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APPLIED RESEARCH
Application of zymetin and super PS probiotics in
hatchery, nursery, and grow-out phases of
Macrobrachium rosenbergii and their impact on
culture environment, production, and economics
Md. Abul K. Azad
1,2
| Shikder S. Islam
1
| Alokesh K. Ghosh
1,3
|
Abul F. Md. Hasanuzzaman
1
| Aaron J. Smith
4
| Joyanta Bir
1,5
|
Mirja K. Ahmmed
6,7
| Fatema Ahmmed
8
| Ghausiatur R. Banu
1
|
Khandaker A. Huq
1
1
Fisheries and Marine Resource Technology
Discipline, Khulna University, Khulna,
Bangladesh
2
Department of Fisheries, Ministry of
Fisheries and Livestock, Dhaka, Bangladesh
3
Faculty of Science, Department of Biology,
Animal Physiology and Neurobiology Section,
Zoological Institute, KU Leuven,
Leuven, Belgium
4
Fisheries and Aquaculture, Institute for
Marine and Antarctic Studies, University of
Tasmania, Launceston, Australia
5
Plentzia Marine Station (PiE-UPV/EHU),
University of the Basque Country, Plentzia,
Spain
6
Department of Food Science, University of
Otago, Dunedin, New Zealand
7
Department of Fishing and Post-Harvest
Technology, Chittagong Veterinary and
Animal Sciences University,
Chittagong, Bangladesh
8
Department of Chemistry, University of
Otago, Dunedin, New Zealand
Abstract
This study evaluated the effect of two commercial probiotics,
zymetin, and super PS, on the culture environment, produc-
tion, and economics of Macrobrachium rosenbergii aquaculture.
The experiment was conducted using three treatments,
including T
1
, probiotics applied in all culture phases; T
2
, during
grow-out; and a control (C). Prawns were fed commercial pel-
leted diets. Earthen ponds were used at a stocking density of
two juveniles/m
2
. Water concentrations of non-ionized
ammonia and nitrite-nitrogen were significantly lower
(p<0.05) inT
1
than in T
2
and C. Probiotic application signifi-
cantly (p< 0.05) reduced phosphate content and balanced soil
pH. Growth performance, condition factor, survival, protein
utilization, and production were significantly higher in T
1
followed by T
2
compared to Cby the end of grow-out. Appli-
cation of zymetin and super PS in all culture phases signifi-
cantly (p< 0.05) reduced the feed conversion ratio in T
1
compared to T
2
and C. Positive allometric growth was
observed in T
1
, whereas growth was isometric in Cand T
2
.
Received: 6 January 2022 Revised: 22 November 2022 Accepted: 13 December 2022
DOI: 10.1111/jwas.12943
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
© 2023 The Authors. Journal of the World Aquaculture Society published by Wiley Periodicals LLC on behalf of World Aquaculture
Society.
J World Aquac Soc. 2023;54:645–665. wileyonlinelibrary.com/journal/jwas 645
Correspondence
Shikder S. Islam, Fisheries and Marine
Resource Technology Discipline, Khulna
University, Khulna, Bangladesh.
Email: shikdersaiful.islam@ku.ac.bd
Funding information
Ministry of Education, Grant/Award Number:
37.01.0000.078.02.018.13-206(38)/6;
Ministry of Science and Technology,
Grant/Award Number:
39.012.002.01.03.022.2015-439
Probiotic treatment during all culture phases achieved the
highest net returns to land, family labor, and management.
Application of probiotics during the entire culture cycle could
be the best practice for improving production and economics
in M. rosenbergii aquaculture.
KEYWORDS
culture cycle, grow-out phase, growth, production, super PS
1|INTRODUCTION
The vast water bodies, agro-climatic conditions, and availability of resources (seed, feed, and cheap labor) in
Bangladesh provide favorable conditions for the cultivation of giant freshwater prawns (Macrobrachium rosenbergii).
The giant freshwater prawn is known for its high nutritional value and provides a good source of vitamins (A, D),
minerals (Ca, Mg, Mn, P), high protein, and omega-3 fatty acid content (Ahmmed, Ahmmed, Tian, et al., 2020) as well
as palatable taste and visual appeal, making it an in-demand product for human consumption (Ahmmed, Ahmmed,
Khushi, et al., 2020; Azad et al., 2019; Sumon et al., 2018). The total production of cultured prawns in Bangladesh
was 359,887 MT in 2015–2016 (FRSS, 2017), which increased to 368,882 MT during 2019–2020 (DoF, 2021).
Because of its export potential, high demand, and high profits in the European Union, USA, Japan, Russia, and China,
freshwater prawn farming is now an important sector in the economy of Bangladesh (DoF, 2021).
Freshwater prawns are susceptible to many diseases, including viral (Anderson et al., 1990; Arcier et al., 1999;
Wang et al., 2008), bacterial (Cheng & Chen, 1998; El-Gamal et al., 1986; Tonguthai, 1997), and fungal (Chen, 1995;
Sung et al., 1998) infection. These diseases cause high mortality in freshwater prawn culture and are responsible for
economic losses (Hameed & Bonami, 2012). In particular, bacterial diseases have received more attention in prawn
cultures and are typically treated with either antibiotics or probiotics (Azad et al., 2019; Defoirdt, 2014). Application
of antibiotics is generally not recommended due to concerns about antibiotic residue bioaccumulation in prawn tis-
sues and bioconcentration into the consumer (Boonsaner & Hawker, 2013; Liu et al., 2018; Zhang et al., 2018,
2021). Moreover, the indiscriminate use of antimicrobial drugs and disinfectants has gradually made them inefficient
against potential pathogens and has led to the evolution of resistant bacterial strains (Esiobu et al., 2002).
Probiotics as an alternative to antibiotics, aid in the control of harmful bacteria through competitive exclusion,
enhancing host immunity, creating unfavorable conditions for pathogens, and exerting antiviral activity (Azad
et al., 2019; Deekshit et al., 2022; Okeke et al., 2022). Probiotics can improve water quality by degrading organic
matter, reducing disease incidence, and increasing aquaculture production (Sahu et al., 2008). Recently, like
other countries in Asia, farmers in Bangladesh have begun using probiotics in Penaeus monodon hatcheries
(Decamp et al., 2008) and grow-out ponds to meet the demand for disease-free, eco-friendly, and sustainable aqua-
culture practices (Rahman et al., 2009; Rico et al., 2013).
Many researchers in different countries have previously reported the potential of probiotic use in aquaculture
for the improvement of aquaculture products and the environment (Edun & Akinrotimi, 2011; Farzanfar, 2006; Hai
et al., 2009; Li et al., 2006; Rahman et al., 2009; Zhou et al., 2009).
Application of gut-derived (e.g., Lactobacillus sp., Clostridium butyricum) and commercial (zymetin) probiotic bac-
teria through diet at the grow-out stage has previously shown antagonistic effects against pathogenic bacteria, like
Vibrio harveyi, Vibrio sp. and Aeromonas sp. in freshwater prawns (Ahmmed, Ahmmed, Khushi, et al., 2020; Azad
et al., 2019; Sumon et al., 2018). Some of the probiotic bacteria, such as Enterococcus faecalis, showed effective
results against Vibrio harveyi at the grow-out phase and enhanced prawn growth, immunity, and digestibility (Khushi
et al., 2020). During the grow-out phase, relative to the application of a single probiotic, the combined effect of
646 AZAD ET AL.
commercial probiotics (zymetin and Super PS) has previously been shown to provide the highest growth perfor-
mance in giant freshwater prawns (Ghosh et al., 2016).
