Estimating ruminal crude protein degradation with
in situ and chemical fractionation procedures
S. Shannak, K.-H. SuÈdekum
*, A. Susenbeth
Institut fuÈr TierernaÈhrung und Stoffwechselphysiologie, Christian-Albrechts-UniversitaÈt, D-24098 Kiel, Germany
Received 28 September 1999; received in revised form 17 March 2000; accepted 24 March 2000
Abstract
The objective of this study was to utilize the fractionation of feed crude protein (CP) of the Cornell net carbohydrate and protein system (CNCPS) as a basis for estimating undegraded dietary protein (UDP) values of feedstuffs obtained from in situ trials. In addition, the experiments comprised a comparison between in situ UDP values of feedstuffs and CP solubility estimated from the protein dispersibility index. Eleven dairy compound feeds and 21 feedstuffs were inserted in polyester bags and incubated in the rumen of three steers. Values for in situ UDP at assumed ruminal passage rates of 2, 5, and 8% hÿ1, respectively, ranged from 63 to 616, 129 to 785, and 167
to 842 g kgÿ1 of CP. When ®sh meal data (n2) were excluded from the data set, multiple regression equations that were based on concentrations of CP and cell wall, and on the A, B, and C fractions of the CNCPS fractionation schedule, explained 87, 93, and 94%, respectively, of the variation in UDP values at assumed ruminal passage rates of 2, 5, and 8% hÿ1. We conclude that in situ UDP values, which serve as one key variable in many protein evaluation systems for dairy cattle, may be reliably and accurately predicted from chemical fractionation of feed CP according to the CNCPS. The coef®cients of determination of estimating UDP values at assumed ruminal passage rates of 2, 5, and 8% hÿ1, respectively, from the protein dispersibility index were only 0.30, 0.29, and 0.33. Hence, the protein dispersibility index was not suitable as a predictor of UDP values for the feedstuffs used in the present study.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Rumen; Protein degradation; Methods; Compound feeds; Feedstuffs 85 (2000) 195±214
*Corresponding author. Tel.:49-431-880-2538; fax:49-431-880-1528. E-mail address: suedekum@aninut.uni-kiel.de (K.-H. SuÈdekum)
1. Introduction
A new system for the estimation of the protein value of feedstuffs for dairy cattle was recently introduced in Germany (Gesellschaft fuÈr ErnaÈhrungsphysiologie, 1997). Key variable in the system is the amount of total crude protein (CP) reaching the duodenum (`nutzbares Rohprotein', nXP), which was estimated from in vivo trials on duodenally cannulated dairy cows (Lebzien et al., 1996). The CP in the digesta at the beginning of the small intestine consists of both the ruminally synthesized microbial CP and the feed CP that has escaped ruminal degradation, i.e. undegraded dietary protein (UDP), besides a varying proportion of endogenous CP. Although UDP values for a large number of feeds are existing, there are considerable gaps in regard to reliable data, in particular for concentrate ingredients. The German feed tables for ruminants (UniversitaÈt Hohenheim-Dokumenta-tionsstelle, 1997) contain values that were obtained by three different approaches:
(a) In vivo from experiments using duodenally cannulated dairy cows; (b) in situ using ruminally cannulated animals, and (c) for feedstuffs where no UDP values were available, these values were estimated from feeds of the same feed class that were similar in chemical composition with known values of UDP.
In vivo measurement of nutrient digestion requires that animals be surgically prepared with cannulas in the rumen and abomasum or duodenum. In addition, suitable markers are required for calculating ¯ow rate of digesta and for differentiation between microbial and dietary nutrients ¯owing to the small intestine (Stern and Satter, 1982). Endogenous contributions of nutrients are dif®cult to measure but they should be assessed to obtain accurate values of digestion; however, these data are limited. In vivo measurement of nutrient digestion is expensive, labour-intensive, time-consuming, and subject to error associated with use of digesta ¯ow rate markers, microbial markers, and inherent animal variation (Stern et al., 1997). In addition, the use of invasive surgical procedures for nutritional research in general is becoming increasingly unacceptable to the public on animal welfare grounds. Therefore, invasive techniques are not suitable for routine estimation of UDP values on a wide range of feeds (Tamminga, 1979).
