Sample Collection And Experimental Site
The experiment was conducted at Department of Genetics and Fish Breeding (GFB) lab and the hatchery complex at Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur, Bangladesh (Fig. 1). In the current investigation, two different types of experiments were conducted: experiment 1, is for reproductive physiology study and Experiment 2, is for broodstock development study using different feeding regimes and stocking densities. For the reproductive biology study, a total of 480 specimens (10.3 ± 1.03 cm TL and 10.6 ± 3.18 g BW) were collected monthly while, for the broodstock development study, a total of 400 P. sophore fish (9.82 ± 0.48 cm TL and 8.64 ± 1.24 g BW) were obtained from the River Brahmaputra and transported to the GFB laboratory of BSMRAU, Bangladesh.
Experiment I: Reproductive Biology Study
On board, a local fishing boat, traditional fishing equipment such as a three-layered trammel net, cast net, scoop net, and trap were used to capture fish samples. After hauling, the catch was taken out, and thoroughly washed, and specimen identification was done using the fishes’ surface markings as well as their morphometric and meristic characteristics. Fish that had been caught were taken to the lab for analysis and put on ice.
Size proportion and sex ratio
Using a measuring scale the fish’s total length (TL, cm) was calculated to the nearest 0.1 cm by measuring from the tip of the snout to the base of the tail end while, an electric balance (Docbel BRAUN) was used to measure the body weight (BW) and gonad weight (GW) up to 0.01 g after adherent water and other debris were removed from the surface of the body (Mazumder et al. 2016). The fish was dissected, and gonads were taken out and put on a glass slide that had been cleaned to investigate sex ratios. Following the addition of a few drops of acetocarmine stain, the gonads were compressed with a cover slip. The plates were examined under a microscope to determine the fish’s sex. While the female gonad has a circular oogonia-like shape, the male gonad has a fine, granular spermatogonia-like structure. After counting the total number of the two sexes in the monthly collected samples, the variation in the sex ratio over time was examined. Using the Chi-square (χ2 test) test, the discrepancies between the sex ratio and the expected ratio of 1:1 were calculated:
$${\left(\chi \right)}^{2}=\sum {(O-E)}^{2}/E$$
1
Where, O = Observed value and E = Expected value.
Condition Factor
The Fulton condition factor (K) of P. sophore, hereinafter referred as fitness, was calculated according to Fulton (1904) which relates the fish's body length to its weight:
Where, BW is the observed individual fish weight in grams (g), TL is the observed individual fish total length in centimeters (cm), and 100 is a factor to bring the value of K near unity.
Gonado Somatic Index (GSI)
Fish ovaries were inspected macroscopically in the lab, and fresh tissue was used to note characteristics such color, texture, form, and turgidity. Each fish was assigned to a reproductive phase based on these characteristics. According to Solomon and Ramnarine (2007), paired gonads were individually weighed to the nearest 0.01 g, and GSI was computed:
$$GSI \left(\%\right)=\left(\frac{GW}{BW}\right)\times 100 \left(3\right)$$
Where, GW is the wet weight of the gonad, BW is the body weight of the fish.
Fecundity Estimation
Using the gravimetric approach, the fecundity of P. sophore was estimated. The weight of freshly dissected ovarian tissue was measured to the nearest 0.01 g. Each pair of ovaries had its exterior connective tissues scraped off. The ovaries were dried out by wiping them with paper. Each ovary had three subsamples collected from the base, middle, and apical regions, and each was weighed to the nearest 0.01 g. Each subsample’s eggs were counted, and the projected total number of oocytes in each ovary was reported as overall fecundity.
$$Fecundity \left(F\right)=(no.\: of\: eggs \times weight\: of\: gonad)/weight\: of\: gonad\: sample\: \left(4\right)$$
The relationships of fecundity (F) to total length (TL), BW and GW were examined by correlation analysis (Simon et al., 2009).
