Effect of dietary prebiotics and probiotics on snakehead (Channa striata) health: Haematology and disease resistance parameters against Aeromonas hydrophila

Accepted Manuscript Effect of dietary prebiotics and probiotics on snakehead (Channa striata) health: Haematology and disease resistance parameters against Aeromonas hydrophila Mohammad Bodrul Munir, Roshada Hashim, Siti Azizah Mohd Nor, Terence L. Marsh PII:

S1050-4648(18)30059-7

DOI:

10.1016/j.fsi.2018.02.005

Reference:

YFSIM 5112

To appear in:

Fish and Shellfish Immunology

Received Date: 20 October 2017 Revised Date:

24 January 2018

Accepted Date: 2 February 2018

Please cite this article as: Munir MB, Hashim R, Nor SAM, Marsh TL, Effect of dietary prebiotics and probiotics on snakehead (Channa striata) health: Haematology and disease resistance parameters against Aeromonas hydrophila, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Abstract: This study examined the effect of dietary prebiotics and probiotics after 16

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weeks, followed by 8 weeks of post feeding trial with the control unsupplemented diet

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on haematological and immune response against Aeromonas hydrophila infection in

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Channa striata fingerlings. Fish were raised on a 40% protein and 12% lipid feed

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containing three commercial prebiotics (β-glucan, GOS or galacto-oligosaccharide,

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MOS or mannan-oligosaccharide); and two probiotics- (Saccharomyces cerevisiae,

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Lactobacillus acidophilus), respectively and a control. Throughout the study,

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supplementation with dietary prebiotics and probiotics led to

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improvement in the red blood cells, white blood cells, packed cell volume, haemoglobin

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concentration and serum protein level and lysozyme activities; and these improvements

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were effective significantly (P<0.05) when the fish were challenged with Aeromonas

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hydrophila at the dose of 2 x 106. The disease resistance against A.hydrophila was

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higher significantly (P<0.05) in fish fed with probiotic feed supplements (L.acidophilus

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was highest) compared to prebiotics and control. The study is the first to report the

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absence of differences in sustaining the efficacies attained after intake of β-glucan, GOS

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and MOS upon post-feeding with an unsupplemented feed, over a prolonged period.

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aquaculture systems has accelarated the outbreak of diseases that are responsible for

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huge fish losses (Bondad-Reantaso et al., 2005). Intensified aquaculture of the Asian

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snakehead, Channa striata (Bloch, 1793) is faced with similar problems associated with

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the deterioration of water quality and diseases outbreak (Dina, 2013; FAO, 2012; Sinh

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and Pomeroy, 2010). The bottom-living habitat of snakehead exacerbates the situation

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since the bottom region boggy water (Sahoo et al., 2012) zone carries 10-20 time higher

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bacterial population compared to the other water column (Lewis and Bender, 1961).

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significant (P<0.05)

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Introduction: The increasing intensification and commercialization of

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ACCEPTED MANUSCRIPT Aeromonas hydrophila is an opportunistic pathogenic bacteria thrives in this habitat and

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produces endotoxins and haemolysins (Pikul, 2009 and Rigney et al., 1978, Pathiratne

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et al., 2007) causing epizootic ulcerative syndrome (EUS) culminating in severe

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ulcerations and mortality (Sahoo et al., 2012). The fish diseases of C.striata are usually

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managed using antibiotics (like other fish species) which have led to antimicrobial

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resistant pathogens, reduction in beneficial microbiota in the gastrointestinal (GI)

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ecosystem, including the accumulation of residual antibiotics in fish muscle making it

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unsuitable for human consumption. Further, these treatments are not suitable for the

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management of parental disease (Sinh and Pomeroy, 2010, Haniffa.and Marimuthu,

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2004) except as nutritional therapy. Supplementation of dietary prebiotics (Gibson and

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Roberfroid, 1995) and probiotics (Fuller, 1989) with fish diet might be a potential

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nutritional therapy that are used as alternatives (Denev, 2008) to overcome the problems

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associated with antibiotics. Among the positive effects of these supplements are the

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enhancement of growth performance, high nutrient protein digestibility, high digestive

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enzymes activities and high expression of immune regulatory genes (Munir et al.,

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2016). Both supplements have led to direct beneficial effects of the hosts in terms of

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growth by improving intestinal microbial balance (Al-Dohail et al., 2009, Dhanaraj et

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al., 2010). Dietary prebiotics and probiotics has also been shown to enhance the quality

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of the haematological and immunological blood parameters of snakehead (Talpur et al.,

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2014). To date, there is no information on the duration of effectiveness of prebiotics and

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probiotics for a period of post-feeding without any supplementation. Hence this study

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evaluated directly the influence of pre- and probiotic feed supplements on blood and

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immunological parameters of Channa striata fingerlings and the duration of their

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effectiveness for a period of post-feeding without any supplementation.

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Methodology:

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2.1

Experimental fish and husbandry conditions: This study was conducted at

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Universiti Sains Malaysia (USM) Aquaculture Research Complex using a stock of

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10,000 healthy snakehead fries (1.5 cm) obtained from a local fish farm, and raised on

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Artemia cysts and tubiflex worms till they achieved 3 cm in length. A total of 4,800

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pieces of Channa striata fingerlings (av. wt. 22.40g + 0.06) were selected from this

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stock and distributed equally (400 fish/ tank) into 12 outdoor cement tanks (2m x 1m x

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0.5m). The fish were maintained with a natural photoperiod where the mean water

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temperature, pH and DO were 27.54°C±0.30, 7.1±0.08 and 6.1±0.18 mg/l respectively.

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The health status of fish were routinely assessed based on movement and response to

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take food.

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protein and 12% lipid were prepared (Table 1) containing three prebiotics, two

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probiotics and a control diet (no supplements), respectively. The fish were fed the

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experimental diets in two phases. Phase 1 involved feeding for 16 weeks to determine

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the effect on health status of Channa striata fingerlings while in Phase 2, feeding was

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switched to the unsupplemented control diet for 8 weeks to evaluate the efficacy of

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prebiotics and probiotic intake in Phase 1.

