Growth and survival of diploid and triploid bata, Labeo bata (Hamilton, 1822)

Aquaculture, Fish and Fisheries: A new home for the Blue Revolution


Inland fishery resources in Bangladesh are under severe pressure from pollution, destructive fishing practices, disease outbreaks and loss of natural breeding grounds (Ali et al., 2015; Islam et al., 2015; Rahi et al., 2021a; Sabbir et al., 2010). Several freshwater fish species have become threatened or endangered, and some have already faced the threat of extinction (Nath et al., 2008; Rahi et al., 2013). The freshwater minor carp, Labeo bata, commonly known as bata, is an important delicacy as a ‘small indigenous fish’ contributing to the nutritional security of the fisher communities in Bangladesh (Ali et al., 2008; Islam et al., 2011; Shah et al., 2011). Bata was previously reported as one of the most threatened fish species in Bangladesh, with a significant decline in its natural abundance because of overfishing, pollution (industrial and agricultural) and habitat destruction. However, successful stock enhancement strategies (essentially establishing sanctuaries in the wild) have helped the conservation status of this species to recover as of ‘least concern’ in Bangladesh (IUCN, 2015).

Triploidy (three chromosome sets) is a ploidy manipulation technique, successfully applied in improving aquaculture production and fisheries management goals over the last few decades (Rahi & Shah, 2012a; Shah et al., 1999; Warner et al., 2018). Triploidy induces sterile populations of fish (Gheyas et al., 2001), and triploids are produced directly by the retention of the second polar body during the second meiotic division. Triploidy is often induced by heat shock, cold shock, pressure shock and chemical shock, but it may also be generated indirectly by the crossing of tetraploid and diploid individuals (Rahi & Shah, 2012b; Tave, 1992). Theoretically, triploid fishes are expected to grow faster than the normal diploids, as triploid cells contain 33% more genetic material due to the presence of an extra chromosome set (Rahi & Shah, 2012a; Warner et al., 2018). Moreover, sterility is another important factor for the faster growth of triploids over their diploid counterparts, as triploids can easily divert their reproductive energy towards somatic growth (Wasow et al., 2004). Triploids are usually sterile because of their three chromosome sets that create difficulty in the cell division process during meiosis due to imbalanced chromosomal distribution (Bramick et al., 1995; Janhunen et al., 2019; Rahi & Shah, 2012a). Triploids also exhibit higher levels of heterozygosity (due to their extra chromosome set), which is known to be associated with better growth performance due to over-dominance and reduced inbreeding depression (Coltman & Slate, 2003; Leary et al., 1985).

Aquaculture of bata has great potential for its higher demand as an edible freshwater fish throughout Bangladesh. However, the small body size and slower growth performance, lack of available broodstock and absence of seed production in hatcheries are major constraints limiting the farming of bata in Bangladesh (Hussain, 2010; Hussain & Mazid, 2005; Sabbir et al., 2017). The induction of triploidy in bata could mitigate some of these disadvantages and further improve its farming potential. Moreover, commercial farming of this species potentially can reduce the fishing pressure in the wild that will ultimately help in the conservation of the natural stocks of bata.

The present study attempted to optimise the parameters for triploidy induction in L. bata by heat shock treatment and subsequently investigate the growth performance of triploids, compared to diploid conspecifics.


Nine experimental trials were performed to determine the optimal combination for triploidy induction in L. bata as outlined in Table 1 (using nine pairs of brood fish from the same parental group and cohort). Therefore, one pair of brood was used for each trial to test four different heat shock temperatures, three heat shock application times post fertilisation and three durations of heat shock treatment.

Experimental design to optimise the treatment combinations for triploidy induction in Labeo bata
No. of trial Temperature (°C) Time after fertilization (AF) (min) heat shock applied Duration of heat shock (min)
Trial-1 38oC 3 1
39oC 3 1
40oC 3 1
41oC 3 1
Trial-2 38oC 3 1.5
39oC 3 1.5
40oC 3 1.5
41oC 3 1.5
Trial-3 38oC 3 2
39oC 3 2
40oC 3 2
41oC 3 2
Trial-4 38oC 4 1
39oC 4 1
40oC 4 1
41oC 4 1
Trial-5 38oC 4 1.5
39oC 4 1.5
40oC 4 1.5
41oC 4 1.5
Trial-6 38oC 4 2
39oC 4 2
40oC 4 2
41oC 4 2
Trial-7 38oC 5 1
39oC 5 1
40oC 5 1
41oC 5 1
Trial-8 38oC 5 1.5
39oC 5 1.5
40oC 5 1.5
41oC 5 1.5
Trial-9 38oC 5 2
39oC 5 2
40oC 5 2
41oC 5 2
Trial-10 Trial with optimum parameters (with five replications) to obtain large number of larvae for growth and survival study

