Effects of sub-lethal doses of miconazole nitrate on Labeo rohita and its curing efficacy against Saprolegniasis

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Effects of sub-lethal doses of miconazole nitrate on Labeo rohita and its curing efficacy against Saprolegniasis
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  Contents lists available at ScienceDirect Aquaculture  journal homepage: www.elsevier.com/locate/aquaculture E ff  ects of sub-lethal doses of miconazole nitrate on  Labeo rohita  and itscuring e ffi cacy against Saprolegniasis Mukta Singh, Ratan Kumar Saha, Himadri Saha ⁎ , Asish Kumar Sahoo, Auroshree Biswal  Department of Aquatic Health and Environment, College of Fisheries, CAU, Lembucherra, Agartala, Tripura 799210, India A R T I C L E I N F O  Keywords: Miconazole nitrate  Labeo rohita HaematologicalBiochemicalAntifungal A B S T R A C T Miconazole nitrate (MCZ) is an imidazole derivative antifungal drug used as therapeutic and prophylactic agentagainst fungal outbreak in human and veterinary animals. However, its use in aquaculture industry has not beenreported yet. The aim of this study was to evaluate the acute toxicity of MCZ in  󿬁 sh and the e ff  ects of sub-lethaldoses of MCZ on the haematological and biochemical parameters in  Labeo rohita . Di ff  erent haematologicalparameters like total blood cell counts, haemoglobin content along with mean corpuscular haemoglobin, meancorpuscular volume and mean corpuscular haemoglobin concentration values were observed. Biochemicalparameters like plasma glucose level, plasma glutamic oxaloacetic transaminase and plasma glutamic pyruvictransaminase activity, alkaline phosphatase activity, plasma Na + and K + levels were also determined. In thisexperiment, 32 treatments (4 dose × 8 exposure times) of four MCZ doses (control- 0.0mgMCZ kgBW − 1 ,T1 – 6.30mgMCZ kgBW − 1 , T2 – 12.61mgMCZ kgBW − 1 and T3 – 25.22mgMCZ kgBW − 1 ) with eight exposuretimes were set up in triplicates. In the second experiment,  󿬁 sh infected with  Saprolegnia parasitica  were fed withMCZ based feeds @ 1% BW daily for 21days and their relative percent survival were recorded. The studyrevealed that the sublethal doses of MCZ have the signi 󿬁 cant (p < 0.05) e ff  ect on the haematological andbiochemical parameters of the  󿬁 sh and also lowered (p < 0.05) the mortalities of saprolegnia infected  󿬁 sh. Fishfed with diet T3 (25.2mgMCZ kg − BW − 1 ) had maximum survivability with highest curing e ffi cacy without anyobservable harmful e ff  ect on the physiology of   󿬁 sh. Thus, MCZ has an e ff  ective role in maintaining the healthstatus of   󿬁 sh and providing protection against oomycetes,  S. parasitica  infection. 1. Introduction Environmental stress causes high mortality among the  󿬁 sh followedby pathogenic attacks causing a tremendous loss to the nation (Kar,2000). Like bacterial, viral and parasitic diseases, diseases caused byfungal infection are also evident in all life stages of   󿬁 sh. Fungal diseasecaused by  󿬁 sh pathogenic fungus,  Saprolegnia parasitica  has been iso-lated from many  󿬁 sh over a period of time (Wood and Willoughby,1986; Puckridge et al., 1989; Soderhall et al., 1991; Hatai and Hoshiai, 1992; Czeczuga and Muszynska, 1999; Czeczuga and Muszynska, 2000; Stueland et al., 2005; Fregeneda-Grandes et al., 2007). Saprolegniasis has also been reported in cultured teleosts in India, recording highmortality in various species of pond-cultured carps (Srivastava, 1980;Jha et al., 1984; Krishna et al., 1990). It is one of the most destructive oomycete pathogens on  󿬁 sh endemic to all freshwater habitats aroundthe world and is also partly responsible for the decline of natural po-pulations of various freshwater  󿬁 sh (VanWest, 2006). Losses in theworld-wide salmon aquaculture due to Saprolegniasis were estimated attens of millions of pounds annually (Hussein and Hatai, 2002; GordonRitchie, personal communication).  “ Winter kill ”  in cat 󿬁 sh caused by  S. parasitica  in the United States results in  󿬁 nancial losses of up to 50%,representing the economic loss of £25 million (Bruno and Wood, 1999).MCZ is a broad-spectrum antifungal agent which is used extensivelyfor the management of dermal (Minghetti et al., 1999), buccal(Bouckaert and Remon, 1993) and vaginal candidiasis (Mandal, 2000). It is among one of the  󿬁 rst azole derivatives synthesized with su ffi -ciently low toxicity to permit intravenous administration for thetherapy of systemic fungal infections (Heel et al., 1980). It disrupts theergosterol synthesis (Schechter et al., 1973) interfering with otherbiological circuits within the fungal cell wall (Kano et al., 2001). Bloodchemistry is considered as an important indicator of patho-physiolo-gical condition of an organism and therefore play important role indiagnosing the structural and functional status of   󿬁 sh exposed to drugsor toxic chemicals (Adhikari et al., 2004). All biochemical and hae-matological indices like red blood cells (RBCs), white blood cells(WBCs), haemoglobin, packed cell volume (PCV) etc. are used to assess https://doi.org/10.1016/j.aquaculture.2018.04.029Received 10 October 2017; Received in revised form 28 February 2018; Accepted 18 April 2018 ⁎ Corresponding author.  E-mail address:  sahacofcau@gmail.com (H. Saha). Aquaculture 495 (2018) 205–213Available online 19 April 20180044-8486/ © 2018 Elsevier B.V. All rights reserved.    the oxygen carrying capacity of blood and hence are the indicator of health status of   󿬁 sh. Thus, the present study was undertaken to studythe changes in haematological and biochemical parameter of   󿬁 sh inresponse to sub-lethal doses of MCZ.Apart from its high antifungal activity, high oral bioavailability,minimal metabolism, predominant renal excretion as unchanged drug,activity against super 󿬁 cial as well as invasive fungal infection and lowtoxicity towards host, MCZ has got some additional properties like anti-parasitic properties (Hess et al., 1986) and bactericidal activity (Zhang et al., 1993). The above-mentioned  󿬁 ndings suggested that the MCZ hasthe immense potential to be utilized as a therapeutic drug in aqua-culture for curing di ff  erent fungal infection and the present study wasconducted to evaluate the above hypothesis. 2. Materials and methods  2.1. Experimental animal Fingerlings of   L. rohita  (average length 16.7 ± 1.5cm and averageweight 24.2 ± 1.5g) were collected from the farm of College of Fisheries, CAU, Lembucherra. Acclimatisation of collected  󿬁 ngerlingswas done for about 20days in circular  󿬁 bre reinforced plastics (FRP)tanks (Plasto Craft, Mumbai) of 500l capacity. These were maintainedwith the routine amount of pelleted feed (from the Dept. of Aquaculture, College of Fisheries, (CAU), Lembucherra) twice a day andwater exchange up to 30% was done daily until the experiments wereconducted. FRP acclimated  󿬁 shes were again acclimated in aquariumtanks for 5days prior to aquarium experiment in respective cases. Priorof the feeding trial,  󿬁 sh were adapted to control feed. However,  󿬁 shwere fasted for one day before feeding them with the experimental dietsto avoid leaching of MCZ from the feeds. Fish has consumed the feedswithin  –  5min.  2.2. Test diets Iso-nitrogenous (35.18 – 35.99% crude protein) and iso-caloric(357 – 365kcal100g − 1 ) diets were prepared (Table 1) and proximatecomposition of the experimental diets was analyzed following standardmethod (AOAC, 2000). Puri 󿬁 ed diets were prepared with the calculatedamount of MCZ (Himedia Laboratories, Mumbai, India) as per treat-ment doses. Feeding was done with the respective test diets at 1% of their body weight to achieve the targeted doses. The above feedingpercentage was selected to maintain the dose of application as well as tomake sure that there was no leftover experimental feeds.  