Effects of dietary FARMARIN® XP supplement on immunological responses and disease resistance of rainbow trout (Oncorhynchus mykiss)

Aquaculture 496 (2018) 211–220

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Effects of dietary FARMARIN® XP supplement on immunological responses and disease resistance of rainbow trout (Oncorhynchus mykiss)

T

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Sevdan Yilmaza, , Sebahattin Ergüna, Murat Yıgıtb a b

Department of Aquaculture, Faculty of Marine Sciences and Technology, Canakkale Onsekiz Mart University, Canakkale 17100, Turkey Department of Marine Technology, Faculty of Marine Sciences and Technology, Canakkale Onsekiz Mart University, Canakkale 17100, Turkey

A R T I C LE I N FO

A B S T R A C T

Keywords: Rainbow trout Humic substances Sulphadiazine/trimethoprim Digestive enzyme Yersiniosis

The study was performed to determine the effects of FARMARIN® XP and INFISH-AQUA® on growth performance, proximate composition, biometric indices, serum biochemical variables, hematological parameters, nonspecific immune responses, digestive enzyme activities and disease resistance of rainbow trout (Oncorhynchus mykiss) juveniles against Yersinia ruckeri. Four experimental groups of fish were fed an additive free basal diet (control) and FARMARIN® XP incorporated test diets at increasing levels (0.1%-F1, 0.2%-F2, 0.4%-F4) for 60 days. Additionally, a fifth group of test diet was antibiotic medicated (0.1%), prepared with the commercial product INFISH-AQUA® (sulphadiazine 20% and trimethoprim 4%). When fish were challenged with Yersinia ruckeri after the 60-days feeding trial, and mortality was recorded over an additional 20-days period, no influence of FARMARIN® XP and antibiotic supplemented diets were observed on growth performance and hematological parameters of rainbow trout. However, the intestinal lipase activities in F1, F2 and AMF groups were significantly higher than the other treatments. Serum glucose level was significantly lower in the F4 group, and triglyceride levels decreased significantly when fish were fed with FARMARIN® XP or antibiotic supplemented diets. The dietary FARMARIN® XP especially at 0.1% and 0.2% significantly increased the respiratory burst activity. A decreasing potential killing activity and phagocytic index were found in the F4 and AMF groups. At the end of the 20-day challenge period the survival rates were significantly higher in the F2 and AMF groups compared to all other treatment groups. Thus FARMARIN® XP can be used as a replacement for antibiotic in rainbow trout diets for the control of yersiniosis.

1. Introduction Rainbow trout (Oncorhynchus mykiss) is one of the most cultured finfish species in the world (Cowx, 2006), reaching 814.091 tons of production with a commercial value of around 3.4 billion USD in 2016 (FAO, 2018). The increased production of rainbow trout in intensive conditions may affect fish health due to decreased fish welfare under stressful culture environments. Yersinia ruckeri is widely seen in O. mykiss causing Enteric Redmouth (ERM) disease (Austin et al., 2003; Austin and Austin, 2016), which can be controlled by vaccination (Ispir and Dorucu, 2010; Chettri et al., 2015) and antimicrobial drugs, particularly sulphamethazine, chloramphenicol or oxytetracycline (Tobback et al., 2007). However, vaccination requires labor efforts and it may cause handling stress. On the other hand, when in excessive, the use of antibiotics may increase the resistance of fish pathogens against antibiotics, since Y. ruckeri strains are capable to developed resistance to a variety of antimicrobial agents in aquaculture facilities (Shah et al.,

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2012; Huang et al., 2014). In order to fight fish diseases, many investigations have been focused on a variety of antibiotics or chemotherapeutics in aquaculture. However, antibiotics or chemotherapeutics are not environmentally sustainable and may have side effects or residues undesired for farmed terrestrial animals and human beings. Humic substances (HUMS), produced by organic materials of rotted or dead plants and animal tissues through microbial activity (Herzig et al., 2001), are important antioxidants with their significant free radical scavenger properties (Ozkan et al., 2015), and also known to have antimicrobial (Van Rensburg et al., 2000), anti-inflammatory (Van Rensburg et al., 2001) and immunostimulatory (Vucskits et al., 2010) effects, through which they can be considered as useful dietary additives in fish feed. Few reports are available on the use of HUMS in poultry (Eren et al., 2000; Kocabağli et al., 2002), however information regarding the effects of dietary HUMS in fish species is scarce and limited to

Corresponding author. E-mail address: [email protected] (S. Yilmaz).

https://doi.org/10.1016/j.aquaculture.2018.07.024 Received 27 April 2018; Received in revised form 16 July 2018; Accepted 17 July 2018 Available online 19 July 2018 0044-8486/ © 2018 Published by Elsevier B.V.

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Thereafter, the pellets were stored in plastic bags and kept in a deep freezer at −20 °C until used.

Dicentrarchus labrax (Soytaş, 2015) and Cyprinus carpio (Sharaf and Tag, 2011; Rousdy and Wijayanti, 2015). Improved fish growth was reported in C. carpio by dietary inclusion of HUMS (Sharaf and Tag, 2011), whereas no growth-promoting effects of dietary HUMS were noted in D. labrax (Soytaş, 2015) or C. carpio (Rousdy and Wijayanti, 2015). There are discrepancies between earlier studies regarding the effects of HUMS in fish diets, therefore it seems necessary to further clarify the effect of dietary HUMS in fish feed. FARMARIN® XP is a commercial product specially developed as a biostimulator, bioregulator and high activity organic performance improver for finfish species by the Scientific Work Group of Farmavet. The content of the product includes mainly humic acids along with fulvic, ulmic and fulfonic acids. However, there is no scientific information so far regarding the effects of commercial products of humus sources produced for finfish species and this study is the first attempt in this regards. Therefore, in the present study the effects of different inclusion levels of dietary FARMARIN® XP on growth performance, chemical composition, serum biochemical variables, haemato-immunologic parameters and survival rate against Y. ruckeri have been investigated in rainbow trout for the first time.

