AFS_V57_1996_FDNcorrigido

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A NIM A L FEEDSC IENC E A NDTECHN O LO G Y

ELSEVIER Animal Feed Science Technology 57 (199 6) 347-358

Standardization of procedures for nitrogenfractionation of ruminant feeds

G. Licitra a, T.M. Hernandez b, P.J. Van Soest bp*aUniuersitci di Catania, I.S.T.PA. - Progetto IBLEO, Via Val di Savoia 5, 95123 Catania, Italyb Cornell University, 324 Morrison Hall, Ithaca, NY 14853 USA

Received 1 November 1994;accepted 26 A pril 1995

AbstractThe Cornell N et Carboh ydrate Protein M odel (Chalupa et al., 1991 ; Sniffen et al., 1992) has

developed the need for uniform procedures to partition feed nitrogen into A, B, and C fractions(Pichard and Van Soest, 1977 ). While carbohyd rate fractions are relatively standardized (based onND F, AD F with corrections for ash, protein, and lignin), the fractionation of plant nitrogen hasbeen open to considerable variation in procedu res. T his has led to non-uniformity among rep ortedvalues for nitrogen fractions. This paper recomm ends reliable pro cedures for nonprotein nitrogen(NP N) and buffer-soluble protein. These procedures have been examined for reproducibility andrelevance to biological expectations. P rocedure s for acid-detergent insoluble nitrogen (AD IN), andneutral-detergent insoluble nitrogen (ND IN) am also included as they are required for the model.Som e alternatives in certain procedures are offered.

1. IntroductionSpecific information on the contents of nonprotein nitrogen (NP N), true protein,

protein degradability, cell wall bound protein, etc. are dependent on preliminarypreparation and separation of nitrogenous components in the sample. T he separation ofprotein and nitrogen fractions as used by the Cornell Net Carboh ydrate Protein Mo del isshown in Table 1. Nonprotein nitrogen is denoted as the A fraction while true protein isbroken down into B,, B, and B, fractions based on decreasing solubility. The respectivefractions are dependent upon the estimation of insoluble nitrogen, true protein, and thenitrogen residual in ADF and NDF. T he nitrogen that is insoluble in acid detergent is

* Corresponding author.

0377-8401/%/$15.00 0 1996 Elsevier Science B.V. All rights reservedSSDI 0377-8401(95)00837-3


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348 G. Licit ra et al./Anim al Feed S cience Technology 57 (1996) 347-358

Table 1Partition of nitrogen and protein fractions in feedstuffsFraction Abbr. Estimation or definition Enzym atic Classification a

Nonprotein N NPN Not precipitableDegradationNot applicable ATrue protein

Tme soluble proteinInsoluble proteinNeutral detergent solubleproteinND insoluble protein, butsoluble in ADInsoluble in acid deter-gent

TP

BSPIPIP-NDIP

NDIP-ADIPADIP orADIN

Precipitate with tungsticacidBuffer soluble but precip- Fast B,itable (TP-IP)Insoluble in bufferDifference between IP Variable B2and protein insoluble inneutral detergent (ND)Protein insoluble in ND Variable to Slow B,but soluble in acid deter-gent (AD)Includes heat-damag ed Indigestible Cprotein and nitrogen asso-ciated with lignin

a According to Pichard and Van Soest, 197 7, and Van Soest, 199 4.

denoted as the C fraction, and is assum ed to be indigestible. A series of preparatoryprocedures to obtain respective NPN and protein components follow with some altema-tives.

2. Nonprotein nitrogen OWN)2.1. M a t e r i a l s a n d m e t h o d s2.1 .I. Def in i t i on

Nonprotein nitrogen is traditionally the nitrogen passing into the filtrate afterprecipitation with a protein specific reag ent. Feed stuffs may contain a wide variety oflow molecular weigh t nitrogenous substances (Hug hes, 1970; Hegarty and Peterson,1973). Another issue is the inclusion of peptides in NP N. Krishnamoorthy et al. (1982)used trichloroacetic acid (TCA) w hich includes them in NP N since they are notprecipitated. The view here is that they are metabolically closer to soluble protein. M anyrumen bacteria (particularly cellulolytics) do not take up single amino ac ids, but requirepeptides, an observation that favors the tungstic acid method which recovers peptides inthe protein precipitate (Greenberg and Shipe, 1979). Certain protein sources particularlyfish meal and blood-feather-meat-bone mix exhibit important differences between thetwo methods indicating presence of peptides. The TCA method is included here as itmay be relevant to a future partition of the B, class into peptides and soluble protein.2.1.2. R ev iew o f p rocedures fo r de t e rm ina t ion

