The ameliorative role of ascorbic acid against blood disorder, immunosuppression and oxidative damage of oxytetracycline in rainbow trout (Oncorhynchus mykiss)

This experiment aimed to determine the possible benecial effects of dietary ascorbic acid (AA) on hematological indices, immune responses, and antioxidative capacity of Oncorhynchus mykiss treated with antibiotic oxytetracycline (OTC). 150 sh were divided evenly among ve experimental groups (30 sh of each, in 3 replicates) receiving diets containing OTC (0 and 100 mg per kg sh weight) and AA (100, 200, 400 and 800 mg per kg sh diet) for 28 days. Treatments include group A or control (100 mg AA without OTC), group B (100 mg AA with OTC), group C (200 mg AA with OTC), group D (400 mg AA with OTC), and group E (800 mg AA with OTC). The results obtained showed that the hematological indices (red blood cells, white blood cells, hematocrit, hemoglobin and neutrophils), immunological parameters (plasma lysozyme, plasma complement and skin mucus alkaline phosphatase activities), and antioxidant enzymes activities (superoxide dismutase and catalase) were signicantly decreased by OTC in O. mykiss fed control diet (P < 0.05). The results also revealed that OTC signicantly increased the activity of biochemical enzymes (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase) in the plasma of O. mykiss fed control diet (P < 0.05). However, in comparison to the control diet, feeding sh with higher amounts of AA (400 and 800 mg/kg diet) signicantly restored the hematological, immunological, and antioxidative responses in OTC treated groups (p < 0.05). These ndings show that the dietary supplementation of AA at 400 or 800 mg/kg diet is benecial in relieving O. mykiss from OTC-induced oxidative stress and immunosuppression.


Introduction
During the previous decade, the outbreak of infectious diseases has emerged as a major limiting factor for aquaculture expansion which causes massive mortality and economic losses (Shalini et al. 2019). An important strategy used to address this problem is the administration of antibiotics (Rico et al. 2013).
Tetracyclines are a common class of antibiotics which are widely used in aquaculture. Oxytetracycline (OTC) is a well-known tetracycline antibiotic that is produced from Streptomyces rimosus fungi (Rodrigues et al. 2017a;FAO 1990). OTC is commonly used as an effective treatment against bacterial pathogens of shes, such as Aeromonas salmonicida, Aeromonas hydrophila, Lactococcus garvieae, Vibrio anguillarum, and Pseudomonas sp. (Leal et al. 2019;Nakano et al. 2018). More utilization of OTC than other antibiotics is due to its low-cost, legal availability, high e ciency and non-speci c selectivity in the treatment of bacterial infection (Pês et al. 2018;Lee et al. 2020;Rodrigues et al. 2017a). OTC can be applied via injection, immersion and oral administration, but is commonly administered through diet in the amount of approximately 75 mg per kg of sh biomass (Yonar 2012).
However, the administration of antibiotic drugs may pose several side-effects such as the occurrence of antibiotic resistance in bacterial pathogens (Lunden et al. 1998), residue in sh tissues (Harikrishnan et al. 2009), and reduction of sh health (Shalini et al. 2019). For instance, OTC is found to induce oxidative stress and liver malfunctioning. OTC can increase the generation of reactive oxygen species (ROS) in the sh body, which cause peroxidation of liver cell membrane lipids resulting in an increased leakage of intracellular enzymes to the blood (Nakano et al. 2018). In addition, negative effects of OTC on immune response, antioxidant defense, hematological parameters, nephrotoxicity, genotoxicity, and histological changes in sh species have been previously documented (Rodrigues et al. 2017b;Hoseini and Youse 2019;Reda et al. 2013;Yonar et al. 2011, Mathew andAmbili 2017). As a result, OTC may increase the sh susceptibility to secondary pathogens and also to environmental stressors. Hence, there is a necessity for the discovery of methods to alleviate the harmful effects of OTC on shes.
Vitamin C or L-ascorbic acid (AA) is an important micro-nutrient for shes, playing a key role in the growth performance and physiological process (Lin and Shiau 2005). It is also a potent natural antioxidant that plays a vital role in scavenging ROS to protect the sh from oxidative stress (Bae et al. 2012;Hajirezaee et al. 2020). Moreover, AA has an important role in hematology (Sandnes et al. 1990), collagen formation (Hunter et al. 1979), reproduction (Emata et al. 2000;Cavalli et al. 2003), immune response (Misra et al. 2007;Roosta et al. 2014;Zhou et al. 2012), and recovery from exposure to toxicant and environmental stressors (Saha and Kaviraj 2009;Kim et al. 2017;Wahli et al. 2003). Dietary supplementation of AA can provide normal growth of sh at low doses; however, a higher dose of AA is required to increase the stress tolerance in sh (Vani et al. 2011).
Nevertheless, up to date, there is no data available on the role of dietary AA on OTC-induced stress in sh.
Therefore, the current study was designed to assess the possible role of dietary AA in mitigating the adverse effects of OTC in terms of hematological parameters, skin mucus and blood immune parameters, and antioxidant capacity of rainbow trout (Oncorhynchus mykiss).

