UNDP/FAO PROJECT THA/89/003
FISH NUTRITION AND AQUACULTURE DIETS

by

Ronald W. Hardy, Ph.D.
Northwest Fisheries Center
Seattle, Washington 98112 USA

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Table of Contents

TERMS OF REFERENCE

Fish Health Management Workshop Session Summary

High-Performance Liquid Chromatography (HPLC) Operation

Gas Chromatography (GC) Operation

Computer Formulation of Feeds

Evaluation of Equipment and Facilities at NIFI

General Nutrition Consultancy

Recommendations

Personnel contacted

Annex I: Fish Health Management and Nutrition in the Asia-Pacific Region

TERMS OF REFERENCE POST II-02:

Description of Duties:

1. To formulate and initiate a research programme to determine the optimum dietary needs of freshwater prawns and marine shrimp including the following:

   1. demonstration of vitamin, Lipid, and amino acid analysis techniques for feeds and tissue samples.

   2. provide assistance in the design of nutritional experiments for juvenile, grow-outs and broodstock fish and crustaceans.

   3. develop the capacity to use computers to formulate and balance feeds.

   4. initiate the compilation of a Thai feed ingredient data base for use in computerized feed formulation.

2. To provide in-service training of national staff on shrimp nutrition with special reference to vitamins.

3. To evaluate the available analytical laboratory equipment and make recommendations concerning their adequacy, installation, and operation.

4. To submit a technical report at the completion of consultancy.

The terms of reference were discussed with the Government Project Manager (Mr. Prasert Sitasit) on arrival and it was decided that most pressing needs were to provide in-service training for government scientists on the methods used to quantify the levels of amino acids and vitamin C in fish feeds and fish tissues. This was made more critical by the departure in August of Nanthiya Unprasert for Mississippi State University, where she will pursue a doctoral program in fish nutrition. Ms. Unprasert was the only government scientist in the Fish Nutrition Project at NIFI who had sufficient training to operate the HPLC equipment. Therefore the progress of research at NIFI was slowed by the lack of trained staff to conduct the required analytical determinations needed to complement the planned feeding trials. Discussions with the GPC and FAO Regional Aquaculture Officer (Mr. Imre Csavas) resulted in a decision to permit the consultant to attend a Fish Health Management Workshop, held in Pusan, ROK, under the sponsorship of the Asian Development Bank and the Network of Aquaculture Centers of Asia (NACA) as an international expert in fish nutrition and its role in fish health management. The consultant attended this meeting from October 7 until October 15. Upon returning to NIFI, work was begun to develop the required technical expertise among the government staff to utilize the HPLC equipment for analysis of amino acids and vitamin C. Demonstration of other procedures used for analysis of thiamin, thiamin pyrophosphate, riboflavin, niacin, pantothenic acid, pyridoxine and its biologically active forms, vitamin A, and vitamin E by HPLC was postponed until next year, at which time the staff will be familiar with HPLC operation. In addition, delays in obtaining the necessary reagents and HPLC supplies made it impossible to demonstrate procedures for determination of these vitamins and biologically active forms. The capability of formulating and balancing feeds by computer using feed ingredients available in Thailand was developed, and the government staff trained in the use of the computer software. A review of equipment and supplies needed for analyzing vitamins was completed, and a list of needs was developed. Short-term and long-term planning and prioritizing with regard to feeding trials designed to determine the nutritional requirements of important farmed fish and crustacean species for essential amino acids, dietary energy, and key essential vitamins was completed.

1. Fish Health Management Workshop Session Summary

1.1 A presentation was made detailing the ways in which fish nutrition interacts with fish health management in aquaculture at the Fish Health Management Workshop in Pusan, ROK, from October 6 through October 16, 1990. The presentation was the only one during the meeting to discuss fish nutrition and its relationship to fish health management, and was a needed component in the workshop. Personnel attending the workshop are listed in Annex I. A summary of the presentation is given below in paragraphs 1.2 – 1.10, with the full text of the presentation given in Annex II.

1.2 Fish nutrition, feed manufacturing, and feeding practices are important considerations in fish health management of farmed finfish and crustaceans. The shift in some countries of the Asia-Pacific region from extensive to semi-intensive and intensive farming of fish demands that nutritionally complete feeds be provides by the farmer. The use of nutritionally inadequate feeds can result in reduced growth and production, but more seriously, the use of such feeds can result in loss of fish from nutritional deficiency syndromes and/or from mortality brought on by increased susceptibility of nutritionally compromised fish to infectious diseases. Poor quality feeds can also contribute to fish losses by reducing rearing water quality, by containing adventitious toxicants, or by serving themselves as sources of infectious agents. Examples of fish losses due to all of these categories of nutritional problems have occurred at one time or another in various countries in the region. Overall, adequate nutrition and high quality feeds are critical elements of fish health management.

