NUTRITIONAL VALUE OF DECAPSULATED CYSTS OF ARTEMIA AND THEIR USE AS PROTEIN SOURCE IN EXPERIMENTAL MICRODIETS FOR FISH LARVAE
Ph.D. thesis by A. Garcia Ortega
Fish Culture and Fisheries Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands
ISBN: 90-5808-151-6, 147 pp.
Author's address: CIAD Unidad Mazatlan, Av. Sabalo Cerritos s/n, Estero del Yugo, Apdo. Postal 711, C.P. 82010, Mazatlan, Sinaloa, Mexico, e-mail: agarcia@victoria.ciad.mx)
Summary:
The general objectives of this thesis were to evaluate the nutritional value of Artemia as starter food for fish larvae and to assess the effects of heat treatment on the nutritional quality of food protein. Furthermore, to determine the importance of proteolytic enzymes on the larval digestion of food and to evaluate the quality of Artemia proteins and its use as protein source in microdiets for larval fish.
For the evaluation of the nutritional quality of Artemia, the biochemical composition and proteolytic enzyme activity of decapsulated cysts and nauplii were determined during the development (Chapter 1). Cysts contain embryos of Artemia in a dormant stage. After cyst hydration and incubation in salt water, the embryo restarted its interrupted metabolism, and after approximately 20 hours of incubation a free-swimming nauplii was hatched. Samples of cysts and nauplii were taken every five hours from the start of incubation until 25 hours. The individual protein and lipid contents during the development of Artemia from cyst to nauplii remained constant until the time of hatching, after which they decreased slightly. However, no
significant incubation effect was found. Small changes in amino acid and fatty acid composition were found during development, but it was assumed that they are too small to be of nutritional importance to the fish larvae. During the first 25 hours of development in Artemia, no major changes were detected in protease activities measured at acid and alkaline pH. Likewise, no significant changes in trypsin activity were observed. It was concluded that, from a point of view of exogenous enzyme contribution, there is no difference in feeding Artemia cysts or nauplii to fish larvae since no difference in qualitative protease composition was found during the first 25 hours of Artemia development.
The effect of heat on the protein quality and protease activity in decapsulated cysts of Artemia as food for fish larvae was evaluated in Chapter 2. Decapsulated cysts of Artemia were subjected to different heat treatments (40, 60, 80 and 96 C) and were fed to African catfish Clarias gariepinus larvae. Heat treatment of cysts increased protein denaturation and decreased protein solubility. The protease activity in the cyst diets decreased with higher heating temperatures. The growth of catfish larvae differed according to the diet, higher fish growth was achieved with nauplii and cysts heated at 40 C. The digestive enzyme activity in larval samples remained similar in all dietary treatments during larval development. The differences in fish growth among the cyst diets might be related to modifications in the structure of the protein during heating. Due to the lack of acid denaturation in stomachless fish larvae, a beneficial effect can be derived from the denaturation caused by heating cysts at 40 C. It was hypothezised that the quality of food protein and the way this protein is processed might be more important for successful larval growth than an exogenous enzyme supply. This idea was further tested in Chapter 4.
The quantification of the ingested food is necessary to determine the contribution of enzymes from dietary sources to the total digestion of food by fish larvae. In Chapter 3, a new method for the estimation of food consumption and gut evacuation in fish larvae was developed and evaluated. The new method consists of measuring the temporary accumulation of ascorbic acid 2-sulfate (AAS) in fish larvae after feeding decapsulated cysts of Artemia. Larvae of C. gariepinus were used to verify whether biosynthesis of AAS by the fish larvae did occur or not. Furthermore, the gut contents in larvae fed ad libitum were calculated as they changed during fish development and the gut evacuation rate was determined during continuous and discontinuous feeding regimes. The experimental period covered the first five days after the start of exogenous feeding in the larvae. The daily food consumption in catfish larvae increased from 46.5% of their body dry weight (BDW) at day 1 to 53.8% (BDW) at day 3. Thereafter, the food consumption decreased to 27.8% (BDW) at day 5. A similar pattern was observed for the gut evacuation rate. The reported method for the determination of food consumption enabled an accurate and precise estimation of the gut contents and the evacuation rate of Artemia cysts in fish larvae.
