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Journal of Animal Science - Animal Nutrition

Ruminally undegradable protein content and digestibility for forages using the mobile bag in situ technique1

 

This article in JAS

  1. Vol. 91 No. 6, p. 2812-2822
     
    Received: Oct 12, 2012
    Accepted: Feb 21, 2013
    Published: November 25, 2014


    2 Corresponding author(s): tklopfenstein1@unl.edu
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doi:10.2527/jas.2012-5982
  1. C. D. Buckner,
  2. T. J. Klopfenstein 2,
  3. K. M. Rolfe,
  4. W. A. Griffin,
  5. M. J. Lamothe,
  6. A. K. Watson,
  7. J. C. MacDonald,
  8. W. H. Schacht and
  9. P. Schroeder
  1. Department of Animal Science, University of Nebraska, Lincoln 68583-0908

Abstract

Four experiments were conducted to evaluate RUP content and digestibility for smooth bromegrass, subirrigated meadow, upland native range, and warm-season grasses. Samples were collected from esophageally cannulated cows or ruminally cannulated steers. Forages were ruminally incubated in in situ bags for durations of time based on 75% of total mean retention time, which was based on IVDMD and rate of passage calculations. One-half of the bags were duodenally incubated and excreted in the feces, and NDIN was analyzed on all bags for RUP calculations. Crude protein was numerically greater early in the growing cycle for grasses compared with later as grasses matured (P ≤ 0.32). The RUP was 13.3%, 13.3%, and 19.7% of CP for smooth bromegrass, subirrigated meadow, and upland native range, respectively. These values tended to be lower early in the growth cycle and increased (linear P ≤ 0.13) as forages matured for warm-season grasses and subirrigated meadows. Because both CP and RUP content change throughout the growing season, expressing RUP as a percentage of DM gives more consistent averages compared with RUP as a percentage of CP. Coefficient of variation values for RUP as a percentage of DM averaged 0.21 over all 4 experiments compared with 0.26 for RUP as a percentage of CP. Average RUP as a percentage of DM was 2.03%, 1.53%, and 1.94% for smooth bromegrass, subirrigated meadow, and upland native range, respectively. Total tract indigestible protein (TTIDP) linearly increased with maturity for subirrigated meadow samples (P < 0.01). A quadratic response (P ≤ 0.06) for TTIDP was observed in smooth bromegrass and warm-season grass samples. Digestibility of RUP varied considerably, ranging from 25% to 60%. Subirrigated meadow, native range, and smooth bromegrass samples tended to have linear decreases (P ≤ 0.11) in RUP digestibility throughout the growing season. The amount of digested RUP was fairly consistent across experiments and averages for smooth bromegrass, subirrigated meadow, and upland native range were 0.92%, 0.64%, and 0.49% of DM, respectively. Warm-season grasses in Exp. 2 had greater RUP (4.31% of DM) and amount of RUP digested (2.26% of DM), possibly because of cattle selecting for leadplant that contains more CP than the grasses. Forages can vary in CP, RUP, TTIDP, and RUP digestibility depending on the forage type, year, and time within year, but RUP digestibility is likely less than what previous sources have reported.



INTRODUCTION

Understanding forage protein quality is important for cattle grazing forages that can be deficient in CP at various times through the growing season. Forages typically have large quantities of RDP with small quantities of RUP (NRC, 1996, 2001). Although cattle need RDP for microbial growth and reproduction, RUP supplementation has improved cattle BW gains during grazing of pastures (Klopfenstein, 1996; Lardy et al., 1999). The digestibility of RUP may differ among feeds, so it is important to quantify RUP content and digestibility because digestible RUP is not hindered by the inefficiencies of using feed protein for rumen microbial N use. The NRC (1996) beef cattle recommendation uses a constant 80% RUP digestibility for all feedstuffs, but the NRC (2001) dairy cattle recommendation uses values of 60% to 75% for grasses and hays. Many research articles report RUP content and digestibility that may not be accurate because the feeds resided in the rumen for 16 h or fewer (DeBoer et al., 1987; Von Keyserlingk et al., 1996; Haugen et al., 2006a). Haugen et al. (2006b) reported that the best rumen incubation time is equal to 75% of total mean retention time (TMRT) to obtain accurate RUP values using the in situ technique. Von Keyserlingk et al. (1996) stated that RUP digestibility measures for forages may be overestimated when rumen incubations are too short. This research addresses the hypothesis that the NRC beef requirement overestimates RUP digestibility for forages.

Estimating protein degradability and RUP digestibility for forages is easier and less expensive if it is not conducted in vivo. Compared with in vitro methods, degradability estimates are more precise with in situ techniques (Vanzant et al., 1996). An analytical advantage to using the in situ technique is directly placing the in situ bags in the duodenum. Therefore, the objectives of this research were to evaluate RUP content and digestibility for several types of forages using the mobile bag in situ technique and to explore relationships between RUP content and other, more easily measured forage characteristics.


MATERIALS AND METHODS

All uses of animals were approved by the University of Nebraska Institutional Animal Care and Use Committee.

Forage samples from 4 experiments were evaluated to document RUP content, RUP digestibility, total tract protein digestibility, and potential variability in these measures.

Forage Sample Description

Experiment 1.