1.3 Nutritional deficiency syndromes can be caused by inadequate levels of essential amino acids, fatty acids, vitamins, or minerals, as well as by imbalanced feeds. However, the deficiency syndromes most likely to result in fish losses are associated with vitamin deficiencies. Vitamin supplementation of fish feeds is an essential practice when fish densities or management practices reduce the amount of natural food available to the fish. Vitamins are available in stable forms for use in fish feeds, but improper feed manufacturing and storage practices can result in vitamin losses in the feed before it is fed to the fish. For many species of farmed fish in the Asia-Pacific Region, there is no information available with which to decide what levels of vitamins are appropriate to add to feeds. Until that information is available, recommendations must be based on information obtained with other species of fish. There is little doubt, however, that all farmed fish require the same vitamins, with a few minor exceptions. The missing information concerns the appropriate levels of each vitamin required in feeds to maintain optimum nutritional status and fish health. Clinical methods have been developed in other regions to measure nutritional status of farmed fish for essential nutrients and these methods can be applied to the determination of the nutritional status of fish farmed in the Asia-Pacific Region. This approach will provide critical information in a relatively short period of time concerning appropriate levels of these nutrients in feeds to reduce losses of fish to nutritionally-related health problems.

1.4 Recommendations for the region were discussed by the country representatives and the regional resource persons. Regional needs identified during the discussion included the development of improved feeds for hatchery and grow-out phases of both freshwater and marine fish and crustaceans. Especially needed are economical pelleted feeds to replace trash fish diets. Another important need is the establishment of standards for feeding experiments conducted throughout the region that will permit comparison and rapid application of the results of different studies. Specific recommendations were as follows:

1.5 Establishment of a feed ingredient data base to collect information on the nutritional value of fish feed ingredients to important fish species in the region. Chemical evaluation should be combined with biological evaluation of ingredients to make the information useful to feed formulators and manufacturers.

1.6 Determination of the nutritional requirements of important farmed fish species. Initial emphasis should be placed on macronutrients, with later emphasis placed on micronutrients.

1.7 Development of feed quality assessment capability. This is needed to guarantee that feeds produced in the region meet the standards required for further development of the aquaculture industry.

1.8 Development of the capability of measuring the nutritional status of fish. This capability is needed for rapid estimation of the nutritional requirements of fish for essential vitamins, and to develop nutritional specifications for feeds that will support maximum nutritional status of fish, thereby eliminating subclinical vitamin deficiencies that contribute to fish losses from disease.

1.9 Training of personnel to accomplish recommendations 1–4.

1.10 Establishment of regional standards for feeding experiments, based on the EIFAC gudelines for fish nutrition studies.

2. High-Performance Liquid Chromatography (HPLC) Operation

2.1 Upon arrival and evaluation of needs in the fish nutrition program at NIFI, it was quickly apparent that with the departure of Ms. Nanthiya Unprasert in August, all capability to utilize the HPLC and GC for analysis of amino acids, vitamins, and fatty acids was lost. Although the remaining individuals had received some training on the equipment, that training was limited to theory of operation, preparation of fatty acid methyl esters, and a demonstration of analysis by Ms. Unprasert. The remaining individuals were unsure of even how to turn on the power and were fearful of damaging the expensive equipment. A program of training of Dr. Wimon, Mr. Pairat, and Ms. Amornrat was immediately implemented, emphasizing at first equipment start-up, operation, and maintenance. Common problems of operation and degassing of solvents was covered, as well as changing parameters for various analysis, and column maintenance and changing. After several weeks of practice, the confidence of the trainees was sufficient to proceed to actual analysis of standards and preparation of samples. Two additional trainees were sent from the National Institute of Coastal Aquaculture, Songkhla, to receive training on the use of HPLC to measure amino acid levels and vitamin C concentrations in fish feeds and tissue samples. The trainees, Mr. Mavit and Mr. Sawet, joined to training course and participated in all facets of HPLC operation.

2.2 Principals of sample preparation for amino acid analysis were reviewed, and the actual preparation of samples was broken down into discrete steps for the trainees, who were given training on one step at a time. Each trainee practiced hydrolyzing intact protein in samples to free amino acids, using casein, gelatin and spirulina as samples, prepared phenylisocyanate derivatives of hydrolyzed samples, injected the samples onto the HPLC, and resolved and identified the amino acids that were separated and identified on the resulting chromatograph.

2.3 Vitamin C analysis by the double C18 column method was demonstrated for the trainees, after the required reagents arrived. One of the required reagents had to be sent by Federal Express courier from the consultants laboratory in Seattle in order for vitamin C analysis to be conducted. Surprisingly, no-one had thought to check the stocks of required reagents prior to the beginning of the consultancy, and this oversight caused a significant delay in developing this assay for the trainees. Nevertheless, the trainees had sufficient practice with vitamin C analysis by HPLC to continue using this procedure on experimental samples, provided by Dr. Mali.

2.4 A second HPLC method of determining vitamin C levels in feeds was presented to the trainees. They were encouraged to order reagents needed for this method and compare results with those obtained using the double C-18 column method.

