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Dep. of Animal Science, Univ. of Arkansas, Fayetteville, AR 72701
* Corresponding author (coblentz{at}comp.uark.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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0.588). Similar results were observed when ruminal escape N was expressed as a proportion of plant DM. Rumen degradable N expressed as a proportion of plant DM was related to indices of heating (P < 0.0001) when harvest years were evaluated together, but the slopes were not significant (P > 0.05) for specific harvest years.
Abbreviations: ADF, acid detergent fiber • ADIN, acid detergent insoluble N • DM, dry matter • HDD, heating degree days >35°C • NDF, neutral detergent fiber • NDIN, neutral detergent insoluble N • NDSN, neutral detergent soluble N
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Bermudagrass has been described for more than a century as one of the most important grasses grown in the southeastern USA (Burton and Hanna, 1995). This warm-season grass is used widely by beef and dairy producers for both grazing and hay production throughout this region. However, efforts to assess rumen degradability of N in perennial warm-season forages grown in the southeastern USA have been very limited; this creates a clear void of needed information for nutritionists serving clients in southern states and clearly complicates the development of supplementation strategies for all livestock classes consuming these forages.
Effects of harvest and storage on the nutritive value of forages such as alfalfa that primarily support confinement dairy enterprises located in areas outside the southeastern USA are well documented (Rotz and Muck, 1994). Several studies have shown that ruminal degradation of forage N in alfalfa hay can be limited by externally applied heat treatment (Yang et al., 1993; Broderick et al., 1993) or by spontaneous heating during storage (Coblentz et al., 1997). However, these increases in rumen escape N can be complicated by concurrent increases in acid detergent insoluble N (ADIN) (Yang et al., 1993; Broderick et al., 1993). Quantification of ADIN has been suggested as an appropriate and sensitive laboratory assay to assess Maillard reaction damage to N in ruminant feedstuffs; this N fraction also is assumed frequently to be indigestible (Licitra et al., 1996).
Recently, Coblentz et al. (2000) reported that concentrations of ADIN in bermudagrass hay were increased from 61.2 to 137.5 g kg-1 N in response to spontaneous heating during the storage period, where the maximum internal bale temperature reached about 62°C. This 125% increase in the concentration of ADIN suggests that N in bermudagrass is more sensitive to Maillard reaction damage than N in alfalfa hay packaged and stored under similar conditions (Coblentz et al., 1996). The relationship between the spontaneous heating incurred by bermudagrass hays during storage and the subsequent susceptibility of N to ruminal degradation has not been evaluated. In addition, it is not clear whether the rumen degradability of N in bermudagrass hays is altered in response to spontaneous heating in a manner similar to that observed for alfalfa hays. For bermudagrass, it should be emphasized that increased resistance to ruminal degradation via spontaneous heating is not necessarily desirable. As discussed previously, proteins in warm season grasses generally exhibit greater natural resistance to ruminal degradation than proteins in cool season grasses or legumes. In addition, spontaneous heating during hay storage can't be controlled; therefore, there are clear risks of combustion. Furthermore, hays that heat spontaneously are likely to exhibit poorer characteristics of nutritive value (Rotz and Muck, 1994) and increased mold growth and associated production of toxins (Roberts, 1995). Despite these considerations, assessing the relationship between spontaneous heating and ruminal degradation characteristics of N in bermudagrass is critical to nutritionists whose clientele are confined primarily to the southeastern USA, where nearly all producers utilize this forage in their livestock enterprises.
Currently, the in situ procedure (Vanzant et al., 1998), corrected for microbial contaminant N, is the most common method used for evaluating relative proportions of rumen degradable N in ruminant feedstuffs; however, in vitro procedures that utilize semipurified proteolytic enzymes have been developed as routine laboratory techniques for the estimation of these N fractions in forages (Krishnamoorthy et al., 1983; Abdelgadir et al., 1997; Coblentz et al., 1999). Our primary objective for this study was to utilize a preparation of Streptomyces griseus protease to assess the relationship between rumen escape or degradable N and spontaneous heating in bermudagrass hays harvested from the same site during 1998 and 1999. A secondary objective was to correlate estimates of rumen escape and degradable N with other measures of forage nutritive value that are evaluated commonly.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Internal bale temperatures were monitored by inserting single thermocouple wires into the center of the bales. Bale temperatures were recorded at 0700 and 1600 h for the first 10 d after baling and once daily (at 1600 h) thereafter, until the end of the 60-d storage period. All temperature data were obtained with an Omega 450 AKT Type K thermocouple thermometer (Omega Engineering, Stamford, CT). For purposes of analysis, the mean internal bale temperature for a given day was considered the same as the observed temperature, except during the first 10 d, when the mean of the two observations was used. Degree days >35°C (HDD) were computed as the summations of the daily increment by which the mean internal bale temperature was greater than 35°C. Therefore, HDD accumulated during storage can be viewed as a single numerical value that represents and combines both the intensity and duration of spontaneous heating in the hay.
