Key Points

BRICS is essentially augmentation of oxytocin

hormone at a precise time after administration of
the commercially available hormonal cocktail
Ovatide™ to elicit voluntary spawning in the
brooders.( )
Oxytocin augmentation aids to sustain gamete
maturation in the male and to induce mating
response resulting in a viable spawning response.
Optimal dose of Ovatide™: of 0.5 ml/Kg body
weight
Optimal dose of Oxytocin: 40 milli international
Unit per kg body weight ( )
Optimum Time gap between ovatide™ and
oxytocin injection: 12 h
Stocking ratio per tank: 1 male and 1 female
BRICS Technology
Air breathing catfishes of the genus Clarias are
excellent candidate species for aquaculture due
BRICS
to their hardy nature and high consumer
preference. A major bottleneck in the seed
Technology for
production of these species was the inability to
induce voluntary spawning in captivity. Magur Breeding
BRICS (Barrier Removal in catfish for Voluntary
Captive Spawning) is a technology developed by
the College of Fisheries, Lembucherra to induce
voluntary captive spawning in air breathing
catfish through hormonal manipulation. The
technology enables the seed production of
Clarias magur in controlled conditions without
necessitating the sacrifice of the male brooder.
This technology will revolutionaries the catfish
seed production and aquaculture.The technology
will also assist in conservation of several fish of
conservation significance.

Published by
Dr. Pramod Kumar Pandey
Dean
College of Fisheries
College of Fisheries
Central Agricultural University (Imphal)
Central Agricultural University (Imphal)
Lembucherra, Tripura-799210
Phone: 0381 2865264 Lembucherra, Tripura (W)- 799210
Email: cofcau.agt-tr@gov.in

Priyadarshi, H, Das, R, Singh, AA, Patel, AB, Pandey,

PK. Hormone manipulation to overcome a major barrier in
male catfish spawning: The role of oxytocin augmentation in
inducing voluntary captive spawning. Aquaculture Research,
2020. DOI: 10.1111/are.14869
Folder-COF-FGR-9-2020

Clarias magur, a high value Asian catfish, fetch
high market price for its taste and medicinal
value.
In India, efforts to breed this fish started with an
AICRP on air-breathing catfish during 1971. The
efforts led to the development of a captive
breeding technology based on artificial
fertilization of stripped eggs using testis extracts
from a killed male. However the inability to induce
voluntary spawning in captivity remained a major
bottleneck so far in propagating magur culture in
India
BRICS (Barrier Removal In Catfish for
Voluntary Captive Spawning) Technology
The BRICS (Barrier Removal in catfish for
Voluntary Captive Spawning) developed at the
College of Fisheries, Tripura) helps through
hormonal manipulation in spawning of desi
magur, resulting in voluntary spawning in
simple tanks. It is in fact the first report of its
kind for any airbreathing catfish. The BRICS
technology has the potential to bring about a
sea change in catfish aquaculture industry.
Selection of Brooders Spawning facilities
 Simple containers such as polystyrene
 Healthy male and female brooders of 100-
boxes, FRP tanks or plastic tubs can be
180 g without external injuries or parasites
used as spawning facilities
 FEMALE: Bulging belly with round genital
 Provision of a lid would be advisable to
papillae
prevent escape of the brooders as well as
 MALE: Slender belly with elongated genital to provide a dark environment for the
papillae brooders
 BREEDING SEASON: May-August  A feeble water current of 0.3-0.5 litre/
minute is provided to ensure aeration and
thereby high/survival of the spawned eggs
and hatchlings.
 The eggs can be incubated within the
spawning tank for hatching, and the
hatchlings are gently siphoned out after
yolk sac absorption is completed for
nursery rearing.
Method
 Around 2000-3500 eggs can be obtained
 OvatideTM injection: to both male and female from a brooder of 100-150 g body weight
@ 0.5 ml per kg body weight above lateral with around 80% fertilization rate
line in line of genital papillae  Hatching occurs 24-30 hours post
 Place both male and female together in the spawning with a hatching rate of 70%
spawning tank
 Oxytocin Injection: After 12 hours of
Ovatide™ injection, inject oxytocin to both
male and female @ 40 milli international Unit
per kg body weight above lateral line on
caudal peduncle
 Release back both male and female together
to the spawning tank
 Spawning starts around 16-18 hours post
injection of Ovatide™.
 Remove brooders after 24 hour of Ovatide™
injection and continue to incubate the eggs in
the tank with a water flow @ 0.3-0.5 liter per
minute
 | |
Received: 3 February 2020    Revised: 5 July 2020    Accepted: 11 August 2020

DOI: 10.1111/are.14869

ORIGINAL ARTICLE

Hormone manipulation to overcome a major barrier in male

catfish spawning: The role of oxytocin augmentation in
inducing voluntary captive spawning

Himanshu Priyadarshi1  | Rekha Das2  | Atom Arun Singh1  | Arun Bhai Patel1  |

Pramod Kumar Pandey1

1
College of Fisheries (CAU, Imphal),
Lembucherra, Tripura, India
2
ICAR Research Complex for NEH Region,
Tripura Regional Centre, Agartala, Tripura,
India
Correspondence
Himanshu Priyadarshi, College of Fisheries
(CAU, Imphal), Lembucherra, Tripura-
799210, India.
Email: priyadarshimanshu@gmail.com
Abstract
A major challenge in Clariid catfish seed production is our inability to induce volun-
tary captive spawning of the brooders. Since voluntary captive spawning had been
previously elicited in a Clarias species by simulating natural breeding environment,
we speculated that the major obstacle in voluntary spawning might be the absence
of an inducing agent that triggers mating response in the brooders. To address the
issue, oxytocin was injected to the brooders along with Ovatide (a commercial in-
ducing agent comprising salmon gonadotropin-releasing hormone analogue and
domperidone). Administration of 40 mIU/kg body weight of oxytocin after 12 hr of
Ovatide injection resulted in voluntary spawning of the fish (81.5%, i.e. 31 out of 38
trials), with more than 70% hatching rate in simple and low-cost spawning facilities.
Histology of male gonads indicated sustained gamete maturation and the gonads
were observed to be visibly turgid in the fish that received both the hormones. In
contrast, gamete maturation slackened after 10 hr in the control fish, injected with
Ovatide only. Administration of oxytocin along with Ovatide was also observed to
induce aggressive sexual behaviour among the males. Neither of the hormones, act-
ing alone, could induce voluntary spawning in the fish or influence sexual behaviour.
Our results clearly show that oxytocin plays a significant role in sustaining the male
gamete maturation induced by Ovatide and trigger the behavioural mating response,
leading to voluntary spawning in C. magur. The results hold potential to make a para-
digm shift in the commercial catfish seed production technology.

