Carlos R. Rojas-García

The supply of amino acids during early feeding stages of marine fish larvae: a review of recent findings

In marine fish larvae, the sum of protein deposition, turnover and catabolism necessary for their rapid growth dictates a high amino acid (AA) requirement. Once the yolk is exhausted, the digestive tract becomes the vital organ that ensures a steady supply of dietary AA to the growing larval tissues. In this paper, we discuss the demand and availability of AA (free and polymerised pools) in relation to larval digestive capacity. The sources of AA from compound and live diets are described, and the early regulatory roles of cholecystokinin (CCK) and a retrograde peristaltic activity are highlighted.

In vivo studies of digestion and nutrient assimilation in marine fish larvae

Abstract This paper describes a method for quantifying the functionality of the digestive system in fish larvae. The system described can provide data for the gut absorption, oxidation and retention (assimilation) of nutrients. A tube-feeding setup (originally described in Aquaculture 116 (1993) (341–352) using 14 C-labelled dietary nutrients formed the basis of a new incubation system. This permitted unabsorbed nutrients evacuated from the gut to be distinguished from molecules originating from catabolism of the absorbed nutrient, both of which are present in the incubation water. The system is based on the release, transfer and entrapment of metabolically produced 14 C–CO 2 through manipulation of the water pH. The efficiency of the trap has been validated and tested, and provides 100.0±1.3% (S.D.) recovery. The usefulness of the method is demonstrated in a study in which Atlantic halibut post-larvae (46 days post first feeding) were fed a 14 C-labelled protein diet. These data show that this protein has a digestibility of 42% for halibut post-larvae. If oxidation had not been measured through the use of the CO 2 trap, digestibility would have been greatly underestimated (at about 25%).

Combined sensitive analytical methods for cholecystokinin levels and tryptic activity in individual fish larvae

A protocol for combined analysis of cholecystokinin (CCK) levels based on radioimmunoassay (RIA) and fluorescence tryptic enzyme activity (FTA) was developed in order to accomplish a sensitive analysis of individual bodies and gut segments of fish larvae. Methanol was used for CCK extraction. The gut of herring larvae contained 8.9±1.2 fmol CCK/mg dry weight and in the post-larval Atlantic halibut the CCK levels varied significantly (p<0.05) from 20.9±15.6 to 101.8±56.7 fmol/mg dry weight for separated intestinal and pyloric segments, respectively. Acid solution, 0.02 mol/l HCl–CaCl2 (pH 1.8), and alkaline solution, 0.1 mol/l Tris–0.02 mol/l CaCl2 (pH 8.0) were tested to prepare crude trypsin extracts from Coregonids and Atlantic halibut larvae. The tryptic activity of crude extracts prepared with acid solution was enhanced by a factor of 3.19±0.52 compared to the tryptic activity of crude extracts prepared with alkaline solution. The larval trypsins (from yolk sac larvae) were stable in methanol, preserving 88% of its starting activity after 6 days of storage. Based on the results, the method of extraction using methanol and acid solution (pH 1.8) was suitable for the combined analysis for CCK levels and FTA in gut segments or single larvae. The potential application of these analytical tools may allow a better understanding of the individual variability of gut functionality, nutritional condition and the feeding activity of developing fish based on their content of CCK and tryptic activity.

Cholecystokinin (CCK) in Atlantic herring (Clupea harengus L.) - ontogeny and effects of feeding and diurnal rhythms.

Neural and alimentary cholecystokinin (CCK) levels in Atlantic herring, Clupea harengus, were analyzed from hatching to 40days after hatching (DAH). The head compartment representing the neural pool was quantitatively dominant (>80% of the total CCK content) while the digestive tract pool represented 6-10%. During ontogeny the CCK level in whole larvae increased almost 15-fold from 0 to 40 DAH, being particularly marked from 14 to 20 DAH. Larvae of 24 to 26 DAH were examined for potential occurrence of a circadian rhythm and to analyze the effects of feeding. Fed and fasted larvae were significantly different, where fed larvae showed higher CCK levels. There were large fluctuations in CCK levels analyzed at 3h intervals without an apparent diurnal pattern. Shorter sampling intervals of 1h in the morning when lights were switched gradually on and food was offered to the larvae demonstrated a marked drop in the relative gut CCK levels and a concurrent increase in the CCK carcass to gut ratio, 1h after introduction of food followed by a return to prefeeding levels after 2h. This response probably results from a release and re-synthesis of CCK in the gut after initiation of feeding. Taken together, these results support earlier reports that CCK participates in the regulation of digestive processes in herring larvae, but CCK does not seem to have a circadian rhythm independent of feeding.

Variability in Digestive Enzyme Capacity in Early Stages of Marine Fish Larvae: Ontogenetic Variations, Biorhythms, Hormonal Control and Nutrient Sensing Mechanisms

In recent years, no substantial progress has been achieved to significantly improve survival rates of cultured marine fish larvae and make the viability in offspring production more predictable. Improving survival and quality of marine larvae in aquaculture industry beyond the present status requires consideration of nutritional quality of the feed but also an in-depth knowledge of larval physiology to account for biorhythms in digestion capacity, appetite and regulation of food ingestion and endocrine control of digestion processes associated to nutrient sensing and feeding activity. Since gut hormones are most likely stimulated and released in response to specific nutrients it is crucial to study the nutrient sensing mechanisms also in the gastrointestinal tract of fish larvae and the importance of nutrients as they are not only substrates, but active regulators of physiological processes. This chapter focuses on the importance of the daily and circadian rhythms in the digestion capacity in larval stages of fish, emphasising the potential variability of enzyme secretion during the day, addresses the specific role of the gut hormones cholecystokinin and ghrelin on appetite and digestive functions in fish larvae and provides a comprehensive consideration of the food sensing mechanisms in the gastrointestinal tract of fish larvae associated to specific nutrients. Knowledge in these research areas, through larval ontogeny, will be specifically beneficial for the development of stage-specific micro diets and replacement of live feed with formulated feed. Recommendations on future research aspects and approaches within these themes are discussed and suggested in the specific sections.