In situ procedures, often based on or similar to the basic studies conducted by érskov and McDonald (1979), are well accepted in many countries for estimating the degree of ruminal CP degradation of feedstuffs (Van der Koelen et al., 1992; Cottrill, 1993; Broderick, 1994; Huntington and Givens, 1995; Michalet-Doreau and NozieÁre, 1998). In situ measures can be used to obtain estimates of UDP values of feedstuffs within a relatively short period of time but still this method requires cannulated animals and there is a continuing need for simpler laboratory methods to estimate the protein value of feeds. There is a revived intensive discussion about the accuracy and relevance of the measurement of soluble CP fractions to predict the rumen CP degradation of feedstuffs. The solvent used must simulate solubilization and degradation in the rumen as closely as possible. The protein degradation in the rumen depends not only on the soluble and insoluble proteins but also on the extent of the slowly digestible and indigestible proteins. Many different procedures to determine soluble and insoluble nitrogen or CP in feedstuffs have been published (e.g. Crawford et al., 1978; Crooker et al., 1978; Krishnamoorthy et al., 1982), yet no single method has so far been accepted as being reliably accurate for predicting the rumen CP degradation in feedstuffs.
The primary objective of this study, therefore, was to utilize the fractionation of feed CP of the Cornell net carbohydrate and protein system (CNCPS; Russell et al., 1992; Sniffen et al., 1992) as a basis of estimating UDP values of feedstuffs. Unlike the CNCPS, our approach aimed at determining one single UDP value for each feedstuff from multiple linear regression equations instead of estimating single UDP values for four different feed CP fractions, which are then summed to provide a single UDP value. In addition, our experiments comprised a comparison between in situ UDP values of feedstuffs and CP solubility measured with the protein dispersibility index (PDIS; American Oil Chemists' Society, 1989), one of the simpler, yet standardized and recently more intensively discussed solubility methods to predict the rumen CP degradation of feedstuffs in practice. A preliminary report including parts of the study has been published previously (Shannak et al., 1999).
2. Materials and methods
2.1. Animals
Five 8-year old Angler Rotvieh steers, ranging in weight from 740 to 940 kg, and one 7-year old HinterwaÈlder steer weighing 660 kg were utilized in the experiment. Each of the six steers was ®tted with a 10 cm i.d. ruminal cannula (Model 1C, Bar Diamond, Parma, ID, USA) and housed indoors in individual tie stalls in a temperature controlled room (188C) under continuous lighting. The steers received a mixed diet consisting of two-thirds of long mixed grass-legume hay and one-third of mixed concentrates. The diet was supplemented with a commercial mineral and vitamin mix. Animals were fed the diets according to the Agricultural Research Council (1980) values for maintenance. The daily allotment of feed was offered in two equal meals at 07:00 and 19:00 hours. The steers had continuous access to water. Prior to the experiment, a period of 2 weeks was allowed for dietary adaptation.
2.2. Feedstuffs
Eleven dairy compound feeds and 21 feedstuffs were selected which should re¯ect a typical range of dairy compound feeds and protein-rich ingredients of commercial dairy compounds in Central Europe. Thus, the ingredients listed below were also components of the selected 11 dairy compound feeds (confer Table 1). Ingredients and compound feeds were obtained from different commercial feed mills and feed suppliers. In addition, three samples of one of the main forages used as a winter feed for dairy cows in major parts of Europe, i.e. wilted grass silage, were used for in situ and laboratory evaluations of ruminal CP degradation (alphabetical order; number of feeds per feed group in parentheses):
commercial dairy compound feeds (11; for ingredient composition see Table 1);
fish meal (2);
grass silage (3);
maize gluten feed (2);
palm kernel meal (2);
rapeseed products (4; rapeseed meal, formaldehyde-treated rapeseed meal, rapeseed expeller, and lignosulphonate-treated rapeseed expeller). The formaldehyde-treated rapeseed meal has been previously studied in situ by SuÈdekum and Andree (1997);
soybean meal (4; two soybean meals, formaldehyde-treated soybean meal, and lignosulphonate-treated soybean meal);
soybeans, crushed (3; untreated soybeans, dry-heat-treated soybeans and moist-heat-treated soybeans);
sunflower seed meal (1).
The chemical composition of the 11 compound feeds and 21 feedstuffs is presented in Table 2. Characteristics of rate and extent of ruminal degradation of CP and organic matter of the feeds as related to degree of synchrony of ruminal CP and carbohydrate degradation will be published elsewhere.
