Collection of fish and commercial probiotic
Healthy juveniles of T. tambroides were collected (Kelah farm SDN BHD. Selangor) and transported to Institute of Tropical Aquaculture and Fisheries Research Hatchery, University Malaysia Terengganu. The fish was acclimatized for 1.5 weeks before running the experiments. Optimum protein requirement for T. tambroides fish is 40% and 5% lipid is sufficient for the best survival and growth performance (Misieng et al. 2011; Ng et al. 2008; Ramezani-fard & Kamarudin, 2012).Thus, 40% protein and 7% fat (Kamarudin et al. 2012) containing commercial feed was selected in this present study. The experimental diet was analysed for proximate composition and fatty acid profile before using as basal feed (Table 1 and Table 2).
Table 1. Proximate composition of basal feed used for formulation of experimental diet throughout the feeding trial.
Proximate analysis (%)
|
|
Moisture
|
10.32±1.03
|
Crude protein
|
44.29±0.74
|
Crude lipid
|
8.76±0.576
|
Ash
|
13.34±0.03
|
Gross Energy kJ g-1
|
22.66±0.88
|
Table 2. Fatty acid composition of basal feed used for experimental diets
Fatty acids
|
% Total fatty acids
|
C14:0
|
1.72±0.08
|
C15:0
|
0.80±0.21
|
C16:0
|
24.04±0.35
|
C17:0
|
1.28±0.58
|
C18:0
|
12.28±0.91
|
C20:0
|
0.45±0.19
|
Total SFA
|
40.60±0.98
|
C16:1n-7
|
2.31±0.35
|
C16:1n-6
|
0.32±0.28
|
C18:1n-7
|
2.04±0.11
|
C18:1n-9
|
22.26±0.92
|
C20:1n-9
|
1.10±0.17
|
Total MUFA
|
28.05±1.05
|
C18:2n-6
|
10.34±1.23
|
C18:3n-6
|
1.06±0.77
|
C20:4n-6
|
1.84±0.24
|
C22:3n-6
|
1.19±0.14
|
Total n6
|
14.61±1.88
|
C18:3n-3
|
1.76±0.24
|
C20:5n-3
|
1.24±0.22
|
C22:5n-3
|
0.63±0.43
|
C22:6n-3
|
13.24±1.40
|
Total n3
|
16.90±2.04
|
Total PUFA
|
31.98±2.0
|
PUFA/SFA
|
0.79±0.06
|
n3/n6
|
1.19±0.27
|
DHA/EPA
|
1.184±0.68
|
C22:3n-6
|
1.19±0.14
|
Total n6
|
14.61±1.88
|
C18:2n-6
|
10.34±1.23
|
C18:3n-3
|
1.76±0.24
|
Most available and used commercial probiotics, “Thohira” containing minimum 1.00×106-1.00×108 CFUml-1 Bacillus sp. was selected for this study. Inclusion in basal diet of this commercial probiotic was followed manufacturers manual where 5g Thohira with 1 litre clean water then probiotic dilution was sprayed into dry basal feed (1 L ThohiraTM to 1 kg dry pellet).
Preparation of Probiotics and Experimental Diets
Two isolated HAPs, Aeromonas sp. strain KT27 were cultured in 50 ml tryptic soya broth (TSB) for 24 hrs. and Enterococcus sp. strain KM07 were cultured for 48 to 72 hrs in 50 ml de Man, Rogosa and Sharpe (MRS) broth at room temperature in an incubator shaker at 100 rpm. Then the culture media was centrifuged at 3,000 rpm for 10 minutes, and pellet was harvested and washed thrice in phosphate buffer solution (PBS, pH 7.4), and harvested pellet subsequently resuspended in the same amount of PBS solution. Solution of the probiotic in PBS was sprayed onto the selected dry commercial pellet (floating pellet code 9961, Thai Lux Enterprise Company Ltd, supplied by local supplier in Terengganu, Malaysia). Two HAPs supplemented diets were prepared by spraying the probiotic solution with PBS onto the basal pellet to achieve a final concentration of 1×108 CFU g−1 feed, determined by plate counting on tryptone soya agar media (TSA) at 25 °C for 24 hours. Bottle sprayer was sterilized by using chlorine and 70 % ethanol after every use (Kuebutornye et al. 2020). Thereafter, experimental diets were dried in aseptic condition under laminar flow, weighed, and stored at 4 °C for use in the feeding trial (Amstrong et al. 2016). The experimental HAP diets were stored for maximum 7 days, and fresh diets were prepared weekly. Experimental diets with commercial probiotics were prepared daily following the protocol described in manufacturer guidelines. Viability of probiotics were tested in feed after inclusion of HAPs and CP by applying in water, after 30 minutes (Asaduzzaman et al. 2018a).
