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Protein degradation of smooth bromegrass, switchgrass, and big bluestem in grazing cattle1
The Professional Animal Scientist 27 (2011):422–427
©2011 American Registry of Professional Animal Scientists
P rbromegrass, otein degradation of smooth switchgrass, and big bluestem in grazing cattle1
B. H. Kirch,*2 L. E. Moser,† S. S. Waller,† T. J. Klopfenstein,‡ and J. Klotz§ *Department of Animal Sciences, Colorado State University, Ft. Collins 80523-1171; †Department of Agronomy, and ‡Department of Animal Science, University of Nebraska, Lincoln 68583-0915; and §USDA-ARS Forage Animal Production Research Unit, Lexington, KY 40546-0091
ABSTRACT The objective of this 2-yr study was to estimate the influence of plant maturity on protein escaping ruminal degradation in steers grazing a cool-season grass, smooth bromegrass (Bromus inermis Leyss.; SB), and 2 warm-season grasses, switchgrass (Panicum virgatum L.; SG) and big bluestem (Andropogon gerardii Vitman; BB). Three ruminally fistulated steers strip grazed monocultures of SB, SG, and BB at 3 stages of plant development (vegetative, stem elongation, and reproductive). Omasal samples were collected after total rumen evacuation to evaluate escape protein. Diet samples were also collected after rumen evacuation and a 45-min grazing period. Purine-N was used to quantify microbial contributions to total omasal-N. Total diet CP was greater (P < 0.01) for SB than for SG and BB. Total diet CP was least (P < 0.01) at the reproductive stage for each grass. Escape protein, calculated as a percentage of DM (EPDM), was similar in SB, SG, and BB, with an average of 5.0% across maturities. The EPDM 1 A contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. 2 Corresponding author: brett.kirch@ colostate.edu
average of SG and BB decreased (P < 0.05) from vegetative to reproductive stages, whereas EPDM of SB remained constant, even slightly increasing at the reproductive stage. Escape protein as a percentage of CP (EPCP) in SG and BB was greater (P < 0.05) than that in SB (43.2 vs. 24.1%). Maturity had little influence on EPCP. Microbial contributions to escape protein averaged 36.4% with this technique, with variation due to species and maturity. Key words: cool- and warm-season grasses, escape protein, grazing, omasal sampling, plant maturity
INTRODUCTION Animal performance from warmseason grass pastures is often greater than would be predicted from common whole-plant forage quality parameters, such as IVDMD, CP, or fiber fractions. One explanation for this discrepancy in performance may be escape protein (EP), which seems to be prevalent at greater levels in warm-season grasses than in cool-season grasses (Hafley et al., 1993). Estimates of EP in warm-season grasses have been primarily studied using in situ techniques (Mullahey et al., 1992; Mitchell et al., 1997), which do not account for the selective ability
of the grazing animal. The grazing animal has demonstrated the ability to enhance its diet in both protein and fiber when compared with forage on offer (Heinemann and Russell, 1969; Kirch et al., 2007). The potential for changes in EP are not only influenced by the animal but also by the stage of development of the grass plant. As the plants mature, EP of grasses tends to increase, while soluble portions of the protein decrease (Hoffman et al., 1993). The objective of this study was to evaluate dietary EP levels in selected cool- and warmseason grasses selected by grazing beef animals at several plant maturities.
MATERIALS AND METHODS This study was conducted at the University of Nebraska Agricultural Research and Development Center near Mead, Nebraska. Established monoculture pastures of smooth bromegrass (Bromus inermis Leyss.; SB), switchgrass (Panicum virgatum L.; SG), and big bluestem (Andropogon gerardii Vitman; BB) were used to evaluate EP and RDP under grazing conditions. The pastures were located on a Sharpsburg silty clay loam soil (fine, montmorillonitic, mesic Typic Argiudoll). The coolseason grass (SB) was fertilized in
423
Forage protein degradation in cattle
late April with nitrogen at 90 kg/ ha, whereas the warm-season grasses (SG and BB) were not fertilized but were burned in late April to simulate common production practices of the area. Grazing of the pastures began in May and extended through August as described previously by Kirch et al. (2007). Each grass was grazed by the same 3 ruminally fistulated steers (295 ± 5 kg) at vegetative, stem elongation, and early reproductive stages of development as defined by Moore et al. (1991). All animals in this study were treated in compliance with the animal use standards of the Animal Use Committee at the University of Nebraska–Lincoln. Each grazing was conducted at comparable stages of morphological development to ensure that evaluation periods were determined by developmental stage and not by calendar date. The 3 grasses were selected for their complementary and varied growth characteristics. Cattle strip grazed each grass at each stage of development for a 6-d acclimation period and samples of the diet were collected on d 7. On d 7 of the grazing period, omasal and diet samples were obtained from each steer after total rumen evacuation. Sampling occurred during early morning (0530 to 0600 h) and was started at the same time for each sampling to minimize diurnal variation. After rumen contents were emptied, samples of the omasal contents were obtained by hand through the reticulo-omasal opening (Carulla et al., 1994). After omasal sampling, animals were allowed to graze for 45 min before a diet sample from the rumen was collected. At each evacuation, rumen contents were mixed and sampled for purine ratio analysis before being returned to the rumen. After the collection, animals were moved to the next grazing treatment or to a holding pasture.
