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AQUACULTURE FEED MICROSCOPY by Lynn S. Bates, PhD. INTRODUCTION Aquaculture, for marine and brackish water species as well as for freshwater species, is expanding rapidly throughout the world. Some of the driving force for this expansion is simply the need for additional food resources. However, in other cases the demand is caused by the recognition of fish oils and other products as healthy substitutes for other traditional products. New potential aquatic species are being studied and cultured each year creating a need for specialized formula feeds and feed ingredients. New food products and processes, especially those from aquaculture, have created by-products that are well suited for aquatic feeds. Many of these products are high moisture and require specialized drying equipment or a carrier on which the product must be dried. The complex infrastructure supporting all these activities, particularly the formula feed industry, is constantly expanding and adjusting to meet demands. Feed microscopy is one of those support areas that is critical to maintaining quality aquaculture feed ingredients and formula feeds. Feed microscopy is source rather than nutrient oriented. Because many aquatic species require rations high in protein, a wide variety of animal and marine meals of very similar appearance are used. Unfortunately these similarities, because they cannot be detected with any routine protein, fat, fiber or other common tests in a laboratory, offer opportunities to adulterate both ingredients and formula feeds. Aquaculture producers and their feed suppliers need to monitor the quality of their ingredients and feeds constantly to assure they are buying the quality of products they are being sold. Similarly, knowledge about an ingredient source allows a feed manufacturer to take advantage of price differentials without risking the quality of their feed. Feed microscopy is the ideal technique to assure the quality of ingredients and formula feeds in a rapid expedient manner. A second factor encouraging contaminated and adulterated feeds is the inability visually to check the cultivated population. Water is generally turbid from high population densities of whatever species is being cultivated. Netting or trapping of the fish, shrimp, crab, etc. is necessary to weigh and follow feed performance. When low nutritional quality ingredients find their way into aquatic feeds, the effects are often not discovered for some time. Even with an extensive quality control program, the few chemical assays available for identifying lower quality materials are not always accurate. Feed microscopy offers a relatively rapid solution for such problem feeds as long as the analyst is familiar with the wide range of aquatic ingredients, their identification and common adulterants and contaminants. It is quite obvious that the rapid expansion of aquaculture has resulted in many profitable opportunities but with its share of difficulties. If we are to sustain a strong aquacultural industry with continued growth, we will need quality feed products, better basic knowledge of the aquatic environment plus improved nutrition and management of the various species cultured. This paper addressed the first of these, the importance of quality feed ingredients and finished feeds, primarily from a feed microscopy viewpoint as it interfaces with routine chemical analyses and the specific analytical problems encountered in aquaculture feeds. AQUACULTURE FEED INGREDIENT QUALITY CONTROL Ingredient quality is routinely measured by a combination of physical and chemical characteristics. Chemical evaluations are nutrient or nutrient availability oriented. For example, we may be concerned with the amount of crude protein present or the pepsin digestibility of that protein in an ingredient. We may also want to know what per cent of the total crude protein comes from a non-protein nitrogen source such as shrimp exoskeleton. We might also request a qualitative yes or no answer confirming if ammonium nitrate, a toxic NPN adulterant, has been added. All these nutrient and toxin related questions are answered via chemical analyses and perhaps NIR. If we are more concerned with sources of the nutrients, adulterants, contaminants or other visible characteristics, we must turn to feed microscopy. Feed microscopy is used to determine if an ingredient meal is derived purely from a single source, fish for example, or if the meal is a mixture of various ingredients. A visible microscopic examination is the preferred method to determine rapidly, in a subjective manner, if the meal exhibits quality problems. Perhaps we need to evaluate ingredients quickly for any potential adulterants and contaminants present before unloading a truck. The range of possible adulterants, chosen to confound analytical tests, would require a long and complicated analytical process. Feed microscopy would again be the method of choice because the adulterants cannot be hidden from a trained microscopist. The analysis is subjective, but it is very rapid and the ideal complement to slower, objective chemical methods. Feed microscopy is thus an essential part of the quality assurance (QA) program for aquatic feeds and their ingredients just as for other feeds with which we are familiar. Remember, as noted earlier, that formula feeds and their ingredients present some unique analytical problems for the quality control chemist and microscopist. Many problems are related to similarities of animal and marine meals. These problems are exacerbated by the very small particle size of the ingredients and their contaminants and adulterants. Still other problems are related to the lack of adequate descriptions of ingredients and the potential for false claims and fraud. For whatever reason, excessive ingredient blending to lower prices or to make composite meal mixtures often results in poor quality products because the blending was done without sufficient knowledge of nutrient availability and feed performance. One should also be aware that some of our routine quality control tests will give erroneous results if not interpreted correctly in view of all these new constituents in composite meals. Shrimp exoskeleton is the most obvious example. It confounds crude protein as well as ADF fiber analyses. It is a source of non-protein nitrogen (NPN) that is not detected by tests designed for urea, biuret and ammonium compounds. Thus shrimp exoskeleton escapes detection when a NPN test is applied to correct the crude protein of an ingredient and a serious error can be made in the formulation relative to nutrient availability and total protein. All these
