Embryonic developmental stages, different doses of inducing hormones and effect of different larval feeds on growth and survival of spiny eel, Macrognathus aculeatus (Bloch, 1786) larvae

Abstract

Induced breeding of Macrognathus aculeatus was carried out in order to determine the most appropriate dose of carp pituitary extract (CPE) and OVAFISH hormone. The best dose of CPE was 100 mg/kg body weight of female and 45mg/kg body weight of male with fertilization rate 81.34% and the best dose of OVAFISH was 1.5 ml/kg body weight of female and 0.75 ml/kg body weight of male with fertilization rate 91.96%. The fertilized eggs were round, sticky, demersal in nature and greenish in color. The perivitelline space of fertilized eggs was observed in 17 min. The First cleavage appeared at 56 min after fertilization (AF), producing two equal blastomeres. The cell division was completed in 4.17h. The fertilized egg took 6.47, 10.54, and 15.12 h to reach morula, blastula, and gastrula stage respectively. The eggs were hatched 34.17h AF at 27-28°C. Five days old post-hatchlings were reared for four weeks in a 160 L tanks using four different diets i.e., artificial feed, mix zooplankton, artemia nauplii and egg custard. The finding showed that hatchlings fed on mix zooplankton had a higher specific growth rate (SGR) (9.60±0.25). The significantly higher mean survival rate was also observed in larvae fed with mix zooplankton (47.67±11.25%) followed by artemia nauplii (38.5±5.4%) and artificial powdered feed (18.17±3.68%) for 28 days experiment. Our finding suggests CPE best dose @ 100mg/kg body weight for female and 45 mg/kg body weight for male and best dose of OVAFISH @1.5 ml/kg body weight of female and 0.75 ml/kg body weight of male for induced breeding. While in larval rearing experiment highest survival and growth rate was obtained in the larvae fed with mix zooplankton for 28 days trial.

Full text

Page 1/24
Embryonic developmental stages, different doses of
inducing hormones and effect of different larval
feeds on growth and survival of spiny eel,
Macrognathus aculeatus (Bloch, 1786) larvae
Bhumika Gamango
Dr. Rajendra Prasad Central Agricultural University
Raj Kamal Mishra
Dr. Rajendra Prasad Central Agricultural University
Aditi Banik
Dr. Rajendra Prasad Central Agricultural University
Shivendra Kumar
Dr. Rajendra Prasad Central Agricultural University
Roshan Kumar Ram
Dr. Rajendra Prasad Central Agricultural University
Prem Prakash Srivastava
Dr. Rajendra Prasad Central Agricultural University
Pravesh Kumar (  pravesh.cof@rpcau.ac.in )
Dr. Rajendra Prasad Central Agricultural University
Research Article
Keywords: Macrognathus aculeatus, Induced breeding, Embryonic development, Larval rearing, Carp
pituitary extract, OVAFISH
Posted Date: June 24th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2805964/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

Page 2/24
Abstract
Induced breeding of
Macrognathus aculeatus
was carried out in order to determine the most appropriate
dose of carp pituitary extract (CPE) and OVAFISH hormone. The best dose of CPE was 100 mg/kg body
weight of female and 45mg/kg body weight of male with fertilization rate 81.34% and the best dose of
OVAFISH was 1.5 ml/kg body weight of female and 0.75 ml/kg body weight of male with fertilization rate
91.96%. The fertilized eggs were round, sticky, demersal in nature and greenish in color. The perivitelline
space of fertilized eggs was observed in 17 min. The First cleavage appeared at 56 min after fertilization
(AF), producing two equal blastomeres. The cell division was completed in 4.17h. The fertilized egg took
6.47, 10.54, and 15.12 h to reach morula, blastula, and gastrula stage respectively. The eggs were
hatched 34.17h AF at 27-28°C. Five days old post-hatchlings were reared for four weeks in a 160 L tanks
using four different diets i.e., articial feed, mix zooplankton, artemia nauplii and egg custard. The nding
showed that hatchlings fed on mix zooplankton had a higher specic growth rate (SGR) (9.60±0.25). The
signicantly higher mean survival rate was also observed in larvae fed with mix zooplankton
(47.67±11.25%) followed by artemia nauplii (38.5±5.4%) and articial powdered feed (18.17±3.68%) for
28 days experiment. Our nding suggests CPE best dose @ 100mg/kg body weight for female and 45
mg/kg body weight for male and best dose of OVAFISH @1.5 ml/kg body weight of female and 0.75
ml/kg body weight of male for induced breeding. While in larval rearing experiment highest survival and
growth rate was obtained in the larvae fed with mix zooplankton for 28 days trial.
1. Introduction
In the last few decades, global aquaculture has entered a new and challenging phase. The continued
demand for animal protein has focused much attention on sheries and aquaculture research (Béné et
al., 2015; Tacon et al., 2009; Finegold., 2009 and Gjedrem., 2012). The development of new technology,
particularly the closing the breeding cycles of new promising aquaculture species, will help in
diversication of aquaculture (Moorhead and Zeng., 2010; Naylor et al., 2021 and Bostock., 2011).
Breeders in India have signicantly increased productivity for many commercially important species of
carps, catshes, and tilapia, but successful breeding (Jayasankar., 2018; Lind et al., 2012) and cultivation
of spiny eel
Macrognathus aculeatus
would be a huge boost to high-value aquaculture and aquaculture
diversication
Macrognathus aculeatus
(Bloch, 1786), popularly known as spiny eel, is a common species in the
Mastacembeliformes. This species is found in a variety of freshwater habitats in India, Bangladesh,
China, Indonesia, Malaysia, Nepal, Singapore, Taiwan, Thailand, Vietnam, and West Africa (Rahman 1989,
Archarya and Iftekhar 2000, and Nguyen et al., 2011). Spiny eels live primarily in medium to large rivers
(Çoban
et al.
,2021; Surasinghe et al., 2019 and Suresh et al., 2006), and are abundant in low-lying
wetlands, freshwater streams, ponds, canals, beels, and tanks (Bhuiyan, 1964; Taki, 1978). They are
benthopelagic and potamodromous in nature and can be found in freshwater and in intertidal zone of
brackish water (Bhuiyan, 1964; Taki, 1978; Riede, 2004). The species prefer freshwater and muddy areas
of small rivers, streams, and canals (Bhuiyan, 1964). It contributes to control population of harmful