Sample Processing and Analysis After collection, all samples were placed immediately on ice and transferred to laboratories in Lincoln,
Nebraska. All samples were stored at −20°C until preparation for analysis. Omasal samples were rinsed with 0.9% saline solution and centrifuged at 25,900 × g for 20 min at 0°C. The effluent was removed each time after centrifugation. This procedure was repeated 4 times. Omasal and diet samples were lyophilized and ground to pass through a 1-mm screen. Rumen content samples collected for purine-to-N ratios were rinsed 2 times with 0.9% saline solution and allowed to stand in saline solution for 24 h at 5°C. Bacterial extracts were then isolated by differential centrifugation (Smith and McAllan, 1974), lyophilized, and ground through a 1-mm screen before analysis. All samples were analyzed for CP by macro Kjeldahl (AOAC, 1990). Omasal and diet samples were analyzed for ADIN (Goering and Van Soest, 1970) and indigestible acid detergent fiber (IADF; Waller et al., 1980). Omasal and bacterial isolates were analyzed for purines using the methods of Zinn and Owens (1986) and modified by using pellet rinsing procedures and standards as prescribed by Aharoni and Tagari (1991).
Calculations Escape protein was calculated using omasal CP to represent protein present after ruminal degradation. The following formula was used to calculate EP as a percentage of the total dietary CP (EPCP): EPCP (%) = [diet IADF]/[omasal IADF] × [omasal CP]/[diet CP] × 100.
To calculate EPCP, omasal samples were corrected for ADIN and microbial N before CP values were calculated. Microbial N contribution to the omasal sample was calculated using ratios for purine to N, which were developed for each grass species and harvest (Table 1). Expression of EP is reported on a percentage basis of the total dietary protein (EPCP) and also on a dietary DM basis (EPDM), which was determined by multiplying the diet CP by the percentage of EPCP. Rumen-degraded proportion of the protein profile was calculated by subtracting EPDM and ADIN from the total diet CP. Insoluble protein in the diet was represented by ADIN values converted to protein values (nitrogen × 6.25) to calculate acid detergent insoluble CP.
Statistical Analysis Maturity effects were tested for vegetative, elongation, and early reproductive stages of grass growth for the 3 species of grasses to determine whether there were differences in protein components. Data from each protein component were analyzed as a split plot design using the mixed model procedure as described in Littell et al. (2006). The split plot was designed with grass species as the main plot, stage of grass development as the subplot, and year as the blocking term. Because of the constraints of monoculture pastures with sufficient carrying capacity for this study, as well as the expense of developing fistulated animals, year was used as the blocking factor and the animal was used as a replicate. The effect of
Table 1. Purine-to-nitrogen ratios Smooth bromegrass
Switchgrass
Big bluestem
Stage1
Yr 1
Yr 2
Yr 1
Yr 2
Yr 1
Yr 2
VG EL RP
0.18 0.17 0.17
0.24 0.25 0.25
0.16 0.15 0.14
0.23 0.22 0.21
0.15 0.18 0.15
0.23 0.24 0.19
1
VG = vegetative; EL = elongation; RP = reproductive.
424 year on variation for most of the variables was small and negated because of the diet selection ability of the animals and similar weather patterns for the 2-yr study. The design of the project was developed to allow the animals to not be constrained in their selection and to express their individual tendencies. The model for this design included year, species, species × year, maturity, and maturity × species. An ANOVA was conducted and comparisons of least squares means (±SEM) were performed using the PDIFF routine (SAS Institute Inc., Cary, NC). The tested effects and comparisons were considered different at P ≤ 0.05 using mean stage counts from the clipped samples presented in Kirch et al. (2007) for defining the grass development.
