2008 Agricultural Microscopy
AM 1: Agricultural Microscopy I
Chair(s): M. McCutcheon, West Virginia Dept. of Agriculture, USA; and G. Kobata, California Dept. of Food & Agriculture Feed & Fertilizer Lab, USA
Admissibility Issues in Forensic Microscopy: Frye and the Daubert Trilogy. M.M. Houck1,2, 1West Virginia University, Morgantown, WV, USA, 2Curtin University of Technology, Perth, Western Australia, Australia
Commentators in the legal community have expressed concerns that forensic microscopical hair comparisons may not be scientific and may not meet admissibility criteria set forth in the Federal Rules of Evidence and guiding legal decisions. Forensic microscopical hair comparisons are founded on the precepts of comparative biology, microscopy, zoology, histology, and anthropology. Empirical testing demonstrates that, while not absolute positive identification, hair comparisons are good evidence of association. The validity and acceptance of forensic hair comparisons are supported by guidelines for hair comparison methodology, papers in peer-reviewed journals, textbooks, and specialized training schools. Hair comparisons are a service offered by both public and private forensic science laboratories. Hair comparison testimony has been accepted in local, state and federal courts for decades. This presentation will demonstrate the logic and structure of an argument for admissibility of microscopical methods.
Feed Microscopy as a Tool to Help Solve Stock Theft Cases. C.W. Cruywagen, Stellenbosch University, Stellenbosch, South Africa
Feed microscopy can be used in stock theft investigations in various ways. Dietary samples, taken on the site where animals have been removed from, can be examined microscopically to identify ingredients. Sample types that can be examined and compared with dietary samples to establish a positive resemblance include rumen content, faecal excretions and material recovered from clothing and shoes. The success rate is higher when samples of rumen content are compared with dietary samples than when faecal samples are used. When only faecal samples are available, weed seed profiles, fibre texture and fibre colour are the most important items to be observed and compared. Positive comparisons have been made in the past when particles of rumen content that have been recovered from clothing and shoes were compared with dietary samples. Animal hair can also be examined microscopically to identify animal type. The success rate to establish positive resemblances depend on the types of diets fed, type of samples taken, condition of samples and ability and experience of the microscopist. It should be taken into account that microscopy is not as individual specific as certain other tests, such as blood typing and DNA finger printing. However, feed microscopy can be a valuable tool to add evidence in stock theft investigations and offers a rapid and less expensive alternative to methods such as DNA analysis.
The Forensic Microscopy of Hair. M.M. Houck1,2, 1West Virginia University, Morgantown, WV, USA, 2Curtin University of Technology, Perth, Western Australia, Australia
Forensic hair examination and comparison is often undervalued as evidence. Significant information can be developed from a thorough microscopic examination and comparison of human and animal hairs that can assist criminal and civil investigations. Animal hairs can be distinguished easily from human hairs and often can be specified to a genus, species, or even breed. Hairs often can be identified as to their body area origin and the racial ancestry of the person from whom they originated. Additionally, damage, disease, or cosmetic treatments can be identified and described. Finally, suitable hairs can be compared microscopically with known hair samples to determine if they could have come from the same source. This application is now being augmented by mitochondrial DNA analysis which enhances the information already available from a microscopic examination of evidentiary hairs.The stage is set, however, for forensic hair comparison to bloom again because hair remains a good marker of human individuality, a microscopic mote bearing a wealth of information and valuable forensic evidence. The information is visual as well as molecular. This prediction will come true if the forensic hair discipline remains true to its science by creating standards for training a new generation of microscopists and for quality laboratory practices.