2.2. Fish and experimental design O. mykiss juveniles, obtained from a trout farm (Keskin Alabalik Co., Canakkale – Turkey) were transported to the research facilities of the Canakkale Onsekiz Mart University. For the visual and external inspection of each experimental fish, the US-EPA (United States Environmental Protection Agency) guidelines for qualitative assessment of fish health were followed (Klemm et al., 1993). Fish were acclimatized to the experimental conditions for a period of two weeks before the start of the experiment, and fed a commercial trout feed (Pınar Camli Co. Turkey, 49% protein/19% lipid, 2 mm) until satiation. A total of 15 experimental fiberglass tanks were stocked with 25 fish per tank (21.73 ± 0.43 g mean ± SD, n = 375), in a triplicate design. Each of the experimental tanks (140 L water volume) was supplied with recirculated freshwater at a flow rate of 145 L/h and aerated with air stones. All experimental groups were fed until satiation two times a day at 08.00 and 17.00 h over a period of 60 days. Water temperature was controlled by a heater/chiller (Tuna Mac®, Çanakkale Turkey) and photoperiod regime followed a 12:12 light:dark cycle throughout the study. Water quality parameters such as temperature, oxygen, conductivity and pH were measured daily, while ammonia, nitrite and nitrate were analyzed weekly. Temperature, pH, dissolved oxygen, conductivity, total ammonia, nitrite, and nitrate were recorded as: 17.5 ± 0.2 °C, 7.8 ± 0.2, 7.7 ± 0.10 mg/L, 450 ± 10.2 μS, 0.015 ± 0.0012 mg/L, 0.05 ± 0.001 mg/L, and 0.5 ± 0.11 mg/L, respectively during the course of the study.

2. Materials and methods 2.1. Experimental diet FARMARIN® XP was obtained from FARMAVET ILAC San Tic AS Co., and incorporated to a laboratory-manufactured feed at levels of 0% (control group), 0.1%, 0.2%, and 0.4% for diets designated as C0, F1, F2, and F4, respectively. These dietary incorporation levels of FARMARIN® XP were followed by the recommended dose inclusion levels of the Farmavet company. In addition, an antibiotic medicated diet (AMD) was prepared with a commercial product of INFISH-AQUA® (sulphadiazine 20% and trimethoprim 4%; Indukern Istanbul Kimya San Tic Ltd. Sti Co.) and 0.1% (corresponding to 50 mg/100 g antibiotic in fish feed) added to the experimental feed. Fish fed on a diet without supplementations of both FARMARIN® XP and antibiotic served as a control group. All ingredients (Table 1) were mixed in a laboratory blender and a pelleting machine (La Monferrina P3, Italy) with a 2-mm die was used to produce the pellets, which were then dried in a drying cabinet at 40 °C until the moisture content of pellets declined to 10%.

2.3. Calculation of growth performance and biometric indices Calculations for the relative growth rate (RGR, %), specific growth rate (SGR, % per day) and feed conversion ratio (FCR) were performed using following equations:

RGR (%) = 100 (final fish weight − initial fish weight)/initial fish weight SGR (%/day) = 100 (ln final fish weight) − (ln initial fish weight) /experimental days FCR = feed intake/weight gain

Table 1 Percentage and proximate composition of the experimental diets.

Ingredients (% dry matter) Fish meal (anchovy meal) Fish oil (anchovy oil) Soybean meal Wheat flour Wheat starch Carboxymethyl cellulose FARMARIN® Antibiotic Vitamin mix Mineral mix BHT Total Chemical analyses (% DM) Protein Fat Ash NFEa Energy (kj/g)b

Right after dissection, the liver, viscera and visceral fat were removed from fish and weighed for the calculated of biometric indices using the formulae given below:

C0

F1

F2

F4

AMF

58 13 12 10 3.99 1 0 0 1 2 0.001 100

58 13 12 10 3.89 1 0.1 0 1 2 0.001 100

58 13 12 10 3.79 1 0.2 0 1 2 0.001 100

58 13 12 10 3.59 1 0.4 0 1 2 0.001 100

58 13 12 10 2.89 1 0 0.1 1 2 0.001 100

Hepatosomatic index (HSI) = {wet weight of liver (g)

44.63 18.27 9.26 16.15 20.50

44.63 18.27 9.35 16.07 20.48

44.63 18.28 9.45 15.99 20.47

44.64 18.28 9.64 15.82 20.44

44.63 18.27 9.35 16.07 20.48

Visceral fat index (VFI) = {wet weight of visceral fat (g)

/[wet body weight (g) − wet weight of liver (g)] × 100}

Viscerosomatic index (VSI) = {wet weight of viscera and associated fat (g) /[wet body weight (g) − wet weight of viscera and associated fat (g)] × 100}

/[wet body weight (g)–wet weight of visceral fat (g)] × 100}

Spleen–somatic index (SSI) = {wet weight of spleen (g)

a Nitrogen-free extracts (NFE) = dry matter − (crude lipid + crude ash + crude protein). b Energy was calculated according to 23.6 kJ/g protein, 39.5 kJ/g lipid, and 17.0 kJ/g NFE.

/[wet body weight (g)–wet weight of spleen (g)] × 100} 212

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2.4. Proximate composition

Activity = Abs 280 (sample) − Abs 280 [blank (without added sample) 1000/10 min

Proximate analyses were conducted using standard methods for both the test diets and whole body fish samples (visceral organs and visceral fats excluded). Moisture analyses were performed by drying the samples to a constant weight at 105 °C for 24 h in an oven, crude protein was analyzed by the Kjeldahl method, and crude ash by incineration at 525 °C in a muffle furnace for 12 h (AOAC, 1998). Methanol/ chloroform extraction method (Folch et al., 1957) was used to analyze crude fat levels of the samples.

× mg protein Enzyme analyses of trypsin in the intestines were conducted with 96-well microplates with minor modifications in the method reported in the earlier by Faulk et al. (2007). Briefly, 100 μL sample was added to the wells in three replicates and the same amount of trypsin assay buffer (0.1 M Trisma, 0.01 M CaCl 2, 1 M NaCl, pH 8.2) containing 1 mM BAPNA substrate was added carefully and quickly with a multi-channel pipettor. Subsequently, spectrophotometric measurements were carried out with a microplate reader (Thermo Multiskan Go) set at 30 °C and followed for 10 min at 410 nm with time intervals of 30 s and the calculated activity was expressed as U/mg protein/min using the following formula:

2.5. Blood sampling Blood samples were collected from the caudal vein after the feeding trial for 60 days. Three fish from each tank (9 fish per group) were used for the samplings. Prior to blood sampling, feeding was withheld and fish were starved for 1 day. Fish were randomly caught, removed from the tanks, and anesthetized using clove oil at 20 mg/L (Iversen et al., 2003). A plastic syringe (2.5 mL) was used to take the blood from the caudal vein as soon as possible from the area behind the anus fin, which was cleaned thoroughly with alcohol in order to avoid any contamination of mucous membrane with the blood. A part of the blood samples were transferred into tubes containing K3EDTA (MiniCollect®Tube, Austria) for manual and automatic hematological (RBC, Hct and Hgb) and immunological (phagocytic activity, phagocytic index, respiratory burst and potential killing activity) measurements. The remaining of the blood was transferred into serum tubes (Z serum sep. Tubes MiniCollect® Tube, Austria) and centrifuged at 5000g for 10 min, then the obtained serum samples were stored at −80 °C for further analyses.

