2.1 Source of broodstock The broodstock used in this study was exclusively grown in captive condition by breeding the base population (P0) in the hatchery.
2.2 Preparation of spawning tanks Depending on the availability of gravid crabs, two different types of spawning tanks were tried for spawning. For individual spawning, nine plastic buckets with a volume of 50 L each was set up in such a way that each bucket had an independent water circulation system through underwater sand filtration (25 cm sand filling) by aeration with final water supply from a general bio-filter. For that of group spawning, 5m2 (4.2 m × 1.2 m) fibre glass tanks were used. The tanks were half-filled with filtered sea water, contained three underwater sand filter tray facilitated with hiding places and setting up of aeration. The tanks were covered with a black plastic net to create darkness.
2.3 Feeding and management of gravid broods The broodstock were fed at the rate of 5 to 10% of body weight twice a day in the morning (09:30 h) and the evening (16:30 h) with chopped raw/natural feeds. The feeding strategy for a week with different feeds was maintained as: Saturday and Sunday> trash fish; Monday>squid; Tuesday> trash fish; Wednesday> blood cockles; Thursday> trash fish; and Friday> small shrimp. The uneaten feeds and excreta were removed in every morning with the help of scoop net. Approximately 75% of the water was replaced with filtered sea water at weekly intervals. Water salinity of the spawning tanks were maintained 30±1.0 ppt and the temperature was 31±1.0 ºC. Gravid and/or mated crabs were reared in the spawning system until they were spawned. When a brood had finished spawning, it was taken out from the spawning tank, weighed and placed in the hatching tank with 30 ppt sea water and aeration.
2.4 Examination of eggs and measurement of size (diametre) Immediately after spawning, a sub-sample of egg was collected with a sterile forcep, placed onto a glass slide and observed under a compound microscope (Magnus Pro., Plan Achromate, Germany). The diameter of 10 eggs was measured with 40X magnifications by holding an ocular micrometer (0.01 mm accuracy) and the average egg diameter was noted against the respective brood.
2.5 Observation of egg fertilization and calculation of fertilization rate Observation of egg fertilization was imposed on the second time collected samples for each brood (egg mass deep orange to orange-red) when the cell division started within the eggs. A sub-sample was placed onto a glass slide and monitored under a compound microscope. Fertilized eggs were counted on the basis of progress in embryonic development. Eggs failed to cell division were regarded as unfertilized. After counting of triplicate samples, fertilization rate was estimated.
2.7 Collection of larvae, estimation of viable larvae, phototaxis larvae, dead larvae and follicle cells Immediately after hatching, the spent brood was taken out and weighed. Aeration of the hatching tank was turned off and one corner of the tank lid was opened to allow light. Phototaxis larvae gathered in the lightened corner were gently collected by a glass beaker and placed into a plastic bucket partially filled with sea water (30 ppt) and aeration. Rest of the larvae indiscriminately swam in the water column (nonphototaxis) were also collected in another bucket. Three sub-samples (100 ml each) of larvae suspension from each bucket were taken, counted separately. Both phototaxis and nonphototaxis larvae were then calculated.
2.9 Proximate composition and amino acid analysis Sub-samples of each type of feeds and newly hatched larvae foreach broods were collected, packed in gipped polybag and stored in the freezer (-20 °C). The samples were then transferred in a low temperature freezer (-180 °C) and kept until analysis. Analysis of proximate composition of crab feeds was done in accordance to the standard protocol. Amino acid was analyzed by using HPLC (High Performance Liquid Chromatography) system. 2.10 Starvation test to the larvae Starvation test was performed in 1 L plastic jars of each under the temperature ranged between 29 ºC to 30 ºC. Each of the jars were filled with 500 ml of 30 ppt sea water and 30 viable larvae were placed. Feeding was ceased and the water of each jar was changed daily with the same salinity and similar temperature water to avoid any secondary stress. Mortality of larvae was monitored after every 12 hours. A larva was considered as dead when it stopped swimming, body movement and even moving in the appendages. The stress test was performed for larvae produced from each brood up to 3rd spawning. The stress test was conducted as according to the standard methods.
2.11 Data analysis All the data and records were separately computed into MSExcel. The data were analyzed through SPSS Version 20 and 22. One way ANOVA (Analysis of Variance) and DMRT (Duncan’s Multiple Range Test) was done to establish the differences and the ranking. A confidence level of 95% was considered and p≤0. 05 was regarded as a significant difference.