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10 Feed Analysis GENERAL CONSIDERATIONS Some laboratories are certified and/or participate in check- sample programs that ensure accuracy among participating Feed analysis is necessary to determine the nutrient con- laboratories. Accuracy of the analytical method and the cho- tent of the feeds used in horse diets. There are many kinds sen laboratory technique should be the focus when selecting of laboratory analysis for feeds: chemical evaluation, in laboratories. vitro digestion systems, and near infrared reflectance (NIR) methods. Analyses using wet chemistry are the most used, but they do not give a direct estimate of nutritive value. Feed ANALYSIS OF CARBOHYDRATES: FIBER analysis could be costly depending on the type of analysis requested. Ideally all feeds should be analyzed, particularly Analytical procedures for fiber are still under develop- those nontraditional feeds or commercial mixed feeds for ment despite a long history (DeVries et al., 1999). Fiber is a which table values are not available. Also, forage chemical unique and complex nutritional entity often defined as the composition varies with state of maturity, climate, season, indigestible or slowly fermenting components of feeds. soil type, and fertilization (Ralston, 1991). Feed analysis Chemically, fiber has been defined as a variable mixture of should be performed if there is a suspicion of nutritional cellulose, hemicelluloses, some pectins, and lignin, plus in- problems based on clinical signs and poor animal perfor- digestible protein and lipids (Mertens, 1992). In human nu- mance. Accurate results depend on good sampling tech- trition, dietary fiber has been defined as the nondigestible niques, proper handling of the samples after collection, and carbohydrates and lignin that are intrinsic and intact in good analytical procedures. The feed composition tables plants (NRC, 2000). Several authors have reviewed the found in this publication represent average values, and vari- methodologies to measure fiber in feeds including Van Soest ations in the composition of some of these feeds are ex- et al. (1991) and Van Soest (1994), who described the ad- pected as indicated above. When balancing feeds for horses, vantages and limitations of the different analytical proce- the most common nutrient concerns are water, energy, fiber, dures for fiber. Most of the methods to measure fiber have protein, calcium, and phosphorus. However, most laborato- been developed for the ruminant animal and for humans, but ries will also provide analysis of other nutrients including not for hindgut fermenters like horses. However, neither the vitamins, essential amino acids, fatty acids, microminerals, ruminant nor the human system fits well with the digestive and sugars (total or single sugars) if needed. Other important physiology and intermediary metabolism of the horse (Hoff- dietary components for the horse, such as fructans, are not man, 2004). Different protocols might be used to measure routinely analyzed by many laboratories because of the fiber content depending on the type of feed. Thus, the actual costs of the equipment required for the analysis and/or diffi- definition of fiber becomes method-dependent. Fiber can be culties of the method. Chemical analytical techniques are al- determined as crude fiber (CF), total dietary fiber (TDF), ways evolving, but most feed testing laboratories will use neutral detergent fiber (NDF), and acid detergent fiber approved procedures recognized by organizations such as (ADF). Associated analyses include: neutral detergent insol- the National Forage Testing Association (NFTA), Associa- uble nitrogen (NDIN), acid detergent insoluble nitrogen tion of Official Analytical Chemists (AOAC), American As- (ADIN), acid detergent lignin (ADL), or ADF Klason lignin. sociation of Cereal Chemists (AACC), and American Oil For further reading on forage fiber analysis, refer to Van Chemists Society (AOCS), among others. The selection of Soest (1994), NRC (1996, 2000, 2001, 2003), and Mertens the laboratory is very important if good results are expected. (1997, 2002). 203

