Time of Daily Supplementation for Steers Grazing Dormant Intermediate Wheatgrass Pasture1t2 R. K. Bartons, L. J. Krysl4, M. B. Judkins, D. W. Holcombe, J. T. Broesdefl, S. A. Guntefl, and S. W. Beam7

School of Veterinary Medicine, University of Nevada, Reno 89557-0104

> .15) forage OM intake; however, total OM intake was greater (P = .01) for supplemented steers (22.3 g/kg of B W than for CON (18.4 g/kg of BW) steers. Supplementation did not affect (P > .151 digesta kinetics. Extent of in situ NDF (96 h) and rate (%/h)of disappearance for supplemented steers was greater (P = .01) than for CON steers. Across all periods, ruminal NH3 N and total VFA concentrations were lower (P = .01)for CON steers than for supplemented steers. Serum insulin (ng/ mL) concentration was lower (P = .03)and concentration of serum growth hormone (ng/mL) was higher ( P = .O2) for CON steers than for supplemented steers. Cottonseed meal supplementation enhanced utilization of intermediate wheatgrass; however, supplementation time had minimal effects on the variables measured. (P

Key Words: Cattle, Ruminal Digestion, Protein Supplements

J. Anim. Sci. 1992. 70:547-558

Introduction Body weight gains, forage intake, and digestibility often increase as a result of feeding small amounts of protein-concentrated meals to cattle

IResearch funded under Hatch Project 315, Univ. of Nevada

Exp. Sta., Reno. 'Appreciation is expressed to the Natl. Hormone and Pituitary Program for providing anti-ovine GH WIADDK anti oGH-2; AFP-CO123080) and biological CNIADDK oGH-12; AFP4015A) and iodination (NIADDK oHG-1-3; U P - 5 2 8 5 0 grade ovine GH used to quantify serum GH. Appreciation is expressed to LiUy Res. Lab. for suppling ovine insulin (615112B108-Dused in the assay. We also thank G. Stabenfelt, Dept. of %prod., Univ. of California, Davis for providing reagents for cortisol amlyses. 3Eastern Oregon Agric. Res. Center, Burns. 4To whom reprint requests should be addressed. 5Montana Coop. Ed., Toole Co., Shelby. 6Anim. Sci. Dept., Oklahoma State Univ., Stillwater. 'Dept. of Anim. Sci., Cornell Univ., Ithaca, NY. Received April 4, 1991. Accepted September 5, 1991.

547

fed low-quality roughages (Lusby and Horn, 1983; Caton et al., 1988; Guthrie and Wagner, 1988). Behavioral CAdams, 1985) and ruminal effects (Hunt et al., 1989; Judkins et al., 1991) of frequency and daily delivery time of protein supplementation have been evaluated with a limited number of forage types. Adams (1985) suggested that energy supplementation during intensive grazing periods interrupted grazing time, thereby reducing forage intake and animal performance. Further, supplementation during intensive grazing bouts may increase ruminal NH3 N concentration at a time when adequate NH3 N concentrations are already present as a result of degradation of forage protein N. Effects of feeding supplemental protein on metabolic hormones have not been investigated extensively; however, this information is paramount to understanding effects of supplementation on grazing animals. The objective of our study was to evaluate the effects of morning LAM;0600) vs afternoon (PM; 1200) cottonseed meal (CSM) supplementation on

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ABSTRACT: To compare the effects of time of daily protein supplementation on grazing behavior, forage intake, digesta kinetics, ruminal fermentation, and serum hormones and metabolites, 12 ruminally cannulated Holstein steers (449 and 378 kg average initial and final BW, respectively) were allotted to three groups. Treatments consisted of CON = no supplement, AM = cottonseed meal (.25% of B W at 0600, and PM = cottonseed meal (.25% of BW) at 1200. Steers grazed a dormant (1.1% Nl intermediate wheatgrass (Thinopyrum intermedium Host) pasture. Sampling trials occurred in December, January, and February. Supplementation altered (P = .01) time spent grazing; CON steers grazed approximately 1.5 h longer than supplemented steers. Supplemented steers lost less Lp = .O2) BW (-40 kg) than CON steers (-75 kg) did. Supplementation did not alter

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BARTON ET AL.

several facets of the response to supplemental protein in cattle grazing dormant intermediate wheatgrass. Variables evaluated included grazing behavior, forage intake, digestibility, ruminal fermentation, digesta kinetics, and serum metabolic hormones and metabolites.