2.3. Acute toxicity test  Firstly, range  󿬁 nding test was conducted to determine the dose of MCZ to be included in de 󿬁 nitive short term test for determining theLD 50  in  󿬁 sh,  L. rohita . The  󿬁 ngerlings of   L. rohita  were fed to series of MCZ at 100, 200, 400, 800mgMCZ kgBW − 1 . Mortality of   󿬁 ngerlingswas observed for 24h. Mortality was observed at 200, 400 and800mgMCZ kgBW − 1 . There was no mortality recorded at 100mgMCZkgBW − 1 . Based on the above  󿬁 ndings, short-term de 󿬁 nitive test wasconducted with MCZ at doses of 0, 100, 120, 140, 180, 200mgMCZkgBW − 1 . The test was conducted in triplicates using 18 glass aquariums(2 ′ ×1 ′ ×1 ′ ) with 5  󿬁 ngerlings in each. Re 󿬂 ex actions, behaviouralchanges and mortality were recorded at every 6h intervals for 96h. Nofeeding was done during this time period. The LD 50  value from thestudy was analyzed using Probit analysis of BIOSTAT 2009 Professional5.8.4 (Analyst Soft Inc., Vancouver, BC V6H 4E4, Canada; Website:www.analystsoft.com) software.  2.4. Feeding trial The experiments were designed to know the e ff  ect of sub-lethaldoses of MCZ on haematological responses of   L. rohita  󿬁 ngerlingsthrough the medicated feed. The experiments were carried out for aperiod of 10days (240h). Three sub-lethal doses of MCZ were (1/20, 1/10 and 1/5 of LD 50  value of 96h) selected for the experiment. Therewere 32 treatments (4 doses of MCZ based medicated feed: T1 (LD 50 /20): 6.3, T2 (LD 50 /10): 12.6, T3 (LD 50 /5): 25.2 and control: 0.00mgkgBW − 1 combined with eight exposure times at 0, 6, 12, 24, 48, 96,120 and 240h) with three replicates each.480  󿬁 ngerlings were distributed randomly in 96 aquariums fol-lowing completely randomized design. Tests were carried out in tri-plicate groups consisting of   󿬁 ve  󿬁 ngerlings each in 45l glass aquaria.The experimental trial was conducted for 10days and the sampling of blood for haematological parameters such as total erythrocyte count(EC), total leukocyte count(LC), packed cell volume (PCV), total hae-moglobin, mean corpuscular volume (MCV), mean corpuscular hae-moglobin (MCH), mean corpuscular haemoglobin concentration(MCHC) and biochemical parameters like plasma glucose level, plasmaglutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvictransaminase (GPT) activity, alkaline phosphatase (ALP) activity,plasma Na + and K + levels were done at 0, 6, 12, 24, 48, 96, 120 and240h. All the  󿬁 sh were harvested from the respective tanks at re-spective sampling occasion and pooled samples from each tank wereused for the study.  2.5. Blood collection for analysis Each  󿬁 sh was anesthetized with clove oil (Merck, Germany) at therate of 50 μ l of clove oil per litre of water before collecting bloodsamples from  󿬁 sh. Blood was drawn from caudal vein of   󿬁 sh by using1.0ml hypodermal syringe & 24 gauge needles. The collected blood(approximately 400 – 500 μ l from each  󿬁 sh) was immediately trans-ferred into vials coated with thin layer of EDTA, an anticoagulant. Vialshaving EDTA coats were shaken gently in order to prevent haemolysis &clotting of blood. Collected blood was also transferred into serologicaltubes with the small amount of anticoagulant and was centrifuged for5min at 3000rpm for separation of serum and plasma. Serum andplasma were collected and stored in microcentrifuge tubes at − 20°Cuntil the assay was done.  2.6. Haematological parameters Counting of RBC was done