Body Weight or Egg Production Losses in Relation to Poultry Feed Formulation. A.S. Dhillon, Avian Health & Food Safety Laboratory-WADDL, Washington State University, Puyallup, WA, USA
Avian Influenza Outbreaks in Poultry and its Impact. A.S. Dhillon, Avian Health & Food Safety Laboratory-WADDL, Washington State University, Puyallup, WA, USA
AM 2: Agricultural Microscopy II
Chair(s): J. Makowski, Windsor Laboratories, USA; and G. Ideus, ADM Alliance Nutrition Inc., USA
Microscopic Characteristics of Corn that Ferment at Different Rates. C.W. Cruywagen, Stellenbosch University, Stellenbosch, South Africa
Corn samples from six suppliers were ground through a 1 mm screen and used to determine fermentation rates. Physical properties were also observed, which included light flotation fractions and fine (≤.42 mm) particle fractions. Fractions were expressed as percentage of the original samples. Variation occurred between the six corn samples regarding fermentation rates (9.2-13.4% per hour), total gas production (536-594 ml/g OM), light flotation fraction (8.8-48.7%) and fine particle fraction (58.6-73.2%). Fractions were also observed under a stereo microscope (12x magnification). A positive correlation was observed between the light fraction and total gas production (0.66; R2=0.43) and between the fine fraction and fermentation rate (0.65; R2=0.42). The low R2 values may be attributed to the small number of samples and the fact that the variation between samples was not very high. Although the samples with the higher fermentation rates exhibited more floury starch than the samples with the lower rates, the microscopic observations are subjective. Feed microscopy therefore does not offer a quantitative base to predict fermentation rates of different corn samples. Particle size distribution of ground samples appears to have some practical value, but more research is required before recommendations can be made.
The Microscopic Examination of Animal Feeds, Herbs, and Spices. H.K. Loechelt-Yoshioka, U.S., Food and Drug Administration, USA
The U.S. Food and Drug Administration (FDA), began its examination of animal feeds using feed microscopy in 2001. The FDA determined that the group to do the feed microscopy would be the filth analysts. The filth analysts are composed mainly of entomologist and biologist. Aftersending the analyst to a training course in feed microscopy, they started examining samples. In our lab to aid in the identification of animal tissues, I started to take photos of authentic animal tissues. Than arranged the photos withnotes into fact sheets. The fact sheets grew into a raining manual. The fact sheets continued to grow and soon included the plant material found in animal feeds. Volume 3, was than added to include herbs and spices. Eventually it was determined that the entire manual would consist of 5 volumes. Minerals would be in volume 4 and volume 5 wouldcover the manufactory process. Over time the fact sheets were becoming more detailed. The animal tissue fact sheets, included hyper links to photos of products which could be mistaken for animal tissue and how particular tissues would react in various test solutions like HCl. Volume 3, also contained a chart, which showed how the herbs and spicescould be adulterated. At the present time volumes 1 and 2 have been placed on an in-house FDA eRoom. Volume 3 is still under construction and it is hoped to start placing it in an eRoom later this year. When volume 3 is finished, work will begin on volumes 4 and 5. Since all 5 volumes are a work in progress, each gets updated as more referencematerial is acquired. The ultimate goal is to get the manual published in at least CD format and made available to the public.
Using Microscopy to Strengthen Mineral Ingredient Quality Programs. T. Costigan, Prince Agri Products, Inc., Quincy, IL, USA
The contamination of pet food with melamine last spring forced the entire feed industry to stop and take a closer look at the safety of their ingredients sources. Quality programs have long utilized chemical analyses as their primary check of ingredient quality. These analyses are invaluable in measuring the levels of nutrients or of known contaminants, but are a less practical method of uncovering unexpected contaminants. Microscopy offers a way to quickly screen samples for contaminants. Samples found to be different in color, particle size, or particle shape can be singled out and submitted for more complete chemical analysis.This presentation will review microscopic images of various mineral ingredients used in animal feed. A brief description of the types of hazards that are likely to occur will be included for selected ingredients.
AM 3 / PCP 3.1: Plant Protein for Aquafeed
Chair(s): N. Vary, Canadian Food Inspection Agency, Canada; and K. Liu, USDA, ARS, USA
NOAA and USDA's Future of Aquaculture Feeds Initiative. M.B. Rust, Northwest Fisheries Science Center, Seattle, WA, USA
To meet the growing consumer demand for seafood in the United States, increasing supplies of finfish and shellfish will be needed. Most experts agree that development of aquaculture will be the only way to sustainably meet this increase in demand. The question that must be answered is how to ensure that aquaculture production increases are sustainable. The development and expansion of farming of carnivorous fish species will be constrained by a limited supply of fish meal and oil for feeds. Fishmeal and oil have traditionally made up a large part of the diet of farm-raised carnivorous fish. Fortunately, there is no dietary requirement for specific amounts of fish meal or fish oil for fish, so feeds that lessen the reliance on these limited feedstuffs—such as alternative protein and oil resources—can, and must, be developed. For this reason, the U.S. Department of Commerce (NOAA) and the U.S. Department of Agriculture (USDA) is in the process of sponsoring expert and public consultations on the future of fish feeds and the benefits to the U.S. by the development of such alternative feeds.
