Nitrogen Partitioning and Estimates of Degradable ... - PubAg - USDA

Published online January 3, 2006

Nitrogen Partitioning and Estimates of Degradable Intake Protein in Wilting Orchardgrass and Bermudagrass Hays Damaged by Simulated Rainfall D. A. Scarbrough, W. K. Coblentz,* R. K. Ogden, J. E. Turner, J. B. Humphry, K. P. Coffey, T. C. Daniel, T. J. Sauer, J. A. Jennings, and D. W. Kellogg drying weather improves, or (iii) subjecting their crop to rain damage. Hay crops harvested in conventional bales at moisture levels .200 g kg21 result in spontaneous heating and decreased forage nutritive value (Coblentz et al., 1996; Turner et al., 2002). The threshold moisture level for acceptable storage is even lower for large-round or large-rectangular hay bales. Alternatively, delaying harvest until drying weather improves can be costly because forage nutritive value is known to decline with advancing plant maturity (Berg and Hill, 1989; Cherney et al., 1993; Ball et al., 2002). Several reports have outlined reductions in the nutritive value of hay crops in response to natural (Gordon et al., 1969; Rotz and Abrams, 1988) and simulated rainfall events (Collins, 1982, 1983; Scarbrough et al., 2005). Reductions in forage nutritive value are associated primarily with losses of water-soluble nonstructural carbohydrates that are leached from plant tissues by rain (Collins, 1982) but also may be associated with continued or reactivated respiration of available carbohydrates by plant enzymes and/or microorganisms after rehydration of plant tissues (Rotz and Muck, 1994). With the advent of new feeding models for ruminant livestock, such as the Cornell Net Carbohydrate-Protein System (Fox et al., 1992; Russell et al., 1992; Sniffen et al., 1992), or systems proposed by the National Research Council (NRC, 1989, 1996, 2001), there is a continuing need for in-depth knowledge of forage proteins, particularly characteristics that describe the partitioning of protein and/or N within the various fiber- and cell-soluble fractions of the plant, and the relative degradability of these forage components within the rumen. Unfortunately, there are relatively few studies that have assessed N partitioning and characteristics of ruminal protein degradation for forages grown in the southeastern USA, especially with respect to the subsequent effects of harvest management on these fractions. The objective of this study was to evaluate the effects of graded increments of rainfall applied with a rainfall simulation system on the N partitioning and ruminal protein degradation characteristics of wilting orchardgrass (Dactylis

Reproduced from Agronomy Journal. Published by American Society of Agronomy. All copyrights reserved.

ABSTRACT This study investigated the effects of simulated rainfall on N partitioning and concentrations of degradable (DIP) or undegradable (UIP) intake protein for wilting orchardgrass (Dactylis glomerata L.) and bermudagrass [Cynodon dactylon (L.) Pers.] hays. Orchardgrass forage was wilted to 674, 153, or 41 g kg21 of moisture (WET-O, IDEAL-O, and DRY-O, respectively) in the field before applying the simulated rainfall (0, 13, 25, 38, 51, 64, or 76 mm). For WET-O, DIP (g kg21 crude protein [CP]) increased cubically (P 5 0.020) with simulated rainfall, but the overall range of response was small (653–673 g kg21 CP). Estimates of DIP (g kg21 CP) for IDEAL-O and DRY-O decreased by 46 and 25 g kg21 CP, respectively, between the 0and 76-mm rainfall increments; for IDEAL-O, these decreases occurred in a linear (P , 0.0001) pattern, whereas quadratic (P 5 0.009) and linear (P 5 0.029) effects were observed for DRY-O. Bermudagrass forage was field wilted to 761, 400, or 130 g kg21 of moisture (WET-B, MID-B, and IDEAL-B, respectively) and evaluated similarly. For WET-B and MID-B, DIP (g kg21 CP) was not affected (P . 0.05) by simulated rainfall. In contrast, quartic (P 5 0.019) and linear (P 5 0.002) effects were observed for IDEAL-B, but these responses were confined primarily to changes between the undamaged (0-mm) control and the initial 13-mm rainfall increment. On a practical basis, concentrations of DIP were, at most, altered only moderately in response to simulated rainfall and relatively little when forages were still too wet to bale.

T

HROUGHOUT MUCH of the southeastern USA, hay harvests can be complicated by climatic conditions that may include high relative humidities and high probabilities of rainfall events during considerable portions of the time that forages are actively growing and/or harvested as hay. The time interval associated with field curing of hay is often prolonged by high relative humidity (Moser, 1995), which increases the probability of rain damage before baling. Therefore, hay producers often must decide between (i) baling before adequate desiccation has occurred, (ii) delaying harvest until curing or

D.A. Scarbrough, Dep. of Animal Science, Univ. of Arkansas, Fayetteville, AR 72701 (current address: 126 Jessie Dunn, Northwestern Oklahoma State Univ., Alva, OK 73717); W.K. Coblentz, R.K. Ogden, J.B. Humphry, K.P. Coffey, and D.W. Kellogg, Dep. of Animal Science, Univ. of Arkansas, Fayetteville, AR 72701; J.E. Turner, Dep. of Animal Science, Univ. of Arkansas, Fayetteville, AR 72701 (current address: North Carolina State Univ. Mountain Res. Stn., Waynesville, NC 28786); T.C. Daniel, Dep. of Crop, Soil, and Environmental Sciences, Univ. of Arkansas, Fayetteville, AR 72701; T.J. Sauer, USDAARS, National Soil Tilth Lab., Ames, IA 50011; and J.A. Jennings, Animal Science Section, Arkansas Coop. Ext. Service, Little Rock 72203. Contribution of the Arkansas Agric. Exp. Stn. Received 28 Mar. 2005. *Corresponding author ([email protected]).