Activity = 410 Abs/min × 1000 × volume of reaction mixture /8800 (extinction coefficient of p−nitroaniline) × mg protein in the reaction mixture For the ɑ-amylase analysis, some modifications were made in the method reported by Jiang and Wang (2012). In brief, 100 μL sample was mixed with amylase assay buffer (pH 6.8, containing 6.7 mM NaCl) containing freshly prepared 2% starch within 100 μL PBS and the mixture was incubated for 1 h at 37 °C. Thereafter, 100 μL color-reactive [prepared with the addition of 16 mL sodium potassium tartrate solution (24 g of sodium potassium tartrate tetrahydrate in 16.0 mL of 2 M NaOH) and 40 mL 96 mM 3, 5 dinitrosalicylic acid solution (0.88 g of 3, 5 dinitrosalicylic acid in 40 mL of distilled water) on 24 mL distilled water] was added and incubated for 5 min in boiling water bath and chilled with ice. Then, 500 μL distilled water was added to the samples and the readings were performed at 540 nm with a spectrophotometer (Optizen POP UV/VIS). The same procedure was applied for the blank tubes, however, in this process, the sample was added just after the incubation. Maltose was used for the standard. Further, mole of maltose was released from starch/min/mg protein at 37 °C. For the lipase analysis, the method reported by German et al. (2004) was used. 287 mL of 5.2 mM sodium cholate, which was dissolved in 250 mM tris-HCl (pH 9), was combined with 20 mL homogenate and 8 mL of 10 mM2-methoxyethanol in a micro-centrifuge tube. It was then incubated at room temperature for 15 min to ensure lipase activation by bile salts. Thereafter, the substrate p-nitrophenyl myristate (18 mL of 10 mM p-nitrophenyl myristate dissolved in 100% ethanol) was added. The assay mixture was incubated at 15 °C for 2 h. At the end of 2 h, the reaction ended with 467 mL of acetone/heptane (5:2, v:v). After this process, the samples were centrifuged at 6100g for 2 min. The absorbance of the resulting lower aqueous layer was read at 405 nm and a pnitrophenol standard curve was used for the detection of the activity. U (1 mmol p-nitrophenol liberated per minute) per mg wet weight of tissue was used to report the lipase activity. Alkaline phosphatase (AP) activity was performed using spectrophotometer on 96-well plates by the previously reported method (Harpaz and Uni, 1999). 200 μL alkaline phosphatase yellow (pNPP) liquid substrate (Sigma-Aldrich, P7998) was added to 40 μL sample wells diluted ten times with distilled water, and the readings were performed at 405 nm with a microplate reader (Thermo Multiskan Go) for 10 min at 24 °C with time intervals of 1 min. The calculated activity was expressed as U/mg protein/min using following equation:

2.6. pH of feed Experimental feeds were weighed as nearly 5 g and diluted with distilled water ten times their weight in a 100-mL plastic tube for the pH analyses (Baruah et al., 2005). Thereafter, the pH measurements of the homogenized samples using a homogenizer (Stuart SHM1, Staffordshire, UK) were carried out with a benchtop pH meter (Hanna, HI 2221) through FC 200 electrode. 2.7. Digestive enzyme analyses The stomach and intestines of 9 fish (3 fish per tank) were used in digestive enzyme analyzes after termination of the feeding trial. Highdose clove oil (200 mg/L) was used to euthanize the experimental fish prior to sampling. Thereafter, surface of the fish was cleaned with 70% alcohol and the stomach and intestines were sampled under aseptic conditions. The extracted fish tissues were quickly and carefully separated into small pieces with a sterile bisturi and weighed in Eppendorf tubes. Subsequently, they were homogenized with a homogenizer (TissueLyser LT) with the addition of cold distilled water ten times their weight. Afterwards, the samples were centrifuged (Hettich Mikro 200R) at 21382g, 4 °C for 30 min. The pH of the clear supernatant was determined according to Nya and Austin (2011) in the same manner as that of dietary pH explained above. Then, the upper phases of the samples were stored at −80 °C until use in digestive enzyme analyzes. The determination of protein amounts in each sample was performed following the method described by Bradford (1976). Pepsin analyses were conducted according to the method reported by Worthington (1993). 500 μL of 2% hemoglobin (Sigma-Aldrich, H2625) solution (pH 2) was added on 100 μL sample and the mixture was incubated at 35 °C for 10 min, afterwards the vortex and the reaction was stopped with 5% TCA. The samples were centrifuged at 12000g for 5 min and the recording of the OD of the upper phases were conducted in a spectrophotometer (Optizen POP UV/VIS) at 280 nm. Thereafter, the activity was calculated using the following equation:

Activity = 405 Abs/min × volume of reaction mixture /18.2 (extinction coefficient of 4−nitrophenol) × volume of sample × mg protein

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air dried. Ethyl alcohol (95%) was used for the fixation of the slides for 5 min. Afterwards the slides were stained with Giemsa solution for 10 min. Accordingly 100 cells were observed per slide as investigated under the light microscope. The calculation of the phagocytic activity and phagocytic index was performed using following equations:

2.8. Hematological parameters Red blood cell count (RBC), hemoglobin concentration (Hgb) and hematocrit ratio (Hct) were performed using an automatic blood count device (Mindray BC 3000 plus) as previously applied and reported in rainbow trout in our laboratory (Yılmaz and Ergün, 2018). The reliability of the automatic results was validated by manual hematological analysis conducted according to Blaxhall and Daisley (1973) on all blood samples immediately after collecting in K3EDTA tubes.

Phagocytic activity (%) = (Number of phagocytic cells with engulfed bacteria /number of phagocytes) × 100

2.9. Biochemical analysis

Phagocytic index = Number of engulfed bacteria/phagocytic cells

Serum glucose, total protein, albumin, globulin, triglyceride, cholesterol, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels were assessed using commercial test kits (Bioanalytic Diagnostic Industry, Germany) as reported earlier by Yilmaz and Ergun (2012). Serum biochemical analysis was performed spectrophotometrically (Optizen POP UV/VIS).