Experimental Procedures

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Study Area. Vegetation in the 16-hastudy pasture was a monoculture of intermediate wheatgrass (Thinopyncm intennedium Host); the pasture did not receive irrigation. Between November 1, 1988 and February 12, 1989, approximately 71.1 c m of precipitation was recorded in the form of snow. Average maximum and minimum ambient temperatures recorded during the study were 6.2 and -7.6"C, respectively. Sample CoZZection Periods. Three 16-d collection periods were conducted following an initial 25-d adaptation period to protein supplement and pasture. The first collection began on December 6 (DEQ, the second on January 3 WAN), and the third on January 28 (FEB). Twelve nupinally cannulated Holstein steers (449 and 378 kg average initial and final BW, respectively; four per treatment) received either no supplement (CONI or supplement at 0600 (AMI or 1200 (PM) daily throughout the study. Steers were supplemented a t .25% BW (as-fed basis1 with CSM (meal form, 7.55%available N [total N minus ADINI, OM basis) and allowed free access to water and trace mineral salt Diamond trace mineral salt; Diamond Crystal Salt, St. Clair, MI; NaCl, 97 to 99%; Zn, .85%;Mn, .22%;Fe, .21%;Mg, .lo%;Cu, .30%;I, .01%,and Co, .006%).All surgical procedures were approved by the University Animal Care Committee, and animal care followed procedures outlined in the Consortium (1988) publication. Steers were weighed at the beginning and end of each collection period. Supplement was given via ruminal cannula and supplementation times were chosen to reflect times when steers typically were not grazing intensively, as determined by preliminary behavioral observations made during the adaptation period. On d 1 and 16 of DEC and JAN and d 1 of FEB (16.5 cm snowfall on d 16 in FEB caused termination of study) behavioral data were collected. Observations were recorded for 24 h at 15-min intervals beginning at 0600 (Gary et al., 1970). Activity (grazing, standing, walking, lying, and consuming water/saltl of each steer was observed from a n established blind. At 0700 on d 2 of each sampling trial, masticate samples were collected by the ruminal evacuation

technique (Lesperance et al., 1960); steers were not fasted before collections. Specifically, the same two steers from each treatment in each period were completely emptied of their reticulo-ruminal contents, walls of the m e n were washed, and steers were allowed to graze for approximately 1 h, after which newly grazed ruminal contents were removed and initial ruminal contents were replaced. Masticate from each steer was allowed to drain through a @mesh screen to remove salivary contaminants, individually mixed, and divided into three subsamples. The first subsample from each steer as lyophilized (Model 600 SL, Virtus Freeze Drier, Virtis, Gardner, NY)and used for dietary quality analysis. The second subsample was composited within treatment, lyophilized, and used as substrate for in vitro and in situ disappearance measurements. The remaining masticate subsample was composited across all steers, rinsed with tap water, followed by distilled water, and labeled with Yb (Teeter et al., 1984) for use as a particular-phase marker. At 0600 on d 6 of each collection period, a measured dose of Yb-labeled masticate was stratified from the ventral to the dorsal part of the rumen of each steer. Steers received approximately 115 g of DM (1.5 g of Ybl. Fecal samples (rectal grab) were collected 0, 6, 12, 18, 24, 30, 33, 36, 42, 48, 54, 60, 72, 84, 96, 108, and 120 h after dosing. Beginning at 0600 on d 11, approximately 3 g of lyophilized ground masticate Wiley mill, 2-mm screen) was placed in polyester bags (9 cm x 16 cm; pore size 27 jun x 47 CLm) and suspended (in duplicate) in the rumen of each steer for 0, 3, 6, 12, 24, 36, 48, and 96 h. One empty bag was included with each set of duplicate bags. After removal, bags were rinsed in cold tap water until the effluent remained clear and subsequently dried in a forced-air oven at 60°C for 48 h, followed by 100°C for 24 h, and weighed. Ruminal sampling also began on d 11. At 0600 (0 hl, 250 mL of whole ruminal contents was removed from each steer and the pH was measured immediately with a combination electrode. Ruminal fluid (100 mL) was strained through four layers of cheesecloth, acidified with 1 mL of 7.2 N H2S04/ 100 mL of fluid, and frozen (-4OOC). Subsequently, ruminal samples were taken at 1, 3, 6, 7, 9, 12, 15, 18, and 21 h and processed in the same manner as the 0-h sample. On d 13, beginning at 0600, 500 mL of ruminal fluid was collected from each steer, strained through four layers of cheesecloth, and used within 1 h to determine in vitro OM disappearance (IVOMD; Judkins et al., 1990). Filtered in vitro residues were ashed and OM disappearance was calculated.

