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Prediction of feed protein degradation in the rumen with bromelain
i ANIMAL FEED SCIENCE AND TECHNOLOGY
L
ELSEVIER
Animal Feed Scienceand Technology53 (1995) 71-80
Prediction of feed protein degradation in the rumen with bromelain 0. Tom&nkova”, J. K~peEn$~~* ‘Institute ofAnimal Production, Prague IO, UhFMves, Czech Republic bInstitute ofAnimal Physiology and Genetics, Czech Academy of Sciences, Prague IO, UhtYnnpVes, Czech Republic
Received 14 February 1994;accepted28 September 1994
First a suitable protease for enzymatic determination of in vitro rumen protein degradation was selected. The actions of Pronase E, papain and bromelain were compared. The highest correlation coefficient for protein degradation estimated by the in sacco method compared with the enzymatic method was obtained with bromelain in a set of 13 feeds. This enzyme was subsequently used for analysis of more than 200 ruminant feed components. Feeds were separated on the basis of their chemical properties into the following live groups: cereals, forages, hays, protein concentrates, &ages. In these groups, correlation coefficients of linear regression equations for rumen protein degradability estimated by the in sacco method and bromelain were 0.839 for concentrates, 0.730 for fresh forage, 0.741 for hays and 0.876 for &ages. Feeds with high starch content had to be treated with cr-amylase to obtain the correlation coefficient r=0.809. The method is going to be used for preliminary testing of ruminant feeds. Keywords: Bromelaiq Protein degradation; Rumen
1. Introdaction
New feeding systems for ruminants are based on exact data of amino acid absorption from the duodenum. It is possible to calculate these values from feed analysis. Calculations have to take into account the effect of rumen fermentation (Russell et al., 1992; Sniffen et al., 1992). In the rumen, nitrogen compounds are degraded and the extent of protein degradation influences the’estimation of the * Corresponding author. 0377-8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZO377-8401(94)00735-7
12
0. Torn&&w&,J. KopeEni,/Animal Feed Scienceand Technology53 (1995) 71-80
amount of amino acids entering the duodenum. One of the most reliable methods to estimate feed protein degradation in the rumen is the in sacco method (0rskov and McDonald, 1979). Unfortunately, this type of measurement is not available to many laboratories and is often replaced by other types of estimation. The most simple and rapid alternative method is the measurement of soluble nitrogen compounds (Madsen and Hvelpund, 1985; Susmel et al., 1993), although various enzymatic methods were found to be more accurate (Poos-Floyd et al., 1985). Mahadevan et al. ( 1987) used isolated rumen proteolytic enzymes for this purpose. Several methods utilize proteinases from Streptomyces griseus (Pronase E ) (Auf&e et al., 1991). Poos-Floyd et al. ( 1985) suggested bromelain as one of the most suitable enzymes for this purpose. Measurements of protein degradability on a small number of samples may lead to inaccurate estimation of linear regression equations and consequently to an incorrect estimation of the feed quality. The aim of this study was to develop an enzymatic method based on bromelain and to evaluate its suitability for predicting protein degradability on a representative set of feeds for ruminants.
2. Materials and methods 2.1. Feeds Feeds were divided into several groups according to their chemical composition and properties of their protein fractions. The ruminant feed components tested are listed in Table 1. 2.2. Animals Two non-lactating crossbred cows (Friesian x Bohemian spotted) fitted with large rumen cannulae (Avon Industrial Polymers, GB) were used for our experiments. Their diet consisted of 14 kg of corn silage, 6 kg of lucerne hay and 1 kg of ground barley with a mix of vitamins and minerals. The diet was offered in two equal meals daily. 2.3. In situ measurements The in sacco rumen degradability was measured according to Orskov and McDonald ( 1979) as adapted by Vencl ( 1986). Air-dried feed samples were milled through a 1.Omm mesh and 1 g was put into nylon bags (6 x 12 cm, pore size 53 pm) and incubated in the rumen for 4,8,16,24 and 48 h. The removed bags were rinsed in cold water and their nitrogen content was determined by Kjeldahl analysis (Association of Official Analytical Chemists (AOAC), 1980). Measurements were done in triplicate. The degradability of feed proteins was
0. Tomcinkovd, J. Kopetnj /Animal Feed Science and Technology 53 (1995) 71-80
73
Table 1 Feed groups and their average chemical composition n
DM (g kg-‘)
OM (gkg-’ DM)
CP (g kg-’ DM)
1. Animal and vegetable proteins Fish meal Blood meal Meat and bone, high oil Feather meal Groundnut meal Rapeseed meal Soyabean meal Cottonseeds meal Sunflower meal Sesame meal Linseed meal Feeding mixtures concentrates
2 2 2 1 4 3 3 2 1 1 1 18
921 922 947 925 930 915 916 911 939 939 915 888
967 952 764 967 925 928 926 921 931 912 943 935
682 283 528 774 439 394 491 474 387 459 404 162
2. Cereals Wheat meal Triticale meal Barley meal Maize meal Wheat bran Oat Rye
3 1 11 4 4 3 1
866 862 889 890 886 897 881
952 940 946 980 959 969 980
140 144 119 113 157 111 111
3. Fresh forages Alfalfa Clover Maize Ryegrass
14 15 7 5
199 172 289 171
889
888 949 899
209 175 190 140
4. Hays Meadow hay Alfalfa
8 24
943 950
907 902
128 170
Silages I: whole plants Maize Alfalfa Oat Meadow grass Barley Wheat + pea Horsebeans
15 8 1 2 5 4 1
945
938 962 920 957 950 949
934 891 867 886 942 903 878
98 181 82 157 90 127 173
9
959
731
127
5. Silages II: leaves Sugar beet
n, number of samples; DM, dry matter; OM, organic matter; CP, crude protein.