The post-prandial activity of alkaline protease in the gut of African catfish larvae was studied in Chapter 4. Different feeding regimes were studied, and the protease activity was monitored at different time ranges from 30 minutes to 4 hours after ingestion of decapsulated cysts of Artemia. In starved larvae the enzymatic activity was low and did not change in time. No significant endogenous secretion of digestive enzymes was detected. The level of alkaline proteolytic activity found in starved larvae was considered as the basal level. In larvae fed only one meal during the day, the enzyme activity significantly increased from 3 hours postfeeding up to a maximum level found 12 hours after feeding. In fish larvae receiving a meal every 4 hours, the total protease activity changed according to the feeding time and fluctuated around a constant level, which was intermediate between the maximum and the basal level. No rhythmic cycle of enzyme production in the fish was observed when the proteolytic activity was studied during a cycle of 24 hours. When specifically trypsin activity was measured, a similar pattern was found as with total protease. Together with the results on food consumption (Chapter 3) and the protease activities found in decapsulated cyst of Artemia, the contribution of digestive enzymes from Artemia to the total digestion of food by the catfish larvae was calculated. This was estimated to be less than 1% of the total amount of the proteolytic activity measured in the larval gut of the catfish.
To assess the importance of the protein structure as a factor to explain the suitability of live food as starter diet for fish larvae, in Chapters 5 and 6 the protein quality in Artemia and fishmeal in microdiets for fish larvae was evaluated by different techniques. In Chapter 5 a method was developed for the determination of the in vitro digestibility of protein of Artemia cysts and nauplii and in microbound diets made with decapsulated cysts and various binders. This method proved useful as an alternative to feeding experiments with live fish and yielded accurate estimations of the protein digestibility in the diets. A microdiet consisting of Artemia decapsulated cysts and carboxymethyl-cellulose as binder gave the best results.
This combination was further tested in a microbound diet and used in a feeding experiment with catfish larvae as described in Chapter 6. It has been suggested that the supply of free amino acids and low molecular weight peptides in starter diets would facilitate their intestinal absorption by fish larvae. This hypothesis was tested in the present thesis. The quality of different protein sources in microbound diets (MBDs) for fish larvae was evaluated, and the differences in diet performance were related to the protein structure. To obtain a more complete picture of the protein quality in fish feeds, studies on the diet amino acid composition, the in vitro protein digestibility, the diet solubility and protein structure were combined with an in vivo feeding trial. The protein quality of MBDs made with decapsulated cysts of Artemia and/or fishmeal as protein source was used as indicator of their quality as starter food for fish larvae. The growth of catfish larvae was higher when the Artemia-based MBDs were fed than when fishmeal-based MBDs were given despite the higher protein and total amino acid content of the latter. The in vitro protein digestibility was high for all the MBDs in comparison to a commercial diet. Differences were found in the molecular weight of the protein among the diets. Most of the proteins in the fishmeal-based diets had low molecular weight in the range between 7.4 and 49.2 kDa. The Artemia-based MBDs had bigger protein fractions between 29.4 and 82 kDa. Decapsulated cysts improved the utilization of the MBDs when used in combination with fishmeal. The explanation for the positive effect of Artemia has yet to be elucidated. However, it is recommended to investigate the interactions between nutrients (e.g. protein-lipid) in live food, because they might have an effect on the functional properties of food proteins.
The results of this thesis confirmed that for the larvae of African catfish C. gariepinus, the supply of exogenous enzymes through Artemia cysts has a small contribution to the total proteolytic activity measured in the gut after feeding. Moreover, it was confirmed that the food ingestion and evacuation in catfish larvae is not constant as previously reported. Our results confirm the high digestibility of Artemia decapsulated cysts and nauplii as food for fish larvae. Similarly, the use of decapsulated cysts as protein source in microdiets improved their performance as starter diet for fish larvae.
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