34 JUNE 2016 • WORLD AQUACULTURE • WWW.WAS.ORG stage 9, eye migration is advanced and reaches the midline, giving larvae an asymmetric appearance. Distinctive skin pigment patterns emerge and larvae rest occasionally on the tank bottom. In post-metamorphic stage 10, eye migration and pigmentation are complete. Stage 10 corresponds, thus, to the juvenile individual and from this point on, the halibut adopts a demersal lifestyle and settles on the tank bottom. In addition to changes in external morphology, coordinated maturation of many tissues and organs occur, including the digestive tract. An important aspect is that larval stages lack a stomach. A fully functional stomach function is only acquired during metamorphosis. The drastic changes in ecology, morphology and physiology that occur when larvae transform into juveniles clearly requires adaptation of zoo-technical production systems for these different life stages. Nonetheless, there are many aspects that remain unexplored when it comes to understanding the biology of the transitional fish in these early stages and also what constraints it poses to the design of culture protocols. The Stomach - a Multifunctional Organ The current large-scale production systems of halibut in Norway rely on the use of short-term enriched Artemia until they are weaned to dry diets 45 to 55 days after first feeding. Thus, as for most marine precocial species (species that develop a stomach during metamorphosis), halibut larvae rely on live prey from first feeding. Designing a feed that is successfully ingested and digested, by taking into consideration the immaturely developed digestive system, including the lack of a functional stomach, continues to pose a major challenge for producers and researchers (Gomes et al. 2014). The stomach fulfills several important functions (Smith et al. 2000, Stevens and Hume 2004). It acts as a short-term reservoir that permits the fish to eat large meals in a short time, in comparison to larvae without a stomach (as well as stomach-less fish) that need to eat much more often. The storage function requires a functional esophagus and strong pyloric sphincters to control retention and passage of food in the stomach. The first phase of mechanical and chemical breakdown of food occurs in the stomach, with strong peristaltic movements caused by smooth muscles in the stomach walls that enable mixing ingested food with secreted gastric acid (HCl) and pepsin. Pepsin, an enzyme that greatly enhances the digestion of proteins, becomes activated in the presence of HCl. The combined effects of HCl and pepsin make the stomach a highly efficient organ for degrading complex proteins. In addition, gastric acid kills many of the bacteria and pathogens ingested with food and represents part of the first line of immune defense that protects the fish. Taken together, these features allow the stomach to increase digestion efficiency by retaining food and initiating degradation and digestion of food before it enters the midgut. This allows the midgut to maximize digestion and absorption. In addition, the stomach produces hormones that are key regulators of appetite and digestion and that also serve as signals to the brain to regulate appetite. The Ontogeny of Protein Digestion in Atlantic Halibut Larvae Fish larvae have huge relative growth potential compared to juvenile and adult fish. Growth is primarily a deposition of muscle protein, which means that the right quality and quantity of amino acids in the form of protein must be supplied to fully realize the growth potential of the fish larvae (Houlihan et al. 1995, Carter and Houlihan 2001, Conceição et al. 2011). In addition, amino acids, the building blocks of proteins, are also a major energy source during the larval stage. Therefore, understanding the digestibility of specific proteins becomes essential to produce optimized diets for fish larvae in culture systems, but the amino acid requirements of fish larvae are poorly understood. The fast growth of fish larvae demonstrates the large and effective processing capacity of the larval digestive tract, given that larvae are feeding highly digestible diets under optimal conditions (Rønnestad and Conceição 2005, Zambonino-Infante et al. 2008). To digest dietary proteins, a range of basic processes in the digestive tract must be coordinated, including enzymatic and fluid secretions and motility. From the onset of exogenous feeding, digestion of dietary proteins in stomach-less fish larvae starts in the midgut where ingested feeds are mixed with pancreatic secretions and bile from the gall bladder. The major pancreatic proteases are trypsin and chymotrypsin that, under the alkaline conditions of the gut lumen, split FIGURE 3. An in vivo method to measure the pH in the lumen of the digestive tract. Drawing is adapted from Rønnestad et al. (2002). FIGURE 2. (A and B) Compartmental distribution of radioactivity given as percent of administered radioactivity: (A) intact protein in proclimax metamorphic (36 dpff) and juvenile (78 dpff) Atlantic halibut; (B) intact and pre-hydrolysed protein in Atlantic halibut prometamorphic stage 6 (25 dpff). Adapted from Tonheim et al. (2004). (C) Distribution of radioactivity in Atlantic halibut prometamorphic stage (24-32 dpff) after tube feeding with intact protein, pepsin hydrolysed, and highly hydrolysed protein. Values are mean±S.E.M. Adapted from Tonheim et al. (2005).
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