2.10.4. Lysozyme activity For the detection of the lysozyme activity, the microtitre-plate method (Nudo and Catap, 2011) used to measure the lysis of a suspension of Micrococcus luteus (Sigma-Aldrich, lot no. # 4698) (0.75 mg/ mL of 0.1 M phosphate/citrate buffer with 0.09% NaCl, pH 5.8) was applied. Bacterial suspension of 175 μL was added at 25 μL of each serum sample to hen egg white lysozyme standard (Sigma-Aldrich, lot no. # L6876; 0–40 μg/mL of 0.1 M phosphate/citrate buffer with 0.09% NaCl, pH 5.8) in flat bottomed 96-well plates which were incubated for 30 min. The rate of lysis was detected against M. luteus blank on a 450 nm plate reader. Hen egg white lysozyme was used as the external standard. A standard curve was used for the conversion of the rate of reduction in absorbance of samples to lysozyme concentration (μg/mL).

2.10. Immune related parameters 2.10.1. Respiratory burst activity The respiratory burst activity of the phagocytes was carried out according to modified Stasiak and Baumann (1996) method, where blood sample around 50 μL was placed into 96 well plates (Thermo Scientific, Nunc, #167008) coated with 50 μl of poly-L-lysine solution (Sigma-Aldrich, lot no. # P4832) and incubated at room temperature for 1 h in order to allow cell adhesion. Thereafter, the supernatant were removed by three-times washing of the wells within HBSS (Sigma-Aldrich, lot no. # H6648), then 100 μL 0.2% NBT (Sigma-Aldrich, lot no. # N5514) was added into the HBSS solution and incubated for another 1 h. Afterwards, the cells were fixed with 100% methanol for 5 min and washed three-times with methanol (70%). The plates were air-dried and 60 μL 2 M potassium hydroxide (KOH, Sigma-Aldrich, lot no. # P5958) and 70 μL dimethyl sulphoxide (DMSO, Sigma-Aldrich, lot no. # D2650) were added into each well. At last, the absorbance (OD) was recorded in a plate reader (Thermo Multiskan Go) at 620 nm.

2.10.5. Myeloperoxidase activity Total myeloperoxidase (MPO) content was measured by the method described by Sahoo et al. (2005) with some slight modifications. In 96well plate, 10 μL serum was diluted with 90 μL of HBSS without Ca2+ or Mg2+. Then, 35 μL of a substrate buffer (0.05 M phosphate-citrate buffer, pH 5.0) containing 0.1 mg/mL 3,3′,5,5′-tetramethylbenzidine dihydrochloride (Sigma-Aldrich, lot no. # T3405) and 0.006% hydrogen peroxide was added into each well. The reaction was stopped after 2 min through the addition of 35 μL of 2 M sulphuric acid, and the absorbance was read in a plate reader 450 nm. 2.11. Bacteria and challenge experiment

2.10.2. Potential killing activity The potential killing activity of blood phagocytic cells were measured according to the techniques modified by Siwicki and Anderson (1993). Initially, 50 μl blood was added into a microtiter plate well coated with 50 μL of poly-L-lysine solution, and incubated for 1 h at room temperature in order to ensure the cells adhered to the plastic surfaces. Then the adhered cells were washed three-times with HBSS in order to gentle remove the supernatant and non-adherent cells. Thereafter, 100 μL of 0.2% NBT in HBSS solution, containing formalin which had a potential of killing 1.5 X l08 cfu/mL of Y. ruckeri E42 cells, was added into the wells. Then the plate was centrifuged for 5 min at 150g to ensure the contact of the bacteria with the adherent cells. Following the incubation which lasted for 30 min at room temperature and the removal of the supernatant, the fixation of the cells were made with 100% (v/v) methanol for 5 min and washed three times with 70% (v/v) methanol. Prior to the addition of 60 μl of 2 M KOH and 70 μl DMSO in order to solubilize the formazan, the plates were air-dried. The recording of the OD of the solution was carried out in a plate reader at 620 nm against a KOH/DMSO blank.

For challenge tests the Y. ruckeri E42 (GenBank accession no. KX388238) was used in the present study. It was previously isolated from diseased trout by Dr. Ertan Emek ONUK (Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun-Turkey). It was produced overnight in Tryptic Soy Broth at 22 °C, and then washed twice with PBS to adjust the density to 3 × 108 CFU/mL. The LD50 value was previously calculated for trout in our previous study (Yılmaz and Ergün, 2018) according to Finney's probit analysis method (Finney, 1971). At the end of the 60-days feeding trial, 100 μL bacterial suspension (3 × 108 CFU/mL in PBS) was intraperitoneally injected into fish (51 fish per group) by an insulin syringe. Fish mortality was recorded 20 days on a daily basis. The mortality related to bacterial infection was confirmed by the re-isolation of Y. ruckeri from spleen, liver and pyloric ceca. The isolates were identified by means of the conventional microbiological tests (Austin and Austin, 2016) and 16S rDNA analysis according to the method described in our previous study (Yılmaz and Ergün, 2018). A total of 276 bacteria were isolated from the dead fish. All of these isolates presented cream-coloured, gram-negative rod, motile, oxidase negative, indol negative and catalase positive characteristics. Out of the isolates, randomly selected 28 strains were analyzed for 16 s rDNA in the laboratories of RefGen (Çankaya-Ankara, Turkey) company and all strains were approved as Y. ruckeri.

2.10.3. Phagocytic activity and phagocytic index A microscopic counting method as reported earlier by Siwicki and Anderson (1993) was followed for the phagocytic activity. Briefly, 100 μL of the blood sample and 100 μL of formalin killed Y. ruckeri E42 (1.5 × 108 in PBS) suspension were mixed in Eppendorf tubes, which were then incubated at 25 °C with continuous mixing on a blood roller mixer for 30 min. Then, a drop of blood was taken on the glass slide and

2.12. Ethics statement Fish experiments were performed in accordance with the guidelines 214

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for fish research from the animal ethics committee at Canakkale Onsekiz Mart University (Protocol Number: 2015/11-02).

show any significant differences (p > .05). Lipase activity in the intestine of fish fed the AMF diet was significantly higher than those in the other treatment groups (p < .05). However, it was significantly lower in the F4 group compared to the C0 and AMF groups (p < .05). Significantly higher alkaline phosphatase activity was noted in the F1 group compared to the F4 treatment group (p < .05), but no significant difference was found between the remaining groups (p > .05). Further, stomach and intestinal pH levels were not affected by the test diets and no significant differences were found among the experimental treatment groups (p > .05).

