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Use of electrophoresis to quantify ruminal degradability of protein from concentrate feeds
• E LS EV I ER
ANIMAL FEED SCIENCE AND TECHNOLOGY
Animal Feed Scienceand Technology49 (1994) 25-35
Use of electrophoresis to quantify ruminal degradability of protein from concentrate feeds M . A . M e s s m a n 1, W.P, Weiss* Department of Dairy Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA
Received 8 June 1993; accepted I March 1994
Abstract
Proteins in soyabean meal, spray-dried and ring-dried blood meals, maize grain, and maize dried distillers grains were extracted before and after ruminal fermentation in vitro. Extracts were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis, and stain intensity of the bands was quantified by densitometry. Undegradability values (proportion of total protein) for soyabean meal, spray-dried blood meal, ring-dried blood meal, maize grain, and distillers grains were 0.40, 0.99, 0.97, 0.56, and 0.82, respectively, after 2 h of fermentation in vitro and 0.09, 0.79, 0.82, 0.49, and 0.59, respectively, after 20 h of fermentation. Soyabean meal and spray-dried blood meal contained slowly degradable, soluble proteins. Individual proteins within feedstuffs showed different degrees of degradation. The basic subunit of glycinin was degraded much slower than were other proteins in soyabean meal. Zein proteins in maize grain and distillers grains were much less degradable than were the glutelin proteins. Electrophoresis has potential as a method for measuring degradability of both particle and fluid associated proteins and to account for bacterial protein contamination.
1. Introduction
The crude protein ( C P ) fraction of feedstuffs is composed of a multitude of proteins. Proteins differ in a m i n o acid composition and sequence and in secondary, tertiary, and quaternary structure. Tertiary ( M a h a d e v a n et al., 1980; Chen et al., 1987) and secondary structure (Wallace and Kopecny, 1983) probably affect rate and extent of protein hydrolysis in the rumen. A more complete char*Correspondingauthor. 1Present address: Growmark, Inc., Bloomington,IL 61702, USA. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10377-8401 (94) 00641-L
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
acterization of the CP fraction of feeds may help explain variation in ruminal degradation of protein within and among feedstuffs. Individual proteins within a feedstuffcan be quantified using electrophoresis and densitometry (Messman et al., 1994). Disappearance of individual proteins during ruminal incubation also has been monitored qualitatively using electrophoresis (Van der Aar et al., 1983; Lynch et al., 1988; Spencer et al., 1988; Newbold and Rust, 1990; Romagnolo et al., 1990; Fahmy et al., 1991 ). Several methods can be used to estimate ruminal degradability of proteins, including fermentation in vitro and in sacco, and enzymatic procedures (National Research Council (NRC), 1985; Nocek, 1988; Michalet-Doreau and Ould-Bah, 1992). Disappearance in sacco is used frequently to estimate protein undegradability, but a major problem is the influence of microbial protein contamination. Nitrogen provided by attached bacteria must be subtracted from residual N or undegradability will be overestimated (Nocek, 1988; Michalet-Doreau and OuldBah, 1992). Another potential problem with the in sacco procedure is the assumption that protein which is soluble and flows out of the bag is degraded instantaneously. Mahadevan et al. (1980) and Spencer et al. ( 1988 ), however, have shown that there is a soluble protein fraction that is not degraded instantaneously. A soluble, but slowly degraded protein fraction may compromise in sacco protein degradability estimates. Objectives of this experiment were: ( 1 ) to determine the ruminal degradability of soluble proteins in concentrate feedstuffs; (2) to quantify extent of ruminal degradation of individual proteins within a feed; (3) to determine if fermentation in vitro followed by electrophoresis and densitometry could be used to estimate ruminal protein degradability.
2. Materials and methods
Feed samples (Table 1 ) underwent fermentation in vitro for 2 h or 20 h (in duplicate). Ruminal fluid used in the invitro system was obtained from a lactating Holstein cow fed a diet consisting of 430 g kg- 1 of alfalfa (Medicago sativa Table 1 Concentrations and extractability of crude protein (CP) Feedstuff
Soyabean meal (solvent) Spray-dried blood meal Ring-dried blood meal Maize grain Maize dried distillers grains
CP ( g kg - 1 DM)
490 960 980 80 300
Extractability a g kg- 1 total CP
SE
600 910 400 630 470
36.8 16.8 58.9 31.6 70.7
aSamples were extracted in duplicate using a borate-phosphate buffer containing sodium dodecyl sulfate.
M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
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L.) silage, 280 g kg -1 of maize ( Z e a m a y s L.) silage, 210 g kg -1 of concentrate, and 80 g kg-1 of alfalfa hay (dry basis). Fluid and particle associated microbes in the ruminal fluid were extracted with cold (4°C) buffer (Craig et al., 1984). Feed samples ( 1 g) were incubated with 50 ml of inoculum at 39°C under CO2. At the desired end point, phenylmethylsulfonyl fluoride and iodoacetate were added (final concentrations of 3 mM and 5 mM, respectively) to stop protease activity (Brock et al., 1982). Contents of the flasks were transferred to centrifuge tubes and centrifuged at 268g (4 °C) for 15 min to separate particles and fluid. A mixture of ground maize cobs without grain (20 g kg- 1 of CP) and urea ( 150 g kg- 1 of total CP) was used to measure microbial proteins present after fermentation in vitro. Particle and fluid portions of the blank were separated by centrifugation. Before fermentation, extracts of blanks contained no electrophoretically identifiable proteins. After fermentation in vitro, proteins found in extracts of residual cob material or the fluid fraction were assumed to be bacterial in origin. Feeds were analyzed for dry matter (DM) and CP (Association of Official Analytical Chemists (AOAC), 1980). Proteins in feeds and the 2 h and 20 h particle fractions were extracted in duplicate using a borate buffer that contained sodium dodecyl sulfate (SDS) and sonication (Messman and Weiss, 1993 ). Extracts of feed and particle fractions, and the fluid fraction were mixed with an equal volume of denaturing buffer, denatured by heating, and electrophoresed on a discontinuous SDS-polyacrylamide gel (See a n d Jackowski, 1989). Stacking and running gels were 4% and 12.5% polyacrylamide, respectively. Following electrophoresis, proteins bands were stained (Coomassie blue R-250) and stain intensity was quantified using a scanning densitometer (BioRad Model 620, Richmond, CA). Disappearance of individual proteins was quantified by scanning gels of feed extracts to determine the relative mobility (Rf) and band density of the major proteins within a feed. Extracts of the fluid and particle fractions from the incubations were electrophoresed and scanned. Densities of bands at the same Rf as in the feed samples were determined. Gels of the extracts of fluid and particulate matter from fermentations of blanks were scanned and any densities found at the Rf of feed proteins were subtracted from the density values obtained from the 2 h and 20 h sample incubations. The blank-corrected densities at each Rf for the 2 h and 20 h samples were then divided by the appropriate density obtained for the original feed. Disappearance of total electrophoretically identified protein (TEIP) was determined by summing the blank-corrected densities of bands at the Rf of interest and dividing by the sum of the densities in the original feeds. Details of electrophoresis and densitometry have been given by Messman et al. (1994). To determine if undegradable soluble proteins made up a significant proportion of total undegradable protein, a t-test (Steel and Torrie, 1980) was used. The amount of undegraded protein in t h e particle fraction was compared with the total amount of protein remaining at each time point. When total and particle associated protein differed ( P < 0.05 ), we concluded that proteins in fluid contributed significantly to the total. To test for differences in degradability of individual proteins within a feed, a completely randomized design was used. The
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M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
model included protein species as the independent variable and undegradability as the dependent variable. Analyses were conducted within a feed and within an incubation period. Fisher's protected least significant difference test (Steel and Torrie, 1980) was used to separate differences in extent of protein hydrolysis within feeds. All statistical calculations were performed using software of the Statistical Analysis Systems Institute, Inc. (SAS) ( 1988 ).