Forage samples from subirrigated meadow and upland native range at the Gudmundsen Sandhills Laboratory near Whitman, NE, were obtained once per month over a 5-mo period from May to September 2000 (Lamothe and Klopfenstein, 2003). The meadow contained forage species, including slender wheatgrass [Elymus trachycaulus (Link) Matte], redtop bent (Agrostis stolonifera L.), Timothy (Phleum pratense L.), Kentucky bluegrass (Poa pratensis L.), smooth bromegrass (Bromus inermus Leyss.), wooly sedge (Carex lanuginose Michx.), spike rush (Eleocharis spp.), white clover (Trifolium repens L.), alsike clover (Trifolium hybridium L.), red clover (Trifolium pretense L.), prairie cordgrass (Spartina pectinata L.), and big bluestem (Andropogon gerardii Vitman; Volesky et al., 2004). The forage species in the upland range included little bluestem [Schizachyrium scoparium (Michx.) Nash], prairie sand reed [Calamovilfa longifolia (Hook.) Fern.], switch grass (Panicum virgatum L.), sand lovegrass [Eragrostis trichodes (Nutt.) Wood], Indian grass (Sorghastrum mutans L. Nash), and grasslike plants (Carex spp. and Cyperus spp.). Forage samples were collected from 2 esophageally fistulated cows, and this procedure was modified from Wallace and Denham (1970). The esophageal cannula was removed and replaced with a screen-bottomed canvas bag, and the cows were allowed to graze for 10 to 45 min until approximately 2 kg of sample was consumed and the cannula was replaced. The samples were hand squeezed slightly to remove excess saliva and frozen at −4°C until they were later lyophilized using a Virtis Freezemobile model 25 SL (The VirTis Company, Gardiner, NY).

Experiment 2.

Forage samples from warm-season grass pastures were obtained in 2005 and 2006 from the University of Nebraska Barta Brothers Ranch near Rose, NE (Rolfe, 2011). Forage species included prairie sand reed [Calamovilfa longifolia (Hook.) Scribn.], sand bluestem [Andropogon gerardii subsp. halli (Hack.) J. Wipff], little bluestem [Schizachyrium scoparium (Michx.) Nash], switch grass (Panicum virgatum L.), sand dropseed [Sporobolus cryptandrus (Torr.) A. Gray], blue grama [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Giffiths], hairy grama (Bouteloua hirsuta Lag.), prairie June grass [Koeleria macrantha (Ledeb.) J. A. Schultes], needle and thread (Hesperostipa comata Trin. & Rupr.), porcupine grass (Hesperostipa spartea Trin.), Scribner panicum [Panicum oligosanthes Schult. var. scribnerianum (Nash) Fernald], Wilcox panicum (Panicum wilcoxianum Vasey), and bluegrass species (Poa compressa L. and Poa pratensis L.). Several species of sedges (Carex spp.) and cactus (Opuntia spp.) also are common. Common forbs and shrubs include western ragweed (Ambrosia psilostachya DC.), cudweed sagewort (Artemisia ludoviciana Nutt.), prairie wild rose (Rosa arkansana Porter ex Porter & J. M. Coult.) and leadplant (Amorpha canescens Nutt. ex Pursh). Eight samples were obtained in 2005 on the single day of June 7, composites from June 17 and 22, July 6 and 13, July 18 and 26, August 1 and 4, August 11 and 17, August 27 and September 3, and the single day of September 24. Eight samples were obtained in 2006 as composites of June 1 and 6, June 14, 25, and 30, July 6 and 13, July 19 and 21, July 29 and August 4, August 10 and 16, August 25 and September 1, and a single day of September 30. Afterward, samples were lyophilized, and ground representative subsamples were taken for composite. These samples were labeled 1 through 8, which correspond to early June, late June, early July, late July, early August, mid-August, early September, and late September. Sample collection with 4 esophageally fistulated cows and drying methods were similar to Exp. 1.

Experiment 3.

Ten subirrigated meadow forage samples were obtained each year for 2 consecutive yr (2007 and 2008) at the Gudmundsen Sandhills Laboratory (20 samples total; Griffin et al., 2012). Forage species in the meadow pastures were similar to Exp. 1. Forage samples in 2007 were obtained on May 23, 29, June 6, 13, 20, 27, July 3, 25, 30, and August 8. Samples in 2008 were obtained on May 20, 28, June 5, 12, 20, 27, July 3, 25, 30, and August 8. These samples were labeled 1 through 10, which correspond with mid-May, late May, early June, June 12–13, June 20, late June, early July, mid-July, late July, and early August. Collection of samples with esophageally fistulated cows and drying methods were similar to Exp. 1.

Experiment 4.

Smooth bromegrass (Bromus inermus Leyss.) samples were obtained across 3 yr from rotationally grazed pastures that had fertilization treatments applied at University of Nebraska–Lincoln Agricultural Research and Development Center near Mead, NE (Greenquist et al., 2009; Watson et al., 2012). Five samples were taken each of the 3 yr (2006, 2008, and 2009) at the midpoint of each of 5 grazing cycles from pastures that were not fertilized (Cont) or fertilized (Fert) with 90 kg N/ha before grazing. The Cont pastures were initially stocked at 6.8 animal unit months (AUM)/ha, whereas Fert pastures were stocked at 9.9 AUM/ha. The 5 sampling time points correspond with May, June, July, August, and September. Samples were collected from 2 ruminally fistulated steers that had been ruminally evacuated before grazing pastures for 30 to 45 min. Diet samples were squeezed, frozen, and lyophilized similar to Exp. 1.

Forage Sample Analysis

Samples were previously evaluated for IVDMD at 48 h using the method described by Tilley and Terry (1963), modified by using 1 g of urea/L of McDougall’s buffer (Weiss, 1994) and a 1-mm Wiley mill grind (Thomas Scientific, Swedesboro, NJ) on the samples. These IVDMD values were used to calculate rate of passage (kp) by kp = 0.07 × IVDMD (%) − 0.20 (Klopfenstein et al., 2001). Mean retention time was calculated as 1/kp and a 10-h passage lag was added to yield TMRT (Ellis et al., 1999). In situ ruminal incubation times used TMRT × 0.75, which Haugen et al. (2006b) stated was accurate in estimating RUP.