ingredient changes and potential adulterations necessitate that feed
manufacturers and aquaculturists look at the many factors affecting
ingredient and feed quality assays that we used to believe were
absolutely accurate. It also requires the analyst to be fully aware
of the range of adulterations and variations in products and their
confounding effects on the various chemical and physical quality
assurance tests used in the routine laboratory. The feed
microscopist is the key to detecting most of these problems and the
complement to the routine QA lab. A second source of variation, related to the above, is introduced at the point of processing. For example, shrimp exoskeleton may be removed to make a table ready product and the waste ground into the next batch of shrimp head meal. That blended or co-dried batch of shrimp head meal will have a higher than normal level of exoskeleton which will add to the relative amount of non-protein nitrogen compared to shrimp head meal from the deheading operations (legs and exoskeleton left on the shrimp for marketing). These can be small batches or large depending on the type of operation involved. Thus the variation can be considerable from one batch to another even when blended if the operations are not based on some QA tests or criteria. The temperature of the meal cookers and driers, the final product temperature going into storage, and the handling of the by-product before processing produce additional variables. Perhaps some meal has been made from processing wastes left from the previous day. This would increase the microbial modification of some nutrients and the potential for disease or other problems. Hammermilling and extrusion introduce other processing and texture variables. These mechanical effects plus those of heat all affect color and texture of the final meal and influence the type of analyses we should choose to measure the ingredient quality. Aquaculture ingredients are high priced and the temptation to adulterate them is great. One of the worst adulterants -- ammonium nitrate -- is cheap and readily available as fertilizer. It was formerly found commonly in feeds but fortunately it is so easily detected both chemically with Nessler's reagent and visually with the microscope that the practice has essentially stopped. Examples of less dangerous but more insidious adulteration is the blending of yeast, fermented plant or animal residues or low grade animal or marine meals into high quality marine meals. These affect the amino acid balance of the meals and reduce their performance although the crude protein may be within the limits of the contract ingredient specifications. This is the one particular area that feed microscopy is invaluable although it is at the limit of practicality for some liquid adulterants. Extracts dried on other matrices may not be fully identifiable. We need to stay well advised and aware of such high moisture by-products, mainly extracts, that are currently entering feed ingredient market channels and the carrier matrices that are being used. Variations from post-process
handling are also quality factors. Ingredient damage can occur from
overheating in unshaded or unprotected storage areas or during
shipping when trucks are delayed at border crossings or check
points. This is a serious problem for fish meals shipped throughout
the tropics in black plastic bags. Increased bacterial or fungal
activity in products stored in high humidity or wetted from
inadequate facilities also causes rapid deterioration. Conversely,
over drying to minimize moisture problems can accelerate oxidative
rancidity and decrease ingredient quality. These defects and
problems that the feed microscopist may be able to see but, not
quantify, are reported to the chemist to verify. The above are some examples of the many variations that occur in aquatic feed ingredients and that may require special testing in a quality assurance program. Microscopy can provide some of the answers but these must interface with spot tests or other chemical tests to be most productive. The QA director has a choice of many single or combinations of tests that can be used to define quality characteristics. It is important that the chosen test(s) measures the characteristic accurately without confounding influences from adulterants or contaminants. A true measure of quality must be assured. Of course the degree of assurance is relative and the benefits must be weighed against established quality standards and the time delays caused by the assay methods. Chemical analyses are the traditional quality control tests. These include Kjeldahl crude protein, crude fat, crude fiber and the more recent acid-detergent fiber and neutral-detergent fiber, ash, moisture, salt and mycotoxins. Most of these standard tests require an average of an hour or more per test. A single analyst can run several tests simultaneously by scheduling the various drying, digesting, distilling, or other time-consuming steps to overlap. These tests give accurate, consistent results for most nutrients. Some adulterants are chosen to