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+ 0.06) was randomly collected before the feeding trial and distributed into five groups

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with equal numbers of fingerlings. Each group contained 3 replicates with 10

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fingerlings in each replicate using glass aquarias (L 60 cm x W 35 cm x H 30 cm). The

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first groups which served as the control, was injected with physiological saline (Koch,

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1994). The following groups were injected with different doses (accurate 1ml of 2 x

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Experimental diets and feeding trial: Six experimental diets contained 40%

Pathogenicity test: A total of 150 Channa striata fingerlings (av. wt. 22.40g

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ACCEPTED MANUSCRIPT 102, 2 x 104, 2 x 106, 2 x 108 CFU / ml) of Aeromonas hydrophila gr-2 obtained from the

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National Fish Health Research Centre (NaFish), Penang, Malaysia, under the sterile

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conditions (Al-Dohail, 2010). The Aeromonas hydrophila gr-2 was verified according

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to Koch (1994) prior to its use. The infected and non-infected (control) fingerlings were

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fed to satiation twice daily with a commercial sea bass pellet feeds contained 43% crude

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protein and 6% crude lipid. Mortality was monitored and recorded twice daily for 2

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weeks.

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weeks) throughout the study using the results of the pathogenicity test determined

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earlier. Thirty fishes were randomly collected from each feeding treatment at the pre-

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determined time and injected with the selected dose of Aeromonas hydrophila.

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blood parameters were determined at the end of the first and second week after injecting

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the pathogenic bacteria. The mortality was recorded daily; and the survival rate of fish

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were recorded as cumulative after 1st and 2nd week using following formula (Talpur et

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al., 2014).

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Survival Rate (%): (Initial stock nos.-Cumulative nos. of death fish/ 30) x 100

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Challenge assay: The challenge assay was performed three times (at 8, 16 nd 24

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Blood collection: The blood samples from pre-challenged and post-challenged

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fingerlings groups were collected from the caudal vein using 1-cc sterile syringe.

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2.6.1

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(WBC) were counted by direct method of Natt-Henrik (Campbel, 1995; Al-Dohail et

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al., 2009).

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Haematological Parameters: Blood cell count: Total red blood cells (RBC) and total white blood cells

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Number of RBC cells per mm3= Number of cells in 5 squares x 5 x 10 x 200

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Number of WBC cells per mm3 = Number of cells in 4 mm2 squares x 500

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2.6.2

Erythrocyte Sedimentation Rate (ESR): The method of Wintrobe (Sinton,

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1948 and Terry, 1950) was used to measure the Erythrocyte Sedimentation Rate (ESR).

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2.6.3

Packed Cell Volume (PCV) or Haematocrit: Packed Cell Volume (PCV) was

measured using Microhematocrit method described by the NCCLS Global Consensus

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Standard.

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PCV (%)= (Height of packed red cells / Total height of packed cells+plasma) x 100 2.6.4

Haemoglobin (Hb) Concentration and status: The Cyanmethaemoglobin

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Method was used (Blaxhall & Daisley, 1973; Schäperclaus et al., 1992).

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Hb (g/ dl)= Absorbance of sample/ Absorbance of standard x Concentration of Standard

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The haemoglobin status was determined by calculating the mean corpuscular

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haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH) and mean

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corpuscular volume (MCV).

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MCHC (g/ dl)= (Haemoglobin Concentration / Haematocrit Volume) x 100

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MCH (pg/ cell)= (Haemoglobin Concentration/ Erythrocyte number) x 10

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MCV (µm3)= (Hematocrit Volume/ Erythrocyte Number) x 10

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2.7

Immunological Parameters:

2.7.1

Total Immunoglobulin Concentration: The method described by Siwicki and

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Anderson in 1993 and Amar et al., in 2000 was used.

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Total Ig (mg/ml)= Total Protein in Serum Sample-Total Protein Treated with PEG;

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where the total protein was determined using the Biuret and Lowry procedure modified

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by Ohnishi and Barr in 1978.

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Protein (mg/ml): (Absorbance of sample/ Absorbance of standard) x Conc. of Standard

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2.7.2

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et al., (2011), with some modification was used.

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Serum Lysozyme: Turbidimetric method described by Salo et al., (2007), Wang

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Statistical Analysis: One-way ANOVA (multiple comparisons) was used to

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analyze the data at P<0.05 confidence level. Two-way ANOVA was performed to

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analyze the influence of feed and duration, and the interaction between these factors.

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3.0

Results:

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3.1

Pathogenicity Test: During the pathogenicity test to determine lethal dose,

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mortalities began on the first day after infection with Aeromonas hydrophila for the 2 x

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108 CFU dosage resulting in the significantly lowest survival (16.67%) of Channa

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striata fingerlings. Mortalities began for the 2 x 104 and 2 x 106 CFU dosage after the

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fourth day of infection with survivals at 73.33% and 60%, respectively, after 14 days.

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The dose of 2 x 106 CFU was selected because of obtaining enough fish survival for the

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subsequent challenge studies.

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3.1.1

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pre-challenged period (for Phases 1 and 2), fish fed with all the dietary prebiotics and

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probiotics tested had a significant influence (P<0.05) on haemalogical parameters

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compared to fish maintained on the control diet, regardless of feeding duration or

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feeding regime (Table 2). Generally fish maintained on the probiotic diets performed

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better compared to those on prebiotic diets throughout Phases 1 and 2. Fish fed with

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LBA supplemented feed resulted in the highest significant (P<0.05) RBC at 8 and 16

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weeks in Phase 1 and this trend continued until the end of Phase 2. This was followed

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by live yeast, which was significantly highest at the 8th week of Phase 1 compared to β-

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glucan and MOS treatmented fish, but RBC value for the live yeast treatment reached

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similar levels to that of the β-glucan and MOS treatments by the end of Phase 1.

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However, when fish were fed the control diet in the Phase 2, RBC level of in the β-

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glucan and MOS treatments were significantly lower than the live yeast. This trend was

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Haematological Parameters during pre-& post-challenged period: In the

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ACCEPTED MANUSCRIPT also similar for the packed cell volume (PCV). In the case of hemoglobin content,

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intake of live yeast continued to be significantly higher at both 8 and 16 weeks and the

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end of Phase 2. The ESR values were tended to be higher in the control treatment at all

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time intervals tested compared to all supplemented diets. Similarly, fish fed with

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probiotics and prebiotics maintained significantly higher WBC content compared to the

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control throughout the study. Among the supplemented treatments, no significant

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differences were observed at 8 weeks of Phase 1 and at the end of Phase 2; differences

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were detected at week 16 of Phase 1 in which values between LBA, live yeast and beta

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glucan were not significantly different (Table 2). The mean corpuscular haemoglobin

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concentration (MCHC), mean corpuscular haemoglobin (MCH) and mean corpuscular

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volume (MCV) were significantly (P<0.05) higher in all supplemented diets compared

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to control (Figure 1).