For the in situ part of the experiment, samples were ground through a 2-mm screen Wiley mill and weighed into 5 × 10 cm Dacron bags (Ankom Technology, Macedon, NY) containing 50-µm pore size (4 bags/sample). Weights were 1.25 g DM for Exp. 1 and 1.5 g DM for Exp. 2, 3, and 4. These procedures were modified from Vanzant et al. (1998). Dacron bags were placed in a polyester mesh (lingerie) bag for ruminal insertion (4 bags per sample) at different initial times within each experiment and were removed from the rumen at the same time and frozen at −4°C. This results in samples being incubated in the rumen for different amounts of time to incubate for an estimated 0.75 × TMRT. Two bags per sample were later thawed and taken through a simulated abomasal digestion. This included incubating the bags in pepsin and HCl solution (1 g pepsin/L and 0.01 N HCl) maintained at 37°C for 3 h. After simulated abomasal digestion, bags were inserted into the duodenal cannula every 5 min (10 to 12 bags/steer daily) of 2 steers fed a mixed diet 2 h after feeding. The mixed diet contained 70.5% bromegrass hay, 14.6% dry-rolled corn, 14.6% soybean meal, 0.3% salt, 0.05% beef trace mineral premix, and 0.03% vitamin A-D-E. Bags that were not incubated immediately were stored at 5°C for 1 to 2 d until duodenal insertion occurred. Bags were collected from the feces up to 24 h postincubation and frozen at −4°C. All bags used in the analysis were recovered within 24 h. Bags collected from the feces and bags from the rumen were machine washed using five 1-min agitation and 2-min rinse cycles with 39°C tap water, similar to Whittet et al. (2002). Deionized water was used to rinse the bags and wash the sample residue to the bottom of the bags. Bags were refluxed in 100°C neutral detergent (ND) solution (Midland Scientific, Davenport, IA) for 1 h. Residue in the bags and the original samples were measured for percent N (in duplicate) using combustion (AOAC, 1999; method 990.03) and were used to calculate CP. Rinsing bags in ND solution and measuring ND insoluble nitrogen accounts for microbial N (Mass et al., 1999). Bags were oven-dried at 60°C for 48 h and weighed and air equilibrated for 24 h and weighed. A laboratory-corrected DM analysis was conducted on the samples by weighing 0.5 g of sample (in duplicate) into a preweighed and predried aluminum pan and drying in a 105°C oven for 16 h, followed by weighing the dried sample plus aluminum pan.

Crude protein on a DM basis was calculated as CP (%)/laboratory-corrected DM (%). Ruminally undegradable protein (DM basis) was calculated using only the rumen incubated bags as CP weight of residue (g/DM) weighed into the bags (g). As a percentage of CP, RUP was calculated as CP weight of residue (g)/CP weighed into the bags (g). Total tract indigestible protein (TTIDP) as a percentage of CP and percentage of DM was calculated from the bags that were incubated in both the rumen and duodenum as CP weight of residue (g)/CP weighed into the bags (g) and CP weight of residue (g)/DM weighed into the bags (g), respectively. Ruminally undegradable protein digestibility (RUP digestibility) was calculated as 1 − [TTIDP (% DM)/RUP (% DM)]. Digested RUP as a percentage of DM was calculated as RUP (% DM) − TTIDP (% DM).

Statistical Analysis

All statistical analyses were conducted by experiment using the glimmix procedure (SAS Inst. Inc., Cary, NC). Individual observations calculated from 4 bags (2 after rumen incubation and 2 after duodenal incubation) for each sample were considered an experimental unit. Cycle, or sampling time, was used as a fixed effect for all experiments. In Exp. 4 treatment was also considered a fixed effect. No interactions were observed between cycle and treatment (P > 0.14), so only main effects are discussed. Because of large differences across years in weather and subsequent effects on forage growth, year was included as a random effect for Exp. 2, 3, and 4. In all 4 experiments orthogonal contrasts were used to examine changes in forage quality over time, with linear and quadratic contrasts reported. Differences were declared to be significant with a P-value of ≤0.10 and tended to be significant at ≤0.15.

Linear regression was used to examine relationships between forage quality measurements. These measurements included IVDMD, CP, RUP, TTIDP, RUP digestibility, and digested RUP. Data were plotted with IVDMD as the independent variable on the x axis and dependent variables on the y axis, which were CP, RUP, TTIDP, RUP digestibility, and digested RUP. Similarly, data were plotted with CP as the independent variable on the x axis and dependent variables on the y axis, which were RUP, TTIDP, RUP digestibility, and digested RUP. Regression equations and r2 values are reported. An r2 value of 0 to 0.20 shows no relationship, 0.20 to 0.40 indicates a weak relationship, and 0.40 to 0.60 is described as a moderate relationship. Measurements with an r2 value greater than 0.60 are considered related.


RESULTS

Experiment 1

Ruminally undegradable protein averaged 1.58% of DM for meadow samples and 1.94% for range samples (P = 0.09; Table 1). There were no differences in RUP content of samples over time (linear P = 0.99, quadratic P = 0.81; Table 2). The RUP digestibility of these samples was 32.8% for meadow and 24.3% for range (P = 0.72) and declined from a high of 41.8% in cycle 1 (May) to 10.8% in cycle 5 (September; linear P = 0.11; Table 2). As a percentage of CP, RUP content was greater for range than meadow (P = 0.02) and tended to increase over time from 13.6% in May to 20.0% in September (linear P = 0.13). Over the same time period, IVDMD declined from 68.9% to 51.4% (quadratic P = 0.11). The IVDMD and RUP digestibility variables were the most closely related (r2 = 0.90) forage quality parameters, followed by IVDMD and digested RUP (r2 = 0.79; Table 3).


View Full Table | Close Full ViewTable 1.