Page 3/24
insects and water pollution up to some extent by eating detritus and insect larvae (Rahman et al., 2009).
M. aculeatus
is commonly known as "ditch eels" (Hora, 1935) and "lesser spiny eels" (Munro, 1955)
because they are frequently found in small puddles with a lot of mud. The upper body of this species is
yellow with a black line down the centre, and the lower body is usually a mix of white and brown. The
dorsal n is preceded by several isolated tiny spines that can be elevated along the eel's backbone; hence
the name is spiny eel (Faridi et al., 2020). There are numerous conspicuous eyespots along the base of
the dorsal n (Froese
et al
., 2007). This species' larvae are frequently seen attached to leafy vegetation in
natural water bodies, whereas adults are mud dwellers (Sahoo et al., 2009).
It is a commercially important and popular food sh species in India due to lack of intramuscular spines,
high nutritive value, good taste, and high protein content (Sahoo et al., 2009). The maximum length and
weight recorded of
M. aculeatus
in wild is 24.5 cm long and 56 gm weight (Das and Kalita, 2003). It is
also having the ornamental value due to its beautiful coloration, body shape, and playful behaviour
(Vidthayanon, 2002). The demand for the species always exceeds the supply, and the supply is highly
dependent on capture resources (Sahoo et al., 2009) The availability of the sh has declined signicantly
from its natural habitat due to habitat destruction and overexploitation, as reported for guchibaim primes
(Afroz et al., 2014). The natural breeding grounds of this species are also under threat due to the drying
up of low-lying areas and the indiscriminate use of fertilizers and pesticides (Rahman et al., 2009).
One of the most effective ways to restore its natural population is to use articial propagation techniques
to breed the sh in captivity and release the ngerlings in natural water systems and second to culture
this species, so the demand will be fullled from aquaculture (Mijkherjee et al., 2002; Rakes et al., 1999;
Mylonas et al., 2010). Captive breeding of
M. aculeatus
has been attempted a few times, either by
stripping or by using aquatic substratum such as water hyacinth (Das and Kalita, 2003). But the articial
propagation and larval rearing are the major production bottlenecks in this species. Currently, information
on this species' larval rearing is scarce. Commercial hormones of various types are used for articial
propagation of various eels. Few studies have been published on the effectiveness of using various
doses of synthetic hormones for articial propagation of various species of freshwater eels. They are
ovaprim, HCG (Human Chorionic Gonadotropin), Pituitary Gland (PG), and Ovulin (Hasan et al., 2016;
Islam
et al.
, 2018; Ali and Taslima, 2013; Chakraborty, 2008).
Artemia nauplii are widely recognized as an excellent starter feed for sh larvae (Radhakrishnan
et al
.,
2020; Das et al., 2012; Syukri et al., 2022). The live zooplanktons are also successfully used for rearing
the larvae of different sh species and particularly spiny eels (Pal et al., 2003; Shifat et al., 2017; Halwart
and Gupta., 2004). The powdered articial feed and egg custard are also effectively used as feed for
larval rearing of freshwater shes (Nik Sin and Shapawi, 2017, Malla and Banik, 2015). So, the aim of the
current study was to nd out the effective dose of Carp Pituitary Extract (CPE) and OVAFISH on the
reproductive response of
M. aculeatus
and assessing the growth performance, and survival of
M.
aculeatus
larvae fed to different types of live and articial feeds.
2. Materials and methods

Page 4/24
The experiment was carried out in the Hatchery complex of the College of Fisheries, Dholi, Dr. Rajendra
Prasad Central Agricultural University, Bihar.
2.1 Brood stock rearing
Around 200
Macrognathus aculeatus
males and females were captured from wild and transported to the
College of Fisheries, Dholi hatchery complex. From January to March, they were stocked and reared in a
cement tank with 4 cm soil at the bottom, and the water level was kept between 2 to 3 feet. The shes
were fed with articial feed containing 30–40 percent protein by weight twice a day at a rate of 4–5
percent of their total body weight. The maturity stages were checked fortnightly from month of April to
June.
2.2 Maturity assessment of brood shes
When the sh are young, determining their sex is challenging. The selected ripe male and female brood
sh were assessed for maturity through physical and visual examination. Females are slightly larger than
males of the same age in general. Females have a bloated belly with a greenish tinge during the breeding
season, while males ooze milt when light pressure is applied to their abdomen. For articial propagation,
only healthy and uninfected brood sh were selected.
2.3 Experimental design
Fishes were collected from the brood stock pond in the early morning hours for induced breeding and
acclimatised in a FRP tank for 6–8 hours by changing the water periodically. The rst experiment was
conducted using carp pituitary extract (CPE) and OVAFISH hormone for nding the best dose of hormonal
injection. The rst experiment consisting of three treatments with CPE (T1 CPE, T2 CPE and T3 CPE) and
three treatments with OVAFISH hormone (T4 OVAFISH, T5 OVAFISH and T6 OVAFISH). The dose of CPE
and OVAFISH is presented below in table. A total 30 pair brood shes were used in this experiment, for
each dose 5 pair of brooders were injected. The induced breeding detail of
M. aculeatus
broodsh is
given in Supplemental document Table 1 and 2.
Sex Treatments
T1 CPE T2 CPE T3 CPE T4 OVAFISH T5 OVAFISH T6 OVAFISH
Female 80mg/kg 90mg/kg 100mg/kg 1ml/kg 1.5ml/kg 2ml/kg
Male 35mg/kg 40mg/kg 45mg/kg 0.5ml/kg 1ml/kg 1.5ml/kg
The second experiment was conducted to determine the weaning strategies of
M. aculeatus
larvae for 4
weeks. Five days old
M. aculeatus
larvae of average weight 1.8 mg and length 6.1 mm were used for
study. Total 12 tanks of 160-liter capacity were set-up and in each tank 200 larvae were stocked. There
were four treatments groups with 3 replicates each in Completely Randomized Design (CRD). The rst
treatment group (T1) was fed with articial powdered feed, second treatment group (T2) with

Page 5/24
Zooplankton, third treatment group (T3) with artemia nauplii, and fourth treatment group (T4) with egg
custard. The feed was given 3 times a day, rst 8.00 AM, second 2.00 PM and third 8.00 PM in all four
groups. Siphoning was done 1 hour after each feeding and 30% water is exchanged daily from all tanks.
The gure below showing transitioning period from test feed to articial powdered feed.
T1-Only articial powdered feed is given.
T2-Zooplankton is given for 14 days then both zooplankton and articial feed is given for 7 days then
only articial powdered feed is given for 7 days.
T3-Artemia nauplii is given for 14 days then both artemia nauplii and articial feed is given for 7 days
then only articial powdered feed is given for 7 days.
T4-Egg custard is given for 14 days then both egg custard and articial feed is given for 7 days then only
articial powdered feed is given for 7 days.
2.4 Preparation of carp pituitary extracts (CPE)
Carp pituitary glands (CPG) in good condition were purchased from the market. During the time of
experiment with a pair of forceps, the pituitary glands were gently taken out from the vial, dried on tissue
paper while the alcohol evaporated, and weighed using an analytical weighing balance as per the
required dose. Based on the total body weight of all the sh, the amount to be weighed out was
calculated as follows:
Weight of CPG (mg) = Wt * Pt/1000
Where, Wt = total body weight (g) of all the shes to be injected and,
Pt = The rate in mg CP to be injected/kg body weight under a particular treatment.
After weighing the CPG, it was homogenized in a tissue homogenizer. The crushed CPG was diluted in
distilled water. The solution is then centrifuged at 5000 rpm for 5 minutes. The supernatant is carefully
transferred in a vail, which can be called as carp pituitary extract (CPE). For injection, 1 ml insulin syringe
was slowly lled with the freshly made clear supernatant solution. The second inducing agent, OVAFISH
(Bhoomi Aqua International, Maharashtra, India), is commercially available in ready to use format.
2.5 Injecting the inducing agents
An insulin disposable syringe was used to inject hormone into the recipient sh. The required amount of
diluted hormone solution was lled into the syringe. The sh were then carefully netted out of the FRP
tank. The sh was placed on a table after being wrapped in a clean, soft, and wet cloth. A suitable dose
of Carp Pituitary Extract (CPE) was administered above the lateral line and at the base of the dorsal n.
Required needle was inserted at a 45° angle to the body. The same protocol was used for OVAFISH
hormone also.