Abbreviations: AIRDRY, forages air-dried in wire cages for 48 h after application of simulated rainfall; ANOVA, analysis of variance; CP, crude protein; DIP, degradable intake protein; DRY, orchardgrass subjected to simulated rainfall at 41 g kg21 of moisture; DM, dry matter; IDEAL, forages subjected to simulated rainfall at ideal moisture concentrations for baling (153 and 130 g kg21 of moisture for orchardgrass and bermudagrass, respectively); MID, bermudagrass subjected to simulated rainfall at the approximate midpoint of dehydration (400 g kg21 of moisture); NDIN, neutral detergent insoluble N; OVENDRY, forages oven-dried at 558C after application of simulated rainfall; UIP, undegradable intake protein; WET, forage subjected to simulated rainfall immediately after mowing (674 and 761 g kg21 of moisture for orchardgrass and bermudagrass, respectively).

Published in Agron. J. 98:85–93 (2006). Forages doi:10.2134/agronj2005.0091 ª American Society of Agronomy 677 S. Segoe Rd., Madison, WI 53711 USA

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AGRONOMY JOURNAL, VOL. 98, JANUARY–FEBRUARY 2006

glomerata L.) and bermudagrass [Cynodon dactylon (L.) Pers.] hays. MATERIALS AND METHODS


Orchardgrass Study A detailed description of the rainfall simulation system and methods used to harvest forages, apply simulated rainfall, and dry forages after rainfall was applied were reported previously (Scarbrough et al., 2005). Briefly, an established stand of ‘Benchmark’ orchardgrass, located at the University of Arkansas Forage Research Area in Fayetteville, AR, served as the source of orchardgrass forage for these studies. A first harvest was taken on 9 May 2001 when forage was mowed, conditioned, and removed from the field as round-bale silage. Second-cutting forage (40 d regrowth) was mowed to a 7.5-cm stubble height on 18 June 2001 with a New Holland Model 1411 disc mower-conditioner (CNH America, Racine, WI) equipped with rubber-covered metal conditioning rollers. Because this was the second harvest for 2001, orchardgrass forage was primarily vegetative regrowth; however, a visual evaluation of the field before mowing indicated that approximately 5% of all tillers were elongated. Freshly swathed orchardgrass forage (674 g kg21 moisture; WET-O) was collected from the entire experimental area, placed onto a tarp, and moved under a barn to minimize desiccation of plant tissues. Once out of direct sunlight, forages were weighed into 42 galvanized wire baskets (15 cm height by 31 cm width by 76 cm length; mesh size 5 1.3 cm) that were filled with forage (485 6 11 g DM m22) so that the forage density and orientation within the wire baskets was comparable to that created by the mower-conditioner in the field. Thirty-six baskets containing orchardgrass forage were placed on a raised (9-cm) wire platform under a rainfall simulator (1.5 by 6.1-m coverage area). Baskets were separated into three experimental blocks (replicates) designated on the basis of location underneath the simulator. The remaining six baskets served as controls and did not receive simulated rainfall (0 mm). Simulated rainfall was applied in graded amounts totaling 13, 25, 38, 51, 64, or 76 mm. The rainfall simulation system was modified from the design of Miller (1987) and consisted of eight TeeJet nozzles (Model 1/2 HH-SS50WSQ; Spraying Systems Co., Wheaton, IL) that were threaded directly into the body of an electrically operated solenoid valve. Solenoids were connected directly to a water supply pipe and were controlled by a custom-built electronic timing system. Solenoids operated on a rapid cycle in which they remained open for 1.0 s and were closed for 0.7 s, resulting in an intermittent rainfall pattern that delivered rainfall at an intensity of 76 mm h21. To apply graded amounts of rainfall, two baskets from each block were removed from under the simulator at 10-min increments. Baskets removed from under the simulator were allowed to drip dry for approximately 0.5 h and were (i) weighed and the forage contents immediately transferred to a 30 by 43-cm paper bag and dried to a constant weight under forced air at 558C (OVENDRY) or (ii) placed outside on grass stubble (5-cm stubble height) and allowed to air dry for 48 h (AIRDRY). After 48 h, AIRDRY forages were transferred into a 30 by 43-cm paper bag and dried to a constant weight as described previously. Baskets containing control forages that received no (0 mm) rainfall were assigned randomly to OVENDRY or AIRDRY treatments. For this study, the OVENDRY method was used to assess changes in N partitioning or concentrations of DIP that oc-

curred in specific response to artificial rainfall; in contrast, the AIRDRY procedure was designed to assess these potential changes plus any additional response that may have occurred during prolonged and/or reactivated plant respiration (Rotz and Muck, 1994) and/or other internal or external biochemical processes. After completing the initial experiment for WET-O, identical experiments were conducted after orchardgrass forage was dehydrated in the field to 153 and 41 g kg21 moisture, which represent nearly ideal (IDEAL-O) and excessively dry (DRY-O) concentrations of moisture for baling, respectively. Simulated rainfall was applied to WET-O, IDEAL-O, and DRY-O forages within one 24-h period. After simulated rainfall was applied to these forages, no natural rainfall fell during the 48-h period that AIRDRY orchardgrass forages were placed outside on grass stubble. Densities of orchardgrass forages weighed into galvanized wire baskets were 406 6 22 g DM m22 for IDEAL-O and 349 6 21 g DM m22 for DRY-O.