Studies of the effects of probiotics in the aquaculture systems of Bangladesh, including the use of zymetin, super
PS, Lactobacillus sp., C. butyricum, and E. faecalis in prawn culture, are limited (Ahmmed, Ahmmed, Khushi,
et al., 2020; Azad et al., 2019,2021; Ghosh et al., 2016; Khushi et al., 2020; Sumon et al., 2018). Previously
published research has focused on the implementation of probiotics during the grow-out stage of prawn culture.
Therefore, knowledge of the application of probiotics over the entire prawn cultivation cycle, including hatchery,
nursery, and grow-out phases, is still limited. The present study aimed to apply probiotics during all stages of the
prawn cultivation cycle to determine the combined effect of soil and feed probiotics on the culture environment,
growth and production, and feed utilization in prawn monoculture systems.
2|MATERIALS AND METHODS
2.1 |Experimental design
Previous studies reported that the combined application of two commercial probiotics, zymetin (Advance
Pharma Co., Ltd., Thailand; Lot No. 09201X) and super PS (CP aquaculture, India) in the grow-out phase of
prawn culture had a higher impact on growth, production, and feed utilization than that of single or no probiotic
application (Azad et al., 2021; Ghosh et al., 2016). In this experiment, both probiotics were applied during all
stages of M. rosenbergii culture, including the hatchery, nursery, and grow-out phases over a period of 9 months.
The experiment was carried out using three treatment groups, including T
1
, probiotics applied in a hatchery,
nursery, and grow-out phase; T
2
, during grow-out culture; and a control group without probiotics (C). In the
hatchery and nursery phases, larvae were reared with and without probiotic treatments. For the grow-out phase
of Cand T
2
, juveniles (2.50 ± 1.18 g) that did not receive probiotic treatment during the hatchery and nursery
rearing phases were used. Thus, juveniles (2.90 ± 0.78 g) used in T
1
for grow-out culture received probiotic
treatment in all its early rearing stages. There were three replicates for each treatment, and nine rectangular
earthen ponds were used for the grow-out culture of the prawn. The size of each pond was 120 m
2
with a
1.5 m water depth.
2.2 |Composition and application of probiotics
Zymetin is a feed probiotic with the bacterial composition of Streptococcus faecalis,Bacillus mesentericus, and
C. butyricum (1.10 10
8
CFU/g), with wheat flour as a carrier. The probiotic zymetin is useful for enhancing the
physical strength and growth of prawns, aids in the stimulation of immune activity, and reduces pathogenic bacteria
by competitive exclusion (Azad et al., 2019,2021; Ghosh et al., 2016). Zymetin helps enzyme equilibrium in the
intestinal tract and enhances carbohydrate, protein, and fat digestibility by increasing amylase, protease, and lipase
enzyme activity (our unpublished data, article under review). Zymetin also improves FCR for shrimp culture and
decomposes organic matter in culture systems (Azad et al., 2021; Ghosh et al., 2016). The probiotic zymetin was
applied as follows: 5 g zymetin with 20 g mutagen (Advance Pharma Co., Ltd., Thailand; Lot No. 0283) per kg feed
for the first 4 days every week (Azad et al., 2019,2021; Ghosh et al., 2016).
Super PS is a soil probiotic with the bacterial composition of Rhodobacter sp. and Rhodococcus sp. in 10
9
CFU/
mL. Super PS significantly improves shrimp health and growth by biodegradation of organic pollutants, reduces
hydrogen sulfide and toxic gases, and maintains optimum water quality and soil parameters (Azad et al., 2021; Ghosh
et al., 2016; Wang et al., 2022). Super PS was applied in tanks or ponds before stocking PL or juveniles by spreading
40 L super PS/ha to enhance the quality of tank or pond bottom organic matter, and to increase the activities of
AZAD ET AL.647
beneficial bacteria (Ghosh et al., 2016). After stocking, super PS was applied at different doses at several
culture stages, such as early-stage (0.5 ppm/week), middle stage (1 ppm/week), and last stage (1–2 ppm/week)
(Ghosh et al., 2016).
2.3 |Hatchery phase
A portion of the hatchery facilities of the Shrimp Research Station of Bangladesh Fisheries Research Institute (BFRI),
Bagerhat were used to produce and rear the larvae of M. rosenbergii following the experimental design (Cand T
1
) for
45 days. The larvae were reared in circular concrete tanks (500 L total volume tank filled with 450 L water) with con-
tinuous aeration and with 20% water exchange every day. The tanks were stocked with 50 larvae/L (larval stage IV
at the age of 4–6 days), and the total number of larvae per tank was 22,500. The salinity of the rearing water was
maintained at 12 ppt. Direct light penetration into the rearing environment was avoided to minimize potential stress
to larvae. The larvae were fed by co-feeding of Artemia nauplii and custard feed (composed of milk 30 ppm, egg
1 piece, corn flour 15 g, cod liver oil 0.1 g, chopped and smashed shrimp 20 g, vitamin premix 2 g) at the rate of
200%–20% body weight, 5–6 times a day. Zymetin probiotic was applied with the custard feed at the previously
described rate, for example, 5 g zymetin and 20 g mutagen (Advance Pharma Co., Ltd., Thailand; Lot No. 0283) per
kg feed for the first 4 days every week. Super PS probiotic was applied in the tanks by spreading at the rate of
40 L/ha before stocking the larvae. After stocking, different doses were applied during the culture stage, such
as 0.5 ppm/week in the early stage, 1 ppm/week in the middle stage, and 1–2 ppm/week in the last stage (Azad
et al., 2021; Ghosh et al., 2016).
2.4 |Nursery and grow-out phase
2.4.1 | Pond preparation
The experimental ponds were dewatered, dried, and excavated to remove organic wastes, aquatic weeds, and unex-
pected fauna. Eroded embankments of the ponds were repaired. After drying for 2–3 days, liming was conducted
with calcium carbonate (CaCO
3
) and dolomite CaMg(CO
3
)
2
at the rate of 1 kg/dec to adjust the pH of the culture
system. After liming, the ponds were filled up to 1.5 m with groundwater (salinity varied from 0 to 2.5 ppt). The total
area of the ponds (each 120 m
2
) was fenced with a nylon net to prevent the entrance of unexpected organisms.
2.4.2 | Experimental animals and acclimation
The experimental animals (M. rosenbergii) were sourced from the hatchery and nursery rearing experimental field at
BFRI, Bagerhat. Post-larvae (PL
45
) were transported carefully to the grow-out experimental research pond complex
of Fisheries and Marine Resource Technology (FMRT) Discipline, Khulna University, Bangladesh. On arrival, the PL
was acclimatized at 30C and at salinity from 0 to 5 ppt for 30 min.
2.4.3 | Culture procedure
Nursery rearing
The post-larvae (PL
45
) were stocked in Cand T
1
separately in the nursery ponds. The stocking density of PL in
the nursery pond was 20 PL/m
2
, and they were cultured in the nursery ponds for 60 days up to becoming juvenile
648 AZAD ET AL.
(2–3 g). During nursery rearing, PL was fed with Mega prawn nursery feed (code. 400/500) with 40% protein, 5%
lipid, and 7% fiber at 20%–10% body weight 3–4 times a day.
