Live Boal fish were collected from the Dhakatia River of Chandpur District. The average size of those fish was 1 - 2 kg. Fisherman catch those catfish from a special fishing process locally called “Jag/Katha” fishing. Close to the bank of the river is surrounded by large bamboo poles or tree branches, and tree branches or thick twigs are then thrown into the surrounding area [14].

The maturity of the Boal fish was examined externally, and their fecundity was identified through dissection. The ovaries of female fishes were collected and preserved in 5% formalin. The weight (g) and status of the gonads were recorded. Moreover, three pieces of ovary weighing 1 g each from the anterior, middle and posterior portions of the ovary were taken and examined for the number of ova present to determine the fecundity.

Anaesthetic overdose was used to humanely euthanize boal fish at the end of the experiment. Fish were immersed in a solution of tricaine methanesulfonate (MS-222), buffered to neutral pH, at a concentration recommended by institutional animal care guidelines. Standard laboratory instruments, including anaesthetic tanks, digital weighing balances, measuring boards, pH meters, and aeration systems, were used during the procedure. Fish are placed and left in a 250 - 500 mg/L buffered MS-222 solution for a minimum of 10 minutes after cessation of opercular movement (anesthesia to respiratory arrest) prior to removal and decapitation (indicate equipment used) to ensure euthanasia.

Selected male and female broodstock were randomly collected and acclimatized in a rectangular tank to avoid the stress of netting or handling for 12 h. Pituitary hormones were subsequently induced in female fish at a dosage of 3 mg/kg body weight as the 1st dose for females. At 6 h after the 1st dose, female fish were injected with the same hormone at a dosage of 12 mg/kg body weight as the 2nd dose, and at that time, males were induced with a dosage of 3 mg/kg body weight. Hormone was injected into deep muscle near the dorsal fin (Figure 1Figure 1).

Figure 1. Hormone injection in male and female Boal fish.

Both male and female fish were subsequently released in a large circular tank for pairing and mating. The spawning behavior of the injected fish was closely observed visually.

The spawning behavior of the injected fish was closely observed visually. Eight hours after the 2nd dose, both female and male fishes were removed from circular tank. To determine ovulation success, females from which no egg emerged were considered fully ovulated. If any egg emerged, the spent females were dissected, and those retained in the abdomen were counted. The number of unreleased eggs was used to calculate the number of eggs released. The eggs and milt were subsequently mixed well for 3 - 5 mins, after which the eggs were washed with water (Figure 2Figure 2).

Figure 2. Breeding of Boal fish. (a) Stripping of female fish; (b) Stripping of male fish; (c) Mixing of eggs and sperm; (d) Release of eggs in a circular tank.

After washing, the eggs were released into a circular tank with gentle water flow. After every hour, the eggs were examined under a microscope, and different development stages were examined. The fertilized eggs were examined for the percentage of fertilization after the formation of blastula, measured and placed in hatching jars with water circulation.

After two months during the daytime in winter, fishers set their nets and enclose the area, and the fish are caught. This is called Jag/Katha fishing. After these live Boal fish were collected, they were carried to a station pond and reared in high-protein commercial feed. Additionally, tilapia is available in this pond, and they also produce fry. This is consumed by the Boal fish. The health and maturity of these fish were monitored periodically. This study of wild fish domesticated under new environmental conditions revealed altered growth, phenotypic and physiological conditions, gonadal maturation, and reproductive performance, similar to previous studies [15]-[17].

Mature female fish were identified on the basis of external characteristics such as a bulging soft abdomen, oval reddish urogenital, and brighter body color, whereas mature male fish were identified on the basis of a slightly pointed genital papilla. A similar study was reported by Gupta et al. [12]. In male and female Boal fish, mature eggs were detected in the ovary, and milt was also detected in the testis (Figure 3Figure 3).

In the present study, the absolute fecundity of W. attu ranged from 35,000 - 45,000 per kg body weight, which is similar to the findings of Prasad and Desai [18]. They reported that the fecundity of W. attu ranged from 16,565 - 29,883 per kg body weight. A similar study was also reported by Absolom [19]. The GSI value in July was 5.32. A similar study reported by Prasad and Desai [20] reported that the GSI of W. attu was 4.56 in July.

Figure 3. Male and female Boal fish identification; (a) Morphology of Boal fish; (b) Genital papilla of male and female Boal fish; (c) Testis and ovary with egg.

Pairing was observed in a tank. During the induced breeding of W. attu, “matting behavior” refers to the courtship and mating rituals that occur, which are stimulated by hormonal injections (Figure 4Figure 4). This behavior is essential for successful reproduction in captivity.

Figure 4. Pairing in a circular tank.

The ovulation rate was 90% - 95%. A similar study was performed by Alam et al. [21] with Liza parsia fish. The fertilization rate ranged from 75% - 85%. Similar findings were reported by Gupta et al. [12].

Embryonic development was observed and is shown in Figure 5Figure 5. Additionally, the present study revealed low survival rates of larvae from the embryonic stage, with impaired cell division observed in the gastrula and organogenesis stages. After fertilization, the embryos progressed through typical teleost developmental stages, beginning with the single-cell zygote and advancing to morula, blastula, gastrula and early organogenesis. Timing of these transitions showed minor deviations, likely reflecting broodstock health and environmental factors. Previous research shows that water temperature is a crucial environmental factor affecting embryogenesis [22][23]. High water temperatures may disrupt enzyme processes for hatching, which leads to hardening of the chorion and prevents the eggs from hatching [24][25]. It is therefore necessary to prepare and handle the parent carefully to obtain good-quality eggs; in this way, embryogenesis can take place optimally and produce good-quality larvae that grow and develop normally. Thus, it was predicted that water temperature would be one of the stress factors involved in the low growth performance of W. attu. However, water may not be as clean as needed. This may be the cause of hampered cell division.

Figure 5. Embryo development of stripped W. attu from fertilized egg; (a) Single-cell formation (fertilization), (b) Morula (2.15 h), (c) Blastula (3.05 h), (d) Gastula (8.30 h), (e) angled from the head and tail (11 h), (f) Nerula (12.15 h), (g) Backbone-forming but affected (13 h).

The present study revealed development stages quite similar to those reported by Prakoso et al. [13]. Figure 6Figure 6 shows the findings of their development stage.

The affected egg is shown in Figure 7Figure 7. The developmental events also do not seem to follow any regular or uniform time intervals from one stage to the other in the present study, and the same was reported by Ahmad [26].

Figure 6. Embryo development of striped wallago catfish Wallago leerii from fertilized egg to larval hatching (a) Single-cell phase (fertilization); (b) Morula (2 h:23 min); (c) Blastula (3 h); (d) Gastrula (9 h:48 min); (e) Angled form, which would be the head and tail (10 h:9 min); (f) Neurula (11 h:34 min); (g) Backbone forming (17 h:19 min); (h) Arting of embryo movement (21 h:28 min); (i) Hatched larvae (26 h:23 min) (Source: Figure from Prakoso et al., [13]).

Figure 7. Observation of affected eggs under a microscope.

The results of this study reveal that hormone-induced breeding of W. attu in captive conditions is possible and could lead to a new era in the country for aquaculture and conservation of this commercially important catfish species. On the basis of the present encouraging findings, further research should be conducted on the efficacy of different hormones at different doses and on the impact of water quality management on the breeding performance of W. attu for the development of complete technology for the breeding and seed production of this fish. Further research is needed for brood management, improvement and/or perfection of induced breeding techniques, larval food and rearing, and mass seed production.