Bermudagrass Study Common bermudagrass was selected from another location at the University of Arkansas Forage Research Area to serve as the forage source for a companion set of studies to those described for orchardgrass. This stand of bermudagrass is like many in northern Arkansas; specifically, it is of unknown origin but has been adapted to the area over several generations. It exhibited dense sod characteristics, considerable upright growth, and excellent cold tolerance. On 2 Aug. 2001, the second cutting of bermudagrass forage was harvested as described previously for orchardgrass, except that the mowing height was set at 5.0 cm. All other experimental procedures associated with the allocation and transfer of swathed forage into wire baskets, application of simulated rainfall, and drying methodology were similar to those described previously. Simulated rainfall was applied to the experimental bermudagrass forage when the concentration of moisture reached 761, 400, or 130 g kg21 (WET-B, MID-B, and IDEAL-B, respectively). Densities of these forages weighed into galvanized wire baskets were 652 6 5, 666 6 10, and 1530 6 14 g DM m22, respectively. The density of the bermudagrass windrows was substantially greater than observed for the orchardgrass forages described previously; therefore, to complete the applications of simulated rainfall on WET-B, MID-B, and IDEAL-B within a 24-h period, it was necessary to invert windrows of IDEAL-B with a side-delivery rake to facilitate drying. Adjacent windrows of bermudagrass forage were rolled against each other, thereby exposing the bottom of each windrow to the air. This practice is used commonly by producers throughout the region, particularly before packaging in large round bales. The density of IDEAL bermudagrass placed into the galvanized wire baskets reflects the amount of forage found in these double windrows and the narrowing of these windrows as a result of raking. Simulated rainfall was applied to all bermudagrass forages within a 24-h time interval, and no natural rainfall fell during the 48-h period that AIRDRY forages were outside on grass stubble.

Chemical Analysis of Forage After drying to a constant weight at 558C, all forage samples were ground through a Wiley mill (Arthur H. Thomas, Philadelphia, PA) equipped with a 1-mm screen. Concentrations of N were quantified by a rapid combustion procedure (AOAC, 1998; Elementar Americas, Inc. Mt. Laurel, NJ), and crude protein (CP) was calculated by multiplying the concentration of N in the forage by 6.25. Insoluble residues remaining after


SCARBROUGH ET AL.: DEGRADABLE INTAKE PROTEIN IN RAIN-DAMAGED WILTING GRASSES

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forages were extracted in neutral detergent were analyzed for N (NDIN) by an identical combustion procedure and reported on a g kg21 N basis. Digestion of forages in neutral detergent was performed by batch procedures outlined by ANKOM Technology Corporation (Fairport, NY); sodium sulfite and heat-stable a-amylase were not included in the NDF solution. Procedures for determination of NDIN were consistent with the guidelines of Licitra et al. (1996), with the exception that the ANKOM filter-bag method was used for digestion of forages in neutral detergent.

analysis of variance (ANOVA) was conducted for each combination of forage and initial moisture concentration. Within each ANOVA, data were analyzed as a randomized complete block design with a 7 3 2 factorial arrangement of treatments. Treatments included seven levels of rainfall (0, 13, 25, 38, 51, 64, or 76 mm) and two post-rainfall drying methods (OVENDRY or AIRDRY). Single-degree-of-freedom orthogonal contrasts (PROC GLM; SAS Institute, 1989) were used to test for linear, quadratic, cubic, and quartic effects of rainfall amount.

In Vitro Incubation in Prepared Protease Solution

RESULTS

The in vitro protease procedures used in this study were based on techniques described by Krishnamoorthy et al. (1983) and Coblentz et al. (1999). Forage samples containing 15 mg of N were incubated for 1 h at 398C in 40 mL (pH 8.0) of borate-phosphate buffer (Krishnamoorthy et al., 1983). One mL sodium azide (1%, w/v) was added to each incubation flask as an antimicrobial agent. After the 1-h buffer incubation, 10 mL of prepared protease solution containing 0.33 activity units mL21 of Streptomyces griseus protease (P-5147; Sigma Chemical Co., St. Louis, MO) were added to each flask, yielding a final enzyme activity concentration of 0.066 activity units mL21 in the incubation medium. Flasks were covered with aluminum foil, incubated in a water bath for 48 h at 398C, and swirled daily. After 48 h, samples were removed from the water bath and immediately placed on ice to suspend enzymatic activity. Residues were filtered through preweighed (dry basis) Whatman #541 filter paper (Whatman International Ltd., Maidstone, England). Each residue was washed with ,400 mL of deionized water (208C) and dried at 1008C to constant weight in a gravity convection oven. Residues were analyzed for N by the combustion technique described previously. Single-time-point estimates of DIP and UIP were calculated as:

For all six experimental forages, regardless of species or concentration of moisture, the interaction of rainfall amount and drying method was rarely significant at P # 0.05. For this reason, only main effect means will be reported and discussed.

DIP ðg kg21 CPÞ 5 ½1 2 ðg residual CP=g initial CPÞ 3 1000 and

UIP ðg kg21 CPÞ 5 ðg residual CP=g initial CPÞ 3 1000 Estimates of DIP and UIP also were expressed on the basis of total plant DM, which quantifies the actual size of each of these pools. Calculations were made by the following:

DIP ðg kg21 DMÞ 5 ½DIP ðg kg21 CPÞ=1000 3 concentration of CP in the forage and

WET-O Forage When simulated rainfall was applied to orchardgrass forage that was wilted to 674 g kg21 moisture, concentrations of CP increased linearly (P 5 0.003) from 132 to 152 g kg21 across the range of applied rainfall (Table 1), but the relative proportion of total forage N partitioned into the NDIN fraction was not affected (P . 0.05). Concentrations of DIP (g kg21 CP) increased from 653 to 673 g kg21 CP with simulated rainfall; although this increase was marginal numerically, a cubic (P 5 0.020) effect was detected. When DIP and UIP were expressed as a proportion of total forage DM, concentrations of both fractions increased with simulated rainfall; however, the overall range for DIP (86–102 g kg21 DM) was about four times greater than that observed for UIP (46–50 g kg21 DM), and these increases were explained by cubic (P 5 0.026) and linear (P 5 0.003) effects for DIP but only by a linear (P 5 0.034) effect for UIP. Drying method had no effect (P . 0.05) on concentrations of CP, but the relative proportions of total forage N associated with the cell wall were greatly affected. The concentration of NDIN in AIRDRY forages was 103 g kg21 N greater (P , 0.0001) than that observed for OVENDRY. Concentrations of DIP and UIP also were affected (P # 0.007) by drying method, but the magnitude of these differences for DIP (34 and 6 g kg21 CP and DM, respectively) and UIP (4 g kg21 DM) was relatively small.

UIP ðg kg21 DMÞ 5 ½UIP ðg kg21 CPÞ=1000 3 concentration of CP in the forage A subsample from each basket (experimental unit) was evaluated by the S. griseus protease method in each of two separate runs, and values from each run were averaged to yield the final DIP value for each experimental unit.

Statistics Because orchardgrass and bermudagrass were harvested at different times and because each of the test forages (WET-O, IDEAL-O, DRY-O, WET-B, MID-B, and IDEAL-B) had to be wilted different lengths of time before applying rainfall, forage type and moisture concentration could not be incorporated into the statistical model. As a result, an independent

IDEAL-O Forage In response to simulated rainfall, CP increased with a quadratic (P 5 0.040; Table 2) effect, but the overall range (137–150 g kg21) was relatively narrow, and responses were erratic across rainfall increments. The proportion of total forage N associated with the cell wall ranged from 562 to 684 g kg21 N and increased with quartic (P 5 0.008), cubic (P 5 0.023), quadratic (P 5 0.0003), and linear (P , 0.0001) effects. In practical terms, most of this response occurred between the 0and 13-mm rainfall increments, and little meaningful change occurred thereafter. Concentrations of DIP (g kg21 CP) decreased linearly (P , 0.0001) by 46 g kg21

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Table 1. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for orchardgrass forage that was damaged by simulated rainfall when the forage was wilted to 674 g kg21 moisture (WET-O). Treatment

CP† 21


g kg Rainfall, mm 0 13 25 38 51 64 76 SEM Contrasts Linear Quadratic Cubic Quartic Drying method Air dried Oven dried SEM§ P.t

DM

132 134 142 143 139 139 152 3.9 0.003 NS‡ NS NS 139 141 2.1 NS

NDIN g kg

21

N

478 469 466 465 486 481 456 17.8

DIP g kg

21

523 420 9.5 ,0.0001

645 679 3.1 ,0.0001

UIP 21

CP

653 662 666 666 654 659 673 5.8 P.F NS NS 0.020 NS

NS NS NS NS

DIP g kg

DM

86 89 95 96 91 92 102 3.0

46 46 47 48 48 48 50 1.4

0.003 NS 0.026 NS

0.034 NS NS NS

90 96 1.6 0.007

49 45 0.7 0.001

† CP, crude protein; DIP, degradable intake protein; NDIN, neutral detergent insoluble N; UIP, undegradable intake protein. ‡ Nonsignificant (P . 0.05). § SEM, standard error of the main effect mean.

Table 2. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for orchardgrass forage that was damaged by simulated rainfall when the forage was wilted to 153 g kg21 moisture (IDEAL-O). Treatment

CP† g kg

Rainfall, mm 0 13 25 38 51 64 76 SEM Contrasts Linear Quadratic Cubic Quartic Drying method Air dried Oven dried SEM§ P.t

21

DM

137 149 145 145 150 138 144 3.1 NS‡ 0.040 NS NS 144 144 1.7 NS

NDIN g kg

21

N

562 650 640 663 660 684 674 9.9 ,0.0001 0.0003 0.023 0.008 647 649 5.3 NS

DIP g kg

21

CP

656 646 636 621 613 609 610 7.9 P.F ,0.0001 NS NS NS 631 623 4.2 NS

DIP

UIP g kg

90 96 92 90 92 84 88 2.6 0.041 NS NS NS 91 90 1.4 NS

21

DM 47 53 53 55 58 54 56 1.3 ,0.0001 0.006 NS NS 53 54 0.7 NS


CP in forage receiving 76 mm of simulated rainfall compared with forage without rain damage (0 mm). For DIP expressed on a DM basis, a linear (P 5 0.041) decrease also was observed, but responses over rainfall increments were erratic, and a difference of only 2 g kg21 DM separated forages receiving 76 mm of rainfall from those receiving no rainfall (0 mm). Concentrations of UIP increased with quadratic (P 5 0.006) and linear (P , 0.0001) effects of rainfall amount; the maximum increase, which was observed for the 51-mm rainfall increment, represented a 23% increase relative to UIP in the control forage receiving no rainfall. Drying method had no effect (P . 0.05) on any response variable.

DRY-O Forage Concentrations of CP were not affected (P . 0.05; Table 3) by rainfall amount, but the proportion of total

forage N associated with the cell wall increased sharply (overall range 5 131 g kg21 N), exhibiting strong quadratic (P , 0.0001) and linear (P , 0.0001) effects. As observed for IDEAL-O, most of this response occurred between the 0- and 13-mm rainfall increments, with limited change thereafter. Concentrations of DIP (g kg21 CP) decreased with quadratic (P 5 0.009) and linear (P 5 0.029) effects, falling from 665 to 623 g kg21 CP between the 0- and 64-mm rainfall increments, but increased slightly at 76 mm of rainfall. On a DM basis, DIP was not affected (P . 0.05) by simulated rainfall. Concentrations of UIP increased with simulated rainfall, exhibiting quadratic (P 5 0.012) and linear (P 5 0.001) effects; UIP increased by 15% at the 38-mm rainfall increment compared with forage receiving no rainfall (0 mm), but responses were relatively static thereafter.