following the method of  Schaperclaus Table 1 Formulation of experimental diets (100g) used in experiment. Ingredients Quantity (in g)Control diet Diet T1(LD 50 /20)Diet T2(LD 50 /10)Diet T3(LD 50 /5)Casein 16.0 16.0 16.0 16.0Gelatin 9.0 9.0 9.0 9.0Dextrin 30.0 30.0 30.0 30.0Fish meal 16.0 16.0 16.0 16.0CMC 2.0 2.0 2.0 2.0Veg oil 4.5 4.5 4.5 4.5Cod liver oil 4.5 4.5 4.5 4.5Vit-min mixture ⁎ 6.0 6.0 6.0 6.0BHT 0.2 0.2 0.2 0.2Cellulose 12.80 12.74 12.67 12.55Drug 0.0 0.063 0.126 0.252 ⁎ Composition of vitamin-mineral premix (PREEMIX PLUS) (quan-tity2.5kg − 1 ): Vitamin A, 5500000IU; Vitamin D 3 , 1,100,000IU; Vitamin B 2 ,2000mg; Vitamin E, 750mg; Vitamin K, 1000mg; Vitamin B 6 , 1000mg;Vitamin B 12 , 6  μ g; Calcium Pantothenate, 2500mg; Nicotinamide, 10g; CholineChloride, 150g; Mn, 27,000mg; I, 1000mg; Fe, 7500mg; Zn, 5000mg; Cu,2000mg; Co, 450mg; L- lysine, 10g; DL- Methionine, 10g; Selenium, 50ppm.  M. Singh et al.  Aquaculture 495 (2018) 205–213 206  (1991) using Hayem's solution (Qualigens, India). Final erythrocytecount (EC) was done using an improved Neubauer haemocytometerunder trinocular microscope (400×) and were reported as 10 6 mm − 3 .White blood cells were also counted using improved Neubauer hae-mocytometer (Shah and Altindag, 2005) using the dilution mixture(WBC diluting  󿬂 uid, Qualigens, Mumbai) and were expressed as10 3 mm − 3 . Estimation of haemoglobin in blood was done by using SahliHaemometer (Marine 󿬁 eld, Germany) following Schaperclaus (1991).Packed cell volume (PCV) was measured following the method of Schaperclaus (1991). The PCV or hematocrit value was expressed as thepercentage fraction of blood cells in the total volume (volume %). MCV,MCH and MCHC was calculated following Dacie and Lewis (2001) andwere expressed in femtoliters ( 󿬂  or 10 − 15 l), pictograms (pg) per celland grams of haemoglobin per 100ml packed cells, respectively.  2.7. Biochemical parameter  For the determination of glucose in plasma, glucose diagnostic kit(Coral Clinical systems, India) was used which is based on Trinder(1969) GOD/POD method. GOT and GPT activity in plasma was de-termined using the diagnostic kit (Accurex Biomedical, India) based onBiuret method (Strickland et al., 1961; Henry, 1974). The kit was based on the principle that GPT converts L-alanine and  α -ketoglutarate topyruvate and glutamate described by Reitman and Franckel (1957).Alkaline phosphatase activity in serum was assayed using alkalinephosphatase a diagnostic kit (Accurex Biomedical, India) based onBiuret method (Strikland et al., 1961; Henri, 1974). The kit was based on the principle that ALP cleaves  p -nitrophenyl phosphate (p-NPP) into  p -nitrophenol and phosphate p-nitrophenol. Na + and K + levels of bloodplasma were assayed using Na + and K + diagnostic kit (Coral ClinicalSystems, India) based on Biuret method (Strickland et al., 1961; Henry, 1974).  2.8. Curing e  ffi cacy of MCZ against Saprolegnia zoospores Experiment was conducted to evaluate the e ffi cacy of MCZ in curing  S. parastica  infection in  L. rohita  󿬁 ngerlings. Excess numbers (about150) of rohu  󿬁 ngerlings (average length 16.7 ± 1.5cm and weight24.2 ± 1.5g) were used for infection. Isolation of fungal strains andinducing sporulation in fungal culture were done using GP (glucose-peptone) PenStrep broth and technical agar following Lilley et al.(1998). Rohu  󿬁 ngerlings were arti 󿬁 cially infected with  S. parasitica following Firouzbakhsh et al. (2014) method with slight modi 󿬁 cationfollowing Saha et al. (2016). To create stress and prevent mucus pro-duction for spore penetration into  󿬁 sh body, method of  Hatai andHoshiai (1993) was followed. Brie 󿬂 y,  󿬁 sh were held in a net (6mmmesh size) with continuous slow shaking and rubbing for 2min fol-lowed by washing of mucus secretions and then placing the  󿬁 sh in theaquarium tanks. The zoospores of   S. parasitica  were added at3×10 5 l − 1 to the tanks and dispersed by suitable aeration. 