Experimental set-up and feeding management
After acclimatization, 360 fish were measured and graded (mean body weight 2.54±0.01g) before randomly distributed into twelve separate 150L capacity fibre glass tanks with a working volume of 120 L water (treated with chlorine and dechlorinated). In a 70-days feeding trial, fish were fed experimental diets supplemented with basal diets as control, HAP Aeromonas sp. A8-29 (T1), HAP Enterococcus sp. strain KM07 (T2), and commercially prepared probiotic ThohiraTM Bacillus sp. (T3), respectively. The fish were fed two times daily at the rate of 2% body weight (9:00 am and 4:30 pm). Unconsumed feed was siphoned out, dried in oven, and taken into account for the accuracy of feed intake weight. Continuous aeration was supplied in every tank by an air stone connected centrally in air compressor. Exchange of water (70%) in every tank was maintained once a week to reduce bacterial growth and ammonia saturation (Andani et al. 2012; Asaduzzaman et al. 2018a). The physicochemical characteristics of tank water, such as dissolved oxygen (DO), pH, temperature, ammonia, alkalinity, nitrate, and nitrite were recorded by using YSI probe (USA) and commercial kit (API water quality test kit) and maintained within optimum level for aquaculture.
Ethical statement
Handling of T. tambroides early juveniles was performed by following the guiding principles for the use and care of laboratory animals were followed in all trials, in addition to Universiti Malaysia Terengganu and European legislation on laboratory animal welfare.
Sampling process
Beginning of the feeding experiment, 12 fish close to mean initial weight (2.54±0.10g) were collected randomly and stored at -800c for initial proximate composition and fatty acid analysis. Triplicate group of experimental diets was stored at -20 0C for proximate composition analysis. At the final period of 70 days feeding trial, 3 fish from each replicant tank were anaesthetized and dissected in aseptically for the collection of mid gut, and muscle tissue. At the same time, 3 fish per replicate were collected and stored at -80 0C for further fatty acid analysis and proximate composition analysis. In addition, a total of 12 healthy and close to mean final weight fish were collected from each tank and transferred to the challenge test laboratory of the Faculty of Fisheries and Food Sciences, Universiti Malaysia Terengganu to be used for challenge study using pathogenic bacteria V. parahaemolyticus through intraperitoneal injection method (Kuebutornye et al. 2020).
Challenge test
Preparation of bacterium inoculum
Pathogenic bacteria V. parahaemolyticus used in this study, were collected from the stock culture of the faculty of fisheries and food sciences, Universiti of Malaysia Terengganu, Malaysia. Briefly, these bacteria were streaked in TSA agar plates and cultured at 35 oC overnight, pure clone were selected and inoculated in TSB broth medium, plated again and incubated at 35 oC for 24 hours in the rotation shaker of 110 rpm. Five (5) ml preculture was taken from the stock solution was transferred into a 50 ml tryptic soya broth (TSB) containing flask and incubated at 35 oC for 24 h. The culture was inoculated in triplicated by maintaining same procedure for this experiment. To identify the concentration as CFUml-1 (Abarike et al. 2018), bacteria was harvested by centrifugation at 3000 rpm for 10 minutes, washed trice by using sterilized phosphate buffer saline (PBS). The concentration was adjusted previously by counting colony forming units on TSA agar medium plates after overnight culture at incubator maintaining temperature 35 oC. It was also adjusted by using TSB to OD 1.40 at web length of 600nm (Spectrophotometer UV1800, Schimadzu, Japan)
Determination of Lethal Dose 50 (LD50)
Malaysian mahseer, T. tambroides is slow growing fish but no reports on serious pathogenic infection in this species (Asaduzzaman et al. 2018a). Prior to the challenge study, the experiment for identification of lethal dose (LD50) for the species was conducted. A lethal dose that kills 50% of challenged fish. LD 50 was determined by using from the same batch of fish (average weight 2.54±0.10 g) by injection method described by (Abarike et al. 2018). Pathogenic bacteria V. parahaemolyticus were used in this experiment. In Brief, 3 groups of fish each group contains 10 fish were anaesthetized by using clove oil (50 mgLitre-1) (Mian et al. 2017) and injected intraperitoneally containing bacterial cell concentration ranging from 1.32×107 to 1.32×109 CFUml-1. Then, the infected fish was maintained with vigorous oxygen supply in a flowing water to bring them normal life before moving to 40 litre water containing culture tank. Three group of same fish were used as a control group. Control group fish where injected same 0.1ml sterilized only PBS solution (Pulpipat et al. 2020). Mortality was monitored for 7 days to determine the LD50 for T. tambroides fish, Then the LD50 was calculated as 108 CFUml-1 for V. parahaemolyticus by the method described by Hossain et al. 2013 with some modification.