SUPPLEMENTATION REGIMEN ON WHEATGRASS PASTURE

Calculations and Statistical Analyses. Fecal Yb extraction curves were fitted to a one-compartment model Bond et al., 1988) using the nonlinear regression option of SAS (1988). Particulate passage rate, retention times, and fecal output were estimated using parameter estimates from the onecompartment model (Krysl et al., 1988). Intake was estimated from fecal output and in vitro OM indigestibility values obtained from the two-stage in vitro fermentation technique. Rate of NDF disappearance was calculated as described by Mertens and Loften (1980) using SAS (1988) nonlinear regression procedures. The model included a coefficient for lag time, but lag time was not included in the statistical analyses. Behavioral (d 1 and 16 were averaged for DEC and JAN; no day x treatment interaction was detected, P > .lo), forage chemical analyses, intake, digesta kinetics, and digestibility data were analyzed as a split-plot design with supplementation time as the main-plot treatment and collection period as the subplot treatment (Snedecor and Cochran, 1980). Treatment @upplementation time) was tested against steer within treatment as the error term (error a);the interaction of collection period x treatment and the main effect of collection period were tested against residual error using the GLM procedure of SAS (1988). Time-sequence data (ruminal pH, NH3 N, VFA, and serum constituents) were analyzed as a split-split-plot design with sampling time and the interaction of sampling time x treatment added to the model (sub-subplot).When a significant F-test was detected, orthogonal contrasts were used to evaluate treatment differences, and sampling period differences were evaluated for linear and quadratic effects.

Results and Discussion Behavior. Collection period x treatment interactions (P > .lo) were not detected for any of the behavioral variables. Supplementation reduced (P = .01) time spent grazing; CON (490 min) steers grazed approximately 1.5 h longer than supplemented (405 min) steers. The two supplement treatments did not differ (P > .15, Table 1) from each other. Similarly, Yelich et al. (1989) reported that cows supplemented with alfalfa hay (3.18 kg/ d) in either the morning (0800) or afternoon (1600) spent less time (.7 and 1.4 h, respectively; P < .05) grazing than did unsupplemented cows. Yelich et al. (1989) supplemented approximately one-third of daily DMI, which is much greater than in our study. As winter progressed during our study, time spent grazing increased (linear, P = .01) from DEC to FEB (54 min). Daily grazing time by CON steers

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On d 14, a t 0600 (0 h), blood samples (approximately 12 mLl were collected from each steer via indwelling jugular catheters that had been inserted 24 h earlier. Subsequently, samples were collected at 1, 2, 3, 6, 7, 8, 9, 12, 15, 18, 21, and 24 h. Blood samples were allowed to clot, centrifuged at 2,300 x g for 15 min at 4OC, and stored at -2OOC. Laboratory Analyses. Ruminal masticate samples for nutrient analyses were ground to pass a 2-mm screen in a Wiley mill. Samples were analyzed for DM, ash, and Kjeldahl N (AOAC, 1984). Neutral detergent fiber, ADF, and ADL were analyzed by nonsequential methods of Goering and Van Soest (1970). The ADIN fraction was determined by Kjeldahl N analysis of ADF residue (Goering and Van Soest, 1970). Residue remaining in polyester bags after in situ digestion was analyzed for DM and NDF. Fecal samples collected for passage rate estimates were dried in a forced-air oven at 6OoCuntil dry, ground in a Wiley mill to pass a 2 - m mscreen, and analyzed for DM and Yb. Ytterbium was extracted using .1 A4 EDTA (Karimi et al., 1986) containing 1 g of KCl/L as an ionization buffer. Ytterbium concentration was measured by atomic absorption spectrometry with a nitrous oxide-plusacetylene flame. A composite fecal sample (24, 48, 72, 96, and 120 h) from each steer at each sample date was analyzed for DM and ash, so that fecal output estimates could be corrected for ash content. Ruminal samples were thawed a t room temperature and centrifuged at 10,000 x g for 10 min. Supernatant fluid was analyzed for NH3 N by the phenol-hypochlorite procedure of Broderick and Kang (1980). After addition of 2-ethylbutyric acid as a n internal standard, fluid was recentrifuged for 10 min at 10,000 x g and VFA concentrations were analyzed by gas chromatography (Goetsch and Galyean, 1983). Serum insulin (Sanson and Hallford, 1984) and growth hormone (GH; Hoefler and Hallford, 1987) were measured using a validated double antibody RIA procedure. Serum cortisol was determined using an ELISA technique (Munro and Stabenfeldt, 1985). Serum thyroxine was determined by use of an ELISA kit (ENDAB-T4 Kit No. 115, Immunotech, Boston, MA). Serum nonesterified fatty acids (NEFA) were determined colorimetrically (NEFA-C,Wako Chemicals, Dallas, TX) using a method described by McCutchen and Bauman (1986). Serum glucose and urea N (SUN) were determined colorimetrically using o-toluidine (Direct [o-toluidine1Glucose Test Set, Stanbio Lab., San Antonio, TX) and diacetylmonoxime (Direct [diacetymonoximel Urea Test Set, Stanbio Lab.) assay procedures, respectively.

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BARTON ET AL.