RESULTS AND DISCUSSION In this grazing trial, levels of protein degradation were primarily species dependent, and effects due to plant maturity were generally the same for all 3 grass species. Dietary CP across vegetative to early reproductive plant maturities was greater (P < 0.01; Figure 1) for cattle grazing SB than for
Kirch et al.
those grazing either SG or BB. Generally, cool-season grasses, such as SB, contain a greater CP concentration than warm-season grasses growing under similar environmental conditions. Mullahey et al. (1992) reported that on a whole-plant basis, SB averaged 11.1% CP compared with 7.7% CP in SG throughout the growing season. Dietary CP of cattle grazing SG and BB declined from vegetative to early reproductive stages of growth, and dietary CP values of SG and BB were similar at the vegetative and elongation stages of maturity but declined at the early reproductive stage when compared with the vegetative stage (P < 0.05). The decline in CP with increasing maturity was characteristic of the decline of whole-plant CP reported by Mitchell et al. (2001) in both SG and BB at similar stages of development. In this study, dietary CP of cattle grazing SG and BB was similar at each stage of growth, which also was observed by Newell and Moline (1978) for harvests of wholeplant SB, SG, and BB under similar conditions. A grass species-by-maturity interaction (P < 0.01) occurred for EPDM (Figure 2). The EPDM of SB was
Figure 1. Diet CP as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
comparable for the first 2 harvests and increased from the elongation to the early reproductive stage, whereas the warm-season grasses decreased from vegetative to early reproduction. Vegetative SB was comparable to the warm-season grasses at the elongation stage of growth. Switchgrass was significantly greater (P < 0.05) in EPDM at the vegetative stage of growth compared with SB, and BB tended to be greater than SB in EPDM (P = 0.10) at the vegetative stage of growth. Both warm-season grasses were similar in EPDM at the elongation stage, and both were lower than SB in EPDM (P < 0.05) at the early reproductive stage of growth. The early reproductive level of EPDM of SB was comparable to the vegetative levels of the warm-season grasses. Although there was an interaction between species and stage of maturity for EPDM, there was none for EPCP (Table 2). Switchgrass and BB had almost twice as much EPCP as SB (P < 0.01). In vivo estimations of EPCP in this study were comparable to in situ results reported by Mullahey et al. (1992), who estimated SG to be 50.9% and SB to be 20.5% EPCP on a whole-plant basis. The lower percentage of the protein escaping degradation of SB was offset by the overall greater CP content of the cool-season grass; thus, EPDM of SB is comparable to warm-season grasses when forage selection is maximized. The EPCP average across all species was unaffected by plant maturity and averaged 38% from vegetative to early reproductive stages (data not shown). Stage of maturity was expected to affect EPCP because in an earlier study Mitchell et al. (1997) reported decreases in totalplant CP and increases in EPCP in SB, SG, and BB with advanced stages of plant development. The inconsistencies noted between these studies in relation to EPCP may be due to differences in evaluation technique. Mitchell et al. (1997) used in situ techniques to evaluate EP of whole-plant material, whereas we used in vivo evaluations that take into account the selection ability of the animal. At various stages of forage growth, steers
Forage protein degradation in cattle
Figure 2. Escape protein as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
had the opportunity to select primarily leaf tissue and avoid stem and other plant matter found in the lower portion of the grass canopy. Rumen-degraded protein in SB was approximately 3-fold higher (P < 0.05) than that of warm-season grasses at the elongation stage and twice that of the warm-season grass at the vegetative and early reproduc-
tive stage harvests (P < 0.05; Figure 3). This was a consequence of greater total protein and greater solubility of protein when digested. Akin and
425 Burdick (1975) noted that warm-season grasses contain a lower percentage of leaf mesophyll tissue than do coolseason grasses, which also degrades at a slower rate than mesophyll tissue from cool-season grasses. In addition, in warm-season grass, protein is found predominantly in the bundle sheath cells, which are slowly degraded. Both warm-season grasses were similar at each stage of harvest. Increasing plant maturity is normally associated with a decline in RDP (Mitchell et al., 1997). Low RDP values at reproductive stages of plant development also have been documented for 8 forages, including both temperate grasses and legumes (Hoffman et al., 1993). The results were similar for this study, where RDP for all 3 species tended (P < 0.10) to decline from vegetative to early reproductive stages of maturity, with the exception of SB, which did not decline from vegetative to elongation stages of maturity. The acid detergent insoluble CP level of the grass species did not differ by species or when compared across all species (Table 3). The lack of species differences may be due to the selection ability of animals to exclude the less
Table 2. Escape protein (% CP) of smooth bromegrass, switchgrass, and big bluestem Species Smooth bromegrass Switchgrass Big bluestem SE
Escape protein,1 % CP 24.1b 43.2a 43.2a 1.9
Mean separation is significant at P < 0.05. 1 Means were determined by pooling data from vegetative, elongation, and early reproductive stages of development for each species. a,b
Figure 3. Rumen-degraded protein as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
426
Kirch et al.