KEYWORDS

aggression, captive spawning, Clarias, GnRH analogue, mating, oxytocin

1 |  I NTRO D U C TI O N
Catfish species of the families Siluridae, Clariidae and Ictaluridae are
commercially significant worldwide as food fish. Seed production of
most of these catfish is done by artificially fertilizing stripped eggs,
using testicular suspension prepared from a sacrificed male. The
technology established during the late 1980s (Manickam & Joy, 1989;
Zonneveld, Rustidja, Viveen, & Mudana, 1988) is still being followed
worldwide due to the absence of any other viable technique. The
major issue, cited in these species, is the inability of the brooders
to spawn voluntarily under captivity even after hormonal induction
(Chowdhury, Chatterjee, Mondal, & Chatterji, 2010). While this con-
straint in catfish seed production might not appear to inflict major
economic losses, there are serious drawbacks with ethical and eco-
nomic consequences. For example, the requirement to sacrifice the
male entails unnecessary animal sacrifice, is cumbersome and limits

Aquaculture Research. 2020;00:1–14. wileyonlinelibrary.com/journal/are© 2020 John Wiley & Sons Ltd     1 |
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2       PRIYADARSHI et al.

genetic improvement programs. Hence, the underlying mechanisms
that govern reproduction and spawning in catfish to elicit voluntary
captive spawning have been extensively explored (Figure 1).
The inducing agents commonly used in seed production of cat-
fish such as Ovatide™, Ovapel™, Ovaprim™ and WOVA-FH™ contain
salmon gonadotropin-releasing hormone analogue (sGnRhA) and a
dopamine antagonist (Brzuska, 2003; Gbemisola & Adebayo, 2014).
In Clarias batrachus, this hormone admixture successfully brings
about the final maturation of eggs, hydration of female gonads
and passage of eggs from the ovary to ovocoel in around 15–18 hr
(Sahoo, Giri, Chandra, & Sahu,  2007, 2009). However, voluntary
spawning is not elicited when a similarly induced male brooder is
placed together with an induced female. It may be argued that the
inducing agent could be physiologically non-compatible to the male
brooders. However, it is worth noting that the injection of Ovaprim™
has been demonstrated to result in progression of gonadal matura-
tion in both male and female C. batrachus (Chowdhury et al., 2010).
Moreover, these earlier mentioned commercial inducing agents have
successfully brought voluntary captive spawning in a variety of fish
such as carp, heteropneustids and perch. Therefore, the hypothesis
that male catfish requires specialized mechanisms for elicitation of
spawning response that are entirely different from that of conspe-
cific females appeared far-fetched. Instead, we suggest that this lack
of voluntary captive spawning was the result of non-synchroniza-
tion of mating response between the brooders, for which additional
hormonal facilitation beyond the usual inducing agent might be
necessary.
Simulation of the natural breeding grounds of the species has
resulted in spontaneous spawning in the case of Clarias species
(Bruton,  1979; Knud-Hansen et  al.,  1990; Priyadarshi et  al.,  2017;
Thakur, 1976). It was also observed that minor deviations in breed-
ing ground design such as lining of the earthen pit with cloth and
replacement of paddy with synthetic egg collectors inhibited spawn-
ing in C. batrachus (Priyadarshi et  al.,  2017). Evidently, failure of
the brooders to spawn in captivity is clearly an offshoot of mating
behaviour rather than a physiological barrier. These observations
show that while the hormonal admixtures currently used in induced
breeding are successful in promoting gametic maturation in both the
sexes, they do not positively influence mating response. Thus, hor-
monal interventions to address this issue in catfish seed production
should aim to remove this barrier in initiating a mating response.
Oxytocin is a highly conserved nonapeptide, which is synthe-
sized in hypothalamic neurons and transported to posterior pituitary
for release in blood circulation in higher vertebrates (Knobloch &
Grinevich, 2014). Homologues of oxytocin are found in a wide group
of animals from invertebrates to mammals under diverse names
such as isotocin in fish, mesotocin in amphibians, reptiles and birds,
annetocinin annelids and conopressin and cephalotocin in mollusc
and pitocin for the synthetic form (Cruz et al., 1987; Knobloch &
Grinevich, 2014; Oumi et al., 1996; Reich, 1992).