74
0. Tomdnkovrt. J. Kopefnj /Animal Feed Science and Technology 53 (1995) 71-80
calculated from the kinetics of in situ degradation using equation of Orskov and McDonald ( 1979): y=a+b(
1 -e-C’)
where a is the immediately degradable nitrogen fraction, b is the potentially degradable nitrogen fraction, c is the constant rate of degradation of the fraction b and t is time (h) of incubation. In situ theoretical degradabilities (TD) were calculated according to the equation: TD=a+
[ (bc)/(c+k)]
The optimal outflow rate (k) for concentrates (Homolka et al., 1990) and fresh forage was calculated to be 0,060 h-’ and for all other types of feeds 0.046 h-‘. 2.4. Enzymatic method Dry feed samples were milled through 0.8 mm mesh and 0.5 g was incubated in a solution (ED solution) of 50 ml 100 mM phosphate buffer pH 7.2 with 5 mM cysteine, 1 mM EDTA, 1 mg ml-’ chloramphenicol, 0.75 mg ml-’ sodium sorbate and 3 mg bromelain (Sigma; 2-4 U mg- ’protein) at 40 ’C for 24 h (Kop&y et al., 1989). The final concentrations of papain and Pronase E (Sigma) were 8.3 pg ml- ’ and 1.4 a ml- ‘, respectively. Papain was incubated in the presence of 100 mM phosphate buffer pH 6 with 5 mM cysteine and Pronase E in the presence of 100 mM phosphoborate buffer pH 8. All other incubation components were the same. Reaction was stopped by 25% trichloroacetic acid (TCA) (final concentration 5% TCA) . Samples were cooled and centrifuged at 5 100 xg for 5 min. The sediment was transferred to filter paper (Whatman No. 54), rinsed twice with 5% TCA and digested using the Kjeldahl procedure. Ammonia was estimated with Nessler reagent (AOAC, 1980). Each sample was done in triplicate. Enzymatic degradability (ED) was expressed as percentage of original crude protein content. Into all series standard samples of alfalfa hay and two blanks without feeds were included. Proteolytic activity was measured using azocasein (Kopetiy and Wallace, 1982). 2.5. Statistical methods Differences between methods were evaluated by analysis of variance and simple linear regression. 3. Results 3.1. Enzyme selection The first step in this study was a selection of the most suitable protease. The selection was done using 13 different types of feeds and three enzymes-papain,
0. Tom&&m&, J. Kopetn$ /Animal Feed Science and Technology 53 (1995) 71-80
75
bromelain and Pronase E. Results were compared with data obtained from the in sacco measurements (Table 2). From the linear regression equations the highest correlation coefficients were obtained for bromelain. 3.2. Bromelain degradation offeedproteins A comparison of in situ (TD) and bromelain degradation (ED) of feed proteins in a set of 186 feed samples is shown in Table 3. Different slopes of linear regression for different types of feeds led us to group feeds with similar properties. Grouping reflected the source and treatment of the feed and protein availability. Data distribution is shown in Fig. 1. Accuracy of the enzymatic estimation from repeat analysis of the same samples was + 3.1%. The effect of final TCA treatment was tested. TCA had no significant effect on protein degradabilityvalues. The only exception were samples of blood meal where Table 2 Linear regressions of feeds rumen protein, degradability lain, papain and Pronase E
estimated in sacco and in vitro with brome-
Enzyme
n
a
b
r
RSD
RSD%
Papain Bromelain Pronase E
13 13 13
33.38 - 19.08 20.04
0.657 1.138 0.714
0.765 0.855 0.627
8.10 6.53 9.81
11.43 9.20 13.85
n, number of samples; a, b, constants of linear regressions; r, correlation standard deviation. Table 3 Comparison proteins Feeds
of in sacco theoretical degradabihty
n
In situ TD (% CP)
(TD) and enzymatic degradability
ED (% CP)
Animal and vegetable concentrates + feeding mixtures 40 68.15k19.16 74.94 f 17.63 Cereals 28 72.81 f 11.08 75.19+ 11.43 Fresh forage 41 62.53f8.15 67.76 kg.90 Hays and silage I 68 63.48 f 14.35 73.38f 13.17 Silage II 9 64.84+ 8.74 58.37+ 10.58 ?? P< 0.05. QP
coefftcient; RSD, residual
(ED) of feed
b
r
0.46
0.914
0.839”
7.072
43.42
0.388
0.423’
10.145
18.65
0.648
0.730-
5.815
- 17.16
1.051
0.741-
7.319
22.55
0.724
0.876*+
4.502
a
RSD
16
0. Tom&nkov&,J. KopeEn$ /Animal Feed Science and Technology 53 (1995) 71-80
TCA treatment caused a 2.8-3.2% decrease of protein degradability (results not shown). An effect of passage rate (in the range 0.02-l .OOh- ’) on in situ degradability and following correlation with enzymatic degradability was tested. It was assumed that the passage rate for concentrates and fresh forage is optimal at the level of 0.06 h- ‘. All other feeds were evaluated at a passage rate of 0.046 h-l. These values gave the highest correlation coefficients between ED and TD. Correlation coefficients for different groups of feeds were at least 0.73. The only exception was the group of cereals where availability of protein for ED was different from sample to sample. In these cases it was necessary to decrease the effect of the starch in the samples (Table 3) on the enzymatic determination. a
.$
40
.G P
L
XI
. .