T h e determination of NPN depends upon precipitation of true protein by a suitableprecipitant, filtration and determination of the insoluble nitrogen in the residue. The


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G. Licitra et al. /Animal Feed Science Technology 57 (1996) 347-358 349

NP N is calculated as the difference between the total crude protein nitrogen and thevalue of the precipitated true protein nitrogen. A variety of precipitating agents forprotein have been employed (Haw k et al., 1947; Greenberg and Shipe, 197 9). Theseinclude tungstic acid, trichloroacetic acid, copper hydroxide, zinc-barium hydroxide andothers. The choice of method depends on the kind of procedure and objectives beingfollowed. Tungstic an d trichloroacetic acids (TCA ) are the most common precipitantsapplied to feeds. As mentioned, these reagents differ in respect to the cut-off inmolecular size. Tungstic acid cuts off at about a peptide size of 3 amino acids, while theTCA cuts off around 10 amino ac ids, depending on the amino ac id profile of the peptide(Greenberg and Shipe, 1979 ; M arais and Evenwell, 1983).2.1.3. R ecom m ended procedure for de t e rmina t ion o f N PN us ing tun gs t i c ac id2.1.3.1. Apparatus. Erlenmeyer flask (125 ml>, W hatm an #54 or 541 filter paper,analytical balance, pH meter, filter funnels, Kjeldahl apparatus.2.1.3.2. Reagen t s .1. Sodium tungstate (Na,W0,2H ,O) (100 g 1-l) solution in water, 0.30 M.2. 0.5 M sulfuric acid (H,SO,).

W eigh 0.5 g dry ground sam ple into a 125 ml Erlenmeyer flask.Add 50 ml of cold distilled water.

2.1.3.3. Procedure.1.2.3.4.5.6.7.

Add 8 ml of 10% sodium tungstate solution .Let flask stand at 20-25C for 30 min.Bring pH to 2 by adding 10 ml of 0.5 M sulfuric acid (check pH w ith pH meter).Let flask stand overnight at room temperature.Fold W hatman #54 or 541 filter paper and place in a conical funnel. Thoroughlywet paper with distilled water before adding any sample. Filter by gravity; or withmild vacuum. If first filtrate is cloudy return to the filter funnel and refilter. Ifvacuum is used, separate filter flasks must be used, so that any cloudy filtrate can berecycled through the funnel.Wash residue twice with cold distilled water.Transfer paper to Kjeldahl flask and determine residual nitrogen.Calculate NPN by subtracting residual nitrogen from total nitrogen. Value of NPNmay be expressed as crude protein (N X 6.25) or as percent of total feed nitrogen.

8.9.10 .

2 .1 .4 . A l t e rna t i ve de t ermina t ion o f N PN us ing t r i ch loroacet i c ac id (TCA )2.1.4.1. Apparatus.1. Erlenmeyer flask (125 ml), Whatm an #54 or 541 filter paper, analytical balance,filter funnels, Kjeldabl apparatus.

Samples of low protein content ( < 20% CP) can be treated with 5 ml of sodium hmgstate solution and6-7 ml of 0.5 M sulfuric acid.


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350 G. Licit ra et al./Anim al Feed Science Technology 57 (1996) 347-358

Table 2Comoarison of NP N values. All values as oercent (N X 6.25) of drv matterFeeds Crude NPN by hmgstic acid NPN by

protein old method l/2 h b ppt 16 h 3 h ppt 16 h trichloro-acetic acidAlfalfa silage 20.8 9.2 9.4 8.9 9.8Blood feather meat bone mix 80.6 3.2 0.2 1.3 5. 6Cottonseed meal 45.1 2.8 3.2 3.3 4. 4Distillers grains 26.4 2.9 2.4 2.2 2. 2Fish meal 64.3 9.8 4.4 6.5 14.1Grass silage 10.9 4.2 3.9 3.1 4. 3Linseed meal 33.8 0.2 0.9 0 2.3Soybean meal 48.4 1.4 1.1 0 1.9Trypticase 84.4 62.5 58.4 52.4 83.9

a 10 ml of 0.3 M hmgstic acid are used (30 m in) pH adjusted to pH 2 with 0.5 M H ,SO,. Let stand 30 minbefore filtering.b Soak in water with 8 ml 0.3 M N a,WO , 30 min, add acid to pH 2, set overnight before filtering. Soak in water with 8 ml 0.3 M N a,WO , for 3 h, add acid to pH 2; set overnight before filtering.

