphan and cyanocobalamin mixture on the immune system and stress in olive flounders, Paralichthys olivaceus. Methods: The mixture was intramuscularly injected into olive flounders at the current recommended dose. Furthermore, to determine the toxicity of overdose, a histological examination was performed after injection of 1-, 2-, and 4-fold higher than the recommended dose. Results: Immunity parameters were altered during the first 2 weeks after a single intramuscular injection of the mixture in olive flounders (average weight 20.5 ± 1.1 g). The levels of all tested items, except glutathione and anti- protease, were higher in the treated group than in the control group in the first week; the levels of all tested items were even higher in the second week in the treated group than in the control group. The level of nitro-blue tetrazolium, myeloperoxidase, and superoxide dismutase between the two groups differed significantly. Changes in the stress response to different seawater temperatures (increase or decrease in seawater temperature by 3–5°C using 50 L heated or cooled seawater tanks) were studied by determining the changes in cortisol and glucose levels on days 1 and 7. Both cortisol and glucose levels were significantly lower in the treated group than in the control group. Histological analysis did not reveal any abnormalities after intramuscular injection of the mixture at doses that were 1-, 2-, and 4-fold higher than the recommended dose. Conclusions: Intramuscular injection of a butaphosphan and cyanocobalamin mixture is safe and effective in reducing stress and improving immunity in olive flounders. Keywords: Butaphosphan, Cyanocobalamin, Immunity, Olive flounder, Stress Background rate, stunted growth, and decline in the capability to Olive flounder is a popular seafood and a commercially maintain homeostasis and necessary immunity are important fish in Korea; it accounted for about 46.2% of reasons why the commercial production of this fish has the total cultured fish domestic production of 37,238 resulted in economic losses. These problems are associ- tons in 2018 (KOSIS 2018). However, increased infection ated with poor water quality and stress caused by high- density culture to meet the excessive demand for this fish (Maule et al. 1989; Kang et al. 2007). * Correspondence: leesj73@korea.kr Most domestic fish farms of olive flounder are set up Seaweed Research Center, National Institute of Fisheries Science, Mokpo by using running water at the natural water temperature 58746, South Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 2 of 9 (Kim et al. 2015). However, large water temperature Immunity and stress markers were quantified in the fluctuations during summer and winter are unsuitable blood and serum. Each analysis was performed in for the optimum growth of olive flounder; consequently, triplicate. this puts the fish under stress (Barton and Iwama 1991). Stress increases the level of cortisol and glucose in fish Test material (Davis and Parker 1990; Demers and Bayne 1997) and BPC mixture, approved by the National Institute of weakens the immune system by reducing the activities Fisheries Science’s as an abdominal and intramuscular of complement, lysozyme, etc. (Sunyer and Tort 1995). injection aquaculture drug (butaphosphan 100 mg and The butaphosphan and cyanocobalamin aquaculture cyanocobalamin 0.05 mg/mL), was used at the recom- drug mixture (BPC) is being used in domestic fish farms mended dose (0.5 mL/kg of fish body weight). to reduce stress and improve growth and immunity and, thus, to reduce the negative effects of the environment. BPC has been used widely in the livestock industry (e.g., Biochemical measurements and safety assessment for the production of cattle and pigs) and reported to (recommended BPC dose) improve liver function, tissue regeneration, and absorp- Olive flounders (n = 180; average weight 20.5 ± 1.1 g) tion of nutrients by increasing hematogenesis (Flasshoff were placed into six (30 fish each/three petitions per 1974) and reduce stress by lowering the cortisol concen- group) 120-L water tanks containing seawater and tration (Deniz et al. 2009). Reduced cortisol levels in fish grouped as follows: control group intramuscularly ad- help to maintain normal immune parameters (Seo et al. ministered 0.01 mL of phosphate-buffered saline and test 2020), and even under high-density culture stress, a groups intramuscularly administered BPC. reduction in the levels of IL-B, AST, and ALT helps to The feeding, behavior, branchial movement, internal prevent loss of immunity function and tissue damage and external features, and mortality were evaluated daily (Mechesso et al. 2019). However, research on the effects until day 14 after BPC administration. Immunity status of BPC in fish is scant. was evaluated on day 7 and 14 after BPC administration BPC is regarded as safe and is free of any residual and and was