,
.o in Go
. r = 0.71
80
degExJationm[%j
y = 18.850
-
+ 0.847X
b
. r = 0.878
-
y = 22.552
+ 0.72x
I
a,
0. Tom&nkznvi,J. Kope&+ /Animal Feed Science and Technology 53 (1995) 71-80
77
d
--y=-0.462=0.91x
mr=0.639
e
A-P
ac
50
60
70
in vitro degradation m r = 0.423
-
81
m
I Irn
[%]
y = 43.426
+ 0.366x
Fig. I. The relationship between measured in situ protein degradability and that estimated from enzymatic degradation for fresh forage (a), hays (b), silage (c), protein concentrates (d) and cereals (e). Linear regression equation and correlation coefficient are added for each data set.
3.3. Amylase treatment Protein degradation in samples with a high starch content did not correlate under in situ conditions with bromelain treatment even for one type of feed. Correlation coefficients for different types of grains and meals were usually lower than 0.4; for a combination of all cereals the result was r= 0.423 (Table 3 ). It was suggested that starch was the main compound limiting the availability of proteins for bromelain. Therefore cu-amylase treatment was included in the procedure. Amylase was tested under different conditions. Initially, sequential incubation was done with cr-amylase, followed by the proteinase treatment. It was found that the degree of amylase inhibition by bromelain was negligible (Fig. 2 ) and thus it was possible to incubate both enzymes together. Therefore 0.5 mg of a-amylase (Bolamylase, specific activity 1.05 mg maltose h-r mg-I; Lachema, Czech Republic) per 1 ml of the ED solution were incubated together with bromelain and samples for 24 h. Blank samples with amylase were introduced to eliminate the effect of the nitrogen content of amylase. After this treatment correlation coefficients of linear regression for cereals in-
78
0. Tom&nkov&,J. KopeCn$ /Animal Feed Science and Technology 53 (1995) 71-80 90
Amylase
added
2 : .g
72
-
E
66
. . No -
amylase
60
0
5
added
1 10
15
Time
(hours]
20
25
Fig. 2. The effect of interval between amylase and bromelain addition on in vitro estimated rumen protein degradation. Amylase was added in a concentration of 0.5 mg ml-’ and incubated for 24 h at 40°C. This was followed by incubation for 24 h with bromelain at the same temperature. The sample tested was barley meal No. 363. Table 4 The effect of amylase on the estimation of the rumen theoretical and enzymatic degradability of cereals Treatment
n
In situ TD (% CP)
ED (% CP)
a
b
r
RSD
No amylase Amylase”
19 19
69.29k11.76 69.29 +_11.76
76.10f 12.33 84.83+ 6.64
65.72 - 52.24
0.159 1.432
0.259* o.s09-
10.066 7.104
‘Amylase (0.5 mg ml- I) was included in the incubation with bromelain. ‘P< 0.05. -P<0.0001. CP, crude protein.