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The data of haematological parameters of one week post infection with 2 x 106 CFU of

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Aeromonas hydrophila are given in Table 3. Generally after 1-week of infection,

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supplementation with probiotics and prebiotics resulted in better blood parameters

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compared to fish maintained on the control diet, with the probiotic based diets

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performing significantly (P<0.05) better than the prebiotic diets (Table 3). A similar

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trend was found for the haematological parameters recorded 2 weeks after injection

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(Table 4). The MCHC, MCH and MCV values of the treated and control fishes in

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Phases 1 and 2) were significantly (P<0.05) lower compared to fish in the pre-

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challenged period. The three red blood cell indices of LBA treated fishes in both Phases

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showed significant highest performance during the post-challenged period, followed by

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live yeast, β-glucan, MOS and GOS (Figure 2).

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ACCEPTED MANUSCRIPT 3.3

Immunological blood parameters during pre-& post challenged period:

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3.3.1

Total Immunoglobulin Content (Ig): Compared with the control, there was a

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significantly higher level of total immunoglobulin in the groups fed the supplemented

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diets. Prolonged intake of dietary prebiotics and probiotics and it was significantly

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(P<0.05) higher over a prolonged use of dietary prebiotics and probiotics. Fish fed with

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LBA supplemented diet produced significantly (P<0.05) highest total immunoglobulin,

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followed by live yeast, β-glucan, MOS and GOS throughout the study in the pre-

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challenged period. Among the prebiotics supplemented treatments, no significant

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differences were observed at the end Phase 1; but the trend was not consistent at the end

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of Phase 2 in the pre-challenged fish. The response of total immunoglobulin was

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significantly (P<0.05) increased in the post-challenged period (Figure 3).

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3.3.2

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probiotics and prebiotics was significantly (P<0.05) higher over control treated fishes

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(Figure 4) which were almost similar to the result of total immunoglobulin during pre

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and post challenged periods.

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3.5

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against the pathogenic bacteria, Aeromonas hydrophila was relatively increased by the

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inclusion of dietary prebiotics and probiotics (Figure 5).

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3.6

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time had an strong effect on haematological and immunological blood parameters as

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well as the enhancement of disease resistance (Table 5). A similar trend was observed

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after 1st and 2nd week of infection with Aeromonas hydrophila. After 1st week of

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infection, time had an strong effect on more blood parameters compared to diets (Table

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6) while the effect of diets and time were slightly changed (Table 7) after 2nd week of

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The Lysozyme Activities: The lysozyme activities in fish groups fed with

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Evaluation of Resistance to pathogenic bacteria infection: The resistance

Factorial Analysis: The two way ANOVA result confirmed that both diet and

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infection. The effect of interaction between feed and time were significantly (P<0.05)

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affected during pre and post challenged period (Table 5, Table 6 and Table 7).

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4.0

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of dietary prebiotics and probiotics significantly enhanced the health status of Channa

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striata fingerlings probable due to a healthier digestive system and increased digestive

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enzyme activities (Munir et al., 2016). Dietary prebiotics and probiotics might have an

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ability to modify the structure and function of the gastrointestinal (GI) tract in the fish

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(Akter et al., 2015; Jian et al. 2012; Carly et al., 2010, Ringø et al., 2007) which usually

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accelerates a healthy digestive system resulting in the optimization of nutrient

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absorption and allowing the efficient passive transfer of nutrients to the blood (John et

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al., 2008). The values of the haematological and immunological blood parameters over

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the 8 weeks of the feeding trial in decreasing order was

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glucan>MOS>GOS>control, whereby MOS treated fish were better than GOS

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compared to a previous study conducted by Talpur et al. (2014) probable due to the

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increment of the MOS (0.5%) dose used. This trend mostly improved over the 16 weeks

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of feeding treatements for all the dietary prebiotics and probiotics tested and it was as

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LBA>live yeast>β-glucan>MOS >GOS>control, which indicated that there were no

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significant differences in efficacy on haematological parameters among the three

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prebiotics tested in the study for prolonged period. But these changes were not

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consistent at the end of Phase 2, when the treated fishes were switched to their

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respective unsupplemented diets.

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Generally, dietary prebiotics and probiotics are active biogenic compounds (Kapka-

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Skrzypczak et al., 2012) having attributes that improve health (Novak and Vetvicka,

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2009). These supplements may enhance the digestion activities (Farnworth, 2008),

LBA>L.yeast>β-

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Discussion: The results of the present study demonstrated that supplementation

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ACCEPTED MANUSCRIPT stimulate the production of blood cells including red blood cells (RBC), white blood

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cells (WBC) and the platelets (Mellors, 2002). Although the present study did not

219

measure platelets, the evaluation of RBC and WBC clearly supported Mellors (2002)

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observation as did the results of PCV, Hb Concentration, MCHC, MCH and MCV. The

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study found that Lactibacillus acidophilus supplementation had the greatest effect

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compared to the controls, similar to the study conducted by Carnevali et al., (2006) and

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Rollo

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acidophilus probiotics and live yeast (Saccharomyces cerevisiae) to fish diet led to

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improve the haematological and immunological parameters in African cat fish, Clarias

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garepinus (Al-Dohail et al., 2009) and Cyprinus carpio (Dhanaraj et al., 2010)

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respectively. The changes to haematological parameters that were observed correlated

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with increased resistance to bacterial infection measured with challenge experiments,

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simlar to what was observed by De Pedro et al. (2005).

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Although the immunostimulant characteristic of dietary prebiotics and probiotics has

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not been shown in fish, human studies have the ability of these supplements to develop

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the blood cells particularly RBC and WBC. The present study also found similarities

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with previous studies on channel catfish in which β-Glucan intake increased RBC

234

values (Duncan and Klesius, 1996) while Cyprinus carpio fish fed the similar prebiotic,

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increased the WBC (Selvaraj et al., 2005). In addition, these feed supplements also

236

modified the haematocrit or PCV (%), erythrocyte sedimentation rate (ESR),

237

haemoglobin concentration, which helps to prevent anemia. Moreover, the MCHC,

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accompanied by MCV test help to evaluate the anemia in living organisms. MCHC is

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the amount of haemoglobin in eash red blood cell. The optimum MCHC is 33%; below

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28% is usually caused for hypochromic anemia. Consequently, when treated fish were

al.,(2006)

on

sea

bream

fish.