3. Results and discussion
Concentrations of CP of feeds ranged from 80 g kg -1 to 980 g kg -1 (Table 1 ). Extractability of CP ranged from 400 g kg- 1 to 910 g kg- 1 of total CP and averaged 490 g kg -1 of total CP. Fish meal and maize gluten meal were included originally in the sample set; however, only 270 g kg -1 and 170 g kg -1, respectively, of total CP were extracted. Extractability of CP from soyabean (Glycine m a x L. ) meal and maize grain was lower than that reported by Krishnamoorthy et al. (1982). Those workers refluxed samples for 1 h in neutral detergent solution, but in our experiment, sonication was used. The extraction procedure used in this experiment was designed to extract forage proteins rapidly (Messman and Weiss, 1993). Green plants contain a high ratio of albumins and globulins to prolamines and glutenins, whereas grains have a low ratio. 3.1. Differences within feeds 3.1.1. Soyabean meal
Eleven proteins were extracted from soyabean meal (Fig. 1; Table 2). Following 2 h of fermentation in vitro, ten proteins were found. Undegradable TEIP (proportion of original TEIP) after 2 h of fermentation in vitro was 0.40 (Table 2 ). The range in undegradability (2 h) for individual proteins was 0-1.0. Glycinin proteins (21, 43, and 48 kDa) were less degradable than were the r- and asubunits of conglycinin (57 and 78 kDa). We found no difference in the ratio of r- to a-conglycinin in the particle associated protein fraction after 2 h. Newbold and Rust (1990) found the ratio of r- to a-conglycinin decreased during 10 h in situ ruminal exposure. The basic subunit of glycinin (21 kDa) showed no disappearance following 2 h of incubation. All other proteins had less than half of their original mass after 2 h of incubation. With the exception of the basic subunit of glycinin, there were few differences between glycinin and conglycinin proteins with respect to 2 h degradability. Our data confirm that the basic subunit of glycinin is less degradable than acidic glycinin subunits (43 and 48 kDa) (Kella et al., 1986; Newbold and Rust, 1990; Romagnolo et al., 1990). Proteins that are degraded at rates less than average will be enriched in the residue at the expense of proteins that degrade at rates faster than average. At 0 h, the 21 kDa protein made up 0.18 of TEIP, but after 2 h of incubation it constituted 0.45 of TEIP (Table 2). The 43 kDa protein made up 0.18 of TEIP at time 0, but only 0.10 following 2 h of incubation. Similar results have been reported
M.A. Messman, W..P. Weiss / Anirnal Feed Science and Technology 49 (1994) 25-35
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Fig. 1. Electrophoretogramof soyabeanmeal. Lane 1 is molecularweight standards; Lane 2, soyabean meal before incubation (0 h); Lanes 3 and 4, particle fraction after 2 h of incubation; Lanes 5 and 6, fluid fraction after 2 h of incubation; Lane 7, particle fraction blank; Lane 8, fluid fraction blank. by others (Van der Aar et al., 1983; Romagnolo et al., 1990). Small changes in the concentration of the 21 kDa protein could have a substantial impact on the overall degradability of soyabean meal. The basic subunit of glycinin (21 kDa) has lower concentrations oflysine and methionine than does total glycinin (Moreira et al., 1979 ); therefore, the nutritional value ofundegradable CP in soyabean meal may be less than that of the original protein. The majority of protein remaining after 2 h of fermentation was found in the particulate fraction, but 0.32 of the remaining proteins was found in the fluid fraction. Most of the 21 kDa protein (basic subunit ofglycinin) and a substantial amount of the 57 kDa protein (fl-conglycinin) was found in the fluid fraction. Ruminal fluid turnover rates in lactating dairy cattle range from approximately 0.1 to 0.15 h-1, which corresponds to residence times of 7-10 h. Even though residence time in the rumen is greater than 2 h, some fluid associated proteins probably escape from the rumen. Others have reported significant amounts of slowly degradable, soluble protein in pea meal (Spencer et al., 1988 ) and bovine serum albumin (Nugent et al., 1983; Spencer et al., 1988 ). After 20 h fermentation in vitro, only four proteins were found in the particle fraction, and no proteins were found in the fluid fraction (Table 2). The 48 kDa protein (heavy acidic subunit of glycinin ) had an undegradability (20 h) of 0.47; however, this protein was found in relatively low amounts. After 20 h, the undegradability of TEIP was 0.09.
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
Table 2 Soyabean meal protein remaining after 2 or 20 h fermentation in vitro Protein ~
Proportion ofTEIP z
Undegradability3
Proportion in fluid fraction 4
(kDa )
21 25 36 39 43 48 57 68 73 78 82 TEIP SE
Oh
2h
20h
2h
20h
2h
20h
0.18 0.06 0.05 0.07 0.18 0.08 0.10 0.06 0.09 0.08 0.05 1.00 -
0.45 0.00 0.03 0.05 0.10 0.07 0.10 0.06 0.03 0.05 0.06 1.00 -
0.21 0.00 0.00 0.00 0.24 0.41 0.15 0.00 0.00 0.00 0.00 1.00 -
1.00 a 0.00 ° 0.29 be 0.34 b 0.23 be 0.39 b 0.29 b~ 0.38 b 0.17 ~ 0.26 b¢ 0.46 b 0.40 0.07
0.10 b 0.00 c 0.00 c 0.00 c 0.12 b 0.47 a 0.14 b 0.00 c 0.00 ¢ 0.00 c 0.00 c 0.09 0.01
0.67* 0.00 0.00 0.00 0.00 0.41" 0.00 0.00 0.00 0.00 0.32* -
0.00 0.00 0.00 0.00 0.00 -
~Known proteins are: 21 k D A - - b a s i c subunit o f glycinin; 43 k D a - - l i g h t acidic subunit o f glycinin; 48 k D a - - h e a v y acidic subunit of glycinin; 57 kDa--fl-subunit of conglycinin; 68 k D a - - ? - s u b u n i t o f conglycinin; 73 kDa----~' subunit of conglycinin; 78 kDa--a-subunit of conglycinin. ZGrams of individual protein per gram of total electrophoretically identified protein ( T E I P ) . 3Gram of protein remaining per gram of protein at 0 h.
