Estimation of Forage Intake by Steers Grazing Three Fescue Types and Determination of Alkaloids in Ruminal Fluid and Forage1

The Professional Animal Scientist 24 (2008):578–587 ©2008 American Registry of Professional Animal Scientists

E sSteers timation of Forage Intake by Grazing Three Fescue Types and Determination of Alkaloids in Ruminal Fluid and Forage1 R. L. Stewart Jr.,*2 G. Scaglia,*3 PAS, O. A. Abaye,† W. S. Swecker Jr.,‡ G. E. Rottinghaus,§ H. T. Boland,* M. McCann,# PAS, and J. P. Fontenot,* PAS *Department of Animal and Poultry Sciences, †Department of Crop and Soil Environmental Sciences, and ‡Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061; §College of Veterinary Medicine, University of Missouri, Columbia 65211-6023; and #College of Agriculture and Life Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061

ABSTRACT During 2 consecutive grazing seasons, DMI of steers grazing ‘Kentucky-31’ endophyte (Neotyphodium coenophialum)infected tall fescue (E+; Festuca arundinacea Shreb.), ‘Kentucky-31’ endophyte-free tall fescue (E–), and ‘Quantum’ tall fescue infected with endophyte AR542, a non-ergot alkaloid-producing strain (Quantum) was estimated using alkanes as markers. The appearance of ergovaline and lysergic acid amide (LSA) also was quantified in forage and ruminal fluid of steers grazing these forages. Estimates of DMI did not differ (P = 1

The research was funded in part by the John Lee Pratt Foundation (Blacksburg, VA) and Virginia Agricultural Council (Richmond, VA); funds and materials were from Wrightson, Ampac Seed Co. (Tangent, OR). 2 Current address: The University of Georgia, 425 River Rd., Athens, GA 30602. 3 Corresponding author: gscaglia@agcenter. lsu.edu

0.88) when based on fecal samples collected at 0800 h, 1700 h, or a composite of the 2 sampling times. Estimation of DMI using hand-plucked samples tended to be greater (P = 0.06) than whole-plant clipped samples. Estimated DMI was greater (P < 0.05) on E– pastures than E+ or Quantum across both years. Both LSA and ergovaline were present in E+ forage throughout the grazing season but were not detectable in E– and Quantum. Similarly, LSA appeared in ruminal fluid of steers grazing E+, but not in steers grazing E– and Quantum. Ergovaline was not detectable in ruminal fluid of steers grazing any of the 3 fescue types. These data suggest time of fecal sampling does not affect DMI estimations, but method of forage sampling does. Lower DMI may affect performance of steers grazing E+ and Quantum. Additionally, the appearance of LSA in ruminal fluid of steers grazing E+ suggests that this ergot alkaloid may contribute to fescue toxicosis.

Key words: beef cattle, dry matter intake, ergot alkaloid, Festuca arundinacea, grazing

INTRODUCTION Tall fescue (Festuca arundinacea Shreb.) is used extensively as a forage crop throughout the southeastern United States, covering approximately 14 million ha (Bacon and Siegel, 1988). The fungal endophyte (Neotyphodium coenophialum), which naturally infects tall fescue, produces alkaloids that have been associated with the deleterious effects on grazing animals generally referred to as fescue toxicosis (Stuedemann and Hoveland, 1988). Non-ergot alkaloidproducing endophyte strains have been developed to potentially eliminate the class of alkaloids associated with fescue toxicosis while maintaining those compounds associated with improved plant persistence (Bouton

Estimated forage intake and alkaloid detection in grazing steers

et al., 2002). Reduced DMI of animals grazing endophyte-infected tall fescue (E+) has decreased animal performance compared with other fescue types (Parish et al., 2003). Literature is limited when comparing both DMI of fescue grasses using alkanes as external markers and new varieties of fescue utilizing non-ergot alkaloidproducing endophytes. Ergovaline, the most abundant ergot alkaloid in tall fescue, has been identified as the toxic compound causing fescue toxicosis (Porter, 1995). However, simpler ergot alkaloids also may contribute to toxicosis, based on evaluation of absorption across the reticulum, rumen, and omasum and appearance in urine (Stuedemann et al., 1998; Hill et al., 2001). Lysergic acid amide (LSA) is a simpler ergot alkaloid in E+ tall fescue (Petroski and Powell, 1991) that has vasoconstrictor activity in bovine vasculature (Oliver et al., 1993). Lysergic acid, an analog of LSA, is an alkaloid that is detectable in ruminal fluid of animals consuming E+ seed or straw (Lodge-Ivey et al., 2006; De Lorme et al., 2007). However, no literature is currently available on the presence of LSA in the ruminal fluid of animals grazing alkaloid-producing E+ tall fescue. The objectives of this research were to estimate the DMI of steers grazing 3 fescue types, and the effect of sampling time of fecal matter and method of forage collection on the estimation. Additionally, the presence of specific alkaloids in forage and ruminal fluid of steers grazing these fescue types was studied.