Page 6/24
2.5.1 Carp Pituitary Extract doses
In females two doses of CPE were administered, while males receive only one dose at the time of second
dose in females. In females, the rst dose was 35, 40, and 45 mg CPE/kg body weight in treatment T1, T2
and T3 respectively, whereas the second dose was 45, 50 and 55 mg CPE/kg body weight. Male shes
were injected with 35, 40, and 45 mg CPE/kg body weight at the time of the second dose in female. The
male and female (1:1) were injected and placed in the different tanks. After 12–13 hours of rst injection,
the female sh was checked periodically for the commencement of ovulation by gently pressing their
abdomen. After 16–17 hours of the rst injection the eggs were coming out easily by slight pressure on
the abdominal region.
2.5.2 OVAFISH hormone doses
Female sh received a dose of 1.0, 1.5 and 2.0ml/kg body weight, whereas male sh received 0.5, 0.75,
and 1.0 ml/kg body weight, in T4, T5 and T6 group respectively. The male and female (1:1) were injected
separately and placed in separate tanks. After 12–13 hours of hormone injection, the brooders were
caught and checked.
2.6 Estimation of spawning, fertilization, and hatching rate
The stripping process was performed in this study. Initially, female sh were stripped in order to collect
eggs in a dry plastic tray. Milt was extracted from the male by pressing gently on its abdomen and pour
on the stripped eggs. The eggs and milt were thoroughly mixed with a soft and clean feather. After 2
minutes of mixing, a few drops of water were added to the tray to ensure proper fertilization, followed by
5 minutes of continuous swirling to ensure homogeneous mixing. The eggs were washed several times to
remove excess milt, bad quality eggs, muscle mass, blood, and dirt. The unfertilized egg was easily
identied as they turned whitish in colour while fertilized egg of green colour. The swollen eggs were
transferred to a hatching tray using a continuous water ow through system. Each hatching tray has 3–4
litre water and can hold up to 1000 eggs. Fertilized eggs were kept in the ow through system of each
group. The ow of water in the tray was controlled during the incubation stage. The eggs hatched in 33 to
35 hours at temperatures ranging from 26 to 280C. To prevent fungal growth, unfertilized eggs, dead
embryos were removed time to time during the incubation period. The number of viable eggs in each
group was determined within 2 to 3 hours of fertilization. The Spawning, fertilization and hatching rate
was calculated by following formula:
Spawning rate = The number of sh ovulated/ Number of sh injected X 100
Fertilization rate = The number of fertilized eggs/ Total number of eggs X 100
Hatching rate = The numbers of hatchlings/ Number of fertilized eggs X 100
2.7 Embryonic developmental stages

Page 7/24
In a Petri plate, 10–20 fertilized eggs were taken out randomly from incubation tray time to time for
recording the different embryonic development stages up to the hatchlings. Alight microscope ZEISS
Primostar 3with attached camera (Carl Zeiss AG, Oberkochen, Germany) was used to observe the
embryonic developmental stages.
2.8 Different live and articial feed used in larval rearing
experiment
The powder starter articial feed of CPF, India Pvt Ltd (A-40) with 40% protein content was used in this
study. The feed was given in the form of small balls by adding little amount of water. The mixed plankton
was collected using plankton nets from Magur cemented pond of College of Fisheries, Dholi and fed
ad
libitum
to the larvae thrice a day. Artemia cysts were purchased from market (Ocean Star International,
Inc., Snowville, USA) and hatched as per manufactures instruction. Briey, in 1 litre of distilled water 30
gm of sodium chloride was added and shake well to dissolve properly. Transfer this water in a conical
shape container/bottle with oxygen input from bottom. Now weight 1.5 g of artemia cyst and pour in
container and stirred continuously for a while. Cover the top of container and start the aeration and put a
100-watt bulb above the container. After 24 hours, switch off the aeration and put the bulb near the
bottom, so all the hatched artemia will come towards the light and then collect the artemia nauplii by
using a pipette. The hatched artemia are of orange-colour and fed
ad libitum
to the sh larvae thrice a
day at same time as the articial feed. Egg custard was prepared by taking one large egg and 27 gm of
LACTOGEN 1 Milk Powder (Nestlé India Ltd, Gurgaon, India), mixed in a blender, and boiled for 15 to 20
minutes in a boiling water bath to get a semi-solid state. Now refrigerate this semi-solid egg custard for
hour or till it required for feeding to larvae. When required remove from refrigerator and crush in small
pieces (0.2-1.0 mm) and fed to larvae. This egg custard is also used to fed 3 times a day as articial feed.
2.9 Physico-chemical parameters
The physico-chemical parameters of water during the egg incubation and larval rearing of
M. aculeatus
were recorded in all the incubation tubs and larval rearing crates regularly in the morning as per the
standard procedure (APHA, 2005). Water temperature (Thermometer), dissolved oxygen (Winkler’s
method), pH (pH meter), alkalinity and hardness (Titration method) were measured in the aquaculture
laboratory, College of Fisheries, Dholi, Muzaffarpur.
2.10 Growth and survival of
M. aculeatus
larvae
After 4 weeks of larval rearing shes were randomly collected from each crate for measuring the length
and weight in millimetre (mm) and milligram (mg) respectively. The weight gain, length gain, weight gain
percentage, length gain percentage and specic growth rate was calculated for each treatment group.
Daily monitoring was done to note down the numbers of dead larvae. The survival was determined by the
counting of remaining larvae in each experimental crate at the end of experimental period.
To determine the growth performance of various experimental shes, the following equations were used:

Page 8/24
Specic growth rate (SGR): -
SGR = (Ln Final weight of sh (mg) - Ln Initial weight of sh (mg))/Total number of days X 100
Weight gain percentage (WG %): -
WG%=Final body wt. (mg)-Initial body wt. (mg)/Initial body wt. X 100
Absolute growth rate (AGR): -
AGR (mg /day) = Final body weight (mg)-Initial body weight (mg)/ Total no. of days
Weekly length gain (LG): -
LG = Final length (mm) -Initial length (mm)
Survivability rate (SR): -
SR = Number of sh survived/ Total number of sh X 100
Mortality rate (MR): -
MR = Number of sh deceased/ Total number of sh X 100
2.11 Statistical Analysis
The data were subjected to a One-way ANOVA by using software (IBM SPSS Statistics version 21) to
determine signicant difference between various treatment groups. Reconciliation was made on 95%
possibility scale (P < 0.05). Data was articulated as mean ± standard error. The one-way ANOVA analysed
which was followed by a post-hoc, Duncan’s, options, Descriptive comparison test if signicant
differences were found.
3. Results
3.1. Effect of different hormonal doses on breeding
performance of
Macrognathus aculeatus
In CPE treatment groups highest average number of stripped eggs (2403) (Supplemental document Table
3) and fertilization rate (81.34 ± 2.05) was observed in T3 group, while in OVAFISH treatment groups the
highest average number of stripped eggs (2110) (Supplemental document Table 4) and fertilization rate
(91.96 ± 1.56) was obtained in T5 group (Fig. 1). The highest hatching rate was observed in T1 group
(52.955 ± 1.14) in CPE but not signicantly different from other groups and T6 group (63.35 ± 1.14) in
OVAFISH (Fig. 2). Stripping was done after 8 hrs of 2nd injection in CPE treatment groups and 10 hrs of
injection in OVAFISH groups. Hatchlings were obtained after 33 to 35 hrs of fertilization in both hormonal
groups. Thus, the T3 was the best treatment in CPE groups and T5 in OVAFISH groups. The water quality

Page 9/24
parameters during the egg incubation were in optimum range and do not signicantly different in
different treatment groups (Table 1).
Table 1
Water quality parameter during the egg incubation period
Treatment Temp. pH DO Alkalinity Hardness
T1 CPE 27.16 ± .76a 8.26 ± .15a 6.43 ± .49a 236.66 ± 2.88a 91.66 ± 5.77a
T2 CPE 27.16 ± .28a 8.30 ± .17a 6.40 ± .36a 245 ± 15a 90.0 ± 5.00a
T3 CPE 27.16 ± .28a 8.23 ± .20a 6.40 ± .10a 238.33 ± 7.63a 86.66 ± 10.40a
T4 OVAFISH 26.66 ± .28a 8.50 ± .26a 6.80 ± .30a 248.33 ± 20.81a 80.00 ± 5a
T5 OVAFISH 27.33 ± .57a 8.30 ± .10a 6.76 ± .35a 246.66 ± 5.77a 83.33 ± 10.40a
T6 OVAFISH 27.16 ± .28a 8.20 ± .10a 6.36 ± .05a 236.66 ± 7.63a 83.33 ± 2.88a
3.2. Embryonic developmental stages of
Macrognathus
aculeatus
The eggs of
M. aculeatus
were green in color, sticky in nature and rounded. The unfertilized eggs (Fig. 3A)
were opaque while the fertilized eggs (Fig. 3B) were transparent with visible egg membrane and yolk. The
formation of perivitelline space was observed in 17 min and hatching of eggs at 34.17 h (Fig. 3C-S).
Fertilized eggs with blastodisc in 28 min, 2-cell stage in 56 min and multicell stage in 4.17 h were
observed. The morula, blastula and gastrulation ring were observed after 6.47 h, 10.54 h and 15.12 h of
fertilization respectively. Complete embryo stage was observed 25.32 h and twitching movement in
embryo observed at 31.13 h of fertilization. As the embryo advances in development, the movement
becomes more and more vigorous. With further twitching movements, the embryo was able to release out
from the surrounding membrane. Eyes were clearly observed. The hatching period was observed 34.17 h
after fertilization. All the details of embryonic development stages are presented in Table 2.

Page 10/24
Table 2
Details of embryonic developmental stages of
M. aculeatus
Stages of
egg Age Developmental detail
Fertilization 0.00 Eggs are round, green color, Clear
Perivitelline
space
formation
17 ±
1.2
min.
Perivitelline space formation around the yolk
Blastodisc 28 ±
2.0
min.
Accumulation of cytoplasm over the animal pole
2 cell-stage 56 ±
2.1
min.
The cytoplasmic disc thickened, and the rst cleavage occurred at a right
angle forming two identical blastomeres
4 cell-stage 1.35
± 0.02
h
2nd cleavage
8 cell-stage 2.11
± 0.03
h
3rd cleavage
16 cell-
stage 2.33
± 0.03
h
4th cleavage
32 cell-
stage 2.55
± 0.02
h
5th cleavage
64 cell-
stage 3.04
± 0.03
h
6th cleavage
128 cell-
stage 3.52
± 0.04
h
7th cleavage
Multicell
stage 4.17
± 0.07
h
8th cleavage
Morula-
stage 6.47
± 0.05
h
The blastomeres were further decreased in size and gathered around the
animal pole, forming a owery cap.
Blastula-
stage 10.54
± 0.1
h
The individual blastomeres were not correctly recognized, the borders of the
peripheral blastomeres were lost and squeezed.

Page 11/24
Stages of
egg Age Developmental detail
Gastrula-
stage 15.12
± 0.2
h
Sheet of cells migrated from animal pole on both sides towards the vegetal
pole is seen in early gastrula. Thin layer of gastrulation ring was observed.
Head and tail are visible but not distinguished and blastoderm covered 3/4th
of the yolk.
Complete
embryo-
stage
25.32
± 0.2
h
Body axis completely encircled the vitelline sphere with attached head and
tail end over the yolk
Twitching-
stage 31.13
± 0.4
h
Twitching movement is seen. Both head and tail clearly visible
Hatching 34.17
± 0.9
h
Larvae hatched out from the egg
3.3 Water quality parameters during larval rearing
The water quality parameters observed during the entire 4 weeks experimental period is represented in the
Table 3. The water temperature was in the range of (28.62 ± 0.51) to (28.81 ± 0.52) 0C throughout the
experiment period. pH ranges from (8.12 ± 0.06) to (8.17 ± 0.04) and dissolved oxygen from (5.71 ± 0.56)
to (5.98 ± 0.53) in the entire experimental period. Total alkalinity was in the range of (239.5 ± 17.86) to
(256.5 ± 16.33) in various treatments groups. Total hardness was determined within the range of (69 ±
18.52) to (75 ± 14.33) while nitrite, nitrate and ammonia were negligible.
Table 3
Water quality parameters during larval rearing ofM. aculeatusin different treatment
groups
Treatments T1 T2 T3 T4
Temp. 28.70 ± .59a 28.64 ± .42a 28.62 ± .51a 28.81 ± .52a
pH 8.12 ± .06a 8.12 ± .10a 8.14 ± .09a 8.17 ± .04a
DO 5.85 ± .46a 5.79 ± .42a 5.98 ± .53a 5.71 ± .56a
Alkalinity 239.5 ± 17.86a 256.5 ± 16.33a 256 ± 14.49a 250.5 ± 21.40a
Hardness 75 ± 14.33a 69 ± 13.49a 70.5 ± 11.41a 69 ± 18.52a
Ammonia 0.23 ± .008bc 0.21 ± .013a 0.22 ± .015ab 0.24 ± .011c
Nitrite 0.002 ± .001a 0.002 ± .001a 0.003 ± .0008a 0.003 ± .0005a
Nitrate 0.03 ± 0.008a 0.04 ± 0.012a 0.03 ± 0.013a 0.03 ± .01a