50% waterexchange with water containing zoospores (3×10 5 l − 1 ) was carriedout on every third day (Fregeneda-Grandes et al., 2001). Daily mor-talities and  󿬁 sh contaminations were monitored during the entire ex-perimental duration. Fungal infections were con 󿬁 rmed using directmicroscopy technique. The fungi at both the internal and external tis-sues were observed (Debnath et al., 2012) using haematoxylin andeosin (H and E) stains. After 6days of infection, infected  󿬁 sh were usedfor the curing e ffi cacy study. There were 5 treatments with two re-plicates each. 5 treatments for the study were infected  󿬁 sh fed withMCZ dose 1(6.3mgMCZ kgBW − 1 ), infected  󿬁 sh fed with MCZ dose 2(12.6mgMCZ kgBW − 1 ), infected  󿬁 sh fed with MCZ dose 3(25.2mgMCZ kgBW − 1 ), infected  󿬁 sh fed with no MCZ (negative con-trol) and uninfected  󿬁 sh fed with no MCZ (positive control) and theywere designated as T1, T2, T3, C –  and C+, respectively. 80 infected and20 uninfected  󿬁 sh were randomly distributed in 8 FRP and 2 FRP tanks,respectively. Feeds prepared during the  󿬁 rst experiment were used inthis experiment too. Feeding of   󿬁 sh with respective feed was done dailyat 1% of the 󿬁 sh biomass to provide the exact dose of MCZ. Feeding wasadjusted daily based on the mortalities of the  󿬁 sh. Evening feeding withcontrol feeds was done at ad-libitum in all the tanks to provide theproper nourishment to the  󿬁 sh (Saha et al., 2016). Water was ex-changed daily at 20% and water quality parameters were found withinthe range for all the tanks. Relative percentage survival was recordedfor 15days. = −× Relative Percentage Survival(1%mortality in treatment/%mortality in control)100  2.9. Statistical analysis Statistical analysis was performed using SPSS-16.0 for windowssoftware (SPSS Inc., Chicago, IL, USA). Results are presented as themean ± standard deviation. Data analysis were done by one-way andtwo- way analysis of variance (ANOVA) and Tukey's comparison of means. Probability level of 0.05 was used to determine the signi 󿬁 cance. 3. Result 3.1. Acute toxicity test  In the range- 󿬁 nding test,  󿬁 ngerlings fed with the medicated diet at100mgMCZ kgBW − 1 were observed under stress for certain time periodbut recovered soon. Based on the result,  󿬁 sh were fed with the MCZ inthe range between 100 and 200mgMCZ kgBW − 1 for the short termde 󿬁 nitive test. The study showed hundred percent mortality of rohu at200mgMCZ kgBW − 1 dose within 96h of feeding. 96h LD 50  value in  L.rohita  󿬁 ngerling for MCZ was found to be 126.145 ± 11.009 mgMCZkgBW − 1 . The mortality rate at di ff  erent doses of MCZ was signi 󿬁 cantly(p < 0.05) di ff  erent from each other. LD 50  values were increasing withdecreasing time period. 3.2. Haematological responses The total erythrocyte count (EC) of di ff  erent treatment groups atdi ff  erent sampling time are given in Table 2. EC of T1, T2 and T3 weresigni 󿬁 cantly (p < 0.05) lower than control from 6 to 24h and showdecreasing trend with respect to time. The total leucocyte count (LC) of T1, T2 and T3 were signi 󿬁 cantly (p < 0.05) higher than the control at6h. However, there was signi 󿬁 cant  󿬂 uctuation in T1, T2 and T3 ascompared to control at di ff  erent sampling hours (Table 2). Packed cellvolume (PCV) of T3 was signi 󿬁 cantly (p < 0.05) lower and show de-creasing trend as compared to control in all sampling hours. At 120h,PCV of T2 was signi 󿬁 cantly (p < 0.05) lower than the T1 and control(Table 2). Haemoglobin content of T1 and T3 were signi 󿬁 cantly(p < 0.05) higher than control