4Gram of protein remaining in fluid per gram of total (fluid + particulate) protein remaining. *Different from zero ( P < 0.05). a,b,CMeans within a column (excluding T E I P ) differ ( P < 0.05 ).
3.1.2. Blood meal Five proteins were found in spray-dried blood meal and three proteins were found in ring-dried blood meal (Table 3). Overall, undegradability values for spray-dried and ring-dried blood meals were very similar; however, the distribution of the proteins was markedly different. No soluble proteins were found after 2 h or 20 h of fermentation for ring-dried blood meal. After 2 h of fermentation, essentially all the original protein was still present. For spray-dried blood meal, no disappearance occurred during 2 h of incubation (Fig. 2), and the majority of the protein was found in the fluid fraction (0.83). Undegradable TEIP (proportion of original TEIP ) was approximately 0.80 for both blood meals (20 h). Undegradability (20 h) of proteins of spray-dried blood meal ranged from 0.46 to 1.00, and for ring-dried blood meal the range was 0.59-0.89 (Table 3 ). In both blood meals, hemoglobin (13 kDa) was the protein least susceptible to degradation. Serum albumin (68 kDa) also was highly resistant to degradation. The residual protein fraction (2 h and 20 h) would be enriched with these proteins. Bovine serum albumin (68 kDa) has been shown by others to be slowly degraded (Mahadevan et al., 1980; Nugent et al., 1983; Spencer et al., 1988). Even after 20 h of fermentation in vitro, substantial amounts of proteins were found in the fluid fraction for spray-dried blood meal. The large amount of soluble protein in
M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
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Table 3 Blood meal proteins remaining after 2 or 20 h of fermentation in vitro Prtoein I (kDa)
Proportion of TEIP 2
Undegradability-1
P r o p o r t i o n in fluid
fraction4 Oh
2h
20h
2h
20h
2h
20h
0.42 0.19 0.17 0.17 0.05 1.00 -
0.50 0.17 0.10 0.20 0.03 1.00 -
1.00 a 1.00 a 1.00 a 1.00 a 0.77 b 0.99 0.037
1.00 a 0.74 b 0.50 b 0.95 a 0.46 c 0.79 0.091
0.75* 0.91" 0.89* 0.84* 1.00" 0.83* -
0.66* 0.82* 0.82* 0.85* 1.00" 0.72* -
0.71 0.20 0.09 1.00 -
0.79 0.14 0.07 1.00 -
0.89 a 0.59 ¢
0.89 a 0.59 c
0.70 b
0.70 b
0.82 0.096
0.82 0.056
0.00 0.00 0.00 0.00 -
0.00 0.00 0.00 0.00 -
Spray-dried blood meal 13 32 59 68 76 TEIP SE
0.41 0.19 0.17 0.17 0.06 1.00 -
Ring-dried blood meal 13 32 68 TEIP SE
0.73 0.19 0.08 1.00 -
~Known proteins are: 13 kDa--hemoglobin; 68 kDa--serum albumin. 2Grams of individual protein per gram of total electrophoretically identified protein ( T E I P ) . 3Grams of protein remaining per gram of protein at 0 h. 4Grams of protein remaining in fluid per gram of total (fluid + particulate) protein remaining. *Different from zero ( P < 0 . 0 5 ) . a,b,CMeans within a column and feedstuff (excluding T E I P ) differ ( P < 0.05 ).
spray-dried blood meal would compromise in sacco disappearance values. Without accounting for soluble proteins, spray-dried blood meal would have an undegradability of approximately 0.20 at 20 h. 3.1.3. Maize and maize distillers grains Five proteins (two zein proteins (25 and 28 kDa) and three glutelin proteins (55, 60, and 65 kDa) ) were found in maize grain (Table 4). Glutelin proteins were degraded rapidly and were not found following 2 h of fermentation in vitro. Most of the zein proteins were still present after 2 h of fermentation. No proteins were found in the fluid fraction. After 20 h of incubation, very little degradation of the 25 kDa protein (a zein subunit) had occurred and about half of the 28 kDa protein remained. The electrophoretic profile of the maize sample in this experiment was similar to that reported by Fahmy et al. ( 1991 ). They reported no differences in the rate of degradation of the two zein proteins in maize grain. We found, however, that the 28 kDa protein was less degradable than the 25 kDa protein. Only two proteins (subunits of zein) were found in maize distillers grains (Table 4). Proteins in distillers grains followed the same general pattern as did proteins in maize grain. Degradation of both proteins in distillers grains was low
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (I 994) 25-35
Fig. 2. Electrophoretogram of spray-dried blood meal. Lane 1 is molecular weight standards; Lane 2, spray-dried blood meal before incubation (0 h); Lanes 3 and 4, particle fraction after 20 h of incubation; Lanes 5 and 6, fluid fraction after 20 h of incubation; Lane 7, particle fraction blank; Lane 8, fluid fraction blank. Table 4 Proteins from maize and maize distillers grains remaining after 2 or 20 h of fermentation in vitro Protein ~ (kDa)
Proportion of TEIP 2
Undegradability 3
Proportion in fluid fraction 4
Oh
2h
20h
2h
20h
2h
20h
0.40 0.28 0.10 0.12 0.10 1.00 -
0.53 0.47 0.00 0.00 0.00 1.00 -
0.69 0.31 0.00 0.00 0.00 1.00
0.74 0.94 0.00
0.83 a 0.52 b 0.00
0.0 0.0
0.0 0.0
-
0.00
0.00
-
0.00
0.00
-
0.56
0.0
0.0
0.11
0.48 0.08
0.66 0.34 1.00
0.68 0.32 1.00
0.80 0.20 1.00
0.85 0.77 0.82 0.11
0.72 a 0.37 b 0.59 0.29
0.0 0.0 0.0
0.0 0.0 0.0
Maize grain 25 28 55 60 65 TEIP SE
Distillers grains 25 28 TEIP SE
~Known proteins are: 25 and 28 kDa--zeins; 55, 60, and 65 kDa--glutelins. 2Grams of individual protein per gram of total electrophoretically identified protein (TEIP). 3Grams of protein remaining per gram of protein at 0 h. 4Grams of protein remaining m fluid per gram of total (fluid + particulate ) protein remaining. a'b'Means within a column and feedstuff (excluding TEIP ) differ (P < 0.05 ).