based on the levels of lysozyme (LZM), nitro- toxic components (MFDS 2013; EPMAR 2014). blue tetrazolium (NBT), superoxide dismutase (SOD), However, there have been reports drawing a correlation myeloperoxidase (MPO), glutathione peroxidase (GPx), between BPC administration and tissue damage and anti-protease (AP), and the total immunoglobulin (TIg) mortality in fish. It is possible that the dose used at some analyses. fish-farming sites is higher than that recommended. However, to get more clarity, we conducted this study to LZM analysis obtain relevant safety and efficacy data of BPC adminis- Serum LZM activity was determined by a turbidometric tration in the cultured olive flounder. assay (Yeh et al. 2008) utilizing lyophilized Micrococcus lysodeikticus cells (Sigma, USA). Briefly, each well of an Methods ELISA plate was filled with 10 μL of serum from individ- Fish ual fish, mixed with 200 μLof an M. lysodeikticus sus- Olive flounders (Paralichthys olivaceus) with no history pension (0.2 mg/mL in 0.05 M sodium phosphate buffer of disease or infection were purchased from a local fish [pH 6.2]), maintained at 25 °C, and the absorbance mea- farm in Jeju Island. Fish were moved to the Marine sured at 530 nm after 1 and 6 min using a plate reader. Environment Research Institute of Jeju National Univer- Each unit of LZM activity corresponded to a 0.001/min sity and were acclimatized for 1 week to the experimen- decrease in sample absorbance. tal environment (Fig. 1). The photoperiod was maintained at 12 h using fluores- cent lamps. The temperature of seawater used for cul- NBT analysis turing was maintained at 20–23 °C. Fish were fed The activation of serum macrophages was determined standard food (approximately 3% of the body weight) by measuring the neutrophil oxidative-radical generation twice daily at 9 am and 6 pm. using the method of Kumari and Sahoo (2005). Samples of whole blood (50 μL) and NBT solution (0.2%) were Sample collection placed in a glass test tube, to which 1 mL of dimethyl Five fish were selected at random from the control and formamide was added to reduce the generated formazan. BPC groups. The fish were anesthetized using 2- After centrifugation at 2000Íg for 5 min, the extent of phenoxyethanol solution (100 ppm). Blood samples were NBT reduction was determined by measuring the ab- taken from the tail vein using a heparin-treated syringe sorbance of the supernatant at 540 nm (Genesys 10UA, and separated by centrifugation (5000 rpm for 10 min). USA). Dimethyl formamide was used as the blank. Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 3 of 9 Fig. 1 Map showing the location of the study area. a Location of Jeju Island and b map of Jeju Island showing topography and location of Hamdeok SOD analysis GPx analysis Serum SOD activity was quantified using the SOD assay Serum GPx activity was measured following the instruc- kit (Sigma, 19160). Samples (20 μL) and 20 μLof WST tions of the GPx activity kit (Biovision, Inc., Milpitase, working solution were placed in a 96-well microplate, CA, USA). Serum samples were placed into a 96-well 15 μL of the enzyme solution was added, the mixture plate; solutions of NADH, glutathione, and glutathione was diluted with 2.5 mL of the dilution buffer, and the reductase were added; and absorbance was measured at content was mixed. After incubation for 20 min at 37 °C, 340 nm using a microplate reader. the absorbance was measured at 450 nm with a micro- plate reader (Thermo, USA). The results were calculated AP analysis using the formula Serum AP activity was determined using the method of Ellis (1990). Serum samples (20 μL) were mixed with ½ Ablank 1 - Ablank 3 -ðÞ Sample - Blank 2 =Ablank 1 20 μL of trypsin solution and reacted at 22 °C for 10 min. - Ablank 3 100 Then, 200 μL of phosphate buffer (0.1 M, pH 7.0) and 250 μL of azocasein (2%) were added to each sample, and the mixtures were reacted at 22 °C for 30 min. The samples were centrifuged at 6000Íg, and 100 μL aliquots MPO analysis of supernatants were placed in the wells of a 96-well Serum MPO activity was determined using the method plate together with 100 μL of NaOH (1 N). The absorb- of Kumari and Sahoo (2005). Each well of a 96-well plate ance was determined at 430 nm. was filled with 80 μL of Hanks balanced salt solution and 20 μL of serum, 20 mM of 3,3′,5,5′-tetramethyben- zidine, and 5 mM H O were added. After a reaction TIg analysis 2 2 time of 2 min, then 15 μLof 4M H SO solution was TIg was determined in the serum using the method of 2 4 added before the absorbance was measured at 450 nm Siwicki and Anderson (1993). Immunoglobulins were using a microplate reader (Thermo, USA). precipitated with 12% polyethylene glycol (Sigma, USA) Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 4 of 9 and quantified using the standard Bradford assay kit solution for 24 h, and dehydrated using 70% EtOH. (Sigma C-690). Tissues