2.3. Sample collection and analysis
Percent weight gain (WG), feed efficiency (FE), hepatosomatic index (HSI), hematocrit (PCV), hemoglobin (Hb) and percent survival were determined at the end of the feeding trial. After the final weighing, six fish were randomly removed from each aquarium; blood samples were obtained using a heparinized syringe from the caudal vein and pooled for hematocrit and hemoglobin determination (Brown, 1980). Ascorbic acid concentrations in diets supplemented with L-ascorbic acid were deter- mined directly by HPLC, and ascorbic acid concentrations in diets supplemented with AMP was calculated after analyzing the AMP contents in diets by HPLC (AMP was produced by Hoffman La Roche, containing 35% ascorbic acid activity). Ascorbic acid concentrations in gill, muscle, liver, and brain of pooled fish (five fish per aquarium) were determined by HPLC (Syknm, German) with a UV detector at 254 nm. The mobile phase was 0.1 M KH2PO4 at pH 2.8 and the flow rate was 0.4 ml/min. Preweighed samples were homogenized in 10% cold metaphosphoric acid. Homoge- nates were centrifuged at 10061 Â g for 25 min and supernatants were analyzed on HPLC after filtered through a 0.45 mm pore-size syringe filter (Sartorius, Go¨ttingen, Germany).
2.3. Sample collection and analysisPercent weight gain (WG), feed efficiency (FE), hepatosomatic index (HSI), hematocrit (PCV), hemoglobin (Hb) and percent survival were determined at the end of the feeding trial. After the final weighing, six fish were randomly removed from each aquarium; blood samples were obtained using a heparinized syringe from the caudal vein and pooled for hematocrit and hemoglobin determination (Brown, 1980). Ascorbic acid concentrations in diets supplemented with L-ascorbic acid were deter- mined directly by HPLC, and ascorbic acid concentrations in diets supplemented with AMP was calculated after analyzing the AMP contents in diets by HPLC (AMP was produced by Hoffman La Roche, containing 35% ascorbic acid activity). Ascorbic acid concentrations in gill, muscle, liver, and brain of pooled fish (five fish per aquarium) were determined by HPLC (Syknm, German) with a UV detector at 254 nm. The mobile phase was 0.1 M KH2PO4 at pH 2.8 and the flow rate was 0.4 ml/min. Preweighed samples were homogenized in 10% cold metaphosphoric acid. Homoge- nates were centrifuged at 10061 Â g for 25 min and supernatants were analyzed on HPLC after filtered through a 0.45 mm pore-size syringe filter (Sartorius, Go¨ttingen, Germany).
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