Page 12/24
3.4. Larval rearing of
Macrognathus aculeatus
with different
feeds
The larvae are bottom feeder and having good swimming behaviour. The cannibalism was noticed during
the experiment trial. The larvae were negatively photo tactic and aggregated in the dark hiding areas of
the larval rearing tanks provided at the bottom. It was observed that the rate of feed intake was higher in
mix zooplankton and artemia fed groups as compared to other feeds tested. The larvae fed on mix
zooplankton showed higher growth rate and attained the highest weight and length as compared to other
treatment groups.
3.4.1 Growth performance of larvae
The Specic Growth Rate (SGR) was calculated to determine the growth performance during the
experimental trial and the results are shown in Fig. 4. The highest SGR (9.60 ± 0.14) was observed in mix
zooplankton feeding group followed by egg custard group (9.46 ± 0.40), articial powdered group (9.21 ±
0.17), However, the difference was not signicant (p < 0.05).
The weight gain percentage was highest in mix zooplankton (1373%) and lowest in larvae fed with
artemia nauplii (1109%). But there was no signicant (p < 0.05) difference in weight gain percentage of
larvae. The absolute growth rate was higher in mixed plankton group (0.86 mg/day) followed by egg
custard group (0.83 mg/day). Growth performance of the larvae like length gain from week 1 to week 4
was also highest in mix zooplankton and lowest in larvae fed with artemia nauplii group (Fig. 4).
3.4.2 Mortality and survival rate
Mortality rate was higher in larvae fed with egg custard (T4) followed by articial powdered feed (T1) and
artemia nauplii (T3). The lowest mortality rate was observed in larvae fed with mix zooplankton (T2). The
higher mortality rate was observed during the 2nd week in all treatment groups (Fig. 5). The larval
mortality rate in different weeks after fed with different live and articial feeds is given in Table 4.
M.
aculeatus
larvae fed with mix zooplankton showed higher survival rate (47.66%) followed by artemia
nauplii (38.5%) and articial powdered feed (18.16%). Least survival (16.6%) was observed in larvae fed
with egg custard (Fig. 5).

Page 13/24
Table 4
Week wise mortality in
M. aculeatus
larvae fed various
feeds
Mortality rate T1 T2 T3 T4
1st week 7.15% 8.83% 9.33% 20.16%
2nd week 40% 34.15% 45.83% 50.33%
3rd week 11.16% 4.66% 4.33% 9.50%
4th week 15.16% 3.66% 1.16% 3.66%
Discussion
In the present experiment, injection of pituitary gland extract at 100 mg/kg body weight of female in two
doses and 45 mg/kg body weight of male in single dose in
M. aculeatus
showed fertilization rate 81.34%
and hatching rate 49.51%. Dose specicity of PGE as observed in the present study is in conformity with
the ndings of Farid et al. (2008) who found better spawning performances of
M. aculeatus
at 90mg
PG/kg body weight with fertilization rate of 86% and hatching rate of 70%. Alam et al. (2009) obtained
91% fertilization rate and 80% hatching rate by using PGE dose of 170 mg/kg female and 60 mg/kg male
in
M. pancalus
which is a species of same family in which current studies sh fall. Similarly, Chakraborty
et al.
(2010) also studied the impact of PGE in
M. pancalus
using the dose of 182 mg/kg body weight of
female and observed 92% fertilization rate and 89% hatching rate. In case of
M. armatus
Ali et al. (2018)
used the PGE dose of 40 mg/kg body weight female and obtained the fertilization rate of 98% and
hatching rate of 58.3%.
In the current study we nd out the most appropriate and economical dose of OVAFISH to be 1.5 ml/kg
body weight of female and 0.75 ml/kg body weight of male with fertilization rate 91.96% and hatching
rate 53.77%. Das and Kalita, (2003) have studied on
M. aculeatus
and injected 0.025 ml and 0.05 ml of
ovaprim for individual male and female respectively and observed 100% spawning performance, 78%
fertilization and 67% hatchability. Sahoo et al. (2009) have also studied on
M. aculeatus
using the same
dose of ovaprim as (Das and Kalita, 2003) and found 83% fertilization rate and 76% hatching rate.
Administration of lower amount of OVAFISH dose at 1.0 ml/kg body weight of female and 0.5 ml/kg
body weight for male resulted in reduced success. The
M. pancalus
was injected with two doses of
ovaprim @0.25 and 0.50 ml/kg body weight of female and 0.5 ml/kg body weight of male observed the
100% spawning rate, 75% fertilization rate and 55% hatching rate (Hasan et al., 2016). The optimum dose
of Ovatide hormone for induced breeding of
Hemibagrus menoda
is 2.5 and 5 ml/kg body weight of male
and female brood sh respectively with ovulation rate of 100%, fertilization rate of 97%, hatching rate of
95% and survival rate of 85% (Hasan et al., 2021).
In the present study, the initiation of the formation of blastodisc at the animal pole was noticed at 28 min
after fertilization (AF) which was almost similar as obtained in study of Sahoo
et al
. (2007) and Islam
et
al
. (2018) in
M. aculeatus
, Rahman et al. (2009) and Islam and rani, (2021) in
M. pancalus
, however Farid

Page 14/24
et al. (2008) also showed the formation of blastodisc started in 15 min in
M. aculeatus
. Further, the
multicell stage was observed in 4.17 h, morula stage in 6.47 h, blastula stage in 10.54 h and Gastrula
stage in 15.12 h after fertilization. In different studies these stages were observed almost in similar time
in same species (Sahoo
et al
., 2007; Islam
et al.
, 2018) but varies with change in species (Rahman et al.,
2009; Islam and rani, 2021). The complete embryo with both head and tail end were clearly visible at
25.32 h, twitching movement in 31.13 h and hatching in 34.17 h AF. The other studies also are in
conrmation of our nding and the variability might be due to the difference in the rate of development
among the species at different water temperatures. Spawning was performed at a water temperature of
26.33 ± 0.88°C between 20 and 24 hours after injection in
M. pancalus
(Borah et al., 2020).
The recorded water quality parameters during larval rearing period such as temperature, dissolve oxygen
(DO), pH, alkalinity and hardness under different treatments were in the range of 28-290C, 5-6mg/l, 8.1–
8.2, 240-250ppm, 70-75mg/l respectively. According to different researchers this is the quite optimum
range of water quality parameters for rearing of larvae of different sh species (Das and Kalita, 2003;
Farid et al., 2008; Sikorska et al., 2018; Saraswathy et al., 2015). Das and Kalita, (2003) also observed the
suitable water temperature between 28–30 0C, pH 7.6–7.8, dissolved oxygen 8–9 mg/L and hardness of
about 1.5 mol/L for the rearing of
M. aculeatus
larvae. Farid et al. (2008) showed that temperature
31.22–31.66 0C, pH 7.7–7.8, dissolved oxygen 3.9–4.1 mg/L, alkalinity 127.2-129.5 mg/L, hardness
121.3-122.9 mg/L is also good for the rearing of
M. aculeatus
larvae. A water temperature 22–25°C for
Muraenesox cinereus
has been described as suitable for growth and survival (Umezawa et al., 1991).
The study showed differences in the growth, SGR, and survival of
M. aculeatus
fed on different diets.
Every tested feed was accepted by
M. aculeatus
larvae with different degree. The feed acceptance was
higher for mixed zooplanktons as compared to other feeds tested might be due to better developed of
their digestive tracts when fed on natural live feed compare to articial feed (Vanhaecke et al., 1990; Shiri
Harzevili et al., 2004; Stejskal et al., 2021). The present study indicated that the post-hatchlings of
M.
aculeatus
successfully weaned on to live and articial diet on an experimental scale. The mix
zooplankton showed to be an excellent feed for post-hatchling rearing of
M. aculeatus
in terms of growth
and survivability in this study. The
Clarias gariepinus
larvae fed on plankton exceeded the other tested
feed like trout starter, soya bean our, Torula yeast, vital yeast, Tetramin, hardboiled egg yolk yeast fed
larvae in growth (Hecht, 1981). Artemia nauplii nutritional properties include a greater crude protein level
of up to 74%, lipids 18% and lower carbohydrates 3% which may be responsible for the improved growth
of sh larvae and fry fed on Artemia nauplii. Powdered articial feed and egg custard are also good feeds
for freshwater sh larval rearing (Nik Sin and Shapawi, 2017, Malla and Banik, 2015). The immature
digestive system was most likely to be responsible for the lower development and survival observed in
larvae fed a prepared meal during the early stages.
In the present study, a higher mean survival (47%) was observed in mix zooplankton followed by artemia
nauplii (38.5%) and articial powdered feed (18.16%). This could be due to the improved food intake by
the post-hatchling from live feeds and because of their nutrient prole, capacity to remain alive in the
rearing environment, and easy digestion and assimilation by the larvae. The live feed such as rotifers,