M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
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after 2 h of incubation. After 20 h, undegradability values for the 25 and 28 kDa proteins were 0.72 and 0.37, respectively. No proteins were found in the fluid fraction.
3.2. Validity of the method In this experiment, the 2 h and 20 h time points were chosen to test for the existence of slowly degradable fluid associated proteins (2 h) and to measure maximal expected extent of degradation when ruminal particle flow rate was 0.05 h -1 (1/0.05=20 h). Values for protein undegradability obtained using our method were correlated ( r = 0.94 for 2 h and r = 0.95 for 20 h) with NRC ( 1989 ) average values (Table 5 ). Protein undegradability in sacco (2 h) for maize, soyabean meal (Michalet-Doreau and Ould-Bah, 1992) and distillers grains (Firkins et al., 1984) are substantially higher than our values (Table 4). Incomplete extraction of proteins from these feeds are probably the reason for the discrepancy. Proteins not extracted from the feeds probably are degraded at a slower rate than proteins that were extracted. Assuming the nonextracted protein was not degraded within 2 h, undegradability estimates for soyabean meal, maize, and distillers grains would be 0.64, 0.72, and 0.91, respectively. These are essentially the same as in sacco values of 0.64, 0.77, and 0.93, respectively (Firkins et al., 1984; Michalet-Doreau and Ould-Bah, 1992). Following 20 h of fermentation in vitro, protein extractability probably is greater because much of the interfering plant matrix has been digested. The 20 h undegradability values obtainedin this study agree with published in sacco values (20-24 h incubation). Overall, the blood meals had the highest undegradability, followed by distillers grains, then maize, and then soyabean meal. Both the relative rankings of the feeds and actual values obtained using this method appear reasonable. Our method is easier and less expensive than determining degradability in vivo. The in sacco method has the disadvantage of not measuring soluble proteins (Nocek, 1988; Table 5 Undegradability of protein determined using electrophoresis and standard estimates of protein undegradability Feedstuff
Soyabean meal Spray-dried blood meal Ring-dried blood meal Corn Distillers grains
Standard estimate a
Electrophoresis estimate 2h
SE
20 h
SE
0.35 0.82
0.40 0.99
0.07 0.04
0;09 0.79
0.01 0.09
0.82
0.97
0.10
0.82
0.06
0.52 0.54
0.56 0.82
0.11 0.11
0.48 0.59
0.08 0.29
~
~)btained from NRC ( 1989, Table 7-3 ).
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
Michalet-Doreau and Ould-Bah, 1992). Spray-dried blood meal and soyabean meal had appreciable quantities of fluid associated proteins, and our method allowed quantification of this fraction. The use of electrophoresis with appropriate blanks can adjust for bacterial contamination. Bacterial proteins can have a considerable effect on undegradability values obtained in sacco. The major limitation to the electrophoretic method is the lack of a efficient extraction method. Protein degradability could not be quantified for maize gluten meal and fish meal because we could not extract sufficient amounts of protein. Once efficient extractions methods are developed, electrophoresis should be tested on additional feedstuffs.
Acknowledgments Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This paper is Manuscript 100-93.
References Association of Official Analytical Chemists (AOAC), 1980. Official Methods of Analysis, 12th edn. AOAC, Washington, DC. Brock, F.M., Forsberg, C.W. and Buchanan-Smith, J.G., 1982. Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors. Appl. Environ. Microbiol., 44: 561-569. Chen, G., Strobel, H.J., Russell, J.B. and Sniffen, C.J., 1987. Effect of hydrophobicity on utilization of peptides by ruminal bacteria in vitro. Appl. Environ. Microbiol., 53:2021-2025. Craig, W.M., Hong, B.J., Broderick, G.A. and Bula, R.J., 1984. In vitro inoculum enriched with particle-associated microorganisms for determining rates of fiber digestion and protein degradation. J. Dairy Sci., 67: 2902-2909. Fahmy, W.G., Aufrere, J., Graviou, D., Demarquilly, C. and E1-Shazly, K., 1991. Comparison between the mechanism of protein degradation of two cereals by enzymatic and in situ methods, using gel electrophoresis. Anim. Feed Sci. Technol., 35:115-130. Firkins, J.L., Berger, L.L., Fahey, Jr., G.C. and Merchen, N.R., 1984. Ruminal nitrogen degradability and escape of wet and dry distillers grains and wet and dry corn gluten feeds. J. Dairy Sci., 67: 1936-1944. Kella, N.K., Barbeau, W.E. and Kinsella, J.E., 1986. Effect of oxidative sulfitolysis of disulfide bonds of glycinin on solubility, surface hydrophobicity, and in vitro digestibility. J. Agric. Food Chem., 34: 251-256. Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J. and van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs. J. Dairy Sei., 65:217-225. Lynch, G.L., van der Aar, P.J., Berger, L.L., Fahey, Jr., G.C. and Merchen, N.R., 1988. Proteolysis of alcohol-treated soybean meal proteins by Bacteroides ruminicola, Bacteroides amylophilus, pepsin, trypsin, and in the lumen of steers. J. Dairy Sci., 71: 2416-2427. Mahadevan, S., Erfle, J.D. and Saner, F.D., 1980. Degradation of soluble and insoluble proteins by Baeteroides amylophilus protease and by rumen microorganisms. J. Anim. Sci., 50: 723-728. Messman, M.A. and Weiss, W.P., 1993. Extraction of protein from forages and comparison of two methods to determine its concentration. J. Agric. Food Chem., 41: 1085-1089. Messman, M.A., Weiss, W.P. and Koch, M.E., 1994. Changes in total and individual proteins during harvest, storage, and luminal fermentation of forages. J. Dairy Sci., 77: 492-500.