Grow-out culture
In the grow-out ponds, juvenile prawns (2.50 ± 1.18 g in Cand T
2
, and 2.90 ± 0.78 g in T
1
) were stocked at a rate of
2 juvenile/m
2
. Before stocking, the dried pond bottom was treated with super PS following the manufacturer's pro-
tocol. During the grow-out phase of 150 days, the animals were fed with Mega prawn grower feed (Code. 404, pellet
diameter =2 mm) with 32% protein at 10%–3% of body weight. Feed protein levels were checked by proximate
composition analysis in the laboratory. The prawns were fed to satiation 2–3 times daily throughout the duration of
the experiment at a slight excess to determine feed ration allocation. Feed was provided in a feeding tray in the
morning, the uneaten feed was collected, and the amount of uneaten feed was estimated. The initial feeding rate
was 10% of the prawn biomass in the pond. The afternoon ration was adjusted according to the amount of feed
given in the morning and the feeding table (Table 1, modified from Tacon et al., 2013). A smaller amount of feed was
given in the morning, and the remaining feed was adjusted in the evening ration at 6 pm.
2.5 |Water quality parameters determination
Water quality parameters, such as pH, dissolved oxygen (DO), alkalinity, hardness, non-ionized ammonia, and nitrite-
nitrogen were measured using the HACH kit (HACH, USA, Model FF-2). A hand refractometer was used to measure
the salinity (ATAGO Co. Ltd, Japan, model no, Master- T 2312, Salinity range 0–100 g/L), and the temperature was
measured using a digital thermometer (China, model no WT-2, Temperature range 20 to 80C). The water quality
parameters were measured fortnightly at 9:00 a.m. in the morning and at 5:00 p.m. in the afternoon.
2.6 |Soil quality parameters determination
Soil pH was determined by a Glass Electrode Soil pH Meter. Walkley and Black's Wet Oxidation Method was used
to determine organic carbon, and Ca and Mg levels were determined through an NH
4
OAc (pH 7.0) based titrimetric
method as previously described (Jackson, 2005). The electric conductivity of the soil was measured at the soil: water
ratio of 1:1.25 using an EC meter and available soil P was measured by Bray's method (Bray, 1948). A soil core sam-
pler was used to collect the samples from different places in the experimental ponds.
2.7 |Growth measurements
The total length (cm) and body weight (g) of prawns were measured at fifteen-day intervals. Fifty-one prawns were
taken for every sampling point. A cm scale was used to measure the length according to Azad et al. (2021) and
Signoret and Brailovsky (2002), and an electronic balance (CSC Balance, YP10002B) was used to measure the
weight. All experimental ponds were dewatered for the final harvest and survival, production, and FCR were
assessed, and a cost–benefit analysis was conducted. The following formulae were used to calculate the growth,
feed utilization, length-weight relationship, and condition factor:
WG¼Wt–W0
In which W
G
is weight gained, W
t
is the final weight and W
0
is the initial weight
AZAD ET AL.649
TABLE 1 Feeding table for the grow-out culture of prawns.
Shrimp body
weight (g) Feed type
Pellet
diameter (mm)
Protein
content (%)
Feeding rate (% of body weight) Feed given in the
morning (%)
Feed given in the
afternoon (%)
Feed given in
the evening (%)19–24C24–28C28–32C
1–3 Prawn grower 2 32 10 8 9 30 20 50
3–5 Prawn grower 2 32 9 7 8 35 15 50
5–7 Prawn grower 2 32 8.5 6.5 7.5 40 10 50
7–9 Prawn grower 2 32 8 6 7 40 5 55
9–11 Prawn grower 2 32 7.5 5.5 6.5 40 0 60
11–13 Prawn grower 2 32 7 5 6 40 0 60
13–15 Prawn grower 2 32 6.5 4.5 5.5 40 0 60
15–17 Prawn grower 2 32 5.5 3.5 4.5 40 0 60
17–30 Prawn grower 2 32 4.5 3.0 3.5 40 0 60
>30 Prawn grower 2 32 3.5 3.0 3.0 40 0 60
650 AZAD ET AL.
DWG ¼WtW0
t
In which DWG is daily weight gain, W
t
is the final weight, W
0
is the initial weight and tis the time in days.
DGR ¼WtW0
t100
In which DGR is the daily growth rate, W
t
is the final weight, W
0
is the initial weight and tis the time in days.
RGR ¼WtW0
W0
100
In which RGR is the relative growth rate, W
t
is the final weight and W
0
is the initial weight.
SGR %ðÞ¼
Ln Wt
ðÞ
Ln W0
ðÞ
t100
In which SGR is the specific growth rate, W
t
is the final weight, W
0
is the initial weight and tis the time in days.
FCR ¼Wf
WtW0
Where, FCR is the feed conversion ratio, W
t
is the final weight, W
0
is the initial weight, and W
f
weight of dry feed
offered (g).
PER ¼WtW0
Wp
Where, PER is the protein efficiency ratio, W
t
is the final weight, W
0
is the initial weight, and W
p
is the weight of pro-
tein offered (g).
PCR ¼Wbpt Wbpi
Wp
Where, PCR is the protein conversion ratio, W
bpt
is the final body protein weight, W
bpi
is the initial body protein
weight, and W
p
is the weight of protein offered (g).
NPUa¼Wbpt Wbpi
Wp
100
Where, NPU
a
is apparent net protein utilization, W
bpt
is the final body protein weight, W
bpi
is the initial body protein
weight and W
p
is the weight of protein offered (g).
SR %ðÞ¼
Nt
N0
100
AZAD ET AL.651
Where, SR is the survival rate, N
t
is the number of live prawns harvested, and N
0
is the initial number of prawns.
GP kg per ha
ðÞ
¼Abw Nt
1000
Where, GP is gross production (kg/ha), A
bw
is average body weight after harvesting (g), and N
t
is the number of
prawns harvested.
NP kg per haðÞ¼
WGNt
1000
Where, NP is net production (kgha
1
), W
G
is average weight gain (g), ad N
t
is the number of prawns harvested.
The length-weight relationship was determined by the formula,
W¼aLb
where W=body weight (g), L=length of pbody (cm), a=regression intercept, and b=slope of the equation.
The formula was linearized by logarithmic transformation and the value of “a”and “b”was estimated,
Log10 WðÞ¼log10aþblog 10 LðÞ
The condition factor was determined by Fulton's condition factor (K) equation:
K¼W100
L3,
where W=wet weight of prawn (g), K=Fulton's condition factor, L
3
=total length of the weighted prawn (cm).
2.8 |Proximate composition determination
The whole body of the prawns, including muscle, appendages, and exoskeleton was used to determine the dry mat-
ter basis of the proximate composition. Association of Official Analytical Chemists (AOAC) standard procedure was
used to analyze crude protein (method 976.06), and ash (method 942.05) (AOAC, 2000). Lipid content was deter-
mined following the method of Bligh and Dyer (1959), and moisture content was measured by following the method
of Reeb, Milota, and Association (1999).