Forage quality measurements of subirrigated meadow and upland native range grass samples taken with esophageally fistulated cows at 5 time points throughout the grazing season for Exp. 1

 
Treatment
Item1 Meadow Range SEM P-value
CP, % DM 11.9 10.0 0.74 0.10
RUP,2 % DM 1.58 1.94 0.130 0.09
TTIDP,3 % DM 1.07 1.46 0.145 0.09
RUP,2 % CP 13.4 19.7 1.49 0.02
TTIDP,3 % CP 9.3 15.0 1.88 0.06
RUP digestibility,4 % 32.8 24.3 6.66 0.72
Digested RUP,5 % DM 0.52 0.49 0.117 0.87
IVDMD, % DM 60.8 60.3 3.18 0.90
1For each variable measured n = 10.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

View Full Table | Close Full ViewTable 2.

Forage quality measurements of subirrigated meadow and upland native range grass samples taken with esophageally fistulated cows at 5 time points throughout the grazing season for Exp. 1

 
Orthogonal contrasts
Item1 May (cycle 1) June (cycle 2) July (cycle 3) August (cycle 4) September (cycle 5) SEM Linear Quadratic
CP, % DM 12.9 10.9 11.2 10.8 8.9 1.23 0.04 0.87
RUP,2 % DM 1.73 1.92 1.56 1.78 1.81 0.294 0.99 0.81
TTIDP,3 % DM 0.98 1.15 1.17 1.44 1.61 0.268 <0.01 0.40
RUP,2 % CP 13.6 17.8 14.2 17.2 20.0 3.62 0.13 0.64
TTIDP,3 % CP 7.7 10.7 10.7 13.9 17.8 3.26 0.01 0.51
RUP digestibility,4 % 41.8 39.6 25.7 19.1 10.8 3.23 0.11 0.34
Digested RUP,5 % DM 0.75 0.77 0.39 0.34 0.21 0.084 0.06 0.27
IVDMD, % DM 68.9 65.5 60.3 56.5 51.4 1.30 0.60 0.11
1For each variable measured n = 10.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

View Full Table | Close Full ViewTable 3.

Scatterplot data showing the relationship between forage quality factors in subirrigated meadow and upland native range grass samples taken with esophageally fistulated cows at 5 time points throughout the grazing season in Exp. 1

 
x axis variable1 y axis variable Regression equation (SE) r2 P-value
IVDMD, % DM CP, % DM y = 0.19(0.07)x − 0.33(4.40) 0.453 0.03
IVDMD, % DM RUP,2 % DM y = 0.001(0.001)x + 1.67(1.07) 0.001 0.94
IVDMD, % DM TTIDP,3 % DM y = −0.03(0.02)x + 3.23(0.95) 0.349 0.07
IVDMD, % DM RUP digestibility,4 % y = 2.00(0.24)x − 95.3(14.5) 0.898 <0.01
IVDMD, % DM Digested RUP,5 % DM y = 0.03(0.01)x − 1.48(0.37) 0.785 <0.01
CP, % DM RUP,2 % DM y = −0.02(0.06)x + 2.02(0.70) 0.018 0.71
CP, % DM TTIDP,3 % DM y = −0.11(0.06)x + 2.47(0.65) 0.306 0.10
CP, % DM RUP digestibility,4 % y = 5.36(1.93)x − 32.6(21.4) 0.492 0.02
CP, % DM Digested RUP,5 % DM y = 0.08(0.04)x − 0.32(0.43) 0.322 0.09
1For each variable measured n = 10.
2RUP = measured using rumen incubations equal to 75% of total mean retention time.
3TTIDP = total tract indigestible protein: CP weight of residue (g)/DM weighed into the bags (g).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

Experiment 2

Both RUP and TTIDP, as a percentage of DM, increased quadratically over time (P ≤ 0.06; Table 4), with maximum values in cycle 7 (early September). These same values, expressed as a percentage of CP, increased linearly over time (P ≤ 0.04). There were no differences over time in in vitro OM disappearance (IVOMD; linear P = 0.24, quadratic P = 0.86). No clear relationships were seen between forage quality variables, with the most closely related being IVOMD and RUP (r2 = 0.24; Table 5), IVOMD and TTIDP (r2 = 0.23), and IVOMD and CP (r2 = 0.22).


View Full Table | Close Full ViewTable 4.

Forage quality measurements of warm-season grass samples taken with esophageally fistulated cows at 8 time points throughout the grazing season for Exp. 2

 
Orthogonal contrasts
Item1 Early June (cycle 1) Late June (cycle 2) Early July (cycle 3) Late July (cycle 4) Early August (cycle 5) Mid-August (cycle 6) Early September (cycle 7) Late September (cycle 8) SEM Linear Quadratic
CP, % DM 13.4 12.6 11.4 10.0 9.8 10.9 11.4 10.4 0.68 <0.01 0.46
RUP,2 % DM 3.09 4.20 5.04 4.13 3.84 5.04 5.30 3.85 0.377 0.21 <0.01
TTIDP,3 % DM 1.36 1.74 2.08 1.86 1.73 2.64 2.79 2.22 0.247 0.20 0.06
RUP,2 % CP 23.5 33.2 43.0 39.4 39.4 50.5 46.3 38.1 5.11 0.02 0.07
TTIDP,3 % CP 10.3 13.8 17.7 17.7 17.8 26.5 24.5 22.0 3.20 0.04 0.25
RUP digestibility,4 % 55.7 58.6 58.9 55.0 55.0 47.5 47.3 42.5 2.92 0.67 0.43
Digested RUP,5 % DM 1.73 2.46 2.96 2.27 2.12 2.40 2.51 1.63 0.206 0.40 0.01
IVOMD,6 % OM 58.3 54.6 51.6 55.2 49.6 55.3 48.8 53.8 0.04 0.24 0.86
1For each variable measured n = 16.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).
6IVOMD = in vitro OM disappearance.

