2021, Vol. 11, No. 2, 141 – 149 http://dx.doi.org/10.11594/jtls.11.02.03
How to cite:
Araf T, Hossen MA, Chowdhury G et al.(2021) Artificial Propagation and Embryonic Growth of Stinging Catfish, Research Article
Artificial Propagation and Embryonic Growth of Stinging Catfish, Heteropneustes fossilis (Bloch, 1794) Using S-GnRHa (Salmon Gonadotropin Releasing Hormone Analogue)
Tasrick Araf 1, Md. Alal Hossen 1, Gourab Chowdhury 1, Mohammad Amzad Hossain 1*, Md. Arifur Rahman 2, Mohammed Mahbub Iqbal 1
1 Department of Fish Biology and Genetics, Sylhet Agricultural University, Sylhet-3100, Bangladesh
2 Department of Fisheries Biology and Genetics, Patuakhali Science and Technology University, Patuakhali-8602, Bangladesh
Article history:
Submission December 2020 Revised January 2021 Accepted March 2021
ABSTRACT
An experiment was performed to examine the potentiality of a synthetic hormone an- alogue in the artificial propagation i.e., the embryonic and larval development of sting- ing catfish (Heteropneustes fossilis). Broodfish were injected with S-GnRHa at the concentrations of 1, 2.5, and 5 ml.kg-1 of body weight (BW) in females and at 0.5, 1.125 and 2.5 ml.kg-1 BW in males treatment groups T1, T2 and T3, respectively, with three replicates in each group. The fishes were induced to spawn, and ovulation oc- curred at about 10-11 hours after the hormone injection. Results showed highest fer- tilization (83.11±1.36) and hatching rates (89.56±1.04) in T2, whereas ovulation rate was 100% in all treatment groups. First cleavage was observed at 30 minutes of post- fertilization. Embryonic developmental period sequentially for 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, 64-cell, Morula (Improve), Blastula, Gastrula, somatic formation, yolk-plug, twisting movement and pre-hatching occurred at 00:30, 00:45, 01:10, 01:30, 02:00, 02:30, 03:00, 04:00, 06:40, 09:00-18:00, 19:00, 20:00-21:00 and 22:00 hours, respectively. Hatching occurred after 23:00 hours of fertilization. Current re- sults suggest the feasibility of S-GnRHa as an effective synthetic hormone in the arti- ficial propagation of H. fossilis.
Keywords:Artificial propagation, Cleavage, Embryonic growth, S-GnRHa, Somatic formation
*Corresponding author:
E-mail: [email protected]
Introduction
The Heteropneustes fossilis (Bloch, 1794), commonly known as stinging catfish, belongs to the family Heteropneustidae [1, 2, 3] and it is treated as one of the recorded commercial fresh- water fish species in Southeast Asia [4, 5]. This fish species is popular because of its taste and high nutritional values, low fat, high iron content, and medicinal point of view, hence, endorsed as a nourishment supplement for convalescing patients [1, 6, 7, 8]. Capability to utilize atmospheric oxy- gen with an accessory respiratory system (air breathing organ), tolerance towards high stocking densities, fast growth, and elevated market de- mand has been recognized as an ideal species for
aquaculture [9, 10]. At present, demand of H. fos- silis culture has increased significantly, however, fry sourced from the wild is not sufficient to meet the commercial supply in aquaculture [11]. There- fore, the hatchery production i of this species is prudent for fish farmers to be assured of a sustain- able supply of H. fossilis for aquaculture.
