2.1 Origin of stock A new founder stock comprising of 30 families having a total of 300 individuals of new generation of GIFT strain have been further introduced from Malaysia through WorldFish Center in March 2005. The founder stock fish were reared in a 100m2 hapas for three months. After three months rearing, the mean weight of female and male were 41.18±5.41 and 30.42±3.47 g, respectively. 2.2 Tagging of founder stock Passive Integrated Transponder (PIT) were used for individually tagging the females and males. A PIT tag was injected into the peritoneal cavity of a fish and the number of the tag was recorded. After tagging all the fish were transferred to a 1000 m2 pond.
2.3 Communal rearing in pond After tagging, the male and female fish were fed with supplementary feed (28% crude protein) six days a week, at a rate of 4-5% of estimated biomass. Fish sampling was done at fortnightly intervals to assess the growth and for feed ration adjustment. Water was supplied once a week to maintain water depth at 1.0 meter. The pond was fertilized fortnightly with Urea and Triple Super Phosphate at the rate of 12.5 and 25.0 kg/ha, respectively. After four months of rearing, the fish were recaptured through seine netting and pond drying. The harvest body weight, sex and tag number of all harvested fish were recorded.
2.4 Estimation of breeding value Breeding values were estimated for individual fish in a full pedigree, using SAS (SAS Inc, 1997) and ASREML. The statistical model included the fixed effect of sex because this effect on size and growth is often found in aquaculture species. Random term in the model was the additive genetic effect of individual fish in the pedigree. Age from birth to harvest was fitted as a linear covariate.
2.5 Breeding in hapas for G1 (Generation 1) production After harvest, genetic evaluation using the statistical model as described above was carried out on 300 founder animals to estimate their breeding values (EBVs). On the basis of breeding values of the founder stock, the best 40 males and best 40 females (from 30 families) were mated for the production of G1 generation. For breeding, 40 breeding hapas (1.0m3 ) were set up in a pond with bamboo poles. A pair of female and male breeders (1:1) was stocked in each breeding hapa. After 12 days from stocking, fertilized eggs were collected from the mouths of brooding females. Collected batches of eggs were transferred to the hatchery for separate incubation. Immediately after hatching, the larvae were transferred to a series of trays (one for each family) and were kept until their yolk sac resorption stage.
2.6 Nursing in hapa From each family, 300 fry were transferred to an individual fine-mesh nursery hapa (2 m3) in a pond. The progeny were fed with nursery feed containing 30% protein at the rate of 30% of estimated body weight. The mean weight of the fry was 2.80±0.42 g after 45 days of nursing.
2.7 Rearing in hapa Subsequently, 150 fry from each progeny group were transferred to an individual rearing hapa (2.0 m3 in size). Supplementary feed (Nursery feed) was applied in all the hapas at the rate of 15% of estimated biomass. After sixty days of rearing the weight range for male and female were 36-43 and 28-32 g, respectively. 2.8 Experimental Procedures Each progeny group 25 male and 25 female fish were randomly sampled and tagged using Passive Integrated Transponder (PIT). Tagged fish from 40 families (2000 fishes) were stocked in a pond having 1000 m2 area for communal rearing. Supplementary feed (25% crude protein) was supplied regularly at the rate of 6% of estimated biomass. The fish were harvested and tag number, weight, sex, body depth were recorded after six months of grow-out in pond. Then, breeding values of G1 generation were estimated from the complete data set, tracing back to the foundation population (G0).
2.12 Evaluation of growth performances between upgraded GIFT strain (G2) and founder population in pond For evaluating the growth performances of upgraded GIFT strain (G2) and founder population of GIFT strain in a pond for a period of five months during June to November 2008. A pond having an area of 1000 m2 was selected for growth performance evaluation. Prior to the evaluation, the pond was cleaned and limed at the rate of 250 kg/ha. After three days of liming, pond was fertilized with urea and Triple super phosphate (TSP) at the rate of 12.50 and 25.0 kg/ha, respectively. Fry of upgraded GIFT strain generation (G2) were stocked together with the progeny of the founder stock in a pond for communal rearing. Fry of founder population of GIFT strain (Original strain) were marked through cauterization of pelvic fin. For each group, 600 fingerlings were stocked. The initial mean weight of upgraded GIFT strain (G2) and founder population were 4.81±0.65 and 4.72±0.82g, respectively. After stocking fingerlings were fed with nursery feed contained 28% crude protein at the rate 5-10% of estimated body weight. Fish were sampled at fortnightly intervals to assess the growth as well as for feed adjustment. In the first month, fish were fed at the rate of 10% of estimated body weight and the consecutive months feed ration was reduced to 8, 6 and 4% in the 2nd, 3rd and 4th month, respectively. After four months of rearing, fish were harvested through repeated netting followed by pond drying. The same protocol was practiced in subsequent generations in 2009, 2010, 2011 and 2012 (corresponding to generations G3, G4, G5 and G6) for growth evaluation.