2.1 Bloodstock maintenance, induced breeding and collection of gametes

Sexually mature fish were initially collected from the Arabpur Fish Hatchery, Jessore, and maintained in broodstock ponds of the Department of Fisheries fish hatchery, Gollamari, Khulna, Bangladesh. The brood fish (200 ± 20 g) were then harvested from the ponds and kept in concrete tanks of 1000 L capacity with continuous water flow for 6 h for acclimatisation. Carp pituitary hormone extract was injected intramuscularly to the female at a dose of 1.5 mg kg–1 body weight, followed by a second injection of 6 mg kg–1 body weight after 6 h. Only one injection was given to the males at a dose of 1.5 mg kg–1 body weight during the second dose to the female. Eggs and milt were collected from brood fishes and mixed well in Petri dishes at ambient temperature (28°C) for fertilisation with a physiological saline solution (5% dextrose and 0.9% sodium chloride).

2.2 Application of heat shock for triploidy induction

Fertilised eggs were transferred from Petri dishes to three replicated scoop nets. The scoop nets were then placed in each of the four thermostatically controlled (± 0.2℃ accuracy) water baths (HHS 4: WS2-133-75, Anjue Equipment Co. Ltd.) maintained at temperatures of 38, 39, 40 and 41°C to apply heat shock treatments. The temperature in each water bath was also further verified by digital thermometers (MT-4320, Sejoy). Four temperatures as above, three heat shock times after fertilisation (viz., 3, 4 and 5 min post fertilisation) and three durations of heat shock application (viz., for 1, 1.5 and 2 min) were tested to induce triploidy with three replications being performed for each treatment combination (Table 1). During each trial, three replicates were also maintained by merely mixing the egg and milt for diploid seed production without heat shock application as control.

2.3 Incubation of eggs, fertilisation and hatching

Heat-shocked eggs and the control (only fertilised eggs) were transferred to conical incubation jars (15-L water holding capacity) supplied with a continuous flow of freshwater. The percentage of triploidy induction, fertilisation and hatching rates of the eggs, as well as survival to the yolk sac absorption stage (yolk-sac fry), were considered as the indicators for the success of heat shock treatments. Immediately after the heat shock treatments, 100 eggs from each treatment were observed using a magnifying glass, and the fertilisation rate was determined by counting the number of fertilised eggs (transparent eggs with an opaque whitish spot in the centre). Eggs hatched within 18 to 20 h. The hatching percentage was determined by incubating 500 fertilised eggs in each hatching jar with three replications (three replicated hatching jars for each treatment) and then counting the number of hatchlings in each jar. Survival of the yolk-sac fry was determined by counting their number (total number of larvae obtained from 1500 fertilised eggs) three days post hatching (Rahi & Shah, 2012a).

2.4 Karyotyping and ploidy determination

Karyotypes were prepared from newly hatched, 1-day-old larvae. From each treatment, 30 individuals (10 from each replicate) were used for ploidy determination (rate of triploidy induction). Larvae were transferred in Petri dishes containing 0.008% colchicine solution for 4–6 h at 28 ± 1°C (larvae were swimming free for this time period). Following this immersion step, larvae were euthanised in ice prior to being fixed in methanol and acetic acid solution (3:1 ratio) for 3 min (Rahi & Shah, 2012a). They were then placed in depression slides with 25 μl of 50% acetic acid and squashed well by using a glass rod. The cell suspension was taken in a micro-hematocrit tube fitted with a rubber bulb and expelled onto a glass slide on a warm plate (50C) to prepare a clean ring of cells. Slides with the ring of cells were kept in freshly prepared 4% Giemsa stain for 20 min; stained slides were washed with freshwater, air-dried and mounted in DPX to prepare permanent slides. Slides were then checked under a microscope to count the number of chromosomes and determine triploidy.