2.1.4.2. Reagents.1. Trichloroacetic acid 10% w/v in water. Keep refrigerated!2.1.4.3. Procedure.1. Weigh 0 .5 g ground dry samp le into a 125 Erlenmeyer flask.2. Add 50 ml of distilled water. Allow to stand 30 min.3. Add 10 ml 10% trichloroacetic acid. Let stand 20-30 min.4. Filter on W hatm an #54 or 541 paper by gravity.5. W ash twice with trichloroacetic acid solution.6. Transfer paper to Kjeldahl flask and determine residual nitrogen.7. Calculate NPN as in the tungstic acid procedure.2.2 . R esul ts and discuss ion

A comparison of trichloroacetic acid and tungstic acid precipitations is shown inTable 2 . A hydrolyzed protein source like trypticase (a tryptic digest of casein) producesno precipitate with TCA, but gives an insoluble fraction of about 52% with tungsticacid. The tungstic acid value is variable depending upon conditions. Several physico-chemical factors can be discerned in this variability. One is the length of time needed inthe mildly alkaline tungstate extraction to solubilize protein. This could be a problem indried feeds, as polymers like proteins need time to swell and dissolve. If this isinadequate, underestimation of soluble protein results. A second factor is the final pHand time of the acidic precipitation. Recom mend ation is to check pH in case of highlybuffered feeds. Overnight precipitation decreases the NPN estimate and signifies morecomplete precipitation.

Precipitates of soluble protein by tungstic acid are finely divided and sometimesdifficult to filter. There is a danger that fine material may pass the filter and time of


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G. Licitra et al./Anim al Feed Science Technology 57 (1996) 347-358 351Table 3Comparison of NPN values obtained by filtration with and without vacuum (value in percent of crude protein)Feeds With

vacuumWithoutvacuum

Withoutvacuumand cover

Mean P value

TrypticaseConcentratesSoybeanRaw soybeanFish mealCorn mealCorn glut. FBrewersHaysAlfalfaVetchSilagesHay silageCorn silageTriticaleGreen ForagesAlfalfaVetchBarley

56.06 55.81 56.17 56.01 0.87542.43 2.05 2.21 2.242.84 3.13 3.05 3.00

11.67 10.38 10.02 10.690.69 0.69 0.93 0.77

11.00 10.62 10.60 10.740.07 0.27 0.22 0.196.21 6.58 6.13 6.29 0.00696.49 6.40 6.43 6.44 0.95087.45 7.40 7.47 7.44 0.55004.32 4.39 4.53 4.42 0.83005.13 5.10 5.21 5.14 0.66906.67 6.16 6.62 6.48 0.26005.53 5.67 5.28 5.50 0.15295.48 5.17 5.29 5.32 0.4378

0.42880.01660.323 10.00010.3400

a Statistical analysis by SAS u sing tbe general linear model.

filtration can be lengthy. A comparison of filtering by gravity and with mild vacuum isshow n in Table 3. Filtration using vacuum can lose up to 10 percent of the solubleprotein, w hich is recoverable if the first filtrate containing any cloudy matter is returnedto the funnel. Thus individual filtration flasks must be used. If samples are filtered bygravity and the time is very long, funnels need to be covered to avoid evaporation whichcan lead to variable results (see Table 3). Centrifugation as an alternative procedurerequires more steps in the preliminary preparation, especially in the case of forages thatdo not form definite pellets. To get a good pellet with forages require pretreatment withultrasonication under m ild vacuum , to take out most of the air trapped in forage structure(P. Schofield personal co mm unication). A swinging bracket centrifuge at 5000 rpm for15 min. at 4C is required. After decan ting the supem atant, the pellet is resuspended indistilled water (15 ml) and recentrifuged.

3. Soluble nitrogen and protein3.1. Materials and methods

3.1 .I. DejnitionSoluble protein is defined here as true protein that is soluble in buffer at rumen pH.This definition differs from others in that NPN components are excluded from the


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fraction. The procedure offered here is for total insoluble nitrogen which in comb inationwith a measu rement of NP N allows estimation of soluble true protein by difference.3.1.2. Review of procedures for determination.