Several efforts have been made to adopt in- duced breeding technique of H. fossilis by re- searchers, especially the application of pituitary extracts from carps and synthetic (S-GnRHa) hor- mone [10, 11, 12]. Even a mixture of both hor- mones was also applied for effective induced breeding [13]. Recently, advanced induced
breeding techniques apart from hand-stripping is more convenient for aquaculture. Because hand- stripping causes weakness, infertility, blood vessel disconnection, eventually, death of female, and sometimes sacrifice of male during sperm collec- tion, results in a reduction of male individuals for the next generation [6]. It is also responsible for the reduction of hatching rates and quality finger- lings. Currently, efficient and commercially rea- sonable seed production of H. fossilis under hor- monal propagation has been performed with some limitations [6]. However, a research suggested that partial ovulation and production of immature eggs by using natural spawning agent, human chorionic gonadotropin (HCG) were observed in some cases, ultimately resulting in a low survival rate low-quality fry of H. fossilis [11]. Nowadays, Gonadotropin Releasing Hormone analogue (GnRHa) is being used as the finest obtainable bi- otechnological component for the fish breeding propagations [14, 15, 16]. The GnRHa has per- formed a successful breeding induction in wide range of carp species (Labeo rohita, Cirrhinus mrigala and Labeo bata), and this could be an ef- fective alternative to Pituitary Gland Extract (PGE) in aquafarm [17 – 20]. Therefore, our pre- sent research's core intention was to reveal appro- priate artificial propagation techniques to detect the chronological embryonic and larval develop- mental stages of H. fossilis for aquaculture im- provement and conservation program.
Material and Methods Study area
The induced spawning using the hormone was held from June to August 2019 at Alalpur Hatch- ery and Fisheries Ltd., Mymensingh, Bangladesh.
Artificial propagation was conducted in the hatch- ery. The embryonic and larval ontogeny phases were observed at the Fisheries Biology and Genet- ics laboratory facility, Bangladesh Agricultural University in association with Sylhet Agricultural University.
Broodstock management
The stocking density of brood fish was 2000- 2500 per acre in the brood rearing pond (earthen pond). The optimum amount of lime, cow dung and fertilizer were administered regularly during the entire rearing period. The proximate composi- tion of the commercial feed was constituted of fish meal 20%, rice bran 25%, wheat bran 15%,
sesame oil cake 10%, meat and bone meal 25%, vitamins and minerals 1% and flour 4% (Bengal Feed and Fisheries Ltd., Bangladesh) and it was fed to broodfish at 4% of total body weight. The ration size was divided into two times a day at 8.30 am and 4.30 pm.
Brood fish selection
Mature broodfish were identified based on secondary sexual characteristics, for instance, flat abdomens, long protruded genital papillae in case of male and soft abdomen, round-swollen urogen- ital opening in case of female, whither milt from male and eggs from female came out when gentle pressure applied on the abdominal region during the breeding season (Figure 1). During the experi- ment, the weight of the matured males and females ranged between 18-22 gm and 40-60 gm, respec- tively.
S-GnRHa hormone injection
A synthetic gonadotropin-releasing hormone (S-GnRHa) was obtained from a commercial source. The required amount of sterilized distilled water was mixed with the hormone powder to di- lute variable doses. Afterward, the prepared hor- mone was carefully taken into a 0.5 ml hypoder- mic syringe and broodfish were intramuscularly injected below the dorsal part and between the lat- eral line. Injected broodfish were kept into breed- ing cistern having a size of 1.5 × 0.5 × 1 m3 with continuous water flow. Treatments of three differ- ent doses of S-GnRHa (T1, T2, and T3), each with three replicates, were carried out in this study. The fish in the control (Tc) group were injected with sterile normal saline solution (Table 1). The ratio of females to males was 1 : 1 in all treatments.
Table 1. Induce breeding trial of H. fossilis with S- GnRHa hormone
Treatment Hapa
Dose (ml/kg) body weight Female Male
T1 H1, H2, H3 1 0.5
T2 H1, H2, H3 2.50 1 .125
T3 H1, H2, H3 5.00 2.50
Tc
(0.9 % NaCl) H1, H2, H3 1 0.5 Water quality measurement
Different water quality parameters like water temperature (ºC), pH, dissolved oxygen (mg/L)
and water transparency (cm), CO2, total hardness, nitrate and ammonia were monitored fortnightly in brood nurturing ponds by using HACH kit box and salinity was measured by refractometer during the study period [21].