Experimental sh
Healthy juveniles of Oncorhynchus mykiss (43 ± 2.6 g) were purchased from a local sh farm and were transported to the wet lab in a berglass aerated tank. They were acclimated to lab conditions for 14 days in 1000-liter berglass tanks lled with tap water under a natural 12-h photoperiod. During the adaptation period, rainbow trout were fed three times a day with a commercial diet containing 43% crude protein, 14% crude fat, 4% crude ber and 11% ash. The water physicochemical parameters were regularly monitored, and water temperature range from 13.64 to 15.47°C, pH from 7.2 to 7.5, and oxygen levels from 7.1 to 7.6 mg L − 1 . Excess feed and fecal waste were siphoned out daily and a quarter portion of water in the tank is replaced with clean water.

Experimental diets
Five experimental diets were provided by mixing a basal diet (Table 1) with varying amounts of OTC (0 and 100 mg/kg body weight) and AA (100, 200, 400 and 800 mg/kg diet). Brie y, feed ingredients were ground into powder with multi-function pulvetizer, and then were passed through a 1.0-mm sieve.
Grounded ingredients were mixed well with the oils, and then distilled water was added into the mixture to form a stiff dough. The dough was passed through grinder, dried for 48 h at 25°C, and nally stored at -18°C until use (Kim et al. 2017

Experimental design
After acclimatization, a total number of 150 rainbow trout were randomly distributed into ve experimental groups (A, B, C, D and E) in triplicates (10 sh per tank) and maintained throughout an experimental period of 28 days. The diet used for all groups (B, C, D and E) were contained OTC (100 mg/kg body weight) except group A (control). The diet used for group A and B were supplemented by the normal dose of AA (100 mg/kg feed), while the diets used for groups C, D and E were prepared by supplementing different high doses of AA (200, 400, and 800 mg/kg, respectively). During the experiment, O. mikiss were fed with one of these diets at 2% of biomass and three times (8:00, 13:00 and 19:00) daily.

Sampling
After 28 days of feeding, three sh were randomly harvested from each replicate and immediately anesthetized with powdered clove (200 ppm). Blood samples were drawn from the lateral tail vein using sterile syringes and become two parts. One part was kept in tubes containing heparin for analysis of blood parameters and the second sample was centrifuged at 1500 ×g for 5 min to separate plasma (supernatant). The plasma was removed and maintained at -70°C until analysis (Yangthong et al. 2016).
Skin mucus was collected according to Shaluei et al. (2017). Brie y, three sh were randomly harvested from each replicate and anaesthetized using clove powder. Each harvested sh was transferred into a separate plastic bag containing 10 ml NaCl (50 mM). The bag was shaken well for 1 minutes and the skin mucus was immediately poured into 15 ml sterile tubes and centrifuged at 1500 ×g for 4 min. The upper layer was collected and maintained at -80°C until use.