2.9 |Cost–benefit analysis
To determine the feasibility of the culture technology, a cost–benefit analysis was conducted for a culture cycle in
this study. All costs associated with the experimental ponds and production systems were recorded. Depreciation
costs, including depreciation on pumps, pump shade, hatchery tanks, and oxygen blowers were considered as fixed
costs. Depreciation was calculated by the straight-line method by following Shang (1990) and Engle (2010).
The considerable variable costs were the purchase cost of seed, feed, pond renovation, probiotics, labor cost,
harvesting cost, and power supply cost. For a single-cycle production cost–benefit analysis, gross return (GR) and
income above variable cost were determined. Finally, the net return to land, family labor, and management was
calculated to determine profit. All the calculations were done by the following formula:
652 AZAD ET AL.
TC ¼VC þFC,
where TC =total cost, VC =variable cost, FC =fixed cost.
GR ¼GP SP,
where GR =gross return, GP =gross production, SP =selling price.
IAVC ¼GR VC,
where IAVC =income above variable cost, GR =gross return, VC =variable costs.
Returns to land, family labor,and management ¼GRTC,
where GR =gross return, TC =total cost. In this study costs for family-owned ponds, management, and labor were
excluded.
2.10 |Statistical analysis
Growth, production, and financial data were analyzed by one-way ANOVA. Significant differences among the vari-
ables were tested using Tukey's post hoc test. Data that did not follow a normal distribution were transformed
through arcsine transformation to satisfy the condition of homogeneity of variance. A non-parametric Kruskal-Wallis
H-test was used to analyze data that did not satisfy the assumptions of a parametric test. Other simple comparative
graphs were developed using Microsoft Excel 2010. All statistical tests were performed using SPSS 27.0 and consid-
ering statistically significant a pvalue of <0.05.
3|RESULTS
3.1 |Water quality parameters
The recorded lowest and highest temperatures were 19.9 ± 0.9 and 30.9 ± 0.7CinC, 19.3 ± 0.9 and 31.0 ± 0.7C
in T
1
, and 19.3 ± 0.8 and 30.9 ± 0.7CinT
2
. pH ranged between 7.5 ± 0.1 and 7.8 ± 0.1 and DO between
3.8 ± 0.1 and 5.7 ± 0.3 mg/L were not significantly different between the experimental ponds. NO
2
-N con-
centration was 0.03 ± 0.02 and 0.18 ± 0.03 mg/L in C, 0.01 ± 0.00 and 0.11 ± 0.04 mg/L in T
1
, 0.01 ± 0.01
and 0.16 ± 0.06 mg/L in T
2
. During the 5 months grow-out phase, NH
3
-N and NO
2
-N were significantly
lower in T
1
compared to Cduring August and September. T
1
also had significantly reduced NH
3
-N concen-
tration compared to Cand T
2
during November and December, it also reduced the NO
2
-N concentration
than the control and T
2
group in December (p< 0.05). Despite this observation, other water quality param-
eters were found within a favorable range (Table 2).
3.2 |Soil quality parameters
Soil pH of the control ponds ranged between 6.3 ± 0.1 and 6.8 ± 0.2, which increased significantly in the probiotic-
treated ponds, ranging from 6.7 ± 0.2 to 7.2 ± 0.3 and 6.7 ± 0.2 to 7.1 ± 0.4 in T
1
and T
2
, respectively (p< 0.05).
AZAD ET AL.653
TABLE 2 Water quality parameters (mean ± standard deviation) of the cultured ponds.
Treatment Month pH DO (mg/L) Salinity (ppt) Temperature (C)
Alkalinity
(MgCaCO
3
mg
/
L) Hardness (mg/L)
Non-ionized
ammonia (mg/L)
Nitrite
nitrogen (mg/L)
CAug 7.7 ± 0.1
a
5.5 ± 0.3
a
0.5 ± 0.0
a
30.9 ± 0.7
a
162.4 ± 27.4
a
237.2 ± 37.2
a
0.011 ± 0.00
a
0.03 ± 0.02
a
T
1
7.7 ± 0.1
a
5.7 ± 0.3
a
0.3 ± 0.3
a
31.0 ± 0.7
a
157.8 ± 25.6
a
245.2 ± 17.1
a
0.006 ± 0.00
b
0.01 ± 0.00
b
T
2
7.8 ± 0.1
a
5.5 ± 0.2
a
0.5 ± 0.0
a
30.9 ± 0.7
a
154.4 ± 22.4
a
219.9 ± 24.7
a
0.009 ± 0.00
ab
0.01 ± 0.01
ab
CSep 7.7 ± 0.2
a
5.3 ± 0.3
a
0.5 ± 0.0
a
30.8 ± 0.8
a
147.5 ± 20.4
a
234.2 ± 38.5
a
0.011 ± 0.01
a
0.08 ± 0.02
a
T
1
7.8 ± 0.1
a
5.3 ± 0.3
a
0.3 ± 0.3
a
30.9 ± 0.8
a
147.5 ± 19.2
a
264 ± 15.7
a
0.004 ± 0.00
b
0.04 ± 0.02
b
T
2
7.7 ± 0.1
a
5.3 ± 0.2
a
0.4 ± 0.2
a
30.8 ± 0.8
a
148.3 ± 15.1
a
242.3 ± 17.7
a
0.010 ± 0.00
ab
0.06 ± 0.02
ab
COct 7.6 ± 0.1
a
4.8 ± 0.2
a
0.3 ± 0.3
a
30.9 ± 0.3
a
135 ± 7.1
a
225.8 ± 44.1
a
0.010 ± 0.01
a
0.13 ± 0.02
a
T
1
7.7 ± 0.1
a
5.0 ± 0.1
a
0.4 ± 0.2
a
31.0 ± 0.5
a
133.3 ± 4.1
a
254.2 ± 14.6
a
0.007 ± 0.00
a
0.08 ± 0.01
a
T
2
7.7 ± 0.1
a
4.9 ± 0.1
a
0.3 ± 0.3
a
30.9 ± 0.3
a
135.8 ± 13.2
a
231.7 ± 27.3
a
0.011 ± 0.01
a
0.10 ± 0.01
a
CNov 7.6 ± 0.1
a
4.1 ± 0.3
a
0.9 ± 0.5
a
23.2 ± 2.0
a
163.3 ± 25.0
a
270.8 ± 22.9
a
0.007 ± 0.00
a
0.11 ± 0.01
a
T
1
7.7 ± 0.1
a
4.3 ± 0.4
a
0.8 ± 0.4
a
23.6 ± 2.4
a
182.5 ± 15.7
a
261.7 ± 36.4
a
0.004 ± 0.00
b
0.07 ± 0.02
b
T
2
7.7 ± 0.1
a
4.1 ± 0.3
a
0.9 ± 0.5
a
23.1 ± 2.1
a
175.8 ± 14.3
a
269.5 ± 23.6
a
0.009 ± 0.00
a
0.09 ± 0.04
ab
CDec 7.5 ± 0.1
a
3.8 ± 0.1
a
2.4 ± 0.8
a
19.9 ± 0.7
a
166.7 ± 19.7
a
320 ± 15.8
a
0.005 ± 0.00
a
0.18 ± 0.03
a
T
1
7.6 ± 0.0
a
3.9 ± 0.2
a
2.3 ± 0.7
a
19.3 ± 0.9
a
179.2 ± 11.1
a
312.5 ± 23.6
a
0.002 ± 0.00
b
0.11 ± 0.04
b
T
2
7.6 ± 0.1
a
3.9 ± 0.2
a
2.4 ± 0.5
a
19.3 ± 0.8
a
175.8 ± 15.9
a
302.5 ± 19.4
a
0.005 ± 0.00
a
0.16 ± 0.06
a
Note: Different superscript letters indicate statistical significance between treatments (one-way ANOVA, p< 0.05).