2.13. Statistical analysis One way analysis of variance (ANOVA) was performed for analyzing the data and values were presented as Means ± Standard Error of Mean (SEM). In case of homogeneity of variances, Tukey's multiple comparison test was applied; otherwise, Tamhane post hoc test was used. Following Dunn's post hoc test, Kruskal-Wallis test was carried out where normality variances were not assumed. Student's t-test was used for the determination of the significance of differences between the manual and automatic hematological values. In each challenge treatment group, Kaplan-Meier analysis was applied to estimate fish survival while the log-rank (Mantel-Cox) test for pairwise comparisons were used for the detection of the differences among the groups. SPSS 19.0 (SPSS Statistics) was used for the analysis of the significance at 0.05 levels.

3.4. Hematological variables Hematological parameters evaluated with both manual and automatic methods did not present any statistical differences (p > .05; data not shown). Hematological variables measured using automatic analyzer is given in Table 5. The RBC count, Hgb concentration, and Hct ratio in the treatment groups did not vary significantly from the values observed for the C0 group (p > .05).

3. Results 3.5. Biochemical variables 3.1. Growth performance and biometric indices At the end of feeding trial, there were no significant differences among all serum biochemical variables with the exception in serum glucose and triglyceride levels as shown in Table 6. Significantly lower serum glucose level was seen in the F4 group compared to all other experimental treatment groups (p < .05). Serum triglyceride level was significantly lower in F2, F2, F4 and AMF groups (p < .05) compared to the value obtained in the C0 group.

All test diets were well accepted by the experimental fish and no mortality or any signs of disease were observed in all treatment groups. Growth performance and biometric indices of rainbow trout fed different experimental diets are given in Table 2. No significant (p > .05) effects of dietary additives were observed on weight gain, feed conversion ratio (FCR), specific growth rate (SGR), viscerosomatic index and spleen–somatic index in rainbow trout. Hepatosomatic index, however, showed significantly higher levels in fish fed the AMF and F4 diets compared to those fed the control and H6 diets (p < .05). Fish fed the F2 and F4 diets presented lowest values of visceral fat index which were significantly different in fish fed the C0 diet (p < .05).

3.6. Immunological variables Findings for the immunological variables are given in Table 7. The respiratory burst activity increased significantly in the F1 and F2 groups (p < .05) compared to the C0, F4 and the AMF treatment groups. Significantly lower potential killing activity and phagocytic index were found in the F4 and AMF groups over those in the C0, F1 and F2 groups (p < .05). The phagocytic index found for the F2 group however was significantly higher than the other experimental groups (p < .05). Not significant differences were seen in terms of phagocytic activity, lysozyme activity and myeloperoxidase activity among the experimental groups (p > .05).

3.2. Whole-body proximate composition The whole-body proximate composition obtained at the end of the experiment is given in Table 3. Whole-body dry matter, protein content and ash content in rainbow trout did not differ significantly between the experimental groups (p > .05). However, significantly higher whole-body lipid contents were found in fish fed diets of F1, F2, F4 and AMF over those fed with the C0 diet (p < .05).

3.7. Challenge test with Y. ruckeri 3.3. Digestive enzyme and pH After the feeding trial for a period of 60 days, experimental fish were challenged with Y. ruckeri and cumulative survival was recorded for 20 days (Fig. 1). Clinically infected fish displayed erratic swimming, darkened color, and redness around the mouth. Internally, petechial

Results obtained for digestive enzyme activities are shown in Table 4. Mean pepsin, amylase and trypsin activities in intestine of fish among all FARMARIN® XP or antibiotic supplemented groups did not

Table 2 Effect of dietary FARMARIN® and antibiotic on growth performance, biometric indices and survival rate in rainbow trout fed different experimental diets for 60 days.

Initial fish weigh (g) Final fish weight (g) RGR (%) SGR (%/day) FCR VSI HSI VFI SSI Survival (%)

C0

F1

F2

F4

AMF

22.21 ± 0.10 54.56 ± 0.31 145.64 ± 2.52 1.50 ± 0.02 1.20 ± 0.02 16.74 ± 0.73 1.22 ± 0.04b 1.82 ± 0.10a 0.13 ± 0.01 100

21.65 ± 0.05 54.35 ± 0.71 150.97 ± 2.71 1.53 ± 0.02 1.19 ± 0.02 15.90 ± 0.41 1.26 ± 0.09ab 1.75 ± 0.16ab 0.12 ± 0.01 100

21.48 ± 0.34 54.65 ± 0.20 154.59 ± 4.73 1.56 ± 0.03 1.18 ± 0.02 15.78 ± 0.86 1.31 ± 0.09ab 1.38 ± 0.08bc 0.10 ± 0.01 100

21.48 ± 0.05 54.37 ± 0.31 153.14 ± 1.76 1.55 ± 0.01 1.18 ± 0.01 15.86 ± 0.44 1.53 ± 0.13a 1.34 ± 0.08c 0.11 ± 0.02 100

21.81 ± 0.34 53.99 ± 0.86 147.51 ± 2.16 1.51 ± 0.01 1.22 ± 0.01 17.46 ± 0.73 1.54 ± 0.06a 1.51 ± 0.06abc 0.12 ± 0.01 100

Values (mean ± SEM, n = 3; n = 9 for biometric indices) with same superscript letters in the same line are not significant different within groups (p < .05). RGR (relative growth rate), SGR (specific growth rate), FCR (feed conversion ratio), HSI (hepatosomatic index), VSI (viscerosomatic index), VFI (visceral fat index), SSI (spleen–somatic index). 215

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Table 3 Effect of dietary FARMARIN® and antibiotic on whole body proximate composition in rainbow trout fed different experimental diets for 60 days.