F I G U R E 1   Chronology of the research attempts to induce voluntary captive spawning in catfish species and major milestones. The
present study reports hormone-based elicitation of voluntary captive spawning (VS) in a Clariid catfish species by administration of
oxytocin. Importantly, natural breeding grounds were not simulated in our study and spawning was conducted in simple containers
amenable to easy management by the breeder
PRIYADARSHI et al.
Oxytocin plays diverse roles in the physiology of animals
such as reproduction, egg laying, pair bonding, courtship, social
behaviour, memory, gut motility and osmoregulation (Feldman,
Monakhov, Pratt, & Ebstein,  2016; Garrison et  al.,  2012). In fish,
oxytocin is known to play significant roles in reproduction includ-
ing induction of physiological responses to spawning (Heller, 1972;
Ohya & Hayashi,  2006; Pickford & Strecker,  1977; Wilhelmi,
Pickford, & Sawyer,  1955). The expression of brain isotocin tran-
scripts was found to be higher during breeding season in female
three-spined sticklebacks (Gasterosteus aculeatus), implying its
role in reproduction (Gozdowska, Kleszczyńska, Sokołowska, &
Kulczykowska, 2006). The ovary and oviduct muscles of ovipa-
rous and ovoviviparous fish were observed to show contractile
responses when treated with oxytocin (Heller,  1972). Ohya and
Hayashi (2006) observed strong immune-blotting signals of isoto-
cin in pre-spawned female medaka fish (Oryzias latipes), indicating
that isotocin had significant role in bringing about the physiological
response to spawning. In killifish (Fundulus heteroclitus), administra-
tion of oxytocin (synthetic as well as natural) elicited spawning re-
flux response similar to neuro-hypophysial preparations (Pickford
& Strecker, 1977; Wilhelmi et al., 1955). With this background, the
study aimed at identifying potential effects of oxytocin on mating
behaviour of C. magur in order to induce voluntary spawning in
captivity.
2 | M ATE R I A L A N D M E TH O DS
2.1 | Brooder collection and housing
Brooders collection, maintenance and experiments were performed
in strict adherence to the ethical standards of the Institutional
Animal Ethics Committee of the College of Fisheries, Central
Agricultural University, India (CAU-CF/48/IAEC/2018). Healthy and
(a)
F I G U R E 2   Spawning facilities used
in the experimental trials: The different
spawning facilities used successfully in
the study. Inner view of the polystyrene
boxes converted into spawning facilities
(a) by fitting a funnel in the inner corner
to act as an outlet (dotted arrow). An inlet
pipe passing through the lid (dotted line)
which provided free flow of water from a
storage tank is visible in the outer view of (c)
the box (b). Successful voluntary spawning
was also elicited in alternate spawning
facilities such as a 200 L FRP tank (c) and
a circular plastic tub (d; data not discussed
due to small sample size). The solid arrows
in each image point to the viable eggs
spawned voluntarily in each tank
|
      3
sexually mature C. magur brooders of 120–180 g body weight (bw)
were procured from farmers’ ponds.
Taxonomical identity of the collected fish was confirmed using
conventional taxonomic keys as well as using their cytochrome oxi-
dase I (COI) gene sequence data. Total genomic DNA was extracted
from fin clippings of the brooders following phenol–chloroform
method (Sambrook & Russell, 2001). COI gene was amplified from
the extracted DNA using the primers and the thermal cycling condi-
tions described by Barman et al. (2018) and subjected to sequencing.
The homology of isolated COI sequence to C. magur was confirmed
by NCBI BLAST homology searching (99.5% similarity observed).
Two representative sequences were submitted to the public domain
(NCBI accession number MT394919 and MT394920).
The experimental fish were housed in indoor cistern tanks with
constant aeration for 60  days. Upturned small earthen pots were
provided as shelter for the fish. The fish were fed ad libitum on pel-
leted feed (procured from the Department of Aquaculture, College of
Fisheries, Tripura), containing 22% crude protein. Uneaten food and
other debris were siphoned out daily, and lost water was replaced.
Around 40% water was exchanged once in a week. Dissolved oxy-
gen, pH and temperature in brooders rearing tanks, overhead tanks
and spawning facilities were maintained at 8.0 ± 1.0 ppm, 7.8 ± 0.2
and 28.0 ± 1.0°C, respectively, throughout the experiments.
2.2 | Spawning tank
Polystyrene boxes (length: 50 cm; breadth: 30 cm & height: 30 cm),
containing water up to 10  cm height (water volume: 15 Litre), and
secured with a lid (Figure 2a,b) were used as spawning tanks for the
study. A water flow of 0.3 Litre/minute was maintained with the help
of a tube in each spawning tank. Care was taken to ensure that the
brooders (male and female), released within a spawning tank, were
of similar size in all the trials.
(b)
(d)
 |
4       PRIYADARSHI et al.
2.3 | Hormonal induction for voluntary spawning
Ovatide™ (Hemmo Pharmaceuticals Pvt. LTD, India; hereafter re-
ferred to as simply ‘Ovatide’), a commercial inducing agent for seed
production in fish, was used as source of sGnRhA (salmon gonado-
tropin-releasing hormone analogue) and domperidone (a dopamine
antagonist) in our study. Each ml of Ovatide contains 20 µg of sGn-
RhA and 10 mg of domperidone.
Ovatide was used uniformly at the recommended dose of 0.5 ml/
kg bw in our experiments (Priyadarshi et  al.,  2017). The male and
female brooders, in every breeding set, were placed together imme-
diately after administration of Ovatide injection and allowed to re-
main together until the end of the respective experiments. Oxytocin
(Novartis, India; 5.0  IU/ml) was diluted, using sterile physiological
saline to attain the desired dose in the experiments while restrict-
ing the final injection volume to 100 µl. To equalize the effect of
injection stress on outcomes, an equal volume of sterile physiolog-
ical saline (blank) was injected to fish in the control groups in place
of oxytocin. For all fish, Ovatide injections were administered just
above the lateral line in the line of the genital organ and oxytocin
was injected just above the lateral line in the caudal peduncle region.
Observations on voluntary captive spawning (VS) were made after
24 hour (hr) of administration of Ovatide.
2.4 | Fertilization and hatching rate estimation
For the estimation of fertilization and hatching rates of the spawned
eggs, 100 eggs were scooped out into a Petri plate from each
F I G U R E 3   Schematic representation
of the experimental set-up: Dotted box
(breeding set), M (male), F (female), Ov
(ovatide injected @ 0.5 ml/kg bw), Ox-20
(oxytocin injected @ 20 mIU/kg bw),
Ox-40 (oxytocin injected @ 40 mIU/kg
bw), Ox-80 (oxytocin injected @ 80 mIU/
kg bw), Sps (sterile physiological saline),
h (hr; time interval between injection of
Ovatide and oxytocin/Sps when read in
combination with the preceding numeral),
R (Replication), ASF (alternate spawning
facility)
spawning tank. The number of fertilized eggs, characterized by their
golden-brown colour, was counted from the total number of eggs.
To enumerate hatching rate, developing eggs were similarly scooped
out after 24 hr of spawning and the number of hatchlings released
from the eggs was counted. The fertilization and hatching rates were