Table 1. Influence of cottonseed meal supplementation time on behavioral variables (MINI for steers grazing dormant intermediate wheatgrass pasturea Treatmentb Behavior

CON

Grazing Standing

490 320 590 25 15

Lying wallring Watering/salting

AM 401 302 678 34 25

ContrastC PM

SET

410 329 861 22 18

20 18 23 2 2

Periodd

Contraste

1

2

DEC

JAN

FEB

SEg

Linear

Quadratic

.01

NS NS NS

399 296 673 54 18

450 364 505 16 15

453 290 661 11 25

12 23 25 6 2

.o 1 NS NS

.ll .01 .01 .02 .03

NS .02

NS .08

.15 .09

.01 .08

fn

= 20.

gPooled SE;DEC and JAN

(n =

241, FEB Cn

E

12).

walking decreased linearly (P = . O l l with advanc-

in our study was greater than that reported in other winter grazing studies on Montana shortgrass prairie (7.2h, Adams et al., 1986) or Kansas tallgrass prairie (7 h, DelCurto et al., 1990). However, grazing time in o w study was less than values reported for winter grazing on Colorado shortgrass prairie (10.1 h, Yelich et al., 1989) or Utah crested wheatgrass-salt desert shrub (9.5 h, Malechek and Smith, 1976). Supplementation time did not alter (P > .15) time spent standing or walking (Table 11; however, supplemented steers spent more (P = .O2l time lying than CON steers did. In addition, supplemented steers spent more Lp = .08l time at water/ salt than did CON steers. As winter progressed, time spent standing was greatest in J A N and least in DEC and FEB (quadratic; P = .01). Correspondingly, lying was greatest in DEC and FEB and least in JAN (quadratic; P = .01). Time spent

ing season (Table 1).

Nutrient Composition. Supplementation time x collection period interactions (P > .lo) were not detected for any masticate variable except IVOMD. Supplementation did not affect (P > .15) masticate OM, NDF, ADF, or ADL content; however, dietary ADF (P = .03) and ADL (P = .04) differed with supplementation time (Table 2). In addition, masticate ADL decreased linearly (P = .01) with advancing season. Total masticate N was not altered (P > .15) by supplementation; however, available masticate N was greater (P = .03)in supplemented steers than in CON steers. In contrast, Judkins et al. (19851 and Caton et al. (1988) reported that protein supplementation did not alter quality of diet selected by steers grazing dormant blue grama rangeland. Total masticate N and available N decreased linearly (P = .01l with

Table 2. Influence of cottonseed meal supplementation time on nutrient composition of forage selected by steers grazing dormant intermediate wheatgrass pasturea Treatmentb Item

CON

AM

OM, I

83.8

82.7 0.6

Total N ADIN Available N NDF ADF ADL

1.0 .2 .8 86.9 63.8 7.9

1.2 .2 1 .o 87.0 62.9 8.2

Contrast'

Periodd

Contraste

PM

SEf

1

2

DEC

JAN

FEB

84.3

A9

NS

NS

82.5

85.9

82.5

.03 .01 .02 .4 .7

NS

NS

NS

1.5 .3 1.2 87.4 62.6 8.7

of OM 1.1 .2 .9 86.8 84.2 7.8

S E ~ Linear .7

Quadratic

.01

.01

9'0 of OM

.5

.03

NS NS

NS

NS

NS NS

.03 .04

.e .2 .7 88.8 63.2 8.2

.9

.2 .7 86.4 65.4 7.1

.06

.01

.02

.01 .04

NS

NS

.01

.4

NS NS .o 1

.02 .01

.8 .5

aNo treatment x period interaction (P > .lo1 was detected. kheatment: CON = no supplement, AM = supplemented at 0600, PM = supplemented at 1200. 'Observed significance level for contrasts: 1 = CON vs AM + PM, 2 = AM vs PM. NS = P > .15. dPeriod: DEC = December 6 to 21, 1988; JAN = January 3 to 19, 1989; FEB = January 28 to February 13, 1989. 'Observed significance level for linear and quadratic effects of period. NS = P > .15. fn = 12.

NS NS

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aNo treatment x period interaction (P ,101was detected. hreatment: CON = no supplement, AM = supplemented at 0800,PM = supplemented at 1200. 'Observed significance level for contrasts: 1 = CON vs AM + PM, 2 = AM vs PM. NS = P > .E. dPeriod: DEC = December 6 to 21, 1988; JAN = January 3 to 19, 1989; FEB = January 28 to February 13, 1989. eObserved significance level for linear and quadratic effects of period. NS = P > .15.