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Table 3. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for orchardgrass forage that was damaged by simulated rainfall when the forage was wilted to 41 g kg21 moisture (DRY-O). Treatment

CP†



21

DM

138 136 134 144 141 139 143 3.7 NS‡ NS NS NS 141 137 2.0 NS

NDIN g kg

21

N

597 679 713 715 714 728 696 13.5 ,0.0001 ,0.0001 NS NS 688 695 7.2 NS

DIP g kg

21

DIP

CP

UIP g kg

665 642 624 633 628 623 640 9.1 P.F 0.029 0.009 NS NS

21

DM

92 87 84 91 89 87 92 3.3

46 49 50 53 52 52 51 1.3

NS NS NS NS

642 632 4.9 NS

0.001 0.012 NS NS

91 87 1.8 NS

51 51 0.7 NS


Table 4. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for common bermudagrass forage that was damaged by simulated rainfall when the forage was wilted to 761 g kg21 moisture (WET-B). Treatment Rainfall, mm 0 13 25 38 51 64 76 SEM Contrasts Linear Quadratic Cubic Quartic Drying method Air dried Oven dried SEM§ P.t

CP†

NDIN

DIP

g kg21 DM

g kg21 N

g kg21 CP

564 541 514 520 541 522 514 12.5

534 540 546 546 535 549 542 9.9 P.F NS NS NS NS

156 158 159 158 152 162 156 2.8 NS‡ NS NS NS

0.022 NS NS NS

155 159 1.5 NS

598 463 6.7 ,0.0001

509 574 5.3 ,0.0001

DIP

UIP g kg21 DM

83 85 87 86 81 89 84 2.1

73 73 72 72 71 73 72 2.2

NS NS NS NS

NS NS NS NS

79 91 1.1 ,0.0001

76 68 1.2 ,0.0001


As observed for IDEAL-O, drying method did not affect (P . 0.05) any response variable.

tween concentrations of NDIN within AIRDRY and OVENDRY forages was large (135 g kg21 N).

WET-B Forage

MID-B Forage

Simulated rainfall did not affect (P . 0.05; Table 4) concentrations of CP, DIP, or UIP. In contrast to the general trends observed for orchardgrass forages, NDIN decreased linearly (P 5 0.022) from 564 to 514 g kg21 N across the range of applied simulated rainfall. These decreases were most pronounced at the 13- and 25-mm rainfall increments, with erratic but mostly static responses thereafter. Drying method affected (P , 0.0001) all response variables except CP (P . 0.05); however, NDIN and UIP were greater (P , 0.0001) in AIRDRY, whereas DIP was greater (P , 0.0001) in OVENDRY forages. As observed for WET-O, the difference be-

Concentrations of NDIN decreased from 583 to 522 g kg21 N between 0 and 38 mm of simulated rainfall but increased at greater rainfall increments (Table 5). Overall, these responses were explained by quadratic (P 5 0.0003) and linear (P 5 0.033) effects. Unlike NDIN, simulated rainfall had no effect (P . 0.05) on concentrations of CP, DIP (g kg21 CP), and UIP. Although DIP (g kg21 DM) exhibited cubic (P 5 0.049) and quadratic (P 5 0.050) responses to simulated rainfall, it is unlikely that these responses were of great biologic significance. The overall range was narrow (9 g kg21 DM), and the estimate of DIP for forage receiving the maximum

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Table 5. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for common bermudagrass forage that was damaged by simulated rainfall when the forage was wilted to 400 g kg21 moisture (MID-B). Treatment

CP†



21

DM

149 149 153 150 154 154 150 1.9

NDIN 21

g kg

N

583 570 548 522 526 546 562 11.0

NS‡ NS NS NS

0.033 0.0003 NS NS

153 150 1.0 NS

572 530 5.9 ,0.0001

DIP g kg

21

DIP

CP

558 541 549 574 584 546 545 13.4 P.F NS NS NS NS 546 567 7.2 0.042

UIP g kg

21

DM

83 81 84 86 90 84 82 2.1

66 68 69 64 64 70 68 2.3

NS 0.050 0.049 NS

NS NS NS NS

83 85 1.1 NS

69 65 1.2 0.016


(76 mm) amount of simulated rainfall differed from the undamaged control by only 1 g kg21 DM. Drying method had no effect (P . 0.05) on concentrations of CP and DIP (g kg21 DM), but concentrations of NDIN (P , 0.0001) and UIP (P 5 0.016) were greater for AIRDRY than for OVENDRY forages. Conversely, DIP (g kg21 CP) was 21 g kg21 CP greater (P 5 0.042) for OVENDRY than for AIRDRY forages.

IDEAL-B Forage Concentrations of CP increased with simulated rainfall, exhibiting quadratic (P 5 0.041) and linear (P 5 0.003) effects; however, these responses were generally static (overall range 5 150–156 g kg21) through the 64-mm rainfall increment, followed by a sharp increase

to 166 g kg21 at 76 mm of rainfall (Table 6). As observed for WET-B, concentrations of NDIN decreased with simulated rainfall, exhibiting quartic (P 5 0.0003) and quadratic (P 5 0.030) effects. Within this context, NDIN decreased by 75 g kg21 N with the first 13-mm increment of simulated rainfall but generally exhibited erratic responses thereafter. Although no relationship between DIP (g kg21 CP) and simulated rainfall was observed for WET-B and MID-B, DIP increased for IDEAL-B, exhibiting quartic (P 5 0.019) and linear (P 5 0.002) effects. This response was confined to a sharp increase of 47 g kg21 CP with the application of the first 13 mm of simulated rainfall, but only minor fluctuations were observed thereafter. For DIP (g kg21 DM), a linear (P 5 0.0003) increase of 17% was observed between forages receiving 76 mm and no (0 mm) simulated rainfall.