creased significantly up to a value of 0.809 (P-C 0.000 1) (Table 4) which is comparable with correlation coeffkients of other types of feeds. 4. Discussion An important factor in the enzymatic prediction of rumen protein degradability is selection of the protease. Many enzymes have been suggested and tested (Krishnamoorthy et al., 1983; Poos-Floyd et al., 1985; KopeEny et al., 1989; Roe et al., 199 1) . Lately the most commonly tested protease has been Pronase E. This product is a mixture of various endoproteinases and peptidases. Their activities are variable, depending on the type of cultivation and proteinase separation. On the other hand, bromelain is a single enzyme with a stable specific activity. Measurements of the rumen protein degradability with proteolytic enzymes
0. Tomdnkov&,J. KopeEnjt/Animal Feed Science and Technology 53 (1995) 71-80
79
depend on the accurate estimation of regression equations for the enzymatic and in situ methods (Roe et al., 199 1). These equations for suitable enzymes have high correlation coelicients when small sets of feeds are analysed (Poos-Floyd et al., 1985; KopeEny et al., 1989; Assoumani et al., 1992). When a larger set of feeds is tested (from our experience) the correlation coefficient usually decreases. To increase the accuracy, it is necessary to group feeds into sets of similar nitrogen fraction properties. This is the reason why hays (Auf&e et al., 1989) and cereals (Susmel et al., 1993) had been tested separately. We went further and separated silages and protein concentrates (Table 2). The properties of the crude protein fraction in these feeds are different when using bromelain as a proteolytic enzyme. It was suggested to treat cereals or feeding mixtures with a mixture of cyamylase and pglucanase to avoid steric inhibition of proteolytic enzymes by starch and cellulose (Assoumani et al., 1992). In our method the cr-amylase treatment was found to be sufficient for the prevention of steric inhibition (Table 3 ). The proposed method is going to be used in this country as a standard method for estimating feed protein degradability.
References Association of official Analytical Chemists, 1980. Method 33.05 1. Offtcial Methods of Analysis of the Association of Official Analytical Chemists, 13th edn. AOAC, Baltimore. Assoumani, MB., Vedeau, F., Jaquot, L. and Sniffen, C.J., 1992. Refinement ofan enzymatic method for estimation the theoretical degradability of proteins in feedstuffs forruminants. Anim. Feed Sci. Technol., 39: 357-368. Auf&e, J., Michalet-Doreau, B., Graviou, D. and Vtrite R., 1989. Predicting in sacco degradability of hay protein by chemical or enzymatic method. XVI Internatiol Grassland Congress, 4-l 1 October 1989, Nice. The French Grassland Society, pp. 887-888. Auf&e, J., Graviou, D., Demarquilly, C., Verite, R., Michalet-Doreau, B. and Chapoutot, P., 199 1. Predicting in situ degradability of feed proteins in the rumen by two enzymatic methods (solubility and enzymatic degradation). Anim. Feed Sci. Technol., 33: 97-l 16. Homo&a, P. and Vencl, B., 1990. Retention time of feed particles in forestomachs. In: B. Vencl and Z. Frydrych (Editors), New systems of energy and protein evaluation for ruminants. International Symposium, 6-7 June 1990, Prague. Czechoslovak Scientific and Technical Society, Prague, pp. 110-115. KopeEny, J. and Wallace, R.J., 1982. Cellular location and some properties of proteolytic enzymes of rumen bacteria. Appl. Environ. Microbial., 43: 1026- 1033. KopeEny, J., Vencl, B., Kyselova, J. and Biezina, P., 1989. Determination of rumen degradable protein with enyzmes. Arch. Anim. Nutr., 39: 549-555. Krishnamoorthy, U., Sniffen, C.J., Stem, M.D. and van Soest, P.J., 1983. Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen undegraded nitrogen content of feedstuffs. Br. J. Nutr., 50: 555-568. Madsen, J. and Hvelpund, T., 1985. Protein degradation in the rumen. Comparison between in vivo, nylon bag, in vitro and buffer measurements. Acta Agric. Stand. Suppl., 25: 103-124. Mahadevan, S., Sauer, F.D., Ertle, J.D., 1987. Preparation of protease from mixed rumen microorganisms and its use for the in vitro determination of the degradability of true protein in feedstuffs. Can. J. Anim. Sci., 67: 55-64. Orskov, E.R. and McDonald, I., 1979. The estimation of protein degradability in the lumen from incubation measurements weighted according to the rate of passage. J. Agric. Sci., 92: 499-503.
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0. Tomdnkowi, J. KopeEnj /Animal Feed Science and Technology 53 (1995) 71-80
Poos-Floyd, M., Klopfenstein, T. and B&ton, R.A., 198.5. Evaluation of laboratory techniques for predicting ruminal protein degradation. J. Dairy Sci., 68: 829-839. Roe, M.B., Chase, L.E. and Sniffen, C.J., 199 1. Comparison of in vitro techniques to the in situ technique for estimation of ruminal degradation of protein. J. Dairy Sci., 74: 1632-l 640. Russell, J.B., O’Connor, J.D., Fox, D.G., van Soest, P.J. and Sniffen, C.J., 1992. A net carbohydrate and protein system for evaluating cattle diets: I. Ruminal fermentation. J. Anim. Sci., 70: 355 l3561. Sniffen, C.J., O’Connor, J.D., van Soest P.J., Fox D.G. and Russell, J.B., 1992. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. J. Anim. Sci., 70: 3562-3517. Susmel, P., Mills, C.R., Colitti, M. and Stefanon, B., 1993. In vitro solubility and degradability of nitrogen in concentrate ruminant feeds. Anim. Feed Sci. Technol., 42: l-l 3. Vencl, B., 1986. Solubility and in situ nitrogen degradability of ruminants feeds. 37 Annual Meeting EAAP, l-4 September 1986, Budapest. Agrinform 80/86. Budapest.
