Collection of broods: The study was carried out in a private hatchery, Puratan Kasba, Jessore during May–June, 2004. Mature brood fishes were collected from the brood stock pond of the farm.
Selection of broods: Nine pairs of male and female broods were selected for the experiment. Female broods were selected with bulged abdomen, soft post-abdominal region, swollen genital pore and comparatively larger in size. Male broods were comparatively slimmer and smaller in size having rough pectoral fins and with the presence of milt with gentle pressure on the abdomen. Weight of reba female and male ranged from 110 to 140 g and 80 to 105 g respectively, the same for bata ranged from 310 to 388 g and 180 to 260 g for female and male respectively.
Conditioning of the brood: Prior to hypophysation, the males and females were kept in two separate tanks for 24 hours under water shower. The tanks were (3m×2m×1m) fitted with outlet pipes and filled with deep tube well water up to 30 cm.
Hormone administration: The broods were taken out from the tank, weighed and amount of hormone required was calculated out. The alcohol preserved PGs were first dried with tissue paper weighed and homogenized with half the quantity of the distilled water. After that it was diluted with rest of the distilled water and centrifuged. The supernatant solution was then drawn out in a syringe for injection. The injection was administered in the muscle at the base of pectoral fin. Time interval between the first and second dose was 6 hours and the doses for males of the respective species were same for all the treatments (1.5 mg for reba and 2.0 mg for bata) The different doses (mg kg-1 body weight) applied for the females.
Spawning: Single pair semi-natural spawning was performed in nine separate hapas under constant water showering. The male was seen chasing the female vigorously and the release of eggs and sperm of reba and bata took place 6 and 8 hours, respectively, after the second dose of injection to the female. The breeders were removed from the hapa after ovulation had been completed. Four hours after ovulation the fertilization rate was reckoned.
Fertilization and hatching: For determining fertilization rate a sample of eggs were taken in a Petri dish and the total number of eggs and the number of fertilized eggs were carefully counted. The fertilized eggs were clear and transparent and the unfertilized eggs were opaque. Thus the percentages of fertilization of each replicate and the average of the percentages within each treatment were determined using the following formula:
Percentage of fertilization = (Number of fertilized eggs/Total number of eggs in the sample) x 100
For determining the rate of hatching a special contrivance was applied. A sample of known number of water-hardened fertilized eggs was transferred to especially hand-made incubation jar. The jars were prepared by cutting 5 cm dia PVC pipe in to 15 cm length. The bottom of the pipe was closed with fine meshed nylon cloth. The jars were made to float in the hatching funnel with polystyrene foam attached to the rim of the pipe. For circulation of water in the hatching jars, a few holes were made on the upper end of the pipe; they were similarly closed with the nylon cloth. The water temperature of the incubation jar was recorded with the help of a centigrade thermometer. After 18±2 hours of fertilization the newly born hatchlings came out from egg cover.
The percentages of hatching from each replicate and the average percentages within each treatment were calculated by counting the number of hatchlings coming out from the fertilized eggs, according to the following formula:
Percentage of hatching = (Number of hatchlings/Total number of fertilized eggs in the sample) x 100
Data analysis: The significance of the differences obtained between the three treatments was tested with one-way ANOVA by using the Microsoft Excel 2002 program.