654 AZAD ET AL.
In addition, organic carbon levels were significantly lower in T
2
compared to C (p< 0.05). Significantly, lower levels
of available P were observed in T
1
and T
2
than that in C (p< 0.05) (Table 3).
3.3 |Growth performance and feed utilization of prawns
3.3.1 | Growth and production performance
After 5 months of rearing in grow-out ponds, the final weight and growth increment were significantly higher in
T
1
and T
2
compared to group C(p< 0.05) (Table 4; Figures S1 and S2). Similarly, specific growth rate, daily
growth rate, gross and net production, and percent survival were significantly higher in the T
1
,followedbyT
2
than that in group C(p< 0.05). However, the highest relative growth rate was observed in T
2
.Incontrast,T
1
and T
2
showed significantly lower FCR in comparison to group C. The apparent net protein utilization (NPU
a
),
protein efficiency ratio, and protein conversion rate were significantly higher in T
1
,followedbyT
2
,comparedto
group C(p<0.05).
3.3.2 | Length-weight relationship
Length-weight regression equations demonstrated that prawns treated with zymetin and super PS during the entire
culture cycle showed higher positive allometric growth (b=3.34). Whereas the growth pattern was isometric in T
2
(b=3.08) and C(b=2.94). The coefficient of determination (r
2
) showed a strong correlation between body weight
and prawn length in all the experimental groups (Table 5, Figure 1).
TABLE 3 Soil quality parameters (mean ± standard deviation) in all the ponds under different treatments.
Treatment Month pH
Organic
carbon (%)
Electrical
conductivity (ds/m) P (meq 100/g) Ca (%) Mg (%)
CAug 6.6 ± 0.3
a
1.12 ± 0.0
a
2.42 ± 0.1
a
0.0264 ± 0.0
a
0.15 ± 0.0
a
0.09 ± 0.0
a
T
1
7.2 ± 0.3
b
1.12 ± 0.0
ab
2.41 ± 0.1
a
0.0234 ± 0.0
b
0.15 ± 0.0
a
0.09 ± 0.0
a
T
2
7.0 ± 0.3
ab
1.11 ± 0.0
b
2.36 ± 0.1
a
0.0231 ± 0.0
c
0.14 ± 0.0
a
0.08 ± 0.0
a
CSep 6.8 ± 0.2
a
1.12 ± 0.0
a
2.38 ± 0.0
a
0.0264 ± 0.0
a
0.15 ± 0.0
a
0.08 ± 0.0
a
T
1
7.2 ± 0.3
b
1.12 ± 0.0
ab
2.36 ± 0.1
a
0.0233 ± 0.0
b
0.15 ± 0.0
a
0.08 ± 0.0
a
T
2
7.0 ± 0.4
ab
1.11 ± 0.0
b
2.37 ± 0.1
a
0.0231 ± 0.0
c
0.14 ± 0.0
a
0.08 ± 0.0
a
COct 6.7 ± 0.1
a
1.13 ± 0.0
a
2.34 ± 0.1
a
0.0263 ± 0.0
a
0.15 ± 0.0
a
0.09 ± 0.0
a
T
1
7.2 ± 0.3
b
1.12 ± 0.0
ab
2.37 ± 0.1
a
0.0232 ± 0.0
b
0.15 ± 0.0
a
0.09 ± 0.0
a
T
2
7.1 ± 0.4
b
1.12 ± 0.0
b
2.37 ± 0.1
a
0.0230 ± 0.0
c
0.14 ± 0.0
a
0.09 ± 0.0
a
CNov 6.4 ± 0.1
a
1.13 ± 0.0
a
6.12 ± 0.3
a
0.0263 ± 0.0
a
0.14 ± 0.0
a
0.09 ± 0.0
a
T
1
6.9 ± 0.2
b
1.12 ± 0.0
ab
5.98 ± 0.3
a
0.0232 ± 0.0
b
0.14 ± 0.0
a
0.08 ± 0.0
a
T
2
6.8 ± 0.2
b
1.12 ± 0.0
b
5.91 ± 0.3
a
0.0230 ± 0.0
c
0.14 ± 0.0
a
0.08 ± 0.0
a
CDec 6.3 ± 0.1
a
1.13 ± 0.0
a
6.16 ± 0.4
a
0.0262 ± 0.0
a
0.15 ± 0.0
a
0.08 ± 0.0
a
T
1
6.7 ± 0.2
b
1.13 ± 0.0
ab
5.89 ± 0.3
ab
0.0231 ± 0.0
b
0.15 ± 0.0
a
0.08 ± 0.0
a
T
2
6.7 ± 0.2
b
1.12 ± 0.0
b
5.70 ± 0.3
b
0.0230 ± 0.0
c
0.14 ± 0.0
a
0.08 ± 0.0
a
Note: Different superscript letters indicate significant differences between the treatments (one-way ANOVA and Kruskal
Wallis Test, p< 0.05).
AZAD ET AL.655
3.3.3 | Condition factor
The Fulton's condition factor Kwas found significantly higher in probiotic-treated prawns (T
1
, followed by T
2
) com-
pared to the non-treated groups during the grow-out period, except in the month of October (p< 0.001, 0.01, and
0.05). The highest value of Kwas in T
1
in December and the lowest value was in Cin July. On average, the Kvalue
was lower in September and higher in December (Table 6).
3.3.4 | Proximate composition
A significantly higher protein content was found in the prawns treated with probiotics in the hatchery, nursery, and
grow-out phases compared to other groups (p< 0.05). Ash content of the probiotic-treated groups was also signifi-
cantly higher than that of the control group (p< 0.001). Interestingly, moisture content reduced significantly in T
2
compared to T
1
and C(p< 0.001). Probiotics did not have an impact on the fat content of prawns (Table 7).
TABLE 4 Growth (g) (mean ± standard deviation), feed utilization (mean ± standard deviation), and production
(mean ± standard deviation) performance of Macrobrachium rosenbergii in different experimental groups.
CT
1
T
2
Initial weight (g) 2.50 ± 1.18 2.90 ± 0.78 2.50 ± 1.18
Final weight (g) 36.27 ± 11.78
a
46.16 ± 16.48
b
42.13 ± 20.45
b
Weight gain (g) 33.37 ± 11.85
a
43.08 ± 16.25
b
39.63 ± 20.34
b
DWG (g/day) 0.22 ± 0.08
a
0.29 ± 0.11
b
0.26 ± 0.14
b
DGR (%) 22.24 ± 7.52
a
28.72 ± 10.83
b
26.42 ± 13.56
b
SGR (%BW/day) 1.67 ± 0.19
a
1.82 ± 0.31
b
1.88 ± 0.42
b
RGR (%) 1177.95 ± 344.28
a
1584.73 ± 742.74
b
1927.61 ± 1257.08
c
Survival rate (%) 86.66 ± 0.84
a
92.91 ± 2.17
b
90.83 ± 0.84
b
FCR 2.08 ± 0.11
a
1.51 ± 0.11
b
1.68 ± 0.05
b
PER 1.48 ± 0.05
a
2.04 ± 0.08
b
1.84 ± 0.05
c
PCR 0.97 ± 0.02
a
1.38 ± 0.04
b
1.22 ± 0.02
c
NPUa (%) 25.33 ± 0.84
a
40.25 ± 0.33
b
29.78 ± 0.81
c
Gross production (kgha
1
150 d
1
) 628.72 ± 26.39
a
858.27 ± 59.84
b
765.45 ± 27.65
b
Net production (kgha
1
150 d
1
) 570.64 ± 31.22
a
796.64 ± 56.69
b
715.50 ± 21.06
b
Note: Different superscript letters indicate statistically significant differences between groups (one-way ANOVA, p< 0.05).