Determination of breeding performance
The breeding performance of H. fossilis was calculated based on the ovulation rate (%) and, then fertilization rate (%) and hatching rate (%).
The ovulation rate was estimated using the for- mula described by Legendre [22].
Ovulation rate (%)= No. of fish ovulated
Total No. of fish injected×100 (1) To determine the fertilization rate, approxi- mately 100 eggs were randomly taken in a Petri dish for each replication and its external structure easily characterized the fertilized ovum? The fer- tilization rate was calculated as follows [23].
Fertilization rate (%)=
No. of fertilized eggs
Total No. of eggs (fertilized + unfertilized) ×100 (2) On the other hand, approx. 100 fertilized eggs were relocated in an isolated hapa with continuous water supply. After hatching, total number of hatchlings was and afterwards, the hatching rate was resolved by adopting the following equation [23].
Hatching rate (%)=No. of fish hatched Total No. of egg ×100
(3) Observation of embryonic and larval develop- ment
Few egg samples were collected in Petri dish with water. Among them, only the fertilized eggs contained even and round yolk masses, and hori- zontal perivitelline surface, was selected for em- bryonic study under a dissecting microscope. Fi- nally, the specific egg was monitored at specific intervals to figure out the timing of development for each embryonic stage (Figure 3). The evolving stages of the eggs were also captured under the mi- croscope (OLYMPUS CX21) with photographic attachment taken by a camera (Rigla-32, Optikam
B3 Digital camera, Italy) and the eggs diameter were measured by using “ImageJ” software.
Data analysis
A one-way analysis of variance (ANOVA) was employed to determine the significance (p <
0.05) level of hormonal treatments. The level of significance of the results was tested following Duncan’s and Tukey using Statistical Package for Social Science (SPSS) programmed (IBM SPSS 26).
Results and Discussions Water quality measurement
The water temperature was recorded as 28 ± 2.7ºC, total hardness 70.2 ± 5.85 mg/L, pH 7.4 ± 0.91, dissolved O2 5.91 ± 0.78 mg/L, transparency (Sechi’s disc) 24.1 ± 2.63 cm, and dissolved CO2
18 ± 2.51 mg/L during the experiment period (Fig- ure 1).
Optimization of S-GnRHa doses and visualiza- tion of fertilized and unfertilized eggs
Three different doses of S-GnRHa i.e., 1.00, 2.50- and 5.00-ml.kg-1 BW for females, and 0.5, 1.25- and 2.50-ml.kg-1 BW for males were verified (Figure 2a). Among those doses, 2.50 ml kg-1 BW for females presented the maximum fertilization and hatching rates, which were significantly dif- ferent (p < 0.05) from the doses 1.00 ml kg-1 BW and 5.00 ml.kg-1 BW of females (Figure 2b). In- stantaneously after fertilization, the embryonic phases commenced and the fertilized eggs were advent watery and slightly transparent in color, while the unfertilized were shifted into cloudy and pale.
Percentage rate of ovulation, fertilization, and hatching
Ovulation of all induced fishes in all treat- ments started at 6 hours and finished at about 10- 11 hours of post-injection. When the highest dose, T3 of S-GnRH was applied, many immature eggs and mature eggs released from the brood nega- tively affect the fertilization, hatching, and finally on the survival rates of treated fishes. In the pre- sent study, the highest fertilization rate was rec- orded in T2 (83.11%) followed by T1 (79.33%) and T3 (60.33%) where T2 was significantly (p < 0.05) higher than that of T1 and T3 (Figure 2B). The hatching rate was significantly (p < 0.05) highest in T2 (89.56%) followed by T3 (74.78%) and T1
(70.11%) (Figure 2B).