confound the tests. [Ammonium nitrate, for example, will be measured as crude protein unless a separate non-protein nitrogen test is run to correct for the adulteration.] Slow objective chemical tests also create a significant time delay from receipt of sample to final determination. This can be critical for larger feed mills that need to maintain the flow of incoming ingredients in unloading areas. Some of these situations require a more rapid, perhaps less accurate and more subjective, type of test. Spot tests and rapid test kits provide answers in this specialized area. Nessler reagent for ammonium ion detection, peroxidase for blood meal adulteration or contamination in animal and marine meals, ELISA (enzyme-linked immunosorbant assays) kits for mycotoxins or drug residues, and rapid test kits for urease activity in soybean meal or full-fat products are some of the commonly used tests. These require limited training and interpretation but they provide extremely rapid answers when needed because quality control begins with the incoming ingredients. If one can minimize the acceptance of poor quality ingredients into the feed mill, the potential quality of the finished feed will have been greatly increased. Rejection or acceptance decisions are often based on qualitative information. Feed microscopy provides some of the fastest possible answers to complex ingredient analysis and quality problems although it does require the most training and practice. A trained feed microscopist can often give more information about an incoming ingredient in 5 minutes than several trained chemists can determine in the lab in several hours. Subjective qualitative answers, particularly from feed microscopy, are the fastest possible values the QA director can obtain to evaluate incoming ingredients. Feed microscopy can thus become
the key front-line defense in an aquaculture quality control
program. Discriminating power and speed are major advantages. The
primary application is to prevent lower quality ingredients from
entering the feed manufacturing process. Of course feed microscopy
can be applied at every stage of the manufacturing process but never
as effectively as at the point of ingredient receipt. Feed microscopy may be divided
into two major types - qualitative and quantitative. Qualitative
feed microscopy is the identification and evaluation of ingredients
and foreign materials, either alone or in mixtures, via surface
features (stereomicroscopy) or via cellular or internal particle
characteristics (compound microscopy). Quantitative microscopy is
the proportioned measurement of each ingredient in finished feeds or
of contaminants and adulterants in ingredients. Most analysts use
the stereomicroscope for a majority of their observations and switch
to the compound microscope for confirmations of observations.
Polarized light, interference contrast, density and particle size
separations, and chemical spot tests add to both qualitative and
quantitative microscopy. And finally, feed microscopy and analytical
chemistry merge at the point of micro-spot testing. Quality control begins with the incoming ingredients before they are unloaded. Natural variations discussed above, filth, adulterants, and contaminants can be observed readily under the microscope and compared to acceptable standards. Acceptance or rejection decisions are made by the QA manager according to the appropriate comparisons to established standards or in-house defect action levels. Routine screening of incoming ingredients, in the absence of a trained microscopist and formal sampling, can be done with a 6x or 8x hand lens in the hands of a trained supervisor at the receiving point of the mill. Approximately 90% of potential formula feed problems can be prevented or corrected at this point. Many feed microscopists train a receiving supervisor or technician to assist them in reviewing ingredients and commodities at the point of ingredient receipt. At the other end of the mill, the importance of rapid post-manufacturing assays is essential to shorten warehouse time and future problems. Each ingredient in finished feeds is verified via qualitative identifications and checked off against the feed tag. Quantitative verifications of ingredients are checked against the feed tag ingredient list as well as against the chemical analyses. Visual QA of finished feeds minimizes any obvious problems that may not be part of the routine chemical lab tests. This is not necessary for computer operated feed mills. Manual batch mills produce varied quality feeds and must be monitored routinely to maintain quality. Ultimately the microscopist sees the problem samples that may be difficult or impossible to analyze chemically. Small amounts of foreign materials or concentrations of microingredients may suggest some solution to processing problems or to a source of contamination. Particle size comparisons or surface features of finished feeds may be evaluated to improve manufacturing processes. Litigation may hinge on testimonies of microscopists in many ways. Types of microscopic analyses are essentially limited only by the presence of definable natural particles based on cellular structures and by the abilities of the microscopist to compare standards and unknowns. Equipment The basic
equipment for feed microscopy is not expensive and consists of: ! A 6 x 12-inch, or slightly larger, piece of duolux hardboard painted with black enamel to lay out sample fractions. Black formica is an ideal surface not affected by chloroform or other heavy solvents used for flotation.MICROSCOPIC ANALYSIS OF INGREDIENTS Feed microscopy
requires a magnification aid to the eye (microscope or hand lens), a
trained mind and a sample set of reference standards. The two types
of microscopes described above and the simple accessory equipment
provide the magnification and tools for manipulation of the samples.