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Michalet-Doreau, B. and Ould-Bah, M.Y., 1992. In vitro and in sacco methods for the estimation of dietary nitrogen degradability in the rumen: a review. Anim. Feed Sci. Technol., 40: 57-86. Moreira, M.A., Hermodson, M.A., Larkins, B.A. and Nielsen, N.C., 1979. Partial characterization of the acidic and basic polypeptides of glycinin. J. Biol. Chem., 254: 9921-9926. National Research Council (NRC), 1985. Ruminant Nitrogen Usage. National Academy of Sciences, Washington, DC, 138 pp. National Research Council (NRC), 1989. Nutrient Requirements of Dairy Cattle, 6th revised edn. National Academy of Sciences, Washington, DC, 157 pp. Newbold, J.R. and Rust, S.R., 1990. Rumen proteolysis of constituent proteins of soyabean meal. Anim. Prod., 50:552-553 (Abstr.). Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci., 71: 2051-2069. Nugent, J.H.A., Jones, W.T., Jordan, D.J. and Mangan, J.L., 1983. Rates of proteolysis in the rumen of the soluble proteins casein, fraction I (18S) leaf protein, bovine serum albumin and bovine submaxillary mucoprotein. Br. J. Nutr., 50: 357-368. Romagnolo, D., Polan, C.E. and Barbeau, W.E., 1990. Degradability of soybean meal protein fractions as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Dairy Sci., 73: 2379-2385. See, Y.P. and Jackowski, G., 1989. Estimating molecular weights of polypeptides by SDS gel electrophoresis. In: T.E. Creighton (Editor), Protein Structure: A Practical Approach. IRL Press, Oxford, pp. 1-19. Spencer, D., Higgins, T.J.V., Freer, M., Dove, H. and Coombe, J.B., 1988. Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis. Br. J. Nutr., 60: 241-247. Statistical Analysis Systems Institute, Inc., 1988. User's Guide: Statistics, Version 6.0. SAS Institute, Inc., Cary, NC. Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics, 2nd edn. McGraw-Hill, New York. Van der Aar, P.J., Berger, L.L., Wujek, K.M., Mastengroek, I. and Fahey, Jr., G.C., 1983. Relationship between electrophoretic band pattems and in vitro ammonia release of soluble soybean meal protein. J. Dairy Sci., 66: 1272-1276. Wallace, R.J. and Kopecny, J., 1983. Breakdown of diazotized proteins and synthetic substrates by rumen bacterial proteases. Appl. Environ. Microbiol., 45:212-217.
ANIMAL FEED SCIENCE AND TECHNOLOGY
Animal Feed Scienceand Technology49 (1994) 25-35
Use of electrophoresis to quantify ruminal degradability of protein from concentrate feeds M . A . M e s s m a n 1, W.P, Weiss* Department of Dairy Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA
Received 8 June 1993; accepted I March 1994
Abstract
Proteins in soyabean meal, spray-dried and ring-dried blood meals, maize grain, and maize dried distillers grains were extracted before and after ruminal fermentation in vitro. Extracts were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis, and stain intensity of the bands was quantified by densitometry. Undegradability values (proportion of total protein) for soyabean meal, spray-dried blood meal, ring-dried blood meal, maize grain, and distillers grains were 0.40, 0.99, 0.97, 0.56, and 0.82, respectively, after 2 h of fermentation in vitro and 0.09, 0.79, 0.82, 0.49, and 0.59, respectively, after 20 h of fermentation. Soyabean meal and spray-dried blood meal contained slowly degradable, soluble proteins. Individual proteins within feedstuffs showed different degrees of degradation. The basic subunit of glycinin was degraded much slower than were other proteins in soyabean meal. Zein proteins in maize grain and distillers grains were much less degradable than were the glutelin proteins. Electrophoresis has potential as a method for measuring degradability of both particle and fluid associated proteins and to account for bacterial protein contamination.
1. Introduction
The crude protein ( C P ) fraction of feedstuffs is composed of a multitude of proteins. Proteins differ in a m i n o acid composition and sequence and in secondary, tertiary, and quaternary structure. Tertiary ( M a h a d e v a n et al., 1980; Chen et al., 1987) and secondary structure (Wallace and Kopecny, 1983) probably affect rate and extent of protein hydrolysis in the rumen. A more complete char*Correspondingauthor. 1Present address: Growmark, Inc., Bloomington,IL 61702, USA. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10377-8401 (94) 00641-L
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
acterization of the CP fraction of feeds may help explain variation in ruminal degradation of protein within and among feedstuffs. Individual proteins within a feedstuffcan be quantified using electrophoresis and densitometry (Messman et al., 1994). Disappearance of individual proteins during ruminal incubation also has been monitored qualitatively using electrophoresis (Van der Aar et al., 1983; Lynch et al., 1988; Spencer et al., 1988; Newbold and Rust, 1990; Romagnolo et al., 1990; Fahmy et al., 1991 ). Several methods can be used to estimate ruminal degradability of proteins, including fermentation in vitro and in sacco, and enzymatic procedures (National Research Council (NRC), 1985; Nocek, 1988; Michalet-Doreau and Ould-Bah, 1992). Disappearance in sacco is used frequently to estimate protein undegradability, but a major problem is the influence of microbial protein contamination. Nitrogen provided by attached bacteria must be subtracted from residual N or undegradability will be overestimated (Nocek, 1988; Michalet-Doreau and OuldBah, 1992). Another potential problem with the in sacco procedure is the assumption that protein which is soluble and flows out of the bag is degraded instantaneously. Mahadevan et al. (1980) and Spencer et al. ( 1988 ), however, have shown that there is a soluble protein fraction that is not degraded instantaneously. A soluble, but slowly degraded protein fraction may compromise in sacco protein degradability estimates. Objectives of this experiment were: ( 1 ) to determine the ruminal degradability of soluble proteins in concentrate feedstuffs; (2) to quantify extent of ruminal degradation of individual proteins within a feed; (3) to determine if fermentation in vitro followed by electrophoresis and densitometry could be used to estimate ruminal protein degradability.
2. Materials and methods
Feed samples (Table 1 ) underwent fermentation in vitro for 2 h or 20 h (in duplicate). Ruminal fluid used in the invitro system was obtained from a lactating Holstein cow fed a diet consisting of 430 g kg- 1 of alfalfa (Medicago sativa Table 1 Concentrations and extractability of crude protein (CP) Feedstuff
Soyabean meal (solvent) Spray-dried blood meal Ring-dried blood meal Maize grain Maize dried distillers grains
CP ( g kg - 1 DM)
490 960 980 80 300
Extractability a g kg- 1 total CP
SE
600 910 400 630 470
36.8 16.8 58.9 31.6 70.7
aSamples were extracted in duplicate using a borate-phosphate buffer containing sodium dodecyl sulfate.