were embedded in paraffin (Leica EG 1150HC, Germany) using an embedder (Leica Jung 820, Statistical analysis Germany), sliced using a microtome, attached to glass SPSS (Ver. 21) statistical analysis program was used to slides, dried, and stained with hematoxylin and eosin. perform the T test. Statistical significance was set at P < Microscopic examination of these tissues was carried 0.05. out using an optical microscope (Zeiss LT60, Germany) to observe any abnormal histological changes that may Analysis of stress caused by changes in seawater have been caused by BPC. temperature Olive flounders (n = 120; average weight 20.5 ± 1.1 g) Results were placed in six (20 fish each/three petitions per Biochemical measurements and safety assessment group) 50-L water tanks that could be heated or cooled (recommended BPC dose) and grouped as follows: control group intramuscularly The safety of BPC was tested in comparison with a con- injected with 0.01 mL of phosphate-buffered saline and trol group. No increased mortality or abnormal findings test groups intramuscularly injected with BPC. Water in food intake, internal/external features, and branchial temperature was cooled and heated (by 3–5 °C) alter- movement were seen (data not shown). LZM, NBT, nately every 24 h by using cooling (17–19 °C) thermo- SOD, MPO, and TIg values, except for GPx and AP ac- stats and heating (20–22 °C) rods, respectively. Cortisol tivity, were higher in the BPC group than in the control and glucose levels were determined on day 1 and day 7. group in the first week, but no significant differences The study was conducted for 7 days only since the possi- were observed except for the LZM activity (Table 1). bility of olive flounders dying because of the changes in The values of all these immunity markers were higher in water temperature needed to be considered; hence, the the second week than in the first week, with higher fish mortality rate was also observed. values being observed in the BPC group than in the con- trol group for all markers. Significant differences were Cortisol analysis observed in NBT, MPO, and SOD activities between the The serum cortisol level was measured by an enzyme two groups (Table 1). immunoassay kit (Bio vision, USA). Reduction of stress caused by changes in seawater Glucose analysis temperature Serum glucose was determined using an automatic Significantly lower levels of cortisol and glucose were biochemical analyzer (IDDXX, USA). found in the BPC group than in the control group (Fig. 2). Fish deaths associated with changes in seawater Statistical analysis temperature occurred from day 5 in the control group Statistical analysis was conducted in the same way as for and reached 20% mortality on the last day of the test, the immunological method using SPSS (Ver. 21) statis- whereas no mortality was observed during the same tical analysis program to perform the T test and to period in the BPC group (Fig. 3). determine the data statistical significance at P < 0.05. Safety assessment (BPC overdose) Safety assessment (BPC overdose) During the 14 days of the study, no abnormality or mor- Olive flounders (n = 240; average weight 20.5 ± 1.1 g) tality was observed in any of the groups (data not were placed in 12 (20 fish each/three petitions per shown). Histological analysis showed similar results for group) 100-L water tanks containing seawater and all the BPC groups. The gill plates and gill filaments grouped as follows: control group intramuscularly showed a constant configuration, and no signs of tox- injected with 0.01 mL of phosphate-buffered saline and icity, such as bleeding, congestion on gills, or necrosis of test groups intramuscularly injected with 0.01 mL, 0.02 epithelial cells, were observed in any of the test groups. mL, and 0.04 mL of BPC. Histological analysis was No fish showed abnormalities in the muscle injected conducted to evaluate the effect of an overdose of BPC with BPC, and while minor degeneration was seen in the on day 7 and day 14 after administration using five muscle fiber, similar minor degeneration was observed randomly collected fish from each group. even in the control group. No bleeding or infiltration of inflammatory cells was observed. No toxic effects such Histological analysis as misalignment of liver cells due to damage to the hep- The liver, kidneys, spleen, gill, intestines, and muscles atic cord or degeneration of the tubular epithelial cells (injected area) were extracted from fish, fixed in Bouin’s in the kidneys caused by the administration of BPC were Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 5 of 9 Table 1 The effect of BPC on immune responses for 1 week and 2 weeks. BPC was injected intramuscularly at a dose of 0.01 mL Groups LZM NBT SOD MPO GPx AP TIg (units/mL) (absorbance) (%, inhibition) (absorbance) (mU/mL) (%, inhibition) (mg/mL) 1 week Control 16 ± 1 0.87 ± 0.01 49 ± 7.46 1.64 ± 0.28 15.1 ± 