3. Gas Chromatography (GC) Operation

3.1 Although fatty acid analysis was not originally included in the terms of reference of the consultancy, it was an area needed badly by the fish nutrition program at NIFI. As was the case with the HPLC, the departure of Ms. Unprasert left no-one at NIFI capable of operating the GC to analyze fatty acid composition of fats and oils, feeds, and fish tissues. This need was acute in the area of larval fish diets. Fortunately, the trainees had received instruction earlier by Dr. Halver on how to extract lipid from samples and how to prepare methyl esters for analysis by GC. What was lacking was instruction on how to operate the GC. This training was provided concurrently with HPLC instruction, during periods in HPLC sample preparation when time was available.

3.2 Operation of the GC was demonstrated, and methods of program timing to satisfactorily separate fatty acid methyl esters found in fish tissues were reviewed. The trainees began by analyzing standards and moved quickly on to analysis of fats from dietary ingredients and from actual samples, including fish oil, vegetable oil, and samples provided by Ms. Amornrat. By the end of the consultancy, the trainees had become proficient in the analysis procedures to the extent that they will be able to continue these analyses without further assistance.

4. Computer Formulation of Feeds

4.1 A computer spreadsheet, designed for use with either Excel or Lotus software by Dr. Hardy, was brought to Thailand and made available to the fish nutrition program at NIFI. This spreadsheet enables the user to develop a feed formulation and obtain instantaneously the proximate composition, amino acid composition, mineral composition, energy content, and price of the feed. It can be used for practical feeds or for experimental feeds, including semi-purified diets.

4.2 The computer file was transferred to the computer in the fish nutrition office at NIFI and instruction was provided for the trainees on how to use it effectively.

4.3 Efforts were begun to modify the data files in the spreadsheet to reflect the proximate and nutrient composition, and price, of feed ingredients available in Thailand. Data generated in the fish nutrition program by Ms. Unprasert were used wherever possible. The use of this spreadsheet by the trainees will greatly improve the speed and accuracy with which they will be able to develop appropriate feed formulations for farmed fish in Thailand and for experimental purposes at NIFI.

5. Evaluation of Equipment and Facilities at NIFI

5.1 The analytical laboratory facilities in the fish nutrition section at NIFI are close to being suitable for the goals and objectives of the agency under project THA/89/003. Delivery and installation of a grinder for preparing samples for analysis and of a ultra-pure water filtration system for preparing water for HPLC analysis in early November further upgraded the capabilities of the analytical laboratory. The recommendations made by the previous consultant (Dr. Halver) in his consultancy report, dated August, 1990, were found to be accurate and no additional needs were identified in the analytical laboratory.

5.2 The wet laboratory facilities will require modification if they are to support appropriate feeding trials with certain freshwater fish species of interest to aquaculturists in Thailand. For example, a recent feeding trial with Pangasius catfish to estimate the dietary vitamin C requirement was a complete failure because the fish wre reared in circular, conical-bottom tanks. This fish will not eat feed in the water column, but only eats feed off the bottom. Thus, the fish did not have access to feed, and growth was negligible during the 8 week feeding trail. Further work with this important fish will require different rearing containers which are flat bottomed. These containers are available at NIFI but require slight modification. Another feeding trial designed to evaluate the protein requirement of Pangasius was marginally successful, although the growth rates of the fish were very low. This situation was caused in part by lack of cover for the flat-bottomed tanks placed on the floor, which caused the fish to become nervous and not feed whenever anyone walked by. Further significant losses of fish from tanks between weekly weighing days occurred, probably the result of predation by cats during the night. While these problems are relatively simple to correct, until they are corrected, the ability of the investigators to conduct meaningful feeding trials will be limited.

5.3 Hand feeding nervous fish, such as Pangasius, is not a desirable practice, since the human activity associated with hand feeding excites the fish and stops feed consumption. Mechanical feeders are recommended for feeding trials with this and similar fish. Mechanical feeders are relatively inexpensive, and their use insures adequate feeding without associated human activity. The use of mechanical feeders will also eliminate the problem of variable feeding practices by the feeding staff.

6. General Nutrition Consultancy

6.1 Several local fish feed manufacturing plants were visited on the weekend.

6.2 Discussions were held with the technical staff of Charoen Pokphand Feedmill Co., Ltd. (CP) during one of the visits during the weekend concerning fish feed ingredient quality assurance and new analytical methods used to measure nutrient levels in feeds.

6.3 A seminar was given to the professional staff at NIFI on the subject of “The Role of Nutrition in Fish Health Management” on November 19, 1990.

6.4 A presentation was given to the aquaculture industry in Thailand on November 23 at The Regent of Bangkok hotel. The occasion was the “International Symposium on Recent Developments in Aquaculture Nutrition”. The presentation was entitled “Considerations in Fish Feed Manufacturing”.

7. Recommendations

7.1 Coordinate ordering of chemicals and supplies necessary for HPLC analysis of vitamins and amino acids so that the supplies are in place before the arrival of the consultant.