View Full Table | Close Full ViewTable 5.

Scatterplot data showing the relationship between forage quality factors in warm-season grass samples taken with esophageally fistulated cows at 8 time points throughout the grazing season in Exp. 2

 
x axis variable1 y axis variable Regression equation (SE) r2 P-value
IVOMD,2 % OM CP, % DM y = 12.5(6.25)x + 4.53(3.35) 0.223 0.06
IVOMD, % OM RUP,3 % DM y = −7.64(3.68)x + 8.39(1.97) 0.235 0.06
IVOMD, % OM TTIDP,4 % DM y = −4.83(2.38)x + 4.63(1.28) 0.228 0.06
IVOMD, % OM RUP digestibility,5 % y = 21.6(32.9)x + 41.1(17.7) 0.146 0.52
IVOMD, % OM Digested RUP,6 % DM y = −2.77(2.47)x + 3.74(1.32) 0.097 0.47
CP, % DM RUP,3 % DM y = −0.21(0.15)x + 6.70(1.67) 0.129 0.17
CP, % DM TTIDP,4 % DM y = −0.16(0.09)x + 3.88(1.04) 0.182 0.10
CP, % DM RUP digestibility,5 % y = 1.43(1.20)x + 36.6(13.6) 0.092 0.25
CP, % DM Digested RUP,6 % DM y = −0.05(0.09)x + 2.82(1.05) 0.022 0.49
1For each variable measured n = 16.
2IVOMD = in vitro OM disappearance.
3RUP = measured using rumen incubations equal to 75% of total mean retention time.
4TTIDP = total tract indigestible protein: CP weight of residue (g)/DM weighed into the bags (g).
5RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
6Digested RUP = RUP (% DM) − TTIDP (% DM).

Experiment 3

Ruminally undegradable protein and TTIDP expressed as percentage of DM or percentage of CP linearly increased over the growing season (P ≤ 0.02; Table 6). Values for RUP digestibility did not indicate any seasonal patterns, and digested RUP was generally 0.5% to 1.0% of DM. There were no differences over time in IVDMD (linear P = 0.43, quadratic P = 0.32), which averaged 56.5%. Relationships between forage quality parameters were moderate, the strongest of which was between CP and RUP as a percentage of DM (r2 = 0.48; Table 7) and CP and TTIDP as a percentage of DM (r2 = 0.40).


View Full Table | Close Full ViewTable 6.

Forage quality measurements of subirrigated meadow grass samples taken with esophageally fistulated cows at 10 time points throughout the grazing season for Exp. 3

 
Orthogonal contrasts
Item1 Mid-May (cycle 1) Late May (cycle 2) Early June (cycle 3) June 12–13 (cycle 4) June 20 (cycle 5) Late June (cycle 6) Early July (cycle 7) Mid-July (cycle 8) Late July (cycle 9) Early August (cycle 10) SEM Linear Quadratic
CP, % DM 16.0 13.2 11.3 11.5 10.8 12.1 11.0 11.2 11.4 11.5 1.29 0.43 0.32
RUP,2 % DM 0.98 1.04 1.74 1.52 1.68 1.64 1.62 1.59 1.77 1.71 0.121 <0.01 0.15
TTIDP,3 % DM 0.40 0.72 0.73 0.94 0.97 0.82 0.85 1.03 1.19 1.11 0.113 <0.01 0.40
RUP,2 % CP 6.2 7.9 15.4 13.3 15.7 13.6 14.8 15.0 16.2 15.5 2.25 <0.01 0.35
TTIDP,3 % CP 2.5 5.4 6.5 8.2 9.1 6.9 7.7 9.6 11.0 10.1 1.69 0.02 0.61
RUP digestibility,4 % 58.5 31.5 57.5 38.5 42.5 50.0 47.5 35.5 33.0 35.0 4.51 0.11 0.34
Digested RUP,5 % DM 0.58 0.32 1.01 0.59 0.71 0.83 0.78 0.56 0.58 0.60 0.080 0.06 0.27
IVDMD, % DM 60.0 64.1 63.7 59.6 56.1 54.3 52.7 47.2 51.3 56.2 4.70 0.43 0.32
1For each variable measured n = 20.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − (TTIDP (% DM).

View Full Table | Close Full ViewTable 7.

Scatterplot data showing the relationship between forage quality factors in subirrigated meadow grass samples taken with esophageally fistulated cows at 10 time points throughout the grazing season in Exp. 3

 
x axis variable1 y axis variable Regression equation (SE) r2 P-value
IVDMD, % DM CP, % DM y = 0.02(0.07)x + 11.0(3.79) 0.004 0.80
IVDMD, % DM RUP,2 % DM y = −0.01(0.01)x + 1.86(0.56) 0.019 0.56
IVDMD, % DM TTIDP,3 % DM y = −0.01(0.01)x + 1.48(0.45) 0.093 0.19
IVDMD, % DM RUP digestibility,4 % y = 0.004(0.003)x + 0.22(0.20) 0.062 0.29
IVDMD, % DM Digested RUP,5 % DM y = 0.004(0.01)x + 0.41(0.37) 0.025 0.51
CP, % DM RUP,2 % DM y = −0.10(0.03)x + 2.76(0.31) 0.475 <0.01
CP, % DM TTIDP,3 % DM y = −0.08(0.02)x + 1.81(0.27) 0.397 <0.01
CP, % DM RUP digestibility,4 % y = 0.02(0.01)x + 0.24(0.15) 0.089 0.20
CP, % DM Digested RUP,5 % DM y = −0.03(0.02)x + 0.96(0.27) 0.068 0.27
1For each variable measured n = 20.
2RUP = measured using rumen incubations equal to 75% of total mean retention time.
3TTIDP = total tract indigestible protein: CP weight of residue (g)/DM weighed into the bags (g).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