The mind is trained by reading literature, by formal studies and
short courses, and by internships and by private study. A set of
standard samples, collected by each microscopist and carefully
stored, is the final key to feed microscopy analyses. This section
suggests ways to obtain and maintain a sample collection, describes
the types of ingredients commonly found in aquaculture feeds, and
identifies the specific characteristics used for routine and
confirmatory analyses. Start your collection with samples supplied by your company or by ingredient suppliers. Seek good quality samples as well as those rejected by your lab and others. A feed microscopy association annual meeting is often a time to exchange unique and odd ingredients or samples of poor quality feed others have rejected. Look around your feed mill and pick up samples from the floor, under old boards behind the loading dock, etc. that represent decomposing feed and sweepings that often get into feeds. Become familiar with concrete, stones, wood splinters, grass, straw, clothing fibers, hairs, and many other items that are around us commonly every day. They will also be found in feeds. You save a lot of time by learning what these look like before you encounter them in feeds. Sample collections
are invaluable and should be treated accordingly. Small clear glass
vials with tight seal caps are preferred to minimize insect
infestation and microbial contamination. A low cost alternative is
used 35 mm film containers. The collection should be stored
protected from light in drawers or in a cabinet in file boxes that
allow easy reviewing. It is common to collect between 500 and 1000
samples. A well organized retrieval system is essential for rapid
analyses. Large samples of ingredients or feeds must be reduced in size by dividing them mechanically with a divider or riffler or by hand via quartering (taking alternate quarters each time). One must be careful to maintain sample integrity during subsequent handling of the sample. Once a representative sample has been taken, the microscopist must decide which type of analysis that will produce the best information. There are two schools of thought for sample particle size reduction: (1) grind all samples to pass through a 40 mesh sieve so that all subsequent observations are on the same approximate size basis, and (2) use a mortar and pestle to break up large agglomerations of samples but try to maintain all homogeneous particles in their original size class. Although most microscopists minimize sample reduction to maximize information about the major constituents of the sample, a trend toward smaller particle sizes in aquaculture feeds may require grinding the sample as in (1) above. Ingredient
samples should be well mixed to prevent separation of fines and
skewed estimates of quality. They can be analyzed directly, in most
cases, without additional sample preparation. Granular formula feed
meals can be analyzed too without further preparation. Pellets,
however, must be mortar and pestle ground to separate the various
constituents. Very hard or high density pellets must be depelletized,
sometimes with water, to separate individual particles. Mortar and
pestle grinding generally separates mineral from organic matter
except for these hard problem pellets. Water depelletizing however
precludes analysis of certain water soluble salts, thus requiring a
separate sample. Extruded materials present particularly difficult
situations. Each sample should be handled to maintain maximum
information for the microscopist. Weigh a 2.0 gram aliquot of the sample, and sieve it using 10, 20 and 40-mesh screens. Weigh each portion on filter paper or in a petri dish before observing. Weights may assist in determining the relative amounts of certain ingredients. Weigh a second 2.0-gram aliquot into a porcelain evaporating dish and flood it with chloroform (use with adequate ventilation) to float off the organic matter from the mineral material. This is also an effective defatting step to allow easier observation of certain fatty animal and plant ingredients. Carefully scoop off the organic matter and pour off the solvent. Allow both organic and mineral fractions to air dry. Each fraction may be sieved and weighed too to gain additional information. Ingredient evaluations are fairly simple once the level of acceptable quality has been established by management. Particular attention is directed to the through the 40 mesh because adulterants are usually ground finely to escape detection. The over 10 mesh is also a common place for fibrous contaminants. Limestone added to soybean meal and certain nitrogen containing salts are found in the mineral fraction after flotation. Of course, all fractions must be examined for extraneous material and the quality of the ingredient estimated against that of the acceptable standard sample. Mixed feed evaluations are more difficult. Pelleted concentrates or mixed concentrates with cracked grains must be separated with coarse sieving before grinding to prevent reduction of the cracked grains. Verification of feed tags is an extension of mixed feed examinations and merely requires the identification of each ingredient. Approximations of major ingredients are sometimes required and can be made by weighing hand isolated particles from the over 10 through 20 sieves that represent most of the ingredients. Sometimes, the over 40 fraction is used when the ingredient in question has been originally ground to a 20 mesh particle size. Quantitative Analysis Feed microscopical techniques are recognized as some of the best and fastest methods to obtain quality assurance information. The presence of contaminants and adulterants in both ingredients and finished feeds; the misidentification of ingredients; the absence of labeled ingredients or the presence of ingredients not included on the label; and approximation