By Partha Bandyopadhyay
Investigations to provide a better understanding of the absorption of amino acids and protein in two species of carps and catfish that will help to develop the right formulation and feeding strategy.
Dietary protein is considered to be of primary importance in fish feeds. Its requirements in fish are higher than those of terrestrial animals. Protein is the basic building nutrient of any growing animal and muscle constituents and it is the major component of the fish body (68—85%dry wt). The degradation products of protein are absorbed from the intestinal content as amino acids or peptides.
Individual amino acids are readily absorbed against concentration gradients and active transport is driven by co-transport with H+ or Na+. These inorganic ions transport according to their electrochemical
To some extent, protein and peptides in the intestinal content are probably also taken up, without previous degradation, by pinocytosis or related processes. A satisfactory level and balance of amino acids in a diet do not guarantee that ingestion of the diet will satisfy the amino acid requirements of the fish. This could be made only by proper digestion and most important is the absorption from the intestinal content.
The use of cheaper but nutritionally balanced artificial diets is becoming common in intensive and semi-intensive culture systems. The following investigation on the intestinal absorption and incorporation
of protein may help to explain to fish nutritionists the difference in
protein utilization between ingredients and pave the way for the use of different protein sources.
Selection of fish
Two fish species were selected for the present investigation The Indian major carp Labeo Rohita is herbivorous and does not have a stomach. The other fish was the air—breathing and carnivorous catfish Clarias Batrachus with a stomach. In India, both are important culture species, although the latter has only emerged recently.
Fry and fingerlings were obtained from a local ﬁsh farm, After acclimatization, these were transferred to a large aquarium, sectioned into four compartments with perforated plastic. There were four groups
each consisting of 10 fish. The fish were maintained with Halver‘s synthetic diet or sometimes with live food i.e.zooplankton.
Absorption of protein
This was analyzed by the everted and non-everted technique. Fish were stewed for 24 hours to empty intestinal (agastric ﬁsh) residues. Thread ligatures were placed at the esophageal (upper and lower),
stomach, duodenal, anterior intestinal and posterior intestinal junctions in anesthetized fish. Thorough and clean washing is essential for a correct estimation of absorption, because the presence of any food
particle may give an erroneous result. Two small incisions were made at the opposite ends of each ligatured section in selected parts of the intestine such a (oesophagus, stomach, duodenum, and ileum etc). These were then thoroughly washed with Krebs—Ringer bicarbonate solution at 30'0.
One ml Kreb—Ringer bicarbonate solution in 100mg of fish saline/per
ug of protein at 30°C was injected into each of the above segment. The vicera and the ligatured segments were kept moist with Krebs—Ringer bicarbonate/fish saline solution at 30°C in an incubator. Absorption was allowed for 30, 60 and 90 minutes, following which the segments were removed from the rest of the gut.
The post absorption fluid from the different segments were collected in vials and wet weight recorded An aliquot of the fluid was taken for spectrophotometric determination of protein and amino acid and was expressed as g protein/ amino acid absorbed hour-1cm-1. This experiment was repeated six times.
The site of absorption of protein in the intestine was observed using Scanning Electron Microscopy (SEM) techniques. Tissues were fixed in primary fixative gluteraldehyde for 15 minutes, kept for 24 hours and then washed by cacodylate buffer with three changes at 15 minutes intervals The tissues were fixed in 1% Osmeum tetroxide tor2 hours. The tissue was then dehydrated with graded ethanol.
Tissues were kept in acetone to a critical point of drying (CPD) These were then coated with gold — palladium malloy (160 thickness) for 5 to 15 minutes in three consecutive series on a sputter coater (Polaron Equipment Ltd, SEM coating unit E 5000) for uniform coating to enhance material density and electrical conductivity. This was necessary for emission of secondary electrons for fine and perfect image. Sampleswere then observed under a Scanning Electron Microscope Philips SEM 515 at varying magnifications. Selected areas were photographed.
Absorption of amino acid
The results showed that the absorption of amino acid was always higher in the posterior intestine of both the carp and catfish. There are no specific differences in the rate of absorption of non-polar, polar, basic or other amino acids in the intestinal serosal layer of both fish. The SEM of different intestinal segments (serosa) of the carp [Figures 1&2 ) and catfish (Figure 3 & 4) revealed that the amino acid could be absorbed in all the intestinal segments starting just below the stomach to the rectum of the catfish (Figure 6). However, amino acid cannot be absorbed in the last part of the posterior intestine to rectum in the case of the carp.
In both fish, satiation time was about 60 minutes after feeding and this may stretch to about 90 minutes for the catfish. However, both fish may continue feeding up to 2 hours, depending upon the physicochemical parameters of water and climatic condition such as temperature.
The feed intake was maximum at 10 am to 12 noon and minimum at midnight for the carp. It was maximum at 6 pm and minimum at 12 noon for the catfish.
Amino acid assimilation
Figure 5 & 6 showed that the maximum amino acid assimilation took place at 10 hours for the carp and 18 hours for the catfish. Maximum incorporation takes place at 2 hours in the liver and 4 hours in muscle, in the case at both fish. However, there are no differences in assimilation rate either in the liver or in the muscle with formulated diets and synthetic diets in both fish.
Differences in protein utilization between diets may be due to the different in rates of digestion and absorption of the constituent amino acids. Optimal protein synthesis requires that all amino acids are present simultaneously and in adequate quantities within the tissues. It is generally accepted that amino acids in excess of requirement, are used for energy purposes or metabolized into fat or glycogen. However, excretion of amino acids cannot be excluded.
The middle intestinal segment had the ability to absorb macromolecules of protein by pinocytosis. A similar situation appeared to exist in both fish, which showed strong alkaline phosphatase activity in the anterior intestine. it was also observed that almost all essential amino acids were absorbed rapidly in the anterior 40 —50% portion of the intestine of tarps and catfishes. Macromolecules of protein such as ferritin was absorbed in the middle segment of the intestine by pinocytosis, i.e. engulfing of macromolecules of protein by the enterocytes.