2.3. In situ procedure
Ruminal CP degradability was determined using polyester bags (R510, Ankom Technology, Fairport, NY, USA) with a pore size of 5015mm. Triplicate samples of each feed were incubated in the rumen of three steers. About 1.3 g of feed ground to pass
Table 1
Ingredient composition (g kgÿ1) of dairy compound feedsa
1 2 3 4 5 6 7 8 9 10 11
Wheat 80 ± ± 220 180 ± ± 230 204 ± ±
Barley ± 135 260 ± ± 200 200 120 105 ± ±
Oats ± ± ± ± ± ± ± ± ± 190 221
Rye 80 ± 120 ± ± ± ± ± ± 192 230
Molasses 60 60 60 40 30 30 45 26 31 30 30
Dried beet pulp ± 100 ± 100 85 250 216 350 305 ± ±
Palm kernel meal ± ± ± 80 80 ± ± ± ± ± ±
Palm kernel expeller 150 140 ± 80 80 ± 35 ± ± ± ±
Maize gluten feed 372 115 85 180 190 170 100 ± ± ± ±
Maize feed meal ± ± ± ± ± ± ± ± ± 225 240
Wheat gluten meal ± ± ± ± 50 ± ± ± ± ± ±
Sun¯ower seed meal 25 ± ± 30 ± ± ± ± ± ± ±
Fish meal ± 30 160 ± ± ± ± ± ± ± ±
Rapeseed meal ± ± 75 ± 90 100 45 ± 339 344 ±
Rapeseed meal, protected ± ± ± ± ± ± 99 ± ± ± ±
Rapeseed expeller ± ± ± 90 140 ± ± ± ± ± ±
Soybean hulls ± 220 30 ± ± 134 100 ± ± ± ±
Soybean oil ± ± ± ± ± ± ± 5 5 ± ±
Soybean meal ± 110 ± 50 60 100 150 250 ± ± 250
Soybean meal, protected ± ± ± 100 ± ± ± ± ± ± ±
Grass meal, dehydrated ± ± 55 ± ± ± ± ± ± ± ±
Citrus pulp 200 80 150 ± ± ± ± ± ± ± ±
Mineral±vitamin mix 33 10 5 30 15 16 10 18 9 18 25
aThe sum of ingredient concentrations in each row may not equal 1000 g kgÿ1due to rounding off numbers.
Table 2
Chemical composition of 11 dairy compound feeds and 21 feedstuffs incubated in situ in the rumen of steersa
DM
Grass silage 1 361 103 167 166 NAc 597
Grass silage 2 639 178 178 178 NA 554
Grass silage 3 550 157 157 171 NA 464
Palm kernel meal 1 901 53 172 469 1 823
Palm kernel meal 2 890 53 173 475 2 854
Maize gluten feed 1 887 67 210 91 209 375
Maize gluten feed 2 889 69 247 99 149 404
Sun¯ower seed meal 911 77 334 312 4 458
Fish meal 1 923 172 679 NA NA 202
Fish meal 2 923 202 766 NA NA 574
Rapeseed meal 920 73 344 220 56 331
Rapeseed meal, formaldehyde-treatedd
904 78 353 236 12 524
Rapeseed expeller 916 68 358 257 7 321
Rapeseed expeller, lignosulphonate-treatede
896 67 322 273 11 538
Soybeans 912 59 398 163 4 217
Soybeans, dry-heat-treated 932 57 398 157 6 207
Soybeans, moist-heat-treated 922 57 397 162 5 202
Soybean meal 1 907 74 546 62 7 140
Soybean meal 2 916 69 512 102 6 166
Soybean meal,
aDM, dry matter; CP, crude protein; ADF, acid detergent ®bre; PNDF, neutral detergent ®bre determined by
manual ®ltration on paper according to the recommendations of Licitra et al. (1996).
bFor ingredient composition of dairy compound feeds see Table 1. cNA: not analysed.
dBiopro®n1R (Biopro®n sales of®ce, Bramsche, Germany).
eRaPass1(Borregaard LignoTech, Sarpsborg, Norway).
fSoyPass1(Borregaard LignoTech, Sarpsborg, Norway).
gBiopro®n1S (Biopro®n sales of®ce, Bramsche, Germany).