7.2 Repeat feeding trials to determine the dietary vitamin C requirement of Pangasius. Make provisions to conduct the feeding trials in containers suitable for a fish that feeds off the bottom of the tank. Make provisions to shelter the fish from human activity and to protect the fish from cats and other predators. Modify flat-bottom containers to allow low levels of flow-through water to provide current and to maintain water quality without changing water every morning and frightening the fish.

7.3 Design and conduct feeding trial to determine the dietary energy requirement of Pangasius. before proceeding with feeding trials to determine the dietary protein requirement. As an alternative, design and conduct a feeding trial in a factorial design, with two levels of dietary energy and four levels of dietary protein. Replicate each dietary treatment three times, for a total of 24 tanks. Conduct analysis of Variance for the Factorial Design on the resulting data, and further test for interactive effects between dietary variables (Protein and energy levels).

7.4 Begin to accumulate data on the nutritional composition of Thai feed ingredients, using the analytical laboratory to conduct the analysis. Use the resulting data to replace existing values in the computer data base of feed ingredients.

7.5 Purchase a sufficient number of mechanical feeders to conduct feeding trials with appropriate numbers of replicate tanks.

7.6 Hire a technician to be responsible for the day to day operation of the analytical equipment, under the supervision of Dr. Wimon.

7.7 Order sufficient HPLC column to provide back-up in the event the current columns fail (which is likely after several months of use). Order at least two C₁₈ columns (30 cm long) and one CN-column for future vitamin analysis needs (Thiamin).

7.8 Purchase a fax machine for use in the project. At present, the trainees have no way to communicate with outside experts, other than by mail. Continued development of analytical facilities and methods will eventually result in technical problems, and having a fax will allow the trainees to quickly contact outside experts, such as Dr. Halver or Dr. Hardy, to assist in quickly resolving their technical problems. This rapid response capability will prevent loss of momentum in the project, should a problem arise.

7.9 Purchase a heating block for hydrolysis of proteins prepared for amino acid analysis by HPLC.

7.10 Purchase a polytron (tissue homogenizer) for preparing samples for vitamin analysis.

7.11 Eventually, the project will need increased access to a refrigerated centrifuge capable of spinning homogenized tissue samples at speeds greater than 10,000 rpm. At present, the project uses a centrifuge in a laboratory of another division. If access to this centrifuge is restricted, vitamin analysis will be also restricted. Consideration should be given to reallocating funds to permit the purchase of an appropriate centrifuge for the project.

Personnel Contacted

Annex I
Fish Health Management and Nutrition in the Asia-Pacific Region

Ronald W. Hardy, Ph. D.
Northwest Fisheries Center
2725 Montlake Blvd. East
Seattle, WA. 98112 USA

October 8–15, 1990
Pusan, ROK

Farmed fish must be provided with all of the essential nutrients from one source or another to maintain health and productivity (the term fish refers to crustaceans as well as finfish). The way in which the essential nutrients are provided differs, depending upon whether the farming is extensive, semi-intensive, or intensive. Feeds used in intensive farming must provide all of the essential nutrients required by the fish to grow and maintain good health, while feeds used in semi-intensive farming need only supply a portion of the nutritional needs of the fish, with the remaining nutrients being provided by the natural food available in the pond. In extensive farming, no feed is provided to the fish directly, rather the pond is fertilized and the fish rely on natural food. The amount of natural food available in ponds can be quite variable, depending upon pond productivity and fish stocking density. Therefore, the proportion of nutrients contributed by natural food to the total nutritional needs of the fish in semi-intensive farming can also be quite variable. This variability brings with it a degree of uncertainty that is difficult for the feed manufacturer to anticipate when formulating feeds for semi-intensive farming, namely that a particular feed formulation will not be appropriate for use in all farms. This fact makes semi-intensive farming the most risky category of aquaculture, from a nutritional point of view. Increases in the amount of prepared feed needed by aquaculture in the future, particularly in the Asia-Pacific Region where semi-intensive farming is widely practiced, will require effort on the part of researchers to provide information on the nutritional requirements of the major farmed species which will be needed to avoid losses of fish due to nutritional deficiency and increased susceptibility to infectious diseases. This will become even more important in the future as productivity in aquaculture increases and as aquaculture shifts from extensive to semiintensive and intensive farming.

