Estimating ruminal crude protein degradation with

Animal Feed Science and Technology 85 (2000) 195±214

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

*

Corresponding author. Tel.: 49-431-880-2538; fax: 49-431-880-1528. E-mail address: [email protected] (K.-H. SuÈdekum) 0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 4 6 - 2

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S. Shannak et al. / Animal Feed Science and Technology 85 (2000) 195±214

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-Dokumentationsstelle, 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.


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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);

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Table 1 Ingredient composition (g kgÿ1) of dairy compound feedsa

Wheat Barley Oats Rye Molasses Dried beet pulp Palm kernel meal Palm kernel expeller Maize gluten feed Maize feed meal Wheat gluten meal Sun¯ower seed meal Fish meal Rapeseed meal Rapeseed meal, protected Rapeseed expeller Soybean hulls Soybean oil Soybean meal Soybean meal, protected Grass meal, dehydrated Citrus pulp Mineral±vitamin mix a

1

2

3

4

5

6

7

8

9

10

11

80 ± ± 80 60 ± ± 150 372 ± ± 25 ± ± ± ± ± ± ± ± ± 200 33

± 135 ± ± 60 100 ± 140 115 ± ± ± 30 ± ± ± 220 ± 110 ± ± 80 10

± 260 ± 120 60 ± ± ± 85 ± ± ± 160 75 ± ± 30 ± ± ± 55 150 5

220 ± ± ± 40 100 80 80 180 ± ± 30 ± ± ± 90 ± ± 50 100 ± ± 30

180 ± ± ± 30 85 80 80 190 ± 50 ± ± 90 ± 140 ± ± 60 ± ± ± 15

± 200 ± ± 30 250 ± ± 170 ± ± ± ± 100 ± ± 134 ± 100 ± ± ± 16

± 200 ± ± 45 216 ± 35 100 ± ± ± ± 45 99 ± 100 ± 150 ± ± ± 10

230 120 ± ± 26 350 ± ± ± ± ± ± ± ± ± ± ± 5 250 ± ± ± 18

204 105 ± ± 31 305 ± ± ± ± ± ± ± 339 ± ± ± 5 ± ± ± ± 9

± ± 190 192 30 ± ± ± ± 225 ± ± ± 344 ± ± ± ± ± ± ± ± 18

± ± 221 230 30 ± ± ± ± 240 ± ± ± ± ± ± ± ± 250 ± ± ± 25

The sum of ingredient concentrations in each row may not equal 1000 g kgÿ1 due to rounding off numbers.

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-heattreated 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 5015 mm. Triplicate samples of each feed were incubated in the rumen of three steers. About 1.3 g of feed ground to pass


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Table 2 Chemical composition of 11 dairy compound feeds and 21 feedstuffs incubated in situ in the rumen of steersa DM Ash CP ADF Starch PNDF (g kg±1) (g kg±1 DM) (g kg±1 DM) (g kg±1 DM) (g kg±1 DM) (g kg±1 DM) Dairy compound feedb 1 2 3 4 5 6 7 8 9 10 11

889 880 879 902 906 899 889 918 915 917 918

79 73 79 81 82 68 71 64 62 60 61

160 182 230 218 220 187 217 217 194 205 212

189 262 165 188 199 177 177 134 185 151 113

140 110 200 139 142 137 137 199 179 280 310

406 554 423 491 410 364 370 323 361 267 212

Grass silage 1 Grass silage 2 Grass silage 3

361 639 550

103 178 157

167 178 157

166 178 171

NAc NA NA

597 554 464

Palm kernel meal 1 Palm kernel meal 2 Maize gluten feed 1 Maize gluten feed 2 Sun¯ower seed meal Fish meal 1 Fish meal 2

901 890 887 889 911 923 923

53 53 67 69 77 172 202

172 173 210 247 334 679 766

469 475 91 99 312 NA NA

1 2 209 149 4 NA NA

823 854 375 404 458 202 574

Rapeseed meal Rapeseed meal, formaldehyde-treatedd Rapeseed expeller Rapeseed expeller, lignosulphonate-treatede Soybeans Soybeans, dry-heat-treated Soybeans, moist-heat-treated Soybean meal 1 Soybean meal 2 Soybean meal, lignosulphonate-treatedf Soybean meal, formaldehyde-treatedg

920 904

73 78

344 353

220 236

56 12

331 524

916 896

68 67

358 322

257 273

7 11

321 538

912 932 922 907 916 893

59 57 57 74 69 69

398 398 397 546 512 504

163 157 162 62 102 92

4 6 5 7 6 10

217 207 202 140 166 491

910

81

386

151

32

260

a DM, 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). b For ingredient composition of dairy compound feeds see Table 1. c NA: not analysed. d Biopro®n1 R (Biopro®n sales of®ce, Bramsche, Germany). e RaPass1 (Borregaard LignoTech, Sarpsborg, Norway). f SoyPass1 (Borregaard LignoTech, Sarpsborg, Norway). g Biopro®n1 S (Biopro®n sales of®ce, Bramsche, Germany).

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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): 1 ÿ DIi ÿ SP WS : CDIi DIi ÿ SP 1 ÿ SP WS Degradation of CP (CDEG) was calculated using the equation of McDonald (1981): CDEG a b1 ÿ eÿctÿL

for t > L;

where CDEG is the disappearance at time t corrected for SP, a an intercept representing the proportion of CP solubilized at initiation of incubation (time 0; soluble fraction), b the fraction of CP insoluble but degradable in the rumen, c the rate constant of disappearance of fraction b, t the time of incubation, and L is the lag phase. The non-linear parameters a, 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): ED a

bc ÿkL e ; ck

where k is the estimated rate of out¯ow from the rumen and a, b, c and L are 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). Correspondingly, values for UDP2 (UDP5, UDP8) (g kgÿ1 of 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


201

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 that aamylase (bacterial crude type XI-A from Bacillus 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 and r2 values for in situ UDP2, UDP5 and UDP8 values versus PDIS values were determined by SAS (1988). Signi®cant relationships were declared at p