SUPPLEMENTATION REGIMEN ON WHEATGRASS PASTURE

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Supplementation time did not affect (P > .15) particulate passage rate (PPR; %/hl, gastrointestinal fill (GIF;g/kg of B W , gastrointestinal mean retention time (GMRT h),or intestinal transit time (ITT, Table 3). In J A N , PPR was slower (quadratic, P = .01) and GMRT was longer (linear, P = .01; quadratic, P = .01) than in DEC or FEB (Table 3). In addition, ITT(linear, P = .01; quadratic, P = .01) and GIF h e m , P = .02) increased with advancing season. Similarly, Judkins et al. (1987)and Hunt et al. (1989) concluded that protein supplementation did not affect PPR for cattle grazing blue grama or fed meadow fescue hay, respectively. In contrast, increased PPR and reduced GMRT over unsupplemented controls have been reported in beef steers supplemented with CSM when fed low-quality prairie hay (McCollum and Galyean, 1985) or grazing blue grama rangeland (Caton et al.,1988). Supplemented steers grazed approximately 1.5 h less than unsupplemented steers; however, forage intake, PRR, GMRT, and GIF were not altered by supplementation (P > .15). Our study only investigated time spent grazing, not bite size or biting rate. However, we recorded grazing behavior in a manner that allowed separation of intense from casual grazing. Evaluation of percentage of time spent grazing intensely as a percentage of the total grazing time indicates that supplemented steers spent a greater percentage of total time grazing intensely (93%; P = .15); CON steers spent 88% of the total grazing time foraging intensely. If one combines information from Tables 1 and 3 and calculates harvesting efficiency (g of forage intake.kg of BW-l.min of grazing1), it is apparent that supplemented (.050) steers were more efficient grazers (approximately 60%; P = .05) than CON t.031) steers. Given the energetic cost of grazing (per unit time), this may explain, in part, the improvement in BW noted for supplemented steers. Although more detailed study is needed to evaluate possible grazing mechanisms involved, our data indicate that steers altered their grazing behavior in response to nutritional and(or1 environment a1 factors , In situ NDF disappearance of lyophilized ruminal masticate at different incubation times (3 h through 48 h) was not altered ( P > .15) by supplementation; however, in situ extent of NDF digestion (96 hl for supplemented steers was greater (P = .01) than for CON steers (Table 4). In addition, supplementation increased (P = .011 rate (%Ad of NDF digestion over CON (Table 4). Neutral detergent fiber disappearance increased (linear, P = . O l l with advancing plant maturity at 3-, 6-, and 12-h incubation times; however, no differences (P > .15) in NDF disappearance were observed at 24- and 36-hincubation times. Finally,

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advancing season. Cook (1972) reported that grasses may lose up to 75% of their N during winter dormancy compared with summer. Low protein values (5.9% CP, 4.4% available CP) observed during J A N and FEB indicate that protein supplementation was warranted based on NRC (1984) equations for protein requirements. Masticate IVOMD was affected (P < .05) by a time of supplementation x sampling period interaction. In DEC, neither supplementation nor time of supplementation altered (P > .15) IVOMD (CON = 39%, A M = 40%, PM = 38%; SE = 1.1). However, during JAN, IVOMD was greater ( P = .01) for supplemented steers than for CON steers (CON = 32%,AM = 41%, PM = 37%; SE = .7). In addition, AM-supplemented steers had greater (P = .011 IVOMD than PM-supplemented steers did. During FEB, supplementation did not alter (P > .151 IVOMD; however, time of supplementation altered (P = .Oll IVOMD (CON = 41%, AM = 39%, P M 44%; SE = .7). Intake and Digestu Kinetics. Collection period x treatment interactions were not observed (P > .lo) for BW, intake estimates, or digesta kinetics. Supplemented steers lost less (P = .02) BW (-40 kg) than CON steers (-75 kg) did during the study (Table 3); all steers lost weight (linear, P = .01; quadratic, P = .01) during the course of the study. Supplementation time did not alter (P > .15) forage OM intake (FOMI, Table 3); however, total OM intake (TOMI) was greater (P = .01) by supplemented steers than by CON steers, reflecting addition of CSM. Likewise, Judkins et al. (1991) found no effect of protein supplementation time on forage intake by Holstein steers consuming a fescue hay (.98% NI diet. Forage OM1 and TOM1 estimates responded quadratically CP = .011 to advancing season. Intake responses to supplementation may differ as a result of differences in forage N content. Rittenhouse et al. (1970), Kartchner (19801, and Judkins et al. (1985) reported that protein supplementation has no effect on forage intake of cattle grazing moderate-quality t.96 to 1.28%N)blue grama rangeland. Similarly, Krysl et al. (1989) noted no alterations in FOMI in proteinsupplemented steers grazing blue grama rangeland; however, forage N content was 1.8%. In contrast, Caton et al. (1988) found that FOMI tended ( P = .121 to be greater in protein-supplemented steers grazing blue grama rangeland (1.31%N).Positive responses to protein supplementation have been reported for subtropical grasses (Hennessy et al., 1983; Lee et al., 19871, bluestem forage (DelCurto et al., 1990; Sanson et al., 19901, Russian wildrye (Adams, 19851, and meadow fescue hay (Hunt et al., 1989) with N levels similar to those noted in our study.