Measurement of blood parameters
Total number of erythrocytes (RBC) and leucocytes (WBC) were determined using a Neubauer chamber by dilution of blood samples in the Natt and Herrick solution (Natt and Herrick 1952). Blood hematocrit (Ht) was measured via micro hematocrit centrifuge at 10500 ×g for 5 min (Baba et al. 2015). The hemoglobin amount (Hb) was estimated spectrophotometrically at 540 nm using cyanmethemoglobin method (Harikrishnan et al. 2009). The mean cell volume (MCV), the mean cell hemoglobin (MCH), and the mean cell hemoglobin concentration (MCHC) were determined from the total RBC counts, Hb concentrations, and Ht values according to the available formulae (Azaza et al. 2020). Neutrophils, lymphocytes, and monocyte numbers were quanti ed by Giemsa stained blood smears under light microcopy (Safari and Sarkheil 2018).

Plasma and skin mucus immunology
The total protein and albumin contents of plasma samples were assayed using quantitative detection kits (Pars Azmun Co, Iran). Plasma total protein content was measured by Piotrowski's assay at 540 nm.
Plasma albumin content was assayed using bromocresol green dye at 630 nm ). Plasma globulin content was measured by subtracting amount of albumin from total protein (Kumar et al. 2005).
The total immunoglobulin content (Total Ig) of plasma and mucus samples was quanti ed according to Siwicki and Anderson (1993). The sample was mixed with 12% polyethylene glycol, incubated at 25°C for 2 hours, centrifuged at 3000 ×g for 15 min, and then the supernatant removed and the remaining protein was measured after its subtraction from total protein content.
Lysozyme activity of plasma and mucus sample was measured according to method of Ellis (1990) using lyophilized Micrococcus lysodeikticus as target in phosphate buffer (pH 6.2). One unit of enzymatic activity was equal to the amount of lysozyme causing a decrease in absorbance of 0.001 at 490 nm.
The method of Yano (1992) involving the use of rabbit red blood cells (RaRBC) as substrate was used to determine the alternative complement activity (ACH50) in plasma. The volume of plasma producing 50% hemolysis of RaRBC was monitored spectrophotometrically at 414 nm.
Protease activity in mucus was measured following Guardiola et al. (2014) using the azocasein hydolysis assay. One unit of protease activity was considered as nanogram of the azo-dye released during 60s at 37°C.

Plasma antioxidant enzymes
Blood superoxide dismutase (SOD) and catalase (CAT) activities were assayed spectrophotometrically Similar to the method of Yonar et al. (2011). SOD activity was measured by checking the amount of enzyme required to prevent the reduction of nitroblue tetrazolium, which determined at 560 nm. The CAT enzyme activity determination was based on measuring the rate constant of hydrogen peroxide decomposition by this enzyme. The conversion of H 2 O 2 to H 2 O, and O 2 was measured at 240 nm.
Plasma malondialdehyde content (MDA) was measured spectrophotometrically at 548 nm according to Placer et al. (1966) using the thiobarbituric acid reaction. The MDA activity was expressed as the nanomol of enzyme per ml of plasma.

Biochemical parameters
The activities of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured spectrophotometrically using quantitative analyses kits (Pars Azmun Co, Iran). ALT activity was expressed as the amount of enzyme needed to form one molecule of pyruvate, which is determined at 340 nm. AST activity was de ned as the amount of enzyme needed to form one molecule of glutamate, which is determined at 340 nm. ALP activity was expressed as the amount of enzyme needed to make one micromole of p-nitrophenol, which is determined at 405 nm.

Statistical analysis
The normality of acquired data was determined by Kolmogorov-Smirnov test and the differences between the means were examined via one-way analysis of variance (ANOVA), followed by Duncan test.
Differences between mean values were considered to be signi cant when the con dence level was 95 % Page 7/21 (P < 0.05). Results are de ned as mean ± standard deviation for each group. All the statistical analyses were done using the SPSS computer software program (Version 24).