Page 15/24
copepods, and Artemia nauplii are the best larval feeds (Damle and Chari, 2011). While post-hatchlings
fed with unconventional feed (egg custard) had comparatively lower survival (16.6%). The low survival
rate (66%) in boiled egg-yolk might be due to impaired feeding of post-hatchlings. (Sharma and
Chakrabarti, 1999) observed 3-5-fold higher growth rate fed with live feed in
Cyprinus carpio
as compared
with the same stocking density for the articial feed. The higher value of SGR (3.92 ± 0.202) was
observed in mix zooplankton feeding group followed by articial powdered group (3.6 ± 0.3), egg custard
group (3.69 ± 0.76). However, SGR was lower in larvae fed on artemia nauplii (3.07 ± 0.29). Among other
growth parameter, weight gain percentage was higher in mix zooplankton (66.72%), followed by egg
custard (65.25%), articial powdered feed (63.52%). However, the weight gain percentage is lower in
larvae fed with artemia nauplii (57.96%).
Conclusion
The results of this study show that the effectiveness of OVAFISH and carp pituitary extract in inducing
M.
aculeatus
breeding was better when the doses were 1.5 ml and 100 mg/kg body weight of female,
respectively. The embryonic developments of
M. aculeatus
was like the other species of eel. This study
provided complete information on the captive breeding and early larval developmental stages of
M.
aculeatus
. But at various stages, the period for development varies. However, as the temperature of the
water uctuated, so did the embryo's rate of development. The development process shows itself faster
as the temperature rises, and vice versa. On the fourth day, the larvae began to eat on external feed after
having completely absorbed their yolk sac. The ndings show the reliability of the procedures for
experimental larval rearing in plastic tanks with a variety of diets. The mix zooplankton might be potential
food for
M. aculeatus
in its early life stages. This study has focused on the idea that prey movement
inuences larvae's choice for live food over articial food. Because of its excellent habitational adaption
and high level of toleration for captivity, the species deserves additional research on its viability for
commercial culture. Zooplankton can be used to successfully raise
M. aculeatus
larvae until they are
ready to transition to formulated diets. Zooplankton can be fed to larval
M. aculeatus
in the early stages
of development, and then progressively articial diets can be added to the mix to improve results until the
larvae are trained to take articial diets. The early fry stage exhibit cannibalism. The results of the current
study open new avenues for investigation into how to improve articial sh breeding, particularly
regarding to environmental parameter and hormone application optimization. However, more effort is
needed to increase survival rates from spawn to fry through diet formulation and live feed culture. These
ndings could be of immense assistance to sheries biologists as researchers manage the shery and
take additional measures toward the captive breeding and mass production of
M. aculeatus
larvae.
Declarations
Ethical Approval
The care and treatment of animals used in this study were in accordance with the guidelines of the
CPCSEA (Committee for Control and Supervision of Experiments on Animals), Ministry of Environment,

Page 16/24
Forest, and Climate Change (Animal Welfare Division), Government of India on care and management of
animals in scientic research. The study was approved by the Animal Ethical Committee of Dr. Rajendra
Prasad Central Agricultural University, Pusa.
Competing interests
The authors declare no conict of interest either of nancial or personal nature.
Authors' contributions
Bhumika Gamango; Writing – original draft, bench work, data curation and validation. Raj Kamal Mishra;
Bench work, data collection. Aditi Banik; Experiment conducting, sampling, data collection. Shivendra
Kumar; Experiment design preparation, corrections in draft. Roshan Kumar Ram; Help in live feed culture.
P.P. Srivastava; Writing - review & editing. Pravesh Kumar - Conceptualization of research idea, Writing -
review & editing.
Funding
This study was supported by the Institutional Project “Domestication and Induced breeding of Indian
Spiny Eel/Gainchi
Macrognathus aculeatus
” which received funding from Director Research, RPCAU,
Pusa. So, the authors sincerely thank the Vice Chancellor, and Director Research, Dr. Rajendra Prasad
Central Agricultural University, Pusa and Dean, College of Fisheries, Dholi, RPCAU for providing nancial
support and all the infrastructure facilities required for conducting the study.
Availability of data and materials
Data may be available on request.
References
1. Afroz, A., Islam, M.S., Hasan, M.R., Hasnahena, M. and Tuly, D.M., 2014. Larval rearing of spiny eel,
Mastacembelus pancalus
in the captivity with emphasis on their development stages. International
Journal of Fisheries and Aquatic Studies,
1
(6), pp.163–167.
2. Alam, M., Alam, M.S., Alam, M.A., Islam, M.A. and Miah, M.I., 2009. Dose optimization with PG
hormone for induced breeding of
Mastacembelus pancalus
. Journal of Ecofriendly Agriculture,
2
(9),
pp.799–804.
3. Ali, M.R. and Taslima, K., 2013. Domestication and observation on induced breeding of spiny eel
Mastacembelus armatus
. American Journal of Food Science and Technology,
1
(4), pp.82–86.
4. Ali, M.R., Mollah, M.F.A. and Sarder, M.R.I., 2018. Induced breeding of endangered spiny eel
(
Mastacembelus armatus
) using PG extract. Progressive Agriculture,
29
(3), pp.267–275.
5. Archarya, P. and Iftekhar, M.B., 2000. Freshwater Ichthyofauna of Maharashtra state: Endemic Fish
Diversity of Western Ghats.
A. NBFGR-NATP Publication. National Bureau of Fish Genetic Resources,