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204 NUTRIENT REQUIREMENTS OF HORSES Total Dietary Fiber β-glucans (Van Soest et al., 1991), which are resistant to mammalian digestive enzymes but rapidly fermented. In ad- Total dietary fiber consists of the remnants of edible plant dition, the NDF fraction will not recover any hemicellulose cells, polysaccharides, lignin, and associated substances re- that is soluble in neutral detergent. sistant to digestion by the alimentary mammalian digestive Some feed laboratories use a filter-bag method com- system (Trowell, 1985). Included in TDF by definition are monly named the ANKOM system for analyzing forages in- cellulose, hemicellulose, oligosaccharides (non-α-glucose cluding NDF, ADF, lignin, and in vitro dry matter di- polymers), lignin, pectins, mucilages, gums, waxes, cutin, gestibility (IVDMD) (Komarek, 1993; Komarek et al., and suberin, among others. Thus, TDF is relevant to most 1994). The filter-bag system can produce results similar to monogastric animals with hindgut fermentation (Van Soest the conventional methods with the advantage that the et al., 1991). The TDF concept arose as a result of interest in method is easier to use and safer than conventional methods fiber and human nutrition and is an analytical method rec- (Vogel et al., 1999). ognized as an official method of the AOAC (method 991.43, 2002) and used extensively in human nutrition (Prosky et al., 1988). TDF has also been recognized as an official Acid Detergent Fiber method by the AACC (1995a,b). The quantification of in- Acid detergent fiber (ADF) analyzed according to Van soluble fibers by the Van Soest methods (NDF, ADF, ADL) Soest and Wine (1967) is intended as a sample preparation is comparable to the insoluble fiber determined by the TDF for the determination of the sum of cellulose, lignin, ADIN, method (Lee et al., 1992; Popovich et al., 1997). Soluble di- acid insoluble ash (AIA), and silica (Van Soest, 1994). ADF etary fiber might be estimated by subtracting NDF from may not be a valid fiber fraction for nutritional use or for TDF (Baer et al., 1997) or by the AOAC method (991.43; predicting digestibility (Van Soest, 1991). Hemicelluloses AOAC, 2002). The analytical method for determining TDF can be estimated by subtracting ADF from NDF (Robertson recovers more fiber components than the NDF method but it and Van Soest, 1981), but the accuracy of the estimations does recover carbohydrates soluble in 78 to 80 percent will depend on the sample (e.g., beet pulp, citrus pulp, etc.) ethanol, such as the fructooligosaccharides (NRC, 2000). and the botanical origin of the sample. To avoid errors, a se- quential analysis for NDF and then ADF on the same sam- Neutral Detergent Fiber ple is recommended (Bailey and Ulyatt, 1970). The Van Soest detergent system (Van Soest et al., 1991) is the most popular method for measuring cell wall con- Cell Wall Phenolics (Lignin) stituents in forages. The detergent system is not a method for isolating cell walls per se, but rather a method of partition- The presence of phenolic compounds within the cell wall ing forage dry matter (DM) into fractions based upon its matrix limits the digestion of polysaccharides (Akin and bioavailability to ruminants (Van Soest, 1994). The original Chesson, 1989). The cell wall phenolics consist primarily of NDF method represents the percentage of cell wall material lignin and phenolic acids chemically bound to lignin or to or plant structure in a feed, including cellulose, hemicellu- cell wall polysaccharides. True lignin appears to be a poly- lose, and lignin as its major components (Van Soest and merized product of phenylpropanoid alcohols and ferulic Wine, 1967; Goering and Van Soest, 1970). Sodium sulfite and para-coumaric acids (Van Soest, 1994). The composi- is used to remove contaminating nitrogen from NDF and tion of lignin preparations varies according to the method of was optional in the method described by Van Soest et al. isolation. It is generally defined as the residue obtained by (1991). However, Hintz et al. (1996) suggested that the use the Klason two-stage sulfuric acid hydrolysis method (Van of sodium sulfite was crucial for the removal of contami- Soest, 1994) or one of its many modifications. However, the nated nitrogen from heat-damaged feeds. Amylase-treated potential presence of nonlignin components in the Klason NDF (aNDF) is a modification of the original method and is residue has been a subject of criticism of the method (Van used to minimize the contamination by starch in all feeds Soest, 1994). The ADL method includes both hydrolytic (Van Soest et al., 1991; Mertens, 2002). The aNDF method (sulfuric acid) and oxidative (potassium permanganate) pro- described by Mertens (2002) includes the use of amylase cedures (Van Soest, 1965). Jung et al. (1997) reported that and sulfite and is applied for the determination of aNDF in the Klason