Dry matter (%) Protein (%) Fat (%) Ash (%)

C0

F1

F2

F4

AMF

25.75 ± 0.09 17.02 ± 0.12 5.35 ± 0.07b 2.82 ± 0.05

25.99 ± 0.19 16.68 ± 0.19 6.07 ± 0.05a 2.58 ± 0.05

26.35 ± 0.15 17.12 ± 0.18 5.88 ± 0.03a 2.55 ± 0.09

26.02 ± 0.27 16.79 ± 0.26 6.22 ± 0.10a 2.77 ± 0.05

26.08 ± 0.10 17.12 ± 0.15 5.93 ± 0.04a 2.78 ± 0.08

Values (mean ± SEM, n = 9) with different superscript letters in the same line are significantly different within groups (p < .05).

treatments groups of F2, F4 and AMF have driven to a decline in visceral fat index. In contrast to our findings, dietary HUMS (61.2%) did not affect abdominal fat pad in broilers (Ozturk et al., 2010). Conversely, the abdominal fat of chicken increased when dietary HUMS offered at 0.5 g/kg level for 42 days, but no significance was recorded at increasing doses (Ozturk et al., 2012). Even though no significant differences were found for fish growth performance in the present study, increasing levels of whole body fat contents were observed in the experimental fish when fed with FARMARIN® XP and antibiotic supplemented diets. These results in terms of the influence of dietary humic acid and sulfadiazine + trimethoprim on proximate composition are the first records, providing important information in this regards. In agreement to our results on fish body composition, increased fat levels were reported in butt meat, when HUMS (61.2%) were added into drink water of broilers at a level of 450 ppm (Ozturk et al., 2010). In contrast however, dietary inclusion of liquid HUMS did not present and effects on dry matter and fat contents, whereas protein and ash rates decreased in chicken fed with 1 g/kg and 0.5 g/kg HUMS, respectively (Ozturk et al., 2012). Based on the discrepancies among different studies, it might be possible that the effects of HUMS in fish diets are species specific and/or there might be a link between the impact time of HUMS and doses or a combination of dose and time. It is known that physiological and biochemical capacity of digestion and transmission of nutrients influence fish growth and feed utilization, further a number of other factors may influence digestion, absorption and transfer of nutrients in the intestine (Esmaeili et al., 2017). The level of nutrient digestibility depends on available enzymes in the digestive system (Suárez et al., 1995). Hence, the inclusion of some additives in fish diets likely to stimulate digestive enzymes, thus improve digestibility, and improving growth performance of fish. HUMS may increase digestibility of nutrients through the formation of protective films against infection and toxins on the mucous epithelium throughout the digestive tract (Islam et al., 2005). However, how the digestive enzymes of fish are affected by dietary HUMS is a question that still remains unclear. A recent study conducted on chicken underlined that dietary humic acid increases the level of pancreatic amylase activity (Lala et al., 2017). Based on the findings of the present study, it can be indicated that increased intestinal lipase activity with 0.1% and 0.2% FARMARIN® XP and antibiotic supplementations has increased whole body lipid, which is in agreement in terms of the correlation between intestinal lipase activity and whole body lipid increments in earlier reports (Knarreborg et al., 2004; Zhao et al., 2012;

haemorrhages on the surfaces of the liver and pyloric ceca, expanded spleen and inflamed intestine were noted. At the end of the 20 day challenge period, fish survival rates were significantly higher in the AMF and F2 groups compared to all other treatment groups (Table 8) (p < .05), but no significant differences were found between the remaining groups (p > .05).

4. Discussion In the present study, FARMARIN® XP and antibiotic supplementations (INFISH-AQUA®) did not affect growth in rainbow trout, which was in agreement with an earlier report (Soytaş, 2015), where humic acid sodium salt was used in diets of Dicentrarchus labrax. Further, Rousdy and Wijayanti (2015) reported best growth performance in Cyprinus carpio fed with 1% humic acid incorporated diet, although no significance was observed and increasing doses over 1% did not affect fish growth. In contrast, significantly improved growth performance was reported in carps fed diets incorporated with humic acid (180 or 360 mg/kg) for a period of 10 weeks (Sharaf and Tag, 2011). High levels of dietary FARMARIN® XP and antibiotic additions increased hepatosomatic index in the present study. Similarly, dietary inclusion of 30 or 60 mL/kg HUMS (humic acid + fulvic acid) increased liver percentage in Japanese quails (Abdel-Mageed, 2012). Biometric indices are useful parameters for the assessment of body condition in fish (Yılmaz et al., 2013), and increased hepatosomatic index due to stress conditions is a general characteristic of fish related to internal detoxification processes (Eisler and Kissil, 1975). Hence, the findings in the present study, in regards to F4 and AMF treatment groups might be accepted as a sign of negative influence on fish health. In contrary, hepatosomatic index were not affected by dietary treatments at low levels (F1 and F2 groups), which is in agreement with the results of Rath et al. (2006), who reported no significant variations in liver percentage in chicken fed dietary humic acid at 1% and 2.5% levels for 5 weeks. Similarly, Ozturk et al. (2014) did not find any differences in liver percentage in chicken when HUMS (61.2% humic acid, 5.1% fulvic acid) where added into the drinking water at levels of 7.5, 15.0, and 22.5 g/kg live weight at the end of a 42-days study. Visceral and abdominal fat contribute to the non-edible part of the fish and are detrimental to dressing yields with disadvantages when evaluating feed conversion efficiency (Quillet et al., 2007). Information on the effects of dietary HUMS on variations in visceral and abdominal fat of farm animals is scarce so far. In the present study, dietary

Table 4 Effect of dietary FARMARIN® and antibiotic on digestive enzyme, feed pH, intestinal pH and stomach pH in rainbow trout fed different experimental diets for 60 days.

Pepsin (U/mg protein/ min) Amylase (mU/mg protein) Lipase (uMol/mg protein/min) Alkaline phosphatase (U/mg protein/min) Tripsin (U/mg protein/min) Stomach pH Intestinal pH Feed pH

C0

F1

F2

F4

AMF

35.06 ± 2.03 2.87 ± 0.36 0.10 ± 0.01a 0.33 ± 0.03ab 0.50 ± 0.06 7.02 ± 0.33 7.09 ± 0.02 5.59

40.50 ± 3.79 3.22 ± 0.46 0.16 ± 0.01b 0.38 ± 0.04a 0.57 ± 0.13 6.76 ± 0.05 7.15 ± 0.02 5.85

34.15 ± 2.39 3.41 ± 0.38 0.17 ± 0.02b 0.29 ± 0.02ab 0.61 ± 0.11 6.90 ± 0.03 7.15 ± 0.02 5.66

28.21 ± 2.46 3.07 ± 0.19 0.12 ± 0.01ab 0.22 ± 0.03b 0.82 ± 0.16 6.86 ± 0.05 7.14 ± 0.03 5.53

39.65 ± 4.80 2.50 ± 0.32 0.15 ± 0.01b 0.37 ± 0.04a 0.69 ± 0.05 6.73 ± 0.05 7.05 ± 0.02 6.07

Values (mean ± SEM, n = 9, except for pH) with different superscript letters in same line are significantly different within groups (p < .05). 216

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Table 5 Effect of dietary FARMARIN® and antibiotic on hematological in rainbow trout fed different experimental diets for 60 days.