The

inclusion

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ACCEPTED MANUSCRIPT injected the pathogenic bacteria of A. hydrophila, it can reduce the frontline

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haematological parameters. Because these pathogenic bacteria produce heat-labile

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enterotoxins incorporated with haemolysin and cytotoxin production (Burke et.al.,

244

1981). The present study observed the similar occurrence.

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Except for the significant increase in WBC values, other basic haematological

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parameters were reduced during post challenged period, these values were not

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significantly (P<0.05) lower than control. Thus suggesting that Channa striata fed with

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supplemented diets responded to infection by developing the WBC making it the first

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line of defense

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Ikhwanuddin (2013). The serum protein content in all feeding regimes decreased

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probable due to infection by the pathogenic bacteria but fish fed with probiotics

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contained the higher serum protein than the fish fed with prebiotics and control,

253

indicating that probiotics are more efficient in boosting the immunological parameters

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of immunoglobulin (Siwicki, 1989). Indeed, immunoglobulin (mg/ml) and lysozyme

255

(U/ml) levels of the fish fed with dietary prebiotics and probiotics increased

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significantly compared to control. Dietary prebiotics and probiotics can modify these

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two immunological blood paramenters and prepare the fish more stronger (immune

258

exclusion, immune elimination and immune regulation) against the pathogenic bacteria

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(Antonio et al., 2010). This statement is also supported by the previous studies of

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Newaj-Fyzul (2007) on rainbow trout; Chiu et al., (2010) on Paralichthys olivaceus,

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Harikrishnan et al., (2010) on Anabas testudineous fish fed with β-glucan being

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challenged with fungus parasite (Das et al., 2013), on Asian cat fish fed with β-glucan

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being challenged with A.hydrophila (Kumari and Sahoo, 2006), on African Catfish fed

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on L. acidophilus being challenged with S.xylosus, A. hydrophila and S. agalactiae (Al-

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against the pathogenic bacteria, as suggested by Talpur and

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ACCEPTED MANUSCRIPT Dohail, 2010). The enhancement of first line defence (WBC number increased by

266

inclusion of pathogenic bacteria), increment of serum immuglobulin and lysozyme

267

activity of the current study have led to increase the survivavility of fish against

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bacterial infection of Channa straita fingerlings.

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5.0

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203/PBIOLOGI/6711308) as well as the USM Global Fellowship for the financial

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support to conduct the research. Special thanks goes to FRI Pulau Sayak, Kedah for

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proving the facility of experimental diets preparation, AllTech(R) for providing free of

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cost (for research) of the Bioactin and Yaa-Sac, as well as to similar to

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FriedlandCampina Domo(R) for Vivinal GOS Syrup and Bio-Origin for Macroguard(R)

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β-glucan.

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6.0

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Al-Dohail M.A.S., 2010. Effects of the probiotics, Lactobacillus acidophilus, on Pathogenic Bacteria, Growth, Haematological Parameters and Histopathology of African Catfish. Clarias gariepinus. A Ph.D Research under School of Biological Sciences, Universiti Sains Malaysia (USM), Penang, Malaysia.

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Al-Dohail, M.A.. Hashim, R., Aliyu_Paiko, M., 2009. Effects of the probiotics, Lactobacillus acidophilus, on the growth performance, haematology parameters and immunoglobulin concentration in African Catfish (Clarias gariepinus, Burchell 1822) fingerling. Aquaculture Research 40. 1542-1652.

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Akter N. Mst., Sutriana A., Talpur A.D., Hashim R., 2015. Dietary supplementation with mannan oligosaccharide influences growth, digestive enzymes, gut morphology, and microbiota in juvenile striped catfish, Pangasianodon hypophthalmus. Aquaculture International DOI 10.1007/s10499-015-9913-8.

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Amar E.C., Kiron V., Satoh S., Okamoto N. & Watanabe T., 2000. E!ect of dietary hcarotene on the immune response of rainbow trout Oncorhynchus mykiss. Fisheries Science 66,1068-1075.

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Antonio A.Z., Giovanni A., and Antonio S., 2010. Prebiotics and Probiotics in Infant Nutrition. In: Bioactive Foods in Promoting Health: Probiotics and Prebiotics. Elsevier Inc. 441-477.

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Acknowledgement: The authors would like to express the thanks to FRGS (Ref:

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De Pedro, N., Guijarro, A.E., Lopez-Patino, M.A., Marinez-Alvarez, R., Delgado, M., 2005. Daily and seasonal variations in haematological and blood biochemical parameters in the tench, Tinca tinca. Aquaculture Research 36, 85–96.

325 326 327 328

Denev SA., 2008. Ecological Alternatives of Antibiotic Growth Promoters in the Animal Husbandary and Aquaculture. DSc. Thesis. Department of Biotechemistry Microbiology. Trakia University Stara Zagora. Bulgaria. pp 294.

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ACCEPTED MANUSCRIPT Dina Muthmainnah, (2013). Growout of Striped Snakehead (Channa Striata) in Swamp Water System Using Fences and Cages. 2013 4th International Conference on Biology, Environment and Chemistry. IPCBEE vol.58. 11: 52-55.

332 333 334 335

Dhanaraj, M., Haniffa, M.A., Arun Singh, S.V., Jesu Arochiaraj, A., Muthu Ramakrishanan, C., Seetharaman, S., Arthimanju, R., 2010. Effect of probiotics on growth performance of koi carp (Cyprinus carpio). J. Appl. Aquac. 22. 202-209.

336 337 338

Duncan, P.L., Klesius, P.H., 1996. Dietary immunostimulants enhance nonspecific immune responses in channel catfish but not resistance to Edwardsiella ictaluri. J.Aquat. Anim. Health 8, 241–248.

339 340

FAO. Food and Agriculture Organization, 2012. The State of World Fisheries and Aquaculture 2014. Rome. 223 pp.

341 342

Farnworth E.R., 2008. The evidence to support health claims. Journal of Nutrition. 138. 1250S-1254S.

343 344

Fuller, R.,1989. Probiotics in man and animals. Journal of Applied Bacteriology 66, 365-378.

345 346

Gibsen G.R. and M.B. Roberfroid, 1995. Dietary Modulation of the Human Colonic Microbiota. In Introducing the Concept of Prebiotics.