Experiment II: Broodstock Development Study
Experimental design
The fish were kept in stocking tanks of equal size (500L) for up to 30 days at a constant ambient temperature (26°C) fed a commercial feed for broodstock development study (Table 1). Every tank was prepared with a good inlet and outlet system to allow for a daily water exchange of at least 20%, with the exception of biofloc. The fish were moved to the experimental circular tank once they began to feed and expel excrement (400L). To satisfy the oxygen requirement, each tank was supported by diffuser air stones linked to 35W air compressors (70L min− 1, > 0.25 Mpa, Hailea Co., Ltd., China). Two experiments were carried out: one is the effect of different regime and another was stocking density effect. For feed regime experiment, fish were subjected to three experimental diets (control D1, diet supplementation in biofloc D2 and probiotic containing diet D3), and in stocking density experiment, three stocking densities (D1: 20, D2: 25, and D3: 30 fish per tank) were used (Fig. 2). Under each treatment, three replicates were used with a good aerator, inlet, and outlet system. Commercial diet was used as a control diet. Fish were fed up to their satiation with twice daily (9.00 and 16.00 h) at the rate of 2–3% of body weight. The experiments were conducted for 60 days. Every 15 days interval fish were sampled and recorded data.
Table 1
Proximate composition of the commercial diet used in the experiment
Proximate composition
|
Percentage (%)
|
Moisture
|
12.98 ± 0.41
|
Protein
|
31.20 ± 0.34
|
Lipid
|
6.72 ± 0.80
|
Ash
|
13.22 ± 0.61
|
Fibre
|
11.58
|
NFE*
|
24.30
|
*Nitrogen free extract (NFE) calculated as: NFE = 100 - %( moisture + crude protein + crude lipid + ash + crude fibre) |
Experimental Feeds And Biofloc
In biofloc treatments, fish were fed with commercial feed (Table 1) and were added molasses and rice powder as an external carbon source to promote the development of flocs considering calculation requirements as recommended by Emerenciano (2011). Water was not changed in these tubs and was maintained constant aeration to avoid sedimentation of flocs.
Experimental Feeds With Probiotic
This work made use of the commercial Lactobacillus plantarum (KC426951) probiotic, which was previously isolated from the intestine of rainbow trout Oncorgynchus mykiss (provided by Zistyar Varna Co, Gilan Province, Iran) (Valipour et al. 2019). To achieve the final conc. of 2×108 CFU kg− 1 of feed (0.2% per 100 g feed), sterile physiological serum (0.9% w/v of NaCl) was mixed in 2 grams of the probiotic (containing 108 CFU per 1 g of cells weight) and sprayed on 1kg of a commercial dry feed. The feed was dried at ambient temperature before stored in a refrigerator (4°C) until use.
Water Quality Parameters
The total ammonia nitrogen (NH3-N) and total hardness were determined on a weekly basis during the study period, whereas the temperature and pH of the water were examined daily. At 9.00h, all measurements were obtained. With the help of a YSI 59 multiparameter water quality probe, temperature and pH were measured (Yellow Spring Instrument Company OH, USA). Utilizing the API Freshwater Master Test Kit, NH3-N and total hardness were measured (SKU: 35647).
Growth And Survival
Fish from each experimental tank were individually weighed at the beginning of the experiment and every two weeks after that. Clobe powder (99% purity, Talya Bitkisel Urunler Ind. Co. Ltd., Antalya, Turkey; 200 mg L− 1) was used to lightly sedate fish prior to sampling. Using a measuring board, the total length and weight were recorded to the nearest 1.0 cm, and an electronic balance (Model: KD-300KC) was used to weigh the total to the nearest 0.01 g precision (Mazumder et al. 2018). These formula were used to calculate food consumption and growth indices.