Table 6. Main effects of rainfall amount and drying method on concentrations of crude protein, neutral detergent insoluble N, degradable intake protein, and undegradable intake protein for common bermudagrass forage that was damaged by simulated rainfall when the forage was wilted to 130 g kg21 moisture (IDEAL-B). Treatment

CP† g kg

Rainfall, mm 0 13 25 38 51 64 76 SEM Contrasts Linear Quadratic Cubic Quartic Drying method Air dried Oven dried SEM§ P.t

21

DM

153 150 155 152 155 156 166 2.8 0.003 0.041 NS‡ NS 157 154 1.5 NS

NDIN g kg

21

N

590 515 526 552 561 509 560 13.3 NS 0.030 NS 0.0003 540 549 7.1 NS

DIP g kg

21

DIP

CP

531 578 565 572 568 590 579 9.0 P.F 0.002 NS NS 0.019 589 549 4.8 ,0.0001

UIP 21

g kg 82 87 88 87 88 92 96 2.4

DM 72 63 67 65 67 64 70 1.5

0.0003 NS NS NS

NS 0.003 NS 0.004

92 85 1.3 0.0003

64 70 0.8 ,0.0001




Quartic (P 5 0.004) and quadratic (P 5 0.003) effects were observed for UIP, but the concentration after 76 mm of rainfall differed from the 0-mm control by only 2 g kg21 DM. Drying method did not affect (P . 0.05) concentrations of CP or NDIN in IDEAL-B. Both measures of DIP (g kg21 CP or DM) were greater (P # 0.0003) in AIRDRY compared with OVENDRY forages, but the opposite response (P , 0.0001) occurred for UIP.

DISCUSSION Crude Protein For WET-O, IDEAL-O, and IDEAL-B, concentrations of CP increased in response to simulated rainfall, exhibiting effects that were quadratic, linear, or both. For forages not exhibiting polynomial effects (P . 0.05), concentrations of CP were often numerically greater when some increment of simulated rainfall was applied relative to control forages receiving no rainfall (0 mm). In either case, the responses to simulated rainfall were generally erratic and limited in magnitude. Several researchers have demonstrated increased concentrations of CP in response to natural or simulated rainfall, but these studies have been limited primarily to legumes (Collins, 1982; Rotz et al., 1991; Smith and Brown, 1994). In other studies (Collins, 1983; Collins, 1985; Fonnesbeck et al., 1986), rainfall has had no consistent effect on concentrations of CP in forages. Rotz et al. (1991) suggested that soluble protein or N was leached from wilting alfalfa during rainfall events, but other components, particularly sugars, were leached at a more rapid rate, thereby causing CP concentrations to increase at a rate of about 0.17 g kg21 mm21 of rainfall. Some evidence suggests concentrations of CP are affected positively by rainfall only when the forage is wet, but the opposite effect occurs when the forage is relatively dry at the time rainfall occurs (Smith and Brown, 1994).

Neutral Detergent Insoluble Nitrogen Any N that is retained within residual forage DM after extraction in neutral detergent (neutral detergent insoluble N [NDIN]) is presumed to be associated with the cell wall (Krishnamoorthy et al., 1982; Licitra et al., 1996) and insoluble in water (Van Soest, 1987). Rotz and Muck (1994) have suggested that any N or CP leached from the forage during rainfall events is highly soluble; therefore, the proportion of more stable, waterinsoluble CP should increase after rainfall events. In addition, a portion of the N that is insoluble in neutral detergent is also insoluble in acid detergent (ADIN), thereby implying indigestibility within ruminant livestock (Licitra et al., 1996). Rotz et al. (1991) reported that concentrations of ADIN in alfalfa increase with rain damage and suggested that this increase could not be explained solely on the basis of the soluble N leached from the forage. It was suggested that rain damage may reduce the detergent solubility of the remaining protein or promote a chemical reaction, such as the Maillard reaction, that increases binding of protein within the ADF matrix.

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Data obtained from the present study were consistent with this premise for relatively dry orchardgrass forages (IDEAL-O and DRY-O) but not when the same forage was wet (WET-O). Concentrations of NDIN were not affected by simulated rainfall (P . 0.05) for WET-O, but they increased within IDEAL-O and DRY-O forages by 112 and 99 g kg21 N, respectively, when 76 mm of simulated rainfall was applied, compared with forages receiving no simulated rainfall. In both of these forages, the vast majority of this response occurred after the first 13 mm of rainfall was applied, and only marginal changes occurred with greater rainfall increments. Based on Rotz and Muck (1994) and Rotz et al. (1991), concentrations of NDIN should increase when large leaching losses of DM occur as a result of rainfall events. This occurred for the orchardgrass forages evaluated in this study. Previously, Scarbrough et al. (2005) reported that IDEAL-O and DRY-O lost a maximum of 88 and 107 g kg21 DM, respectively, in response to these graded applications of simulated rainfall. In the present evaluations of N partitioning for these same forages, large and concomitant increases (P # 0.023) in the concentrations of NDIN (Tables 2 and 3, respectively) were observed. However, the response for WET-O was static; a maximum DM loss of only 19 g kg21 was observed in response to simulated rainfall (Scarbrough et al., 2005), and the concentrations of NDIN were not affected (P . 0.05; Table 1). For bermudagrass, all moisture levels exhibited single or multiple polynomial effects in response to simulated rainfall (Tables 4, 5, and 6, respectively), but these responses were inconsistent across moisture levels, and the biologic significance of these responses remains unclear. For WET-B, MID-B, and IDEAL-B, NDIN decreased numerically in all rain-damaged forages relative to the corresponding undamaged controls, but these responses varied somewhat erratically across rainfall increments. In addition, the total range across all three bermudagrass hays was relatively narrow (509–590 g kg21 N). Scarbrough et al. (2005) showed that these WET-B, MID-B, and IDEAL-B forages lost a maximum of only 1, 38, and 21 g kg21 DM, respectively, and exhibited only minor changes in forage nutritive value in response to simulated rainfall. The limited and somewhat erratic decreases for concentrations of NDIN within bermudagrass forages may illustrate the relatively inert nature of bermudagrass during rainfall events rather than the clearly contrasting response to that observed for orchardgrass forages.