347 348

Haniffa.M.A., Marimuthu.K., 2004. Seed Production and Culture of snakehead. INFOFISH International 2 , 16-18.

349 350 351 352

Harikrishnan, R., Balasundaram, C., Heo, M.S., 2010. Lactobacillus sakei BK19 enriched diet enhances the immunity status and disease resistance to Streptococcosis infection in kelp grouper, Epinephelus bruneus. Fish Shellfish Immunology 29, 1037–1043.

353 354 355 356

Jian Z., Yongjian L., Lixia T., Huijun Y., Guiying L., Donghui X., 2012. Effects of dietary mannan oligosaccharide on growth performance, gut morphology and stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei. Fish and Shell Fish Immunology 33. 1027-1032.

357 358

John S., Arkadios D., Simon D. and Silvia T., 2008. Nutrient Uptake. Gut morphologya key to efficient nutrition. International Aquafeed: 26-30.

359 360 361

Kapka-Skrzypczak L, Niedźwiecka J, Wojtyła A, Kruszewski M., 2012. Probiotics and prebiotics as a bioactive component of functional food (Article in Polish). Pediatr Endocrinol Diabetes Metabolism 2012;18(2):79-83.

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329 330 331

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ACCEPTED MANUSCRIPT Koch, A., 1994. Growth measurement. In: P. Gerhardt. R. Murray, W. Wood, N.Krieg (eds.). Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C., p. 249-277.

365 366 367

Kumari, J., Sahoo, P.K., 2006. Dietary beta-1,3 glucan potentiates innate immunity and disease resistance of Asian catfish, Clarias batrachus (L.). Journal of Fish Disease 29, 95–101.

368 369 370

Lewis, W.M., Bender, M., 1961. Free-living ability of a warm-water fish pathogen of the genus Aeromonas and factors contributing to its infection of the golden shiner. Program Fish Culture 23, 124–126.

371 372

Mellors, R. C., 2002. Immunopathology; hypersensitivity reactions. Retrieved May 8, 2003, from http://www.med.cornell.edu/education.

373 374 375 376

Munir, M.B., Roshada, H., Yam, H. C., Terence, L. M., Azizah, S., 2016. Dietary prebiotics and probiotics influence growth performance, nutrient digestibility and the expression of immune regulatory genes in snakehead (Channa striata) fingerlings. Aquaculture. Volume 460, 1 July 2016, Pages 59–68.

377 378 379

Newaj-Fyzul, A., Adesiyun, A.A., Mutani, A., Ramsubhag, A., Brunt, J., Austin, B., 2007. Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum). J. Appl. Microbiol. 103, 1699–1706.

380 381 382 383

NCCLS. Procedure for Determining Packed Cell Volume by the Microhematocrit Method: Approved Standard- Third Edition. Vol. 20. No 18. NCCLS document H7-A3 (ISBN 1-56238-413-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA 2000.

384 385

Novak, M. And Vetvicka, V., 2009. Glucans as Biological Response Modifiers. Endocrine, Metabolic and Immune Disorders- Drag Target.9: 67-75.

386 387

Ohnishi, S.T., and Barr, J.K., 1978. A simplified method of quantitating proteins using the biuret and phenol reagents. In Analysis Biochemistry, 86, 193 (1978).

388 389 390 391

Pathiratne, A., Widanapathirana, G. S. And Chandrakanthi, W. H. S. , 2007. Association of Aeromonas hydrophila with epizootic ulcerative syndrome (EUS) of freshwater fish in Sri Lanka. Journal of Applied Ichthyology. Volume 10, Issue 2-3, pp 204–208, October 1994.

392 393 394

Pikul, J., & et al., 2009. A highly virulent pathogen, Aeromonas hydrophilia from fresh water crayfish Pacifastacus leniusculus. Journal of Invertebrate Pathology., 101(1), 5666.

395 396

Rigney, M. M., Zilinsky, J. W., & Rouf, M. A., 1978. Pathogenicity of Aeromonas hydrophila in Red Leg Disease in Frogs. Current Microbiology, 1, 175179.

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362 363 364

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ACCEPTED MANUSCRIPT Ringo E, Myklebust R, Mayhew TM and Olsen RE. 2007. Bacterial translocation and pathogenesis in the digestive tract of larvae and fry. Aquaculture, 268, 251264.

400 401 402

Rollo, A., Sulpizio, R., Nardi, M., et al., 2006. Live Microbial Feed Supplement in Aquaculture for Improvement of Stress Tolerance. Fish Physiological Biochemistry 32: 167-177.

403 404

Schäperclaus, W., Kulow., H. And Schreckenbach, K., 1992. Fish Disease (5th Ed.) Volume 1. 594 pp. Published by A. A. Balkema/ Protterdam.

405 406 407 408

Selvaraj, V., Sampath, K., Sekar, V., 2005. Administration of yeast glucan enhances survival and some non-specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila. Fish Shellfish Immunology 19, 293–306.

409 410 411

Sahoo, P.K., Mohanty, J., Das, P.C., Mohanta, K.N., Barik, N.K., Jayasankar, P., 2012. CIFA: 25 Years of Freshwater Aquaculture Research. Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India. 195 pp.

412 413 414

Salo HM, Hebert N, Dautremepuits C, Cejka P, Cyr DG, Fournier M., 2007. Effects of montreal municipal sewage effluents on immune responses of juvenile female rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology 84:406–414.

415 416 417 418

Selvaraj, V., Sampath, K., Sekar, V., 2005. Administration of yeast glucan enhances survival and some non-specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila. Fish Shellfish Immunology 19, 293–306.

419 420 421

Sinh, L.X. and Pomeroy, P.S., (2010). Current situation and challenges for the farming of Snakehead fish (Channa micropeltes and Channa striata) in the Mekong Delta, Vietnam Aquaculture Asia Volume XV No. 4: pp 11-18.

422 423

Sinton JR. Clinical value of some methods of estimating erythrocyte sedimentation rate. Br Medical Journal 1948 Feb 28;1(4547):391–393.

424 425 426

Siwicki, A.K., 1989. Immunostimulating influence of levamisole on nonspecific immunity in carp (Cyprinus carpio). Developmental and Comparative Immunology 13, 87–91.

427 428 429 430 431

Siwicki A.K. & Anderson D.P.,1993. Non-Specific Defence Mechanisms Assay in Fish: II. Potential Killing Activity of Neutrophils and Macrophages, Lysozyme Activity in Serum and Organs and Total Immunoglobulin Level in Serum. Disease Diagnosis and Prevention Methods, pp. 105-12. FAO-Project GCP/INT/JPA, IFI, Olsztyn, Poland.