Soluble crude protein (nitrogen) is a simplistic concept that evolved out of theobservation that most soluble nitrogen com ponents were rapidly degraded in the rumen,and therefore, reduced protein that could be passed to the lower tract. The earlyprocedures of W ohlt et al. (1973); W aldo and Goering (1979); Crooker et al. (1978)claim to measure soluble protein, the soluble fraction in fact includes NPN, whichsupports the need for a determination of NP N as well as soluble protein and peptides(Section 2) (Krishnamoorthy et al., 1982; Roe et al., 1990). These procedures wereapplied from a biological point of view, which attempted to mim ic rumen environm ent,i.e., use of rumen type buffers (M cDo ugalls solution) high in bicarbonates, in additionto incubation at physiological temperature (37C). Bicarbonate buffers are unstableevolving C O, with pH rise. Krishnamoo rthy et al. (1982) introduced a borate-phosphatebuffer to insure pH stabilization. This buffer is used in this study. Incubation atphysiological pH of nonsterile feeds provides the opportunity of microbial growth an dutilization of nitrogenous feed compon ents, as well as activation of indigenous enzymespresent in the sample. None of these factors were examined in the original work for theireffectiveness or their necessity. In these papers it wa s generally assum ed that adherenceto physiological conditions of the rumen w ould ensure a correct biological measuremen t.3.1.3. Recommended procedure for soluble protein (buffer-soluble nitrogen)3.1.3.1. Apparatus.1. Erlenmeyer flask (125 ml>, W hatm an #5 4 or 541 filter paper, analytical balance,

waterbath, vacuum source, filter manifold fitted with conical funnels (50 ml),Kjeldahl apparatus.

3.1.3.2. Reagents.1. Borate-phosphate buffer, pH 6.7-6.8 including

1.1. monosodium phosphate (NaI-I,PO,.H,O) 12.20 g 1-i1.2. sodium tetraborate (Na,B,O,.lOH,O) 8.91 g 1-l1.3. tertiary butyl alcohol 100 ml 1-l

2. So dium az ide 10 % solution freshly prepared.3.1.3.3. Procedure.1.2.3.4.5.6.7.

W eigh 0.5 g ground dry sample into a 125 ml Erlenmeyer flask.Ad d 50 ml borate-phosphate buffer.Ad d 1 ml of sodium azide solution.Let stand at room temperature for 3 h.Filter through W hatm an #54 or #541 filter paper using mild vacuum.Wash the residue with 250 ml cold distilled water.Estimate N in residue by Kjeldahl. This gives the insoluble protein fraction. Solubleprotein is calculated by difference from total crude protein. The soluble true protein


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Table 4Comparison of protein solubility obtained by varying the temperature and the hours of incubation (value inpercent of crude protein) Soak time 3h 3h 3h lh Mean 2 P valueIncub. temp. room temp temp 4C temp 37C temp 37CFEEDSCorn silage 702 29.53 28.06 30.67 31.03 29.80 0.023Corn silage 7 14 48.91 50.69 49.54 51.60 50.18 0.012Corn silage 570 50.15 49.39 49.28 50.15 49.74 0.45 ICorn silage 627 62.27 62.27 62.45 62.73 62.43 0.975Corn silage 70 1 61.66 61.72 62.12 60.56 61.51 0.479Brewers 30.24 29.55 31.92 30.67 30.59 0.010Soybean meal 44% 6.43 5.49 10.65 7.97 7.64 0.001Raw soyean 60.35 61.32 59.50 63.17 61.08 0.094Linseed whole 24.45 21.91 26.48 26.44 24.83 0.001Corn gluten feed 67.40 67.90 67.06 66.71 67.26 0.009Barley green forage 31.29 28.83 29.40 30.52 30.01 0.001Alfalfa foragegreen 31.23 31.58 31.92 30.54 31.32 0.058Vetch foragegreen 35.98 34.57 36.80 36.76 36.03 0.001Overall experiment 41.39 a 41.07 a 42.26 b 42.37 b 41.81 0.0001 The fmal wash has been done with water for all the samples; Tertiary butanol and sodium azide have beenincluded in the buffer.2 Statistical analysis by SAS using the general linear model.ab Mean values with different superscripts are significantly different.

can be obtained by subtracting the NPN by tungstic acid procedure, Section lc. Notethat when tungstic acid is used the soluble protein will include the shorter peptides.3.2. Resu l t s and d i scuss ion

A comparison of the effect of temperature and time upon protein solubility is shownin Table 4. Borate-phosphate buffer was used to minimize drift in pH during incuba tion.Protein solubility was significantly different amon g treatments (P < 0.001). Som ewha thigher values w ere obtained at 37C . There was no statistical difference between theprotein solubilities obtained at room temp erature compared to 4C .