of the per cent protein or fiber present in ingredients are all examples of rapid determinations possible via feed microscopy. Unfortunately, quantification is not as easy as qualification. Any subjective technique is empirical and most microscopists develop techniques that work best for them individually. Consequently no one quantifying technique has been standardized and many variations exist depending on the type of sample analyzed. However, results from collaborative studies are remarkably close and guidelines can be established to minimize errors. The feed microscopist should determine the best method(s) to use for quantifying constituents in particular feeds and ingredients and develop an effective course of action. The primary quantification methods with some variations and examples are presented. Pick and Weigh The most meticulous and accurate quantifying is done by physically picking out all particles of some material and weighing the separated fraction. This is a very difficult and laborious task and should be performed on small samples to minimize fatigue (assuming that they are sufficiently homogeneous to permit small sub-samples). It works best on colored material (e.g. cottonseed hulls) or crystals (e.g. salt) that are easily identified at low magnification (10 to 30X). It also works on whole seeds like oats in horse rations when a larger sample has been taken. Only the easier removed constituents should be hand picked unless time is not a factor. A reasonably accurate determination of a ground constituent (e.g. maize) can be made too if the starchy endosperm dust is estimated and added to the larger particles, separated by pick and weigh, as a correction. Ratio Standards A second method of quantifying involves the use of standards prepared to simulate the same density of material that one is trying to quantify and of known rations. For example, a mixture of 1 g of salt, 5 g of copper sulfate, and 94 g of dicalcium phosphate can be used to establish the per cent of minor ingredients of "heavier-than-chloroform" fractions after flotation. A whole series of similar standards (using 2 constituents) for rations of 10:90, 20:80, 30:70, 40:60, 50:50 or of spreads of 5 units (35:65, 40:60, 45:55, etc.) can also be prepared to do the same thing. The eye cannot readily distinguish between ratios less than 5 units apart so don't expect to make the determinations more accurate. One must count several fields of view of the unknown and the ratio standards to establish the most correct percentage. Standards are prepared also for lighter than chloroform fractions and for specific problem determinations (poultry meal and meat meal). Counting Cells A third method, one that is rather mechanical, involves a counting cell and the compound microscope. Counting cell slides are available commercially (blood counting with Neubauer grid; ocular grids) or a grid pattern can be etched on a microscope slide with a diamond glass marking pen. The slide must be calibrated with standards carefully weighed and distributed in mounting medium. Unknowns are treated similarly and several slides are counted per determination. The counting cell method is described in the AAFM Manual of Microscopical Analysis of Feedstuffs. Analysis or Verification from Chemical Analysis A fourth method uses crude protein data to determine per cents of ingredients (directly or by difference) or to verify the percentages derived from other methods. When ingredient percentages derived from other methods, multiplied by their "book" values and summed, do not equal the total crude protein, then the percentages are incorrect, or the products "book" value is not consistent with the quality of the ingredient used, or one or more proteinaceous ingredients have been missed, or some other problem exists. Consequently the microscopist must reconfirm the relative ratios of ingredients and/or otherwise find the source of the inequality. The numbers should be within +/- 10% and preferably +/- 5% for an acceptable level of confidence. Spot and Other Chemical Tests Although the microscopist uses both the physical surface features of particles (color, sheen, irregularities, etc.) and cellular structures (cell walls, hairs, starch grains, etc.) to identify ingredients, chemical tests are an important adjunct to identifications. They eliminate extraneous possibilities and allow more exacting analyses. Drugs and other feed additives are tested for presence or absence as a quick check of correct formulating and mixing. Minerals and vitamins, often purchased as premixes, need only be verified in the finished feed for quality control. Often, only one or two key ingredients of the premixes are checked. The following are easy tests to identify groups of compounds. A small amount of "through 40 mesh" particles may be sprinkled on the surface of two drops of reagent in a white spot plate, or a single drop of reagent may be added to an isolated crystal of unknown. Observe the reaction at approximately 20x. ! Hydrochloric acid: 0.5 N gives effervescence with carbonates of all types.! Quimociac reagent gives effervescence but no precipitate with carbonates as a typical acid on carbonate reaction. A yellow precipitate with effervescence indicates phosphates such as dical made with the CaCO3 process and some bone meals. A yellow precipitate without effervescence indicates di- or monosodium phosphates, bone meals (generally), and dical made via the CaO process.! Silver nitrate: 0.1 N indicates salt (chloride) is present if the white precipitate is insoluble in nitric acid but soluble in concentrated ammonium hydroxide. All chlorides will react with silver nitrate.! Distilled water gives a milky white solution in the presence of milk products. Extruded and some pelleted feeds will not give this simple reaction.by Lynn S. Bates, PhD.
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