Induced Breeding of Striped Spiny Eel, Mastacembelus Pancalus: Considering Various Doses of Pituitary Gland Hormone

Study Site

The study was conducted in the laboratory of Department of Fisheries and Marine Bioscience, Jessore University of Science and Technology, Jessore, Bangladesh. All the preparations needed for induced breeding of M. pancalus were done in the laboratory. The aquarium set up, water supply facilities; instruments required for hormone administration and working space were assured before the breeding program. The experiment was done from April to July, 2016.

Aquariums setup

The rectangular glass aquariums (36 inches x 15 inches x 14 inches) were used for both rearing and spawning of fish. The aquariums were supported with sand, gravel and bamboo slits, used as fish hiding place. The water hyacinth was supplied to hold the sticky eggs. All spiny eels appreciate floating plants.

Measurement of Water Quality

The water quality of the experimented aquarium were maintained and recorded carefully. Water parameters such as dissolved oxygen (DO), pH and water temperature were measured weekly during brood fish rearing and daily during induces. A pH meter (EZo DO; Model-7200; Made in Taiwan) was used for the measurement of pH and temperature; a DO meter (LT Lutron; Model- YK-22 DO; Made in Taiwan) was used for the measurement of DO. The water was exchanged daily at 20%-30% by siphoning so that excessive amount of iron, carbonate and other wastes were not accumulated at the bottom of the tank.

Collection and Rearing of Brood Fish

Samples of M. pancalus for induced breeding were collected from local fishermen at Joghati baor, Jessore Sadar. The fishes were reared in aquarium about one month (Figure 1). Tubefex was given twice as feed daily at early morning and evening according to their demand.

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Figure1: Rearing of broods M. pancalus in the aquarium condition. Used water hyacinth and aeration (inset).

Selection of Broods and Pairing

Selection of brood fish is the main task on which successful spawning mostly depends. Improper selection of brood may result poor quality or fish may not spawn at all. Mature and healthy fishes were selected by observing morphological symptoms like swollen and enlarge abdomen for female and slender-shaped, smaller in size at the same batch with whitish belly for male. The body weight of individuals was measured by using electric weight balance (A&D Company. Ltd; Model-EK 1200I; Made in Japan) and noticed average body weight 9.00 ± 1.2 g. The selected broods were kept in separated experiment aquarium at sex ratio 1:1 for male and female for pairing and conditioning for 3-4 days.

Experimental Design

The experimental fish was induced with pituitary gland (PG) hormone in two different trials and each trial had three treatments (T1, T2, and T3) with three replications of each. In first trial (double dose) fishes were injected with two separate doses (Table 1). The resolving dose (2nd dose) received double amount of PG of the preliminary dose (1st dose). In the 2nd trial, fishes were induced with single dose of hormone and the amount of PG was the equivalent to the total amount of PG applied in 1st trial (Table 1).

Hormone Administration

The brood fish were collected for hormone administration very carefully and wrapped with soft and moist cloth. The needle was pushed at the basal part of dorsal fin at an angle of 45º with the body (Figure 2). The amount of hormone for each fish was determined before according to the body weight of the broods. After the administration of hormones, the fishes were kept in separate aquarium at the sex ratio of 1:1 for natural propagation. The fishes were observed carefully at every 15 min with naked eye for any behavioral changes and spawning activity i.e. their body coloration, change of position, chasing behavior up to spawning time.

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Determination of Fertilization Rate

The eggs were attached with the roots of water hyacinths in the aquarium. To determine the fertilization rate 10 eggs was taken from each replication of different treatment and placed in a Petridis containing water. Then the collected eggs were observed under a microscope (Carl Zesis microscopy GMBH, S.N. MKG8639, made by Germany) and fertilized eggs were counted and fertilization rate was calculated by following formula. The fertilized eggs were recognized as their transparent shell from the unfertilized eggs which were opaque.