Degradable Intake Protein (g kg21 CP) Estimates of DIP averaged over all rainfall increments for WET-O, IDEAL-O, and DRY-O were 662, 627, and 637 g kg21 CP, respectively. These estimates of DIP were somewhat lower than those determined by in situ methodology for orchardgrass harvested in Wisconsin at the second node (817 g kg21 CP), boot (752 g kg21 CP), and full inflorescence (714 g kg21 CP) growth stages (Hoffman et al., 1993). Balde et al. (1993) used similar methods to evaluate DIP for orchardgrass


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harvested in Maryland at the vegetative (780 g kg21 CP), early head (787 g kg21 CP), full heading (719 g kg21 CP), and anthesis (696 g kg21 CP) growth stages. These studies were confined to the initial growth of orchardgrass, and neither study considered regrowth. Balde et al. (1993) reported concentrations of NDIN in forages ranging from 288 to 370 g kg21 N, which were considerably lower than observed for orchardgrass in the present study and are consistent with higher estimates of DIP. Protein that is insoluble in neutral detergent but soluble in acid detergent is expected to degrade slowly because of its association with the cell wall, and a high proportion is expected to escape degradation in the rumen (Sniffen et al., 1992). For WET-B, MID-B, and IDEAL-B, mean estimates of DIP over all rainfall application levels were 542, 557, and 569 g kg21 CP, respectively, which agreed closely with previous estimates (Scarbrough et al., 2002; Coblentz et al., 2004). Generally, CP from perennial warmseason grasses have exhibited greater resistance to ruminal degradation than perennial cool-season grasses (Coblentz et al., 2004; Mitchell et al., 1997; Mullahey et al., 1992). This has been explained, in part, on the basis of anatomic differences between plants fixing carbon by C3 and C4 photosynthetic pathways (Mullahey et al., 1992). In the present study, the mean estimate of DIP over all orchardgrass forages (WET-O, IDEAL-O, and DRY-O; 642 g kg21 CP) was 86 g kg21 CP higher than that observed for all bermudagrass forages (WET-B, MID-B, and IDEAL-B; 556 g kg21 CP). Single or multiple polynomial effects (P # 0.029) relating simulated rainfall amount and concentrations of DIP were observed for WET-O, IDEAL-O, DRY-O, and IDEAL-B. From a practical standpoint, these effects described only minor changes in concentrations of DIP. For example, DIP decreased in quadratic (P 5 0.009) and linear (P 5 0.029) patterns for DRY-O, but the difference between the 0- and 76-mm rainfall increments was only 25 g kg21 CP (Table 3), which likely differs only marginally from the sensitivity of the measurement itself. In addition, the concentration of moisture within the forage at the time simulated rainfall was applied had little practical effect; the overall mean estimates of DIP for WET-O, IDEAL-O, and DRY-O ranged narrowly from 627 to 662 g kg21 CP, and a similar narrow range (542–569 g kg21 CP) was observed for WET-B, MID-B, and IDEAL-B. Based on increased resistance to ruminal degradation by proteins associated with the cell wall (Sniffen et al., 1992), the range in DIP for orchardgrass forages is exceptionally narrow. Overall mean concentrations of NDIN in WET-O, IDEAL-O, and DRY-O were 472, 648, and 692 g kg21 N, respectively, which represents a wide range relative to estimates of DIP. In contrast, overall mean concentrations of NDIN were consistent across WET-B, MID-B, and IDEAL-B (531–551 g kg21 N). These concentrations were lower numerically than observed for IDEAL-O and DRY-O, yet protein within bermudagrass forages was more resistant to enzymatic attack in all cases than in orchardgrass forages.

Degradable Intake Protein and Undegradable Intake Protein (g kg21 DM) Rotz and Muck (1994) have suggested that any N or CP leached from the forage during rainfall events is highly soluble and that the proportion of more stable, water-insoluble CP should increase after rainfall events. Logically, UIP in forages should be largely or completely water insoluble and probably associated closely with the cell wall (Sniffen et al., 1992). Therefore, the leaching of soluble components during rainfall events should theoretically concentrate UIP as a function of the remaining plant DM. For orchardgrass forages, this premise proved to be true; concentrations of UIP increased for WET-O, IDEAL-O, and DRY-O. However, a simple linear (P 5 0.034) effect was observed for WET-O, while quadratic (P # 0.012) and linear (P # 0.001) effects were observed for both IDEAL-O and DRY-O. In contrast to orchardgrass, concentrations of UIP for bermudagrass forages were largely unaffected by simulated rainfall; no polynomial effects (P . 0.05) were observed for WET-B and MID-B, and UIP for IDEAL-B differed by only 2 g kg21 DM between the 0- and 76-mm rainfall increments. This is consistent with the relatively stable nature of bermudagrass during rainfall events discussed previously with respect to concentrations of NDIN and for other measures of nutritive value (Scarbrough et al., 2005). Concentrations of DIP (g kg21 DM) did not exhibit any clear trend in response to simulated rainfall across the six orchardgrass and bermudagrass forages. Two forages (DRY-O and WET-B) exhibited no polynomial effects (P . 0.05), whereas other forages exhibited various polynomial effects that generally represented somewhat erratic, but limited, linear or curvilinear changes. The overall effect of these changes on DIP was positive (WET-O, IDEAL-B), negative (IDEAL-O), or neither (MID-B). Overall, these results were somewhat unexpected because water-soluble N or CP is likely to be highly rumen degradable; therefore, DIP should be preferentially leached during rainfall events.