Table 3. Acid detergent insoluble CP (ADICP) of the vegetative, elongation, and early reproductive stages of growth Stage of growth
ADICP1
Vegetative Elongation Early reproductive SE
1.26a 1.12ab 1.05b 0.30
Mean separation is significant at P < 0.05. 1 Means were determined by pooling data from smooth bromegrass, switchgrass, and bigbluestem at each grass maturity. a,b
digestible or less desirable portions of forage on offer, and this may be an expression of increased selectivity as the grass became more mature (Kirch et al., 2007). Microbial augmentation of EP did not differ across harvests for SB (Figure 4) and averaged 37.1%. The SG also showed no difference when compared with the warm-season
grasses. The microbial protein for the warm-season grass averaged across all harvests was greater for SG when compared with BB (P < 0.05; Figure 4). Estimation of EP by omasal sampling techniques requires adjustment for microbial augmentation of the rumen-degraded samples. Differential centrifugation in the rinsing phase contributed to artificially high levels of microbial augmentation in the omasal CP, causing the values to be significantly larger than in in situ evaluations of forage EP. The values of microbial protein, which were 33 to 39% of omasal CP, were much greater than the 2.6 to 13.9% reported by Mitchell et al. (1997) and Mullahey et al. (1992). This technique difference is apparently due to the vigorous rinsing used in the in situ procedures. The estimation of EPCP in grazing animals demonstrated values that were similar to those determined in the laboratory. However, when EPDM of each grass was determined, differences between cool- and warm-season grasses were not significant. These results may indicate that, in grazing animals, the performance differential
of warm-season grasses may be due to factors other than protein. Differences in fiber digestion may explain animal performance on warm-season grasses. Slightly increased rumen fill capabilities of warm-season grasses may contribute to increased performance. The selection ability of the animal cannot be overlooked as a possible explanation for the performance differential of warm-season grasses. In this study the selection ability of the animal was able to lessen the effects of declines in forage quality that are a result of increasing plant maturity.
IMPLICATIONS Estimations of protein degradation by in situ techniques are adequate to evaluate general trends relative to actual production situations. However, selection ability of grazing livestock must be considered when evaluating production situations. Cool- and warm-season grasses seem to differ little in escape protein on a DM basis; thus supplementation for grazing warm-season grasses should be directed at RDP to increase performance. It is essential to ascertain the stage of plant development that the animal is offered to maximize performance of supplement management.
LITERATURE CITED Aharoni, Y., and H. Tagari. 1991. Use of nitrogen-15 determinations of purine nitrogen fraction of digesta to define nitrogen metabolism traits in the rumen. J. Dairy Sci. 74:2540. Akin, D. E., and D. Burdick. 1975. Percentage of tissue types in tropical and temperate grass leaf blades and degradation of tissues by rumen microorganisms. Crop Sci. 15:661. AOAC. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA. Carulla, J. E., V. A. Wilkerson, D. H. Shain, T. J. Klopfenstein, and R. A. Britton. 1994. Omasal sampling to estimate escape protein of forages. J. Anim. Sci. 72(Suppl. 2):85. (Abstr.)
Figure 4. Microbial protein as a percentage of escape protein (EP) for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS, USDA, Washington, DC.
Forage protein degradation in cattle Hafley, J. L., B. E. Anderson, and T. J. Klopfenstein. 1993. Supplementation of growing cattle grazing warm-season grass with proteins of various ruminal degradabilities. J. Anim. Sci. 71:522. Heinemann, W. W., and T. S. Russell. 1969. Evaluation of rotation grazed pastures from esophageal and hand gathered forage samples. Agron. J. 61:547. Hoffman, P. C., S. J. Sievert, R. D. Shaver, D. A. Welch, and D. K. Combs. 1993. In situ dry matter, protein, and fiber degradation of perennial forages. J. Dairy Sci. 76:2632. Kirch, B. H., L. E. Moser, S. S. Waller, T. J. Klopfenstein, G. E. Aiken, and J. R. Strickland. 2007. Selection and diet quality of beef cattle grazing smooth bromegrass, switchgrass, and big bluestem. Prof. Anim. Sci. 23:672. Littell, R. C., G. A. Milliken, W. W. Stroup, R. D. Wolfinger, and O. Schabenberger. 2006.
SAS for Mixed Models. 2nd ed. SAS Inst. Inc., Cary, NC. Mitchell, R. B., J. Fritz, K. Moore, L. Moser, K. Vogel, D. Redfearn, and D. Wester. 2001. Predicting forage quality in switchgrass and big bluestem. Agron. J. 93:118. Mitchell, R. B., D. D. Redfearn, L. E. Moser, K. J. Moore, R. J. Grant, K. J. Moore, and B. H. Kirch. 1997. Relationships between in situ protein degradability and grass developmental morphology. J. Dairy Sci. 80:1143. Moore, K. J., L. E. Moser, K. P. Vogel, S. S. Waller, B. E. Johnson, and J. F. Pedersen. 1991. Describing and quantifying growth stages of perennial forage grasses. Agron. J. 83:1073. Mullahey, J. J., S. S. Waller, K. J. Moore, L. E. Moser, and T. J. Klopfenstein. 1992. In situ ruminal protein degradation of switchgrass and smooth bromegrass. Agron. J. 84:183.