Observation of embryonic and larval develop- ment
Table 2 and Figure 3 present detailed infor- mation about each of the embryonic developmen- tal stages of H. fossilis just after fertilization. The fertilized eggs were transparent, spherical in shape, reddish green, and size accounted as 514.29
± 0.3 μm. Fertilized eggs exhibit a prominent red- dish spot (blastodisc) on their pole and was easily identifiable (Figure 3a). After twenty minutes of post-fertilization, a reddish spot appeared in the animal pole as blastodisc (Figure 3b) and egg di- ameter was measured as 550.05 ± 0.22 μm. At the first stage of cleavage, the blastodisc was divided into two parts within 00:30 h of post-fertilization (Figure 3c) with a diameter of 558.84 ± 0.28 μm.
The second cleavage (4-cell stage) appeared ap- proximately at 00:45 h after fertilization with 560.4 ± 0.19 μm of diameter (Figure 3d). The third division formed 8 blastomeres was noticed with
increased cell numbers and passed through 16- cells, 32-cells, afterward, multi cells within 1:10 h, 1:30 h, 2:00 h, and 2:30 h of post-fertilization, respectively and diameter were observed 569.23 ± 0.39 μm, 580.04 ± 0.44 μm, 586.83 ± 0.49 μm and 5.88±0.54 μm, respectively (Figure 3e-h). Blasto- meres were being lessened in size and the morula phase was stretched within 3:00 h of post-fertili- zation while the average diameter was 589.82 ± 0.38 μm (Figure 3i). The blastula stage appeared at about 4:00 h of post-fertilization and was recog- nized by levelling of the blastodermal cells (Figure 3j). The blastomeres were entirely missing their individuality and stirred in both sides of the animal pole, conquering 30% area over the yolk mass while the mean diameter was 593.84 ± 0.23 μm.
Then gastrulation ring was observed after 6:40 h of fertilization. At that stage, the blastoderm with 598.86 ± 0.51 μm of diameter was further spread in both side which covered about 60-70% area (Fi- Figure 1. Water quality parameters recorded during the hormone treatment
A B
Figure 2. Body biometrics (A) and breeding performance (B) of H. fossilis with different treatments of S- GnRHa. *Different superscripts in the same row show significant differences (p < 0.05)
gure 3k) following development of the germinal ring. Somatic formation started around 9:00 h and then proceeded to 18 somites within 18:00 h after fertilization. At the beginning of a somatic stage, the diameter was 907.69 ± 1.68 μm (Figure 3l-n).
Yolk-plug formed after 19:00 h of fertilization with 915 ± 1.19 μm of diameter (Figure 3o). The
twisting movement was observed at about 20:00- 21:00 h after fertilization, occupying a diameter of 920 ± 1.34 – 1065 ± 1.17 μm (Figure 3p-q). Wrig- gling movement amplified as chorion walls sur- rounded the embryo, heartbeat rose to 68 folds per minute. Pre-hatching started at 22:00 h of post-fer- tilization. The diameter of that stage was 1108.54 Figure 3. The embryonic developmental stages of H. fossilis: (a) fertilized egg; (b) blastodisc formation; (c) 2-
cell; (d) 4-cell; (e) 8-cell; (f) 16-cell; (g) 32-cell stage; (h) multi-cell (about 64-cell); (i) morula; (j) blastula; (k) gastrula; (l) somatic formation; (m) 12 h old somites; (n) 18 h old somites; (o) yolk-plug;
(p-q) twisting movement; (r) pre-hatching; (s) newly hatched larvae; (t) 1 h after hatching.
a b c d
e f g h
i j k l
m n o p
q r s t
± 1.79 μm (Figure 3r). Hatching occurred after 23:00 h of fertilization. The diameter of newly hatched larvae was 1116.48 ±1.63 μm (Figure 3s).
Rapture of the chorion occurred as embryo con- tracting to give rise to the emerging tail first, the trunk and head region (Figure 3t).