In these comparisons it is assumed that the analyses of feed introduced into afermentation system will represent the feed as presented to the rumen. Therefore, anybiological activity whether microbial or enzymatic that will influence the laboratoryvalue is irrelevant to the biological interpretation. The analytical requirement is toprovide an estimate of what was actually introduced into the rumen.

Roe et al. (1990) chose incubation at 37C because it was assumed that conditions ofthe assay need to conform to that of the rumen. This leads to a conflict betweenanalytical criteria and biological relevance. Incubation at 37C in a non sterile systemwill lead to confounded results from (a) fermentation during the incubation and (b)indigenous enzymatic activity provided by the samples. Sodium azide was used tocontrol microbial g rowth. However, there is no easy way to inhibit indigenous enzymesother than incubation a t lower temperatures. Incubation at physiological temperature


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Table 5Comparison of protein solubility obtained by washing the residue with cold distilled water and with buffersolution (all values ate in percent of total nitrogen)Feeds Dist. water a SD Buffer solution a SDCorn silage 62.21 1.33 63.41 1.06Soyabean meal 60.35 2.22 59.26 0.95Corn gluten meal 67.40 0.16 68.56 0.06Alfalfa foragegreen 31.23 0.25 31.03 0.29Vetch foragegreen 35.98 0.21 36.21 1.11 Each combination represents the average results of three determinations. Samp les were incubated for 3 h atroom temperature.

may not be stable relative to indigenous biological activity. As noted feeds may containenzymes as well as microorgan isms. Attem pts to sterilize by heat will induce furtherproblems by altering the sample through denaturation reactions involving proteins thatcan lead to unrealistic resu lts. Proteolytic activity will increase protein solub ility, whilemicrobial activity in most plant products would use soluble nitrogen by using it formicrobial gro wth. As with the NP N determination there is a need to maxim izeextraction, leading to a longer (3 h) extraction time that risks the biological hazards ofhydrolysis and microbiological growth.

There is no advantage in using buffer in the final wash (Table 5). Variation betweenthe wa sh treatments is not statistically significant. The procedure of choice is theincubation at room temp erature (2 0-25C ) wh ich involves the least work and equip-ment. Tertiary butanol has been included to facilitate wetting of the feed although it mayalso serve as an inhibitor in some feeds.

4. Determ ination of acid-detergent insoluble nitrogen (ADIN)4.1. Materials and metho&4.1.1. Definition

It is not possible to completely extract all nitrogen from plant cell w all. A residualcore appears to be resistant, indigestible and associated with lignin even in fresh foragesthat do not contain tannins. Tannins, if present, are one possibility for increasedinsoluble protein associated with plant cell wall. Another is the Maillard or nonenzy-matic browning reaction caused by heating and drying. These fractions have lowbiological availability and tend to be recovered in acid-detergent fiber (Van Soest, 1965;Van Soest and Mason, 1991).

Heat-drying of forages at temperatures above 6&C sho ws analytically significantincreases in yield of lignin and fiber. The increased yield of acid-detergent fiber (ADF)can be accounted for largely by the production of artifact lignin via the nonenzymicbrowning reaction (Van Soest, 1965). The nitrogen content of the ADF is suggested as asensitive assay for nonenzymic browning due to overheating of feeds (Van Soest andMason, 1991).


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5. Leave in the forced draft oven for 4 h, or better overnight.6. Weigh the hot crucible with AD residue, record the weight.7. Wash residue from crucible to W hatm an #5 4 or #541 filter paper with distilled

water. Suck dry and transfer to a Kjeldahl flask.8. Return empty crucible to oven, dry and reweigh. Calculate sample weight as thedifference b etween weight in step 6-step 8.9. Estimate nitrogen in residue by standard Kjeldahl.

10. Titrate distillate with 0.01 N standard acid.4.2. Discuss ion

The procedure for ADIN has not been studied for analytical variation in this paper;however, it is included here because original publication US DA Handbo ok 379 (Goeringand Van Soest, 1970) is no longer in print. In addition these procedures combine therecomm endations of the AO AC study (Van Soest, 1973) with that of the nitrogendetermination.