Hematological parameters
The hematological parameters of rainbow trout are presented in Table 2. The RBC, WBC, Hb, Hct, and neutrophils values were signi cantly changed with the incorporation of OTC in the diet of rainbow trout (P < 0.05). The highest numbers of RBC and WBC were recorded in group A (100 mg AA without OTC) while the group B (100 mg AA with OTC) showed the lowest values. Feeding sh in the E group (800 mg AA with OTC) signi cantly increased these parameters as compared to the B group (P < 0.05). Hemoglobin, hematocrit and neutrophils values were signi cantly lower in group B than in group A (control) and the group which were fed with 800 mg/kg diet of AA (group E) recorded signi cantly increased Hct value as compared to group B (P < 0.05). There was no signi cant alteration in the MCV, lymphocyte and monocyte values between the ve groups. Values presented as mean ± standard deviation (SD). The absence of letters indicates the absence of signi cant differences between treatments (P < 0.05) for n = 3; P values are given A-diet contained 100 mg AA without OTC; B-diet contained 100 mg AA with OTC; C-diet contained 200 mg AA with OTC; D-diet contained 400 mg AA with OTC; E-diet contained 800 mg AA with OTC

Biochemical enzymes
As shown in Fig. 1 the plasma AST, ALT, and ALP activities were signi cantly elevated by the addition of OTC to the diet of sh (P < 0.05). The highest activities of these enzymes were recorded in the group B. Fish in the group E exhibited signi cantly decreased ALT and ALP activity compared with those in the group B. Dietary supplementation with different levels of AA did not cause signi cant changes in the AST activity (P > 0.05).

Plasma immune responses
The measurements of the humoral immunity parameters are presented in Table 3. The lysozyme and complement activities were signi cantly affected with the incorporation of OTC in the diet (P < 0.05). The highest values of these indicators were revealed in the group A whereas the lowest were in the group B. Dietary supplementation of AA signi cantly improved the lysozyme and complement activities in the group D (400 mg AA/kg diet). There were no signi cant changes in the total immunoglobulin, total protein, albumin, and globulin levels between the study treatments (P > 0.05). Values presented as mean ± standard deviation (SD). The absence of letters indicates the absence of signi cant differences between treatments (P < 0.05) for n = 3; P values are given A-diet contained 100 mg AA without OTC; B-diet contained 100 mg AA with OTC; C-diet contained 200 mg AA with OTC; D-diet contained 400 mg AA with OTC; E-diet contained 800 mg AA with OTC

Mucus immune responses
The measurements of the mucus immunity parameters are presented in Table 4. The mucus lysozyme, alkaline phosphatase, and protease activities in the group B (diet contained 100 mg AA with OTC) decreased signi cantly compared to the control group (P < 0.05). The lowest activities of lysozyme and protease were observed in group B and dietary supplementation with additional amounts of AA had no signi cant change for these parameters (P > 0.05). The lowest alkaline phosphatase activity was observed in group B. The dietary supplementation with AA signi cantly improved alkaline phosphatase activity in the group E (800 mg AA/kg diet) (P < 0.05). The total immunoglobulin content did not signi cantly vary among the different study groups (P > 0.05). As seen in Fig. 2 the activities of SOD and CAT were signi cantly altered by the incorporation of OTC in the diet of sh (P < 0.05). The lowest SOD and CAT activities were observed in group B and the groups which were supplemented with higher levels of AA (group D and E) showed signi cantly increased SOD and CAT activities. The blood level of MDA found to be signi cantly increased upon OTC administration in group B compared to control group (P < 0.05). The highest activity of MDA was recorded in group B and dietary supplementation with additional levels of AA (200, 400 and 800 mg/kg diet) signi cantly decreased the level of blood MDA compared to the group B.