Several kinds of information are required to properly formulate fish feeds. First, there must be information on the availability, price, and nutritional value of the ingredients used to manufacture the feed. Second, there must be information on the appropriate minimum and maximum levels of each ingredient for the species of fish being fed or the type of pellet being produced. Third, there must be information on the nutritional requirements of the fish being fed so that the feed will contain the desired levels of essential nutrients. Using the information listed above the best combination of ingredients must be found that will result in a feed that has the correct physical and nutritional characteristics at a cost that is realistic. Other considerations, such as the goals of production, must also be considered in order for appropriate feeds for a specific application to be formulated and manufactured. In the Asia-Pacific Region, there is insufficient information concerning the nutritional value of ingredients for the species of fish being raised, and insufficient information concerning the nutritional requirements of many species of fish to permit scientifically-formulated feeds to be produced. In many cases, feed manufacturers are estimating the ingredient nutritional values and fish nutritional requirements, using information developed for other species of fish studied in other regions, or they are relying on feed formulations that have been derived empirically and have been shown to work reasonably well. This is a sensible practice, but it should not be mistaken for how things ought to be done. Until the needed information is obtained, fish farmers using prepared feeds will be at risk and production levels and returns to the farmer will not be as high as they could be if feeds were based on scientifically-derived information.

There are approximately 40 essential nutrients required by all fish. These include dietary protein, which contains essential and nonessential amino acids, essential fatty acids, dietary energy, vitamins, certain minerals, and, in the case of crustaceans, a source of dietary sterols. The amount of each of these nutrients that must be in the diet to maintain health and desirable growth rates depends on the species of fish and whether the fish is a carnivore, omnivore, or herbivore, and whether the farming is semiintensive or intensive. However, regardless of the species, fish in the fry stage are carnivorous, and thus require diets high in protein and energy. As fish reach juvenile and post-juvenile stages, their dietary protein requirement decreases (Wilson, 1989). The degree to which the dietary protein decreases depends upon whether the fish is a carnivore, omnivore, or herbivore. Fish do not actually require dietary protein per se, but rather, they require the essential amino acids found in protein (Wilson, 1989). Fish require a dietary source of ten amino acids (Table 1), because they cannot synthesize these amino acids in the body. Fish require certain fatty acids in the diet to prevent fatty acid deficiency signs, although the exact kinds of fatty acids seems to differ with the species of fish, and whether the fish is a freshwater or marine species (Watanabe. 1982). A source of dietary energy is required by fish, and fish can derive energy from protein, fat and carbohydrates. However, using protein as a source of energy is an expensive practice, and in a well balanced feed, most of the energy comes from fat and carbohydrates. Fish require 12–15 vitamins (depending upon the species) in the diet in relatively small amounts (Halver, 1989). Each of these vitamins has specific functions in the fish. Some vitamins act an essential cofactors for specific metabolic processes, while others have structural roles in cell membranes (Table 2). Elimination of these vitamins from the diet results in vitamin deficiency signs in the fish. It is standard practice to supplement the diets of intensively grown fish with vitamins, and the levels of supplementation are designed to completely supply the vitamin needs of the fish without considering the contribution of vitamins from the other ingredients in the feed (NRC, 1983). Vitamins are by nature relatively unstable compounds, but the chemical forms of most vitamins that are used to supplement feeds are quite stable under most conditions (Table 3). The principle exception is ascorbic acid, or vitamin C. which is very unstable in feeds due to its function as a sacrificial antioxidant. Crystalline ascorbic acid is rapidly lost in feeds during pelleting and storage, particularly during storage in tropical climates. There are now a number of forms of ascorbic acid which have been developed for use in feeds and are protected against oxidation (Table 4). Fish also require certain minerals in the diet (Table 5), although the amount of these minerals that must be present in the feed depends upon their concentration in rearing water, because fish can absorb some minerals directly from the rearing water (Lall, 1989). The metabolic functions of dietary minerals in fish generally correspond to their functions in birds and mammals.

NUTRITIONAL DEFICIENCY SIGNS

Nutritional deficiency signs are the visible signs of dietary nutrient deficiency, and are useful in the diagnosis of feed problems. However, the absence of visible signs of nutrient deficiencies does not necessarily mean that a fish is in optimal nutritional health. Increasing dietary levels of all essential nutrients result in an increasing physiological response, up to the point at which the dietary requirement has been met (Figure 1). Beyond a certain dietary level of a nutrient, a concomitant increase in physiological response is not seen. This is the dietary level, that supports optimal nutritional status. At some dietary level below the requirement, visible deficiency signs appear in the fish. The range of dietary intake between that resulting in visible signs of deficiency and that resulting in no concomitant increase in physiological function is an area referred to as sub-clinical deficiency. Fish receiving this level of an essential nutrient do not show signs of deficiency, yet they may be nutritionally compromised and less able to resist infectious disease or water quality problems.

Visible, or clinical, signs are specific for several vitamin deficiencies, and can be categorized as those affecting eyes, gill tissue, blood, kidney, or cartilaginous structures (Halver, 1989). For most vitamins, however, the visible signs of dietary deficiency are non-specific, with anorexia (loss of appetite) being the most obvious and common sign. It is important to realize that by the time signs of nutrient deficiency are visible, fish have usually stopped eating, and feeding fortified diets will not completely reverse the deficiency condition in the fish population and prevent fish losses.