Experiment 4

Except for CP content, fertilization treatment had no effect on any variable measured (P ≥ 0.20; Table 8), and no treatment by cycle interactions were observed (P ≥ 0.25). Thus, the main effects of cycle are shown in Table 9. A quadratic decrease (P < 0.01) in RUP as a percentage of DM was observed with maximum values of 2.64 and 2.14 in cycles 1 and 5 (May and September, respectively) and a minimum value of 1.59 in cycle 2 (June). A similar quadratic decrease (P < 0.01) was observed in TTIDP as a percentage of DM. Over the growing season RUP digestibility and IVDMD decreased linearly (P < 0.01). Several factors, including IVDMD and CP, CP and RUP as a percentage of DM, and CP and digested RUP, appeared to be weakly related, with r2 ≥ 0.34 (Table 10).


View Full Table | Close Full ViewTable 8.

Forage quality measurements of smooth bromegrass samples taken with ruminally fistulated steers at 5 time points throughout the grazing season for Exp. 4

 
Treatment1
Item Cont Fert SEM P-value Treatment × cycle
CP, % DM 14.2 16.5 0.71 0.03 0.14
RUP,2 % DM 1.98 2.08 0.128 0.56 0.84
TTIDP,3 % DM 1.10 1.11 0.074 0.94 0.90
RUP,2 % CP 14.0 12.5 0.79 0.20 0.82
TTIDP,3 % CP 7.9 6.8 0.56 0.20 0.61
RUP digestibility,4 % 44.3 46.7 1.86 0.62 0.25
Digested RUP,5 % DM 0.87 0.97 0.074 0.35 0.34
IVDMD, % DM 59.4 60.1 1.83 0.76 0.99
1Treatments consisted of Cont [control pastures with no treatment applied and cattle initially stocked at 6.8 animal unit months (AUM)/ha] or Fert (pastures that were fertilized in the spring with 90 kg N/ha and stocked at 9.9 AUM/ha). For each variable measured n = 30.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

View Full Table | Close Full ViewTable 9.

Forage quality measurements of smooth bromegrass samples taken with ruminally fistulated steers at 5 time points throughout the grazing season for Exp. 4

 
Orthogonal contrasts
Item1 May (cycle 1) June (cycle 2) July (cycle 3) August (cycle 4) September (cycle 5) SEM Linear Quadratic
CP, % DM 18.6 13.7 13.7 15.3 15.5 1.01 0.11 <0.01
RUP,2 % DM 2.64 1.59 1.78 1.99 2.14 0.203 0.33 <0.01
TTIDP,3 % DM 1.33 0.82 0.94 1.16 1.30 0.117 0.42 <0.01
RUP,2 % CP 14.4 11.7 13.1 13.2 14.1 1.25 0.83 0.19
TTIDP,3 % CP 7.4 6.0 7.0 7.6 8.6 0.88 0.15 0.19
RUP digestibility,4 % 50.2 48.3 46.8 41.7 39.0 2.93 <0.01 0.77
Digested RUP,5 % DM 1.31 0.78 0.84 0.84 0.84 0.117 0.03 0.03
IVDMD, % DM 68.1 59.6 56.1 59.0 56.0 2.35 <0.01 0.06
1For each variable measured n = 30.
2RUP = measured using rumen incubations equal to 75% of total mean retention time: CP weight of residue after rumen incubation (g)/DM weighed into the bags (g) = RUP (% DM). CP weight of residue after rumen incubation (g)/CP weighed into the bags (g) = RUP (% CP).
3TTIDP = total tract indigestible protein: CP weight of residue after duodenal incubation (g)/DM weighed into the bags (g) = TTIDP (% DM). CP weight of residue after duodenal incubation (g)/CP weighed into the bags (g) = TTIDP (% CP).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).

View Full Table | Close Full ViewTable 10.

Scatterplot data showing the relationship between forage quality factors in smooth bromegrass samples taken with ruminally fistulated steers at 5 time points throughout the grazing season in Exp. 4

 
x axis variable1 y axis variable Regression equation (SE) r2 P-value
IVDMD, % DM CP, % DM y = 0.25(0.06)x + 0.67(3.90) 0.339 <0.01
IVDMD, % DM RUP,2 % DM y = 0.01(0.02)x + 1.38(0.91) 0.018 0.48
IVDMD, % DM TTIDP,3 % DM y = −0.01(0.01)x + 1.41(0.51) 0.013 0.55
IVDMD, % DM RUP digestibility,4 % y = 0.01(0.002)x + 0.15(0.12) 0.195 0.01
IVDMD, % DM Digested RUP,5 % DM y = 0.02(0.01)x − 0.02(0.52) 0.108 0.08
CP, % DM RUP,2 % DM y = 0.12(0.03)x + 0.13(0.43) 0.419 <0.01
CP, % DM TTIDP,3 % DM y = 0.04(0.02)x + 0.50(0.30) 0.134 0.05
CP, % DM RUP digestibility,4 % y = 0.01(0.01)x + 0.28(0.07) 0.164 0.03
CP, % DM Digested RUP,5 % DM y = 0.08(0.01)x − 0.37(0.23) 0.542 <0.01
1For each variable measured n = 30.
2RUP = measured using rumen incubations equal to 75% of total mean retention time.
3TTIDP = total tract indigestible protein: CP weight of residue (g)/DM weighed into the bags (g).
4RUP digestibility = 1 − [TTIDP (% DM)/RUP (% DM)].
5Digested RUP = RUP (% DM) − TTIDP (% DM).