METHODS AND MATERIALS Treatments and Design The experiment was conducted at Virginia Tech’s Kentland Farm located west of Blacksburg, Virginia (37°11′ N, 80°35′ W). Treatments were defined as forage type and included ‘Kentucky-31’ E+, ‘Kentucky-31’ endophyte-free tall fescue (E–), and ‘Quantum’ tall fescue infected with endophyte AR542, a

non-ergot alkaloid producing strain (Quantum). Treatments were arranged in 2 replicates of a randomized complete block design with soil type as the blocking factor and pasture as the experimental unit. All procedures were accepted by the Virginia Tech Animal Care Committee.

Pasture and Animal Management Pastures were managed under rotational stocking during the grazing seasons of 2004 (135 d) and 2005 (136 d). Grazing began on May 5, 2004, and May 3, 2005. Each treatment pasture was subdivided into 6 paddocks (approximately 0.20 ha), and movement of steers from one paddock to the next was determined by available forage (based on residual height of approximately 7 to 10 cm). Pastures were seeded September 20 to 25, 2002, at seeding rates of 25 kg/ha. Because of stand failure, the E– treatment was reseeded on March 30, 2003. All pastures were fertilized according to soil test recommendations and were similar for all paddocks. Before the beginning of the grazing season of both years, 33.6 kg/ha of liquid N was applied, and on August 20, 2004, an additional 56 kg/ha of 46-0-0 fertilizer was applied to all treatments. Hay was harvested from 3 of the 6 paddocks in each treatment pasture on June 6, 2004, and May 15, 2005. The remaining paddocks were clipped to eliminate reproductive tillers after animals were moved to new paddocks in late spring. For the 2004 grazing season, steers were purchased on December 1, 2003, at a Virginia feeder cattle sale and shipped to Smithfield Farm, Virginia Tech, Blacksburg, Virginia. Steers were kept in drylot from date of purchase until May 4, 2004 (d 1 of the grazing period). From January 6 to May 4, steers were fed a diet consisting of 51% barley straw, 37% corn, and 5% molasses, with the remaining portion containing soybean meal, feather meal, and urea. Average daily gain for steers during the drylot period was 0.64 kg. In 2005, steers

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were purchased on April 19, 2005, at a Virginia feeder cattle sale and shipped to Kentland Farm. Eighteen crossbred steers with initial average BW of 272 ± 19 kg in 2004 and 244 ± 17 kg in 2005 were blocked by BW and randomly allotted within block to the 3 treatments. Three steers were in each of the pastures, 2 of which were randomly assigned as tester animals for sampling. Each year, steers were vaccinated for infectious bovine rhinotracheitis, bovine viral diarrhea, bovine respiratory syncytial virus, and parainfluenza3 with Pyramid 4 (Fort Dodge Animal Health, Fort Dodge, IA) and for clostridial disease with Vision 7 (Intervet, Millsboro, DE). Steers were treated with Cydectin (moxidectin; Fort Dodge Animal Health) for internal and external parasites on d 0 and 56 of the grazing season. On approximately d 56 of both years, steers were tagged with Co-Ral Plus insecticide ear tags (Diazinon and Coumaphos; Bayer HealthCare, Shawnee Mission, KS).