Drying Method Effects of drying method were most obvious for NDIN within forages that were still well hydrated (WET-O, WET-B and MID-B) at the time simulated rainfall was applied. In each of these cases, AIRDRY exhibited greater concentrations of NDIN (P , 0.0001) than OVENDRY forages; in two cases (WET-O and WET-B), these differences were substantial (103 and 135 g kg21 N, respectively). In each of these three forages, DIP (g kg21 CP) was greater (P # 0.042) in OVENDRY than in AIRDRY forages, which is consistent with the concept that NDIN is less degradable in the rumen than cell-soluble protein (Sniffen et al., 1992). In contrast, drying method had no effect (P . 0.05) on any response variable for IDEAL-O and DRY-O, and AIRDRY increased (P , 0.0001) for DIP (g kg21 CP) within IDEAL-B. Reasons for these contrasting responses in relatively dry forages remain unclear, but the proteolytic activity within these wilting forages was


probably suspended before the application of rainfall. Proteolysis within wilting forages is thought to be negligible when the forage is dehydrated to ,600 g kg21 moisture (Rotz and Muck, 1994).


SUMMARY As previously described for dry matter losses and changes in nutritive value (Scarbrough et al., 2005), concentrations of DIP (g kg21 CP) within orchardgrass and bermudagrass forages exhibited the greatest changes when forages were dry enough to bale at the time simulated rainfall was applied. Within this context, responses were diverse across forages; DIP decreased in response to simulated rainfall for orchardgrass forages but increased for bermudagrass. However, the magnitude of these changes was relatively small, and the difference between the 0- and 76-mm rainfall increments was #48 g kg21 CP for any forage dry enough to bale at the time rainfall was applied. In contrast, forages that were relatively wet when simulated rainfall was applied exhibited remarkably consistent concentrations of DIP (g kg21 CP) across rainfall increments. On a practical basis, concentrations of DIP were, at most, altered only moderately in response to simulated rainfall and not at all when forages were still too wet to bale. Based on these and previous findings, forage grasses are resistant to reductions in nutritive value if they are still well hydrated at the time the rainfall event occurs, but damage is maximized when forages are dry enough to bale. REFERENCES AOAC, Association of Official Analytical Chemists. 1998. Official method 990.03. Official methods of analysis. 16th ed. AOAC, Gaithersburg, MD. Balde, A.T., J.H. Vandersall, R.A. Erdman, J.B. Reeves, and B.P. Glenn. 1993. Effect of stage of maturity of alfalfa and orchardgrass on in situ dry matter and crude protein degradability and amino acid composition. Anim. Feed Sci. Technol. 44:29–43. Ball, D.M., C.S. Hoveland, and G.D. Lacefield. 2002. Southern forages. 3rd ed. Potash and Phosphate Inst. and the Foundation for Agronomic Research, Norcross, GA. Berg, C.C., and R.R. Hill, Jr. 1989. Maturity effect on yield and quality of spring harvested orchardgrass forage. Crop Sci. 29:944–948. Cherney, D.J.R., J.H. Cherney, and R.F. Lucey. 1993. In vitro digestion kinetics and quality of perennial grasses as influenced by forage maturity. J. Dairy Sci. 76:790–797. Coblentz, W.K., I.E.O. Abdelgadir, R.C. Cochran, J.O. Fritz, W.H. Fick, K.C. Olson, and J.E. Turner. 1999. Degradability of forage proteins by in situ and in vitro enzymatic methods. J. Dairy Sci. 82: 343–354. Coblentz, W.K., J.O. Fritz, K.K. Bolsen, and R.C. Cochran. 1996. Quality changes in alfalfa hay during storage in bales. J. Dairy Sci. 79:873–885. Coblentz, W.K., J.E. Turner, R.K. Ogden, K.P. Coffey, F.W. Pohlman, A.H. Brown, Jr., M.B. Daniels, J.L. Gunsaulis, and M.L. Thomas. 2004. Case study: Estimating degradable intake protein in warmand cool-season forages grown on producer farms in northern Arkansas. Prof. Anim. Sci. 20:443–452. Collins, M. 1982. The influence of wetting on the composition of alfalfa, red clover, and birdsfoot trefoil hay. Agron. J. 74:1041–1044. Collins, M. 1983. Wetting and maturity effects on the yield and quality of legume hay. Agron. J. 75:523–527. Collins, M. 1985. Wetting effects on the yield and quality of legume and legume–grass hays. Agron. J. 77:936–941. Fonnesbeck, P.V., M.M. Garcia de Hernandez, J.M. Kaykay, and M.Y. Saiady. 1986. Estimating yield and nutrient losses due to rainfall on field-drying alfalfa hay. Anim. Feed Sci. Technol. 16:7–15.

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