DISCUSSION

Crude Protein, Undegradable Protein, and Total Tract Indigestible Protein

Average CP values for smooth bromegrass, subirrigated meadow, upland range pastures, and warm-season grasses were 15.4%, 12.0%, 10.0%, and 11.2%, respectively. Forages typically have greater CP at the beginning of the growing season when plants are in the vegetative form, and CP decreases as forages become more mature (NRC, 1996). This was true in each of these experiments (Exp. 1 through 4) for smooth bromegrass, subirrigated meadow, and warm-season grasses.

Some grain sources such as corn and some protein supplements such as blood meal and feather meal have CP that is greater than 50% RUP (DeBoer et al., 1987; NRC, 1996), but forage protein is often degraded rapidly in the rumen and is typically 10% to 40% RUP, as a percentage of CP (NRC, 1996, 2001). Average RUP as a percentage of CP for smooth bromegrass (Exp. 4), subirrigated meadow (Exp. 1 and 3), upland native range (Exp. 1), and warm-season grasses (Exp. 2) were 13.3%, 13.3%, 20.0%, and 39.2%, respectively, including ranges of 6% to 50%. Results for RUP content in Exp. 2 were numerically greater than the other experiments, possibly due to cattle selecting for leadplant [Amorpha canescens (Nutt.) Pursh.]. Large amounts of CP are attributed to leadplant (above 20% early in the growing season; Myron, 1986), which would likely be greater than the grasses. The RUP content and digestibility of leadplant is unknown. Amount of RUP as a percentage of CP was typically the lowest at the first collection time point of the growing cycle in these experiments and increased as plants matured, which agrees with the NRC (1996) for early bloom and late bloom forages. However, there were no statistical differences in RUP as a percentage of CP over time in Exp. 1 and 4 (P ≥ 0.13). Ruminally undegradable protein is important because cattle respond to RUP supplementation with increased BW gain (MacDonald et al., 2007). However, as CP content decreases and RUP as a percentage of CP increases as forages mature, the amount of RUP that the animal consumes changes. Therefore, expressing RUP as a percentage of DM better illustrates changes in RUP.

When RUP was expressed as a percentage of DM, averages for smooth bromegrass, subirrigated meadow, upland native range, and warm-season grasses were 2.03%, 1.54%, 1.94%, and 4.31%, respectively. Variation in RUP due to changes in seasonality and forage growth stage decreased when RUP was represented as a percentage of DM rather than as a percentage of CP (average CV over all 4 experiments = 0.21 compared with 0.26). These values were lower at the beginning of the season in Exp. 3 compared with later in the growing season. A quadratic response was observed in both Exp. 2 and 4. However, in Exp. 2 it was a quadratic increase and subsequent decrease over the growing season, whereas a quadratic decrease followed by some rebound late in the growing season was observed in Exp. 4. This could be due to different stages of growth occurring at different times for cool-season smooth bromegrass in Exp. 4 and warm-season grasses in Exp. 2. There were no differences in RUP, expressed as a percentage of DM, over time in Exp. 1 (linear P = 0.99, quadratic P = 0.81). Therefore, the variation in these RUP effects may be partially due to environmental conditions and analytical variation. The exception is the warm-season grass samples from Exp. 2 that contained 3.1% to 5.3% RUP, as a percentage of DM, possibly because of inclusion of leadplant species (Schroeder, 2007).

Haugen et al. (2006a) observed 1.4% to 2.1% RUP as a percentage of DM for smooth bromegrass and bird’s-foot trefoil using the in situ technique with rumen incubation time calculated as 75% TMRT (ranging from 18.7 to 41.2 h). After rumen incubation of alfalfa hay for 16 h, Erasmus et al. (1994) reported 32.7% disappearance of CP from the intestinal tract, which is equal to 5.3% of DM. DeBoer et al. (1987) reported 22.2% intestinal disappearance of CP after 8 h rumen incubation and 8.4% after 24 h. Von Keyserlingk et al. (1996) reported intestinal disappearance of CP after 12 h rumen incubation. The average was 18.1% for alfalfa hay and 22.0% for grass hay, when assuming no microbial contamination. This is equal to 3.8% and 3.5% of DM. Microbial contamination is a large source of potential error when measuring RUP in forages because the CP of forages is typically quite small, less than 15%, to begin with. Measuring ND insoluble N of the residue accounts for microbial N contamination (Haugen et al., 2006b).

Total tract indigestible protein as a percentage of CP had similar trends as RUP as a percentage of CP. Values for TTIDP (percentage of CP) were typically the least early in the growing season in each experiment and increased over time. As forage CP decreased throughout the growing season, TTIDP values as a percentage of CP increased. Haugen et al. (2006a) observed an increase in TTIDP, as a percentage of DM, in smooth bromegrass and bird’s-foot trefoil samples as forages reached maturity later in the growth cycle. This seasonal effect was similar in Exp. 1 through 3, with TTIDP as a percentage of DM numerically lowest early in the growing season and increasing over time; however, this was not statistically significant. Smooth bromegrass samples in Exp. 4 were an exception with TTIDP, as a percentage of DM, the same at the beginning and end of the growing season (1.3%) and lower throughout the summer months.

Digestibility of Ruminally Undegradable Protein

An important parameter to measure was RUP digestibility. Because RUP contains both digested and undigested protein, greater RUP values could imply the forage is a good source of MP for the animal. However, if the RUP is indigestible, it supplies no MP to the animal. The RUP digestibility varied considerably, but most values across all forage types ranged from 25% to 60%. Ruminally undegradable protein digestibility averages for smooth bromegrass, subirrigated meadow, upland native range, and warm-season grasses were 45.2%, 42.0%, 24.3%, and 52.6%. In Exp. 1, 3, and 4, a tendency for a linear decrease in RUP digestibility was observed over time. More variation in RUP digestibility was observed in Exp. 2, but overall values declined from cycle 2 to cycle 8. It is important to realize that RUP digestibility may not be the same for all forages. Forage quality and digestibility vary considerably and depend on many factors such as species, maturity, level of fertilization, season, soil type, and weather conditions (Von Keyserlingk et al., 1996).

