30 JUNE 2016 • WORLD AQUACULTURE • WWW.WAS.ORG These results with fish larvae indicate that, in all cases, even under a forced artificial environment with continuous feeding and illumination, feed intake and/or digestion have a resting period. From a practical point of view, alternating periods of dark and light appear to be necessary to recover the level of enzymes and other metabolic compounds required for proper functioning of the digestive system and to synthesize new tissues. Studies by other researchers have demonstrated that the best quality of larvae, fastest development and lowest degree of deformities were achieved under rearing conditions resembling those of the natural aquatic environment of the species. Therefore, a least a short dark period is recommended in visual-feeding species. Overall, the few species that have been studied do not indicate notable differences in the time required to reach weaning size when comparing a dark-light cycle with permanent illumination. A second finding is that each species has its particular way to process food over the daily cycle but unfortunately only few species have been explored and the information is still very scarce to allow a clear picture. In the juvenile stage with discrete feeding, we have to look at whether the postprandial response is affected by feeding protocol. Digestion in juvenile fish with a size above one gram has two steps: first acidic digestion in the stomach followed by alkaline digestion in the intestine (Fig. 4), although many fish species lack a stomach and therefore the first digestion step. Generally grow-out of juvenile fish is made under a natural light-dark photoperiod. The simplest situation is one meal a day, which allows us to understand without interferences what is happening during the whole process from ingestion to the excretion of undigested debris. In this case, the stomach is filled immediately after food is supplied. After some time, food is passed towards the intestine until the stomach is emptied. The main hydrolytic enzyme in the stomach is pepsin, activated in an acidic environment. Two main strategies have been described for acidic digestion in the animal kingdom; a permanent acidic luminal environment in the stomach or neutral pH during the resting period between meals. Both models can be found in teleosts, although most species have the second model, as is the case of the species we have studied: gilthead seabream, white seabream Diplodus sargus, thicklip grey mullet Chelon labrosus and meagre Argyrosomus regius. In these species, the gastric pH, neutral when the stomach is empty, declines quickly after food ingestion and the stomach is filled, while pepsin activity increases as an immediate response. All parameters recover to initial values during the second part of the daily cycle, to restart again the following day when food is added. At the molecular level, expression of pepsinogen and the proton pump, responsible for luminal acidification, increases during the night, as a preparatory maneuver for the next meal on the coming day (Fig. 5, Yúfera et al. 2012). Several daily meals, the most common practice in juvenile ongrowing, may produce an overlapping of subsequent postprandial responses and in general a modulation of the feeding and digestion pattern over the daily cycle. In gilthead seabream, the temporal pattern of feed intake and digestion changes when several meals are offered. Thus, while a single daily meal forces fish to eat immediately when food is supplied, several meals or continuous feeding allow fish to choose the better moments to eat (Fig. 6). At the gastric level, the most striking finding is that the daily period in which the luminal pH is within the adequate range to activate pepsin (below pH 4) varies considerably with the feeding protocol, being longer with a greater feeding frequency (Fig. 7). These differences in digestion capacities are behind the fact that different weight gain is reached with the same daily ration when the feeding frequency changes (Yúfera et al. 2014). On the other hand, the luminal pH in the intestine seems to be less affected by feeding frequency and always shows an increase to alkaline values in the central hours of daylight, returning to neutral values at night. This alkaline environment is necessary to the appropriate functioning of intestinal alkaline proteases. We have yet to analyze enzyme activities throughout the day in the intestine but FIGURE 5. Complete gastric digestion process in white seabream fed a single daily meal. The stomach is filled immediately after food supply (green frame); the gastric pH declines quickly, activating pepsin that increases in the following hours. Pepsinogen and proton pump mRNA is overexpressed during the night (grey frame) to prepare the digestion machinery for the next daily cycle. Modified from Yúfera et al. (2012). FIGURE 4. Different steps of food digestion in juvenile and adult gilthead seabream.
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