2.5 Growth performance of triploid fish

The optimum treatment combination for triploidy induction in L. bata was determined from the initial trials described above (Table 1) prior to testing the growth performance of triploid fish. The heat shock applied at 39℃ for 1 min duration, 3 min after fertilisation, was found to be the optimal combination (see Table 2 below). Therefore, another trial (Trial 10) was conducted using the optimal conditions to produce larger quantities of triploid larvae to evaluate the growth performance against a diploid control. This trial was conducted using one pair of brood; fertilised eggs were incubated in five replicated hatching jars for both the diploid controls and triploids. Triploid and diploid larvae were reared in the hatchery for 5 days prior to stocking in the experimental ponds. Triploidy was again confirmed by counting chromosome numbers during the rearing period in the hatchery.

Fertilisation rate, hatching rate and triploidisation efficiency in bata (L. bata) obtained in each treatment
Temperature (°C) Time AF (min) heat shock applied Duration of heat Shock (min) Fertilisation rate (%) Hatching rate (%) Triploidy induced (%)
38oC 3 1 90aA ± 5 29aA ± 4 4
1.5 86bA ± 3 22bA ± 6 4
2 81cA ± 7 18cA ± 5 3
4 1 92aA ± 2 23bA ± 4 2
1.5 91aA ± 4 20cA ± 5 0
2 89abA ± 6 19cA ± 5 0
5 1 93aA ± 1 25bA ± 3 0
1.5 91aA ± 3 19cA ± 6 0
2 88abA ± 6 13dA ± 5 0
39oC 3 1 87abA ± 4 25aB ± 5 100
1.5 88abA ± 3 21bA ± 4 100
2 85aA ± 5 19bA ± 6 75
4 1 95cA ± 1 20bB ± 4 100
1.5 90bA ± 3 19bA ± 5 80
2 91bA ± 3 18bA ± 5 80
5 1 96cA ± 1 21bB ± 4 0
1.5 94cA ± 1 19bA ± 5 10
2 89bA ± 4 15cB ± 7 20
40oC 3 1 92aA ± 3 19aC ± 6 86
1.5 90aA ± 4 18aB ± 4 63
2 82bA ± 6 12aB ± 2 24
4 1 92aA ± 3 21abAB ± 5 57
1.5 90aA ± 4 17aB ± 4 40
2 89aA ± 5 13aB ± 4 60
5 1 94aA ± 1 23bB ± 3 50
1.5 91aA ± 3 23bB ± 5 40
2 86bA ± 4 13aB ± 5 75
41oC 3 1 81aB ± 5 14aD ± 2 43
1.5 76bB ± 6 11aC ± 3 84
2 71cB ± 4 7aC ± 2 58
4 1 86 dB ± 3 12aC ± 3 50
1.5 82aB ± 5 10aC ± 3 40
2 80aB ± 2 7aC ± 1 80
5 1 90dA ± 2 13aC ± 4 73
1.5 79bB ± 7 13bC ± 2 64
2 78bB ± 4 6aC ± 1 38
  • Note: ‘±’ indicates standard deviation from the mean while N = 100 samples/individuals were considered. Superscripts in small letters indicate comparisons among different variables (between time after fertilisation and duration of heat shock) within temperature, while capital superscripts indicate comparisons among the temperature treatments.

Ten rectangular rearing ponds each of 20 m2 area (5 x 4 m), and 1-m water depth were used for evaluating growth performance between triploid and diploid bata (five replicated ponds for each group). All of the rearing ponds were protected by fencing with small mesh nets to avoid the entry of any pests. The ponds were prepared well before the stocking of larvae following standard practices by cleaning the bottom, sun drying, liming and filling with water. The stocking density of larvae was maintained at 1200 larvae in each pond (60 m–2): Equal numbers of larvae were stocked for both experimental groups (diploids and triploids). The larvae were reared in the experimental ponds for 12 weeks. Commercially available supplementary feed (25% crude protein content) was provided to the experimental larvae at the rate of 5% of total biomass. Water temperature, pH and dissolved oxygen levels were checked daily during the whole growth trial. Fish samples were collected once a week (100 individuals in each sampling: 20 from each replicated pond) to investigate the growth rate (weekly change/increase in body weight) between diploids and triploids. Other growth parameters including daily weight gain, specific growth rate (SGR), feed intake (FI) and feed conversion ratio were determined according to the methods outlined in Rahi et al. (2021c). Survival was determined from the difference between the total number of larvae stocked and the number of fish obtained at the end of the culture period in each replicate.