estimated by the standard method (Manickam & Joy, 1989).
2.5 | Experiments to identify most effective dose-
timing interval combination
On the basis of the earlier literature relating to other animals
(Argiolas, Melis, & Gessa,  1986; Argiolas, Melis, Stancampiano,
& Gessa, 1989; Arletti, Bazzani, Castelli, & Bertolini,  1985; Melis,
Argiolas, & Gessa, 1986; Thackare, Nicholson, & Whittington, 2006;
Wilhelmi et al., 1955), a preliminary estimate of dosage of oxytocin
in C. magur was arrived at as 40 milli-international units per kilogram
body weight (mIU/kg bw). To identify the most suitable dose of
oxytocin and appropriate time interval between Ovatide and oxy-
tocin administration, experiments were conducted by altering the
dose (dose determination experiments) and time interval between
Ovatide and oxytocin injections (time interval experiments). In these
experiments, a breeding set consisting of one male and one female
brooder were maintained per spawning tank (Figure 3).
In the time interval experiments, a fixed oxytocin dosage of
40  mIU/kg bw was used for the brooders (both male and female)
in the treatment group, administered simultaneously with Ovatide
(0 hr), and after 4, 8, 12 and 16 hr of Ovatide injection. Brooders in
control group were administered 100 µl sterile physiological saline
PRIYADARSHI et al.
(blank) in place of oxytocin at each specified time interval after
Ovatide. For each chosen time interval, breeding sets in quadru-
plicates were maintained and the experiment was repeated twice.
Thus, a total of eight replicates (breeding set) for each time interval
were maintained for the treatment group and eight replicates for
each corresponding control. Cumulatively there were a total of 40
treatment and 40 control breeding sets in the time interval exper-
iments (Figure 3).
On the basis of the results of time interval experiments, three
different oxytocin doses 20, 40 and 80 mIU/kg bw were tested for
both male and female at the time interval between Ovatide and oxy-
tocin injection identified as the most suitable. In the control group,
the brooders were administered Ovatide, followed by injection of
100 µl sterile physiological saline (blank) at the identified time inter-
val. These experiments were also conducted in quadruplicate and
repeated twice. Therefore, cumulatively there were a total of 24
treatment and 24 control breeding sets in the dose determination
experiments (Figure 3).
An additional control was also used (in quadruplicates, repeated
twice) where the brooders were administered only oxytocin at the
dose of 40 mIU/kg bw.
2.6 | Experiments to confirm most effective dosage-
timing combination
The identified most effective dose-timing combination was fur-
ther tested in ‘confirmatory experiments’. In these experiments,
minor modifications to the experimental set-up were made to
check the robustness of the identified dose-timing combination
including breeding set structure, spawning facility used as well as
use of brooders collected from different locations. Considering
the recommendation for use of different sex ratios for optimal
hatchery operations in several fish species (Pillay & Kutty, 2005),
two breeding set combinations—type I combination (one male
and one female in each spawning tank) and type II combination
(two males and one female in each spawning tank)—were tested
in these experiments. For each of the breeding set combination,
9 replicates were conducted, using the identified dosage-timing
combination. In respective control group (9 replicates for each
breeding set combination), brooders were injected with Ovatide,
followed by injection of 100 µl sterile physiological saline (blank)
after 12 hr. In addition, identified dose–time combination was
tested in C. magur brooders, collected from three different loca-
tions of Tripura (Khowai, Teliamura and Gomati), to observe the
efficacy of the treatments for voluntary spawning response. For
brooders from each location (hereafter referred to as stock), tri-
als were conducted with five replicates in both control (brooders
received only Ovatide) and treatment groups. Only type I breed-
ing set combination was used for these experiments in both the
treatment and control. The effectiveness of the identified dose
and time combination was also tested, using a different (altered)
spawning facility (FRP tanks of area: 1.0  m2 and water depth:
|
      5
10 cm with simple aeration and egg collectors; Figure 2 c) using
five replicates for each treatment and control.
2.7 | Male gonad histological studies
In a separate experiment, 25 males (treatment animals) were ad-
ministered Ovatide and oxytocin (0.5 ml/kg bw and 40 mIU/kg bw,
respectively) simultaneously and held in a cement tank, containing
freshwater with constant aeration. The control animals (25 num-
bers) were administered only Ovatide at the rate of 0.5 ml/kg bw
and held in a different tank. Five animals from each group were
sampled randomly at 0, 5, 10, 20 and 24 hours post injection(hpi).
Testis and seminal vesicles of the sampled individuals were dis-
sected aseptically and preserved in 10% neutral buffered formalin
for histological examination. Tissue samples were also collected
from spent live and morbid individuals of the trials, (24 hr after
injection of Ovatide) from type II combination of confirmatory
experiment and preserved in 10% neutral buffered formalin for
further study.
The fixed tissues were dehydrated in ascending grades of alco-
hol and embedded in paraffin wax to form blocks. Sections of 4 µm
thickness were cut from these blocks, using a microtome and placed
on a glass slide. The tissue sections were rehydrated, using descend-
ing grades of alcohol and stained with haematoxylin and eosin. The
sections were then mounted with a coverslip and DPX mountant and
observed under a light microscope (Bell & Lightner, 1988). The slides
were photographed, using Zeiss Axio ScopeA1 microscope camera
(Germany).
For semi-quantitative staging of spermatogenic maturity pro-
gression, histology slides imaged at 20× magnification were used
for manual cell counting using ImageJ 1.52v software. Five sections
from each ribbon were mounted per slide, and two slides were pre-
pared per individual. Therefore, 10 sections per individual were
counted. The Grid function in ImageJ was used to place a 154 points
grid uniformly over each image. The counts per tissue category pres-
ent in the upper right corner of the intersection point of each grid
were noted manually from the images. This tissue category count
was used to estimate the Spermatogenic Maturity Index (SMI) fol-
lowing the procedure of Tomkiewicz, Kofoed, and Pedersen (2011).
2.8 | Statistical Analysis
Since the outcomes of the experiments were categorical, two-
tailed Fisher's exact test at 5% level of significance was employed
to test the effect of oxytocin dosage, time interval for oxytocin in-
jection and breeding set combination on voluntary captive spawn-
ing response and sexual competition-mediated fatal injury. The
outcomes were scored as 'voluntary captive spawning (VS) or no
voluntary captive spawning (NVS)” and “fatal injury or no injury”.
Fisher's exact test has been shown to provide more accurate results
in the case of categorical data with expected cell frequency being
 |
6       PRIYADARSHI et al.