The Use of Corn Gluten in Salmonid Diets: Issues and Opportunities. G. Vandenberg1, G. Dagenais1, M. de Francisco2, D. Bureau2, 1Université Laval, Quebec, QC, Canada, 2University of Guelph, Guelph, ON, Canada
The main goal of this study was to test the use of white corn gluten in experimental diets to counter the problems associated with the undesirable colouring of trout flesh. The second objective is supplementation of feed with lysine, an essential amino acid naturally found in corn gluten, in order to improve carotenoid binding in fish flesh via protein deposition. A 6-month factorial study (2 x 2; yellow and white corn gluten by presence or absence of supplemental lysine) was undertaken. Rainbow trout (Oncorhynchus mykiss; 250 g initial body mass) were fed with diets that contain 30% of corn gluten and 1.7% of lysine. The colorimetric analyses (CIE L*a*b*) were compared to HPLC xanthophylls analyses. The fish showed a better retention of astaxanthin (p<0,05) when they were fed with lysine-supplemented diet. There are also significant differences in flesh colour according to the type of corn gluten (yellow or white) present in fish feed (p<0,05). A significant effect of the fish weight on the yellow flesh colour (b*) with colorimetric analyses (p<0,001) was shown. The results of this study will enable researchers and aquaculturists to gain a better theoretical understanding of the pigmentation of trout flesh as a result of the effect of lysine, while promoting the use of white corn gluten as a low-polluting, non-colouring, high protein feed ingredient
Selecting Salmonids to Better Utilize Plant Based Diets. K. Overturf, T.G. Gaylord, F. Barrows, USDA, RS, Hagerman Fish Culture Experiment Station, Hagerman, Idaho USA
Evaluation of genotype by diet interactions in aquaculture species for specific dietary components has only recently begun on a limited basis. Initial studies have examined such species as rainbow trout and Atlantic salmon. Because of the high-protein diet these species consume in the wild, commercial diets have relied heavily on fish meal and oil as protein and energy sources. Other omnivorous fish species such as tilapia and catfish have demonstrated a greater proclivity for utilizing plant feedstuffs and carbohydrate for energy, but little research has been performed on these species in regards to precise physiological changes in accord with nutrient modification or selection to alter performance on specific feed types. Research performed in other agriculture animal systems, such as cattle and poultry, have found alterations in specific physiological traits for different strains. These changes have also been found to significantly correlate with individual genes in research done with murine and laboratory fish models. This research is now being applied to aquaculture. Initial findings have varied between stocks and with different diets, but as the technology and experimental designs improve, it appears that this research will prove important for optimizing diets and carnivorous fish species for enhanced utilization of sustainable plant and oil products.
Chemistry and Technology of Processing Canola Proteins for Aquafeed. J. Wanasundara, Agriculture & Agri-Food Canada, Saskatoon, SK, Canada
Processing of canola/rapeseed is primarily to obtain oil. Seed proteins remain in the meal and account up to 42% of the drymatter. Canola meal is a widely used protein source in animal feeds including farmed fish. Processing of meal protein ingredients is not yet an established industry. Biofuel and fuel additive generation from oil increases the domestic canola crushing so does the generated meal volume. A record volume of 3.42 MilT of canola was processed in Canada during 05/06 and is expected to increase in 06/07. Developing high value end uses such as protein ingredients from meal biomass will help the economics of biofuel production and enhance the value of meal components. Canola protein processing includes generation of concentrates and isolates. Composition of proteins and minor components of canola is different than other oilseeds thus approaches to obtain protein products are different. Approaches that are different as well as similar to soy protein processing have been tested for canola. Two of the processes developed in Canada are underway for large scale canola protein ingredient processing. This presentation will provide scientific insight into canola protein processing technologies with reference to the seed chemistry, process technology advances, potential product applications and the significance of protein ingredient development in total seed biomass utilization.