L
ELSEVIER
Animal Feed Scienceand Technology53 (1995) 71-80
Prediction of feed protein degradation in the rumen with bromelain 0. Tom&nkova”, J. K~peEn$~~* ‘Institute ofAnimal Production, Prague IO, UhFMves, Czech Republic bInstitute ofAnimal Physiology and Genetics, Czech Academy of Sciences, Prague IO, UhtYnnpVes, Czech Republic
Received 14 February 1994;accepted28 September 1994
First a suitable protease for enzymatic determination of in vitro rumen protein degradation was selected. The actions of Pronase E, papain and bromelain were compared. The highest correlation coefficient for protein degradation estimated by the in sacco method compared with the enzymatic method was obtained with bromelain in a set of 13 feeds. This enzyme was subsequently used for analysis of more than 200 ruminant feed components. Feeds were separated on the basis of their chemical properties into the following live groups: cereals, forages, hays, protein concentrates, &ages. In these groups, correlation coefficients of linear regression equations for rumen protein degradability estimated by the in sacco method and bromelain were 0.839 for concentrates, 0.730 for fresh forage, 0.741 for hays and 0.876 for &ages. Feeds with high starch content had to be treated with cr-amylase to obtain the correlation coefficient r=0.809. The method is going to be used for preliminary testing of ruminant feeds. Keywords: Bromelaiq Protein degradation; Rumen
1. Introdaction
New feeding systems for ruminants are based on exact data of amino acid absorption from the duodenum. It is possible to calculate these values from feed analysis. Calculations have to take into account the effect of rumen fermentation (Russell et al., 1992; Sniffen et al., 1992). In the rumen, nitrogen compounds are degraded and the extent of protein degradation influences the’estimation of the * Corresponding author. 0377-8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZO377-8401(94)00735-7
12
0. Torn&&w&,J. KopeEni,/Animal Feed Scienceand Technology53 (1995) 71-80
amount of amino acids entering the duodenum. One of the most reliable methods to estimate feed protein degradation in the rumen is the in sacco method (0rskov and McDonald, 1979). Unfortunately, this type of measurement is not available to many laboratories and is often replaced by other types of estimation. The most simple and rapid alternative method is the measurement of soluble nitrogen compounds (Madsen and Hvelpund, 1985; Susmel et al., 1993), although various enzymatic methods were found to be more accurate (Poos-Floyd et al., 1985). Mahadevan et al. ( 1987) used isolated rumen proteolytic enzymes for this purpose. Several methods utilize proteinases from Streptomyces griseus (Pronase E ) (Auf&e et al., 1991). Poos-Floyd et al. ( 1985) suggested bromelain as one of the most suitable enzymes for this purpose. Measurements of protein degradability on a small number of samples may lead to inaccurate estimation of linear regression equations and consequently to an incorrect estimation of the feed quality. The aim of this study was to develop an enzymatic method based on bromelain and to evaluate its suitability for predicting protein degradability on a representative set of feeds for ruminants.
2. Materials and methods 2.1. Feeds Feeds were divided into several groups according to their chemical composition and properties of their protein fractions. The ruminant feed components tested are listed in Table 1. 2.2. Animals Two non-lactating crossbred cows (Friesian x Bohemian spotted) fitted with large rumen cannulae (Avon Industrial Polymers, GB) were used for our experiments. Their diet consisted of 14 kg of corn silage, 6 kg of lucerne hay and 1 kg of ground barley with a mix of vitamins and minerals. The diet was offered in two equal meals daily. 2.3. In situ measurements The in sacco rumen degradability was measured according to Orskov and McDonald ( 1979) as adapted by Vencl ( 1986). Air-dried feed samples were milled through a 1.Omm mesh and 1 g was put into nylon bags (6 x 12 cm, pore size 53 pm) and incubated in the rumen for 4,8,16,24 and 48 h. The removed bags were rinsed in cold water and their nitrogen content was determined by Kjeldahl analysis (Association of Official Analytical Chemists (AOAC), 1980). Measurements were done in triplicate. The degradability of feed proteins was
0. Tomcinkovd, J. Kopetnj /Animal Feed Science and Technology 53 (1995) 71-80
73
Table 1 Feed groups and their average chemical composition n
DM (g kg-‘)
OM (gkg-’ DM)
CP (g kg-’ DM)
1. Animal and vegetable proteins Fish meal Blood meal Meat and bone, high oil Feather meal Groundnut meal Rapeseed meal Soyabean meal Cottonseeds meal Sunflower meal Sesame meal Linseed meal Feeding mixtures concentrates
2 2 2 1 4 3 3 2 1 1 1 18
921 922 947 925 930 915 916 911 939 939 915 888
967 952 764 967 925 928 926 921 931 912 943 935
682 283 528 774 439 394 491 474 387 459 404 162
2. Cereals Wheat meal Triticale meal Barley meal Maize meal Wheat bran Oat Rye
3 1 11 4 4 3 1
866 862 889 890 886 897 881
952 940 946 980 959 969 980
140 144 119 113 157 111 111
3. Fresh forages Alfalfa Clover Maize Ryegrass
14 15 7 5
199 172 289 171
889
888 949 899
209 175 190 140
4. Hays Meadow hay Alfalfa
8 24
943 950
907 902
128 170
Silages I: whole plants Maize Alfalfa Oat Meadow grass Barley Wheat + pea Horsebeans
15 8 1 2 5 4 1
945
938 962 920 957 950 949
934 891 867 886 942 903 878
98 181 82 157 90 127 173
9
959
731
127
5. Silages II: leaves Sugar beet
n, number of samples; DM, dry matter; OM, organic matter; CP, crude protein.
