For many fish species, further growth of the farming industry is hampered by the absence of a reliable production of young fish needed for stocking grow-out tanks. This constraint is strongly related to an insufficient knowledge of the nutritional aspects in rearing the larval stages of those species. Most fish species require live food organisms as first food, hampering thereby the development of more dependable larval production techniques. To overcome this problem, more appropriate (formulated) feeds and feeding procedures for larval fish are required. Because in fish larvae, the development of organs and functions is not yet completed, larval rearing and feeding procedures can only be developed when the biological properties of the developing young animal are taken into account. In the present thesis, this philosophy was applied for the case of the African catfish, Clarias gariepinus (Burchell). The study aimed (a) at the development of appropriate rearing and feeding procedures and of a suitable dry starter diet, which could also be used in nutritional research with fish larvae and (b) to elucidate the relation between problems in larval nutrition and the associated ontogenetic aspects. As many other fish species, larvae of the African catfish have no stomach at the onset of exogenous feeding but develop it later on. The basic assumption behind the study was that differences in nutritional requirements between larval and juvenile fish would provide the cues for the development of such a formulated diet. As a working hypothesis the mentioned differences between larvae and juveniles were supposed to be induced both by the small size of larvae (a quantitative scaling effect) and by qualitative differences in digestive and metabolic properties because of ontogenetic effects. The research project was subdivided in three phases, which dealt respectively with the development of a standardized husbandry system, the ontogeny of the digestive and metabolic properties of the growing larvae and with the lipid metabolism in the early life stages. During the first phase of the study, standardized rearing and feeding conditions for nutritional experiments with larval catfish were developed. At the start of the project, the nutritional value of several dry test diets for larvae of C. gariepinus was determined. Dried and decapsulated cysts of Artemia gave consistently the best growth and survival rate. Micro-encapsulated (chicken)-egg diets, especially when enriched with casein and a vitamin/mineral mixture, resulted in high survival rates but low growth rates. Analysis of the hepatocyte ultrastructure in the different test groups suggested that the egg diets contained all essential nutrients. Apparently not only the quality but also the intake of the larval diets is important. In comparative studies with dry formulated diets, behavioral influences on feed intake can be avoided by using dried and decapsulated cysts of Artemia , instead of just hatched nauplii. This first study did not include a dry diet composed of Single Cell Protein (yeast cells) which has been reported to produce good growth and survival in larval C. gariepinus (Appelbaum and van Damme 1988; Van Damme et al. 1990). However, later and unreported tests with the latter diet in our laboratory yielded consistently lower growth rates than in groups fed with Artemia . Hence, the conclusion that C. gariepinus larvae need live food organisms as first food to maximize growth rates, is still valid. For nutritional experiments, Artemia was suggested as a reference diet. In a second study, the growth performance of larval C. gariepinus under different conditions of feeding rates and temperature was investigated. A method for determining feeding levels in larval nutrition studies was developed. It entails a daily adjustment of the feeding level to changes in relative growth rate and in dry matter content of the larvae. Using this method, a classic dosage-response relation between feed ration and growth was obtained. Larval growth rate was maximized (growth coefficient 9 = 0.30-0.31) when food rations were calculated according to predicted growth coefficients of 0.3-0.5. The optimal temperature was set at 27.5 °C. In a third study, the length of the experimental duration was investigated. The study showed that larvae of C. gariepinus can be weaned from Artemia to crumbles of a commercial trout diet without loss of growth and of survival, provided that they had first received live food for four days. Taking into account that weaning occurred gradually, these results make it clear that eight days after the start of exogenous feeding, the larvae are not restricted by a larval diet, and therefore may be regarded as small juvenile fish. This finding gives a good support to the duration of the experiments which was used throughout the present study, i.e. 10 days. During the second phase of the research project the hypothesis that in C. gariepinus, the larval period is ending at an age of approximately 5-8 days was further elaborated. In two separate studies, the morphofunctional development of the digestive system and the metabolic development of C. gariepinus was elucidated. At the onset of exogenous feeding, the intestine is subdivided into three