MAHMUD HASAN
Department of Fisheries, University of Dhaka, Dhaka-1000, Bangladesh
ARIFA RAHMAN
Department of Fisheries, University of Dhaka, Dhaka-1000, Bangladesh
S. M. MAHBUBUR RASHID
Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000,Bangladesh
SALIM AHMED
Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000,Bangladesh
Tertiary Amyl Alcohol, 2 Phenoxyethanol, Quinaldine, Benzocaine, DNA:RNA ratio, Catla fingerlings
“Maa Fatema Fish Hatchery”, Chachra, Jessore, Bangladesh
Animal Health and Management
Catla fingerlings (10.46 ± 2.51 cm; 12.82 ± 1.37 g; mean ± SE) used in this experiment were obtained from “Maa Fatema Fish Hatchery”, Chachra, Jessore, Bangladesh. Before loading for transport simulation, fingerlings were held in a flow-through-tank system and feeding was stopped 12 h before loading into the plastic drum used as the experimental system. Efficacy test: To determine the optimal dosages of sedatives with least or no mortality, efficacy test was undertaken before doing simulated transport experiment. The safe and optimal sedative dosage required for inducing sedation with least mortality of catla fingerlings were determined by a series of preliminary tests. Immobilization and recovery period including cost and availability of the anesthetics with least or no mortality were considered to determine the optimal dosages. Several aluminum vessels of 10 liters capacity with 1 L well water were loaded with 15 catla fingerlings. Several dosages of 2 PE (100, 150, 200, and 250 μL L-1), benzocaine (25, 30, 35, 40 and 45 mg L-1); TAA (1.0, 1.5, 2.0, 2.5 and 3.0 mL L-1) and quinaldine (4%) (150, 175, 200, and 225 μL L-1) were applied to determine the optimal dosages. DO concentration and temperature of the water in the vessels were monitored at 1, 3 and 6 hour of exposure. Time required for immobilization was recorded. Behavioral responses of fingerlings were also observed and recorded during 1, 3, and 6 hour duration. Recovery periods of fingerlings were observed too. Finally, considering cost and availability, 2 PE, quinaldine and benzocaine were used in the transport simulation experiment. Transport experiment: The experiment was simulated by using a truck. A 4 × 3 factorial design was used with three replicates. The experimental variables were four transport methods and three transport durations. The control did not receive any sedative. The indicator variables were immediate and delayed mortality, and RNA: DNA ratio. Water quality variables such as dissolved oxygen concentration and temperature were also measured. Fingerlings were placed into 12 200 L plastic drums. Each drum was filled with 100 L subsurface clean water and mixed with sedatives and given oxygen injection by two air stones. Before loading, for simulation transport purpose, each drum was stocked with 40 kg fingerlings at the rate of 400 g L-1 loading density. TAA, 2 PE and benzocaine were obtained from Sigma-Aldrich, Germany while quinaldine 4% as TRANCE was collected from Argent Laboratories, Redmond, WA, USA. Solutions of all four sedatives were prepared by adding to the respective transport drum until the desired concentration was achieved. Oxygen cylinder containing 9.8 m3 oxygen at 2200 PSI with 99.9% purity was used as the source of pure oxygen. Oxygen was released at 8 PSI into transport drum water. Dead fingerlings were removed and counted to determine the immediate mortality rate at 1, 3 and 6 h after transport. Samples were also drawn for RNA: DNA at the time of dead fingerling counting. Water quality variables were also measured at the time of fingerling sampling for RNA: DNA quantification. After collection, samples were held in frozen condition at -200 C. For determination of delayed mortality after simulation experiment, dead fingerlings were removed from stocking hapa and counted 24 hours after the experiment began. Dissolved oxygen concentration and temperature of transport water were measured using HACH portable DO (HACH sensionTM 6, USA) after 1, 3 and 6 h of transport simulation. To determine RNA:DNA ratio, total nucleic acid (DNA and RNA) was isolated by CTAB (Cetyltrymethylammoniumbromide, a non-ionic detergent) method. The original protocol was modified by not using RNase at the last step of DNA isolation so that RNA remains intact with genomic DNA. Moreover, the overall laboratory setup and experimental conditions were ensured as DNase and RNase free. Since a single experiment was undertaken for quantification of both DNA and RNA from a single weighed sample, there was almost zero chance of any biasness in quantification as well as the ratio of DNA and RNA. Quantity of DNA and RNA was measured by Flurometry method by using a Qubit® 2.0 Flurometer (Invitrogen, USA). Qubit® 2.0 flurometer is sensitive than UV mediated technologies, accurate and reproducible. All percent data were transformed into square root before statistical analysis. Treatments were compared by anova followed by Tukey’s HSD post hoc for multiple comparisons. Data were analyzed by using spss software version 10.0 with the level of significance at p<0.05.
J. Asiat. Soc. Bangladesh, Sci. 40(1): 121-131, June 2014
Journal