a 2 mm screen were placed in each bag, which was anchored with a 20 cm length of cable binder. Prior to incubation, the bags were soaked in warm water (408C) for 10 min. On Day 1 of incubation, the bags were clamped to an 800 g cylindrical plastic weight, which was tied to an 80 cm long main line tied outside the ®stula. All bags were inserted into the ventral sac of the rumen at 07:00 hours immediately before the morning feeding. Incubation periods were 2, 4, 8, 16, 24, and 48 h. Immediately after removal from the rumen, bags were immersed in ice-water to stop or minimize microbial activity and then washed with cold water in a washing machine for 35 min. Zero time disappearance values (0 h) were obtained by washing pre-soaked, unincubated bags in quadruplicate in a similar fashion. Water-soluble material (WS) was estimated by washing duplicate samples through a folded ®lter paper (No. 5951/2, Schleicher and Schuell, Dassel, Germany). All washed bags and ®lter paper residues were freeze-dried. Water-insoluble CP escaping in small particles (SP) from the bags during washing were estimated by subtracting water-soluble CP from 0 h values. The single values obtained for CP disappearance (DIi) were then corrected (c) for SP by the equation (Weisbjerg et al., 1990):
CDIiDIiÿSP
1ÿ DIiÿ SPWS 1ÿ SPWS
:
Degradation of CP (CDEG) was calculated using the equation of McDonald (1981):
CDEGab 1ÿeÿc tÿL for t>L;
where CDEG is the disappearance at timetcorrected for SP,aan intercept representing the proportion of CP solubilized at initiation of incubation (time 0; soluble fraction),bthe fraction of CP insoluble but degradable in the rumen,cthe rate constant of disappearance of fractionb,tthe time of incubation, andLis the lag phase. The non-linear parametersa, b, c, and L were estimated by an iterative least squares procedure (SAS, 1988). The effective degradability (ED) of CP was calculated using the following equation (McDonald, 1981):
EDa bc
cke
ÿkL
;
wherekis the estimated rate of out¯ow from the rumen anda,b,candLare the same parameters as described earlier. The ED of CP was estimated as ED2, ED5 and ED8 assuming rumen solid out¯ow rates of 2, 5, and 8% hÿ1, which is representative for low, medium, and high feeding amounts (Agricultural Research Council, 1984). Correspond-ingly, values for UDP2 (UDP5, UDP8) (g kgÿ1of CP) were then calculated as 1000-ED2 (ED5, ED8).
2.4. Analytical procedures
2.4.1. General methods
The dry matter of the grass silages and the residues after ruminal exposure was estimated by freeze-drying and subsequent oven-drying at 1058C overnight. The dry
matter of all other feeds was estimated by oven-drying at 1058C overnight. All feedstuffs and freeze-dried residues after ruminal incubation were successively ground in mills with 3 and 1 mm screens and, for starch analysis, with a 0.2 mm screen. Nitrogen was determined using the standard Kjeldahl procedure with Cu2 as a catalyst. Ash was determined by ashing at 5508C overnight. The ADF was analysed according to the Association of Of®cial Analytical Chemists (1990). Starch content was determined by enzymatic hydrolysis of starch to glucose as described by Brandt et al. (1987). The PDIS was analysed on all samples except the three grass silages as described by the American Oil Chemists' Society (1989).
2.4.2. Fractionation of crude protein
The CP of all feedstuffs was partitioned into ®ve fractions (A, B1, B2, B3, and C; Table 3) according to the CNCPS (Russell et al., 1992; Sniffen et al., 1992), using standardisation and recommendations published by Licitra et al. (1996) except thata -amylase (bacterial crude type XI-A fromBacillus subtilis; Sigma, St. Louis, MO) was used on all feeds in the NDF procedure to facilitate ®ltration through the ®lter paper with the exception of the three silage samples. As neutral detergent ®bre (NDF) values of the feed samples that were determined within the CP fractionation schedule by manual ®ltration on paper according to the recommendations of Licitra et al. (1996) may deviate from those obtained with the conventional NDF method, the cell-wall fraction obtained as a residue on ®lter paper was named PNDF. All analyses of CP, CP fractions and PNDF were carried out at least in duplicate.
2.5. Statistical methods
Linear and non-linear regression equations andr2values for in situ UDP2, UDP5 and UDP8 values versus PDIS values were determined by SAS (1988). Signi®cant relationships were declared atp<0.10 unless otherwise stated.
Table 3
Partition of nitrogen and protein fractions of feedstuffs according to the Cornell net carbohydrate and protein systema
Fraction Protein-fraction Enzymatic degradation
Estimation
A NPN (Non protein N) Not applicable Soluble in sodium tungstate B1 True protein Fast True protein soluble in buffer B2 True protein Variable Difference between buffer protein and
protein insoluble in NDb
B3 Cell wall associated true protein Variable to slow Protein insoluble in ND but soluble in ADc
C Includes heat-damaged protein and nitrogen associated with lignin
Indigestible Protein insoluble in AD
aFrom Licitra et al. (1996). bND: neutral detergent. cAD: acid detergent.
Stepwise linear multiple regression (SAS, 1988) was employed to predict in situ UDP2, UDP5, and UDP8 values from CP fractions A, B1, B2, B3 and C, and from PNDF and CP values of feedstuffs. Variables and two- and three-way interactions were incorporated in the models atp<0.10 and when the model r2was improved by at least 0.01. In the regression equations, the termedenotes the residual error.