In the Asia-Pacific Region, it is not uncommon for fish to exhibit signs of vitamin deficiency. The most common vitamin deficiency encountered is ascorbic acid deficiency, followed by thiamin deficiency, and perhaps, pantothenic acid deficiency. Ascorbic acid deficiency occurs due to its unstability in feeds, particularly in feeds containing oxidizing fish oils, and in feeds stored in hot, humid conditions. It is common practice in many areas to add ascorcbic acid to the pellets immediately before feeding to insure adequate ascorbic acid intake by the fish. The development of protected forms of ascorbic acid ought to reduce the likelyhood of the development of ascorbic acid defiency in farms in the future. Thiamin is generally a stable vitamin in fish feeds, except when the feeds contain fresh fish. Many fish contain an enzyme called thiaminase, which hydrolyzes thiamin, and the use of these fish in moist feeds and trash fish diets can result in the development of thiamin deficiency. The species of fish that contain thiaminase are listed in the NRC Bulletin (NRC, 1983). Development of other vitamin deficiency conditions in practical feeds in the Asia-Pacific Region is most likely the result of improper feed manufacturing and storage, and is usually avoidable by following proper diet preparation and handling procedures (Hardy, 1989).

NUTRITION AND INFECTIOUS DISEASE

Feeding trash fish in place of pellets is a common practice in many parts of the Asia-Pacific Region. While this practice is economical, it is not without cost to the fish's environment. Water quality deterioration due to feeding raw fish is known to be stressful to fish and thereby lowers their susceptibility to infectious disease. In terms of economy, farms are often more productive when pelleted feeds replace trash fish diets, and the farmer actually sees a higher return despite the increased cost of the pellets.

Malnutrition is known to cause profound changes in the immune response of all vertebrates (Landolt, 1989). Defiencies of protein, energy, vitamins, and certain minerals have all been shown to reduce the ability of animals to resist disease. Fish have several defense against disease, which can be categorized as non-specific immune response, which includes phagocytosis, complement, and non-specific cytotoxic cells, and as specific immune response, which includes antibody production, B-cell response, and T-cell response. One of the first reports demonstrating a relationship between the nutritional status of fish and resistance to disease was by Hardy et al. (1979), who showed that mortality of chinook salmon (Oncorhynchus tshawytscha) experimentally infected with Vibrio anguillarum was higher in fish fed diets containing low levels of the vitamin pyridoxine than in fish fed diets containing high levels of pyridoxine. Fish fed the diets low in pyridoxine did not exhibit clinical signs of deficiency, but were subclinically deficiency, as judged from the results of serum alanine amino transferase activity. Fish fed high protein diets required higher dietary levels of pyridoxine to resist disease compared to fish fed low protein diets. A relationship between dietary ascorbic acid level and disease resistance has been demonstrated in catfish (Li and Lovell, 1985) and rainbow trout challenged with bacteria (Navarre and Halver, 1989) and a virus (Anggawati-Satyabudhy et al, 1989). Dietary levels approximately 10 times higher than those required to prevent deficiency signs were required to provide maximum disease resistance. What these and other studies show is that fish suffering from subclinical deficiencies, which cannot be detected by observing the fish, are more likely to be lost from disease than are those in optimal nutritional condition. Landolt (1989) has recently reviewed the subject of the relationship between nutrition and disease resistance in fish.

MEASURING NUTRITIONAL STATUS IN FISH

Nutritional status assessment in fish is an area receiving increasing attention from researchers. particularly in connection with subclinical deficiency states. Early researchers in fish nutrition relied on growth rates, feed conversion ratios, and the absence of deficiency signs to indicate adequate intake of an essential nutrient. Adaptation of clinical tests used in human medicine and measuring liver storage levels of vitamins by microbiological assay provided the first measurements of subclinical vitamin status in fish.

Measuring nutritional status is the only way to insure that farmed fish receive the correct dietary nutrient levels, and are therefore prepared to resist disease. Defining optimal nutritional status is a subject of debate among nutritionists, but generally it refers to a condition at which additional dietary levels of a nutrient do not result any change in the physiological status of the fish. Current approaches to determining nutritional status rely on relatively new developments in human clinical medicine, and utilize advanced equipment and methods. The three general measurements that are used to determine nutritional status in fish are by measuring the activity of specific enzymes that are dependant upon a specific vitamin as a cofactor, to measure directly with HPLC or RIA the actual tissue levels of vitamins or their active forms, or, in the case of minerals, to measure the level of the mineral in indicator tissues or in the whole body of the fish. For same nutrients, there are other clinical measures of nutritional status, such as serum lavels, erthrocyte fragility, iron-binding capacity of the serum, and so on. Measuring nutritional status of fish is a rapidly advancing field, and undoubtably there will be new, sensitive, and relatively simple methods developed in the future.

OTHER NUTRITIONALLY-RELATED HEALTH MANAGEMENT PROBLEMS

Feeds used in aquaculture can contribute to fish losses from disease in several other ways. First, the use of unpasteurized trash fish or fish offal as a feed for fish presents the possibility of transmitting disease to the fish directly via the feed. There are many examples of this happening in aquaculture throughout the world, most notably in North America during the 1950's in Pacific salmon enhancement hatcheries (Wood, 1979). The problem was eliminated by requiring that all fish products used in fish feeds must go through a heating step to destroy any fish pathogen that might be present.