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ACCEPTED MANUSCRIPT Talpur A.D., Mohammad Bodrul Munir, Anna Marry and Roshada Hashim, 2014. Dietary probiotics and prebiotics improved food acceptability, growth performance, haematology and immunological parameters and disease resistance against Aeromonas hydrophila in snakehead (Channa striata) fingerlings. Aquaculture journal 426-427: 14-20.

437 438 439 440

Talpur, A.D., Ikhwanuddin, M., 2013. Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass, Lates calcarifer challenged with Vibrio harveyi. Fish Shellfish Immunology 34, 254– 264.

441 442

Terry R. Erythrocyte sedimentation in anaemia. Br Medical Journal 1950 Dec 9;2(4692):1296–1299.

443 444 445 446

Wang GX, Wang Y, Wu ZF, Jiang HF, Dong RQ, Li FY, Liu XL., 2011. Immunomodulatory effects of secondary metabolites from thermophilic Anoxybacillus kamchatkensis XA-1 on carp, Cyprinus carpio. Fish Shellfish Immunology 30:1331–1338

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EP

TE D

M AN U

SC

RI PT

432 433 434 435 436

17

Control 534 340 5 60 11 10 20 20 0

β- Ga 0.2% 534 340 5 60 9 10 20 20 2

GOSb 1% 534 340 5 60 1 10 20 20 10

MOSc 0.5% 534 340 5 60 6 10 20 20 5

M AN U

Ingredients Danish Fish Mealf Korean Corn Starch Fish oil Soyabean oil Cellulose CMCg Vitamins mixh Minerals mixi Supplement

SC

Table 1: Feed Ingredients and Proximate Composition of the Formulated Diet (g/ kg, dry matter)

RI PT

ACCEPTED MANUSCRIPT

L.acidophiluse 0.01% 534 340 5 60 10.9 10 20 20 0.1

β-G= Beta-Glucan from Macrogard(R) GOS= Galactooligosaccharides from Vivinal(R) GOS syrup, Friesland Campina Domo, Netherland c MOS= Mannan-oligosacchafrides from Alltech(R), Actigen 1, USA d S.cerevisae = Saccharomyces cerevisiae or Live yeast from Alltech(R), YEA-SACC 1026, USA e L.acidophilus = Lactobacillus acidophilus powder from International Laboratories, USA f Danish Fish Meal Kg-1 =Crude Protein 746.6 and Crude Lipid 101.6 g CMC= Carboxymethyl Cellulose h Vitamin Mix Kg-1 = Rovimix 6288, Roche Vitamins Ltd. Switzeland; Vit A 50 million i.u., Vit D 310 million i.u., Vit E 130 g, Vit B1 10g, Vit B2 25g, Vit B6 16g, Vit B12 100 mg, Biotin 500mg, Pantothenic acid 56g, Folic Acid 8g, Niacin 200g, Anticake 20g, Antioxident 200mg, Vit K3 10g and Vit C 35g i Vitamin Mix Kg-1 = Calcium phosphate (monobasic) 397.65g, Calcium lactate 327g, Ferous sulphate 25g, Magnessium sulphate 137g, Potassium chloride 50g, Sodium chloride 60gm, Potassium iodide 150mg, Copper sulphate 780mg, Manganese oxide 800mg, Cobalt carbonate 100mg, Zinc oxide 1.5g and Sodium selenite 20g.

EP

AC C

b

TE D

a

S.cerevisaed 1% 534 340 5 60 1 10 20 20 10

ACCEPTED MANUSCRIPT

Phase 1_16W*

4.03+0.02a

4.35+0.02c

4.25+0.02b

Phase 2_8W**

3.95+0.03a

4.33+0.02d

4.15+0.04b

Phase 1_8W*

37.04+0.41a

42.03+0.40d

40.00+0.31b

Phase 1_16W*

40.52+0.77a

46.76+0.55c

Phase 2_8W**

37.79+0.15a

Phase 1_8W*

RI PT

3.88+0.03b

LBA

3.94+0.03c

4.03+0.05d

4.08+0.03e

4.35+0.01c

4.37+0.2c

4.43+0.02d

4.29+0.02c

4.36+0.02e

4.42+0.01f

41.21+0.36c

43.18+0.50e

44.22+0.30f

45.63+1.23b

46.65+0.62c

47.05+1.02c

48.16+0.39d

44.38+0.13d

41.10+0.23b

43.62+0.30c

45.04+0.06e

46.57+0.36f

9.26+0.10a

12.34+0.33d

10.64+0.09b

11.23+0.16c

13.14+0.15e

14.35+0.13f

Phase 1_16W*

9.38+0.11a

14.20+0.08c

13.52+0.31b

14.07+0.21c

15.39+0.21d

15.84+0.11e

Phase 2_8W**

9.26+0.17a

13.64+0.10d

11.32+0.12b

12.47+0.06c

14.19+0.07e

15.03+0.10f

Phase 1_8W*

0.55+0.04c

0.40+0.03ab

0.41+0.04b

0.40+0.02ab

0.38+0.02ab

0.37+0.02a

Phase 1_16W*

0.46+0.01d

0.36+0.01c

0.37+0.01c

0.36+0.01c

0.35+0.01b

0.32+0.01a

Phase 2_8W**

0.49+0.01e

0.38+0.01bc

0.40+0.01d

0.39+0.01c

0.37+0.01b

0.34+0.01a

Phase 1_8W*

22.67+0.61a

24.50+1.52b

24.17+0.82b

24.17+1.08b

24.58+0.20b

25.08+0.97b

Phase 1_16W*

23.17+1.13a

25.67+1.94bc

24.75+0.99b

25.33+0.82b

26.08+0.80bc

27.08+0.55c

Phase 2_8W**

22.92+1.11a

24.75+0.52b

24.67+1.25b

24.67+0.88b

24.75+0.88b

25.58+1.16b

TE D

M AN U

SC

3.96+0.04c

AC C

EP

(106 mm-3) (10 mm )

PCV

(%) (g dl ) (mm h )

-1

Hb

-3

ESR

3.75+0.05a

3

WBC

Phase 1_8W*

-1

RBC

Table 2: Mean (+SD, n=6) haematological parameters of Channa striata fingerlings fed a single dose of supplemented diets and a control Parameters Fish Group Control β-Glucan GOS MOS Live Yeast

*Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each column represents different feeding trial and row represents the time. Superscripts in each row represent significant differences among the treatement tested.

