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Department of Animal Sciences, University of Kentucky, Lexington 40546-0215
3 Correspondence:
805 WP Garrigus Bldg. (phone: 859-257-9438; fax: 859-257-3411; E-mail:
evanzant{at}uky.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Beef Cattle • Diet • Festuca arundinacea • Nutrient Content • Protein Degradation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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From April 23, 2000 to October 9, 2000, hand-clipped and ruminal masticate samples were collected at 28-d intervals from each pasture. Six hand-clipped samples were taken from each pasture using a 0.25-m2 quadrat. Hand shears were used to cut the forage at approximately 2 cm above soil level. Samples were immediately placed into individual paper bags and transported to the laboratory. Within each collection period, botanical composition was estimated for descriptive purposes by manually separating 10 samples into tall fescue, red clover, other grasses, weeds, and senescent material. Dry sample weights were recorded after drying samples for 96 h in a 55°C forced-air oven, and the biomass of each plot was calculated on a kg/ha basis. Samples were ground through a 2-mm screen using a Wiley mill (No. 4, Arthur H. Thomas, Co., Philadelphia, PA). Pasture composite samples were made by combining an equal weight of ground sample from each of the six samples within a pasture. These composite samples were then ground through a 1-mm screen using a Wiley mill.
Four ruminally-cannulated crossbred steers (average BW = 288 kg) were used to collect masticate samples. Steer cannulations and daily care were approved by the University of Kentucky Animal Care and Use Committee. Steers’ rumens were manually emptied and rinsed clean with water once in the morning and once in the evening on two consecutive days. Both times, each steer was turned out to graze within a randomly selected pasture for approximately 30 min, at which time about 1 kg of wet masticate sample was retrieved from each steer’s rumen. Ruminal contents were then returned to each steer. Samples were placed on ice for transport to the laboratory, where they were stored at -34°C. Collection times were established such that each pasture was sampled by a steer once in the morning and once in the evening over the course of the 2 d (using different steers for the two collections within a pasture). Masticate samples were lyophilized, ground through a 2-mm screen using a Wiley mill, and composited by combining an equal weight of ground sample from the morning and evening samples within a pasture. One-half of each pasture composite sample was ground through a 1-mm screen using a Wiley mill. All ground samples were stored in sealable plastic bags at room temperature until further analysis.
All hand-clipped and ruminal masticate samples were analyzed in duplicate for DM, OM, CP, NDF, ADF, and CP degradability. Dry matter was determined by drying 1 g of sample in a vacuum oven for 24 h at 85°C. Organic matter was measured as weight loss following combustion for 8 h at 500°C. Crude protein concentration was determined by the combustion method using a Leco FP-2000 N analyzer (Leco Corp., St. Joseph, MI). Concentrations of ash-free NDF and ADF were determined using an Ankom 200 fiber analyzer (Ankom Technology Corp., Fairport, NY). No sodium sulfite or decalin were used for fiber analyses (Van Soest et al., 1991). Estimates of CP degradability for all samples were generated using Streptomyces griseus protease (Coblentz et al., 1999). Additionally, CP degradability estimates of masticate samples were generated with in situ techniques for comparison with the enzymatic procedure.
In Vitro Crude Protein Degradation.
Crude protein degradation of both hand-clipped and ruminal masticate samples was determined using Streptomyces griseus protease (SGP; Coblentz et al., 1999). The SGP (P-5147; Sigma Chemical Co., St. Louis, MO) used contained 4.4 enzyme activity units/mg of solid; one activity unit of enzyme was able to hydrolyze casein to produce color equivalent to 1.0 µmol (181 µg) of tyrosine/min at pH 7.5 and 37°C. Samples containing the equivalent of 15 mg of N were weighed into 125-mL Erlenmeyer flasks in duplicate. Samples were incubated at 39°C in 40 mL of borate-phosphate buffer (pH 7.8 to 8.0) for 1 h. After the initial incubation, 10 mL of freshly prepared SGP solution containing 0.33 activity units/mL (to give a final enzyme activity concentration of 0.066 units/mL of incubation medium) was added to each flask and incubated at 39°C for 48 h. At the end of the 48-h incubation, the samples were washed with 400 mL of distilled water and filtered through Whatman #541 filter paper using vacuum suction. Filter paper with residue was placed in a 100°C forced-air oven and dried for 24 h. The filter paper plus residue was analyzed for N content by the combustion method using a Leco FP-2000 N analyzer. Estimates of DIP were calculated as follows:
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In Situ Crude Protein Degradation.