427 Newell, L. C., and W. J. Moline. 1978. Forage quality evaluations of twelve grasses in relation to season for grazing. Univ. Nebraska Agric. Exp. Stn. Res. Bull. 283. Univ. Nebraska, Lincoln. Smith, R. H., and A. B. McAllan. 1974. Some factors influencing the chemical composition of mixed rumen bacteria. Br. J. Nutr. 31:27. Waller, J., N. Merchen, T. Hanson, and T. Klopfenstein. 1980. Effect of sampling intervals and digesta markers on abomasal flow determinations. J. Anim. Sci. 50:1122. Zinn, R. A., and F. N. Owens. 1986. A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66:157.
©2011 American Registry of Professional Animal Scientists
P rbromegrass, otein degradation of smooth switchgrass, and big bluestem in grazing cattle1
B. H. Kirch,*2 L. E. Moser,† S. S. Waller,† T. J. Klopfenstein,‡ and J. Klotz§ *Department of Animal Sciences, Colorado State University, Ft. Collins 80523-1171; †Department of Agronomy, and ‡Department of Animal Science, University of Nebraska, Lincoln 68583-0915; and §USDA-ARS Forage Animal Production Research Unit, Lexington, KY 40546-0091
ABSTRACT The objective of this 2-yr study was to estimate the influence of plant maturity on protein escaping ruminal degradation in steers grazing a cool-season grass, smooth bromegrass (Bromus inermis Leyss.; SB), and 2 warm-season grasses, switchgrass (Panicum virgatum L.; SG) and big bluestem (Andropogon gerardii Vitman; BB). Three ruminally fistulated steers strip grazed monocultures of SB, SG, and BB at 3 stages of plant development (vegetative, stem elongation, and reproductive). Omasal samples were collected after total rumen evacuation to evaluate escape protein. Diet samples were also collected after rumen evacuation and a 45-min grazing period. Purine-N was used to quantify microbial contributions to total omasal-N. Total diet CP was greater (P < 0.01) for SB than for SG and BB. Total diet CP was least (P < 0.01) at the reproductive stage for each grass. Escape protein, calculated as a percentage of DM (EPDM), was similar in SB, SG, and BB, with an average of 5.0% across maturities. The EPDM 1 A contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. 2 Corresponding author: brett.kirch@ colostate.edu
average of SG and BB decreased (P < 0.05) from vegetative to reproductive stages, whereas EPDM of SB remained constant, even slightly increasing at the reproductive stage. Escape protein as a percentage of CP (EPCP) in SG and BB was greater (P < 0.05) than that in SB (43.2 vs. 24.1%). Maturity had little influence on EPCP. Microbial contributions to escape protein averaged 36.4% with this technique, with variation due to species and maturity. Key words: cool- and warm-season grasses, escape protein, grazing, omasal sampling, plant maturity
INTRODUCTION Animal performance from warmseason grass pastures is often greater than would be predicted from common whole-plant forage quality parameters, such as IVDMD, CP, or fiber fractions. One explanation for this discrepancy in performance may be escape protein (EP), which seems to be prevalent at greater levels in warm-season grasses than in cool-season grasses (Hafley et al., 1993). Estimates of EP in warm-season grasses have been primarily studied using in situ techniques (Mullahey et al., 1992; Mitchell et al., 1997), which do not account for the selective ability
of the grazing animal. The grazing animal has demonstrated the ability to enhance its diet in both protein and fiber when compared with forage on offer (Heinemann and Russell, 1969; Kirch et al., 2007). The potential for changes in EP are not only influenced by the animal but also by the stage of development of the grass plant. As the plants mature, EP of grasses tends to increase, while soluble portions of the protein decrease (Hoffman et al., 1993). The objective of this study was to evaluate dietary EP levels in selected cool- and warmseason grasses selected by grazing beef animals at several plant maturities.
MATERIALS AND METHODS This study was conducted at the University of Nebraska Agricultural Research and Development Center near Mead, Nebraska. Established monoculture pastures of smooth bromegrass (Bromus inermis Leyss.; SB), switchgrass (Panicum virgatum L.; SG), and big bluestem (Andropogon gerardii Vitman; BB) were used to evaluate EP and RDP under grazing conditions. The pastures were located on a Sharpsburg silty clay loam soil (fine, montmorillonitic, mesic Typic Argiudoll). The coolseason grass (SB) was fertilized in
423
Forage protein degradation in cattle
late April with nitrogen at 90 kg/ ha, whereas the warm-season grasses (SG and BB) were not fertilized but were burned in late April to simulate common production practices of the area. Grazing of the pastures began in May and extended through August as described previously by Kirch et al. (2007). Each grass was grazed by the same 3 ruminally fistulated steers (295 ± 5 kg) at vegetative, stem elongation, and early reproductive stages of development as defined by Moore et al. (1991). All animals in this study were treated in compliance with the animal use standards of the Animal Use Committee at the University of Nebraska–Lincoln. Each grazing was conducted at comparable stages of morphological development to ensure that evaluation periods were determined by developmental stage and not by calendar date. The 3 grasses were selected for their complementary and varied growth characteristics. Cattle strip grazed each grass at each stage of development for a 6-d acclimation period and samples of the diet were collected on d 7. On d 7 of the grazing period, omasal and diet samples were obtained from each steer after total rumen evacuation. Sampling occurred during early morning (0530 to 0600 h) and was started at the same time for each sampling to minimize diurnal variation. After rumen contents were emptied, samples of the omasal contents were obtained by hand through the reticulo-omasal opening (Carulla et al., 1994). After omasal sampling, animals were allowed to graze for 45 min before a diet sample from the rumen was collected. At each evacuation, rumen contents were mixed and sampled for purine ratio analysis before being returned to the rumen. After the collection, animals were moved to the next grazing treatment or to a holding pasture.