Discussion
Vitamin C or L-ascorbic acid (AA) is a water-soluble micronutrient needed for multiple physiological process of aquatic animals (Shahkar et al. 2015). Previous studies showed the anti-stress (Misra et al. 2007), antioxidant (Wan et al. 2014), immuno-stimulatory (Zhou et al. 2012), and growth promoting (Liang et al. 2017) properties of AA in shes. Considering the bene cial effects of AA, the current study evaluated the possible role of dietary AA in the reduction of OTC-induced stress in O. mykiss.
The measurement of hematological indicators is an important tool to monitor the physiological and the pathological alterations in sh (Burgos-Aceves et al. 2019;). In the existing experiment, the RBC number and the Ht and Hb values were signi cantly declined by the inclusion of OTC in the diet of O. mykiss fed with low dose of AA (100 mg/kg diet). These results suggested that OTC has caused anemia condition in O. mykiss, which may be a result of erythropoiesis inhibition and also increased erythrocyte lysis (Ramesh et al. 2018;Yonar et al. 2020). Similarly, signi cant reductions in RBC, Ht, and Hb values were observed by Omoregie and Oyebanji (2002) in Oreochromis niloticus fed diets incorporated with OTC. However, in our study, administration of AA at 800 mg/kg diet markedly restored the RBC and Ht values in sh treated with OTC, which were comparable with control group. This may be due to the anti-oxidative properties of AA which prolongs the life span of erythrocytes by its potent ROS scavenging activity (Nayak et al. 2007). Our nding agrees with the observation of Affonso et al. (2007), who suggested that feeding with higher levels of AA signi cantly increased the RBC and Ht values in Brycon amazonicus.
The study of white blood cell (WBC) pro le can provide useful information regarding the general health status of sh. The white blood cells and differential leukocytes number are useful tools to evaluate the immune response in sh (De Pedro et al. 2005;Vali et al. 2020). In this study, OTC administration signi cantly decreased the WBC and neutrophils values with no signi cant effect on monocytes and lymphocytes. The reduction in the WBC and neutrophils values may indicate immunosuppressive effects of OTC and subsequently increased sh's susceptibility to infectious diseases. These obtained results are in accordance with the nding of Dobšíková et al. (2013), who observed that OTC decreases the WBC value in Cyprinus carpio. On contrary, a signi cant increase in WBC value was recorded with the supplementation of AA in group D and E (400 and 800 mg/kg diet respectively) compared to group B, which indicates that the immune system has been restored. Similarly, Misra et al. (2007) suggested that higher levels of dietary AA could induce a signi cant elevation in WBC count in Labeo rohita.
The innate immunity in sh includes two parts of mucosal and humoral immunity which both defense lines play an vital role in the ght against pathogens (Harikrishnan et al. 2012;Hoseinifar et al. 2016). The presence of various factors such as lysozymes, complement, and other lytic components in plasma prevents/or kills microorganisms (Alexander and Ingram 1992). The result of the current study revealed that OTC administration signi cantly decreased the activities of lysozyme and complement in the plasma of sh fed low dose of AA, suggesting that innate immunity of sh was suppressed following treatment with OTC antibiotic. This OTC-induced immunosuppression is attributable to its high tissue penetration capacity, making OTC capable of interfering with immune system organs, such as the liver and kidney (Yonar et al. 2011). Our results are in accordance with Hoseini and Youse (2019) that indicated OTC treatment in rainbow trout signi cantly decreased lysozyme and complement activities. The results of our experiment also revealed that the plasma lysozyme and complement activities were signi cantly increased in the D group (fed 400 mg AA/kg diet) compared with those in the group B (fed 100 mg AA per kg diet), which may indicate an improved immune response in sh. In line with our results, several studies showed that dietary AA elevated the innate immune responses in shes (Li and Lovel 1985;Hardie et al. 1991;Dunier et al. 1995).
The skin mucus includes various factors such as lysozyme, protease, immunoglobulins, and lectins that play a vital role in the primary defense and protection of sh against pathogens (Hoseinifar et al. 2015;. The results of the present experiment revealed that the mucus lysozyme, protease, and alkaline phosphatase activities markedly decreased with the incorporation of OTC in the diet of rainbow trout fed with low dose of AA (100 mg/kg diet), which may indicate a weakened ability to cope with pathogens. While many experiments have focused on the immunosuppressive effects of OTC on the humoral immunity of sh, these effects in mucosal immunity have not been investigated. Our results also showed that the mucus alkaline phosphatase activity of sh treated with OTC increased signi cantly in the group E (800 mg AA/kg diet), which may be attributed to an increased mucosal immune response by the AA supplementation at higher doses. Similar to our results, Roosta et al. (2014) observed an increase in the activity of mucus alkaline phosphatase in Rutilus rutilus caspicus following dietary supplementation with high levels of AA.