at 12h. However, after 24h no sig-ni 󿬁 cant di ff  erence in haemoglobin content was observed in all the ex-perimental groups.MCV of T3 experimental group show statistically signi 󿬁 cant(p < 0.05) decreasing trend with respect to time as compared tocontrol group. MCH value of T1 and T3 were signi 󿬁 cantly (p < 0.05)higher than control at 6h to till 48h. After 120h, no signi 󿬁 cant dif-ference in MCH was observed between di ff  erent experimental groupsMCHC of all the experimental groups does not show any signi 󿬁 cant(p < 0.05) di ff  erences among each other up to 12h. However, MCHCof T1 and T2 were signi 󿬁 cantly (p < 0.05) higher than control at 120and 240h (Table 2). From the results of two way ANOVA analysis, wefound that there is a statistically signi 󿬁 cant di ff  erence in haematolo-gical parameter of all treatment diets with respect to di ff  erent samplinghours (p < 0.05).  M. Singh et al.  Aquaculture 495 (2018) 205–213 207  3.3. Biochemical responses The plasma glucose levels of di ff  erent treatment groups were sig-ni 󿬁 cantly higher and show increasing trend as compared to controlgroup with di ff  erent sampling hours (Table 3). Alkaline phosphatase(ALP) of di ff  erent treatment groups were signi 󿬁 cantly (p < 0.05)higher than control upto 24h. However, in experimental group T3,increasing trend in alkaline phosphatase activity was observed till 240hof sampling. GPT and GOT level of T2 and T3 group were signi 󿬁 cantly(p < 0.05) higher than control during the entire experimental duration Table 2 Haematological parameters (Erythrocyte count, leukocyte count, packed cell volume, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin andmean corpuscular haemoglobin concentration) of   Labeo rohita  󿬁 ngerlings of di ff  erent experimental groups at various sampling hours with di ff  erent doses of MCZbased medicated feed. Hours Control T1 T2 T3Erythrocyte count (EC×10 6 mm − 3 )0h 3.61 ± 0.01 a 3.59 ± 0.02 a 3.59 ± 0.03 a 3.58 ± 0.03 a 6h 3.55 ± 0.01 a 3.30 ± 0.02 b 3.30 ± 0.01 b 3.13 ± 0.01 c 12h 3.56 ± 0.02 a 3.02 ± 0.01 b 2.98 ± 0.04 b 3.01 ± 0.02 b 24h 3.54 ± 0.01 a 3.16 ± 0.01 bc 3.09 ± 0.06 b 3.03 ± 0.05 c 48h 3.11 ± 0.01 a 3.03 ± 0.01 ab 2.97 ± 0.07 b 3.08 ± 0.01 ab 96h 3.02 ± 0.01 a 3.15 ± 0.01 b 2.85 ± 0.03 c 2.99 ± 0.04 a 120h 3.07 ± 0.03 a 3.21 ± 0.05 b 3.09 ± 0.01 ab 2.91 ± 0.04 c 240h 3.36 ± 0.03 a 3.06 ± 0.02 b 3.32 ± 0.01 a 3.14 ± 0.01 b Leucocyte count (LC×10 3 mm − 3 )0h 244.76 ± 0.88 a 244.33 ± 1.76 a 244.33 ± 0.8 a 244.28 ± 0.57 a 6h 243.89 ± 0.57 a 247.67 ± 1.76 b 250.67 ± 1.20 bc 252.67 ± 0.88 c 12h 241.00 ± 0.57 a 239.33 ± 2.18 a 247.67 ± 2.72 b 243.33 ± 1.20 ab 24h 242.00 ± 0.57 a 244.33 ± 2.02 a 257.00 ± 0.57 b 257.33 ± 2.72 b 48h 241.00 ± 0.57 a 232.33 ± 3.52 b 243.67 ± 1.45 a 252.33 ± 0.88 c 96h 241.33 ± 1.76 a 241.00 ± 3.46 a 254.33 ± 1.85 b 249.33 ± 0.88 b 120h 242.33 ± 2.02 a 236.67 ± 1.85 b 243.67 ± 1.45 a 252.67 ± 1.20 c 240h 241.67 ± 2.33 a 230.33 ± 1.20 b 242.00 ± 0.57 a 243.33 ± 2.72 a Packed cell volume (%)0h 34.33 ± 1.20 a 34.33 ± 1.20 a 33.66 ± 0.66 a 33.33 ± 0.88 a 6h 34.33 ± 1.20 a 34.26 ± 0.12 a 31.83 ± 0.60 a 30.00 ± 0.28 b 12h 33.33 ± 0.66 a 31.16 ± 0.72 b 32.16 ± 0.60 ab 28.16 ± 0.44 c 24h 33.00 ± 0.57 a 32.66 ± 1.20 a 33.33 ± 2.60 a 25.66 ± 2.02 b 48h 30.00 ± 1.04 a 30.66 ± 1.45 a 30.66 ± 1.20 a 24.88 ± 2.08 b 96h 31.33 ± 1.20 a 30.50 ± 1.25 a 30.16 ± 0.72 a 24.33 ± 2.40 b 120h 31.16 ± 0.44 a 30.33 ± 0.72 a 25.66 ± 1.45 b 25.83 ± 2.16 b 240h 30.66 ± 0.16 a 28.16 ± 0.60 a 27.60 ± 0.87 a 23.50 ± 0.76 b Haemoglobin content (gdl − 1 )0h 4.46 ± 0.088 a 4.46 ± 0.033 a 4.43 ± 0.120 a 4.56 ± 0.145 a 6h 4.33 ± 0.28 a 4.7 ± 0.057 a 4.33 ± 0.088 a 4.53 ± 0.185 a 12h 4.53 ± 0.03 a 4.8 ± 0.057 b 4.68 ± 0.044 ab 4.76 ± 0.088 b 24h 5.10 ± 0.115 a 5.2 ± 0.057 