2.6 Statistical analysis

The results of the fertilisation, hatching and survival rates of triploid and diploid bata were statistically analysed using a three-way analysis of variance (ANOVA) test (between temperature treatments, time after fertilisation heat shock applied and duration of heat shock), while a two-way ANOVA was conducted for the growth data (comparison between ploidy levels and sampling times). Pairwise comparisons between triploid and diploid individuals were made using Duncan’s multiple range and Tukey’s post hoc tests. Results obtained from Trial 10 were further analysed using t-test (for comparing fertilisation, fertilisation and survival rates between diploids and triploids). All statistical analyses were performed using SPSS (version 23) at 5% level of significance.


3.1 Heat shock treatment and triploidy induction

The results of triploidy induction, fertilisation and hatching rates of L. bata are presented in Table 2.

In several trials, 100% triploidy was induced, but the hatching rates were very low. At 38°C, the heat shock was found to be ineffective in inducing triploidy with different intervals post fertilisation (3, 4, 5 min) and durations of heat shock (1, 1.5, 2 min) tested. At 39°C, the heat shock applied 3 min after fertilisation for 1.5 min duration, and 4 min after fertilisation for 1 min duration revealed 100% triploidy; while the fertilisation (88% and 95%, respectively) and hatching rates (21% and 20%, respectively) were relatively lower.

No significant interaction was obtained from a three-way ANOVA test (F (2, 33) = 6.39, p < 0.05) among the independent variables (between four temperature treatments, three times after fertilisation heat shock applied and three durations of heat shock). Pair-wise comparisons among the independent variables also revealed no significant differences (p > 0.05) for fertilisation and hatching rates among 38, 39 and 40°C. Significantly lower (p < 0.05) fertilisation and hatching rates were obtained at 41°C, compared to the other three temperature treatments. The thermal shock treatment at 39°C, 3 min after fertilisation and for 1-min duration was considered as the optimal combination for triploidy induction in bata because it revealed 100% triploidy with comparatively higher fertilisation (87%) and hatching (25%) rates (Table 2).

Cold shock was the first technique to induce polyploidy (triploidy) in fish, while application of heat shock treatment started later on (Purdom, 1983). Therefore, the use of heat shock was far less studied than cold shock for chromosome manipulation in different aquatic species. Effectiveness of shock treatment in arresting second polar body for triploidy induction depends on the time after fertilisation shock treatment applied; shock treatment becomes ineffective to induce triploidy after a specific time of fertilisation once the second polar body of eggs is destroyed (Hussain & Mazid, 2005; Malison et al., 1993; Rahi & Shah, 2012a; Scheerer & Thorgaard, 1987; Wolters et al., 1981). The success of thermal shock treatment for triploidy induction also depends on the type of species; there is evidence that a highly effective treatment combination for one species does not work for another species (Gheyas et al., 2001; Mia et al., 2001). Warm water species have been found to be more susceptible to cold shock (Rahi et al., 2021b; Wolters et al., 1981) than to heat shock for survival; while heat shock was found to be more effective for cold water species for ploidy induction and a reverse pattern is observed for warm water species (Chourrout, 1980; Pujolar et al., 2006). The observed variability in triploidy induction by the application of heat shock treatment in different fish species, namely, rohu and mrigal (Rahi & Shah, 2012a), salmon and trout (Shah et al., 1999) and loaches (Yoshikawa et al., 2007) corroborates this study. As a warm water inhabitant, bata was thought to be highly susceptible to heat shock treatment for triploidisation because warm water fishes inhabit nearly close to their highest temperature tolerance limit (Aziz et al., 2018; Moshtaghi et al., 2018; Rahi et al., 2021b). Although supposed to be vulnerable to high temperatures, triploidy was successfully induced in bata using the heat shock treatments in this study. There is still potential to investigate the effectiveness of heat shock treatment earlier (e.g., 1 or 2 min after fertilisation as 3 min post fertilisation was the earliest one tested in this study). Moreover, shock treatments could be applied beyond 5 min post fertilisation to determine the most prolonged interval after fertilisation that is effective in arresting the second polar body for triploidy induction in L. bata.

