The objective of this study was to evaluate the effect of water temperature, gut pH values, and postprandial time on the activity of gastric (pepsin) and pancreatic (trypsin, chymotrypsin, alpha-amylase, and lipase) enzymes. For this purpose, two hundred rainbow trouts were equally distributed among twenty four quadrangular 2000 L tanks. Then, fish were divided on two experimental groups that were kept for 20 days at 13 °C and 20 °C, respectively. All tanks were connected to a recirculation unit (IRTAmar™) equipped with mechanical, biological and UV-filters and a temperature controller. Fish were hand fed twice per day during the trial. At the end of the trial, before determination gut pH and activity of digestive enzymes, fish were fasted for 72 h. Then, fish were normally fed and they were sacrificed in 2, 5, 9, 13, 18, 26, 36, 48, 60, 84, 96, 120 hours after feeding. Fish were euthanized by a blow on the head and individually weighed and their guts removed. The values of pH were measured with a HI 8314 portable pH meter with a HI 1083 B microelectrode of 3 mm of diameter (Hanna Instruments, USA). Then, the stomachs and intestines were lyophilized and stored at -80 °C.
The activity of pepsin, trypsin, chymotrypsin, alpha-amylase, and lipase were quantified. In the present study we used “standard conditions” i.e. room temperature (25°C) and constant buffer pH (pH 2.0 for pepsin and pH 8.0 for trypsin, chymotrypsin, alpha-amylase, and lipase) and “actual conditions” i.e. actual physiological pH values in fish stomach and intestine and temperature in tanks (13 and 20 °C respectively). Moreover, for all mentioned enzymes the pH optimums were assayed as well.
The pH maximum level of pepsin activity was found at pH 2.0-2.5 whereas at pH higher than 5.0 the activity was completely absent for both groups of fish. The pH maximum level for trypsin was found at pH 9.0 and 10.0-10.5, for chymotrypsin at pH 8.0-9.0, for alpha-amylase at pH 7.5-8.0, and for lipase at pH 8.0-8.5 and 9.5 for both groups of fish. The time after feeding, actual pH values in gut, and water temperature in tanks were significantly influenced on the activity of studied enzymes (ANOSIM, p ?0.05). This study showed the important role of three key factors (temperature, pH, and time after feeding) on the activity of main digestive enzymes in gut of rainbow trout.