a 5.1 ± 0.057 a 5.23 ± 0.088 a 48h 4.63 ± 0.120 a 5.26 ± 0.120 b 5.43 ± 0.120 b 5.06 ± 0.088 b 96h 4.66 ± 0.088 a 5.3 ± 0.057 b 4.56 ± 0.088 a 4.23 ± 0.088 c 120h 4.70 ± 0.152 a 5.26 ± 0.088 b 4.93 ± 0.145 ab 4.36 ± 0.145 c 240h 4.70 ± 0.057 a 5.06 ± 0.088 b 5.00 ± 0.057 b 4.63 ± 0.120 a Mean corpuscular volume (10 − 15 l)0h 95.90 ± 3.66 a 95.42 ± 2.79 a 94.82 ± 1.51 a 94.37 ± 3.32 a 6h 96.50 ± 2.93 a 96.64 ± 0.99 a 96.26 ± 1.75 a 95.85 ± 1.36 a 12h 93.44 ± 1.27 a 102.98 ± 2.5 b 107.93 ± 0.74 b 79.19 ± 1.12 c 24h 93.03 ± 1.36 ab 103.19 ± 4.18 a 107.57 ± 7.71 a 78.03 ± 7.49 b 48h 96.35 ± 3.32 a 94.49 ± 4.69 a 103.3 ± 5.19 a 77.90 ± 7.16 b 96h 103.63 ± 3.82 a 96.82 ± 3.96 a 109.38 ± 2.73 a 77.84 ± 7.32 b 120h 101.46 ± 2.53 a 94.48 ± 1.37 ab 82.99 ± 4.86 b 89.00 ± 8.55 ab 240h 91.29 ± 1.18 a 91.88 ± 2.5 a 83.036 ± 2.39 b 74.67 ± 2.19 c Mean corpuscular haemoglobin (pg)0h 12.37 ± 0.30 a 12.36 ± 0.05 a 12.32 ± 0.20 a 12.36 ± 0.50 a 6h 12.19 ± 0.86 a 14.2 ± 0.09 b 13.10 ± 0.29 ab 14.48 ± 0.63 b 12h 12.71 ± 0.07 a 15.85 ± 0.12 b 15.71 ± 0.07 b 13.40 ± 0.33 c 24h 14.38 ± 0.35 a 16.42 ± 0.20 b 16.47 ± 0.13 b 15.87 ± 0.50 b 48h 14.88 ± 0.37 a 17.36 ± 0.48 bc 18.28 ± 0.48 b 16.43 ± 0.28 c 96h 15.43 ± 0.25 a 18.41 ± 0.16 b 16.03 ± 0.47 a 14.15 ± 0.48 c 120h 15.29 ± 0.53 a 15.42 ± 0.52 a 15.15 ± 0.49 a 15.00 ± 0.38 a 240h 13.98 ± 0.02 a 16.51 ± 0.12 b 15.04 ± 0.1 c 14.72 ± 0.31 c Mean corpuscular haemoglobin concentration (gdl − 1 )0h 0.12 ± 0.0025 a 0.12 ± 0.0035 a 0.13 ± 0.0027 a 0.14 ± 0.0014 a 6h 0.12 ± 0.0013 a 0.13 ± 0.0021 a 0.13 ± 0.0031 a 0.15 ± 0.0050 a 12h 0.13 ± 0.0024 a 0.15 ± 0.0048 a 0.14 ± 0.0013 a 0.16 ± 0.0053 a 24h 0.15 ± 0.0031 a 0.15 ± 0.0070 a 0.15 ± 0.0011 a 0.19 ± 0.0015 b 48h 0.15 ± 0.0026 a 0.18 ± 0.0013 ab 0.17 ± 0.0040 ab 0.19 ± 0.0015 b 96h 0.14 ± 0.0037 a 0.15 ± 0.0098 a 0.15 ± 0.0049 a 0.18 ± 0.0017 b 120h 0.15 ± 0.0054 a 0.17 ± 0.0061 b 0.17 ± 0.0058 b 0.19 ± 0.0018 c 240h 0.15 ± 0.0020 a 0.18 ± 0.0058 b 0.18 ± 0.0068 b 0.19 ± 0.0057 c All data are presented as MEAN ± SE. Mean values with di ff  erent superscript within a row for a parameter are signi 󿬁 cantly di ff  erent (p < 0.05).  M. Singh et al.  Aquaculture 495 (2018) 205–213 208  (Table 3). Plasma Na + ion of di ff  erent treatment groups was sig-ni 󿬁 cantly (p < 0.05) higher than control. At 240h, no signi 󿬁 cantdi ff  erence between T2 and T3 group were observed as compared tocontrol. Plasma K + ion concentration of T1 was signi 󿬁 cantly(p < 0.05) higher than control from 6h onwards (Table 3). Statisti-cally signi 󿬁 cant di ff  erence in the biochemical parameter of all treat-ment diets with respect to di ff  erent sampling hours (p < 0.05) wasobserved. 3.4. Curing e  ffi cacy  Results of the experiments conducted to evaluate the e ffi cacy of MCZ through feed against  S. parasitica  in  L. rohita  are given in the Fig. 1.Infected  󿬁 sh fed with diet T1, T2 and T3 dose of MCZ showed highersurvival percentage as compared to infected  󿬁 sh fed with control diet.Among the treatments, infected  󿬁 sh fed with T3 diet showed relativelyhigher survival than T1 and T2. There were no mortalities recorded inuninfected  󿬁 sh fed with control diet. 4. Discussion Determination of a lethal dose of any drug greatly depends on itssusceptibility to particular biological population. For the  󿬁 rst experi-mental study, MCZ was fed to  󿬁 sh at 100, 200, 400, and 800mgMCZkgBW − 1 of   󿬁 sh. Fish fed at 100mgMCZ kgBW − 1 accepted the medi-cated feed completely and showed complete survival. However, little Table 3 Biochemical parameters (plasma glucose level, plasma glutamic oxaloacetic transaminase activity, plasma glutamic pyruvic transaminase activity, alkaline phos-phatase activity, plasma Na + and K + levels) of   L. rohita  󿬁 ngerlings