3.2 Fertilisation and hatching rates

Diploid bata showed significantly higher (p < 0.05) fertilisation (94%) and hatching (53%) rates over the triploids (Figure 1). Lower fertilisation, hatching and early survival rates of triploids, compared to their diploid counterparts have been previously reported (David & Pandian, 2006; Gheyas et al., 2001; Kim et al., 1995; Maxime, 2008; Rahi & Shah, 2012a; Yoshikawa et al., 2007). The lower fertilisation (85%) and hatching (25%) rates of triploids (Figure 1) obtained in the present study might be due to an impact of the heat shock treatment on the developing zygotes that are highly susceptible to any environmental stressor (e.g., heat shock in the current study). This likely reduced the viability of heat-shocked (triploid) zygotes. In each replicated tank, several fertilised eggs (embryos) in the triploid group were found to be deformed or destroyed (ruptured external cell wall) indicating that the shock treatment potentially reduced the viability of the fertilised eggs and embryos.

Mean fertilisation rates, hatching rates, survival rates up to yolk sac absorption stage (obtained from Trial 10: heat shock only with optimum parameters) and final survival rates (after 12 weeks of rearing in experimental ponds) of diploid and triploid Labeo bata. Error bars represent + 1 SE, where n = 100 individuals were considered. ‘*’ inside the bars indicate significant difference at 5% level of significance (p < 0.05)

3.3 Survival up to yolk sac absorption stage

This study revealed significantly higher (p < 0.05) survival rates of the diploid hatchlings over the triploids up to the yolk sac absorption stage (49% for diploids and 38% for triploids; Figure 1). Survival rate at earlier life stages was also observed to be lower in a wide range of triploid fish species including Nile tilapia (Hussain et al., 1991), mosaic loaches (Yoshikawa et al., 2007), stinging catfish (Gheyas et al., 2001), triploid tetrads (David & Pandian, 2006), hybrid loaches (Kim et al., 1995), salmon and trout (Chourrout, 1980; Wolters et al., 1981; Scheerer & Thorgaard, 1987; Shah et al., 1999) rainbow trout (Janhunen et al., 2019). The higher ploidy level could potentially make the embryonic developmental stages shorter or faster (Fopp-Bayat et al., 2007a; McKay et al., 1992). Therefore, triploids are most likely to survive less at their early life stages as triploids do not get adequate time for proper embryonic development as well as to complete metamorphosis (Gray et al., 1993; Rogl et al., 2018).

3.4 Chromosome karyotyping and ploidy determination

The chromosome numbers for both the diploid and triploid bata and their metaphase preparations are presented in Figures 2 and 3. In diploid bata, 48 (2n) chromosomes were recorded in every trial, while in triploids, the 3n chromosome number was found to be 72. The number of chromosomes varies from species to species, while closely related species are likely to share a closer (or same) number of chromosomes with each other (Janhunen et al., 2019; Rahi & Shah, 2012b). Diploid chromosome numbers in other closely related species of L. bata were found to be 48–52 (e.g., L. rohita = 50).


Diploid chromosome numbers of L. bata, 2n = 48


Triploid chromosome numbers of L. bata, 3n = 72

3.5 Growth performance in culture ponds

The weekly mean body weight and weight gain of the experimental fishes are presented in Figure 4. The initial mean body weights (before stocking the fry in nursery ponds) of the diploid and triploid groups were 0.10 and 0.11 g, respectively. At the end of 12 weeks of the rearing in experimental ponds, triploids (10.04 g) showed significantly higher (F (11, 99) = 61.8, p < 0.05) body weight than the diploids (5.87 g). Significantly higher weekly weight gain performance (F (11, 99) = 45.6, p < 0.05) was also observed for the triploids over their diploid counterparts. Although an equivalent amount of FI was observed (no significant difference; p > 0.05) between diploid and triploid groups, triploids exhibited significantly higher performance (p < 0.05) for all other growth parameters (Table 3). These results indicate that triploids were more efficient in diverting feed energy for somatic growth.


Weekly (a) mean body weight and (b) growth rate (weight gain) of diploid and triploid L. bata. Error bars represent + 1 SE, where n = 100 samples were checked during each sampling

Comparative growth and survival performance between diploid and triploid groups of bata (L. bata). Different superscripts indicate significant differences at α = 0.05
Parameters Diploids Triploids
Initial weight (BWi; g) 0.10a ± 0.04 0.11a ± 0.03
Final weight (BWf; g) 5.87a ± 0.43 10.04b ± 0.76
Daily weight gain (%) 6.41a ± 0.71 11.03b ± 0.79
Specific growth rate (%) 3.8a ± 0.14 6.15b ± 0.26
Feed intake (g g–1day–1) 0.159a ± 0.04 0.161a ± 0.03
Feed conversion ratio 1.76a 1.34b
Survivability (%) 56a 69b