6

−3

RBC (x10 mm Hgb (g dL−1) Hct (%)

)

C0

F1

F2

F4

AMF

1.40 ± 0.05 9.01 ± 0.17 27.29 ± 0.53

1.43 ± 0.04 9.08 ± 0.18 27.56 ± 0.71

1.40 ± 0.04 8.99 ± 0.25 26.99 ± 0.85

1.29 ± 0.07 8.24 ± 0.39 24.48 ± 1.30

1.45 ± 0.03 9.28 ± 0.23 26.61 ± 0.56

Values (mean ± SEM, n = 9) with different superscript letters in the same line are significantly different within groups (p < .05). RBC = red blood cell count, Hct = hematocrit, Hgb = hemoglobin.

fish (Yılmaz and Ergün, 2012; Yılmaz et al., 2016). In our study, significantly lower serum glucose level was found in the F4 group. The triglyceride levels decreased significantly in fish fed FARMARIN® XP or antibiotic supplemented diets. Similar findings were reported in chicken (Yalcin et al., 2006; Maysa and Sheikh, 2008; Lala et al., 2017), Japanese quail (Ipek et al., 2008), and pig (Písaříková et al., 2010). In contrast, significant decline of total plasma protein were found in O. mykiss treated with sulfamerazine (Saglam and Yonar, 2009). In sheep fed diets incorporated with Bovifarm® (containing humic, fulvic, ulvic and fulphonic acids and synergistic combinations of organic minerals, phytoenzymes and phytohormones) at increasing levels of 0.1, 0.2, and 0.4% did not pose any differences in terms of glucose, triglycerides, total protein and albumin concentrations in the serum by the end of a 14-day feeding experiment, whereas serum cholesterol and serum HDL levels increased significantly with a decrease in serum LDL levels (Tunc and Yörük, 2012). The addition of liquid HUMS in chicken diets (0.5, 1 and 1.5 g/kg body weight) did not cause any changes in plasma glucose, total protein and triglycerides levels, however cholesterol levels decreased in the 1 and 1.5 g/kg dietary supplement groups and HDL decreased in the 1 g/kg treatment group (Ozturk et al., 2012) at the end of a 42-day feeding trial. In another report on rats, humic acid incorporated diets decreased serum total lipids, total cholesterol and glucose levels and increased HDL and globulin levels (Banaszkiewicz and Drobnik, 1994). Despite the approach that the discrepancies among different studies might be attributed to different experimental animals used, a strong possibility lies in the effective-mechanism of HUMS from different sources and/or various components. Vucskits et al. (2010) reported that peat extracts were used for prophylaxis or healing wound infections during the World WareI. Hence, the immunomodulatory effects of HUMS have been known for many years. Recently, the antimicrobial, anti-inflammatory and antiviral effects of HUMS were also reported by Groven (2013). An earlier research carried out in our laboratory, underlined antiparasitic and immunomodulatory effects of humic acid sodium salt in fish (Ergün et al., 2014; Soytaş, 2015). In our study, increased phagocytic index and respiratory burst activity particularly in the F2 treatment group endorsed the immunomodulatory effects of FARMARIN® XP in fish. Along with the increased immune parameters, the survival rate against Y. rukeri observed in the F2 group was significantly higher over the control

Zhang et al., 2015). For the assessment of stress conditions, disease and health status in fish, hematological parameters are important criteria (Campbell, 2004; Yılmaz et al., 2016). The reference values of hematological parameters used for rainbow trout in the present study, were similar to those reported in earlier studies (RBC 1.55–4.45x106mm−3, Hgb 7.33–11.5 g dL−1, and Hct 25.78–37.40%; Fazio et al., 2016; Yılmaz and Ergün, 2018). Additionally, dietary incorporation of FARMARIN® XP or antibiotics in fish feeds did not pose any negative changes in hematological parameters in this study, which is in agreement with an earlier report (Sampaio et al., 2016) where no hematological differences were observed between the control and antibiotic sulfadiazine exposed Oreochromis niloticus. In contrast to our findings however, decreased levels of RBC, Hct and Hgb were reported in sulfamerazine-treated O. mykiss (Saglam and Yonar, 2009). The discrepancies between studies could be linked to the application length of time and dose of the applied antibiotics, or a combination of the both including other factors such as husbandry and environmental conditions. No differences in the RBC amounts were found in C. carpio when fed diets supplemented with HUMS at 1, 3 and 5% levels, whereas increased Hct ratio in the 1% group, and significant decreased Hgb in the 3% and 5% groups were recorded after a 21-day feeding trial (Rousdy and Wijayanti, 2015). These results might indicate that the addition of HUMS in fish diets exceeding 3% might lead to anemia. Similarly, earlier reports in chicken (Maysa and Sheikh, 2008; Lala et al., 2017), Japanese quail (Ipek et al., 2008), and pig (Wang et al., 2008) underlined that the dietary incorporation of HUMS did not change hematological parameters in chicken, which is in close agreement with our findings in the present study on rainbow trout. In contrast, however, the dietary inclusion of 0.15% HUMS in chicken feed increased RBC and Hgb amounts significantly after a 60-days feeding trial (Çetin et al., 2006). A recent study conducted in our laboratory, evaluated humic acid supplementation levels (0.25, 0.50, 0.75, and 1.5%) in diets of D. labrax for 20 days and anemia was detected in fish naturally infested with Amyloodinium spp., however it was found that anemia was prevented in fish, especially in the group of 0.5% humic acid (Soytaş, 2015). Serum biochemical parameters are known as important indicators for the determination of impacts of feed additives on health status of

Table 6 Effect of dietary FARMARIN® and antibiotic on serum biochemical parameters in rainbow trout fed different experimental diets for 60 days.