Abbreviations: DGR, daily growth rate; DWG, daily weight gain; FCE, feed conversion efficiency; FCR, feed conversion ratio;
NPU
a
, apparent net protein utilization; PER, protein efficiency ratio; RGR, relative growth rate; SGR, specific growth rate.
TABLE 5 Length-weight relation in coefficient of functional regression parameters, and growth pattern of
probiotic treated Macrobrachium rosenbergii.
Treatment n
Body length (cm) Body weight (g) Linear regression parameters
Growth patternMin. Max. Median Min. Max. Median abr
2
C614 4.10 22.30 12.80 1.30 97.50 19.70 1.90 2.94 0.95 Isometric
T
1
614 5.30 20.40 14.50 1.00 102.50 28.29 2.41 3.34 0.94 Positive allometric
T
2
667 5.2 20.2 14 1.9 94.4 24.9 2.08 3.08 0.94 Isometric
656 AZAD ET AL.
3.4 |Cost–benefit analysis
This study used an institute's hatchery facilities and earthen experimental ponds. The estimated fixed cost for one
cycle of production per hectare was significantly higher in T
1
(US$179.38 ± 0.00) compared to Cand T
2
(US
$141.25 ± 0.00, respectively) (p< 0.05). Significantly higher variable cost and total cost per hectare were estimated
in T
1
(US$3567.79 ± 49.37 and US$3747.17 ± 49.37), followed by T
2
(US$3006.38 ± 58.26 and US
$3147.63 ± 58.26) than that of C(US$2347.86 ± 26.19 and US$2489.11 ± 26.19) (p< 0.001). The highest net return to
land, family labor, and management was observed in T
1
(US$3778.33 ± 61.08), followed by T
2
(US$3263.02 ± 173.30)
and C(US$2619.21 ± 188.22) (p<0.01)(Table8). In T
1
, probiotics zymetin and super PS were applied during the entire
BW = 2.9358BL - 1.9002BW = 2.9358BL - 1.9002
R² = 0.946, n = 614
R² = 0.946, n = 614
00
0.50.5
11
1.51.5
2
2
2.52.5
0.5 0.7 0.9 1.1 1.3 1.50.5 0.7 0.9 1.1 1.3 1.5
Log10(weight (g))Log10(weight (g))
Log10(length (cm))Log10(length (cm))
BW = 3.3449BL - 2.4091BW = 3.3449BL - 2.4091
R² = 0.9447, n = 614R² = 0.9447, n = 614
00
0.50.5
11
1.51.5
2
2
2.52.5
0.5 0.7 0.9 1.1 1.3 1.50.5 0.7 0.9 1.1 1.3 1.5
Log10(weight (g))Log10(weight (g))
Log10(length (cm))Log10(length (cm))
BW = 3.0782BL - 2.079BW = 3.0782BL - 2.079
R² = 0.944, n = 667R² = 0.944, n = 667
00
0.50.5
11
1.5
1.5
2
2
2.52.5
0.5 0.7 0.9 1.1 1.3 1.50.5 0.7 0.9 1.1 1.3 1.5
Log10(weight (g))Log10(weight (g))
Log10(length (cm))Log10(length (cm))
(a)
(c)
(b)
FIGURE 1 Length-weight relationship of Macrobrachium rosenbergii cultured under, (a) no probiotic treatment,
(b) zymetin and super PS probiotic treatment during hatchery, nursery, and grow-out phase, and (c) zymetin and
super PS treatment during grow-out culture.
TABLE 6 Fulton's condition factor (K) (mean ± standard deviation) of Macrobrachium rosenbergii over the culture
period.
Treatments
Grow-out culture period (months)
Jul Aug Sep Oct Nov Dec
C0.84 ± 0.23
a
0.88 ± 0.13
a
0.87 ± 0.11
a
0.99 ± 0.17
a
1.09 ± 0.19
ac
1.12 ± 0.17
a
T
1
1.17 ± 0.70
b
1.03 ± 0.17
b
0.97 ± 0.15
b
0.98 ± 0.20
a
1.15 ± 0.19
b
1.23 ± 0.15
b
T
2
1.02 ± 0.21
c
0.97 ± 0.19
c
0.94 ± 0.17
b
1.01 ± 0.21
a
1.14 ± 0.16
bc
1.14 ± 0.14
a
Note: Different superscript letters over the six-month grow-out period in different treatments (Kruskal-Wallis test, p< 0.05).
AZAD ET AL.657
TABLE 7 Proximate composition (dry weight basis) of prawn in % of body weight (mean ± standard deviation).
Treatments
Proximate compositions
Protein Lipid Ash Moisture
C65.27 ± 0.65
a
4.52 ± 0.08
a
12.66 ± 0.04
a
9.10 ± 0.29
a
T
1
67.22 ± 0.60
b
4.44 ± 0.23
a
13.47 ± 0.07
b
9.06 ± 0.49
a
T
2
66.07 ± 0.68
ab
4.25 ± 0.08
a
14.31 ± 0.15
c
6.06 ± 0.27
b
Note: Different superscript letters indicate a significant difference between the treatments (one-way ANOVA, p< 0.05).
TABLE 8 Cost–benefit analysis (mean ± standard deviation) of probiotic-treated prawns during different culture
stages.