Chemical Analysis of Forage
After the storage period, at least two cores (Star Quality Samplers, Edmonton, AB, Canada) were taken from the ends of each bale (35-cm depth). Core samples were dried to constant weight under forced air at 50°C. Dry forage samples were ground through a Wiley mill (Arthur H. Thomas, Philadelphia, PA) equipped with a 1-mm screen and subsequently analyzed for N, neutral detergent fiber (NDF), acid detergent fiber (ADF), neutral detergent insoluble N (NDIN), acid detergent lignin (ADIN), and whole-plant ash. Respective concentrations of neutral detergent soluble N (NDSN) and ADIN were calculated and reported as proportions of total plant DM and N. Similarly, NDSN was calculated on a total DM and N basis, where NDSN = total N - NDIN. Total plant N and the concentration of N in NDF and ADF residues were determined using a macro-Kjeldahl procedure (Kjeltec Auto 1030 Analyzer, Tecator, Inc., Herndon, VA). Neutral detergent fiber, ADF, acid detergent lignin, and cellulose were determined by batch procedures outlined by ANKOM Technology Corp. (Fairport, NY). Whole-plant ash was determined by combusting forage samples in a muffle furnace at 500°C for 8 h. Concentrations of hemicellulose were calculated mathematically as the difference between NDF and ADF after nonsequential analysis of NDF and ADF.
In Vitro Incubation in Prepared Protease Solution
The in vitro protease procedures used in this study were similar to those described by Krishnamoorthy et al. (1983), Roe et al. (1990), and Coblentz et al. (1999). The Streptomyces griseus protease (P-5147; Sigma Chemical Co., St. Louis, MO) used in this study contained 5.5 enzyme activity units/mg of solid, and one activity unit of enzyme was able to hydrolyze casein to produce color equivalent to 1.0 µmol (181 µg) of tyrosine/minute at pH 7.5 and 37°C. Bermudagrass hay samples containing 15 mg of N were incubated for 1 h at 39°C in 40 mL of borate-phosphate buffer (pH 8.0) (Krishnamoorthy et al., 1983). One milliliter sodium azide (1%, w/v) was added to each incubation flask as an antimicrobial agent. Following the 1-h buffer incubation, 10 mL of prepared protease solution containing 0.33 activity units/mL of Streptomyces griseus protease were added to each flask, yielding a final enzyme activity concentration of 0.066 activity units/mL in the incubation medium. Flasks were covered with aluminum foil and incubated in a water bath for 48 h at 39°C. Following incubation, samples were immediately placed on ice to suspend enzymatic activity and then filtered through preweighed (dry basis) Whatman #541 filter paper (Whatman International Ltd., Maidstone, England). Residues then were washed with 400 mL of deionized water (20°C) and placed in a gravity convection oven (100°C) until dry. Residues and associated filter papers were then analyzed for N by the same macro-Kjeldahl procedure described previously. Blank filter papers also were analyzed to correct for any contaminant N associated with the filter papers. Single time point estimates of rumen escape N were calculated as: rumen escape N (g kg-1 N) = (residual N/total N) x 1000. Estimates of rumen escape and degradable N also were expressed on the basis of total plant DM. All forty samples of bermudagrass hay were evaluated in each of three separate runs; therefore, estimates of rumen escape and degradable N are the means of three individual evaluations.
Statistical Analysis
Estimates of rumen escape and degradable N were regressed linearly on three indices of spontaneous heating; these included HDD, maximum internal bale temperature, and the average internal bale temperature over the first 30 d of storage (PROC REG; SAS Institute, 1989). For purposes of analysis, bale temperatures were averaged over the initial 30 d of storage because little evidence of heating occurred beyond this time interval in a previous study (Coblentz et al., 2000). An independent test of homogeneity (PROC GLM; SAS Institute, 1989) was included to determine if a common line could be used to describe the data from both years. Correlation analysis relating rumen escape and degradable N with various indices of nutritive value was conducted with the PROC CORR option of SAS (SAS Institute, 1989).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Relating Rumen Degradability Characteristics to Indices of Spontaneous Heating
Concentrations of rumen escape N for bermudagrass hays harvested in 1998 exhibited a range of 113 g kg-1 N or 4.1 g kg-1 DM (Fig. 1 and 2, respectively). Slightly smaller ranges were observed for bales harvested in 1999 (90 g kg-1 N or 3.7 g kg-1 DM), which can probably be explained on the basis of the narrower range of heating characteristics in these bales. Mean concentrations of rumen escape N were numerically greater for hay harvested in 1999 than in 1998 (571 vs. 446 g kg-1 N or 11.8 vs. 9.9 g kg-1 DM). Conversely, the mean concentration of degradable N was generally greater for bales harvested in 1998 (12.3 vs. 8.8 g kg-1 DM). Estimates of rumen escape N for warm-season grasses can exceed 50% of the total N pool, and concentrations of rumen escape N for both harvest years were consistent with those determined for other warm-season grasses by various enzymatic and in situ methodologies (Vanzant et al., 1996; Abdelgadir et al., 1997; Coblentz et al., 1998; Coblentz et al., 1999).