A second potential nutritionally-related fish health management problem is connected with the use of ingredients which are contaminated with microorganisms. This is primarily a problem with fish meals and other animal byproduct meals, which are known to be frequently contaminated with Salmonella. Avoiding this problem involves the establishment of standards for fish feed ingredient quality which include maximum acceptable levels of microbial contamination.

A third potential nutritionally-related fish health management problem concerns the possibility of having adventitious toxicants, such as aflatoxin, present in a fish feed. At even low levels in the feed, aflatoxin can cause liver tumors and eventually death of the fish (Hendricks and Bailey, 1989). These toxicants generally are produced in feed ingredients before they are used to make fish feeds, so the establishment of standards for feed ingredients used in aquaculture must include examination of suspected feed ingredients for aflatoxin. In addition, feed must be manufactured and stored in such a way as to prevent mold formation during storage. In feeds susceptible to mold formation during storage, the addition of mold inhibitors is a recommended practice. There are many mold inhibitors approved for use in feeds (Hardy, 1989), and the choice of a mold inhibitor-for feed use must be based on the particular conditions required for the inhibitor to be most effective, and on the potential effects of addition of the mold inhibitor on the palatability of the feed to fish. Moldy feed should never be fed to fish.

A fourth potential nutritionally-related fish health management problem concerns the use of feed ingredients that may contain antinutritional factors. Most antinutritional factors are found in feed ingredients of plant origin. The most common antinutritional factors in feed ingredients are trypsin inhibitors, found in soybean meal. Many other feed ingredients of plant origin also contain antinutritional factors (Roberts and Bullock, 1989). While most of them do not directly cause fish losses, they may contribute to fish losses by reducing the health of the fish, making them more susceptible to disease. The solution to this problem is to establish feed ingredient standards that specify acceptable levels of these antinutritional factors, and to test the effects of various dietary levels of antinutritional factors on the important farmed species in the Asia-Pacific Region. At present, there is no information available in the Region upon which to base such standards.

A final potential nutritionally-related fish health management problem concerns the use in feeds of fats and oils that are undergoing oxidation. This is a particular danger when feeds contain highly unsaturated fish oils that have not been stabilized with antioxidants. It is well known that oxidizing fish oils reduce the vitamin E status of the fish (Hung et al. 1981), thereby increasing the likelyhood of causing clinical signs of vitamin E deficiency, and potentially affecting the immune response of the fish and increasing the chances of fish losses due to disease.

LARVAL FISH FEEDS

Many fish species of interest in aquaculture go through a larval stage where they require live food at first feeding. There have been many attempts to make artificial larval feeds, and some of these attempts have been quite successful, notably the feeds developed in Japan (Kanazawa et al, 1982). However, in practical farming, there are still large losses of fish occurring at the larval stages in hatcheries, and these losses are often directly connected with nutrition. Larval fish must ingest and digest feed particle, and the released nutrients must then be absorbed and utilized by the larval fish. Most research on larval feeds has been focussed on the first part of this process, namely feed ingestion. Only now are researchers beginning to look at digestion, absorption, and utilization. Many species of larval fish do not possess a fully functional digestive system, and cannot utilize ingested feed particles because they cannot break them down into usable components. It has been suggested that live food is needed because the prey themselves contain digestive enzymes, and it is these enzymes that digest the prey in the gut of the larvae. Another suggested reason for the general superiority of live food is that the prey often contain relatively high levels of free amino acids that are easily absorbed by the larvae. Research now being conducted in many laboratories to characterize the development of the digestive system of larval fish ought to result in improvements in larval feeds in the next few years, thereby reducing the losses of fish that occur at this critical stage.

SUMMARY

There are various ways that fish nutrition interacts with fish health management, and it is clear that optimum nutrition is needed to reduce losses of fish from both nutrition deficiency and from infectious diseases. It is also clear that further development of aquaculture in the Asia-Pacific Region will require the development of high quality feeds, and that the production of high quality feeds is only possible through the use of high quality feed ingredients. Knowledge of the nutritional requirements of the main fish species of interest in aquaculture is a necessity to produce appropriate feeds for the Region.

RECOMMENDATIONS

1. Feed Ingredients. A data base of feed ingredients available in the Asia-Pacific Region must be obtained. Information obtained by chemical evaluation, measuring such things as nutrient composition, the presence of toxicants, and variability among supplies must be coupled with biological evaluation, including digestibility, to enable fish feed manufacturers to formulate and produce high quality feeds.

2. Nutritional Requirements of Major Species of Fish

Efforts must be started to determine the quantitative requirements of various species of fish important to aquaculture for various essential nutrients. Efforts should concentrate first on protein and energy requirements of these fish, followed by evaluation of vitamin requirements.