Differences in growth performance between triploids and diploids (superior growth performance of triploids in most cases) are hypothesised to be due to: differences in cell volume and heterozygosity, presence of an extra chromosome set and the induced sterility in triploids due to an imbalanced distribution of chromosomes during meiosis (Carter et al., 1994; Malison et al., 1993; O’Keefe & Benfey, 1997; Rahi et al., 2017; Warner et al., 2018; Withler et al., 1998; Yamazaki & Goodier, 1993). Triploids usually contain 33% additional genetic material in each nucleus (Rahi & Shah, 2012a; Tave, 1992; Vargas et al., 2015). This could potentially increase their heterozygosity (Altimiras et al., 2002; Pujolar et al., 2006; Rahi, 2017) and favour the superior growth performance of triploid individuals (Borell et al., 2004; Garner et al., 2008; Islam et al., 2014; Leary et al., 1985; Malison et al., 1993; Rahi et al., 2019). Sterility and the presence of extra genetic material are reported to be the main reasons for the better growth performance of triploids over their diploid counterparts (Felip et al., 2001; Johnson et al., 2007; Leary et al., 1985; Moshtaghi et al., 2017; Shah et al., 1999; Tave, 1992; Utter et al., 1983). The sterility probably enables triploids to divert their extra energy from reproduction to growth.

In the present study, the survival rates of pond-reared bata were significantly higher (p < 0.05) in triploids (69%) over the diploid fish (56%; Figure 1). The survival rate of triploids was superior over the diploids due to the heat shock treatment that probably made the triploids to acclimate well (due to their ability to tolerate thermal stress at early stage of life) with culture conditions and minimise stress. It is also thought that the presence of extra genetic materials may also be a potential reason for higher survivability in triploids, which increase their immune functions and hormonal secretion for adaptability (Li et al., 2009; Rahi et al., 2020).

The higher survival rate of triploids over their diploid counterparts was also observed in other fishes, namely, European sea bass (Felip et al., 2001), rainbow and brown trout (Altimiras et al., 2002) and brook trout (Fopp-Bayat et al., 2007b) although reduced survival rate of triploids was observed in case of tiger trout (Mckay et al., 1992). The results obtained from the present study indicate that triploidisation in L. bata increased its growth, survival rate and adaptability in culture conditions. The induction of triploidy could be used as a strategy to enhance the aquaculture potential of bata as well as a means of conserving their natural stocks from overfishing.


In the present study, triploidy was successfully induced in L. bata by applying heat shock treatment. With different parameters tested, the application of heat shock at 39°C for 1 min duration, 3 min post fertilisation resulted in 100% triploidy induction as well as optimal fertilisation (87%) and hatching rates (25%). Although the triploid L. bata had lower fertilisation and hatching rates, compared with their diploid counterparts, the triploids showed significantly higher growth and survival rates over diploids in a 12-week grow-out trial in earthen ponds. The superior growth of triploid individuals could be beneficial to overcome the slow growth performance of this fish in culture conditions. This would also help facilitating the integration of triploid bata in the inland aquaculture sector of Bangladesh. The results of the present study could form the basis of further trials to test the growth performance of triploid bata for extended culture periods and bigger harvested sizes, while optimising the heat shock treatment to enhance the fertilisation and hatching rates of triploids for the commercial production of triploid seed for aquaculture. The induced sterility from triploidisation could also be further studied as a tool in achieving the desired conservation and management goals in the aquaculture of bata.


We are grateful to the staff of Fish Biology Laboratory, Fisheries and Marine Resource Technology Discipline, Khulna University and the staff of the Government Fish Seed Multiplication Farm, Gollamari, Khulna under Department of Fisheries, Bangladesh for facilitating this research.


The authors declare no conflict of interest.


We have the necessary animal clearance from Khulna University authority (KUAEC-2021/09/21)


Conceptualisation, data curation, formal analysis, methodology, software, validation, writing-original draft: Khan Benzir Afroz. Conceptualisation, funding acquisition, investigation, methodology, project administration, supervision, writing-review and editing: Md Saifuddin Shah. Conceptualisation, methodology, writing-review and editing: Salin Krishna. Conceptualisation, formal analysis, project administration, supervision, writing-review and editing: Md Lifat Rahi.

Source: Online Library, Wiley

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