Sample Processing and Analysis After collection, all samples were placed immediately on ice and transferred to laboratories in Lincoln,
Nebraska. All samples were stored at −20°C until preparation for analysis. Omasal samples were rinsed with 0.9% saline solution and centrifuged at 25,900 × g for 20 min at 0°C. The effluent was removed each time after centrifugation. This procedure was repeated 4 times. Omasal and diet samples were lyophilized and ground to pass through a 1-mm screen. Rumen content samples collected for purine-to-N ratios were rinsed 2 times with 0.9% saline solution and allowed to stand in saline solution for 24 h at 5°C. Bacterial extracts were then isolated by differential centrifugation (Smith and McAllan, 1974), lyophilized, and ground through a 1-mm screen before analysis. All samples were analyzed for CP by macro Kjeldahl (AOAC, 1990). Omasal and diet samples were analyzed for ADIN (Goering and Van Soest, 1970) and indigestible acid detergent fiber (IADF; Waller et al., 1980). Omasal and bacterial isolates were analyzed for purines using the methods of Zinn and Owens (1986) and modified by using pellet rinsing procedures and standards as prescribed by Aharoni and Tagari (1991).
Calculations Escape protein was calculated using omasal CP to represent protein present after ruminal degradation. The following formula was used to calculate EP as a percentage of the total dietary CP (EPCP): EPCP (%) = [diet IADF]/[omasal IADF] × [omasal CP]/[diet CP] × 100.
To calculate EPCP, omasal samples were corrected for ADIN and microbial N before CP values were calculated. Microbial N contribution to the omasal sample was calculated using ratios for purine to N, which were developed for each grass species and harvest (Table 1). Expression of EP is reported on a percentage basis of the total dietary protein (EPCP) and also on a dietary DM basis (EPDM), which was determined by multiplying the diet CP by the percentage of EPCP. Rumen-degraded proportion of the protein profile was calculated by subtracting EPDM and ADIN from the total diet CP. Insoluble protein in the diet was represented by ADIN values converted to protein values (nitrogen × 6.25) to calculate acid detergent insoluble CP.
Statistical Analysis Maturity effects were tested for vegetative, elongation, and early reproductive stages of grass growth for the 3 species of grasses to determine whether there were differences in protein components. Data from each protein component were analyzed as a split plot design using the mixed model procedure as described in Littell et al. (2006). The split plot was designed with grass species as the main plot, stage of grass development as the subplot, and year as the blocking term. Because of the constraints of monoculture pastures with sufficient carrying capacity for this study, as well as the expense of developing fistulated animals, year was used as the blocking factor and the animal was used as a replicate. The effect of
Table 1. Purine-to-nitrogen ratios Smooth bromegrass
Switchgrass
Big bluestem
Stage1
Yr 1
Yr 2
Yr 1
Yr 2
Yr 1
Yr 2
VG EL RP
0.18 0.17 0.17
0.24 0.25 0.25
0.16 0.15 0.14
0.23 0.22 0.21
0.15 0.18 0.15
0.23 0.24 0.19
1
VG = vegetative; EL = elongation; RP = reproductive.
424 year on variation for most of the variables was small and negated because of the diet selection ability of the animals and similar weather patterns for the 2-yr study. The design of the project was developed to allow the animals to not be constrained in their selection and to express their individual tendencies. The model for this design included year, species, species × year, maturity, and maturity × species. An ANOVA was conducted and comparisons of least squares means (±SEM) were performed using the PDIFF routine (SAS Institute Inc., Cary, NC). The tested effects and comparisons were considered different at P ≤ 0.05 using mean stage counts from the clipped samples presented in Kirch et al. (2007) for defining the grass development.