The water quality parameters have direct in- fluence on induced breeding and fertilization rate, hatching rate and survival rate if aquatic organism.
The water temperature recorded in this study ranged between 22-30°C and such ranges have been reported as optimal for the hatchery culture of and of H. fossilis [24]. The suitable level for pH should be between 6.5 to 7 and dissolved O2
should be > 5 mg/l [25, 26]. Observed values for other parameters i.e., total hardness, transparency and CO2 also found within favorable range for op- timal culture and reproduction of H. fossilis.
Application of synthetic and non-synthetic hormones in the induced breeding of H. fossilis are increasing to avail quality eggs as well as milt pro- duction in commercial farming [6, 29]. In most of the cases, PG extract [11, 28], ovaprim [29, 30, 31], ovasis [32], ovatide and ovulin [27] were us- ing in artificial propagation strategies. At present, S-GnRHa is also showing a proper response in fish induced breeding techniques, especially, in carp species [17]. In the current study, the result showed marked differentiation effectiveness of the doses in induced fertilization. All individuals ovu- lated in all treatments from 6-12 hours post-injec- tion. The best optimized dose of S-GnRHa was T2
(2.50 ml kg-1 BW in female) according to present study which was little bit higher compared to Mondol et al., [17], where 0.5 ml.kg-1 BW was ef- fective in carp species. Srivastava et al. [33] used Ovaprim 1.0-2.0 ml kg-1 BW where the doses were effective in Asian Catfish, Clarias batrachus. An- other research observed that 2.5 ml.kg-1 BW of fe- males in Mystus gulio showed maximum ovula- tion and a hatching rate of 80% injected with Ovaprim [34]. The spawning occurred in Clarias batrachus within 21–22 h after Pituitary extract fertilization administration [35] and 24 hours after ovaprim injection [36].
In the present research, the eggs were transpar- ent, spherical, and reddish green which to finding of Raji et al. [37], who stated that the eggs of H.
fossilis were green, translucent, round and moder- ately sticky with a diameter of 1.00 mm and sur- rounded by chorion wall. Ovulation rate in the pre- sent study was similar with Mondol et al. [17], who succeeded to obtain 100% ovulation using S- GnRHa in major carp which was better than Ali et al. [11] observed 82.67% with HCG and PG injec- tion in H. fossilis. Again, El-Hawarry et al. [38]
found 70.76% ovulation rate with mixture of GnRHa and domperidone (Dom) treatment in Af- rican catfish (Clarias gariepinus), while Mosha, [27] observed highest fertilization rate of 87.34%
in African Catfish, C. gariepinus with Ovaprim in- duction. Above several research seems quite simi- lar with our current study where the highest ferti- lization rate (83.11%) was in T2 whereas it was Table 2. Detailed information of embryonic stages after fertilization of H. fossilis
Figure 3 Development stages Egg diameter (μm) Development time (hour: minutes)
a Fertilized egg 514.29 ± 0.30 00:00
b Blastodisc formation 550.05 ± 0.22 00:20
c 2-cell 558.84 ± 0.28 00:30
d 4-cell 560.4 ± 0.19 00:45
e 8-cell 569.23 ± 0.39 1:10
f 16-cell 580.04 ± 0.44 1:30
g 32-cell 586.83 ± 0.49 2:00
h 64-cell 588.82 ± 0.54 2:30
i Morula 589.82 ± 0.38 3:00
j Blastula 593.84 ± 0.23 4:00
k Gastrula 598.86 ± 0.51 6:40
l-n Somatic formation 907.69 ± 1.68 9:00-18:00
o Yolk-plug 915 ± 1.19 19:00
p-q Twisting movement 920 ± 1.34-1065 ± 1.17 20:00- 21:00
R Pre-hatching 1108.54 ± 1.79 22:00
S Newly hatched larvae 1116.48± 1.63 23:00
reduced (60.33%) with the increase of hormone dose. Ghosh et al., [30] found a lower fertilization rate of 61.82% by using Ovaprim, 1 ml kg-1 in Cy- prinus carpio. However, higher fertilization rate (95%) in H. fossilis in PG-PG treatment group and 96.15% in Macrognathus pancalus with Ovasis treatment were also observed [11, 32]. Initiation of hatching at 23-24 hrs after fertilization and 97- 98% of hatching rate have been recorded for same species [39, 40, 41], while hatching started at 55 hours of post feralization in case the Korean East- ern Catfish, Silurus asotus [42]. However, re- duced hatching rate of 85%, after 17 hours of fer- tilization occurred at 28.5 ± 0.5°C for African cat- fish Clarias gariepinus [43]. This variation might be because of the differences in the maturity of eggs used or methods of activations, season, and other environmental conditions. Our current ex- periment also revealed highest hatching rate in T2
(89.56%) which was quite closer of Borah et al., [32] found 92.49% in M. pancalus with Ovasis treatment.