M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
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L.) silage, 280 g kg -1 of maize ( Z e a m a y s L.) silage, 210 g kg -1 of concentrate, and 80 g kg-1 of alfalfa hay (dry basis). Fluid and particle associated microbes in the ruminal fluid were extracted with cold (4°C) buffer (Craig et al., 1984). Feed samples ( 1 g) were incubated with 50 ml of inoculum at 39°C under CO2. At the desired end point, phenylmethylsulfonyl fluoride and iodoacetate were added (final concentrations of 3 mM and 5 mM, respectively) to stop protease activity (Brock et al., 1982). Contents of the flasks were transferred to centrifuge tubes and centrifuged at 268g (4 °C) for 15 min to separate particles and fluid. A mixture of ground maize cobs without grain (20 g kg- 1 of CP) and urea ( 150 g kg- 1 of total CP) was used to measure microbial proteins present after fermentation in vitro. Particle and fluid portions of the blank were separated by centrifugation. Before fermentation, extracts of blanks contained no electrophoretically identifiable proteins. After fermentation in vitro, proteins found in extracts of residual cob material or the fluid fraction were assumed to be bacterial in origin. Feeds were analyzed for dry matter (DM) and CP (Association of Official Analytical Chemists (AOAC), 1980). Proteins in feeds and the 2 h and 20 h particle fractions were extracted in duplicate using a borate buffer that contained sodium dodecyl sulfate (SDS) and sonication (Messman and Weiss, 1993 ). Extracts of feed and particle fractions, and the fluid fraction were mixed with an equal volume of denaturing buffer, denatured by heating, and electrophoresed on a discontinuous SDS-polyacrylamide gel (See a n d Jackowski, 1989). Stacking and running gels were 4% and 12.5% polyacrylamide, respectively. Following electrophoresis, proteins bands were stained (Coomassie blue R-250) and stain intensity was quantified using a scanning densitometer (BioRad Model 620, Richmond, CA). Disappearance of individual proteins was quantified by scanning gels of feed extracts to determine the relative mobility (Rf) and band density of the major proteins within a feed. Extracts of the fluid and particle fractions from the incubations were electrophoresed and scanned. Densities of bands at the same Rf as in the feed samples were determined. Gels of the extracts of fluid and particulate matter from fermentations of blanks were scanned and any densities found at the Rf of feed proteins were subtracted from the density values obtained from the 2 h and 20 h sample incubations. The blank-corrected densities at each Rf for the 2 h and 20 h samples were then divided by the appropriate density obtained for the original feed. Disappearance of total electrophoretically identified protein (TEIP) was determined by summing the blank-corrected densities of bands at the Rf of interest and dividing by the sum of the densities in the original feeds. Details of electrophoresis and densitometry have been given by Messman et al. (1994). To determine if undegradable soluble proteins made up a significant proportion of total undegradable protein, a t-test (Steel and Torrie, 1980) was used. The amount of undegraded protein in t h e particle fraction was compared with the total amount of protein remaining at each time point. When total and particle associated protein differed ( P < 0.05 ), we concluded that proteins in fluid contributed significantly to the total. To test for differences in degradability of individual proteins within a feed, a completely randomized design was used. The
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M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
model included protein species as the independent variable and undegradability as the dependent variable. Analyses were conducted within a feed and within an incubation period. Fisher's protected least significant difference test (Steel and Torrie, 1980) was used to separate differences in extent of protein hydrolysis within feeds. All statistical calculations were performed using software of the Statistical Analysis Systems Institute, Inc. (SAS) ( 1988 ).
3. Results and discussion
Concentrations of CP of feeds ranged from 80 g kg -1 to 980 g kg -1 (Table 1 ). Extractability of CP ranged from 400 g kg- 1 to 910 g kg- 1 of total CP and averaged 490 g kg -1 of total CP. Fish meal and maize gluten meal were included originally in the sample set; however, only 270 g kg -1 and 170 g kg -1, respectively, of total CP were extracted. Extractability of CP from soyabean (Glycine m a x L. ) meal and maize grain was lower than that reported by Krishnamoorthy et al. (1982). Those workers refluxed samples for 1 h in neutral detergent solution, but in our experiment, sonication was used. The extraction procedure used in this experiment was designed to extract forage proteins rapidly (Messman and Weiss, 1993). Green plants contain a high ratio of albumins and globulins to prolamines and glutenins, whereas grains have a low ratio. 3.1. Differences within feeds 3.1.1. Soyabean meal
Eleven proteins were extracted from soyabean meal (Fig. 1; Table 2). Following 2 h of fermentation in vitro, ten proteins were found. Undegradable TEIP (proportion of original TEIP) after 2 h of fermentation in vitro was 0.40 (Table 2 ). The range in undegradability (2 h) for individual proteins was 0-1.0. Glycinin proteins (21, 43, and 48 kDa) were less degradable than were the r- and asubunits of conglycinin (57 and 78 kDa). We found no difference in the ratio of r- to a-conglycinin in the particle associated protein fraction after 2 h. Newbold and Rust (1990) found the ratio of r- to a-conglycinin decreased during 10 h in situ ruminal exposure. The basic subunit of glycinin (21 kDa) showed no disappearance following 2 h of incubation. All other proteins had less than half of their original mass after 2 h of incubation. With the exception of the basic subunit of glycinin, there were few differences between glycinin and conglycinin proteins with respect to 2 h degradability. Our data confirm that the basic subunit of glycinin is less degradable than acidic glycinin subunits (43 and 48 kDa) (Kella et al., 1986; Newbold and Rust, 1990; Romagnolo et al., 1990). Proteins that are degraded at rates less than average will be enriched in the residue at the expense of proteins that degrade at rates faster than average. At 0 h, the 21 kDa protein made up 0.18 of TEIP, but after 2 h of incubation it constituted 0.45 of TEIP (Table 2). The 43 kDa protein made up 0.18 of TEIP at time 0, but only 0.10 following 2 h of incubation. Similar results have been reported
M.A. Messman, W..P. Weiss / Anirnal Feed Science and Technology 49 (1994) 25-35
29
Fig. 1. Electrophoretogramof soyabeanmeal. Lane 1 is molecularweight standards; Lane 2, soyabean meal before incubation (0 h); Lanes 3 and 4, particle fraction after 2 h of incubation; Lanes 5 and 6, fluid fraction after 2 h of incubation; Lane 7, particle fraction blank; Lane 8, fluid fraction blank. by others (Van der Aar et al., 1983; Romagnolo et al., 1990). Small changes in the concentration of the 21 kDa protein could have a substantial impact on the overall degradability of soyabean meal. The basic subunit of glycinin (21 kDa) has lower concentrations oflysine and methionine than does total glycinin (Moreira et al., 1979 ); therefore, the nutritional value ofundegradable CP in soyabean meal may be less than that of the original protein. The majority of protein remaining after 2 h of fermentation was found in the particulate fraction, but 0.32 of the remaining proteins was found in the fluid fraction. Most of the 21 kDa protein (basic subunit ofglycinin) and a substantial amount of the 57 kDa protein (fl-conglycinin) was found in the fluid fraction. Ruminal fluid turnover rates in lactating dairy cattle range from approximately 0.1 to 0.15 h-1, which corresponds to residence times of 7-10 h. Even though residence time in the rumen is greater than 2 h, some fluid associated proteins probably escape from the rumen. Others have reported significant amounts of slowly degradable, soluble protein in pea meal (Spencer et al., 1988 ) and bovine serum albumin (Nugent et al., 1983; Spencer et al., 1988 ). After 20 h fermentation in vitro, only four proteins were found in the particle fraction, and no proteins were found in the fluid fraction (Table 2). The 48 kDa protein (heavy acidic subunit of glycinin ) had an undegradability (20 h) of 0.47; however, this protein was found in relatively low amounts. After 20 h, the undegradability of TEIP was 0.09.