Crossbred steers (360 kg), housed in individual pens, were used to determine characteristics of in situ CP degradation of masticate samples. Steers were fed twice daily (0630 and 1800 h) a 70:30 forage:concentrate diet (DM basis) at 1.5% of BW/d. Tall fescue hay from an endophyte-infected pasture (8.6% CP, 70.0% NDF, 40.3% ADF) made up the forage component. The concentrate consisted of cracked corn, molasses, vitamins, and minerals (8.6% CP). Water was available ad libitum. Diet adaptation took place for 10 d prior to initiation of the in situ incubation. Dacron bags (10 x 20 cm; 53 ± 10-µm pore size; Ankom Co., Fairport, NY) containing 4 g of 2-mm ground sample to allow for a sample size:surface area ratio of 10 mg/cm2 were heat-sealed. Single dacron bags for each pasture composite sample were incubated in each of the four steers. Approximately 50 dacron bags were placed into a large mesh laundry bag (43 x 55 cm) along with 20 rubber stoppers (No. 8; to provide weight to ensure ventral location within the rumen). Dacron bags were either not incubated (0 h) or incubated in the rumen for 6 or 96 h. Incubated bags were inserted into the ventral rumen at 96 h and 6 h before removal. The bags were inserted just before feeding. At termination of incubation, all bags were removed simultaneously, rinsed in a 38-L bucket of ice-cold water to stop microbial activity, placed on ice, and transported to the laboratory. All bags, including 0-h bags, were rinsed using five rinse cycles (1-min agitation and 2-min spin per rinse) in a top-loading washing machine (Kenmore, heavy duty, Sears, Roebuck and Co., Chicago, IL). Zero-hour bags were rinsed separately from the ruminally incubated bags. After rinsing, all bags were placed in a 55°C forced-air drying oven and dried to a constant weight. Dried sample bags were allowed to equilibrate with atmospheric conditions before analysis for residual DM and N. A subsample from each bag was analyzed for N content by the combustion method using a Leco FP-2000 N analyzer.
At termination of ruminal incubation, after all in situ bags were removed, ruminal contents within each steer were thoroughly mixed and a 2,000-g sample was combined with 1 L of cold (4°C) 10% formaldehyde/9% NaCl mixture and frozen. Thawed ruminal content samples were blended for 1 min at high speed in a 1-L blender (Waring commercial blender 700, Dynamics Corp. of America, New Hartford, CT), and then strained through four layers of cheesecloth. Strained ruminal fluid was centrifuged at 1,000 x g for 10 min to remove protozoa and feed particles. The supernatant was then centrifuged three times at 30,000 x g for 15 min to sediment bacteria. The bacterial pellet was resuspended at each stage with distilled water. Isolated bacteria were lyophilized and ground by mortar and pestle for analysis. Bacterial N concentration averaged 8.22% for all four steers. The purine content of residual forage for ruminally incubated samples was determined by the procedures outlined by Zinn and Owens (1986) with the following modifications: 2 M HClO4 was substituted for 12 M HClO4 (Makkar and Becker, 1999), and the precipitating solution was used to wash the sedimented pellet (Obispo and Dehority, 1999). Both modifications were incorporated because results in our laboratory (unpublished) indicated slightly greater recovery of purines with these modifications. All in situ N fractions were reported on a purine-corrected basis.
The total N pool was partitioned into three fractions (A, B, and C) based on relative susceptibility to ruminal degradation. The A fraction represented the immediately soluble N, the B fraction represented N degraded at a measurable rate, and the C fraction represented ruminally undegradable N. The A and C fractions were determined experimentally. The A fraction equaled the N washed out of the 0-h bags during the washing machine rinses, and the C fraction equaled the residual N in the 96-h bags. All other N was considered to represent the "B" fraction. The B fraction degradation rate was calculated from 0- and 6-h disappearance values using the "double-point estimation" approach described by Vanzant et al. (1996). Crude protein degradability (DIP) was calculated according to Ørskov and McDonald (1979):
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where kd = rate of degradation of the B fraction and kp = rate of passage from the rumen, which was assumed to be 4.0%/h for all forages in this study.
Statistical Analysis.
Data were analyzed using a model for repeated measures within a split-plot design with a completely randomized whole-plot by the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). Forage type, sampling method, and month were included as fixed effects and pasture (forage type) as a random effect. Forage type was considered the whole plot and sampling method the subplot. Errors of repeated measures were modeled using an autoregressive correlation structure. Orthogonal polynomial contrasts were used to identify linear, quadratic, and cubic time effects. When significant time x treatment interactions existed, the SLICE option of the LSMEANS statement was used to identify significant treatment effects within months and contrasts were used to identify time effects within individual treatments.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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). However, differences due to forage type were only detected (P 
0.05) for April and June. At the start of the season, rapid forage growth occurred. However, from May to October a steady decline in biomass occurred, resulting in a cubic response (P < 0.01) across the entire grazing season.