functionally different regions, possessing substantial activities of digestive enzymes, both from the pancreas and from the enterocytes. The intestine is capable to digest and absorb lipids. Immunohistochemical evidence for the presence of pancreatic hormones, e.g., insulin and glucagon, and for the presence of a gastrin/CCK-Iike hormone demonstrated the capacity for a coordinated digestive and metabolic process from the start of exogenous feeding onwards. Apparently, at the start of exogenous feeding, the animals are capable to ingest, digest, and absorb nutrients. A major difference with the juvenile period is the absence of a functional stomach at the start of exogenous feeding. The morphological and functional development of the stomach is completed about 5 days after the start of exogenous feeding. Other studies which are not included in this thesis (Segner et al. 1993; Verreth et al. 1993) revealed that the onset of stomach functions coincided with a switch from an alkaline proteolytic digestion to a combined acid and alkaline proteolytic digestion. Pepsin-Iike activity reached a maximum, eight days after the start of exogenous feeding. The stomach differentiation seems to coincide with the earliest possible weaning time as determined in the previous study, e.g., after an Artemia feeding period of about 5 days at 27.5 °C or at a size of about 20 mg. Also the next study, e.g., on the ontogeny of metabolic functions as exemplified by enzyme activities, showed that this size of 20 mg is a kind of "turning point" in the development of C. gariepinus. All investigated enzymes of the central intermediary metabolism were present from the start of exogenous feeding onwards. Important ontogenetic changes were found in the activities of those enzymes which are characteristic for glycolysis, glyconeogenesis, glycogen storage and amino acid catabolism. All these changes were quantitative of nature and occurred at a size of about 20 mg, i.e. after 3-5 days of Artemia feeding. These changes in metabolism coincide with changes of muscle organization, gill morphology, respiration, and as shown earlier, with changes in stomach functions. The combined change of the various morphological and physiological parameters indicates that the larval period in C. gariepinus ends at a body size of approximately 20 mg. The third phase of the research project focused on the assessment of nutrient requirements in larval C. gariepinus. The present thesis includes three studies on the lipid metabolism in the early life stages of this species. The aim was to analyze the relative importance of endogenous and of exogenous sources of lipids and fatty acids for larvae of the African catfish. The first of these three papers deals with the conversion of the different neutral and polar lipids in the yolk to the body tissues of the growing yolk sac larvae and their fate in subsequently starving larvae. The eggs of C. gariepinus can be characterized as having moderate amounts of lipid (approximately 5%) of a highly polar nature (80% of the lipid), with phosphatidylcholine as the most dominant lipid class (about 70-75% of the total lipid). Phosphatidylcholine was catabolized proportionally to total lipid, demonstrating its role as main energy supplier. All yolk phosphatidylethanolamine (PE) was converted into body PE. The neutral lipids consisted of triglycerides (TAG), cholesterol and cholesteryl esters. All TAG were depleted before complete yolk absorption. The most abundant fatty acids in the eggs were 16:0, 22:6n-3, 18:1n-9, 18:0, 20:5n-3 and 18:2n-6 (respectively about 29, 19, 17, 11, 6 and 5% of the total fatty acid composition). Most yolk fatty acids were absorbed proportional to the total fatty acids, e.g., at approximately 0.5% of their initial amount per time unit (physiological day degree). Apparently during the yolk sac period there is no strong preference for individual fatty acids to be catabolized or to be converted into body tissue. The overall conversion efficiency of most fatty acids from yolk to body tissue was lower than 60%. Nevertheless, during the yolk sac phase, some fatty acids were clearly synthesized, showing conversion efficiencies above 100%: 20:4n-6; 20:5n-3 and 22:6n-3. These three fatty acids are essential for marine fish and crucial for a good organogenesis and functional development of any fish (Sargent 1994). At the end of the yolk sac period, 22:6n-3 is the most abundant fatty acid in the lipid (about 28% of the total fatty acids), suggesting a crucial role of this fatty acid for C. gariepinus as well. Nevertheless in a feeding experiment with Artemia , enriched with either low or high levels of highly unsaturated fatty acids (HUFA), dietary n-3 HUFA levels did not affect growth or survival rates in C. gariepinus. Irrespective of the dietary levels, the tissue levels of 22:6n-3 in the fish decreased from about 17 mg/g dry weight at the start of exogenous feeding to 2-4 mg/g dry weight at the end of the experiment (at a body size of about 50 mg wet weight). Fatty acid retention percentages revealed a capacity to synthesize HUFA. The dietary fatty acid levels seem of less importance to the growing larvae because of abundant levels of essential fatty acids resulting from the yolk reserves. |