GLU (mg/dL) Tprot (g/dL) ALB (g/dL) GLO (g/dL) TRIG (mg/dL) COL (mg/dL) GOT (U/L) GPT (U/L) LDH (U/L) ALP (U/L)

C0

F1

F2

F4

AMF

117.43 ± 9.77a 4.24 ± 0.32 0.69 ± 0.04 3.55 ± 0.28 103.21 ± 8.65a 165.41 ± 9.72 21.61 ± 2.39 7.31 ± 1.13 549.70 ± 45.78 109.03 ± 11.70

130.10 ± 10.10a 4.11 ± 0.13 0.76 ± 0.02 3.35 ± 0.12 58.12 ± 5.81b 173.46 ± 9.83 30.151 ± 2.71 11.53 ± 1.72 640.30 ± 57.05 127.84 ± 18.86

112.53 ± 5.53a 4.13 ± 0.27 0.73 ± 0.05 3.40 ± 0.23 83.23 ± 7.27b 162.60 ± 12.83 17.13 ± 2.44 7.86 ± 1.25 552.37 ± 63.04 147.20 ± 23.72

82.27 ± 3.17b 3.99 ± 0.28 0.71 ± 0.05 3.28 ± 0.23 74.31 ± 9.62b 151.56 ± 10.37 17.28 ± 3.21 9.23 ± 2.39 567.05 ± 40.82 112.33 ± 18.95

109.73 ± 2.62a 4.33 ± 0.18 0.79 ± 0.04 3.54 ± 0.16 56.09 ± 6.66b 170.98 ± 8.41 24.57 ± 3.23 12.67 ± 2.35 559.20 ± 37.13 109.95 ± 11.90

Values (mean ± SEM, n = 9 with different superscript letters in the same line are significantly different within groups (p < .05). GLU = glucose, Tprot = total protein, ALB = albumin, GLO = globulin, TRIG = triglyceride, COL = cholesterol, GOT = glutamic oxaloacetic transaminase, GPT = glutamic pyruvic transaminase, LDH = lactate dehydrogenase and ALP = alkaline phosphatase. 217

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Table 7 Effect of dietary FARMARIN® and antibiotic on immune related parameters in rainbow trout fed different experimental diets for 60 days. C0 Respiratory burst activity (OD at 620 nm) Potential killing activity (OD at 620 nm) Phagocytic activity (%) Phagocytic index Lysozyme activity (μg/mL) Myeloperoxidase activity (OD at 450 nm)

F1 b

0.10 ± 0.010 0.28 ± 0.014a 26.86 ± 0.36 2.14 ± 0.09b 11.62 ± 1.91 0.61 ± 0.09

F2 a

0.17 ± 0.007 0.29 ± 0.005a 28.05 ± 0.88 2.95 ± 0.23b 11.26 ± 2.99 0.46 ± 0.06

F4 a

0.18 ± 0.010 0.27 ± 0.012a 33.66 ± 1.00 4.41 ± 0.26a 10.53 ± 2.01 0.47 ± 0.06

AMF b

0.11 ± 0.007 0.12 ± 0.007b 22.01 ± 0.47 1.08 ± 0.12c 11.30 ± 1.47 0.37 ± 0.07

0.10 ± 0.008b 0.15 ± 0.008b 27.09 ± 1.66 1.04 ± 0.15c 15.20 ± 1.72 0.39 ± 0.07

Values (mean ± SEM, n = 9) with same superscript letters in the same line are not significantly different within groups (p < .05).

seen against Y. ruckeri. Some earlier studies reported an increasing effect of dietary HUMS on fish survival against fish pathogens, and these were influenced by dose level or application time in different fish species. In C. carpio for instance, respiratory burst activity and survival rate against Aeromonas hydrophilla increased when fish were fed a diet with 1% humic acid inclusion for 20 days, compared to the 3% or 5% humic acid-containing feeds (Rousdy and Wijayanti, 2016). None the less, in C. carpio fed with humic acid extracts of increasing levels (0.2, 1, 5, 10, and 20%) for 30 days, enhanced survival rates were observed particularly in the 5% and 10% treatment groups exposed to A. salmonicida infection, whereas no significant difference were seen over or below these doses (Kodama and Nakagawa, 2007). Similarly, Ayu (Plecoglossus altivelis) exposed to Flavobacterium psychrophilum, showed significant increase in survival rate when fish were fed with 1, 5, and 10% humus extract for 30 days (Nakagawa et al., 2009). The use of sulfonamides, such as sulfadiazine and sulfamethoxazole in combination with trimethoprim is quite common in veterinary for fighting pathogenic bacteria (Riviere and Spoo, 2001). In this study, we observed a decline of potential killing activity and phagocytic index via oral administration of sulfadiazine+trimethoprim. The immunosuppressive effects of sulfadiazine (Yonar et al., 2010) and sulphadiazine+trimethoprim (Didinen et al., 2005) have been reported earlier. However, no effects of oral administration of 30 mg/kg sulfadiazine along with trimethoprim on the immune response were observed of rainbow trout (Lundén and Bylund, 2000; Lundén et al., 2002). These results provide information that the immune response of fish to antibiotics is species specific and may differ according to drug type, drug concentration and the time of exposure to the drugs.

Fig. 1. Kaplan–Meier survivorship curves (cumulative % survival over time; days 0, 5, 10, 15, 20) for rainbow trout after challenge with Yersinia ruckeri; the fish were fed with FARMARIN® [0, 0.1, 0.2, or 0.4 g FARMARIN® 100 g−1 feed; control diet (C0), F1, F2 and F4, respectively] and antibiotic (AMF) supplemented diets prior to bacterial challenge.

5. Conclusions Table 8 Mortality rate, survival and relative percentage survival (RPS) of infected rainbow trout fed with FARMARIN® at different ratios.

Control F1 F2 F4 AMF

No. of challenged fish

Mortality Rate (%)

Survival Rate (%)

RPS

51 51 51 51 51

50.98 45.10 27.45 37.25 5.88

49.02b 54.90b 72.55a 62.75b 94.12a

11.54 46.15 26.92 88.46

In conclusion, findings of the present study indicate that feeding rainbow trout with a diet containing 0.2% FARMARIN® XP over a period of 60 days might be adequate to improve fish biometric conditions, meat quality, intestinal enzymes, immune parameters, serum biochemical variables, as well as survival rate against Y. ruckeri, similar to antibiotic treatment without any adverse effect on growth performance of fish. Thus FARMARIN® XP can be suggested as a dietary substitute for antibiotic to prevent yersiniosis in rainbow trout.

RPS = (1 − (% experimental mortality/% control mortality)) × 100.

Acknowledgments

and the other FARMARIN® XP-treated groups, as it was also the case for the antibiotic treatment group. This was in agreement with Soytaş (2015), who found significantly decline in respiratory burst activity of D. labrax fed with humic acid at different dietary inclusion levels (0.25, 0.50, 0.75, and 1.5%) for 20 days; after the infestation of Amyloodinium spp., however immunosuppressive effect of parasite was not seen in fish fed with 0.5% humic acid diet and survival rate against the parasite increased significantly. In the present study, supplementing high dose FARMARIN® XP in fish diets evoked an increase of potential killing activity and phagocytic index, while no affects were observed on the other immunological parameters. Additionally, no signs of improvement in survival were

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