lignin method showed higher values than the forages, grains, grain byproduct feeds, oil seeds, oilseed ADL method for grasses (2 to 4 times higher) and up to 30 meals, and mixed feeds. The NDF concentrations for dairy percent higher in legumes. However, both methods were feeds in the NRC (2001) nutrient composition table were equally correlated with digestibility of forages. ADL has determined using amylase and sulfite. The aNDF method is been criticized for underestimating the lignin content of for- the recommended method by the National Forage Testing ages due to the ADF step of ADL, losing acid soluble lignin Association (Undersander et al., 1993). The NDF method (Lowry et al., 1994). does not recover fructans, pectins, gums, mucilages, and

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FEED ANALYSIS 205 Crude Fiber NFC values for feeds are calculated by subtracting from 100 the addition of CP, NDF, ether extract (EE), and ash. The Crude fiber is one of the components analyzed when per- nonstructural carbohydrate (NSC) fraction is measured by forming the proximate analysis, the others being moisture, hydrolytic methods using a modified procedure by Smith ash, crude protein, ether extract, and nitrogen-free extract (1981). The terms NSC and NFC are not synonymous and (NFE). Henneberg and Stohmann of the Weende Experi- should not be used interchangeably. Some fiber components mental Station in Germany developed the “Proximate such as pectins and gums are included in NFC but not in Analysis” or “Weende system” of feed analysis more than a NSC (Mertens, 1997). Recently, there has been an interest in century ago. For analyzing CF, samples are treated with di- determining specific NSC components such as fructans lute acid and then with alkali to mimic digestion of gastric (oligo- and polyfructosyl sucrose) in fresh forages fed to secretions (Method 962.09; AOAC, 2002). The Weende CF horses because of their potential effects on laminitis (Long- procedure is still being used by regulatory agencies despite land et al., 1999). Methods to determine oligofructans and the errors in the determination of the structural and non- fructan polysaccharides in foodstuffs have been reported structural carbohydrates. The most significant error of the by Cairns and Pollock (1988), McCleary et al. (2000), and Weende system is due to the solubilization and loss of part McCleary and Rossiter (2004). of the lignin, cellulose, and hemicellulose in the preparation of CF and its inclusion in the NFE (Van Soest, 1994). The Weende system is still being used in Europe for the evalua- Water-Soluble Carbohydrates (WSC) tion of feeds for horses (Martin-Rosset et al., 1994; Gud- Water-soluble carbohydrates (WSC) include compounds mundsson, 1998). soluble in cold water or in gastrointestinal contents such as monosaccharides (e.g., glucose, fructose, galactose), disac- ANALYSIS OF CAROBOHYDRATES: NONFIBER charides (e.g., sucrose, melibiose), oligosaccharides (e.g., FRACTIONS raffinose, stachyose), and some polysaccharides (Van Soest, 1994). Fructans are included within the WSC fraction (Fig- ure 10-1).Glucose and fructose generally constitute virtually Nonfiber Carbohydrates (NFC) all of the free-reducing sugars and, with sucrose and malt- The more readily digestible carbohydrates in animal ose, all of the free total sugars found in grass and legume feeds lack a satisfactory system of classification, particularly species (Smith, 1981). Extraction with 80-percent ethanol is for the horse, but they constitute a major energy-yielding the most common method used to remove sugars such as component of feeds. They comprise those carbohydrates not glucose, fructose, and sucrose, and the extracts can then be included in the cell wall matrix and not recovered in NDF as analyzed using nonspecific colorimetric or titrimetric assays described for the ruminant system of analysis (Van Soest et (Smith, 1981; Sturgeon, 1990). Further fractionation of al., 1991; Mertens, 1997; Hall and Van Horn, 2001; Hall, these carbohydrates can be accomplished by chromato- 2003). The NFC are calculated by difference: 100 – (pro- graphic techniques (Fales et al., 1982) or by enzyme- tein + (NDF – NDICP) + fat + ash), where NDICP = neutral coupled assays (Avigad, 1990). A WSC analysis can be also detergent insoluble crude protein, and include sugars, determined by NIR spectoscopy (NIRS) as reported by Ja- starches, fructans, galactans, pectins, β-glucans, and organic fari et al., (2003). From an analytical perspective, the NSC acids (Van Soest