Fertilization rate (%) = No. of fertilized eggs Total no. of eggs × 100

Determination of Hatching Rate

The hatching rate was the portion of the fertilized eggs that came out as developing larvae and was determining by counting the number of larvae from predetermined number of fertilized eggs. The hatching rate was calculated by the following formula:

Hatching rate % = No of hatchlings Total number of fertilized eggs × 100

Statistical Analysis

The results found in the study were subjected to statistical analysis, chi-square test that showed the significance (P<0.05) level of differences between the treatments. This statistical analysis was performed with the aid of the Microsoft Excel. Results

Water Quality

The physico-chemical condition such as temperature, pH and dissolved oxygen (DO) of water in experimental aquariums under different treatments of the two trials is presented in table 2. The water temperature, pH and DO were ranged from 26.5 to 31.1ºC, 7.06 to 8.04 and 4.1 to 5.6 mg·L-1 respectively in 1st trial and 28.7 to 31.2°C, 7.56 to 7.98 and 4.2 to 5.4 mg·L-1 at 2nd trial. The recorded water parameters were not significantly (p<0.05) different between treatments of the both trials.

Breeding Behavior and Spawning

The different behavior of the species showed during copulation and some of them are presented in the figure 3. Both male and female changed from whitish-grey to yellowish body color after injected hormone the fish in case of both trials (Figure 3 inset right-down). Courtship begins with the male chasing the female and swimming in a tight circle at 9-10 h of administration of hormone (1st dose) irrespective of different trials (Figure 3 black arrow). The female and male moved together in anti-clockwise and the female was hold by the male and later bending its body, rubbing, knocking and nudging her (Figure 3 white arrow). Then the pair encircles each other around the roots of the water hyacinths for spawning and it takes place within 15 to 20 h after administration of PG hormone. Male releases sperm simultaneously with female and the eggs were fertilized externally. The eggs stick to the root of water hyacinths due to adhesive in nature (Figure 4). They spawned in different batches and took rest about 30 min after one span of spawning. The swimming movements of the female become slow at the end of the spawning activity and parent take rest at the bottom. The parents were removed from the spawning tank after spawning and noticed that their body color became gradually faded away. The fertilized eggs are spherical and transparent when unfertilized eggs are opaque (Figure 5). The eggs were started to hatch after 36-41 h of spawning.

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Fishes spawned 100% in all the treatments at 16-20h and 15-19h after hormone administration at 1st trial and 2nd trial respectively (Table 3). Incubation period also noticed more or less similar in both trials. The 100% survival rate of broods noticed in all the treatments of both trials.

Table4: The fertilization rate of M. pancalus at double doses (1st trial) and single dose (2nd trial) of PG extract. (Mean ± SD); values of the fertilization rate with different superscripts (a,b,c) differ significantly (p<0.05) in each treatments and trials Hatching rate was found 41.1%, 67.85% and 57.93% in treatments of T1, T2 and T3 respectively in 1st trial and 41.1%, 60.31% and 55.53% in T1, T2 and T3 respectively in 2nd trial (Table 5). The highest hatching rate was recorded 67.85 ± 9.44 % in T2 at 1st trial. The significant (p<0.05) variation noticed in T2 in both trial compare to other treatments. There was no significant (p<0.05) variation in terms of hatching rate between 1st and 2nd trial.

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Water Quality Parameters

Water quality parameters play an important role in successful breeding. Among them water temperature plays a vital role in gonadal development and timing of the spawning and hatching. If the optimum temperature of the species cannot be maintained the maturation may delay or the eggs may release spasmodically and the total number of egg may also be reduced [19]. The fertilization rate, hatching rate, survival rate, and embryonic development is closely dependent upon the oxygen supply and pH. The dissolved oxygen content of the water should not be below 4 - 5 ppm. Successful breeding of M. pancalus was performed at an ambient temperature ranged from 26.5 to 31.1ºC (Table 2) that was suitable for the breeding of M. pancalus which is supported by [20, 21] who reported that temperature ranged from 27 to 31ºC is favored by most of the indigenous small fishes for breeding. This range of temperature is also supported by [3, 6, 18] for breeding of M. pancalus and they all reported successfully breed within 26-31 ºC. Besides this, M. aculeate us was successfully bred by [22] at 27 to 33ºC and by [23] at 28 to 30ºC. Thus it may say that water temperature 27 to

31ºC could be the optimum temperature for breeding of eel fishes. The recorded water pH and dissolved oxygen varied from 7.06 to 8.04 and 4.10 to 5.6 mg·L-1 in both trials this also supported by [17, 18]. Afroz, et al. [17] conducted their experiment at 7.13 to 8.69 and 3.6 to 4.9 mg·L-1 of pH and DO respectively. Alam, et al. [18] successfully breed M. pancalus at 6.5 to 7.4 and 6.5 to 7.5 mg·L-1 of pH and DO respectively. Hasan, et al. [6] recorded pH value 7.13 to 8.69 and DO value 3.3 to 5 mg·L-1.

