MATERIALS AND METHODS

Materials: The Department of Fisheries, Aquaculture Extension Center Perlok, Jerantut, Pahang, Malaysia has established a collection of Hampala macrolepidota broodstock caught from Pahang river. Therefore, the induced breeding was carried out at this center. Other materials used in this study were net, digital balance, clove oil, ovaprim (Syndel, USA), syringe, basins, bowl, towel, mud, tanks, rearing trays, dissecting microscope and petri dish.

Methods
Broodstock selection: H. macrolepidota broodstock were collected from pond by using net. Five females and ten males (1 F:2 M) were selected and used for this study.

Induced breeding: Body weight of each fish were measured by using a digital balance. Fishes were then anesthetized by using clove oil. Dosages were 0.6 and 0.3 mL ovaprim kg^-1 female and male, respectively. Injections were given intramuscularly. This study was carried out at 4:00 p.m. on 9 February 2012.

Stripping: Fish were monitored for ovulation from time to time by applying a slight pressure on the abdomen. At 10:00 pm on the same day, eggs and milt were stripped from females and males, respectively. This stripping was carried out 6 h after the injection. Eggs stripped from females were weighed individually. Eggs samples were then collected for fecundity estimation.

Fertilization: Remaining eggs were then inseminated with sperm. Eggs were washed several times with mud to remove its adhesiveness. Eggs were then transferred onto trays placed in a flow through incubation tanks.

Fecundity and eggs diameter: Fecundity was estimated using 1 g of egg sample and then multiplied with total weight of the stripped eggs. Diameter of eggs was determined by taking randomly 30 samples of eggs from each female broodstock.

Embryonic development: The embryonic development was observed and photographed under a dissecting microscope. The observation on the embryonic stages was carried out until eggs hatched.

Water quality parameters: Four water quality parameters measured in this study were temperature, dissolved oxygen, pH and Total Dissolved Solids (TDS). The measurements were taken during the incubation period.

Statistical analysis: Data for eggs diameter were statistically analyzed by one-way ANOVA using SPSS 17.0 and Duncan Multiple Range Test (DMRT) used to determine the significant difference between the means.

RESULTS

H. macrolepidota with weight ranged from 180-280 g are able to produce 34,985-75,646 eggs/female. Mean egg diameter for H. macrolepidota injected with ovaprim is from 0.55-1.06 mm. There was no significant different (p>0.05) for the eggs diameter produced between the females. Newly fertilized eggs were spherical and adhesive. Fertilization rate ranged from 18-28%. Majority of the eggs hatched about 24 h after fertilization. Results for eggs fecundity, diameter and fertilization of H. macrolepidota are as shown in Table 1. Embryonic development of H. macrolepidota lasted for about 24 h. Description of the developmental stages is summarized in Table 2 and shown in Fig. 1.

Fig. 1:	Embryonic stages of Hampala macrolepidota

Table 1:	Fecundity, fertilization rate and eggs diameter of H. macrolepidota
Mean with the same superscript letters are not significantly difference at p>0.05

Table 2:	Description of the embryonic development of H. macrolepidota until hatching with each stage
*Eggs inseminated with sperm at 11:00 p.m. on February 9, 2012

Table 3:	Water quality parameters during the eggs incubation period for H. macrolepidota

Four water quality parameters measured were temperature, pH, Dissolved Oxygen (DO) and Total Dissolved Solids (TDS) with readings ranged from 24.29-24.90°C, 6.10-6.42, 7.57-7.77 mg L^-1 and 0.165-0.169 g L^-1, respectively Table 3. Comparatively, these readings are within the acceptable ranges for fish culture.

DISCUSSION

Proper identification mature fishes to be used as broodstock is very crucial. Therefore knowing the life cycle of fishes is important (Hosseinzadeh et al., 2001). Matured females H. macrolepidota were identified by their rounded bellies while males slim and prominently colored. Previous study by Rosli (1987) mentioned that H. macrolepidota with body weight between 180-470 g, induced with pituitary extract and Human Chorionic Gonadotropin (HCG), produced to a maximum of 11,992 eggs/female. However, in this study at a much lower cost with the commercially available ovaprim, can produced higher fecundity (34,985-75,646 eggs/female). The use of anaesthesia reduced the stress during handling of fish thereby, producing better fecundity.

Zohar and Mylonas (2001) stated that ovaprim produced better results as compared to ovatide, another commercial hormone used in the induced breeding of fish. Several other factors may affect the fish fecundity, are age and size of the female (Thorpe et al., 1984), the strategy of life history (Morita and Takashima, 1998), temperature and food (Fleming and Gross, 1990). The fecundity of this H. macrolepidota is low when compared to others cyprinid. For example, Labeo dero fecundity is about 90,000 (Biswas et al., 1984). According to Rideout and Burton (2000), degeneration or malfunction of ovaries reduces the female fecundity and reproductive potential. Fecundity increases with body size maybe because of energy amount of the fish available for egg production and the body cavity accommodating the eggs increases with fish size (Jonsson and Jonsson, 1999).

Egg diameter for H. macrolepidota produced in this study was between 0.55-1.06 mm. Comparatively, these eggs are larger than 0.52473-0.52492 mm produced by Rosli (1987). As for the embryonic development it is very similar to the others cyprinids. In this study, majority of the eggs hatched 24 h after fertilization. The blastomeres are regular in shape and size and the morphology of this blastomere has been used as a predictive indicator of egg viability within a clutch in most species (Shields et al., 1997).

Different species of fish have different water quality requirements. In this study, the incubation period lasted for 24 h at water temperature of 24°C. Incubation period was only 16-18 h in Heteropneustess fossilis at a temperature of 26°C (Kohli and Vidyarthi, 1990). It is possible to manipulate the temperature to reduce the incubation period. However, care must be taken to ensure that the species studied are able to tolerate higher temperature. Water parameters measured through out this study were within acceptable range for fish culture.

CONCLUSION

It can be concluded that H. macrolepidota can be induced breed with a less costly and commercially available hormone, ovaprim. Fertilized H. macrolepidota eggs are able to hatch in just 24 h, therefore this information can be useful for the larval rearing of this species. The finding of this study can be used to establish and improve the fry production of this species so that in the near future, this species can be introduced as an aquaculture species in Malaysia.

ACKNOWLEDGMENTS

Thanks to the Department of Fisheries Malaysia (Aquaculture Extension Center Perlok) for providing the broodstock, facilities and assistance and Universiti Putra Malaysia for supporting this study.