This is well established that increased reactive oxygen species (ROS) levels will lead to oxidative stress, which may negatively affect the sh and crustaceans' health and cause structural damage in tissue cells (Akbary and Aminikhoei 2018;Tavabe et al. 2020). Antioxidant defense system includes some pivotal enzymes such as superoxide dismutase (SOD) and catalase (CAT) that tend to prevent ROS formation (Yonar et al. 2014). In our study, a signi cant decline in the activity of SOD and CAT as well as a signi cant increase in the level of MDA were observed in the plasma of sh in the group B (100 mg AA with OTC) compared to the control group (100 mg AA without OTC). These recorded changes in SOD and CAT activities can be attributed to an excessive formation of ROS in the sh body, which resulted in the high consumption of these enzymes during elimination of ROS (Rahman et al. 2020). Moreover, the increase in MDA level similarly shows this situation, knowing that MDA is the main product of lipid peroxidation (Yonar 2018). Previously, similar results were obtained from dietary administration of OTC in O. mykiss by Nazeri et al. (2017). On the other hand, feeding sh in the D and E treatments (400 and 800 mg/kg diet, respectively) signi cantly reversed the activity of SOD and CAT enzymes and as well as the MDA level in the sh plasma, indicating the ameliorative effects of dietary AA on OTC-induced oxidative damage. This can be due to the high anti-oxidative capacity of AA which makes it capable of neutralizing ROS and reduction of oxidative stress (Rouhier et al. 2008;Verma et al. 2007). This is in agreement with Wan et al. (2014) who found that higher levels of vitamin C could effectively suppress oxidative stress induced by the high levels of pH.
The measurement of the activity of biochemical enzymes (AST, ALT and ALP) in the plasma can provide useful information regarding the health condition of sh and crustaceans' liver tissue (Nakano et al. 2018;). In the current study, our results showed signi cantly increased plasma AST, ALT, and ALP activities in OTC treated sh (group B) compared to the group A (control). This increase may be linked to the OTC-induced oxidative stress, which affect the permeability of hepatocyte through oxidative damage resulting in leakage of these enzymes to the sh blood (Yonar 2012;Banaee et al. 2011). In line with our ndings, the increased activities of biochemical enzymes (AST and ALT) in sh treated with OTC have been previously observed also in Oncorhynchus kisutch (Nakano et al. 2018). The results of present study also showed that feeding sh in the group E (diet contained 800 mg AA) signi cantly decreased these parameters as compared to the B group (diet contained 100 mg AA). These changes may indicate that dietary supplementation with AA helped to reduce the OTC-induced liver damage, resulting in the decreased leakage of these enzymes from liver to the blood. In similar study conducted by Nazeri et al. (2017) feeding with different levels of rutin (a avonoid) signi cantly alleviated the activities of plasma enzymes in OTC treated Oncorhynchus mykiss.
In conclusion, the current results showed that the dietary administration of OTC in rainbow trout signi cantly affected hematological pro le, innate immune response, and antioxidant capacity of O. mykiss. The low dose of dietary AA was not capable of alleviating the OTC-induced stress. However, supplementation of sh with higher levels of AA found to restore the suppressed immune response as evidenced by increased activities of plasma lysozyme and complement, skin mucus alkaline phosphatase, and augmented WBC and neutrophils values. Moreover, attenuation of OTC-induced oxidative stress by increased activity of anti-oxidative enzymes (SOD and CAT) and decreased activity of plasma biochemical enzymes (AST, ALT and ALP) was also found in sh fed with higher levels of AA. Hence, dietary administration of AA at higher levels could be an effective strategy to decrease the negative effects of OTC on O. mykiss.

Declarations
Funding: Current research was funded by the University of Tehran under grant number 26713.
Con icts of interest: The authors are fully satis ed with all stages and there are no con icts of interests to declare.
Ethics approval: The trial protocol was approved by the Ethics Committee for the Animal Research, University of Tehran; none of the sh suffered starvation, trauma or electrical shock and all the sh were completely anesthetized before tissue sampling.
Consent to participate: All names in author list have been involved in various stages of experimentation or writing and agree with being a part of this work.
Consent for publication: All authors agree with submit the paper for publication in the journal of Fish

Physiology and Biochemistry
Availability of data and material: All data and materials used in the experiment are available and achievable upon request to the corresponding author Sina.javanmardi@ut.ac.ir.