lower than 5 (Han, Yanagisawa, Kato, Park, & Nakamura,  1993;
McDonald,  2009; Schumacher, Tait, & Holmes,  1981). SMI data
were normalized by arcsine transformation before analysis by one-
way ANOVA, followed by Duncan's multiple comparison post hoc
tests. Significance was accepted as p < .05. Statistical comparisons
for fertilization rate and hatching rate between different treat-
ment groups of the respective experiments were carried out, using
Kruskal–Wallis test (p < .05).
3 | R E S U LT S
3.1 | Effective dose–time interval combination
In the time interval experiments, the proportion of breeding sets
with a voluntary spawning response was significantly higher when
oxytocin was administered at a dose of 40 mIU/kg  bw after 12  hr
of Ovatide injection (Figure  4 A) compared with the rest of the

F I G U R E 4   Spawning response outcome in the experiments: The proportion of breeding sets where voluntary spawning occurred in each
experimental trial is depicted by the dark portion of the bars in (a) experiments to identify most effective dose-timing combination and (b)
confirmatory experiments. The statistically highest proportion (p < .05) of VS was observed when oxytocin was administered 12 hr after
ovatide and at the dose of 40 mIU/kg bw (indicated by the bars highlighted with asterisk symbol; a). This dose-timing combination yielded
higher VS proportion than control groups irrespective of the stock of brooders, breeding set combination type or spawning facility alteration
(bars highlighted with different superscripts to denote statistically significant difference; b). Statistical comparison was made within the
respective experiment as summarized in the text. Treatment group in confirmatory experiments received Ovatide at dose of 0.5 ml/kg and
oxytocin at the rate 40mIU/kg bw, 12 hr apart; control group received Ovatide at dose of 0.5 ml/kg and blank physiological saline after
12 hr. Legend: type I: type I combination breeding set (one male and one female in each spawning tank); type II: type II combination breeding
set (two males and one female in each spawning tank); VS: voluntary captive spawning; NVS: no voluntary captive spawning; T: treatment
(received both Ovatide and oxytocin) and C: control (received Ovatide and sterile physiological saline)
PRIYADARSHI et al.
treatment groups (p < .05). In the group that was administered oxy-
tocin 16 hr after Ovatide injection, the brooders spawned unviable
eggs and so the response was counted as null. In addition, spawn-
ing was observed to be partial in our breeding trials where oxytocin
was administered ≤8 hr after Ovatide injection (only about 200 eggs
spawned as against 3,500–4,000 eggs in breeding trials where oxy-
tocin was administered 12 hr after Ovatide). In dose determination
experiments, when three different doses of oxytocin (administered
12 hr after Ovatide injection) were further tested for efficiency, the
dose of 40 mIU/kg bw yielded significantly higher (p < .05) instances
of voluntary spawning (VS: 7 out of 8) over both 20 mIU/kg bw and
80 mIU/kg bw doses (VS: 2 out of 8 and 1 out of 8 respectively;
Figure 4a). No significant difference in fertilization rate or hatching
rate was observed among the tanks where voluntary spawning was
observed (p > .05). The fertilization and hatching rates in the tanks
ranged from 75%–85% and 65%–80% respectively (Figures 5 and 6).
3.2 | Confirmatory experiments
Since the voluntary spawning response was highest when 40 mIU/
kg bw oxytocin was administered to the brooders after 12 hr of
Ovatide injection, this dose and time interval combination was fur-
ther tested in different breeding set combinations, stock and differ-
ent spawning facilities. There was no significant difference in the
voluntary spawning response between the two breeding set com-
binations (p > .05). Voluntary spawning was recorded in 7 out of 9
trials in type I and 8 out of 9 trials in type II breeding sets (Figure 4b)
with fertilization and hatching rates of 83%–85% and 75%–81% re-
spectively (Figures 5 & 6). Voluntary spawning occurred at statisti-
cally similar rates (p  >  .05) between the brooders, collected from
F I G U R E 5   Comparison of fertilization rate among the
treatment groups: Bars represent mean fertilization rate of
voluntarily spawned eggs in the respective treatment group of
the experiments. The mean values were compared only within the
respective experiments. No significant difference in fertilization
rate was observed (p > .05)
|
      7
different locations (Khowai, Teliamura and Gomati; Figure 4b) with
70%–75% hatching (Figures 5 & 6). All the five breeding sets in the
altered spawning facility also spawned voluntarily with 70%–75%
hatching rate (Figures 5 & 6). Cumulatively, out of 38 treatment trials
conducted, using this dose and time combination, voluntary spawn-
ing of viable eggs was observed in 31 trials (Figure 4b).
No voluntary captive spawning was observed in any of the con-
trol groups in the dose–time interval combination and confirmatory
experiments. In the controls, where only Ovatide was administered,
there was clear swelling of belly and free oozing of eggs in females,
but voluntary spawning could not be observed in any of the sets.
In contrast, no change was observed in the control group brooders
which received oxytocin only.
3.3 | Aggression in brooders
Fatal injury was consistently observed in one of the male brooders
in type II breeding sets when the fish received both Ovatide and
oxytocin (Figure 7). Both the males, in these breeding sets, were of
similar size. The occurrence of this outcome was significantly higher
in the type II breeding set combinations (7 out of 9 trials) than the