Page 17/24
Lucknow, UP, India
.
. Béné, C., Barange, M., Subasinghe, R., Pinstrup-Andersen, P., Merino, G., Hemre, G.I. and Williams, M.,
2015. Feeding 9 billion by 2050–Putting sh back on the menu. Food Security, 7(2), pp.261–274.
7. Bhuiyan, A. L. 1964. Fishes of Dacca, Asiatic Society of Pakistan, Dacca, East Pakistan, p.118–119.
. Borah, R., Sonowal, J., Nayak, N., Kachari, A. and Biswas, S.P., 2020. Induced breeding, embryonic,
and larval development of
Macrognathus pancalus
(Hamilton, 1822) under captive condition.
International Journal of Aquatic Biology,
8
(1), pp.73–82.
9. Bostock, J., 2011. The application of science and technology development in shaping current and
future aquaculture production systems. The Journal of Agricultural Science,
149
(S1), pp.133–141.
10. Chakraborty, B.K. 2008. Induction of spawning in spiny Tara baim,
Macrognathus aral
(Bloch &
Schneider) in Bangladesh. Aquaculture India Vol.9(1): 37–44.
11. Chakraborty, T. and Bhattacharjee, S., 2008. The ichthyofaunal diversity in the freshwater rivers of
south Dinajpur district of West Bengal, India. Journal of the Bombay Natural History Society,
105
(3),
pp.292–298.
12. Çoban, M.Z., Eroğlu, M. and Düşükcan, M., 2021. Some biological properties of spiny eel
(
Mastacembelus mastacembelus
, Banks & Solander, 1794) living in the Upper Euphrates River Basin,
Turkey. Scientic Reports,
11
(1), pp.1–9.
13. Damle, D.K. and Chari, M.S., 2011. Performance Evaluation of Different Animal Wastes on Culture of
Daphnia sp.
Journal of Fisheries and Aquatic Science
,
6
(1), p.57.
14. Das, P., Mandal, S.C., Bhagabati, S.K., Akhtar, M.S., and Singh, S.K., 2012. Important live food
organisms and their role in aquaculture. Frontiers in aquaculture,
5
(4), pp.69–86.
15. Das, S. K. and Kalita, N. 2003. Captive breeding of peacock eel,
Macrognathus aculeatus
. Aquac.
Asia,
8
: 17–21.
1. Farid SM, Miah MI, Akter M, Saha D, Rahman MM. 2008. Embryonic and larval development of
tarabaim,
Macrognathus aculeatus. Journal of Agroforestry and Environment 2(2), 123–129.
17. Faridi, A.A., Bano, A. and Serajuddin, M., 2020. Aspects of reproductive biology of the lesser spiny eel
Macrognathus aculeatus
(Bloch, 1786) from river Ganga, Uttar Pradesh, India. Indian Journal of
Fisheries,
67
(2), pp.15–22.
1. Finegold, C., 2009. The importance of sheries and aquaculture to development. The Royal Swedish
Academy of Agriculture and Forestry.
19. Froese, Rainer and Pauly, Daniel, eds. (2007). "
Macrognathus aculeatus
" in FishBase. May 2007
version.
20. Gjedrem, T., Robinson, N. and Rye, M., 2012. The importance of selective breeding in aquaculture to
meet future demands for animal protein: a review. Aquaculture,
350
, pp.117–129.
21. Halwart, M. and Gupta, M.V., 2004.
Culture of sh in rice elds
. FAO; WorldFish Center.
22. Hasan, M.R., Islam, M.S., Afroze, A., Bahdur, P. and Akter, S., 2016. Captive breeding of Striped Spiny
Eel,
Mastacembelus pancalus
(Hamilton, 1822) considering the various hormonal responses.

Page 18/24
International Journal of Fisheries and Aquatic Studies,
4
(3), pp.07–11.
23. Hasan, M.Z., Islam, M.F., Haque, S.A., Islam, M.S., Rahman, M.M. and Miah, M.I., 2021. Dose
Optimization of Ovatide Hormone for Induced Breeding of Freshwater Gang Magur,
Hemibagrus
menoda
(Hamilton, 1822). Research in Agriculture Livestock and Fisheries,
8
(1), pp.171–179.
24. Hecht, T., 1981. Rearing of sharptooth catsh larvae (
Clarias gariepnus
Burchell, 1822: Clariidae)
under controlled conditions. Aquaculture,
24
, pp.301–308.
25. Hora, S. L. 1935. Physiology, bionomics and evolution of the air-breathing shes of India.
Trans. Nat.
Inst. Sci. India, 1: 1–16.
2. Islam, M.S., 2018. Stages of lesser spiny eel,
Macrognathus aculeatus
in captive condition:
embryonic development. International Journal of Biosciences,
13
(01), pp.18–26.
27. Islam, S. and Rani, T., 2021. Embryonic development of the striped spiny eel,
Mastacembelus
pancalus
(Hamilton, 1822) in captive condition. Jordan Journal of Biological Sciences,
14
(4).
2. Islam, S., Sultana, R. and Paul, P., 2017. Larval rearing of lesser spiny eel,
Macrognathus aculeatus
in
the captivity with emphasis on their development stages. Int. J. Biosci,
11
, pp.93–103.
29. Jayasankar, P., 2018. Present status of freshwater aquaculture in India-A review. Indian Journal of
Fisheries,
65
(4), pp.157–165.
30. Kandathil Radhakrishnan, D., AkbarAli, I., Schmidt, B.V., John, E.M., Sivanpillai, S. and Thazhakot
Vasunambesan, S., 2020. Improvement of nutritional quality of live feed for aquaculture: An
overview. Aquaculture Research,
51
(1), pp.1–17.
31. Lind, C.E., Ponzoni, R.W., Nguyen, N.H. and Khaw, H.L., 2012. Selective breeding in sh and
conservation of genetic resources for aquaculture. Reproduction in domestic animals,
47
, pp.255–
263.
32. Malla, S. and Banik, S., 2015. Larval rearing of an endangered catsh,
Ompok bimaculatus
(Bloch,
1794) with live and articial diets: A preliminary study in Tripura, India.
Int. J. Fauna Biol. Stud
,
2
,
pp.16–21.
33. Mijkherjee, M., Praharaj, A. and Das, S., 2002. Conservation of endangered sh stocks through
articial propagation and larval rearing technique in West Bengal, India. Aquaculture Asia,
7
(2),
pp.8–11.
34. Moorhead, J.A. and Zeng, C., 2010. Development of captive breeding techniques for marine
ornamental sh: a review. Reviews in Fisheries Science,
18
(4), pp.315–343.
35. Munro, I. S. R. 1955. The marine and freshwater shes of Ceylon Department of External Affairs,
Canberra, Australia, 267 pp. https://trove.nla.gov.au/version/41071505
3. Mylonas, C.C., Fostier, A. and Zanuy, S., 2010. Broodstock management and hormonal manipulations
of sh reproduction. General and comparative endocrinology,
165
(3), pp.516–534.
37. Naylor, R.L., Hardy, R.W., Buschmann, A.H., Bush, S.R., Cao, L., Klinger, D.H., Little, D.C., Lubchenco, J.,
Shumway, S.E. and Troell, M., 2021. A 20-year retrospective review of global aquaculture. Nature,
591
(7851), pp.551–563.