et al., 1991). Nonstarch polysaccharides is approximated to the sum of WSC and starch, depending (NSP, non-α-glucan polysaccharides) are broadly defined in on the methodology used (Figure 10-1). human nutrition as the fiber in plant material that includes cellulose, hemicellulose, and pectic substances, without in- Starch clusion of nonfibrous entities (Englyst and Cummings, 1990). From an analytical perspective, the NSP component Starch in forages can be measured by either acid or en- does not include lignin, which might present a limitation for zyme hydrolysis on residues that have been previously ex- horse diets although a filtration step of the residue after cell tracted with ethanol to remove soluble sugars (Smith, 1981). wall hydrolysis gives a value for Klason lignin, which with Enzymatic hydrolysis is preferred because of its specificity. the total NSP gives a total fiber value. The NSP does not in- The enzyme α-amylase hydrolyzes the starches to glucose clude fructan but does include galactan (Theander and without hydrolyzing the fructosans. Glucose concentrations Aman, 1982). Starch is measured by an enzymatic method are then converted into equivalent starch concentrations (Salomonsson et al., 1984) modified by Herrera-Saldana et (Smith, 1981; Moore and Hartfield, 1994). Determination of al. (1990). Mertens (1997) and Hall and Van Horn (2001) di- starch in animal feeds is described by the AOAC method vided the nonfibrous or non-NDF carbohydrates into four 920.40 (AOAC, 2002) and in plants by the method 948.02 general categories: organic acids, sugars (mono and oligo- (AOAC, 2002). Starch analysis in a variety of plant material saccharides), starch, and neutral detergent soluble fiber. The is described also by Hall et al. (1999).

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206 NUTRIENT REQUIREMENTS OF HORSES Plant Carbohydrates1 Cell Contents Cell Wall Organic Sugars- Oligosaccharides 3 - Pectins Fructan Starch mono & including β-Glucans §- Glucans and Gums Hemicelluloses Cellulose Lignin 2/Phenolics 2 Acids2 polysaccharides 3 disaccharides fructooligosaccharides Nutritional/Digestive Starch4 NSP TDF 4 CHO-H4 CHO-F R 5 CHO-FR CHO-F S5 NFC/NDSC 6 NDF 6 7 Starch4 WSC ADF Analytical 8 NSC 7 CF 9 NSP SDF 10 TDF 10 FIGURE 10-1 Fractionation of plant carbohydrates and related compounds Current and proposed systems for partitioning dietary carbohydrates based on current analytical methods (lower/shaded bracket; solid/dashed lines) and nutritional or physiologic definitions (upper bracket; dotted lines) relative to equine digestive function. Adapted from Hall, 2003 and Hoffman, 2004. In the analytical bracket, dashed lines indicate that recovery of included compounds may be incomplete. Abbreviations: ADF = acid detergent fiber; CF = crude fiber; CHO-H = hydrolyzable carbohydrates; CHO-FS = slowly fermentable carbo- hydrates; CHO-FR = rapidly fermentable carbohydrates; NDF = neutral detergent fiber; NDSC = neutral detergent soluble carbohydrates; NFC = nonfiber carbohydrates; NSC = nonstructural carbohydrates; NSP = nonstarch polysaccharides; SDF = soluble dietary fiber; TDF = total dietary fiber; WSC = water-soluble carbohydrates 1Major categories of carbohydrates and associated substances are shown. These categories may not include all carbohydrates produced by plants. 2Some noncarbohydrate components are included here as they are components of the specific analytical fractions. 3Specific fructans can be categorized as either fructooligosaccharides or fructan polysaccharides depending on degree of polymerization. 4A variable fraction of total starch can be resistant to enzymatic hydrolysis and thus some starch may appear in other nutritional fractions. 5Fermentability of gums may be variable. 6Some hemicellulose may be soluble in neutral detergent and thus recovered in the NFC/NDSC fraction, rather than the NDF fraction. 7Recovery of compounds in the analytical WSC fraction (and thus the NSC fraction when NSC is approximated as starch + WSC) may de- pend on methodology used. 8Amount of cell wall constituents included in CF analysis varies by feed. 9From a nutritional perspective, NSP includes all polysaccharides except starch. However, the analytical method for NSP may recover a vari- able amount of fructan polysaccharide. 10From a nutritional perspective, TDF includes all carbohydrates resistant to mammalian digestion. However, the analytical method for TDF (and SDF) does not recover oligosaccharides and may recover a variable amount of fructan polysaccharides.