type I breeding set combinations (0 out of 9 trials) by Fisher's exact
test (p < .05). Fatal injury was also recorded on the body of female
brooders in type I breeding sets when the brooders were adminis-
tered an oxytocin dose of 80 mIU/kg bw (6 out of 8 trials). In con-
trast, no such injury on female was observed when the brooders
were administered an oxytocin dose of 20 or 40 mIU/kg bw, which
was significantly lower than 80 mIU/kg bw (p < .05).
Notably, no aggression or injury was noted in any control group,
where oxytocin was either not administered or administered alone.
3.4 | Gonadal histology
In a separate experiment, the effect of hormonal treatment on the
gonadal tissues (testis and seminal vesicles) was examined through
histology. Sampling was performed at 0, 5, 10, 20 and 24 hpi for both
the treatment and control groups. Sampling was also performed
from live but spent brooders and morbid male brooders after 24 hr
of Ovatide injection from type II set of confirmatory experiments.
The testicular histology clearly indicated a progression in go-
nadal maturation, which was apparent even in the control group
fish, administered Ovatide only (Figure 8). However, this progression
slackened after 10 hr, with the gametes at advanced stages replaced
by more primary stages in most of the lobules in the control group
(Figure 9a, c). At the end of 24 hr, testis sections from these individ-
uals (control group) appeared similar to the zero (0) h sections. In
contrast, when oxytocin was injected along with Ovatide, the tes-
tis tissues sustained the progression of gamete maturation beyond
10 hr (Figure 9b), and at 24 hr, the lobules were primarily occupied by
spermatozoa (Figure 9d). In addition, the testicular epithelial lining
of the individuals that received both the hormones appeared more
 |
8       PRIYADARSHI et al.
turgid than those that did not (Figure S1). In the seminal vesicles, the
cells lining the lobules showed signs of increased secretory activity
over time in both the groups (Figure  10). However, this appeared
relatively more pronounced in the treatment groups.
The histological appearance of the gonads was also compared
between the spent live male brooders and morbid male brooders
from the type II breeding sets of confirmatory experiments. The
testis histology of the spent live individuals featured mostly empty
lobules except for scanty loose packets of spermatids. Some lob-
ules also contained large beehive-like secondary spermatocyte
cysts. In comparison, the testes of the morbid individuals from
these trials were filled with spermatozoa (Figure S2), indicating that
sperm release did not happen in these individuals. The semi-quan-
titative staging and SMI estimation from the histology slides also
F I G U R E 6   Comparison of hatching rate among the treatment
groups: Bars represent mean hatching rate of voluntarily spawned
eggs in the respective treatment group of the experiments.
The mean values were compared only within the respective
experiments. No significant difference in hatching rate was
observed (p > .05)
(a) (c)
(b)
corroborate these observations (Figure S3, S4). The differences in
the seminal vesicle histology also were dramatic between the spent
live and morbid fish. While the sections from the spent live brooder
displayed large round lobules filled with secretions, the lobules from
the morbid males had disintegrated walls with no sign of epithelial
repair activity (Figure S5).
4 | D I S CU S S I O N
Seed production technology for many catfish species of Siluridae,
Clariidae and Ictaluridae families still rely on the artificial fertiliza-
tion of stripped eggs, using the grounded testis extracts from a killed
male (Brzuska,  2003; El-Hawarry, El-Rahman, & Shourbela,  2016;
Hogendoorn, 1979; Su, Perera, Mu, & Dunham, 2013; Tsadu, Yisa,
& Etuh,  2012; Zonneveld et  al.,  1988). This procedure is adopted
since these fish species do not spawn voluntarily in captivity even
after hormonal induction (Chowdhury et al., 2010). This require-
ment of having to sacrifice a brooder of potential genetic worth at
every cycle imposes enormous limitations in developing genetic im-
provement protocols for these species. Based on the observations
that females of these species respond to hormonal inducing agents
and release eggs on stripping, we speculated that this inability to
spawn in a restricted environment is the result of barriers in mat-
ing behaviour. We report for the first time the use of oxytocin to
remove barriers to the mating response and induce voluntary cap-
tive spawning in C. magur, a Clariid catfish preferred in the Indian
subcontinent for its fast growth rate, hardy nature, taste and me-
dicinal value.
Captive seed production in food fish including cyprinids and
catfish is carried out using natural or synthetic analogues of go-
nadotropin-releasing hormone to trigger final maturation of the
gametes and spawning (Brzuska,  2003; Kahkesh, Feshalami, Amiri,
& Nickpey,  2010). Previous research has demonstrated physiolog-
ical responses to Ovatide administration in catfish (Chowdhury
et al., 2010; Sahoo, Giri, Chandra, & Sahu, 2007, 2009), and suc-
cessful spawning following Ovatide injection has been elicited in
F I G U R E 7   Aggression induced
by sexual competition in the treated
brooders: Images of actual breeding sets
captured after completion of the trial
showing injured brooders due to sexual
aggression. The arrows in the images
show an injured male brooder in type II
breeding set (a) and an injured female in a
type I breeding set where 80 mIU/kg bw
oxytocin was administered. The close-
up image of the injury inflicted is also
shown (c)
PRIYADARSHI et al. |
      9