Breeding Behavior

The present study recorded yellowish body color of both male and female after hormone administration (Figure 3). This may be due to hormonal effect for the attraction of both sexes each other that help the female to spawn and the male to release milt successfully. Body coloration also has an important role in mate choice. Though there is no such other evidence in color changes of guchiaim, it may compare to other species like [24] recorded orange-red color spot on the operculum of both male and female after hormone administration in case of Puntius chola. Fishes showed a courtship behavior that is supported by [3, 6] for the same species. Fishes showed such behavior because the reproductive success of fish depends partly on ability to perform correct courtship behavior. The courtship begins with the male chasing the female and swimming in a tight circle. The similar observation was recorded [6, 25, 26]. In the present study, female and male moved together in anti-clockwise and the female was hold by the male and later bending its body, rubbing, knocking and nudging her. Male hold the female and then bending its body and rubbing to prepare her for spawning and that behavior pattern is supported by [3, 6, 22]. It was also found that during spawning, the male bent its body close to the female and released its milt simultaneously with female and the eggs were fertilized externally.

Breeding Performance

Pituitary gland (PG) extract was used as the inducing agent for breeding of M. pancalus and all the experimental fishes breed successfully (Table 3). Effectiveness of PG in induced breeding of spiny eel is supported by [3, 6, 18, 22]. The 100% spawned noticed in the present study by inducing different doses of PG in different phases confirm the effectiveness of PG hormones for the induce breeding of M. pancalus. The courtship and spawning began within 9-10 h and 16-20 h of hormone administration which was earlier than [16]. They noticed these behaviors at 29-32 h and 33-35 h after injecting of different hormones for the same species. The quick spawning occurred in T3 when the high dose (200 mg·kg-1) was applied at a time in the 2nd trial.

The incubation period of the species was more or less similar in all treatments of both trials and it was within 36-41 h. Rahman et al. [3] recorded incubation period 33- 35 h by carrying out study on induced breeding of M. pancalus with PG hormone. However, the little bit shorter incubation period noticed in T2 in both trials where PG used as medium dose in the present study. So it can be said that secretion of gonadotropins in female occurred quickly at higher dose than that of lower doses but the incubation is depended on other factors. In average of two trials, it was showed that 100% spawning performance, 70% fertilization rate and 64% hatching rate of M. pancalus in medium dose, 180 mg·kg-1 of PG rather than other two doses (160 mg·kg-1 and 200 mg·kg-1). The findings of the present study showed higher spawning but lower fertilization and hatching rate compare to other such studies on M. pancalus like [3, 18]. Alam et al. [18] showed 77% spawning rate, 91% fertilization rate and 80% hatching rate while [3] found 90% spawning rate, 90% fertilization and 80% hatching rate (Table 6). Farid et al. [22] reported high fertilization (86%) but low hatching rate (50%) for the same species using same hormones at different dose. However, low performance reported compare to present findings by [6] and recorded 50% spawning, 35% fertilization and 26% hatching performances by using PG at 60 mg·kg-1 body weight of M. pancalus. There was no prominent variation in terms of spawning and incubation time between 1st and 2nd trials. However, a bit higher fertilization and hatching rate in 1st trial (double doses) could be suggested for the inducing of the species.

Table 6: The comparison of breeding performance at different PG dose of the present study with other such studies. The present findings indicated that the PG extract is effective in induction of M. pancalus under captive condition. The fish responded in all experimental doses but respect to fertilization (73.33%) and hatching 67.85% , PG extract at the double dose 60 mg•kg-1 + and 120 mg•kg-1 body weight) could be used as an appropriate dose for induced breeding and seed production of the species.