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
Table 2 Soyabean meal protein remaining after 2 or 20 h fermentation in vitro Protein ~
Proportion ofTEIP z
Undegradability3
Proportion in fluid fraction 4
(kDa )
21 25 36 39 43 48 57 68 73 78 82 TEIP SE
Oh
2h
20h
2h
20h
2h
20h
0.18 0.06 0.05 0.07 0.18 0.08 0.10 0.06 0.09 0.08 0.05 1.00 -
0.45 0.00 0.03 0.05 0.10 0.07 0.10 0.06 0.03 0.05 0.06 1.00 -
0.21 0.00 0.00 0.00 0.24 0.41 0.15 0.00 0.00 0.00 0.00 1.00 -
1.00 a 0.00 ° 0.29 be 0.34 b 0.23 be 0.39 b 0.29 b~ 0.38 b 0.17 ~ 0.26 b¢ 0.46 b 0.40 0.07
0.10 b 0.00 c 0.00 c 0.00 c 0.12 b 0.47 a 0.14 b 0.00 c 0.00 ¢ 0.00 c 0.00 c 0.09 0.01
0.67* 0.00 0.00 0.00 0.00 0.41" 0.00 0.00 0.00 0.00 0.32* -
0.00 0.00 0.00 0.00 0.00 -
~Known proteins are: 21 k D A - - b a s i c subunit o f glycinin; 43 k D a - - l i g h t acidic subunit o f glycinin; 48 k D a - - h e a v y acidic subunit of glycinin; 57 kDa--fl-subunit of conglycinin; 68 k D a - - ? - s u b u n i t o f conglycinin; 73 kDa----~' subunit of conglycinin; 78 kDa--a-subunit of conglycinin. ZGrams of individual protein per gram of total electrophoretically identified protein ( T E I P ) . 3Gram of protein remaining per gram of protein at 0 h.
4Gram of protein remaining in fluid per gram of total (fluid + particulate) protein remaining. *Different from zero ( P < 0.05). a,b,CMeans within a column (excluding T E I P ) differ ( P < 0.05 ).
3.1.2. Blood meal Five proteins were found in spray-dried blood meal and three proteins were found in ring-dried blood meal (Table 3). Overall, undegradability values for spray-dried and ring-dried blood meals were very similar; however, the distribution of the proteins was markedly different. No soluble proteins were found after 2 h or 20 h of fermentation for ring-dried blood meal. After 2 h of fermentation, essentially all the original protein was still present. For spray-dried blood meal, no disappearance occurred during 2 h of incubation (Fig. 2), and the majority of the protein was found in the fluid fraction (0.83). Undegradable TEIP (proportion of original TEIP ) was approximately 0.80 for both blood meals (20 h). Undegradability (20 h) of proteins of spray-dried blood meal ranged from 0.46 to 1.00, and for ring-dried blood meal the range was 0.59-0.89 (Table 3 ). In both blood meals, hemoglobin (13 kDa) was the protein least susceptible to degradation. Serum albumin (68 kDa) also was highly resistant to degradation. The residual protein fraction (2 h and 20 h) would be enriched with these proteins. Bovine serum albumin (68 kDa) has been shown by others to be slowly degraded (Mahadevan et al., 1980; Nugent et al., 1983; Spencer et al., 1988). Even after 20 h of fermentation in vitro, substantial amounts of proteins were found in the fluid fraction for spray-dried blood meal. The large amount of soluble protein in
M.A. Messman, W.P. Weiss / Animal Feed Science and Technology 49 (1994) 25-35
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Table 3 Blood meal proteins remaining after 2 or 20 h of fermentation in vitro Prtoein I (kDa)
Proportion of TEIP 2
Undegradability-1
P r o p o r t i o n in fluid
fraction4 Oh
2h
20h
2h
20h
2h
20h
0.42 0.19 0.17 0.17 0.05 1.00 -
0.50 0.17 0.10 0.20 0.03 1.00 -
1.00 a 1.00 a 1.00 a 1.00 a 0.77 b 0.99 0.037
1.00 a 0.74 b 0.50 b 0.95 a 0.46 c 0.79 0.091
0.75* 0.91" 0.89* 0.84* 1.00" 0.83* -
0.66* 0.82* 0.82* 0.85* 1.00" 0.72* -
0.71 0.20 0.09 1.00 -
0.79 0.14 0.07 1.00 -
0.89 a 0.59 ¢
0.89 a 0.59 c
0.70 b
0.70 b
0.82 0.096
0.82 0.056
0.00 0.00 0.00 0.00 -
0.00 0.00 0.00 0.00 -
Spray-dried blood meal 13 32 59 68 76 TEIP SE
0.41 0.19 0.17 0.17 0.06 1.00 -
Ring-dried blood meal 13 32 68 TEIP SE
0.73 0.19 0.08 1.00 -
~Known proteins are: 13 kDa--hemoglobin; 68 kDa--serum albumin. 2Grams of individual protein per gram of total electrophoretically identified protein ( T E I P ) . 3Grams of protein remaining per gram of protein at 0 h. 4Grams of protein remaining in fluid per gram of total (fluid + particulate) protein remaining. *Different from zero ( P < 0 . 0 5 ) . a,b,CMeans within a column and feedstuff (excluding T E I P ) differ ( P < 0.05 ).
spray-dried blood meal would compromise in sacco disappearance values. Without accounting for soluble proteins, spray-dried blood meal would have an undegradability of approximately 0.20 at 20 h. 3.1.3. Maize and maize distillers grains Five proteins (two zein proteins (25 and 28 kDa) and three glutelin proteins (55, 60, and 65 kDa) ) were found in maize grain (Table 4). Glutelin proteins were degraded rapidly and were not found following 2 h of fermentation in vitro. Most of the zein proteins were still present after 2 h of fermentation. No proteins were found in the fluid fraction. After 20 h of incubation, very little degradation of the 25 kDa protein (a zein subunit) had occurred and about half of the 28 kDa protein remained. The electrophoretic profile of the maize sample in this experiment was similar to that reported by Fahmy et al. ( 1991 ). They reported no differences in the rate of degradation of the two zein proteins in maize grain. We found, however, that the 28 kDa protein was less degradable than the 25 kDa protein. Only two proteins (subunits of zein) were found in maize distillers grains (Table 4). Proteins in distillers grains followed the same general pattern as did proteins in maize grain. Degradation of both proteins in distillers grains was low
32
M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (I 994) 25-35
Fig. 2. Electrophoretogram of spray-dried blood meal. Lane 1 is molecular weight standards; Lane 2, spray-dried blood meal before incubation (0 h); Lanes 3 and 4, particle fraction after 20 h of incubation; Lanes 5 and 6, fluid fraction after 20 h of incubation; Lane 7, particle fraction blank; Lane 8, fluid fraction blank. Table 4 Proteins from maize and maize distillers grains remaining after 2 or 20 h of fermentation in vitro Protein ~ (kDa)
Proportion of TEIP 2
Undegradability 3
Proportion in fluid fraction 4
Oh
2h
20h
2h
20h
2h
20h
0.40 0.28 0.10 0.12 0.10 1.00 -
0.53 0.47 0.00 0.00 0.00 1.00 -
0.69 0.31 0.00 0.00 0.00 1.00
0.74 0.94 0.00
0.83 a 0.52 b 0.00
0.0 0.0
0.0 0.0
-
0.00
0.00
-
0.00
0.00
-
0.56
0.0
0.0
0.11
0.48 0.08
0.66 0.34 1.00
0.68 0.32 1.00
0.80 0.20 1.00
0.85 0.77 0.82 0.11
0.72 a 0.37 b 0.59 0.29
0.0 0.0 0.0
0.0 0.0 0.0
Maize grain 25 28 55 60 65 TEIP SE
Distillers grains 25 28 TEIP SE
~Known proteins are: 25 and 28 kDa--zeins; 55, 60, and 65 kDa--glutelins. 2Grams of individual protein per gram of total electrophoretically identified protein (TEIP). 3Grams of protein remaining per gram of protein at 0 h. 4Grams of protein remaining m fluid per gram of total (fluid + particulate ) protein remaining. a'b'Means within a column and feedstuff (excluding TEIP ) differ (P < 0.05 ).