ACCEPTED MANUSCRIPT

β-Glucan

Control a

d

GOS

RI PT

Fish Group

MOS

b

c

Live Yeast

LBA

2.75+0.020

e

3.27+0.023

3.40+0.041f

3.45+0.018b

3.70+0.052c

4.03+0.130d

2.58+0.015

3.17+0.021

2.66+0.035

Phase 1_16W*

3.09+0.051a

3.49+0.010b

3.44+0.012b

Phase 2_8W**

3.58+0.024a

4.12+0.027c

3.86+0.032b

3.91+0.023b

4.20+0.032d

4.39+0.112e

Phase 1_8W*

25.33+0.159a

33.49+0.232d

27.33+0.373b

28.46+0.251c

34.74+0.204e

36.51+0.420f

Phase 1_16W*

30.31+1.685a

40.77+1.287c

36.67+0.628b

36.76+1.053b

43.33+0.628d

47.95+1.799e

Phase 2_8W**

32.28+0.063a

40.26+0.318c

35.77+0.421b

36.31+0.238b

41.32+0.350d

44.69+1.145e

Phase 1_8W*

5.38+0.273a

7.91+0.187d

6.63+0.137b

7.22+0.192c

9.24+0.120e

10.51+0.107f

Phase 1_16W*

6.98+0.171a

10.66+0.181c

9.56+0.157b

9.59+0.146b

12.36+0.240d

14.10+0.293e

Phase 2_8W**

6.98+0.071a

10.25+0.105d

8.88+0.065b

9.41+0.092c

11.53+0.102e

14.05+0.089f

Phase 1_8W*

0.66+0.028d

0.60+0.035b

0.66+0.025d

0.64+0.018cd

0.62+0.023bc

0.46+0.038a

Phase 1_16W*

0.54+0.011e

0.45+0.011c

0.47+0.009d

0.46+0.015c

0.44+0.010b

0.38+0.014a

Phase 2_8W**

0.51+0.014d

0.45+0.010c

0.45+0.015c

0.44+0.015c

0.41+0.013b

0.38+0.010a

Phase 1_8W*

38.25+2.115b

34.50+1.949a

34.25+2.382a

34.67+0.931a

34.33+0.983a

33.33+1.252a

Phase 1_16W*

37.92+1.744b

26.75+1.475a

27.75+1.440a

27.08+1.530a

26.42+2.940a

26.17+1.941a

Phase 2_8W**

37.58+1.772c

28.67+2.206ab

30.75+1.917b

29.67+2.066ab

28.33+2.317ab

27.75+1.440a

EP

TE D

M AN U

SC

Phase 1_8W*

3

WBC

-3

(10 mm )

ESR (mm h-1)

Hb (g dl-1)

PCV (%)

RBC (106 mm-3)

Parameters

Mean (+SD, n=6) haematological parameters of Channa striata fingerlings fed the experimental diets 1-week post challenged with 2 x 106 CFU of Aeromonas hydrophila

AC C

Table 3:

*Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each column represents different feeding trial and row represents the time. Superscripts in each row represent significant differences among the treatments tested.

ACCEPTED MANUSCRIPT

Fish Group

β-Glucan

Control a

d

GOS

MOS

b

3.30+0.060b

3.34+0.048bc

3.40+0.094c

3.69+0.035c

3.78+0.031d

3.91+0.039e

24.11+0.247b

25.28+0.251c

29.64+0.186e

30.82+0.420f

37.47+0.811c

34.73+1.078b

34.73+0.643b

38.68+0.743cd

39.72+1.341d

23.37+0.498a

35.31+0.594d

32.43+0.319b

33.81+0.106c

36.89+0.886e

39.18+0.397f

Phase 1_8W*

3.96+0.195a

6.34+0.171d

5.83+0.217b

6.09+0.149c

7.25+0.134e

8.37+0.133f

Phase 1_16W*

5.52+0.106a

9.69+0.174d

8.45+0.166b

8.72+0.152c

9.89+0.159d

10.98+0.312e

Phase 2_8W**

4.95+0.256a

8.76+0.125d

6.86+0.105b

7.78+0.154c

9.03+0.137e

9.90+0.134f

Phase 1_8W*

0.69+0.040e

0.60+0.022b

0.66+0.031de

0.65+0.028cd

0.62+0.044bc

0.48+0.018a

Phase 1_16W*

0.64+0.012f

0.53+0.010c

0.57+0.008e

0.55+0.007d

0.50+0.015b

0.45+0.008a

Phase 2_8W**

0.66+0.017d

0.60+0.010c

0.61+0.012c

0.61+0.010c

0.55+0.018b

0.52+0.013a

Phase 1_8W*

58.77+1.943c

41.90+2.807ab

43.50+1.789b

42.83+2.601ab

41.00+3.000ab

40.17+2.183a

Phase 1_16W*

58.50+2.646e

32.25+1.666c

35.00+1.265d

33.67+0.931cd

30.17+0.983b

27.83+2.090a

Phase 2_8W**

58.42+1.158e

37.08+2.084bc

39.17+o.931d

38.25+1.605cd

35.75+1.636b

33.75+1.129a

2.39+0.026

Phase 1_16W*

2.48+0.013a

3.32+0.016b

3.30+0.065b

Phase 2_8W**

2.65+0.099a

3.69+0.019c

3.55+0.044b

Phase 1_8W*

18.84+0.346a

28.74+0.299d

Phase 1_16W*

24.06+1.519a

Phase 2_8W**

M AN U

TE D

EP

2.50+0.024

SC

2.78+0.030

e

LBA 2.87+0.039f

1.97+0.037

c

Live Yeast 2.82+0.018

Phase 1_8W*

AC C

WBC (103 mm-3)

ESR (mm h-1)

Hb (g dl-1)

PCV (%)

RBC (106 mm-3)

Parameters

RI PT

Table 4: Mean (+SD, n=6) haematological parameters of Channa striata fingerlings fed the experimental diets 2-weeks post challenged with 2 x 106 CFU of Aeromonas hydrophila

*Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each column represents different feeding trial and row represents the time. Superscripts in each row represent significant differences among the treatments tested.