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FEED ANALYSIS 207 CRUDE PROTEIN AND AMINO ACID ANALYSES leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine. The AOAC method 999.13 (AOAC, Crude Protein 2002) is applicable to the determination of the free non- protein-bound amino acids lysine, methionine, and threo- The percent of crude protein (CP) reflects the total nitro- nine in feeds and premixes. Tryptophan in feeds can be de- gen (N) content of feeds (N × 6.25). Official methods for ni- termined by the AOAC method 988.15 (AOAC, 2002). trogen determinations in feeds include several modifications of the Kjeldahl method for plants and animal feeds (methods 976.05, 976.06; 977.02; AOAC, 2002). Total nitrogen pro- ANALYSES OF FAT AND FATTY ACIDS vides a good estimate of digestible protein in forages that are not heat damaged. For heat-damaged feeds, adjustments Ether Extract (Crude Fat) of the total protein content are necessary to account for the The ether extract (EE) or crude fat is one of the compo- bound protein unavailable to the animal. nents of the Proximate Analysis of feeds and commonly is used to represent the total fat in a diet or ingredient. How- Acid Detergent Insoluble Nitrogen ever, using the term EE might be an oversimplification, as EE also contains organic acids, oils, pigments, alcohols, and The acid detergent insoluble nitrogen, also called ADFN the fat-soluble vitamins. Crude fat is determined by a sol- in forages, represents heat-damaged browning (Maillard) vent extraction method using a Soxhlet apparatus (method products or bound protein that are indigestible or poorly di- 920.29; AOAC, 1990) or modifications of the latter such as gested by animals (Van Soest, 1965; Goering and Van Soest, the Soxtec method (Randall, 1974). Another method to de- 1970; Goering et al., 1972; Weiss et al., 1986; Van Soest and termine crude fat used mainly with extruded feeds is the Mason, 1991). Acid detergent insoluble nitrogen can also be gravimetric method 954.02 (AOAC, 2002). measured in forages that have not been exposed to heat and is related to lignin and to a fraction of protein in forages (Mertens, 1979). This author found that between 5 and 12 Fatty Acid Analysis percent of the nitrogen in nonheat-damaged forages is iso- Free fatty acid (FFA) content is measured after extraction lated as ADIN, representing the truly indigestible protein in of fat by petroleum ether. The method includes fatty acid feeds. The ADIN fraction is determined as the nitrogen in methyl ester derivatization followed by gas chromatography the ADF residue using sodium sulfate. Acid detergent insol- (GC) analysis (AOAC method 996.06, 2002). The profiles uble crude protein (ADICP) is ADIN expressed as crude will include saturated FFA such as C12 (lauric), C14 (myris- protein on a dry matter basis. The quantitative subtraction of tic), C16 (palmitic), and C18 (stearic), and unsaturated FFA ADIN from crude protein to estimate available nitrogen such as C18:1,ω–9 (oleic), C18:2,ω–6,9 (linoleic), and seems justified (Waters et al., 1992). C18:3,ω–3,6,9 (α-linolenic), among others. Neutral Detergent Insoluble Nitrogen CARBOHYDRATE ANALYSIS IN HORSE NUTRITION The neutral detergent insoluble nitrogen, or NDICP, Neither the ruminant (Van Soest et al., 1991) nor human measured without the use of sodium sulfite represents ADIN (Englyst and Cummings, 1990) systems of carbohydrate plus insoluble fibrous proteins and cell wall protein. The dif- classification and analysis fit well with the digestive physi- ference between NDIN and ADIN represents an estimate of ology and metabolism of horses (Hoffman, 2004). The ru- the slowly degraded protein in the rumen or bypass protein minant system of carbohydrate analysis will divide carbohy- that can be digested in the lower digestive tract (Van Soest, drates as cell wall (measured as NDF) and cell contents 1994; Licitra et al., 