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). However, no differences due to forage type (P > 0.10) were detected in the proportion of any of the other measured botanical components. The major seasonal effect detected was an increase in the proportion of the biomass comprised of senescent material through July, followed by a decrease through October (quadratic, P < 0.01). Apparently because of the relatively small influence on botanical composition of the pastures, very little difference was observed in chemical composition of pastures with or without interseeded red clover. Forage type x sampling method x month interactions were not detected (P > 0.10) for any of the chemical constituents evaluated (data not shown). Forage type x month interactions were detected (P < 0.10) for CP concentration and CP degradability (estimated with the SGP procedure). However, the magnitude of differences between forage types was small. For CP, differences were only detected (P < 0.10) for samples collected during the months of April, August, and September (data not shown), when the fescue/red clover pastures had 1.0 percentage unit less, 1.5 percentage units less, and 1.0 percentage unit more CP than the fescue pastures. For CP degradability, differences (P < 0.10) were only detected for September and October samples (data not shown), when the maximal difference in CP degradability was a 2 percentage units greater DIP value for samples from the fescue/red clover pastures. Except for DIP estimates, the forage type x sampling method interaction was nonsignificant and the main effect of forage type was nonsignificant for any of the forage quality variables. Because of the minimal influence of forage type on measurements of forage quality, data summarized in Figures 3 to 8 were averaged across forage type and emphasis was placed on differences due to season and sampling methods.
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). Lesperance et al. (1974) suggested that the increase in ash content from salivary contamination can range from 1 to 4% in masticate samples. In order to separate effects due to salivary contamination from effects of selective grazing, Barth and Kazzal (1971) fed samples of clipped tall fescue to steers and harvested these through an esophageal fistula. In their study, samples that had been exposed to saliva had 4.6 percentage units more ash than hand-clipped samples. No difference in ash content was detected in masticate samples by allowing steers to collect samples while grazing vs. feeding hay. Thus, the difference of about 5.0 percentage units OM between clipped and masticate samples in our study was attributed to salivary ash contamination. To account for this difference, all other constituents have been expressed on an OM basis.
On average, masticate samples had 4.5 percentage units higher CP concentrations from April to September compared with clipped samples and both responded cubically (P < 0.01) across time with maximal values in April and minimal values in June (Figure 4). No difference (P > 0.10) was seen between clipped and masticate CP concentrations during October. Our April data correspond with values reported by Elizalde et al. (1998; 1999) from esophageal masticates collected from tall fescue pastures during mid-April. In our study, a decline was seen in CP concentration during May and June, followed by a relatively constant CP concentration from July to October. Crude protein concentrations of the clipped samples agree with values reported for tall fescue by Smith (1977) for May (12.8%), Fritz and Collins (1991) for June and July (10%), and Daura and Reid (1991) for October (16.1%). Various researchers have demonstrated that salivary contamination has very little influence on the CP concentration of a wide variety of consumed forages (Harris et al., 1967; Galt and Theurer, 1976; Hart, 1983). However, diets selected by grazing cattle have been reported to contain greater protein concentrations than clipped forage, particularly when little or no effort was made to simulate the grazing behavior of cattle when clipping, as in the present study (Shumway et al., 1963; Theurer et al., 1969). With low- to moderate-CP forages (clipped samples range from 3.6 to 9.2% CP), Shumway et al. (1963) reported an average difference of 5.4% CP (DM basis) between clipped and masticate samples, in close agreement with our results. Weir and Torell (1959) also reported that seasonal forage samples obtained from esophageally fistulated sheep contained 4.1 percentage units more protein compared with hand-clipped samples. Similar results were shown by McCracken et al. (1993) with steers. The lack of difference between clipped and masticate samples during October was likely related to low forage availability, which could have inhibited selectivity.
On average, clipped samples contained 5.5 percentage units greater (P < 0.01) NDF concentrations from April to September than masticate samples whereas no difference (P > 0.10) existed in October (Figure 5). This difference corresponds to that of Papachristou and Nastis (1994), who obtained a 4.2 percentage unit difference in NDF concentration between hand-clipped and esophageal masticate samples from Mediterranean shrubland. Clipped samples responded quadratically (P < 0.01) and masticate samples responded cubically (P < 0.01) across time. Clipped sample NDF results in our study are supported by data reported by Fritz and Collins (1991; 66.2%) and Forcherio et al. (1995; 72.0%) for May, and Rayburn et al. (1980; 71.8%) and Turner et al. (1996) for September (71.8% NDF). Masticate sample NDF concentrations are similar to values reported by Elizalde et al. (1998; 60.4%) for April, Martz et al. (1999; 67.3%) and Elizalde et al. (1998; 71.2%) for May, and Hess et al. (1996; 74.0%) for June.