Manual filtration on paper facilitates transfer to Kjeldahl digestion. However, analternative method is provided using the Fibertec apparatus where transfer from asintered g lass crucible h as to be accom plished. If this is done w eighing the crucible aftertransfer is necessary because a complete transfer is hardly possible.4.3. Determina t ion o f neu t ra l -det e rgen t i n so lub le n i t rogen (N DIN )4.3.1. Dejinit ionT h e nitrogen associated with NDF is normally cell wall-bound protein which alsoincludes the indigestible nitrogen found in the acid-detergent residue. The proteininsoluble in the neutral-detergent solution, but soluble in acid-detergent is digestible, butslowly degradable and has been termed the B, fraction in the Cornell Net CarbohydrateProtein M odel. Generally the cell wall-associated protein is extensin covalently linked tohemicellulosic carbohydrate (glycoproteins) that are involved in cross linking carbohy-drate chains in plant cell wa lls (Fry, 1988). Hea ting dena tures B , proteins an d mayrender them insoluble thu s increasing the B, fraction as well as the C fraction obtainedas the ADIN.4.3 .2 . R eview of procedures

Krishnamo orthy et al. (1982) determined ND IN by preparing ND F filtration on paperfollowed by Kjeldahl determination of nitrogen. The variations in this procedure dependon which procedure for NDF was followed. There are no less than 14 publishedvariations on ND F procedures (Mascarenhas-Ferreira et al., 1983; Van Soest et al.,1991). The procedures published here follow that of Van Soest et al. (1991).

The determination of ND F has the optional use of sodium sulfite to reduce proteincontent of ND F. Sulfite cleaves disulfide bridges in cystine. B ecause this is not abiological p ossibility, use of sodium sulfite is precluded. For example, sulfite willdissolve keratins (skin, hair etc. from anima l products tha t are completely indigestible)(Van Soest and W ine, 1967). Similarly the use of urea-amylase to remove resistant


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starch cannot be used, because the chaotropic character of 8 molar urea will dissolveproteins belonging in the B, fraction (Van Soest et al., 1991).4.3.3. R ecom m ended procedure us ing m anua l j l t ra t ion on paper

T h e manual procedure follows exactly as that for acid-detergent insoluble nitrogenexcept that neutral-detergent solution is substituted for acid-detergent reagent (Van Soestet al., 1991). Sodium sulfite is omitted. Pretreatment with urea-amylase should not bedone, although amylase may be used in the boiling step to aid filtration. Filtration ispreferable on Whatman 54 paper, although the alternative procedure using sintered glasscrucibles may be followed as described in Section 4.4.3 .4 . A l ternat e procedure for neut ral -detergent insolu ble n i t rogen u s ing Fiberteca p p a r a t u s

T h e procedure using the Fibertec apparatus for insoluble nitrogen in neutral-detergentsolution is the same as described for AD IN except that neutral-detergent reagent issubstituted for acid detergent. Amylase is added at the beginning in the case of starchyfoods. This procedure has the disadvantage that removal of sample from crucible may beincomplete and it is necessary to reweigh the crucible after transfer to paper as inprocedure 4.3.2.4.3.5. Discussion

This procedure unlike that for ADIN was not included in the Agriculture handbook379. How ever, it wa s conducted by Krishnamo orthy et al. (1982). The proceduredescribed here utilizes the latest recomm endations for ND F (Van Soest et al., 1991 1,filtration on paper followed by nitrogen determination by Kjeldahl.

AcknowledgementsThe help of Stefania Carpino, Francesca Lauria, Elisa Tumino, Patrizia Campo and

other Staff of the Progetto IBLEO, I.S.T.P.A. Facolta di Agraria, University of Cataniaat Rag usa, Sicily who performed some of the comparative analyses and helped on thestatistical evaluation of the results, is gratefully acknow ledged. This work w as supportedin part by the Sicilian Government Agriculture Department who funded the ProgettoIBLEO.

ReferencesChalupa, W., Sniffen, C.J., Fox, D.G. and Van Soest, P.J., 1991. Model generated protein degradation

nutritional information. Proc. Cornell Nutr. Conf., Dept.Animal Science, Cornell University, Ithaca, NY.pp. 44-51.

Crooker, B.A., Sniffen, C.J., Hoover, W.H. and Johnson, L.L., 1978. Solvents for soluble nitrogen measure-ments in feedstuffs. J. Dairy Sci., 61: 437-447.

Fry, S.C., 1988. The Growing Plant Cell Wall: Chemical and Metabolic Analysis. Wiley, 352 pp.Goering, H.K. and Van Soest, P.J., 1970. Forage Fiber Analyses (Apparatus reagents, procedures, and someapplications). Agric. Handbook No. 379. ARS USDA, Washington DC, pp. 20.


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