RESULTS AND DISCUSSION In this grazing trial, levels of protein degradation were primarily species dependent, and effects due to plant maturity were generally the same for all 3 grass species. Dietary CP across vegetative to early reproductive plant maturities was greater (P < 0.01; Figure 1) for cattle grazing SB than for
Kirch et al.
those grazing either SG or BB. Generally, cool-season grasses, such as SB, contain a greater CP concentration than warm-season grasses growing under similar environmental conditions. Mullahey et al. (1992) reported that on a whole-plant basis, SB averaged 11.1% CP compared with 7.7% CP in SG throughout the growing season. Dietary CP of cattle grazing SG and BB declined from vegetative to early reproductive stages of growth, and dietary CP values of SG and BB were similar at the vegetative and elongation stages of maturity but declined at the early reproductive stage when compared with the vegetative stage (P < 0.05). The decline in CP with increasing maturity was characteristic of the decline of whole-plant CP reported by Mitchell et al. (2001) in both SG and BB at similar stages of development. In this study, dietary CP of cattle grazing SG and BB was similar at each stage of growth, which also was observed by Newell and Moline (1978) for harvests of wholeplant SB, SG, and BB under similar conditions. A grass species-by-maturity interaction (P < 0.01) occurred for EPDM (Figure 2). The EPDM of SB was
Figure 1. Diet CP as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
comparable for the first 2 harvests and increased from the elongation to the early reproductive stage, whereas the warm-season grasses decreased from vegetative to early reproduction. Vegetative SB was comparable to the warm-season grasses at the elongation stage of growth. Switchgrass was significantly greater (P < 0.05) in EPDM at the vegetative stage of growth compared with SB, and BB tended to be greater than SB in EPDM (P = 0.10) at the vegetative stage of growth. Both warm-season grasses were similar in EPDM at the elongation stage, and both were lower than SB in EPDM (P < 0.05) at the early reproductive stage of growth. The early reproductive level of EPDM of SB was comparable to the vegetative levels of the warm-season grasses. Although there was an interaction between species and stage of maturity for EPDM, there was none for EPCP (Table 2). Switchgrass and BB had almost twice as much EPCP as SB (P < 0.01). In vivo estimations of EPCP in this study were comparable to in situ results reported by Mullahey et al. (1992), who estimated SG to be 50.9% and SB to be 20.5% EPCP on a whole-plant basis. The lower percentage of the protein escaping degradation of SB was offset by the overall greater CP content of the cool-season grass; thus, EPDM of SB is comparable to warm-season grasses when forage selection is maximized. The EPCP average across all species was unaffected by plant maturity and averaged 38% from vegetative to early reproductive stages (data not shown). Stage of maturity was expected to affect EPCP because in an earlier study Mitchell et al. (1997) reported decreases in totalplant CP and increases in EPCP in SB, SG, and BB with advanced stages of plant development. The inconsistencies noted between these studies in relation to EPCP may be due to differences in evaluation technique. Mitchell et al. (1997) used in situ techniques to evaluate EP of whole-plant material, whereas we used in vivo evaluations that take into account the selection ability of the animal. At various stages of forage growth, steers
Forage protein degradation in cattle
Figure 2. Escape protein as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
had the opportunity to select primarily leaf tissue and avoid stem and other plant matter found in the lower portion of the grass canopy. Rumen-degraded protein in SB was approximately 3-fold higher (P < 0.05) than that of warm-season grasses at the elongation stage and twice that of the warm-season grass at the vegetative and early reproduc-
tive stage harvests (P < 0.05; Figure 3). This was a consequence of greater total protein and greater solubility of protein when digested. Akin and
425 Burdick (1975) noted that warm-season grasses contain a lower percentage of leaf mesophyll tissue than do coolseason grasses, which also degrades at a slower rate than mesophyll tissue from cool-season grasses. In addition, in warm-season grass, protein is found predominantly in the bundle sheath cells, which are slowly degraded. Both warm-season grasses were similar at each stage of harvest. Increasing plant maturity is normally associated with a decline in RDP (Mitchell et al., 1997). Low RDP values at reproductive stages of plant development also have been documented for 8 forages, including both temperate grasses and legumes (Hoffman et al., 1993). The results were similar for this study, where RDP for all 3 species tended (P < 0.10) to decline from vegetative to early reproductive stages of maturity, with the exception of SB, which did not decline from vegetative to elongation stages of maturity. The acid detergent insoluble CP level of the grass species did not differ by species or when compared across all species (Table 3). The lack of species differences may be due to the selection ability of animals to exclude the less
Table 2. Escape protein (% CP) of smooth bromegrass, switchgrass, and big bluestem Species Smooth bromegrass Switchgrass Big bluestem SE
Escape protein,1 % CP 24.1b 43.2a 43.2a 1.9