The Utilization of Carotenoids to Improve Fish Health in Aquafeeds. T. Nakano, T. Yamaguchi, M. Sato, Marine Biochemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
Fish oils used in aquaculture is known to contain plentiful highly unsaturated fatty acids (HUFAs). Oxidized HUFA could lead to oxidative stress in fish. Some fish diseases are thought to be catalyzed by oxidative damage to tissue. Hence improvement of the defensive ability of fish against oxidative stress would be important. Carotenoids have been suggested to show many kinds of biological activities in mammals. However, most research on dietary carotenoids, especially astaxanthin (ASX), in aquafeeds has been done in terms of muscle pigmentation, so that information on the biological effects of carotenoids in fish is lacking. Here, we will present several findings of the effect of ASX, including red yeast Phaffia rhodozyma, which is rich in ASX, on the health in rainbow trout#. Diets supplemented with ASX could decrease the levels of lipid peroxides in serum and liver of fish. The elevation of tocopherol concentration in the liver was also observed in fish fed ASX. Furthermore, red yeast is known to contain many kinds of bioactive compounds such as vitamins, trace elements and polysaccharides. Thus we might conclude that ASX and red yeast have many possibilities to improve fish health in aquaculture.#T. Nakano: Dietary Supplements for the Health and Quality of Cultured Fish (Nakagawa, Sato and Gatlin III, Eds), CABI, UK, 86, 2007.
Replacement of Fish Meal and Oil by Canola Protein Concentrate and Vegetable Oils in Diets Fed To Rainbow Trout. M.D. Drew, University of Saskatchewan, Saskatoon, SK, Canada
Replacement of fishmeal and fishoil with plant proteins and oils requires the development of feed ingredients with nutrient profiles similar to marine ingredients. However, such feed ingredients have been difficult to develop. Most plant protein ingredients contain antinutritional factors that limit their use in salmonid diets. An exception to this is canola protein concentrate (CPC). CPC is produced by aqueous extraction of canola meal and has a crude protein content and amino acid profile similar to fishmeal and is also low in antinutritional factors. Rainbow trout fed diets containing CPC had growth rates comparable to those fed diets containing fishmeal. Furthermore, the addition of a 1% of a soluble fraction of CPC to diets fed to rainbow trout significantly increased feed consumption compared to controls. On the oil side, canola and linseed oils have desirable fatty acid composition, due to their high content of n-3 fatty acids and appear to be the best candidates for replacing fishoil in salmonid diets. Recent studies in our laboratory have confirmed that the replacement of fishoil with linseed and canola oils does not diminish growth performance and results in fish fillets with acceptable fatty acid composition, sensory properties and decreased levels of organochlorine contaminants.
AM 4 / PRO 4: Processing Methods and Concerns for Fish Oil and Fish Meal
Chair(s): N. Dunford, Oklahoma State University, USA; K. Koch, Northern Crops Institute, USA; and S. Metin, Cargill, Inc., USA
Improvement of Stability and Quality of Food Grade Fish Oil. W.M. Indrasena, C.J. Barrow, J.A. Kralovec, Ocean Nutrition Canada, Dartmouth, Nova Scotia, Canada
Beneficial effects of essential fatty acids such as EPA and DHA have been broadly recognized during the past two decades, and the global demand for dietary supplements containing these miraculous bio-active compounds has been increasing exponentially. These highly unsaturated fatty acids are vulnerable for rapid oxidation resulting in the production of off odours and flavours. Therefore, it is imperative to produce oil that has bland taste and odour.It is essential to protect the oil from oxidation during the process as well as to remove the compounds that give off flavour to the oil, and compounds that are detrimental to the oxidative stability. Significant reduction of chemical contaminants such as heavy metals is also essential. Several purifying steps such as refining, bleaching and deodorization are required for the removal of most objectionable and deleterious substances that contribute to off-flavours.Fish oils with varied amounts of EPA and DHA were bleached and deodorized with natural antioxidants and some selected antioxidants were added after deodorization. The quality of the oil including sensory properties was monitored during the storage using both subjective and objective analyses. This presentation will emphasize the possible improvement of the quality of oil using various antioxidants during bleaching and deodorization processes.