SUPPLEMENTATION REGIMEN ON WHEATGRASS PASTURE

tion. The greater NH3 N concentration detected after supplementation in AM steers followed a pattern similar to that reported by several researchers (FJritchard and Males, 1982, 1985; Judkins et al., 1991) for morning supplementation of cattle consuming low- to moderate-quality forages. The PM steers did not exhibit as high an NH3 N concentration peak, or maintain an elevated concentration for as long, as AM steers did. This may reflect increased protein escape for PM steers associated with either water intake or decreased ruminal proteolysis. Alternatively, ruminal microbes in PM steers may have used NH3 N at a rate similar to release. With the influx of forage during the morning grazing bout, cellulolytic bacterial populations may have been growing and sequestering NH3 N. Lack of a prominent peak in the PM group may further suggest that afternoon supplementation could create a more stable ruminal environment, elevating ruminal NH3 N concentrations without the potential for excessive N waste. Similar ruminal NH3 N curves were reported by Judkins et al. (1991).Leng (1990)reported that even though fiber digestion was maximized by ruminal concentrations of 5 mg/dL, forage intake was not increased until ruminal NH3 N concentrations reached 20 mg/dL, which may help explain the lack of a forage intake response observed in our study. In all periods, total ruminal VFA concentrations in CON steers were lower (P = .01) than in supplemented steers, which did not differ from each other (P > .15; Table 5). These results agree with those of Hunt et al. (1989) and DelCurto et al. (1990), who found that VFA concentration was enhanced by protein supplementation. Likewise, Judkins et al. (1991) reported increased VFA concentration in steers consuming fescue hay and receiving CSM in a n early- or mid-morning supplementation regimen however, no improvement was observed with PM supplementation. In contrast, McCollum and Galyean (19851, Caton et al. (1988) and Krysl et al. (1989) found no increase in VFA concentration with protein supplementation of either cattle fed prairie hay or grazing dormant blue grama rangeland. Within treatment, VFA concentrations for AM, PM, and CON steers declined W e a r , P = .011 with advancing season; this decline was most pronounced for CON steers. Lower VFA concentrations may further indicate a suboptimal ruminal environment in CON steers. Ruminal molar proportions of acetate and propionate (Table 51, within sampling period, were not altered (P > .15) by supplementation. Similar results were reported by Krysl et al. (19891, who found that soybean meal supplementation either lowered or had no effect on acetate proportions in

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NDF disappearance increased with season at 48-h (linear, P = .02; quadratic, P = .03) and 96-h (linear, P = .05; quadratic, P = .01; Table 4) incubation times. Rate of NDF digestion was not altered (P > .15) by advancing plant maturity. Caton et al. (1988) reported increased (P < .05) NDF disappearance after 4, 8, 12, 18, and 36 h of incubation as a result of protein supplementation; however, no differences in extent (72 h) or rate of digestion were detected in CSM-supplemented steers grazing blue grama rangeland. Rumina2 Fermentation. Ruminal pH, NH3 N, and VFA concentrations exhibited a collection period x treatment interaction (P < .05; Table 5). In addition, a sampling time x supplementation interaction (P < .05) was observed for ruminal NH3 N and butyrate concentration (data not shown); however, the nature of this interaction did not preclude evaluation of main effects. Supplementation time did not affect (P > .15) ruminal pH during DEC and JAN sampling periods; however, FEB ruminal pH was greater (P = .01) for CON steers than for the two groups of supplemented steers, which did not differ (P > .15) from each other. Within treatments, ruminal pH increased (linear, P = .01) with advancing season. DelCurto et al. (1990) reported a similar pH response to various levels and types of protein supplementation with lowquality tallgrass prairie hay diets. In contrast, results from other studies have shown that CSM supplementation of low- to moderate-quality forages does not alter ruminal pH (McCollum and Galyean, 1985; Caton et al., 1988; Hunt et al., 19891. In all periods, ruminal NH3 N concentrations in CON steers were lower CP = .01) than in supplemented steers. Between the two supplement treatments, no differences (P > .15) were noted in any period except FEB, when AM steers had greater (P = .03) ruminal NH3 N concentrations than PM steers did (Table 5). Within treatment, CON steers exhibited a decrease Llinear, P = .Ol) in NH3 N concentrations with advancing season (DEC = 4.2, JAN = 3.1, FEB = .8 mg/dW. A quadratic (P = .01) response was observed across sampling dates with PM supplementation (DEC = 8.7,J A N = 10.3, FEB = 5.8 mg/dL); however, NH3 N concentrations were not altered by season (P > .15) with AM supplementation (DEC = 9.3,JAN = 9.6,FEB = 8.8 mg/dW. At 3 h after AM supplementation, a ruminal NH3 N concentration peak (13.8 mg/dW was noted in AM steers; this value declined to presupplementation levels (P > .15) by 15 h after supplementation. The PM steers exhibited a lower NH3 N concentration peak (10.0 mg/m at 1 h after supplementation, which declined to presupplementation levels Lp > .15) by 9 h after supplementa-