Nesa et al., [7] identified first cleavage in 00:21 – 2:09 h after fertilization to form 32-cell by using PG extract in H. fossilis, while the present study found first cleavage up to 32-cell formation in 00:30 – 2:00 h of post-fertilization. Borah et al., [32] resembled our current result where first cleav- age was identified in 30-40 min after fertilization in M. pancalus with Ovasis injection. A study by Udit et al., [44] also got similar results with Ovatide, where first cleavage occurred 30 min af- ter fertilization. Moreover, first cleavage was identified within 45 min post-fertilization in Labeo bata with PG extract injection [45]. How- ever, Puvaneswari et al., [4] observed 2-cell for- mation within 15-20 min post-fertilization but 32- cell formed within 2:00 h in H. fossilis using Ovaprim. The 4, 8, 16 and 32 multiplication phases within 38-58 minutes of fertilization for Heterobranchus Bidorsalis in Nigeria [46], while same cleavages initiated within 49 ± 3 min in case of Asian sun catfish, Horabagrus brachysoma [47]. In the current study, morula stage appeared in 3:00 h post-fertilization. Puvaneswari et al., [4]
and Nesa et al., [6] using Ovaprim and PG extract, respectively, in H. fossilis found the same stage quite earlier within 2:35 h post-fertilization. The mitotic cleavage has been reported at 69 min post- fertilization and blastopore closed at 11 h 8 min for African giant catfish Heterobranchus bidorsa- lis [48]. In the present study, the embryo reached
into blastula stage at around 4:00 h post-fertiliza- tion which was identical by the formation of a ger- minal ring. Same stage was also reported by Nesa et al., [7] in H. fossilis at 3:25-4:10 h. This time variation might be due to the presence of diverse strains, conditions, and the female fish size [49].
Gastrula formed after 6:40 h of fertilization where Nesa et al., [7] found around 6:20-6:35 h after fer- tilization to start gastrulating in H. fossilis.
The somatic stage gradually appeared after the gastrulation stage proceed to 18 somites which re- sembled to obserbation of Nesa et al., [7], who rec- orded 18-20 somites in H. fossilis. The twisting movement of embryo before of hatching has been reported by Puvaneswari et al. [4]. However, pow- erful twisting movements were shown by C. car- pio at 67:15 h after fertilization [30], while the time gap was so much higher compared to our pre- sent study which because of two completely dif- ferent species with different physiological nature.
A 72 beats/min heartbeats has been observed at 17 h of post fertilization in African giant catfish Heterobranchus bidorsalis [48]. In our current ob- servation, hatching occurred at 23 h post-fertiliza- tion, likewise, Marimuthu et al., [50] found hatch- ing time 23-24 h. However, Nesa et al., [7] ob- served hatching time around 20-24 h in H. fossilis.
Conclusion
Our current study elucidated the potentiality of S-GnRHa hormone as an alternate to currently available hormones in the artificial propagation to- gether with embryonic and larval development of stinging catfish.
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