74
0. Tomdnkovrt. J. Kopefnj /Animal Feed Science and Technology 53 (1995) 71-80
calculated from the kinetics of in situ degradation using equation of Orskov and McDonald ( 1979): y=a+b(
1 -e-C’)
where a is the immediately degradable nitrogen fraction, b is the potentially degradable nitrogen fraction, c is the constant rate of degradation of the fraction b and t is time (h) of incubation. In situ theoretical degradabilities (TD) were calculated according to the equation: TD=a+
[ (bc)/(c+k)]
The optimal outflow rate (k) for concentrates (Homolka et al., 1990) and fresh forage was calculated to be 0,060 h-’ and for all other types of feeds 0.046 h-‘. 2.4. Enzymatic method Dry feed samples were milled through 0.8 mm mesh and 0.5 g was incubated in a solution (ED solution) of 50 ml 100 mM phosphate buffer pH 7.2 with 5 mM cysteine, 1 mM EDTA, 1 mg ml-’ chloramphenicol, 0.75 mg ml-’ sodium sorbate and 3 mg bromelain (Sigma; 2-4 U mg- ’protein) at 40 ’C for 24 h (Kop&y et al., 1989). The final concentrations of papain and Pronase E (Sigma) were 8.3 pg ml- ’ and 1.4 a ml- ‘, respectively. Papain was incubated in the presence of 100 mM phosphate buffer pH 6 with 5 mM cysteine and Pronase E in the presence of 100 mM phosphoborate buffer pH 8. All other incubation components were the same. Reaction was stopped by 25% trichloroacetic acid (TCA) (final concentration 5% TCA) . Samples were cooled and centrifuged at 5 100 xg for 5 min. The sediment was transferred to filter paper (Whatman No. 54), rinsed twice with 5% TCA and digested using the Kjeldahl procedure. Ammonia was estimated with Nessler reagent (AOAC, 1980). Each sample was done in triplicate. Enzymatic degradability (ED) was expressed as percentage of original crude protein content. Into all series standard samples of alfalfa hay and two blanks without feeds were included. Proteolytic activity was measured using azocasein (Kopetiy and Wallace, 1982). 2.5. Statistical methods Differences between methods were evaluated by analysis of variance and simple linear regression. 3. Results 3.1. Enzyme selection The first step in this study was a selection of the most suitable protease. The selection was done using 13 different types of feeds and three enzymes-papain,
0. Tom&&m&, J. Kopetn$ /Animal Feed Science and Technology 53 (1995) 71-80
75
bromelain and Pronase E. Results were compared with data obtained from the in sacco measurements (Table 2). From the linear regression equations the highest correlation coefficients were obtained for bromelain. 3.2. Bromelain degradation offeedproteins A comparison of in situ (TD) and bromelain degradation (ED) of feed proteins in a set of 186 feed samples is shown in Table 3. Different slopes of linear regression for different types of feeds led us to group feeds with similar properties. Grouping reflected the source and treatment of the feed and protein availability. Data distribution is shown in Fig. 1. Accuracy of the enzymatic estimation from repeat analysis of the same samples was + 3.1%. The effect of final TCA treatment was tested. TCA had no significant effect on protein degradabilityvalues. The only exception were samples of blood meal where Table 2 Linear regressions of feeds rumen protein, degradability lain, papain and Pronase E
estimated in sacco and in vitro with brome-
Enzyme
n
a
b
r
RSD
RSD%
Papain Bromelain Pronase E
13 13 13
33.38 - 19.08 20.04
0.657 1.138 0.714
0.765 0.855 0.627
8.10 6.53 9.81
11.43 9.20 13.85
n, number of samples; a, b, constants of linear regressions; r, correlation standard deviation. Table 3 Comparison proteins Feeds
of in sacco theoretical degradabihty
n
In situ TD (% CP)
(TD) and enzymatic degradability
ED (% CP)
Animal and vegetable concentrates + feeding mixtures 40 68.15k19.16 74.94 f 17.63 Cereals 28 72.81 f 11.08 75.19+ 11.43 Fresh forage 41 62.53f8.15 67.76 kg.90 Hays and silage I 68 63.48 f 14.35 73.38f 13.17 Silage II 9 64.84+ 8.74 58.37+ 10.58 ?? P< 0.05. QP
coefftcient; RSD, residual
(ED) of feed
b
r
0.46
0.914
0.839”
7.072
43.42
0.388
0.423’
10.145
18.65
0.648
0.730-
5.815
- 17.16
1.051
0.741-
7.319
22.55
0.724
0.876*+
4.502
a
RSD
16
0. Tom&nkov&,J. KopeEn$ /Animal Feed Science and Technology 53 (1995) 71-80
TCA treatment caused a 2.8-3.2% decrease of protein degradability (results not shown). An effect of passage rate (in the range 0.02-l .OOh- ’) on in situ degradability and following correlation with enzymatic degradability was tested. It was assumed that the passage rate for concentrates and fresh forage is optimal at the level of 0.06 h- ‘. All other feeds were evaluated at a passage rate of 0.046 h-l. These values gave the highest correlation coefficients between ED and TD. Correlation coefficients for different groups of feeds were at least 0.73. The only exception was the group of cereals where availability of protein for ED was different from sample to sample. In these cases it was necessary to decrease the effect of the starch in the samples (Table 3) on the enzymatic determination. a
.$
40
.G P
L
XI
. .