M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
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after 2 h of incubation. After 20 h, undegradability values for the 25 and 28 kDa proteins were 0.72 and 0.37, respectively. No proteins were found in the fluid fraction.
3.2. Validity of the method In this experiment, the 2 h and 20 h time points were chosen to test for the existence of slowly degradable fluid associated proteins (2 h) and to measure maximal expected extent of degradation when ruminal particle flow rate was 0.05 h -1 (1/0.05=20 h). Values for protein undegradability obtained using our method were correlated ( r = 0.94 for 2 h and r = 0.95 for 20 h) with NRC ( 1989 ) average values (Table 5 ). Protein undegradability in sacco (2 h) for maize, soyabean meal (Michalet-Doreau and Ould-Bah, 1992) and distillers grains (Firkins et al., 1984) are substantially higher than our values (Table 4). Incomplete extraction of proteins from these feeds are probably the reason for the discrepancy. Proteins not extracted from the feeds probably are degraded at a slower rate than proteins that were extracted. Assuming the nonextracted protein was not degraded within 2 h, undegradability estimates for soyabean meal, maize, and distillers grains would be 0.64, 0.72, and 0.91, respectively. These are essentially the same as in sacco values of 0.64, 0.77, and 0.93, respectively (Firkins et al., 1984; Michalet-Doreau and Ould-Bah, 1992). Following 20 h of fermentation in vitro, protein extractability probably is greater because much of the interfering plant matrix has been digested. The 20 h undegradability values obtainedin this study agree with published in sacco values (20-24 h incubation). Overall, the blood meals had the highest undegradability, followed by distillers grains, then maize, and then soyabean meal. Both the relative rankings of the feeds and actual values obtained using this method appear reasonable. Our method is easier and less expensive than determining degradability in vivo. The in sacco method has the disadvantage of not measuring soluble proteins (Nocek, 1988; Table 5 Undegradability of protein determined using electrophoresis and standard estimates of protein undegradability Feedstuff
Soyabean meal Spray-dried blood meal Ring-dried blood meal Corn Distillers grains
Standard estimate a
Electrophoresis estimate 2h
SE
20 h
SE
0.35 0.82
0.40 0.99
0.07 0.04
0;09 0.79
0.01 0.09
0.82
0.97
0.10
0.82
0.06
0.52 0.54
0.56 0.82
0.11 0.11
0.48 0.59
0.08 0.29
~
~)btained from NRC ( 1989, Table 7-3 ).
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M.A. Messman, W.P. Weiss/Animal Feed Science and Technology 49 (1994) 25-35
Michalet-Doreau and Ould-Bah, 1992). Spray-dried blood meal and soyabean meal had appreciable quantities of fluid associated proteins, and our method allowed quantification of this fraction. The use of electrophoresis with appropriate blanks can adjust for bacterial contamination. Bacterial proteins can have a considerable effect on undegradability values obtained in sacco. The major limitation to the electrophoretic method is the lack of a efficient extraction method. Protein degradability could not be quantified for maize gluten meal and fish meal because we could not extract sufficient amounts of protein. Once efficient extractions methods are developed, electrophoresis should be tested on additional feedstuffs.
Acknowledgments Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This paper is Manuscript 100-93.
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Michalet-Doreau, B. and Ould-Bah, M.Y., 1992. In vitro and in sacco methods for the estimation of dietary nitrogen degradability in the rumen: a review. Anim. Feed Sci. Technol., 40: 57-86. Moreira, M.A., Hermodson, M.A., Larkins, B.A. and Nielsen, N.C., 1979. Partial characterization of the acidic and basic polypeptides of glycinin. J. Biol. Chem., 254: 9921-9926. National Research Council (NRC), 1985. Ruminant Nitrogen Usage. National Academy of Sciences, Washington, DC, 138 pp. National Research Council (NRC), 1989. Nutrient Requirements of Dairy Cattle, 6th revised edn. National Academy of Sciences, Washington, DC, 157 pp. Newbold, J.R. and Rust, S.R., 1990. Rumen proteolysis of constituent proteins of soyabean meal. Anim. Prod., 50:552-553 (Abstr.). Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci., 71: 2051-2069. Nugent, J.H.A., Jones, W.T., Jordan, D.J. and Mangan, J.L., 1983. Rates of proteolysis in the rumen of the soluble proteins casein, fraction I (18S) leaf protein, bovine serum albumin and bovine submaxillary mucoprotein. Br. J. Nutr., 50: 357-368. Romagnolo, D., Polan, C.E. and Barbeau, W.E., 1990. Degradability of soybean meal protein fractions as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Dairy Sci., 73: 2379-2385. See, Y.P. and Jackowski, G., 1989. Estimating molecular weights of polypeptides by SDS gel electrophoresis. In: T.E. Creighton (Editor), Protein Structure: A Practical Approach. IRL Press, Oxford, pp. 1-19. Spencer, D., Higgins, T.J.V., Freer, M., Dove, H. and Coombe, J.B., 1988. Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis. Br. J. Nutr., 60: 241-247. Statistical Analysis Systems Institute, Inc., 1988. User's Guide: Statistics, Version 6.0. SAS Institute, Inc., Cary, NC. Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics, 2nd edn. McGraw-Hill, New York. Van der Aar, P.J., Berger, L.L., Wujek, K.M., Mastengroek, I. and Fahey, Jr., G.C., 1983. Relationship between electrophoretic band pattems and in vitro ammonia release of soluble soybean meal protein. J. Dairy Sci., 66: 1272-1276. Wallace, R.J. and Kopecny, J., 1983. Breakdown of diazotized proteins and synthetic substrates by rumen bacterial proteases. Appl. Environ. Microbiol., 45:212-217.