Items
Experimental groups
CT
1
T
2
A. Items of cost
1. Fixed cost (FC) (US$/hec)
1.1 Depreciation costs on
1.1.1 Pumps 40.63 ± 0.00a 40.63 ± 0.00
b
40.63 ± 0.00
a
1.1.2 Pump shade 62.50 ± 0.00
a
62.50 ± 0.00
a
62.50 ± 0.00
a
1.1.3 Hatchery tank 25.00 ± 0.00
a
50.00 ± 0.00
b
25.00 ± 0.00
a
1.1.4 Oxygen blower 13.13 ± 0.00
a
26.25 ± 0.00
a
13.13 ± 0.00
a
Total FC 141.25 ± 0.00
a
179.38 ± 0.00
b
141.25 ± 0.00
a
2. Variable cost (VC) (US$/hec)
2.1 Seed 812.50 ± 0.00
a
1062.50 ± 0.00
b
812.50 ± 0.00
a
2.2 Feed 1064.58 ± 9.55
a
1204.17 ± 19.09
b
1064.58 ± 9.55
a
2.3 Probiotics 0.00 ± 0.00
a
807.50 ± 6.61
b
658.17 ± 32.25
c
2.4 Pond renovation and preparation 92.71 ± 1.80
a
93.92 ± 6.26
a
94.38 ± 1.08
a
2.5 Bamboo, spade, pipe, net bucket, and so on 46.88 ± 0.00
a
46.88 ± 0.00
a
46.88 ± 0.00
a
2.6 Labour costs 116.67 ± 1.91
a
116.25 ± 1.65
a
108.96 ± 3.15
b
2.7 Harvesting costs 76.17 ± 4.54
a
79.79 ± 4.61
a
79.13 ± 2.49
a
2.8 Power supply/ cost for electricity 31.90 ± 0.61
a
34.50 ± 3.68
a
32.21 ± 0.73
a
2.9 Miscellaneous 64.58 ± 5.91
a
75.25 ± 4.51
a
65.00 ± 5.45
a
2.10 Other maintenance cost 41.88 ± 1.88
a
46.04 ± 2.95
a
44.58 ± 3.55
a
Total VC 2347.86 ± 26.19
a
3567.79 ± 49.37
b
3006.38 ± 58.26
c
Total cost (TC) (FC +VC) 2489.11 ± 26.19
a
3747.17 ± 49.37
b
3147.63 ± 58.26
c
B. Returns (US$/hec)
Gross production (kg/hec) 628.72 ± 26.39
a
858.27 ± 59.84
b
765.45 ± 27.65
b
Gross returns 5108.32 ± 214.41
a
7525.50 ± 110.44
b
6410.64 ± 231.56
c
Income above variable cost 2760.46 ± 188.22
a
3957.70 ± 61.08
b
3404.27 ± 173.30
c
Net returns to land, family labor,
and management
2619.21 ± 188.22
a
3778.33 ± 61.08
b
3263.02 ± 173.30
c
Note: Different superscript letters indicate the significant difference between the treatments (one-way ANOVA and
Kruskal-Wallis test, p< 0.05).
658 AZAD ET AL.
culture period. Therefore, the application of probiotics from the hatchery-rearing stage to the end of the culture cycle can
significantly increase the earnings of the farmers.
4|DISCUSSION
4.1 |Effect of zymetin and super PS on the culture environment of M. rosenbergii
Optimum water quality in aquaculture is essential for the survival and growth of fish and shellfish. The water quality
was found within the suitable range during this study (Table 2; dos Santos et al., 2022; New, 2002). The range of
pH 7.0–8.5 is considered optimum for a healthy aquaculture environment (Boyd & Zimmermann, 2010; New, 2002).
In this study, the pH of all ponds ranged from 7.3 to 8.1, which reflected the optimum range for the growth of
M. rosenbergii (Boyd & Zimmermann, 2010; New, 2002). Similarly, although not significant, other water quality
parameters, including dissolved oxygen, hardness, alkalinity, and water salinity were found within the optimal cultiva-
ble range (New, 2002; Zafar et al., 2015). In contrast, NH
3
-N and NO
2
-N concentration was found lower in T
1
com-
pared to Cand T
2
from August to December, except for October 2015. Rhodobacter sp. present in the super PS
probiotic is a potential denitrifying bacterium, entering two or more denitrification steps, causing nitrous oxide
reduction or removal of nitrogen (LaSarre et al., 2022; Pang et al., 2022). On the other hand, Rhodococcus sp. is
a nitrifying-aerobic denitrifying bacteria directly convert pollutant associated nitrogen into nitrogen, effectively
used as biological nitrogenremover(Wangetal.,2022). Several authors have reported that probiotic
(e.g., Bacillus,Lactobacillus) application in fish and shrimp farming reduce the NH
3
-N (Hang et al., 2008;
Sakkaravarthi et al., 2010)andNO
2
-N level (Jha, 2011). Apart from the growth and production performance of
M. rosenbergii, we also report reduced non-ionized ammonia and nitrite-nitrogen in T
1
,aswellasT
2
,whichis
indicative of the activity of zymetin and super PS as environmental modulators (Kumar et al., 2016;Matias
et al., 2002). However, this study was conducted in a low stocking density culture system. We suggest con-
ducting an experiment in a highly crowded semi-intensive or intensive prawn culture system, where probiotic
treatment may exert greater control over water quality parameters.
Farm soil quality is essential to provide a suitable aquatic environment for prawn production (Boyd &
Zimmermann, 2010;New,2002). Soil quality significantly influences water pH (Mosley et al., 2014). In this study, signifi-
cantly lower pH was observed in the control group compared to the treatment groups. This suggests that soil probiotic
bacteria in super PS, especially Rhodococcus spp., may improve soil quality by degrading bottom pollutants thereby provid-
ing an environment more suitable for aquatic organisms (Table 3; Aislabie et al., 2013). In addition, Rhodobacter spp. fixes
the CO
2
and reduces the oxygen tension by photoautotrophic and photoheterotrophic mechanisms (McEwan, 1994).
Moreover, feed probiotics (Bacillus spp.) have been reported to have the capability to degrade plant polysaccharides as
well as fix or denitrify nitrogen, whereas Clostridium spp. is well-known in fermenting sugars, starch, pectin, and cellulose
(Aislabie et al., 2013).
In this study, organic carbon was found lower in T
2
compared to C(p< 0.01), which indicates that probiotics
might have some effect on organic carbon assimilation. Moreover, the percent organic carbon in soil was found to
vary from 1.11% to 1.13%, which is similar to 0.82% ± 0.03% recorded in southwest Bangladesh (Zafar et al., 2015).
The electric conductivity of soil is regulated by its pH and aggregated ion concentrations (Carmo et al., 2016). The
results of the current study presented a fluctuation in EC which ranged from 2.36 to 2.42 ds/m from August to
October, which increased up to 5.70–6.16 ds/m during November and December (Table 3). Fluctuations in electric
conductivity in later months might be due to the increase of ionic concentration in soil due to evaporation and a lim-
ited supply of freshwater (e.g., low precipitation). Adhikari et al. (2007) observed that EC (ds/m) increases in
0.33–0.94 ds/m with the increase of calcium hardness (mg/L as CaCO
3
).
Nevertheless, the phosphorus concentration in the soil was found significantly lower in T
1
and T
2
compared to
C, which means that soil probiotic bacteria, for example, Rhodococcus spp. and Rhodobacter spp., might convert
AZAD ET AL.659
inorganic phosphorus to organic phosphorus or may reduce it by other biogenic mechanisms. It has been reported
that Rhodococcus spp. has P solubilization (Chen et al., 2006; Sharma et al., 2013) and bio-filtering capacity (Gamal-
Eldin & Elbanna, 2011).
4.2 |Effect of zymetin and super PS on the growth and feed utilization of prawn
Probiotics have roles in controlling pathogenic bacteria, enhancing growth rates, and contributing to the welfare of
fish and shellfish in aquaculture production (Martínez Cruz et al., 2012; Yanbo & Zirong, 2006). This study investi-
gated the effect of two commercial probiotics on the growth, production, and feed utilization of M. rosenbergii during
a production cycle. The results revealed a significant difference in several growth parameters (i.e., final weight,
weight gain, DWG, DGR, and survival) in T
1
, followed by T
2
compared to C. Relatively higher growth performance in
T
1
than that of T
2
might be due to the application of probiotics during all culture stages rather than only in the grow-
out phase. Animals under T
1
are potentially linked to better digestion and assimilation of feed, and improved quality
of soil and water in treated ponds. T
2
, prawn treated with probiotics at the grow-out phase also showed higher
growth performance compared to the control group. A similar observation was claimed in a previous report, that the
combined effect of zymetin and super PS in the grow-out stage of prawns was higher than their individual applica-
tion (Ghosh et al., 2016). Combined application of these probiotics also supports prawn production at higher stocking
densities up to 4–5 juveniles/m
2
(Azad et al., 2021). These observations are in accordance with previous reports, for
example, shrimp fed with B. subtilis and Bacillus sp. showed improved growth (El-Dakar & Goher, 2004; Gullian
et al., 2004). Commercial probiotics zymetin and super PS, and combined application of B. subtilis and S. cerevisiae
have a positive impact on the digestion and growth of prawn (Ghosh et al., 2016; Seenivasan et al., 2015).