Values reported here do not agree with the NRC (1996) beef recommendation, which uses a common 80% RUP digestibility for all feedstuffs. The NRC (2001) dairy recommendation uses 60% to 75% RUP digestibility for grasses and hays. Numerous research articles have reported RUP digestibility for different feedstuffs between 35% and 98% (Hvelplund, 1984; DeBoer et al., 1987; Erasmus et al., 1994). Soybean meal and cottonseed meal are at the top end of this range, and hay would be at the lower end. Most previous work was conducted using 16-h rumen incubations. This may be typical for grains in a total mixed ration for dairy cattle; however, forages in a forage-only diet result in rumen retention time greater than 16 h (Von Keyserlingk et al., 1996; Haugen et al., 2006a). The amount of protein in a feed that becomes RUP depends on both passage and degradation of the protein, which are competing mechanisms (Orskov and McDonald, 1979). Haugen et al. (2006a) validated a RUP determination method in which feeds are incubated for 75% of TMRT. These RUP values were highly correlated (r2 = 0.95) with values calculated from Broderick (1994) equations with a 10-h lag. Calculated RUP values without a passage lag period (Broderick, 1994) overestimated RUP content by 32% (Haugen et al., 2006a). The RUP was underestimated when feeds were incubated for 100% TMRT rather than 75% TMRT. Therefore, the RUP content of forages in the NRC (2001) dairy recommendation, which reports values with 16-h incubation times or less, may be overestimated, resulting in inflated RUP digestibility values.

The amount of digested RUP as a percentage of DM for forages was relatively consistent across experiments and declined over time as forage matured. Warm-season grasses in Exp. 2 appeared to have numerically greater digested RUP (average = 2.26% ± 0.53%) than native range samples in Exp. 1 (average = 0.49% ± 0.21%); however, these were analyzed as separate experiments. The warm-season grasses in Exp. 2 may have been different than the warm-season grasses in Exp. 1, but our methods did not allow us to directly compare forages across experiments. Additionally, the greater amount of digested RUP in Exp. 2 may be due to leadplant consumption (Schroeder, 2007). For smooth bromegrass and subirrigated meadow samples the amount of CP and RUP did not have a large impact on the amount of digested RUP as a percentage of DM.

Level I of the NRC (1996) model is a simple method for calculating MP supplies for grazing cattle. However, the model is designed more to model protein concentrates than forage protein. The inputs required for modeling MP of feed are CP and RDP as a percentage of CP. The model then assumes all RUP is 80% digestible. The NRC (2001) dairy recommendation and the data reported herein show clearly that forage RUP is much less digestible than 80%. Therefore, adjustments need to be made to the specific forage inputs to provide the model with the correct amounts of RDP and digested RUP. An example follows.

Average digested RUP for smooth bromegrass was 0.92%. Because the NRC (1996) model assumes 80% digestibility of RUP, the value used by the model for RUP (as a percentage of DM) would be 1.15% (0.92/0.80). The average RDP for smooth bromegrass was 13.8% of DM. Adding the RDP and RUP gives an adjusted CP of 14.9%. The CP is adjusted for the TTIDP in excess of 20% of RUP. The RDP value (percentage of CP) for the model would be 92.6% (13.8/14.9).

Predicting Ruminally Undegradable Protein

Variables measuring protein quality and digestibility that result in interactions involving year can be expected because of varying environmental conditions between year and/or analytical error among in situ bags. Year was included as a random effect in statistical analysis because of the truly random nature of environmental conditions across year. These experiments indicate that although RUP content of forages can vary, it is typically 5% to 36% of CP. Although CP is greater at the beginning of the forage growth cycle, the RUP content of that forage is typically greater when the forages mature. Total tract indigestible protein can vary considerably, but the amount of RUP digested is fairly similar among forage species. One exception is when a forb, such as leadplant, may be consumed in substantial amounts with warm-season grasses.

Relationships between several forage quality variables were evaluated in x-y scatterplots in an attempt to define another variable that is more easily and accurately measured than RUP and highly correlated to RUP content. Within each experiment either IVDMD or CP was weakly related (r2 ≥ 0.24) to at least one of the other variables measured (RUP, TTIDP, RUP digestibility). However, there was no consistency across experiments. The closest relationship was between IVDMD and RUP digestibility in Exp. 1 with an r2 = 0.90 and slope of the regression of 2.0. These 2 variables were not related in Exp. 2, 3, and 4 (r2 = 0.15, 0.06, and 0.20, respectively). In Exp. 3, RUP was most closely related to CP (r2 = 0.48). This relationship was moderate in Exp. 4 (r2 = 0.42). However, no relationship was observed in Exp. 1 and 2 (r2 = 0.02 and 0.13, respectively).


Conclusions

Each experiment had a unique set of environmental conditions, weather patterns, and forage species. This led to variation within IVDMD, CP, RUP, and digested RUP content of samples. We conclude that neither IVDMD nor CP is a good indicator of RUP or digestible RUP content of different forage species over time. Measuring RUP content of feeds and RUP digestibility by analyzing ND insoluble nitrogen after incubation for 75% of TMRT, estimated from IVDMD plus a 10-h passage lag (Haugen et al., 2006a,b), continues to be a relatively simple procedure. The RUP content of feeds can be highly variable, and RUP digestibility adds further variation to these measures. Forages, in particular, can have large variation in these parameters throughout the grazing season. However, it is clear that the 80% RUP digestibility assumed for all feedstuffs by the NRC (1996) beef recommendation overestimates digestibility of forage RUP.

 

References

Footnotes


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