44 DECEMBER 2024 • WORLD AQUACULTURE • WWW.WAS.ORG spawning season. During the first 7 days of acclimation, the fish received a 25-ppm formalin bath for 30 minutes every day. Initially, the fish were not accepting any feed. However, after about a week in the quarantine tanks they started to feed on freshly prepared wet feed consisting of 150g/Kg wheat flour, bread, 100g/ Kg omega algae powder (Bern Aqua-Belgium), 3g/kg Bactosafe (Bern Aqua, Belgium), 2 g/kg Vitamin C, 2g/kg Vitamin E, 2g/kg Vitalyte (Vitamin mix-ANUPCO, UK) and 15ml/kg fish oil and mixed with 500 ml hot water and made into a dough and dried after cutting into small pieces. The prepared feed was fed at a rate of 1 percent of the total body weight once daily for 3-4 days. After this, 6mm formulated pellet feed (Sketting, Turkey) enriched with 2g/kg monocalcium phosphate, 15 ml fish oil, 2 g vitamin C, 2g/kg vitamin E and 2g vitalyte 2 g/Kg was provided ad libitum. Spawning Induction, Egg Incubation and Hatching During middle of March 2024, the fish were segregated by sex and female maturity evaluated by ovarian biopsy using cannulation. Males were checked using abdominal pressure and milt-expressing males were separated. AQUI-S® at 20 ppm was used to anaesthetize the fish during handling, cannulation and hormone injection. The cannulated eggs were checked under the microscope and egg diameter measured. All females having egg size more than 625µm were selected and used for the spawning induction trials. The selected mature fish were transferred to the spawning tanks by third week of March 2024 in 35 ppt seawater and three trials were undertaken with different hormone therapies administered intramuscularly to four groups of fish between March and April 2024. In the first trial 6 females (with an average egg diameter of 644±79.5µm) were given a priming dose of 20mg/Kg carp pituitary and 100 µg/Kg LH-RHa. A resolving dose of 100µg/Kg LH-RHa was administered 24 hours later. The males were not given any hormone injection in this trial. In the second trial 5 females (with an average egg diameter of 635±39.7µm) were given a priming dose of 20mg/Kg carp pituitary and 1000 IU/Kg HCG and 24 hours later a resolving dose of 100µg/ Kg of LH-RHa was administered. The 7 males in this group were given GnRH (Syndel USA) at 10µg/Kg on the day when the females were given the priming dose. The third trial had two groups, one whose average egg diameter was 776±20.4 µm and the other group whose average egg diameter was 632±45.3µm. Both the groups were given a single combined dose of 200µg/Kg LH-RHa plus 250µg/Kg GnRH. The males received 10µg/Kg of GnRH. Spawning was observed within 48 hours of the resolving dose of the hormone injections. The spawning success in the first two trials was very low with only 2 and 1 fish spawning, respectively and the fertilization and hatching rates were also low in both groups. In the third trial, the group with the lower egg size of 632±45.3µm, none of the 18 females spawned while all 5 females having an average egg diameter of 776±20.4 µm spawned and the total number of fertilized eggs collected was 480 x103 (Table 1). The fertilized eggs were incubated in hatching baskets and the embryonic development was closely monitored during the incubation period and photographed using light microscope, Nikon Eclipse Ci, Japan (Figure 2). The eggs hatched in about 26-28 hours of incubation in 35 ppt at a water temperature of 23±1.0 0C and the hatching rate was 48 percent (Table 1). Larval Rearing The newly hatched larvae, averaging 2.87 ± 0.05 mm in total length, were counted and stocked in six 3-T capacity rectangular concrete larval rearing tanks at a density of 20 larvae/L (2 tanks) and 40 larvae/L (4 tanks). The salinity was 35±1 ppt and the water temperature was 22±1.0 0C. The larval rearing protocol generally conformed to that followed earlier at the aquaculture center (AMSC) for M. cephalus (Yousif et al. 2010, Yousif et al. 2016) with minor modifications. The green algae, Nannochloropsis sp. was added to the tanks at a concentration of 40,000 cells/ml from day 2 to day 22 post hatch (dph). First feeding started with enriched rotifers at 3-5/ml. Starting 12 dph artemia nauplii were fed at 2.5 nauplii/ml and continued till 25 dph. From 22 dph the larvae were fed ad libitum until 30 dph with the micro-diet Gemma wean 0.1, 100-300 µm (62 percent crude protein, 14 percent lipid, 8 percent ash and 0.2 percent fiber) and from 30 dph to 40 dph they were fed ad libitum Gemma wean 0.2, 200-400 µm (Skretting, France). Tank bottoms were siphoned daily from 15 dph onwards to remove faeces and unconsumed feed. Water exchange was carried out by flow-through of filtered and disinfected sea water of 35 ppt. In the first 14 days of rearing the daily water exchange was only 5-20 percent. At 15 dph the daily water exchange was increased to 50 percent and after 20 dph a continuous flow-through was maintained at 80-100 percent daily. The photoperiod was maintained at 12h:12h (light: darkness). Ammonia levels were maintained at less than 0.5mg/L and nitrite less than 0.02 mg/L. Dissolved oxygen was always above 5 mg/L. Fry numbers on day 25 were around 22,000 (an estimated survival of 8.69 percent). However, when all the tanks were harvested on day 40, the total number collected was 3102 fish, with an average survival of only 1.23 percent (Table 2). There was substantial mortality between day 16 and 20 dph. The variation in body size was very distinct at harvest; the body length and weight ranged from 2.1 – 3.1 mm (a mean of 2.66 ± 0.29 mm) and 0.22- 0.42 g (a mean of 0.32± 0.05 g), respectively. FIGURE 2. Embryonic development of V. seheli: (A) Earlier cell division, (B) late Blastula, (C) Early Gastrula, (D) Pre Neurulla, (E) Middle Neurulla, (F) Late Neurulla, (G) Eye development, (H) Fully developed embryo and (I) Hatched Larvae.
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