2.02 60.3 ± 3.51 21.2 ± 2.26 BPC 18.3 ± 4.04* 0.88 ± 0.005 53 ± 0.73 1.73 ± 0.32 13.3 ± 3.27 59.3 ± 1.53 21.3 ± 0.66 2 weeks Control 22.3 ± 3.78 0.93 ± 0.002 51.2 ± 3.52 1.81 ± 0.11 28.2 ± 2.89 75 ± 3.61 25.6 ± 2.64 BPC 25 ± 4.58 0.95 ± 0.007* 56.1 ± 5.32* 2.01 ± 0.09* 30.1 ± 3.08 77 ± 2 27.6 ± 1.93 Values are mean ± SE (n =3) LZM lysozyme, NBT nitro-blue tetrazolium, SOD superoxide dismutase, MPO myeloperoxidase, GPx glutathione peroxidase, AP anti-protease, Tig total immunoglobulin *Significant difference between the control group and the BPC group based on the t test (P < 0.05) observed. Red blood cells were observed in the spleen An increase in non-specific immune response reduces tissue of olive flounders in both the treated and control mortality and improves fish health (Ji et al. 2007; Yuan groups, but no increase in white blood cells was ob- et al. 2007; Citarasu 2010). LZM is a widely distributed served. BPC injection did not cause any inflammation or enzyme that plays a major role in the defense mechan- other toxic effects. There were no changes over time to ism of higher animals as it functions as an opsonin and the normal histology after BPC administration (Fig. 4). protects against microbes (e.g., viruses and bacteria) and disease conditions (e.g., cancer) (Jollès and Jollès 1984). Discussion NBT increases reactive oxygen species (ROS) formation BPC is used in domestic fish farms of olive flounder as a by hydrolyzing invading organisms such as viruses and registered aquaculture drug to improve immunity and bacteria. MPO plays a role in the inflammatory response; reduce stress in fish (Mechesso et al. 2019; National In- it induces toxicity in the deceased host when destroying stitute of Fisheries Sciences 2018; Seo et al. 2020). Al- the invading organisms (Palić et al. 2005). Activation of though BPC is shown to be effective based on its SOD and GPx antioxidant enzymes is known to prevent application in the livestock industry (Pereira et al. 2013a, ROS-mediated cell damage (Lopes et al. 2001). TIg im- 2013b; Tabeleao et al. 2017), its effects on fish remain proves immunity as it increases acidophilic granulocytes unclear. In this study, BPC was intramuscularly injected (Picchietti et al. 2007). Our study showed higher im- into olive flounders at the current recommended dose, munity in fish treated with BPC than in control fish; this and tests were conducted to determine the effects of was inferred as all measured parameters improved in the BPC on immunity and stress. Furthermore, the toxic ef- BPC groups whereas only one the control group that im- fects of BPC overdose (1-, 2-, and 4-fold higher than the proved in the second week. Hasi et al. 2005a, 2005b recommended dose) were studied based on histological reported that butaphosphan improved humoral and cell- examination. mediated immune responses in mice; we observed the Fig. 2 Effect of BPC on stressed responses. BPC was injected intramuscularly at a dose of 0.01 mL. a Cortisol and b glucose Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 6 of 9 Fig. 3 Cumulative mortality in the control group and the BPC group in 1 week Fig. 4 Histological results of the gills (a), muscles (b), kidneys (c), liver (d), spleen (e), and intestine (f) after injecting different concentrations of BPC for 1 week. Hematoxylin and eosin stating (scale bar = 20 μm). Ch, chondrocyte; Gc, goblet cell; Gf, gill filament; Gl, gill lamella; Glo, glomerulus; He, hepatocyte; Mb, muscle bundle; Mc, mucous cell; Ml, mucosal layer; MMC, melanomacrophage; Pc, pillar cell; RBC, red blood cell; Rt, renal tubule; Si, sinusoid; Sl, submucosal layer; WBC, white blood cell-enriched region Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 7 of 9 same in the present study in fish. We observed that fish energy metabolism dynamics that occur when fish are treated with BPC showed excellent feeding behaviors under stress (Barton and Iwama 1991). We determined than control fish when similar feed amounts were whether the reduced glucose concentration in the BPC supplied. This result may also have some effect on group was related to the energy consumed under stress. immunity. However, we could not assess the possible ac- Although our results match those by Chang et al. (2001) tion mechanisms of BPC that would promote appetite and show a close correlation with the decreased cortisol and immunity; additional tests will be needed to identify levels, further studies might be beneficial to elucidate such mechanisms. the mechanism connecting stress, glucose, cortisol, and Factors that could activate stress in fish farms might BPC. include physical (selection or transport), chemical The permitted residual quantities of veterinary drugs (changes in seawater temperature, salinity, or dissolved used in domestic animals are determined by combining oxygen), and mental and social stress (high-density cul- toxicological evaluation