ADF concentrations followed the same general trends seen with NDF concentrations, with clipped samples containing on average 3.0 percentage units higher (P < 0.01) ADF concentrations from April to September compared with masticate samples (Figure 6). As with NDF, clipped samples responded quadratically (P < 0.01), while masticate samples responded cubically (P < 0.01) across the season. As seen with CP, October clipped and masticate NDF and ADF values were not different (P > 0.10). Both clipped and masticate samples contained the lowest NDF and ADF concentrations during vegetative growth (April, September, and October). Neutral detergent fiber and ADF concentrations reached a maximum during June, July, and August, coinciding with a peak in the proportion of mature, senescent forage in the pastures.
In Vitro CP Degradation.
Forage type and sample collection method interacted (P < 0.01) for SGP CP degradation estimates, but differences due to forage type were minimal when compared to differences seen between sample collection methods. For fescue pastures, DIP estimates averaged 59.4 and 71.2% of CP, whereas for fescue/red clover pastures, DIP estimates averaged 61.4 and 70.6% of CP for clipped and masticate samples, respectively. Thus, as with other variables, emphasis has been placed on effects of collection method and season. Crude protein degradability of masticate samples responded cubically (P < 0.01) across the season, and were, on average, 10.5 percentage units greater (P < 0.01) across the grazing season than with hand-clipped samples, which changed quadratically (P < 0.01) across the season (Figure 7). Degradable intake protein concentrations for masticate samples were between 72.4 and 74.6% of CP during April and October, when vegetative growth was occurring, and declined to 65.0% in June when the forage was at maturity. Similar trends were seen with hand-clipped samples, in that the highest DIP (percentage of CP) values were seen during vegetative growth (April, May and October), and a decline in DIP (percentage of CP) was seen in June. However, hand-clipped sample DIP (percentage of CP) concentrations tended to decline until August. Unlike the other constituents, where differences between clipped and masticate samples were relatively consistent across months, differences in S. griseus DIP varied from 4 percentage units in May to 20 percentage units in August. Thus, although it may be reasonable to apply standard correction values to clipped samples for estimating CP and fiber concentrations of selected diets, it may be difficult to make similar extrapolations for CP degradability. On average, across the entire grazing season, 60% of the total CP in clipped samples and 70% of the total CP in masticate samples were estimated to be ruminally degradable. Estimates for the clipped sample DIP (percentage of CP) are lower than values obtained by Janicki and Stallings (1988) with orchardgrass hays (71.5%) and Abdalla et al. (1988) with tall fescue (78.5%). However, the concentration of the SGP solution used by Abdalla et al. (1988) was not reported. Our clipped sample DIP values were comparable to those reported by Coblentz et al. (1999) in vegetative, boot, and mature switchgrass (65.9, 60.4, and 58.6%, respectively). Larger average differences were seen between clipped and masticate DIP values compared with CP, NDF, or ADF values. This could be related to differences in sample processing. The clipped samples were oven-dried, whereas masticate samples were lyophilized. Although oven-drying would be expected to slightly increase concentrations of fibrous components, it would be expected to have a greater effect on protein susceptibility to enzyme breakdown.
Comparison of In Situ vs In Vitro Techniques.
Comparison of in situ and SGP procedures are shown in Figure 8. A technique x month interaction existed (P < 0.01) in which no differences (P > 0.10) were seen between the two procedures early in the season (April to June), whereas from July to October the SGP procedure gave larger (P < 0.01) estimates of DIP than the in situ procedure. These results are supported by those of Coblentz et al. (1999), who used similar procedures to estimate the DIP concentration of 18 different forages and found that the SGP procedure did an acceptable job of estimating DIP of most grasses harvested at boot stage, but inconsistent results were found when using the SGP procedure to estimate DIP of vegetative and mature grasses. Mathis et al. (2001), in a collaborative study involving seven institutions, showed a high correlation (R2 = 0.80) between the mean DIP values from 48-h SGP-incubated (0.33 activity units/mL) and in situ evaluation of five forage hays (alfalfa, bermudagrass, brome, forage sorghum, and prairie hay). In the present study, even though significant differences were seen late in the season, the SGP procedure did mimic the trends of the in situ procedure. The overall average difference between the two procedures across the entire grazing season was only 3.0 percentage units DIP (percentage of CP). Procedural differences with respect to incubation time, maintenance of ruminally fistulated animals, and applicability to commercial laboratories favor the SGP over the in situ procedure.
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Footnotes |
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2 Current address: 5639 Evergreen Farms Ln., Greenback, TN 37742.
4 Current address: P.O. Box 342, Buckeystown, MD 21717.
5 Current address: 203 Animal Sciences Bldg., Stillwater, OK 74078.
6 Current address: 154 Kinkaid Dr., Lafayette, IN 47909.
Received for publication June 27, 2002. Accepted for publication December 3, 2002.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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