F I G U R E 8   Comparison of progression
(a) (b)
of gamete maturation in testis of C. magur
in response to oxytocin administration:
Clear progression in the extent of
gamete maturation in comparison with
0 hr samples can be observed in both
the groups. It is clear that spermatids
increasingly replace the spermatocyte
cysts in the lumen of these samples.
This rate of replacement is visibly higher
in the fish injected with both Ovatide
and oxytocin. a, b, c: Testis micrograph
of Clarias magur injected with Ovatide (c) (d)
alone and sampled at 0, 5 and 10 hpi
respectively; d, e, f: testis micrograph
of Clarias magur injected with Ovatide
and oxytocin, and sampled at 0, 5 and
10 hpi respectively. Sc, spermatocyte
cysts; St, spermatids; Sz, spermatozoa; Sg,
spermatogonia; GE, germinal epithelium;
Se, Sertoli cells; Le, Leydig cells; CT,
connective tissue; Tc, tunica albuginea;
Vc, vacuole. The scale bar represents
2 µm (e) (f)

(a) (b)

(c) (d)

F I G U R E 9   Stalling of gamete maturation after 10 hpi in testis of Ovatide induced C. magur in the absence of oxytocin: Prominent
germinal epithelia containing spermatogonial cells and Sertoli cells can be observed in both the groups at 20 hpi (arrowheads), indicating
an ongoing process of gamete maturation. However, the testicular lobules of fish that received only Ovatide (control) featured large
spermatocyte cysts at 20 hpi (a) and 24 hpi (c) as against spermatozoa in fish injected with both Ovatide and oxytocin (b and d respectively).
Sc, spermatocyte cysts; St, spermatids; Sz, spermatozoa; Sg, spermatogonia; GE, germinal epithelium; Se, Sertoli cells; Le, Leydig cells; CT,
connective tissue; Tc, tunica albuginea; Vc, vacuole. The scale bar represents 2 µm
 |
10       PRIYADARSHI et al.

(a) (b)

(c) (d)

F I G U R E 1 0   Seminal vesicle micrographs in C. magur after hormonal induction: At 0 hr (a), the lobules have well-defined boundaries
lined by flattened epithelial cells. Five hours post injection (b), the epithelial cells are visibly turgid, with a cuboidal appearance. Vacuole-
like structures filled with unstained secretions are seen secreted into the lumen. Over time, the lobules get distended due to the secretions
(10 hpi, c). At 20 hpi (d), the lobules are highly distended and epithelial walls between lobules disintegrate to allow mixing of the contents
between the lobules (block arrows). A major portion of the lobules are occupied by the unstained secretions from epithelia (indicated by
asterisk)