Page 19/24
3. Nguyen TDP, Nguyen THT, Do VT, Nguyen TT, Nguyen HD. 2011. Freshwater ecosystem services and
biodiversity values of Phu Yen District, Son La, Viet Nam. 1–49.
39. Nik Sin, N.N. and Shapawi, R., 2017. Innovative egg custard formulation reduced rearing period and
improved survival of giant freshwater prawn,
Macrobrachium rosenbergii
, larvae. Journal of the
World Aquaculture Society,
48
(5), pp.751–759.
40. Pal, S., Rashid, H., Tarafder, M.A.K., Narejo, N.T. and Das, M., 2003. First record of articial spawning
of
Nandus nandus
(Hamilton) in Bangladesh using carp pituitary gland: An endangered species bred
in captivity. Pakistan Journal of Biological Sciences,
6
(18), pp.1621–1625.
41. Rahman, A.A., 1989.
Freshwater shes of Bangladesh
. Zoological Society of Bangladesh.
42. Rahman, M. M., Miah, M. I., Taher, M. A. and Hasan, M. M. 2009. Embryonic and larval development
of guchibaim,
Mastacembelus pancalus
(Hamilton).
J. Bang. Agri. Univ., 7: 193–204
43. Rakes, P.L., Shute, J.R. and Shute, P.W., 1999. Reproductive behavior, captive breeding, and
restoration ecology of endangered shes. Environmental Biology of Fishes,
55
(1), pp.31–42.
44. Riede, K. 2004. Global register of migratory species from global to regional scales. Final Report of the
R&D-Project 808 05 081. Federal Agency for Nature Conservation, Bonn, Germany, 329 pp.
45. Sahoo, S.K., Giri, S.S., Chandra, S. and Sahu, A.K., 2009. Larval rearing of the spiny eel,
Macrognathus aculeatus
(Lacepede) under different rearing conditions: a preliminary study. Indian J.
Fish,
56
(1), pp.47–49.
4. Saraswathy, R., Muralidhar, M., Sundaray, J.K., Lalitha, N. and Kumararaja, P., 2015. Water quality
management in sh hatchery and grow-out systems. In
Advances in Marine and Brackishwater
Aquaculture
(pp.217–225). Springer, New Delhi.
47. Sharma, J.G. and Chakrabarti, R., 1999. Larval rearing of common carp
Cyprinus carpio
: A
comparision between natural and articial diets under three stocking densities. Journal of the world
aquaculture society,
30
(4), pp.490–495.
4. Shifat, J., Rahman, M.M. and Mollah, M.F.A., 2017. Effect of stocking density and different feed on
growth and survival of
Mastacembelus armatus
larvae. Fundamental and Applied Agriculture,
2
(2),
pp.237–242.
49. Shiri Harzevili, A., Dooremont, I., Vught, I., Auwerx, J., Quataert, P. and De Charleroy, D., 2004. First
feeding of burbot,
Lota lota
(Gadidae, Teleostei) larvae under different temperature and light
conditions. Aquaculture research,
35
(1), pp.49–55.
50. Sikorska, J., Kondera, E., Kamiński, R., Ługowska, K., Witeska, M. and Wolnicki, J., 2018. Effect of four
rearing water temperatures on some performance parameters of larval and juvenile crucian carp,
Carassius carassius
, under controlled conditions. Aquaculture Research,
49
(12), pp.3874–3880.
51. Stejskal, V., Gebauer, T., Sebesta, R., Nowosad, J., Sikora, M., Biegaj, M. and Kucharczyk, D., 2021.
Effect of feeding strategy on survival, growth, intestine development, and liver status of maraena
whitesh
Coregonus maraena
larvae. Journal of the World Aquaculture Society,
52
(4), pp.829–842.
52. Surasinghe, T., Kariyawasam, R., Sudasinghe, H. and Karunarathna, S., 2019. Challenges in
biodiversity conservation in a highly modied tropical river basin in Sri Lanka.
Water
,
12
(1), p.26.

Page 20/24
53. Suresh, V.R., Biswas, B.K., Vinci, G.K., Mitra, K. and Mukherjee, A., 2006. Biology and shery of barred
spiny eel,
Macrognathus pancalus
Hamilton. Acta ichthyologica et piscatoria,
36
(1), pp.31–37.
54. Syukri, F., Hassan, N.H. and Ani, A.S.N., 2022. Performances of fs feed, artemia nauplii and
commercial diet on early development of
Clarias gariepinus
larvae. Journal of Sustainability Science
and Management,
17
(2), pp.35–45.
55. Tacon, A.G., Metian, M., Turchini, G.M. and De Silva, S.S., 2009. Responsible aquaculture and trophic
level implications to global sh supply. Reviews in sheries science,
18
(1), pp.94–105.
5. Taki, Y. 1978. An analytical study of the Mekong basin as a biological production system in nature.
Research Institute of Evolutionary Biology. Special Publication
No. 1: 77, Tokyo, Japan.
57. Umezawa, A., Otake, T., Hirokawa, J., Tsukamoto, K. and Okiyama, M., 1991. Development of the eggs
and larvae of the pike eel, Muraenesox cinereus. Japanese Journal of Ichthyology,
38
(1), pp.35–40.
5. Vanhaecke, P., Vrieze, L.D., Tackaert, W. and Sorgeloos, P., 1990. The use of decapsulated cysts of the
brine shrimp Artemia as direct food for carp
Cyprinus carpio
L. larvae. Journal of the World
Aquaculture Society,
21
(4), pp.257–262.
59. Vidthayanon, C. 2002. Peat swamp shes of Thailand. Oce of Environmental Policy and Planning,
Bangkok, Thailand, 136
pp.
Figures
Figure 1
Fertilization rate of
M. aculeatus
by using different doses of carp pituitary extract and OVAFISH

Page 21/24
Figure 2
Hatching rate of
M. aculeatus
by using different doses of carp pituitary extract and OVAFISH

Page 22/24
Figure 3
Embryonic developmental stages of
Macrognathus aculeatus
:- A) Unfertilized egg, B) Fertilized egg C)
Formation of perivitelline space, D) Blastodisc, E) 2 cell stage, F) 4 cell stage, G) 8 cell stage, H) 16cell
stage, I) 32 cell stage, J) 64 cell stage, K) 128 cell stage L) Multicell stage, M) Morula, N) Blastula, O)
Early gastrula, P) Late gastrula, Q) Complete embryo, R) Twitching movement, S) Hatching

Page 23/24
Figure 4
Growth performance of
M. aculeatus
larvae fed with different feeds during 4-week larval rearing
experiment.

Page 24/24
Figure 5
Mortality and survival percentage of
M. aculeatus
larvae fed with different feeds after 4-week larval
rearing experiment.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
Supplementaldocumenttables.docx
Graphicalabstract.docx