3. Results
3.1. In situ crude protein degradation
Table 4 shows the UDP values as derived from in situ incubations. A wide range of UDP values was observed, ranging from 63 g kgÿ1of CP for untreated soybeans at an assumed rumen solid out¯ow rate of 2% hÿ1 (UDP2) to 842 g kgÿ1 of CP for the formaldehyde-treated rapeseed meal at a passage rate of 8% hÿ1(UDP8). As a general observation, soybean products contained less UDP than rapeseed commodities. The CP of maize gluten feed and palm kernel meal was degraded ruminally to a similar extent. The highest UDP concentrations were observed for extensively processed feedstuffs (e.g. ®sh meal and formaldehyde- and lignosulphonate-treated expeller and meals), which is in line with the expectations. The UDP concentrations of dairy compound feeds lay within the range of values observed for the ingredients.
3.2. Protein dispersibility index
The PDIS values for 29 feeds ranged from 21 g kgÿ1of CP for a palm kernel meal to 762 g kgÿ1of CP for the untreated soybeans (Table 4). The data plot of in situ UDP8 values against PDIS values is depicted in Fig. 1. From this ®gure it becomes clear that the relationship between the two measurements was weak. Even the exponential regression equation, which yielded the best ®t of data to the linear and non-linear regression models that were tested, gaver2values of only 0.30, 0.29, and 0.33, respectively, for estimates of UDP2, UDP5, and UDP8, from the PDIS values.
3.3. Chemical fractionation of crude protein
The results of partitioning feed CP into A, B, and C fractions of the 11 dairy compound feeds and 21 feedstuffs under investigation are presented in Table 5. All CP fractions varied widely among feeds.
3.4. Chemical fractionation versus in situ estimates
Fig. 2 illustrates the effects of chemical (lignosulphonate) treatment of soybean meal and rapeseed expeller on CP fractions and PNDF contents and on UDP2, UDP5, and UDP8 values. Treatment elevated the more slowly degradable CP fractions (B2 and B3) at the expense of the A and B1 fractions, which represent nonprotein nitrogen and true soluble protein, respectively. As a result, UDP values at all three assumed passage rates of
the treated feeds were more than twice the values of the respective untreated or moderately treated feeds.
Fig. 3 depicts CP fractions, PNDF contents, and UDP values of four selected dairy compound feeds. The general pattern that was observed for rapeseed expeller and soybean meal (Fig. 2) was also found for the dairy compound feeds. Higher UDP values
Table 4
Values for in situ undegraded dietary protein (UDP) and the protein dispersibility index (PDIS) of 11 dairy compound feeds and 21 feedstuffsa
UDP2
Grass silage 1 194 276 335 NAb
Grass silage 2 133 189 229 NA
Grass silage 3 121 170 207 NA
Palm kernel meal 1 182 264 336 21
Palm kernel meal 2 165 255 325 80
Maize gluten feed 1 187 282 329 482
Maize gluten feed 2 168 260 324 322
Sun¯ower seed meal 123 200 261 242
Fish meal 1 398 578 662 161
Fish meal 2 616 773 834 142
Rapeseed meal 161 262 337 194
Rapeseed meal, formaldehyde-treated 615 785 842 45
Rapeseed expeller 178 247 303 214
Rapeseed expeller, lignosulphonate-treated 468 656 742 77
Soybeans 63 129 167 762
Soybeans, dry-heat-treated 89 182 252 498
Soybeans, moist-heat-treated 116 240 330 249
Soybean meal 1 84 182 258 298
Soybean meal 2 95 197 276 217
Soybean meal, lignosulphonate-treated 355 573 677 42
Soybean meal, formaldehyde-treated 338 512 610 35
aUDP and PDIS values were calculated as outlined in Section 2; UDP2, UDP5, and UDP8, respectively,
refer to UDP at assumed ruminal out¯ow rates of 2, 5, and 8% hÿ1. bNA: not analysed.
Fig. 1. The relationship between the protein dispersibility index (x) and in situ undegraded dietary protein (y) at an assumed rumen out¯ow rate of 8% hÿ1(UDP8) for a
total of 29 dairy compound feeds and feedstuffs. The best ®t of linear and non-linear regression equations was attained with the equation y0.5131eÿ1.4926x
(r20.3333).
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were related to lower soluble or rapidly degradable CP fractions and to higher slowly or less degradable CP fractions.