,
.o in Go
. r = 0.71
80
degExJationm[%j
y = 18.850
-
+ 0.847X
b
. r = 0.878
-
y = 22.552
+ 0.72x
I
a,
0. Tom&nkznvi,J. Kope&+ /Animal Feed Science and Technology 53 (1995) 71-80
77
d
--y=-0.462=0.91x
mr=0.639
e
A-P
ac
50
60
70
in vitro degradation m r = 0.423
-
81
m
I Irn
[%]
y = 43.426
+ 0.366x
Fig. I. The relationship between measured in situ protein degradability and that estimated from enzymatic degradation for fresh forage (a), hays (b), silage (c), protein concentrates (d) and cereals (e). Linear regression equation and correlation coefficient are added for each data set.
3.3. Amylase treatment Protein degradation in samples with a high starch content did not correlate under in situ conditions with bromelain treatment even for one type of feed. Correlation coefficients for different types of grains and meals were usually lower than 0.4; for a combination of all cereals the result was r= 0.423 (Table 3 ). It was suggested that starch was the main compound limiting the availability of proteins for bromelain. Therefore cu-amylase treatment was included in the procedure. Amylase was tested under different conditions. Initially, sequential incubation was done with cr-amylase, followed by the proteinase treatment. It was found that the degree of amylase inhibition by bromelain was negligible (Fig. 2 ) and thus it was possible to incubate both enzymes together. Therefore 0.5 mg of a-amylase (Bolamylase, specific activity 1.05 mg maltose h-r mg-I; Lachema, Czech Republic) per 1 ml of the ED solution were incubated together with bromelain and samples for 24 h. Blank samples with amylase were introduced to eliminate the effect of the nitrogen content of amylase. After this treatment correlation coefficients of linear regression for cereals in-
78
0. Tom&nkov&,J. KopeCn$ /Animal Feed Science and Technology 53 (1995) 71-80 90
Amylase
added
2 : .g
72
-
E
66
. . No -
amylase
60
0
5
added
1 10
15
Time
(hours]
20
25
Fig. 2. The effect of interval between amylase and bromelain addition on in vitro estimated rumen protein degradation. Amylase was added in a concentration of 0.5 mg ml-’ and incubated for 24 h at 40°C. This was followed by incubation for 24 h with bromelain at the same temperature. The sample tested was barley meal No. 363. Table 4 The effect of amylase on the estimation of the rumen theoretical and enzymatic degradability of cereals Treatment
n
In situ TD (% CP)
ED (% CP)
a
b
r
RSD
No amylase Amylase”
19 19
69.29k11.76 69.29 +_11.76
76.10f 12.33 84.83+ 6.64
65.72 - 52.24
0.159 1.432
0.259* o.s09-
10.066 7.104
‘Amylase (0.5 mg ml- I) was included in the incubation with bromelain. ‘P< 0.05. -P<0.0001. CP, crude protein.