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steers grazing blue grama rangeland. Our results are different from those of McCollum and Galyean (19851, who found that CSM supplementation increased molar proportions of propionate; however, other researchers have reported that propionate was not altered by protein supplementation (Topps et al., 1965; Wagner et al., 1983; Krysl et al., 1989). Ruminal butyrate proportions (Table 5) were not altered (P > .15)during DEC; however, supplementation increased (P = .01) ruminal butyrate concentrations during JAN and F'EB. Krysl et al., (1989)reported no increase in molar proportions of butyrate with soybean meal supplementation of steers grazing blue grama rangeland. Ruminal isobutyrate proportions (Table 51, within sampling period, were not altered (P > .15) by supplementation. Ruminal isovalerate and valerate concentrations were not altered (P > .15) by supplementation during DEC; however, supplementation increased isovalerate and valerate during JAN and FEB (P = .05; P = .01; P = .01; P = .01, respectively; Table 5). Serum Hormones. Sampling time x supplementation interactions (P > .lo) were not observed for serum cortisol and thyroxine; however, a collection period x treatment interaction was detected for both hormones (P e .05; Table 6). Serum cortisol concentrations'were not altered (P > .15) by supplementation. Within treatment, serum cortisol concentrations increased (linear, P = .02) in PM-supplemented steers, whereas CON steers exhibited a quadratic (P = .01) response with advancing season. Serum cortisol concentrations were not altered (P > .151 with advancing season in AM-supplemented steers (Table 6). Serum cortisol concentrations observed in our study were similar to those reported in beef bulls that were gaining BW (2.8 ng/mL; Henricks et al., 1984). Because increased cortisol concentrations are generally associated with increased stress, steers in our study seemed to be adapting to changing nutrition and(or1 environmental conditions. Serum thyroxine concentrations were not altered (P > .15) by supplementation; however, AM steers had a greater (P = .03)thyroxine concentration than PM steers did during DEC (Table 6). In addition, within treatment, serum thyroxine concentrations decreased (quadratic, P = .01) with advancing seaSon in AM steers. Thyroxine concentrations have been shown to range from 50.9 to 78.4 ng/mL in cattle consuming diets containing greater than 1.6% N (Verde and Trenkle, 1987; Williams et al., 1987; Anderson et al., 1988). Values from our study tended to be at the lower end of the reported ranges for cattle. Sampling time x supplementation or supplementation x collection period interactions (P > .lo)

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been shown to be negatively correlated with energy balance (Erfle et al., 19741, and increased NEFA are an indication of mobilization of body fat (Kronfield, 1982; Blauwiekel and Kincaid, 1986). Our data indicate that the greater GH concentrations observed in CON steers reflected liberation of fatty acids to meet nutritional demands.

Implications Cottonseed meal supplementation enhanced utilization of low-quality intermediate wheatgrass forage. Morning (0600) supplementation created greater spikes in ruminal ammonia concentration than afternoon (12001supplementation did. However, no improvement in intake, digestion, or digesta kinetics was observed with time of supplementation. Blood metabolites were altered by supplementation but were not markedly altered by time of supplementation. Our data suggest that cattle producers should not be unduly concerned about the time of day when they provide protein supplements to cattle grazing dormant rangeland.

Literature Cited Adams, D. C. 1985. Effect of time of supplementation on performance, forage intake and grazing b e h v i o r of yearling beef steers grazing Russian wild ryegrass in the fall. J. Anim. Sci. 61:1037. Adams, D. C., T. C. Nelsen, W. L. Reynolds, and B. W. Knapp. 1986. Winter grazing activity and forage intake of range cows in the Northern Great Plains.J. Anim. Sci. 62:1240. Anderson, P. T., W. G. Bergen, R. A. Merkel, W. J. Enright, S.A. Zinn, K. R. Refsal. and D. R. Hawkins. 1988. The relationship between composition of gain and circulating hormones in growing beef bulls fed three dietary crude protein levels. J. Anim. Sci. 66:3059. AOAC. 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Washington, DC. Blauwiekel, R., and R. L. Kincaid. 1986. Effect of crude protein and solubility on performance and blood constituents of dairy cows. J. DnjIv Sci. 69:2091. Broderick, G. A., and J. H. Kang. 1980. Automated simultaneous determinations of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63:64. Caton, J. S.,A. S.Freeman, and M. L. Galyean. 1988. Influence of protein supplementation on forage intake, in situ forage disappearance, ruminal fermentation and digesta passage rate in steers grazing dormant blue grama rangeland. J. Anim. Sci. 66:2262. Consortium. 1988. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, Champaign, IL. Cook, C. W. 1972. Comparative nutritive value of forbs, grasses and shrubs. In: C. M. McKell, J. P. Blaisdell, and J. R. Goodwin (Ed.) Wildland Shrubs-Their Biology and Utilization. pp 303-310. USDA Forest Services General Tech. Rep. INT-1. DelCurto, T., R. C. Cochran, T. G. Ngaraja, L. R. Corah, A. A.