spawning facilities that simulated their natural breeding grounds
(Priyadarshi et al., 2017). This indicates that the hormone receptors
that respond to these allochthonous hormonal agents (sGnRhA and
dopamine antagonist) are expressed in these species at this time, in
both the sexes. With this background, we hypothesized that the bar-
rier to spawning in the species is mediated by non-synchronization
or mating behaviour of rather than physiological gonadal maturation
issues.
Our predicted optimal dose of 40 mIU/kg bw was confirmed to
be effective in the dosage and timing interval combination exper-
iments. Lower dosages resulted in a poor response, while higher
doses resulted in severe aggressive behaviour in the males.
Oxytocin alone did not elicit spawning in our studies. In fact,
in the absence of Ovatide, oxytocin failed to induce final matura-
tion of gonads even in the females and free oozing of eggs was not
observed. This was not surprising, since previous studies have in-
dicated that oxytocin exerts its effects during the spawning phase
rather than the initial maturation stages. For instance, in vitro treat-
ment of gold fish pituitary gland with isotocin caused release of
luteinizing hormone (LH) but not the follicle-stimulating hormone
(FSH; Mennigen, Volkoff, Chang, & Trudeau, 2017). While FSH pre-
dominates and control early stage of gametogenesis, LH is expressed
and prominently regulates ovulation and spermiation (Kazeto
et al., 2008; Palermo, 2007; Suzuki, Nagahama, & Kawauchi, 1988;
Tyler, Sumpter, Kawauchi, & Swanson, 1991). Brain and ovarian iso-
tocin transcript expression in Heteropneustes fossilis was shown to
peak in preparation to spawning and during spawning, quickly de-
clining thereafter (Banerjee, Chaube, & Joy, 2015). In vitro incuba-
tion of C. gariepinus testis slices in a medium containing oxytocin
caused release of sperms into the medium (Viveiros, Jatzkowski, &
Komen, 2003). Interestingly, however, in vivo treatment with oxyto-
cin in hypophysed males did not improve the strippability of males
in the study conducted by Viveiros et al. (2003). In that study, the
authors injected oxytocin at a dose of 5 IU/kg bw intravenously into
male C. gareipinus, that had already been induced using carp pituitary
gland extract, and the semen samples were collected by stripping
30 min later. It is possible that the lack of significant improvement
after oxytocin injection was due to insufficient time available for ac-
tion before sampling.
From our histological results, the progression of gametic mat-
uration in males declined after 10 hr in fish that received only
Ovatide. In sharp contrast, gonadal tissues of the fish that received
both the hormones simultaneously showed sustained advance-
ment of maturation over time. This advancement also culminated
in spawning when the injected males were housed together with
a similarly induced female. Administration of oxytocin after 12 hr
of Ovatide injection was observed to yield the best response in
terms of voluntary captive spawning. It coincided with the begin-
ning of the decline in gamete maturation after Ovatide adminis-
tration. Further, voluntary spawning was partial where oxytocin
was administered ≤8 hr after Ovatide injection. These results fur-
ther strengthen the hypothesis that oxytocin primarily exerts its
actions in the later stage of the reproductive processes, after the
gamete maturation has been completed (Mennigen et al., 2017).
Further, on the basis of our results, we suggest that oxytocin is
also important for sustaining the gamete maturity advancement in
male gonads until mating.
Curiously, the seminal vesicle histology of males with a 16-hr
gap between the injections looked similar to fully spawned males.
However, the spawned eggs were not fertilized and no hatching
PRIYADARSHI et al.
was observed in these cases. This may be the result of a mismatch
between spawning readiness in the male and female brooders. The
average time for release of eggs into ovocoel in C. magur females
is around 15–18  hr post Ovatide injection. The eggs are known
to begin rapid deterioration once released to the ovocoel (Sahoo
et al., 2007, 2009). It is possible that due to the larger time gap be-
tween the injections, the spawning response in the males was not
synchronized with the females in these breeding sets. This probably
resulted in the female gametes undergoing degeneration well before
the release of the sperm.
A common observation in the fish dissected for histological
studies was the stark difference in the appearance of the gonads.
The fish that received both oxytocin and Ovatide had markedly tur-
gid gonads, especially apparent at 20 hpi. This was also reflected in
the appearance of the gonadal cells in the histology of this group.
Hydration is understood to be one of the most important factors
in the expulsion of matured eggs from the ovocoel or release of
gametes (egg and spermatozoa) in fish (Adebayo & Fagbenro, 2004;
Brown-Peterson, Wyanski, Saborido-Rey, Macewicz, & Lowerre-
Barbieri, 2011; Rainis, Mylonas, Kyriakou, & Divanach, 2003).
Oxytocin is known to influence tissue osmoregulation in verte-
brates, possibly through a signalling pathway involving arginine
vasopressin (AVP) and aquaporins (Li et  al.,  2008). Considering
the close structural and functional similarity demonstrated be-
tween arginine vasopressin and it is piscine homolog arginine va-
sotocin (AVT; Sangiao-Alvarellos et  al.,  2006; Warne, Harding, &
Balment, 2002), the potential role of oxytocin–AVT signalling in go-
nadal hydration and spawning warrants further study. Interestingly,
the testes of individuals that suffered injury or died in the experi-
mental trials were found not to be turgid. It was also evident from
the tissue histology that these individuals had not spawned.
Severe/fatal injuries to a male brooder were a consistent fea-
ture in all the tanks where two males were housed with a female
when both Ovatide and oxytocin were administered. This was a
striking observation throughout the study. Tissue histology sug-
gests that both the males in such a scenario were of similar sexual
maturity levels and ready to spawn. The possibility that aggression
was due to the differences in size can be ruled out as both the
male brooders were of the same size. Importantly, no injury/death
was observed in the male when the tanks contained one female
and one male. In addition, such aggressive interactions were not
observed in the tanks when only one of the two hormones was
administered alone. It is evident from these results that the ag-
gression, resulting in fatal injuries in the study, was the result of a
sexual competition for mating since both males appeared physio-
logically capable of mating.
The pattern of aggression observed also clearly underlines the
role of oxytocin in mating behaviour of male C. magur. Oxytocin is
implicated in a range of reproduction-related behaviours in ver-
tebrates. Interestingly, some crosstalk between the receptors of
oxytocin and AVP has been demonstrated in vertebrates (Song &
Albers, 2018) that significantly influences the social neurocircuits
|
      11
in the brain (Baribeau & Anagnostou,  2015) and affect multiple
physiological and behavioural responses. In dominant Oreochromis
mossambicus males that exhibit territorial behaviour and actively
court females (Oliveira & Almada,  1998), isotocin levels in the
telencephalon (forebrain) have been found to be higher than in
the subordinate males (Almeida, Gozdowska, Kulczykowska, &
Oliveira, 2012). Oxytocin has been associated with sexual arousal
in human beings (Blaicher et al., 1999; Carter, 1992; Thackare
et al., 2006; Veening, De Jong, Waldinger, Korte, & Olivier, 2015)
and elicits the spawning reflex response in killifish, Fundulus het-
eroclitus (Pickford & Strecker, 1977; Wilhelmi et al., 1955). Notably,
aggression was directed towards the female when oxytocin levels
were significantly elevated in males, in a 1:1 breeding pair. It can
be concluded elevated levels of the hormone led to exaggerated
and unsynchronized sexual arousal in the male which could not be
complemented by the female.
This is the first report of the use of oxytocin for inducing volun-
tary captive spawning in any catfish species. Our results indicate that
oxytocin works both by sustaining the gamete maturation process in
male catfish after GnRhA-domperidone induction and by eliciting
behavioural responses needed for mating. The molecular mecha-
nisms and potential signalling pathways that mediate these effects
would be an exciting avenue for future work in this direction. While
this work was performed on C. magur, it might be equally applicable
in inducing voluntary spawning and achieving higher efficiency in
hatchery operations for several other fish species. Our protocol has
the potential for widespread change in the seed production tech-
nology for C. magur, one of the most important aquaculture species
in Asia.
AC K N OW L E D G M E N T S
The authors are grateful to Professor M. Premjit Singh, Vice-
Chancellor, CAU, Imphal, for providing necessary facilities. The first
author would like thank to Dr. Rekha Das (second author) for her
enormous and unfatigued support and compromise at family front
for devoting her time to analyse data, review literature and in overall
shaping the entire study.
C O N FL I C T O F I N T E R E S T
The author(s) declare(s) that there is/are not conflict(s) of interest.
AU T H O R C O N T R I B U T I O N S
H.P. conceptualized the idea; H. P., R.D. and A. A. S. involved in
methodology and primary data acquisition; H. P., A. A. S. and A. B.
P. contributed to field management of experiments; R.D. and H.P.
involved in statistical analysis and histological examination; R. D. and
P.K.P. involved in manuscript drafting and editing; P. K. P. provided
administrative support.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
 |
12       PRIYADARSHI et al.
ORCID
Himanshu Priyadarshi  https://orcid.org/0000-0003-2350-9734
Rekha Das  https://orcid.org/0000-0002-8901-8797
Atom Arun Singh  https://orcid.org/0000-0002-2525-6497
Arun Bhai Patel  https://orcid.org/0000-0003-3216-0693
Pramod Kumar Pandey  https://orcid.org/0000-0001-9629-4549
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