1996). The NDIN or NDICP as a per- (measured as NSC). On the other hand, the human system centage of NDF for certain feeds can be as high as 40 per- uses fractions that can be applied to the horse such as TDF cent (Weiss et al., 1989) and between 8 and 12 percent of the and NSP. However, NSP ignores the lignin and lignocellu- NDF for unheated forages (NRC, 2001). lose fractions present in horse diets that might have an effect on retarding the rate of fermentation. Hoffman et al. (2001) suggested that it would be helpful to develop a scheme of Amino Acid Analysis proximate analysis components for the horse that better re- Most amino acids in feeds and feed ingredients are mea- lates to the digested fractions. The authors proposed to use sured by AOAC methods (e.g., 994.12; AOAC, 2002). Sam- the following terminology: (1) hydrolysable carbohydrates ples are derivatized and amino acids measured using high (CHO-H) measured by direct analysis (e.g., Davis, 1976; performance liquid chromatography (HPLC). This method Smith, 1981); and (2) fermentable carbohydrates (CHO-F) can be applied to measure alanine, arginine, aspartic acid, that can be divided into rapidly fermentable carbohydrates cystine, glutamic acid, glycine, histidine, isoleucine, (CHO-FR) and slowly fermentable carbohydrates (CHO-FS).

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208 NUTRIENT REQUIREMENTS OF HORSES Values for CHO-FR are calculated as the difference between by NIRS due to association with organic molecules (Shenk NFC and CHO-H, representing gums, mucilages, β-glucans, and Westerhaus, 1994). Biological components such as pectins, and/or storage polysaccharides such as fructans. In ADIN might be more difficult to measure using NIRS be- the Hoffman et al. (2001) system, CHO-FS is equated with cause of its complex composition. the NDF values of the Van Soest et al. (1991) ruminant system. The breakdown of dietary carbohydrates is given in Figure 10-1 (adapted from Hall, 2003 and Hoffman, REFERENCES 2004). AACC. 1995a. Method 32-05: Total Dietary Fiber. Approved Methods of the American Association of Cereal Chemists, 9th ed. St. Paul, MN: American Association of Cereal Chemists. FEED ANALYSIS USING NEAR INFRARED AACC. 1995b. Method 32-07: Determination of Soluble, Insoluble and REFLECTANCE Total Dietary Fiber in Foods and Food Products. Approved Methods of the American Association of Cereal Chemists, 9th ed. St. Paul, MN: Near infrared reflectance spectroscopy (NIRS) relates a American Association of Cereal Chemists. sample’s reflectance of near infrared light to its chemical Abrams, S. M., J. S. Shenk, M. O. Westerhaus, and F. E. Barton II. 1987. composition. It relies on prediction equations of nutrient Determination of forage quality by near infrared reflectance spec- levels rather than direct measurements. The technique was troscopy (NIRS): efficacy of broad based calibration equations. J. Dairy Sci. 70:806–813. first applied to measure moisture content and was devel- Akin, D. E., and A. Chesson.1989. Lignification as the major factor limit- oped at the U.S. Department of Agriculture, Beltsville, ing forage feeding value especially in warm conditions. Pp. 1753–1760 Maryland (Norris, 1964). In the last 20 years, NIRS has in Proc. 16th Intl. Grassl. Congr., October 4–11, Nice, France, French gained acceptance as an analytical tool for feeds and for- Grassl. Soc. ages because of its quickness, low operating costs, safety, Andrieu, J., and W. Martin-Rosset. 1995. Chemical, biological and physi- cal (NIRS) methods for predicting organic matter digestibility of for- and accuracy to measure chemical composition. The near ages in horse. Pp. 76–77 in Proc. 14th Equine Nutr. Physiol. Soc. infrared region of the spectrum (700 nm to 2,500 nm) in- Symp., Ontario, CA. cludes molecular absorptions of overtone and combination Andrieu, J., M. Jestin, and W. Martin-Rosset. 