and permitted residual quantity ture or sound) (Donaldson 1981). Unlike fish grown in evaluation considering international standards. However, their natural environment, cultured fish may always be butaphosphan and vitamins are classified as safe and are exposed to stress as they are cultured in a closed and exempt from the evaluation of the residual quantity and limited space (Pickering 1990). toxicity limits (MFDS 2013; EPMAR 2014). Fish cultured under stress for a long time may have in- Aquaculture drugs used in fish farms are excreted by creased disease incidence owing to their decreased abil- or disappear naturally in fish if the dose and usage ity to maintain homeostasis, lesser resistance to stress, comply with the existing standards. This can ensure the and lower immunity, all of which contribute to increased safety of sea foods. However, some fish farms tend to mortality (Heo et al. 2002; Yang et al. 2016). administer drugs in excessive amounts over the recom- This study adopted the anesthetic approach suggested mended dose. We conducted histological analysis after by Wer et al. (2009), which involved limiting the time BPC was administered at 1-, 2-, and 4-fold higher than under anesthesia to 3 min and the recovery time to 5 the recommended dose to determine its safety. The min to suppress a possible long-term physiological stress results showed that BPC did not alter the histological due to the applied anesthetic. findings and that it was safe for olive flounders even at a We observed significantly lower levels of cortisol and dose 4-times higher (0.04 mL) than the recommended glucose in the BPC groups than in the control group. dose. No side effects were observed with the high dose Seo et al. (2020) tested stress in BPC-treated olive floun- of BPC. ders kept under high-density culture conditions and re- Seo et al. (2020) reported that the AST level signifi- ported that cortisol levels decreased sharply in the BPC cantly increased within 24 h after injecting 0.3 mL/kg group compared to those in the control group (phos- BPC into olive flounders without any toxicity. Histo- phate-buffered saline-treated fish); the results of our logical examination in this study showed normal liver study are similar. BPC is an amino acid phosphonic acid tissues, which is a natural consequence of a long-term derivative; while its exact mechanism of action has not investigation as the drug is excreted from the body or been elucidated (EMEA 1999), it was reported that it disappears naturally over time with no complications can accelerate the production of ATP by supplying inor- seen histologically. ganic phosphate to support oxidative phosphorylation The results of our study indicate that intramuscular that is an important metabolic process in ATP gener- injection of BPC improves the immunity of, and reduces ation (Hasi et al. 2005a, 2005b). Seo et al. (2020) re- stress in, olive flounders. The relationships between im- ported that ATP concentration in olive flounders that munity and appetite and between immunity and stress received an intramuscular injection of BPC increased are still to be elucidated. In addition, field test data on compared with that in the control group (phosphate- BPC use will be required. buffered saline-treated fish). It is likely that the increase in ATP production contributed to the stress reduction Conclusion by compensating for the stress-induced energy loss; BPC effectively and safely reduces stress levels by redu- elucidation of the exact underlying mechanisms here cing the production of cortisol and glucose. BPC would be of interest. improves the health of olive flounder, especially when Hematological factors are used as indicators of physio- administered before or during stressful situations. logical stress in fish in response to changes in water temperature (Adam 1990; Cataldi et al. 1998). Glucose is Abbreviations BPC: Butaphosphan and cyanocobalamin mixture; LZM: Lysozyme; one of the factors that, via a secondary reaction, in- GPx: Glutathione peroxidase; AP: Anti-protease; NBT: Nitro-blue tetrazolium; creases cortisol level; increased blood glucose acts as an MPO: Myeloperoxidase; ROS: Reactive oxygen species; SOD: Superoxide energy source and compensates for the changes in dismutase; TIg: Total immunoglobulin Kim et al. Fisheries and Aquatic Sciences (2020) 23:26 Page 8 of 9 Acknowledgements Flasshoff FH. Clinical and chemical blood serum investigations in cattle and This work was supported by a grant from the National Institute of Fisheries treatment studies with ornithine-aspartate product HMV20 and with catosal Science (R2020060). for the reduction of fertility and health disorders. Germany: Tierarztliche Hochschule Hannover; 1974. Hasi S, Du X, Jiang J, Zhu B. Study on immune stimulating properties of Authors’ contributions compound butafosfan solution in mice. 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