Diversity in Fish Oils and Fish Meals Derived from Alaska Seafood By-Products. A.C.M. Oliveira1, P.J. Bechtel2, S. Smiley1, S. Plante1, 1Univeristy of Alaska Fairbanks, Kodiak, AK, USA, 2ARS, USDA, Fairbanks, AK, USA
Alaska annually processes roughly 2.2 million mt (Mmt) of fish harvested for human food, generating some 1.5 Mmt of fish waste, depending on season, species composition and product form. In the western Gulf of Alaska and along the Bering Sea, larger seafood processing operations are mandated by regulation to effectively handle the by-products of seafood processing. These concerns employ wet- reduction processing to manufacture co-products such as fish meal, bone meal, fish oil and stickwater from this material. In 2003, it was estimated that the total amount of Alaska byproducts produced on a dry matter basis was 208 599 Mmt, and that about 40% of the solids were reported to be recovered as fish meal and fish oil. Alaskan fish oil production, while difficult to document, is probably between 30,000 and 45,000 T per annum. Nonetheless, the volume of oil that could be extracted from Alaska seafood by-products could be upwards of 70,000 T. In this presentation some important chemical and nutritional characteristics of commercial Alaska groundfish meals and salmon fish meals will be discussed. Furthermore, the composition of commercial fish oils produced from the processing byproduct streams of walleye pollock, pink salmon, sockeye salmon, Pacific Ocean perch, and sablefish will be presented.
Processing of Fish Oil to Minimize Deterioration. E. Hernandez, Omega Pure, Houston, TX, USA
Omega 3 fatty acids, especially from fish oil, have been widely studied for their health promoting properties. As a result consumption of Omega 3 oils or PUFAS have increased and are now commonly applied to foods and used as dietary supplements. Fish oils are normally processed for the removal of impurities, contaminants and to make them palatable for human consumption. Processing steps include chemical or physical refining, bleaching, winterizing and deodorizing. All polyunsaturated oils, including omega-3 oils, are inherently unstable and prone to oxidation. Consequently fish oil is susceptible to rapid deterioration during processing, cooking or storage. Threfore special precautions have to be taken to minimise extreme processing conditions such as excessive heating, exposure to oxygen and reactive metals. This presentation will review techniques commonly followed to prevent deterioration of fish oil during processing, such as lower temperatures in refining, use of antioxidants, molecular distillation, and special packaging.
Overview of Fish Protein and Lipid Recovery from Processing Waste. R.B. Johnson, Northwest Fisheries Science Center, Seattle, WA 98112, USA
Use of Lipases for the Production of N-3 Polyunsaturated Fatty Acid Concentrates from Fish Oil. T. Okada , M. Morrissey, Oregon State University, Portland, OR, USA
The Pacific sardine (Sardinops sagax) is a coastal pelagic fish found from the Gulf of California to Southeastern Alaska and have made a strong biological comeback in the Oregon and Washington coastal area. Currently, the majority of the sardine catch is frozen whole and sold at prices to Asian markets for both the bait fishes as well as human food. Because of the recent interest in the health benefits of marine oils this study was initiated to determine different methods of oil extraction from the Pacific sardine. Commercially available microbial lipases, from Candida Rugosa (CR), Candida cylindracea (CC), Mucor javanicus (MJ), and Aspergillus niger (AN) were used for enzymatic hydrolyses with extracted sardine oil, run at 37ºC with constant stirring for 1.5, 3, 6, and 9 h. Fatty acid composition analysis by gas chromatography showed that the refined unhydrolyzed oil contained 26.86% of eicosapentaenoic acid (EPA) and 13.62% of docosahexaenoic acid (DHA) (wt/wt%). CR lipase was the most effective in concentrating n-3 PUFA. Hydrolysis with 250 U CR lipase increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62 to 29.94% with 500 U after 9 h. Compared to CR and CC lipases, MJ and AN lipases resulted in low n-3 PUFA concentration. TG levels decreased significantly as reaction time progressed. An additional study was conducted to develop an immobilized-enzyme system to entrap lipase in chitosan-alginate-CaCl2 beads for the purpose of concentrating n-3 polyunsaturated fatty acids (n-3 PUFAs) from sardine oil. Lipase was immobilized by an ionotropic gelatin method, and its characteristics were determined. Optimum pH of immobilized lipase shifted from 7.0 to 6.0, and immobilized lipase showed higher stability against pH and temperature changes. Immobilized lipase significantly increased eicosapentaenoic acid from 25.21% to 39.64% and docosahexaenoic acid from 7.18% to 15.33%. This study demonstrated a simple method to immobilize lipase and concentrate n-3 PUFA with a solvent-free process.