1996. Prediction of the or- bands (Birth and Hecht, 1987). Since 1973, with the appli- ganic matter digestibility (OMD) of forages in horse by near infrared cation of NIRS to the analysis of cereals and oilseeds spectrophotometry (NIRS). P. 299 in Proc. of the 47th European Assoc. (Williams, 1975), NIRS technology has become widely ac- of Animal Production, Lillehammer, Norway AOAC Method 920.29. 1990. Fat (Crude) or Ether Extract in Animal Feed. cepted in food analysis. Its potential for rapidly evaluating Official Methods of Analysis, 15th ed. Gaithersburg, MD: Assoc. Offi- forage quality was first demonstrated by Norris et al. cial Anal. Chem. (1976). A complete review of the NIRS application to for- AOAC Method 989.03. 1990. Fiber (Acid Detergent) and Protein (Crude) age analysis has been given by Shenk and Westerhaus in Animal Feed and Forages: Near-infrared Reflectance Spectroscopy (1994). Most feed testing laboratories will offer analysis of Method. Official Methods of Analysis, 15th ed. Gaithersburg, MD: Assoc. Official Anal. Chem. feeds and forages by NIRS and at a lower cost. The AOAC AOAC Method 920.40. 2002. Starch in Animal Feed. Official Methods of official method of analysis 989.03 (AOAC, 1990) describes Analysis, 17th ed. Gaithersburg, MD: Assoc. Official Anal. Chem. the application of NIRS to measure protein and ADF in AOAC Method 948.02. 2002. Starch in Plants. Official Methods of Analy- feed and forages. NIRS is also applied to measure NDF or sis, 17th ed. Gaithersburg, MD: Assoc. Official Anal. Chem. aNDF of feed and forages (Martin et al., 1989). Because all AOAC Method 954.02. 2002. Fat (Crude) or Ether Extract in Pet Foods. Of- ficial Methods of Analysis, 17th ed. Gaithersburg, MD: Assoc. Official energy-yielding components of feeds absorb in the near in- Anal. Chem. frared region of the spectrum, NIRS has the potential to be AOAC Method 962.09. 2002. Fiber (Crude) in Animal Feed and Pet Food. a fast alternative method to predict metabolizable energy in Official Methods of Analysis, 17th ed. Gaithersburg, MD: Assoc. Offi- feeds. Thus, Valdes and Leeson (1992, 1994) successfully cial Anal. Chem. applied NIRS to measure apparent metabolizable energy in AOAC Method 976.05. 2002. Protein (Crude) in Animal Feeds and Pet Foods. Official Methods of Analysis, 17th ed. Gaithersburg, MD: poultry feeds and feed ingredients, reducing the need and Assoc. Official Anal. Chem. the cost for bioassays. NIRS has been used to estimate ani- AOAC Method 976.06. 2002. Protein (Crude) in Animal Feeds and Pet mal response and, according to Abrams et al. (1987), NIRS Foods. Official Methods of Analysis, 17th ed. Gaithersburg, MD: could be more accurate in predicting animal response than Assoc. Official Anal. Chem. AOAC Method 977.02. 2002. Nitrogen (Total) (Crude Protein) in Plants. any single reference method or combination of these meth- Official Methods of Analysis, 17th ed. Gaithersburg, MD: Assoc. Offi- ods. The application of NIRS to predict organic matter di- cial Anal. Chem. gestibility of forages fed to horses has been reported by An- AOAC Method 988.15. 2002. Tryptophan in Foods and Food and Feed drieu and Martin-Rosset (1995) and Andrieu et al. (1996). Ingredients—Ion Exchange Chromatographic Method. Gaithersburg, NIR predictions of hydrolysable carbohydrates in forages MD: Assoc. Official Anal. Chem. AOAC Method 991.43. 2002. Total Soluble and Insoluble Dietary Fiber in have been reported by Hoffman et al. (2001) and by Jafari Foods. Official Methods of Analysis, 17th ed. Gaithersburg, MD: et al. (2003). Ideally, minerals should not be analyzed using Assoc. Official Anal. Chem. NIRS as they do not absorb light energy in the near infrared AOAC Method 994.12. 2002. Amino Acids in Feeds. Official Methods of region. However, some minerals can be measured indirectly Analysis, 17th ed. Gaithersburg, MD: Assoc. Official Anal. Chem.

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