Effects of different feeding protocols on daily proteolitic enzyme activity in gilthead seabream juveniles

Abstract

Many studies have assessed digestive enzymes activity in response to diverse feed ingredients and water environmental conditions. However, the circadian rhythm of the digestion process and the factors influencing these rhythms have scarcely been examined. The aim of the present work was to compare the daily activity pattern of two key proteolytic enzymes (acid proteases and trypsin) in the gilthead seabream (Sparus aurata) fed with different protocols. Juvenile fish (17.66 ± 0.38 g) were distributed into 4 groups (250-L tanks, 19.5 ± 1.0 °C, photoperiod 11L/13D). All groups were fed a daily ration of 2 % body mass during the daylight with different feeding protocols: (a) 1 meal (08:30 h); (b) 3 meals (08:30, 13:30, and 18:30 h); (c) 5 meals (08:30, 10:30, 13:30, 16:30 and 18:30 h); and (d) continuous feeding. Fish were acclimatized during 2 weeks and then were sampled every 4 hours during a 24 h cycle. Total acid protease activity in the stomach was analyzed using hemoglobin, at pH 2.0. Trypsin activity in the intestine was analyzed using BAPNA at pH 8.5. Not significant variations were observed in the pepsin daily activity in none of the feeding protocols, excepting the fish fed 3 times that exhibited a peak at the beginning of the dark cycle (Fig. 1A). However, juveniles fed 5 daily meals showed higher trypsin activity during daylight with a considerable decrease during the dark period. Similarly, in fish with continuous feeding, trypsin activity increased gradually during the daylight, reached maximum levels around 21:00 h, and decreased drastically afterwards. On the other hand, fish fed 1 and 3 times a day did not show significant variations in the trypsin activity (Fig. 1B). According to our findings, feeding frequency and timing have a major influence on the daily pattern of enzymatic activity, especially during the intestinal phase of digestion. It seems that splitting the daily ration in several meals leads to higher trypsin activity during the daylight and the beginning of the dark period.