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0.416) between slopes for hays made in 1998 and 1999, thereby suggesting that heat affected concentrations of rumen escape N consistently across years. Across the entire range of spontaneous heating, estimates of rumen escape N were generally greater in 1999 than in 1998, and intercepts were dissimilar (P < 0.0001) for these regression lines; therefore, no single line could be used to describe the relationship between rumen escape N and indices of spontaneous heating in any case. The r2 statistics for these relationships were substantially greater for hays harvested in 1998 (range = 0.588–0.706) than in 1999 (range = 0.291–0.439); it remains unclear why these relationships were weaker for hay harvested in 1999. In an effort to remove variability from the relationship between rumen escape N and indices of spontaneous heating, estimates of rumen escape N also were converted to an organic matter basis to remove any variability created by different proportions of ash in the experimental forage samples. Linear regressions of rumen escape N (g kg-1 OM) on HDD for bales made in 1998 (Y = 0.0063 X + 9.4; r2 = 0.698; P < 0.0001) and 1999 (Y = 0.0076 X + 10.5; r2 = 0.376; P = 0.004) did not show any meaningful improvement in fit relative to regressions on the basis of rumen escape expressed on a DM basis. Similar trends were observed when the maximum internal bale temperature and 30-d average internal bale temperature were used as independent variables. In 1998, respective regression equations using these predictor variables were Y = 0.092 X + 5.9 (P < 0.0001; r2 = 0.717) and Y = 0.189 X + 2.9 (P < 0.0001; r2 = 0.703). For 1999, relationships again were poorer than observed in 1998; these linear relationships were defined by Y = 0.119 X + 5.4 (P = 0.002; r2 = 0.437) when the maximum internal bale temperature was designated as the independent variable and by Y = 0.228 X + 2.7 (P = 0.004; r2 = 0.374) when the 30-d average temperature was used.
There was no practical advantage for using HDD or 30-d average internal bale temperature as the predictor variable. These indices incorporate information obtained from the entire period during bale storage in which spontaneous heating occurred. In contrast, the maximum internal bale temperature is a single measurement taken at one point in time during the storage period, yet regression equations based on this predictor variable generally had the highest r2 statistics within each harvest year. The simplicity of making this measurement further enhances its potential value as a predictor of changes in rumen escape N and other measures of forage nutritive value (Coblentz et al., 2000).
Rates of increase for rumen escape N were 2.76 and 2.65 g kg-1 N per 1°C increase in the maximum internal bale temperature in 1998 and 1999, respectively; these slopes were significant (P 
0.01), but did not differ from each other (P = 0.643). On the basis of HDD, rates were 0.18 and 0.15 g kg-1 N per HDD in 1998 and 1999, respectively. These slopes were significant (P 0.01), but did not differ (P = 0.638), and correspond closely to the slope relating rumen degradable N and HDD reported for alfalfa hay (Coblentz et al., 1996). In that study, the effective in situ rumen degradability of N decreased at about the same rate (0.18 g kg-1 N per HDD). Some caution should be used in comparing these results because experimental conditions surrounding the generation of these slopes were not totally consistent across studies. In the alfalfa study, effective rumen degradability of N was assessed by the in situ method and HDD was based on a 30°C, rather than 35°C, threshold. The later discrepancy was mediated, in part, by an early fall harvest date (9 September) and cooler temperatures during the time interval in which bales were stored. However, these findings do suggest that effective rumen degradability of N may change at a relatively constant rate across forage species in response to spontaneous heating; this needs to be verified in studies using other forages and common methodologies.