3.5. Regression equations
The results of the development of linear regression equations for estimating in situ UDP2, UDP5, and UDP8 values from CP fractionation data are presented below. Due to dif®culties in ®tting the ®sh meal data to any one of the employed regressions equations,
Table 5
Values for crude protein fractions of 11 dairy compound feeds and 21 feedstuffsa
A (g kgÿ1
Grass silage 1 283 58 306 311 44
Grass silage 2 518 45 280 121 37
Grass silage 3 578 50 287 46 40
Palm kernel meal 1 62 12 127 661 137
Palm kernel meal 2 65 17 137 641 139
Maize gluten feed 1 503 82 306 81 29
Maize gluten feed 2 341 65 328 205 62
Sun¯ower seed meal 70 296 532 55 47
Fish meal 1 106 63 704 119 8
Fish meal 2 41 32 404 519 5
Rapeseed meal 38 226 613 59 62
Rapeseed meal, formaldehyde-treated 44 0 616 267 71
Rapeseed expeller 72 341 519 08 59
Rapeseed expeller, lignosulphonate-treated 29 22 362 487 99
Soybeans 53 509 402 5 31
Soybeans, dry-heat-treated 49 206 708 6 31
Soybeans, moist-heat-treated 80 90 788 14 28
Soybean meal 1 51 212 716 10 12
Soybean meal 2 66 91 817 8 19
Soybean meal, lignosulphonate-treated 23 7 397 512 60
Soybean meal, formaldehyde-treated 24 36 858 54 28
aFor explanation of crude protein fractions, see Table 3.
Fig. 2. Effects of lignosulphonate (L) treatment of rapeseed expeller (RSE) and soybean meal (SBM) on values for crude protein fractions (A, B1, B2, B3, and C according to the Cornell net carbohydrate and protein system; g kgÿ1of crude protein), cell-wall constituents retained on ®lter paper (PNDF, g kgÿ1of dry matter), and
in situ undegraded dietary protein at assumed rumen out¯ow rates of 2, 5, and 8% hÿ1(UDP2, UDP5, and UDP8; g kgÿ1of crude protein).
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Fig. 3. Values for crude protein fractions (A, B1, B2, B3, and C according to the Cornell net carbohydrate and protein system; g kgÿ1of crude protein), cell-wall constituents retained on ®lter paper (PNDF, g kgÿ1of dry matter), and in situ undegraded protein at assumed rumen out¯ow rates of 2, 5, and 8% hÿ1(UDP2, UDP5, and UDP8; g kgÿ1of crude protein) of dairy compound feeds (feed numbers refer to numbers in Tables) of varying ingredient and chemical composition (see Tables 1 and 2).
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195±214
which was likely due to the fact that ®sh meal contains no cell-wall (NDF) though PNDF values of ®sh meal were rather high (see Table 2), estimates of UDP values reported below were conducted without ®sh meal, i.e. 30 out of 32 feeds Ð 11 dairy compound feeds and 19 feedstuffs Ð were included in the data set. Values of UDP, CP, and PNDF concentrations of feeds are given as g kgÿ1of dry matter, whereas A, B, and C fractions are given as g kgÿ1of feed CP.
The general form of the regression equations was identical for UDP2, UDP5, and UDP8. The CP fraction per se did not contribute in explaining the observed variation of UDP values, and did therefore, not appear in any one of the regression equations.
Parameter estimates of b0±b7 with their probabilities and the model r2 values are presented in Table 6. The coef®cient of determination (r2) was >0.9 for the models that estimated UDP5 and UDP8 values, and was slightly lower for the UDP2 estimation.
Table 7 shows the differences between UDP values as obtained from ruminal incubations of dairy compound feeds and feedstuffs in situ and those predicted
Table 6
Statistical model parameter estimates with their levels of signi®cance and coef®cient of determination (r2) values of regression equations for estimating undegraded dietary protein (UDP) values at assumed rumen out¯ow rates of 2, 5, and 8% hÿ1from chemical fractionation of feed crude protein
UDP2a UDP5 UDP8
Parameter pb Parameter p Parameter p
b0 ÿ243.576 *** ÿ189.682 ** ÿ98.663
aUDP2, UDP5, and UDP8, respectively, refer to UDP at assumed ruminal out¯ow rates of 2, 5, and 8% hÿ1. b,p<0.10; **,p<0.01; ***,p<0.001.
from chemical partitioning of feed CP plus PNDF values. In accordance with the coef®cients of determination (Table 6), the differences between in situ and chemical fractionation values were smallest for UDP8 and largest for UDP2. The range of differences, expressed as g kgÿ1 of CP, was ÿ84 to 145, ÿ69 to 79, and ÿ64 to 78 for UDP2, UDP5, and UDP8, respectively. Only six out of 30 UDP8 values differed by more than 50 g kgÿ1 of CP and more than half of the feeds (16) had differences of
30 g kgÿ1of CP.