of di ff  erent experimental groups at various sampling hours with di ff  erent doses of MCZ basedmedicated feed. Hours Control T1 T2 T3Plasma glucose (mgdl − 1 )0h 32.74 ± 0.36 a 32.95 ± 0.183 a 32.47 ± 0.037 a 32.98 ± 0.316 a 6h 31.64 ± 0.23 a 35.69 ± 0.301 b 36.11 ± 0.900 bc 37.94 ± 0.900 c 12h 32.95 ± 0.03 a 42.23 ± 0.068 b 49.64 ± 0.013 c 50.20 ± 0.043 c 24h 32.74 ± 0.03 a 48.44 ± 0.023 b 49.45 ± 0.030 b 51.63 ± 0.034 c 48h 32.74 ± 0.03 a 41.62 ± 0.054 b 46.11 ± 0.235 c 51.62 ± 0.256 d 96h 31.64 ± 0.03 a 45.85 ± 0.004 b 46.38 ± 0.185 b 46.79 ± 0.007 b 120h 32.95 ± 0.03 a 41.92 ± 0.074 b 41.69 ± 0.046 b 42.34 ± 0.058 b 240h 31.64 ± 0.03 a 40.41 ± 0.324 b 41.54 ± 0.283 b 40.64 ± 0.314 b Alkaline phosphatase (IUl − 1 )0h 2.988 ± 0.0052 a 3.004 ± 0.0028 a 2.948 ± 0.0378 a 2.943 ± 0.0380 a 6h 2.943 ± 0.0380 a 3.287 ± 0.0115 b 3.813 ± 0.0354 c 4.893 ± 0.0596 d 12h 2.943 ± 0.0380 a 3.804 ± 0.0993 b 4.651 ± 0.2821 c 4.996 ± 0.0107 c 24h 2.943 ± 0.0380 a 3.694 ± 0.1916 b 3.947 ± 0.0283 bc 4.187 ± 0.0922 c 48h 2.943 ± 0.0380 a 3.486 ± 0.1390 b 2.789 ± 0.0888 a 3.970 ± 0.0302 c 96h 2.943 ± 0.0380 a 3.265 ± 0.073 ab 3.620 ± 0.1718 b 3.671 ± 0.2762 b 120h 2.896 ± 0.0702 a 2.925 ± 0.037 a 3.402 ± 0.2119 b 3.795 ± 0.1297 b 240h 2.943 ± 0.0380 a 1.288 ± 0.0175 b 2.009 ± 0.0065 c 3.360 ± 0.0814 d Glutamate pyruvate transaminase (Ul − 1 )0h 41.22 ± 0.5 a 41.65 ± 0.3 a 40.70 ± 0.3 a 40.52 ± 0.2 a 6h 40.38 ± 0.2 a 41.06 ± 0.5 a 42.70 ± 1.3 a 46.80 ± 1.2 b 12h 41.02 ± 0.0 a 42.61 ± 1.6 a 47.59 ± 0.7 b 49.36 ± 0.3 b 24h 40.17 ± 0.1 a 40.82 ± 0.4 a 45.23 ± 1.6 b 52.07 ± 1.1 c 48h 38.69 ± 0.6 a 41.05 ± 0.0 ab 44.03 ± 1.0 b 57.84 ± 1.4 c 96h 40.55 ± 0.2 a 39.82 ± 0.8 a 48.39 ± 0.3 b 52.49 ± 0.8 c 120h 39.33 ± 0.6 a 42.27 ± 1.1 ab 43.02 ± 1.3 b 49.48 ± 0.7 c 240h 40.61 ± 0.3 a 41.70 ± 0.7 ab 43.69 ± 0.9 b 49.70 ± 0.3 c Glutamate oxaloacetate transaminase (Ul − 1 )0h 22.506 ± 0.33 a 23.803 ± 0.63 a 23.216 ± 0.87 a 22.863 ± 0.56 a 6h 23.246 ± 0.31 a 24.871 ± 1.41 a 28.320 ± 1.20 b 35.054 ± 0.94 c 12h 22.506 ± 0.33 a 23.31 ± 0.334 ab 24.683 ± 0.08 b 33.803 ± 1.14 c 24h 22.506 ± 0.33 a 23.610 ± 0.27 a 25.589 ± 0.22 b 30.550 ± 0.72 c 48h 22.506 ± 0.33 a 24.665 ± 0.27 ab 25.857 ± 1.34 b 33.333 ± 0.40 c 96h 22.506 ± 0.33 a 22.827 ± 0.49 a 26.758 ± 0.13 b 29.710 ± 0.35 c 120h 22.506 ± 0.33 a 23.072 ± 0.51 ab 25.851 ± 1.76 b 30.033 ± 0.60 c 240h 22.506 ± 0.33 a 21.444 ± 1.51 a 23.335 ± 1.75 a 25.745 ± 2.06 a Na + (mmoll − 1 )0h 148.24 ± 0.6 a 147.64 ± 0.80 a 148.36 ± 0.3 a 148.65 ± 0.23 a 6h 148.61 ± 0.3 a 198.66 ± 0.06 b 192.75 ± 0.74 c 182.4 ± 0.40 d 12h 148.36 ± 0.3 a 209.08 ± 1.13 b 207.42 ± 0.3 b 204.96 ± 1.6 b 24h 148.36 ± 0.3 a 188.31 ± 3.23 b 191.1 ± 0.48 b 181.69 ± 0.35 c 48h 148.36 ± 0.3 a 183.7 ± 2.41 b 178.34 ± 1.1 c 167.94 ± 0.61 d 96h 148.36 ± 0.3 a 171.07 ± 0.04 b 165.96 ± 0.5 c 154.58 ± 1.29 d 120h 148.36 ± 0.3 a 163.28 ± 0.98 b 157.06 ± 1.0 c 151.4 ± 0.58 a 240h 148.36 ± 0.3 a 158.7 ± 0.36 b 151.1 ± 0.61 a 149.44 ± 1.16 a K + (mmoll − 1 )0h 7.45 ± 0.068 a 7.49 ± 0.012 a 7.58 ± 0.005 a 7.49 ± 0.008 a 6h 7.49 ± 0.005 a 9.50 ± 0.012 b 5.03 ± 0.026 c 3.75 ± 0.023 d 12h 7.55 ± 0.018 a 9.65 ± 0.023 b 7.56 ± 0.240 a 4.44 ± 0.020 c 24h 7.55 ± 0.018 a 9.61 ± 0.020 b 7.57 ± 0.018 a 4.74 ± 0.011 c 48h 7.55 ± 0.018 a 9.60 ± 0.016 b 7.01 ± 0.014 c 4.06 ± 0.028 d 96h 7.55 ± 0.018 a 9.58 ± 0.052 b 6.96 ± 0.025 c 4.85 ± 0.021 d 120h 7.55 ± 0.018 a 9.44 ± 0.017 b 6.06 ± 0.029 c 4.66 ± 0.031 d 240h 7.55 ± 0.018 a 8.70 ± 0.130 b 6.9 ± 0.026 c 6.58 ± 0.005 d All data are presented as MEAN ± SE. Mean values with di ff  erent superscript (a, b and c) within a row for a parameter are signi 󿬁 cantly di ff  erent (p < 0.05).  M. Singh et al.  Aquaculture 495 (2018) 205–213 209
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