Mean separation is significant at P < 0.05. 1 Means were determined by pooling data from vegetative, elongation, and early reproductive stages of development for each species. a,b
Figure 3. Rumen-degraded protein as a percentage of DM for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
426
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Table 3. Acid detergent insoluble CP (ADICP) of the vegetative, elongation, and early reproductive stages of growth Stage of growth
ADICP1
Vegetative Elongation Early reproductive SE
1.26a 1.12ab 1.05b 0.30
Mean separation is significant at P < 0.05. 1 Means were determined by pooling data from smooth bromegrass, switchgrass, and bigbluestem at each grass maturity. a,b
digestible or less desirable portions of forage on offer, and this may be an expression of increased selectivity as the grass became more mature (Kirch et al., 2007). Microbial augmentation of EP did not differ across harvests for SB (Figure 4) and averaged 37.1%. The SG also showed no difference when compared with the warm-season
grasses. The microbial protein for the warm-season grass averaged across all harvests was greater for SG when compared with BB (P < 0.05; Figure 4). Estimation of EP by omasal sampling techniques requires adjustment for microbial augmentation of the rumen-degraded samples. Differential centrifugation in the rinsing phase contributed to artificially high levels of microbial augmentation in the omasal CP, causing the values to be significantly larger than in in situ evaluations of forage EP. The values of microbial protein, which were 33 to 39% of omasal CP, were much greater than the 2.6 to 13.9% reported by Mitchell et al. (1997) and Mullahey et al. (1992). This technique difference is apparently due to the vigorous rinsing used in the in situ procedures. The estimation of EPCP in grazing animals demonstrated values that were similar to those determined in the laboratory. However, when EPDM of each grass was determined, differences between cool- and warm-season grasses were not significant. These results may indicate that, in grazing animals, the performance differential
of warm-season grasses may be due to factors other than protein. Differences in fiber digestion may explain animal performance on warm-season grasses. Slightly increased rumen fill capabilities of warm-season grasses may contribute to increased performance. The selection ability of the animal cannot be overlooked as a possible explanation for the performance differential of warm-season grasses. In this study the selection ability of the animal was able to lessen the effects of declines in forage quality that are a result of increasing plant maturity.
IMPLICATIONS Estimations of protein degradation by in situ techniques are adequate to evaluate general trends relative to actual production situations. However, selection ability of grazing livestock must be considered when evaluating production situations. Cool- and warm-season grasses seem to differ little in escape protein on a DM basis; thus supplementation for grazing warm-season grasses should be directed at RDP to increase performance. It is essential to ascertain the stage of plant development that the animal is offered to maximize performance of supplement management.
LITERATURE CITED Aharoni, Y., and H. Tagari. 1991. Use of nitrogen-15 determinations of purine nitrogen fraction of digesta to define nitrogen metabolism traits in the rumen. J. Dairy Sci. 74:2540. Akin, D. E., and D. Burdick. 1975. Percentage of tissue types in tropical and temperate grass leaf blades and degradation of tissues by rumen microorganisms. Crop Sci. 15:661. AOAC. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA. Carulla, J. E., V. A. Wilkerson, D. H. Shain, T. J. Klopfenstein, and R. A. Britton. 1994. Omasal sampling to estimate escape protein of forages. J. Anim. Sci. 72(Suppl. 2):85. (Abstr.)
Figure 4. Microbial protein as a percentage of escape protein (EP) for smooth bromegrass, switchgrass, and big bluestem grazed at vegetative (VG), elongation (EL), and early reproductive (RP) stages of grass development.
Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS, USDA, Washington, DC.
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SAS for Mixed Models. 2nd ed. SAS Inst. Inc., Cary, NC. Mitchell, R. B., J. Fritz, K. Moore, L. Moser, K. Vogel, D. Redfearn, and D. Wester. 2001. Predicting forage quality in switchgrass and big bluestem. Agron. J. 93:118. Mitchell, R. B., D. D. Redfearn, L. E. Moser, K. J. Moore, R. J. Grant, K. J. Moore, and B. H. Kirch. 1997. Relationships between in situ protein degradability and grass developmental morphology. J. Dairy Sci. 80:1143. Moore, K. J., L. E. Moser, K. P. Vogel, S. S. Waller, B. E. Johnson, and J. F. Pedersen. 1991. Describing and quantifying growth stages of perennial forage grasses. Agron. J. 83:1073. Mullahey, J. J., S. S. Waller, K. J. Moore, L. E. Moser, and T. J. Klopfenstein. 1992. In situ ruminal protein degradation of switchgrass and smooth bromegrass. Agron. J. 84:183.
427 Newell, L. C., and W. J. Moline. 1978. Forage quality evaluations of twelve grasses in relation to season for grazing. Univ. Nebraska Agric. Exp. Stn. Res. Bull. 283. Univ. Nebraska, Lincoln. Smith, R. H., and A. B. McAllan. 1974. Some factors influencing the chemical composition of mixed rumen bacteria. Br. J. Nutr. 31:27. Waller, J., N. Merchen, T. Hanson, and T. Klopfenstein. 1980. Effect of sampling intervals and digesta markers on abomasal flow determinations. J. Anim. Sci. 50:1122. Zinn, R. A., and F. N. Owens. 1986. A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66:157.