Table 7
Difference between in situ undegraded dietary protein (UDP) values of 11 dairy compound feeds and 19 feedstuffs at assumed ruminal out¯ow rates of 2, 5, and 8% hÿ1a and the predicted UDP values based on
chemical fractionation of feed crude protein
UDP2 UDP5 UDP8
Difference (g kgÿ1of CP)
Dairy compound feed
1 2 25 36
2 ÿ84 ÿ69 ÿ52
3 145 79 46
4 ÿ64 50 ÿ39
5 ÿ13 ÿ3 7
6 ÿ44 ÿ7 16
7 ÿ23 ÿ7 ÿ5
8 ÿ34 ÿ41 ÿ46
9 ÿ34 ÿ30 ÿ28
10 77 77 71
11 45 35 18
Grass silage 1 ÿ21 ÿ22 ÿ22
Grass silage 2 ÿ24 ÿ39 ÿ45
Grass silage 3 44 26 12
Palm kernel meal 1 22 12 7
Palm kernel meal 2 ÿ21 ÿ31 ÿ35
Maize gluten feed 1 19 49 57
Maize gluten feed 2 ÿ32 ÿ45 ÿ39
Sun¯ower seed meal ÿ31 ÿ24 ÿ25
Rapeseed meal ÿ8 ÿ26 ÿ29
Rapeseed meal, formaldehyde-treated 9 ÿ11 ÿ27
Rapeseed expeller 95 74 65
Rapeseed expeller, lignosulphonate-treated 23 40 44
Soybeans ÿ30 ÿ13 ÿ11
Soybeans, dry-heat-treated ÿ7 ÿ21 ÿ27
Soybeans, moist-heat-treated ÿ13 ÿ10 ÿ3
Soybean meal 1 9 18 24
Soybean meal 2 ÿ35 ÿ56 ÿ64
Soybean meal, lignosulphonate-treated 1 8 6
Soybean meal, formaldehyde-treated 26 61 78
aFor equations, see Section 3.5 and Table 6.
4. Discussion
In this study, an attempt was made to predict UDP values from simple laboratory measurements, using in situ UDP values as reference values. Data for the disappearance of CP from polyester bags in situ were corrected by subtraction of the proportion of small particles that theoretically would still be in the bag at speci®c incubation times according to Weisbjerg et al. (1990). No attempt was made, however, to correct for microbial contamination of undegraded residues. The error caused by ignoring microbial contamination may be large with ®brous or starchy feeds of lower protein content, and clearly there is a need for a microbial correction of in situ residues for these feeds (Alexandrov, 1998). Microbial contamination exerts probably only a slight in¯uence upon undegraded in situ residues of protein-rich feeds (Varvikko, 1986), which made up the majority of samples used in this study. Therefore, we assume that microbial contamination did not signi®cantly bias our estimates of in situ ruminal CP degradation characteristics.
Extensive research efforts have been directed towards estimating ruminal protein degradation in ruminant feeds by in vitro methods which require no access to ruminally cannulated animals, yet, no simple laboratory method has evolved which conveniently yields reliable UDP values. Mahadevan et al. (1987) discussed the advantages of the use of a protease from mixed rumen micro-organisms for the in vitro determination of the degradability of true protein over other in vitro methods. These authors found that substituting the fungal protease fromStreptomyces griseusfor the rumen protease gave results which were very different from those obtained with the rumen enzyme. Roe et al. (1991) have reported that the use of bacteria or enzymes to predict degradability has not been fully exploited. Broderick (1994) reported that commercial protease do not give reliable results. Licitra et al. (1998) compared three in vitro methods based on the use of
S. griseusprotease, and showed that amount of protease, ratio of enzyme to substrate and
buffer pH signi®cantly affected the estimate of degradable CP, and, consequently also UDP in a feed. In a subsequent study, Licitra et al. (1999) showed that by standardizing
theS. griseusmethod progress towards a reliable estimate of in situ CP degradability and,
accordingly, UDP values may be achieved.
Studies using laboratory procedures based on the use of chemical buffer solutions (Burroughs et al., 1975; Crooker et al., 1978) have led to the view that there seems to be a strong relationship between the solubility of CP in a feedstuff and its degradability in the rumen. Most of these studies have attempted to predict the rumen degradability of CP by measuring the solubility of CP using only one buffer solution. Subsequent studies, with a wider range of feedstuffs, have suggested that these relationships may only hold true for a limited range of feedstuffs. The main reason for this is likely to be that feeds contain several different CP fractions which vary considerably in their rates and extents of degradation (Cottrill, 1993). Nevertheless there are many studies that yielded high correlation coef®cients between solubility or enzymatic methods and in situ data (e.g. AufreÁre et al., 1991; Susmel et al., 1993; De Boever et al., 1997; Kandylis and Nikokyris, 1997). The low coef®cients of determination between PDIS and the in situ UDP values clearly indicates that this method was not suitable to predict UDP values for the range of dairy compound feeds and feedstuffs under investigation in the present study.