creased significantly up to a value of 0.809 (P-C 0.000 1) (Table 4) which is comparable with correlation coeffkients of other types of feeds. 4. Discussion An important factor in the enzymatic prediction of rumen protein degradability is selection of the protease. Many enzymes have been suggested and tested (Krishnamoorthy et al., 1983; Poos-Floyd et al., 1985; KopeEny et al., 1989; Roe et al., 199 1) . Lately the most commonly tested protease has been Pronase E. This product is a mixture of various endoproteinases and peptidases. Their activities are variable, depending on the type of cultivation and proteinase separation. On the other hand, bromelain is a single enzyme with a stable specific activity. Measurements of the rumen protein degradability with proteolytic enzymes
0. Tomdnkov&,J. KopeEnjt/Animal Feed Science and Technology 53 (1995) 71-80
79
depend on the accurate estimation of regression equations for the enzymatic and in situ methods (Roe et al., 199 1). These equations for suitable enzymes have high correlation coelicients when small sets of feeds are analysed (Poos-Floyd et al., 1985; KopeEny et al., 1989; Assoumani et al., 1992). When a larger set of feeds is tested (from our experience) the correlation coefficient usually decreases. To increase the accuracy, it is necessary to group feeds into sets of similar nitrogen fraction properties. This is the reason why hays (Auf&e et al., 1989) and cereals (Susmel et al., 1993) had been tested separately. We went further and separated silages and protein concentrates (Table 2). The properties of the crude protein fraction in these feeds are different when using bromelain as a proteolytic enzyme. It was suggested to treat cereals or feeding mixtures with a mixture of cyamylase and pglucanase to avoid steric inhibition of proteolytic enzymes by starch and cellulose (Assoumani et al., 1992). In our method the cr-amylase treatment was found to be sufficient for the prevention of steric inhibition (Table 3 ). The proposed method is going to be used in this country as a standard method for estimating feed protein degradability.
References Association of official Analytical Chemists, 1980. Method 33.05 1. Offtcial Methods of Analysis of the Association of Official Analytical Chemists, 13th edn. AOAC, Baltimore. Assoumani, MB., Vedeau, F., Jaquot, L. and Sniffen, C.J., 1992. Refinement ofan enzymatic method for estimation the theoretical degradability of proteins in feedstuffs forruminants. Anim. Feed Sci. Technol., 39: 357-368. Auf&e, J., Michalet-Doreau, B., Graviou, D. and Vtrite R., 1989. Predicting in sacco degradability of hay protein by chemical or enzymatic method. XVI Internatiol Grassland Congress, 4-l 1 October 1989, Nice. The French Grassland Society, pp. 887-888. Auf&e, J., Graviou, D., Demarquilly, C., Verite, R., Michalet-Doreau, B. and Chapoutot, P., 199 1. Predicting in situ degradability of feed proteins in the rumen by two enzymatic methods (solubility and enzymatic degradation). Anim. Feed Sci. Technol., 33: 97-l 16. Homo&a, P. and Vencl, B., 1990. Retention time of feed particles in forestomachs. In: B. Vencl and Z. Frydrych (Editors), New systems of energy and protein evaluation for ruminants. International Symposium, 6-7 June 1990, Prague. Czechoslovak Scientific and Technical Society, Prague, pp. 110-115. KopeEny, J. and Wallace, R.J., 1982. Cellular location and some properties of proteolytic enzymes of rumen bacteria. Appl. Environ. Microbial., 43: 1026- 1033. KopeEny, J., Vencl, B., Kyselova, J. and Biezina, P., 1989. Determination of rumen degradable protein with enyzmes. Arch. Anim. Nutr., 39: 549-555. Krishnamoorthy, U., Sniffen, C.J., Stem, M.D. and van Soest, P.J., 1983. Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen undegraded nitrogen content of feedstuffs. Br. J. Nutr., 50: 555-568. Madsen, J. and Hvelpund, T., 1985. Protein degradation in the rumen. Comparison between in vivo, nylon bag, in vitro and buffer measurements. Acta Agric. Stand. Suppl., 25: 103-124. Mahadevan, S., Sauer, F.D., Ertle, J.D., 1987. Preparation of protease from mixed rumen microorganisms and its use for the in vitro determination of the degradability of true protein in feedstuffs. Can. J. Anim. Sci., 67: 55-64. Orskov, E.R. and McDonald, I., 1979. The estimation of protein degradability in the lumen from incubation measurements weighted according to the rate of passage. J. Agric. Sci., 92: 499-503.
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Poos-Floyd, M., Klopfenstein, T. and B&ton, R.A., 198.5. Evaluation of laboratory techniques for predicting ruminal protein degradation. J. Dairy Sci., 68: 829-839. Roe, M.B., Chase, L.E. and Sniffen, C.J., 199 1. Comparison of in vitro techniques to the in situ technique for estimation of ruminal degradation of protein. J. Dairy Sci., 74: 1632-l 640. Russell, J.B., O’Connor, J.D., Fox, D.G., van Soest, P.J. and Sniffen, C.J., 1992. A net carbohydrate and protein system for evaluating cattle diets: I. Ruminal fermentation. J. Anim. Sci., 70: 355 l3561. Sniffen, C.J., O’Connor, J.D., van Soest P.J., Fox D.G. and Russell, J.B., 1992. A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. J. Anim. Sci., 70: 3562-3517. Susmel, P., Mills, C.R., Colitti, M. and Stefanon, B., 1993. In vitro solubility and degradability of nitrogen in concentrate ruminant feeds. Anim. Feed Sci. Technol., 42: l-l 3. Vencl, B., 1986. Solubility and in situ nitrogen degradability of ruminants feeds. 37 Annual Meeting EAAP, l-4 September 1986, Budapest. Agrinform 80/86. Budapest.