3. Feed Quality. Efforts must be started to develop standards for feed quality. This should include both development of pelleted feeds for species of fish are not now being fed pellets, and improvement in existing feeds to insure nutritional quality and to reduce water quality problems. Regional capability to chemically evaluate commercial feeds must be developed.

4. Nutritional Status Determination. Efforts should be made in the Region to establish the capability of measuring the status of fish for many essential nutrients. This would allow farmers and diagnosticians to have fish tested for the presence of sub-clinical nutrient deficiencies that may be contribute to losses from disease. It would also allow the industry to judge the efficacy of micro-nutrient fortification of feeds, and to stop unnecessary and costly supplementation that is above levels needed to support optimal nutritional status of fish.

5. Training of Personnel to accomplish the items listed above. Training of personnel at key institutions must be continued to insure that the first four recommendations can be accomplished. The benefits to the aquaculture industry in the region will greatly outweigh the cost of such training.

6. Guidelines for Nutrition Studies. Guidelines for the execution of fish nutrition studies in the Region must be developed to increase the rate of progress in nutrition research. Often it is difficult to evaluate the results of feeding studies carried out in the Region because of inadequate replication of experimental treatments, incomplete reporting of the conditions of the experiment, or other reasons. Guidelines based in those of EIFAC (Castell and Tiews, 1978) could be easily adapted and made available to the various investigators in the Region.

LITERATURE CITED

Anggawati-Satyabudhy, A. M., Grant, B. F., and Halver, J. E. 1989. Effects of L-ascorby1 phosphates (AsPP) on growth and immunoresistance of rainbow trout (Oncorynchus mykiss) to infectious hematopoietic necrosis (IHN) virus. Proc. Third Int. Symp. on Feeding and Nutr. in Fish. Toba, Japan, Aug. 28-Sept. 1, pp. 411–426.

Castell, J. D. and Tiews, K. 1980. EIFAC Tech. Paper (36).

Halver, J. E. 1989. The Vitamins. pp 31–109 In Fish Nutrition, Second Edition, J. E. Halver (ed). Academic Press, New York.

Hardy, R. W., Halver, J. E., and Brannon, E. L. 1979. Effect of dietary protein level on the pyridoxine requirement and disease resistance of chinook salmon. In: J. E. Halver and K. Tiews (eds), Finfish Nutrition and Fishfeed Technology, Vol. 1. Heenemann, Berlin, pp. 253–260.

Hardy, R. W. 1989. Diet Preparation. pp 475–548 In Fish Nutrition, Second Edition, J. E. Halver (ed). Academic Press, New York.

Hung, S.S.O., Cho, C.Y., and Slinger, S.L. 1981. Effect of oxidized fish oil, DL-alpha-tocopherol acetate and ethoxyquin supplementation on the Vitamin E nutrition of rainbow trout (Salmo gairdneri) fed practical diets. J. Nutr., 111:648–657.

Kanazawa, A., Teshima, S., Sosada, H., and Rahman, S. A. 1982. Bull. Jpn. Soc. Sci. Fish., 48:195–199.

Lall, S. P. 1989. The Minerals. pp 219–257 In Fish Nutrition, Second Edition, J. E. Halver (ed). Academic Press, New York.

Landolt, M. L. 1989. The relationship between diet and the immune response of fish. Aquaculture, 79:193–206.

Li, Y. and Lovell, R. T. 1985. Elevated levels of dietary ascorbic acid increases immune response in channel catfish. J. Nutr., 115:123–131.

National Research Council (NRC). 1983. Nutrient requirements of Warmwater fishes and shellfishes. National Academy of Sciences, Washington, DC, 102 pp.

Navarre, Q., and Halver, J. E. 1989. Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C. Aquaculture, 79:207–221.

Roberts, R. J. and Bullock, A. M. 1989. Nutritional Pathology. pp 423–473 In Fish Nutrition, Second Edition, J. E. Halver (ed). Academic Press, New York.

Watanabe, T. 1982. Lipid nutrition in fish. Comp. Biochem. Physiol., 738:3–15.

Wilson, R. P. 1989. Amino Acids and Proteins. pp 111–151 In Fish Nutrition. Second Edition, J. E. Halver (ed). Academic Press, New York.

Wood, J. W. 1979. Diseases of Pacific salmon - their prevention and treatment, 3rd Ed., State of Washington, Department of Fisheries, Olympia, Washington, 82 pp.

Table 1. Classification of Amino Acids for fish.

Table 2. Essential Vitamins for Fish.

Table 3. Chemical Forms and Stability of Vitamins.

1 Stability of some vitamins is reduced in the presence of Choline chloride. Choline is not mixed with the vitamin premix, but rather added separately at pelleting.

Table 4. Forms of Ascorbic Acid used in fish feeds.

Table 5. Minerals required by fish¹.

1 May be supplied by the rearing water. Absolute dietary requirement may depend on the levels of other minerals or ingredient used in the feed.