Accordingly, Tremblay et al. (1996) reported that the PDIS was poorly correlated (r20.28) to UDP estimates based on an inhibitor in vitro method.
The partitioning of feed CP according to the CNCPS yields fractions which differ in their degree of ruminal degradation from 100 (fraction A, nonprotein nitrogen) to almost zero (fraction C, bound true protein), hence, a diversity of protein characteristics can be utilized to estimate UDP values of feeds. Theoretically, this attempt should yield estimates that could be consistent across feed classes. In the present study, general CP solubility characteristics as derived from the fractionation procedure indicate both similarity to and deviations from data of earlier work conducted with a variety of CP solubility methods. For example, Krishnamoorthy et al. (1983) have obtained higher concentrations of the B3 fraction and lower A and B2 fractions for soybean meal than observed in this study. Crawford et al. (1978) have assayed CP solubility by the use of Wise Burroughs mineral buffer and sodium chloride solutions, and reported values that were similar to the (AB1) fractions we found in sun¯ower meal, soybean meal, and maize gluten feed. Data for sun¯ower meal, soybean meal and maize gluten feed presented by Sniffen et al. (1992) were similar to our results for the soluble (AB1) CP fractions and the CP fractions insoluble in neutral (B3C) and acid detergent (C) solution. De Boever et al. (1997), using a borate-phosphate buffer, reported CP solubilities of soybean meal and formaldehyde-treated soybean meal that were also similar to our data. Kusumanti et al. (1996) investigated rapeseed meal, sun¯ower meal and maize gluten feed and found fraction C (insoluble in acid detergent) values that were similar to those observed in the present trial.
In contrast to the CNCPS, in which the UDP of a feedstuff is derived from CP fractions B1, B2, and B3, each having its own `fractional' UDP value, the current investigation employed the feed CP fractionation with the objective to predict one single UDP value for each feed. In addition to the CP fractions, the CP content per se and the PNDF values were included in the regression equations. It is noteworthy, however, that the CP fraction per se did not contribute in explaining the observed variation of UDP but only interacted with the PNDF and CP fractions. Although ana-amylase treatment was part of the PNDF determination, values as high as 854 g kgÿ1of dry matter for a palm kernel meal indicate that more reliable cell-wall values could have been achieved by additionally applying a protease treatment to the samples, which has been shown to facilitate ®ltration of protein-rich feedstuffs further (Dorleans et al., 1996). As all samples were identically treated for the PNDF determination except for the three silages, it is assumed that no effect was exerted on UDP estimates by the PNDF method.
From the results presented in Table 7, it becomes obvious that the difference between predicted and in situ UDP values generally was <50 g kgÿ1of CP. For UDP2, UDP5, and UDP8, respectively, only 17, 20, and 20% of the feeds had differences of >50 g kgÿ1of CP. The coef®cient of determination was highest for the equation which estimated UDP8 values. In other words, the smallest difference between predicted and in situ UDP values was obtained for cattle with an assumed high feed intake, which, when accompanied by a high milk yield, requires elevated UDP concentrations in the diet, because ruminal microbial synthesis is limited due to restricted fermentable energy intake. Hence, the need for safe and reliable UDP estimates is of paramount importance when intake and out¯ow from the rumen are high. Animals consuming less feed and yielding less milk
also have low UDP requirements, that can be met by almost all protein sources and the need for estimating appropriate UDP values at low UDP requirements is much less urgent than at high intakes.
5. Conclusions
The results of the present study indicate that in situ UDP values, which serve as one key variable in many protein evaluation systems for dairy cattle, can be reliably and accurately predicted from chemical fractionation of feed CP according to the CNCPS. Studies are underway to extend the current database with the goal to improve accuracy and precision of the estimates. Further examinations of feedstuffs may lead also to simpler regression equations. Furthermore, when a larger data base is available, separate equations for forages and concentrate ingredients can be developed and might improve estimates of UDP concentrations of feedstuffs further.
Acknowledgements
The authors thank C. Lewin for taking care of the steers, D. Nibbe and A. Wessels for helping with parts of the in situ trials, and W. KuÈhl and E. Saggau for assistance in laboratory analyses. The authors thank Prof. S. Tamminga and an unknown reviewer for their comments on an earlier version of this manuscript. Financial support was provided by `Stiftung Schleswig-Holsteinische Landschaft' under a research programme `Nitrogen Cycle on a Specialized Dairy Catte Farm'. Special thanks are expressed to the following individuals or company representatives for providing feedstuffs for the study: A. Helmedach, Dr. T. KoÈhler, Dr. U. Runge, O. Schade, Dr. H. Spiekers and Dr. K. Werner.
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