When all forages (n = 40) were considered together, all measures of spontaneous heating negatively affected concentrations of rumen degradable N expressed on the basis of plant DM (P < 0.0001; data not shown). The slopes of linear regression lines did not differ (P 0.428) between harvest years, regardless of which index of spontaneous heating was used as the predictor variable. Generally, estimates of rumen degradable N were higher in 1998 than in 1999. Therefore, the intercepts of regression lines differed (P < 0.0001) between years, regardless of which index of heating was used as the predictor variable, and in no case could a single regression line be used to explain the responses of forages harvested across both years. However, linear regression lines for individual harvest years did not have significant (P 0.085) slopes (data not shown), indicating that heating had relatively little effect on estimates of rumen degradable N within specific harvest years. The overall significant (P < 0.0001) relationship between rumen degradable N and indices of spontaneous heating for all (n = 40) forages is likely an anomaly created by (i) the generally consistent numerical differences in concentrations of rumen degradable N between harvest years and (ii) the narrower range of spontaneous heating for bales harvested in 1999.
Within a given harvest year, it is possible that concentrations of rumen escape N expressed on the basis of plant DM could increase while concentrations of rumen degradable N remained relatively static. This can occur because overall concentrations of N usually increase with spontaneous heating during the short-term (
60 d) type of storage used to generate these sample sets (Rotz and Muck, 1994). For the bales evaluated in this study, concentrations of N ranged from 19.8 to 25.8 g kg-1 in 1998, and from 19.0 to 23.9 g kg-1 in 1999; these ranges are considerably greater than would be expected for normal variation within the field and were probably exacerbated greatly by the spontaneous heating that occurred during bale storage (Coblentz et al., 2000).
Correlations with Indices of Nutritive Value
Correlation coefficients relating typical measures of forage nutritive value with estimates of rumen escape N (Table 1) illustrate the complex nature of associating rumen escape N expressed as a proportion of total plant N with any individual measure of forage nutritive value. In many cases, indices of nutritive value were highly correlated with rumen escape N for specific harvest years, but not when years were combined. Specifically, this was true for ADIN (g kg-1 DM or g kg-1 N); correlation coefficients were high for specific harvest years (r 0.762; P < 0.001), but were lower (r 0.412), although significant (P 0.05), when years were combined. Conversely, correlation coefficients were high for NDSN (g kg-1 DM and g kg-1 N) (r -0.802; P < 0.001) and for NDIN (g kg-1 N) (r 0.802; P < 0.001) when years were combined, but these relationships were nonsignificant (P > 0.05) for at least one specific harvest year. Acid detergent fiber (r 0.716; P < 0.001) and lignin (r 0.595; P < 0.01) were the indices of nutritive value that had the closest relationships with rumen escape N expressed as a proportion of total plant N for both individual harvest years and harvest years combined. These fractions would be expected to increase in concentration with spontaneous heating in bermudagrass hay (Coblentz et al., 2000), but would also reflect any inherent differences in the quality characteristics of the forages before harvest.
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0.490; P 0.05) with rumen escape N for individual harvest years and for harvest years combined, but other measures of nutritive value were not. Rumen degradable N, NDF, ADF, lignin, NDIN (g kg-1 N), and NDIN-ADIN (g kg-1 N) were all negatively correlated (r -0.514; P < 0.001) when harvest years were combined, but none of these indices of nutritive value exhibited a significant (P < 0.05) correlation in both individual harvest years. Conversely, both N and NDSN (g kg-1 DM and g kg-1 N) exhibited strong positive correlations (r 0.711; P < 0.001) with rumen degradable N, but only N was significantly correlated (P < 0.05) in both individual harvest years. |
SUMMARY |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Within years, some correlations relating typical measures of forage nutritive value with estimates of rumen escape N and degradable N were significant (P < 0.001), but these relationships were not necessarily maintained when harvest years were combined. In other cases, specific measures of forage nutritive value were not correlated (P > 0.05) with our in vitro estimates of rumen escape N and degradable N within years, but these correlations were significant (P < 0.001) when years were combined. For bermudagrass hay, spontaneous heating appears to increase the estimated proportion of plant N escaping the rumen.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Received for publication October 18, 2000.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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  R. K. Ogden, W. K. Coblentz, K. P. Coffey, J. E. Turner, D. A. Scarbrough, J. A. Jennings, and M. D. Richardson Ruminal in situ disappearance kinetics of nitrogen and neutral detergent insoluble nitrogen from common crabgrass forages sampled on seven dates in northern Arkansas J Anim Sci, March 1, 2006; 84(3): 669 - 677. [Abstract] [Full Text] [PDF]
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; data from 1999 are represented by 
. In each case, intercepts associated with regression lines for 1998 and 1999 differed (P < 0.0001), but slopes did not (P 