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were not observed for serum insulin, GH, or insulin:GH ratio (Table 7). Serum insulin concentrations were greater (P = .03)for supplemented steers than for CON steers. In addition, PM steers tended (P = .08)to have a greater insulin concentration than did AM steers (Table 7).Serum insulin concentrations increased (Linear, P = .01;quadratic, P = .03) with advancing season. Serum GH (Table 7) c oncentrations were greater (P = .O2) in CON steers than in supplemented steers, which did not differ from each other ( P > .15).Serum GH increased (linear, P = .01;quadratic, P = .01)with advancing season. A greater (P = .021 insulin:GH ratio was maintained by supplemented steers than by CON steers; however, sampling period did not alter (P > .15) insulin:GH ratio. In general, published GH and insulin concentrations are available only for cattle that are in a positive N balance. Cattle generally have GH concentrations ranging from 4.1 to 7.4 ng/mL (Trenkle and Topel, 1978; Martin et al., 1979; Verde and Trenkle, 19871, with insulin concentrations ranging from .14 to .96 ng/mL (Williams et al., 1987;Anderson et al., 1988).Serum GH concentrations in our study were substantially higher than values that have been reported previously and reflect the low plane of nutrition these steers experienced during late dormancy. Greater concentrations of GH in our steers may indicate mobilization of body energy stores and liberation of free fatty acids. Serum Metabolites. Sampling time x supplementation interactions ( P > .lo)were not observed for serum glucose, SUN or NEFA concentrations; however, a supplementation x collection period interaction was detected for all metabolites (P e .05;Table 6). Serum glucose concentrations were greater (P = .01)for supplemented steers than for CON steers during DEC (Table 6); however, no differences (P > .05)in serum glucose concentrations were detected during JAN and FEB. Within treatment, serum glucose declined (Linear, P = .05) with advancing season. Serum urea N was not altered ( P > .15)with supplementation during DEC: however, during JAN and FEB, SUN was greater (P = .01) in supplemented steers than in CON steers (Table 6). Serum urea N concentration exhibited a pattern similar to that observed with ruminal NH3 N concentrations for these steers. Serum NEFA concentration was not altered ( P > .15)with supplementation during DEC; however, during JAN and FEB, serum NEFA concentration was greater (P = .011 in CON than in supplemented steers. In addition, PM steers had greater (P = .01) serum NEFA concentrations than AM steers did during JAN and FEB. Serum NEFA have

SUPPLEMENTATION REGIMEN ON WHEATGRASS PASTURE Beharka, and E. S.Vanzant. 1990. Comparison of soybean meal/sorghum grain, alfalfa hay and dehydrated alfalfa pellets as supplemental protein sources for beef cattle cons u m i n g dormant tallgrass-prairie forage. J. Anim. Sci. 68: 2901.

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Judkins, M. B., J. D. Wallace, M. L. Galyean, L. J. Krysl, and E. E. Parker. 1987. rates* rumen fermentation and weight change in protein supplemented grazing cattle. J. Rang* Manage. 40:lOO. Karimi, A. R., F. N. Owens, and G. W. Horn. 1986. Simultaneous extraction of Yb, Dy and Cr from feces with DCTA, DTPA, Or EDTA. Anim. sei. Res. Oklahoma &nC.EXP. MP-119:118.

Kartchner, R. J. 1980. Effects of protein and energy supplementation Of cows grazing native winter range On intake and digestibility. J. Anim. Sci. 51:432. Kronfield, D. S. 1982. Major metabolic determinants of volume, mammary efficiency, and spontaneous ketosis in dairy cows. J. Dairy Sci. 85:2204. Krysl, L. J., M. E.Branine, A. U. Cheema, M. A. Funk, and M. L. Galyean. 1989. Influence of soybean meal and sorghum grain supplementation on intake, digesta kinetics, ruminal fermentation, site and extent of digestion and microbial protein synthesis in beef steers grazing blue grama rangeland. J. Anim. Sci. 67:3040.

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