ACCEPTED MANUSCRIPT

Factor

PCV

Hb

MCMH

F-Value

461.96

16.62

131.25

479.05

2859.17

605.39

P-value

<0.001 1712.18

<0.001 11.51

<0.001 49.30

<0.001 645.30

<0.001 1254.96

<0.001 58.25

F-Value

<0.001 10.32

<0.001 0.58

<0.001 2.45

<0.001 8.20

<0.001 70.76

P-value

<0.001

0.825

0.012

<0.001

F-Value P-value

Interaction

ESR

<0.001

MCH

MCV

S.Protein

Total Ig

Lysozyme

1558.25

128.34

35320.95

791.75

473.32

<0.001 281.77

<0.001 176.29

<0.001 20521.23

<0.001 1802.85

<0.001 77.67

<0.001 21.89

<0.001 44.07

<0.001 3.42

<0.001 725.39

<0.001 45.41

<0.001 1.76

<0.001

<0.001

0.001

<0.001

<0.001

0.08

SC

Time

WBC

M AN U

Diet

RBC

RI PT

Table 5: Two Way ANOVA analysis showing the F and P values (the mean difference is significant at the p<0.05) depending on diet and time

AC C

EP

TE D

Note: RBC= Red Blood Cell; WBC= White Blood Cell; ESR= Erythrocyte Sedimentation Rate; PCV= Packed Cell Volume; MCMH= Mean Corpuscular Haemoglobin Concentration; MCH= Mean Corpuscular Haemoglobin; MCV= Mean Corpuscular Volume; S.Protein= Serum Protein; Total Ig= Total Immunoglobulin. Individual feeding trial (Diet), Rearing Weeks (Time) and both were influenced significantly (p<0.05) higher except the interaction between feed and time did not influence signifcantly for WBC, Lysozyme (presented in bold).

RI PT

ACCEPTED MANUSCRIPT

Table 6: Two Way ANOVA analysis showing the F and P values (the mean difference is significant at the p<0.05) depending on diet and time after 1st week of infection PCV

Hb

705.51

55.39

133.75

690.32

3320.28

P-value

<0.001 1644.88

<0.001 111.70

<0.001 790.10

<0.001 1155.10

<0.001 2904.01

F-Value

<0.001 21.06

<0.001 4.07

<0.001 11.83

<0.001 12.72

P-value

<0.001

<0.001

<0.001

<0.001

P-value Interaction

ESR

F-Value F-Value

Time

WBC

MCHC

MCH

MCV

S.Protein

Total Ig

Lysozyme

132.64

685.50

142.30

55508.14

1274.33

650.14

<0.001 161.05

<0.001 465.31

<0.001 596.42

<0.001 39980.41

<0.001 4432.68

<0.001 391.51

<0.001 36.36

<0.001 9.97

<0.001 14.85

<0.001 13.91

<0.001 1099.98

<0.001 47.79

<0.001 7.81

<0.001

<0.001

<0.001

0.001

<0.001

<0.001

<0.001

M AN U

Diet

RBC

SC

Factor

Factor F-Value

1261.39

403.84

158.39

P-value

<0.001 5123.61

<0.001 175.51

<0.001 128.43

F-Value

<0.001 33.74

<0.001 7.35

P-value

<0.001

<0.001

F-Value P-value

Interaction

ESR

PCV

Hb

MCHC

MCH

MCV

S.Protein

Total Ig

Lysozyme

393.58

1726.06

65.64

320.30

163.24

61501.43

2377.56

1217.56

<0.001 610.43

<0.001 2017.23

<0.001 49.74

<0.001 572.06

<0.001 575.91

<0.001 27184.78

<0.001 3754.60

<0.001 556.78

<0.001 7.42

<0.001 4.64

<0.001 24.43

<0.001 8.66

<0.001 16.51

<0.001 5.93

<0.001 534.93

<0.001 125.04

<0.001 14.89

<0.001

<0.001

<0.001

<0.001

<0.001

0.001

<0.001

<0.001

<0.001

EP

Time

WBC

AC C

Diet

RBC

TE D

Table 7: Two Way ANOVA analysis showing the F and P values (the mean difference is significant at the p<0.05) depending on diet and time after 2nd week of infection

Note: RBC= Red Blood Cell; WBC= White Blood Cell; ESR= Eruthrocyte Sedimentation Rate; PCV= Packed Cell Volume; MCMH= Mean Corpuscular Haemoglobin Concentration; MCH= Mean Corpuscular Haemoglobin; MCV= Mean Corpuscular Volume; S.Protein= Serum Protein; Total Ig= Total Immunoglobulin.

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Figure 1: Effect of dietary prebiotics and probiotics on red blood cells indices (MCHC, MCH and MCV) in Channa striata fingerlings over different periods in two phases. *Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each value is the mean (+ SD, n=6). Superscripts in represents significant (P<0.05) differences among the treatments tested.

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Figure 2: Status of red blood cells indices (MCMH, MCH and MCV) in Channa striata fingerlings after challenged with Aeromonas hydrophila . *Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each value is the mean (+ SD, n=6). Superscripts represents significant (P<0.05) differences among the treatments tested.

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Figure 3. Effect of dietary prebiotics and probiotics on total immunoglobulin in Channa striata fingerlings over different periods in two phases in pre- and post-challenged with Aeromonas hydrophila *Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each value is the mean (+ SD, n=6). Superscripts represents significant (P<0.05) differences among the treatments tested.

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Figure 4. Effect of dietary prebiotics and probiotics on lysozyme activities in Channa striata fingerlings over different periods in two phases in pre- and post-challenged with Aeromonas hydrophila *Feeding treatments with supplementation and control diet, respectively. **Treated fish fed with a control diet only for the next 8 weeks. Each value is the mean (+ SD, n=6). Superscripts represents significant (P<0.05) differences among the treatments tested.

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Figure 5: Evaluation of survival status in Channa striata fingerings after feeding with dietary prebiotics and probiotics in the post-challeneged period *Feeding Treatments with supplementation and a control. **Treated fish fed with a control for next following 8 weeks. Superscripts represents significant (P<0.05) differences among the treatments tested.

ACCEPTED MANUSCRIPT HIGHLIGHTS: Dietary pre- and probiotics triggered the good health in Channa striata;

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The efficacies of β-glucan, GOS and MOS were NULL over a prolonged period of use;

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Dietary pre- and probiotics demonstrated protective barrier against pathogen;

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Expression of immunological blood parameters upregulated against A.hydrophila.

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