Infection test
All the above-mentioned treatments were subjected to experimental infection in triplicate. A control group was added to this challenge experiment, and the fish in this group were injected. Previously collected 12 healthy fish close to average weight (4.66±0.02 to 5.64±0.07 g) from each treatment were challenged with pathogenic bacteria by intraperitoneal injection with 0.1ml pathogen consisting LD50 (1×108 CFUml-1) (Tachibana et al. 2020), clinical sign and mortality pattern were observed and recorded for 7 days after challenge. To evaluate the disease resistance capabilities displayed by each treatment with those of the control group, cumulative mortality percent and relative percent of survival (RPS%) were calculated using by the following formula (Liu et al. 2017).
Growth performance
After 70 days of the feeding trial, the total amount of feed administered to each of the experimental groups and the control group were computed. Thereafter growth parameter were calculated by following the formula, a) apparent feed conversion ratio (FCR)=feed fed (g)/weight gain (g), b) weight gain (g)=final weight (g)-initial weight (g), and c) specific growth rate (SGR% day-1) =100 (ln W2-lnW1) ⁄ T; where W1 and W2 are the initial and final weights (g), respectively, and T is the number of days in the feeding period (Abarike et al. 2018; Tukmechi et al. 2011).
Proximate composition and fatty acids analysis
Dietary composition and whole-body composition as determined by the AOAC (2006), Moisture analysis of sample was determined by drying in oven at 105°C temperature until constant weight. Ash was determined by muffle furnace by keeping sample at 550°C for 12 hours. Crude protein was extracted using an automatic Kjeldahl machine, and crude lipids are extracted using a Soxhlet extractor and petroleum ether. Gross energy of experimental diet was measured by using bomb calorimeter. Triplicate freeze dried samples of whole body T. tambroides specimen weighing 200 mg were collected for a one-step method of fatty acids (FAs), which was combined the extraction and esterification processes in a single tube one step method (the fatty acid methyl esters-FAMEs) (Abdulkadir & Tsuchiya, 2008). Fatty acids (FAs) were separated and quantified by gas chromatography with Mass spectrometer (GC-MS-QP2010 Ultra). Qualitatively as percentage individual FAs as fatty acid were calculated by comparing peak area of each FA with total peak area of all FAs in the sample.
Histological analysis
After 70 days of probiotic feeding experiments all fish were starved for 24 hours, weighed, data were recorded, and 9 fish (close to the mean treatment weight) from each treatments tank were randomly chosen, anaesthetized (using clove Oil; 50mgLitre-1), dissected aseptically for the collection of transverse section of muscle and mid gut sample by following the method described by Asaduzzaman et al. 2018a.
Muscle morphology analysis
Immediate after dissection skeletal muscle sample were taken transversely from near dorsal fin region and immersed in Bouins solution for 24 hours, the ratio of fixative and sample was 10:1 and later the sample was moved to 70% alcohol for further histological process. To evaluate the muscle growth, number of muscle fibers and diameters in the cross-sectional area of transverse section compartmental area of the section using PC and software attached with the microscopes (Kuebutornye et al. 2020) to investigate the effects of HAPs supplements on muscle hypertrophy and muscle hyperplasia. Compound microscope Nikon Eclipse 80i was used to measure individual fibre area which was attached with computerized image analyse system/software (NIS elements illustrator D5 10.00 64 bit). Six (6) fish per treatment group and 6×100=600 fibre was measured per treatment and classified into 4 group, D10: d ≤10, D30: 10 < d ≤ 30, D50: 30< d ≥50 and D70: 50< d ≥70. and the muscle fibre frequency was measured by diameter relative to total number of fibers (Asaduzzaman et al. 2017; Nebo et al. 2013).
Gut Histology Analysis
Mid intestine was dipped in Bouins solution (24 hours as 1:10), was transferred to 70% alcohol for further histological processing (Sawatari et al. 2010). Fixed gut samples were processed in a LeicaTM automatic tissue processor, embedded in paraffin wax, and sliced into 5-7 µm thickness by using microtome machine and placed in a glass slide. The glass slides were placed in hot plate for drying overnight. Before staining with haematoxylin-eosin (H&E) solution, the paraffin slices were dewaxed and dehydrated by running them through a xylene-ethanol series. The slides were dried at room temperature for 30 minutes before mounting with DPX. Evenly located villi ranging 5-10 villi/slide were measured. A total 6 fish sample were used to measure the gut morphological parameters, such as villus height, width of villi and area of villi, and maximum possible number of villi were selected from each sample were selected to measure the parameters.
Statistical analysis
Earlier than analysis, the assumptions of normal distribution and variance homogeneity were double-checked. Each treatment's data was subjected to a one-way analysis of variance (ANOVA) using SPSS (version 25; IBM, Chicago, IL, USA), followed by a Tukey multiple range test to evaluate the differences between treatments, when significant differences (p < 0.05) were found.