We observed that T
1
had a lower FCR than T
2
, as well as C, which potentially indicates better utilization of feed
due to the use of probiotics in supplied feed during all-life stages compared to no probiotic treatment or probiotic
treatment only during the grow-out stage. Probiotics have also been reported to have an impact on enhanced feed
utilization by improving digestive enzyme activities (e.g., amylase, protease, lipase, cellulase, and alginase), increasing
nutrient absorption, lowering FCR, and increasing protein utilization (Assan et al., 2022; El-Dakar & Goher, 2004;
Ghosh et al., 2016; Hossain et al., 2013). The results also showed that PER, PCR, and NPUa (%) were significantly
higher in T
1
compared to T
2
and C, which is indicative of the influence of probiotic treatment in all culture stages on
the protein utilization capacity of M. rosenbergii. Higher PER was reported in ponds supplemented with feed pro-
biotics L. plantarum, and commercial probiotics “Improval”(Dash et al., 2014; Gupta & Dhawan, 2013). Likewise,
Seenivasan et al. (2015) showed significantly higher PER in 3% B. subtilis and yeast S. cerevisiae incorporated diet-fed
PL group. Dietary probiotics L. sporogenes (Prasad et al., 2012), Biogen
®
(EL-Zahar Veterinary Trading Company,
Taiwan) (Saad et al., 2009), and B. subtilis and L. rhamnosus (Devi et al., 2015) were reported to have a significant
impact on PER and NPUa in M. rosenbergii.
We observed higher production in T
1
, followed by T
2
compared to C, which might relate to faster growth rate,
higher survival, and better feed utilization due to the support of probiotics throughout their culture cycles, compared
to probiotic use in single-stage or no probiotic support. Recently, we also reported higher production of prawns from
zymetin and super PS treated group compared to single probiotic treatment or the control group during their grow-
out stage (Ghosh et al., 2016). The increased survival, as reported from probiotic-treated prawn and shrimp (Hossain
et al., 2013; Rinisha et al., 2010; Seenivasan et al., 2011), might be due to the change in Bacillus bacterial community
in the gut microflora, likely excluding pathogenic bacteria, especially in the larval stage (Rengpipat et al., 1998). A
higher bvalue in T
1
(b=3.34, b> 3 is positive allometric) indicated higher growth pattern of prawn treated with
zymetin and super PS throughout their entire culture cycle. On the other hand, isometric growth in Cand T
2
also
indicated a reasonable index of shape (Riedel et al., 2007). Positive allometric, isometric, as well as negative allo-
metric (in females) growth pattern of prawn have been also recorded in previous studies (Azad et al., 2021;
Lalrinsanga et al., 2012; Rocha et al., 2015). Similarly, higher condition factor (K)inT
1
throughout the entire
660 AZAD ET AL.
culture cycle indicated the striking impact of probiotics to maintain their steady growth. In this study we found
higher condition factor (K)inT
1
(0.97–1.23) with the zymetin and super PS treatment during their entire culture
cycle than that of our recent study (Azad et al., 2021). Higher condition factor in T
1
and T
2
indicated that
zymetin and super PS enhanced the physical health of cultured prawn, well managed culture systems and
healthy culture environment (Araneda et al., 2008; Gopalakrishnan et al., 2014; Kuberan et al., 2022;
Rochet, 2000). Azad et al. (2021) described the condition factor of prawnusingthesameprobioticsonlyatthe
grow-out stage at stocking density 2 juvenile/m
2
ranged from 0.93 to 1.16, which is like T
2
(0.94–1.14) in this
study. The range of condition factor in this study was found higher than that of some other previous studies
(Karim & Uddin, 2008; Kunda et al., 2008; Lalrinsanga et al., 2012). Thus, this study suggests that zymetin and
super PS probiotics enhanced the nutritional and health status of shrimp through a series of beneficial activities,
including improving digestive system, protecting from pathogens, microbial balance in the gut, nutrient absorp-
tion, and supplying essential vitamins (El-Kady et al., 2022).
4.3 |Application of probiotics throughout the entire culture cycle of prawn and its
impact on economics
In this study, seed and feed were two major variable costs in the control and treatment groups, for example, it was
32.64% and 42.76% of the total cost in C, 28.35% and 32.14% in T
1
, and 25.81 and 33.82% in T
2
, respectively.
Moreover, the addition of probiotics during a production cycle significantly increased costs in T
1
(21.55% of the total
cost) and T
2
(20.91% of the total cost). Although this study demonstrated that T
1
was more expensive compared to
the Cand T
2
, the cost–benefit assessment indicated that using probiotics in the hatchery, nursery and grow-out
phase was more profitable than without probiotic culture, or culturing prawn using probiotics only in the grow-out
phase. Gross return, income above variable cost, and net returns to land, family labour, and management was
47.82%, 43.37%, and 44.25% higher in T
1
than that of C. However, it was 17.39%, 16.26%, and 15.78% higher in T
1
compared to T
2
(Table 8).
In conclusion, this study suggests that the application of zymetin and super PS in the entire culture cycle of
M. rosenbergii increases growth and production, improves feed utilization, and provides control over the culture envi-
ronment. Notably, the probiotic application only in the grow-out phase also significantly increases prawn production
and keeps the culture environment healthy. However, considering the higher net returns to land, family labor,
and management, this study suggests that using probiotics in the hatchery, nursery, and grow-out phase of giant
freshwater prawn culture may be more profitable than the application of probiotics only during the grow-out phase.
Further research may investigate the impact of other probiotics on the growth and immunity during their application
in the entire culture cycle of prawns.
ACKNOWLEDGMENTS
The authors' sincere acknowledgment goes to Shrimp Research Station, Bangladesh Fisheries Research Institute,
Bagerhat, Bangladesh for their support to allow us to use their prawn hatchery and nursery tanks.
FUNDING INFORMATION
This research project was funded by the Ministry of Education (Grant ID: 37.01.0000.078.02.018.13-206(38)/6) and
the Ministry of Science and Technology (Grant ID: 39.012.002.01.03.022.2015-439), Government of the People's
Republic of Bangladesh.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
AZAD ET AL.661
ORCID
Shikder S. Islam https://orcid.org/0000-0001-7134-6736
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SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this
article.
How to cite this article: Azad, M. A. K., Islam, S. S., Ghosh, A. K., Hasanuzzaman, A. F. M., Smith, A. J., Bir, J.,
Ahmmed, M. K., Ahmmed, F., Banu, G. R., & Huq, K. A. (2023). Application of zymetin and super PS probiotics
in hatchery, nursery, and grow-out phases of Macrobrachium rosenbergii and their impact on culture
environment, production, and economics. Journal of the World Aquaculture Society,54(3), 645–665. https://
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