$$Body\:weight\:gain\:\left(BWG,\:g\right)=\left(W2-W1\right)\times n\dots \dots \dots \dots \dots \dots .\dots \dots \dots \dots \dots \dots \dots \dots \dots \left(5\right)$$
$$Food\:consumption\:\left(FC,\:\frac{g}{d}\right)=\left(\frac{g\:food\:consumed}{day}\right)\dots \dots \dots \dots \dots \dots \dots \dots \dots \dots ....\left(6\right)$$
$$Food\:cconversion\:ratio \left(FCR\right)=\left(\frac{F}{W2-W1}\right)\dots \dots ..\dots \dots \dots \dots \dots \dots .\dots \dots \dots \dots \dots ....\left(7\right)$$
$$Food\:cconversion\:efficiency \left(FCE\right)=\left(gain\:in\:fish\:mass/g\:food\:consumed\right)..\left(8\right)$$
$$Specific\:grwoth\:rate \left(SGR, \%day-1\right)=100\times \left(LnW2-LnW1/t\right)\dots \dots \dots \dots \dots .\left(9\right)$$
$$Relative\:grwoth\:rate \left(RGR, \%\right)=100\times \left(W2-W1/W1\right)\dots \dots \dots \dots \dots \dots ..\dots \dots \dots .\left(10\right)$$
$$Daily\:grwoth\:rate \left(DGR,\:\%\right)=100\times \left(W2-W1/t\right)\dots \dots \dots \dots \dots \dots \dots \dots \dots \dots \dots \dots .\left(11\right)$$
$$Survival\:rate \left(\%\right)=100\times \left(Final\:no.\:of\:fish /initial\:no.\:of\:fish\right)\dots \dots \dots \dots \dots .\left(12\right)$$
Here, F represents total amount of food consumed during the experiment, W1 & W2 represent the starting (0 day) and the final (60 day) mean individual weights for each treatment (to the closest 0.01 g), n represents the total no. of fish, and t represents length of the experiment.
Surgical Protocol And Haematological Analysis
Five fish were randomly selected from each tank at the end of the experiment to assess various haematological factors. Fish were anesthetized with clove oil (99% purity, Talya Bitkisel Urunler Ind. Co. Ltd., Antalya, Turkey; 200 mg L− 1) until ventilator movements stopped in order to lessen handling stress (Mazumder et al. 2019). Fish were rapidly caught, and blood samples were collected from the caudal vein using a heparinized syringe and transferred to tubes that contained the anticoagulant substance EDTA. The haematological profile of the blood samples collected in EDTA tubes was determined using automated haematology analysis (HeCo Vet C; SEAC, Florence, Italy) and a special fish lysing reagent (SEAC, Code 71010460), which had previously been used to investigate the haematological profile in other fish species (Fazio et al. 2013). The determination of the red blood cell count (RBC), haemoglobin conc. (Hb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin conc. (MCHC), thrombocyte (TC), and white blood cell count (WBC) were all included in the evaluation of the haematological parameters. Animal ethics committee of BSMRAU approved the protocol with the code BSMRAU/Research Wing/Innovation Project/128/2019/903.
Statistical analysis
Prior to statistical analysis, the Kolmogornov-Smirnov test was used to determine whether all data had normal distribution, and the Bartlett’s test was used to see whether the variances were homogenous (Sokal and Hohlf 1995). Throughout the sampling period, a chi-square (χ2 test) was used to look for any associations between month and sex. To examine the impact of monthly variation in condition factor (K) and GSI, analysis of variance and Tukey’s post hoc tests were performed. Fecundity was found to be linearly related to TL, BW, and GW. A one-way ANOVA analysis was performed on the data to determine the impact of the feeding experiment and stocking density on the haematological responses of P. sophore and the BWG, FCR, FCE, SGR, RGR, AND DGR. P < 0.05 was used as level of significance for each analysis. Every outcome was presented as mean ± S.E. Software programs Origin™ v 2016 and Minitab v 17 were used for all statistical analyses.