Sample Collection On d 0 of the grazing season and every 28 d thereafter, steers were gathered between 0700 and 0900 h and ruminal fluid was collected via stomach tube. Ruminal fluid was placed on dry ice, transported to the laboratory, and stored at −20°C until further analysis. Samples for DMI estimation and forage nutritive analyses were collected during 3 periods throughout each grazing season (Table 1). During these periods, animals were dosed (d 0) with a controlled release device (CRD) containing the even-chained alkane hentriacontane (C32) fabricated to be used in large cattle (300 to 700 kg), as indicated by the manufacturer (Captec Ltd., Nufarm, Auckland, NZ). Seven days were allowed for the alkane marker concentration to reach equilibrium. From d 7 to 14, fecal samples were collected twice daily (0800 and 1700 h) from each dosed steer. Steers were observed until they defecated, at which point a sample of feces was collected from

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580 the ground, with care taken to avoid contamination by foreign material. If a fecal sample was not obtained in the pasture, steers were moved to a nearby working facility where grab samples were taken. Forage sampling (representing what the animal consumed that particular day) began on d 5 and continued through d 12 of each period. A 48-h retention time of the forage material in the gastrointestinal tract of the animal was assumed (McCracken et al., 1993); hence, fecal samples began on d 7 and continued through d 14. This represents what the animal consumed 48 h before the sampling day. Forage samples were collected before or during the a.m. fecal collection by walking an “X” in the paddock and stopping every 10 steps to collect a sample. Samples collected in each paddock consisted of a whole-plant sample and a handplucked sample, harvesting approximately the top 8 cm of the canopy to mimic the selectivity of the forage exerted by the grazing animal. Both fecal and forage samples were placed on ice for transport to the laboratory, where they were frozen at −20°C until analyzed. Air temperature (°C) and relative humidity (RH, %) were recorded on an hourly basis (Virginia Agricultural

Experiment Station, 2006). Temperature humidity index (THI) was calculated as a measure of heat stress using the following equation (National Oceanic and Atmospheric Administration, 1976) and was subsequently converted to °C: THI = °F − (0.55 − (0.55 × RH%/100) × [air temp. (°F) – 58.8]. Level of heat stress previously evaluated in dairy cattle using the following THI values were used in the current research to determine periods of heat stress: <22 = no stress, 22 to 26 = mild stress, 27 to 32 = moderate stress, and >32 = severe stress (Armstrong, 1994).

Laboratory Analyses Forage Characteristics. Forage samples collected for nutritive value were dried in a forced-air oven at 60°C for 48 h and ground to pass a 1-mm screen in a stainless steel Wiley mill (Thomas Wiley Laboratory Mill Model 4, Arthur H. Thomas Co., Philadelphia, PA). Ash was determined after placing samples in a 500°C muffle furnace for 3 h (AOAC, 2000). Crude protein was determined by analyzing N content with a Ni-

Table 1. Periods and dates of DMI estimation Year

Period

Dosing or sampling

Dates

2004

1

Dose Forage Fecal Dose Forage Fecal Dose Forage Fecal Dose Forage Fecal Dose Forage Fecal Dose Forage Fecal

May 21 May 26 to June 1 May 28 to June 3 July 2 July 7 to 13 July 9 to 15 August 27 September 1 to 7 September 2 to 9 June 8 June 13 to 19 June 15 to 21 July 27 August 1 to 7 August 3 to 9 September 16 September 20 to 26 September 22 to 28

2

3

2005

1

2

3

trogen Auto-analyzer (2410 N Analyzer, Perkin-Elmer, Norwalk, CT) by the combustion method (AOAC, 2000). Neutral detergent fiber and ADF were analyzed with an Ankom 200/220 Fiber Analyzer (Goering and Van Soest, 1970). All samples were obtained before animals entered the paddock in which DMI was estimated. Before each grazing season, plant tillers from the tall fescue treatments were collected from the paddock and packed on ice, and a subsample was shipped to a laboratory for analysis of endophyte infection level (Agrinostics Ltd. Co., Watkinsville, GA). The endophyte infection level of E+ and Quantum pastures was 85 and 90%, respectively, and endophyte infection was not detected in E− pastures. Herbage allowance (HA) was determined for each pasture using the double sampling technique described by Dubeux et al. (2006). It was calculated as the herbage mass (kg/ ha) divided by the average BW of the steers during that 28-d period. Alkane Analysis. All samples were analyzed in duplicate. Forage samples were lyophilized in a FreeZone 12 L lyophilizer (Labconco Co., Kansas City, MO) or a VirTis Genesis 25 EL lyophilizer (SP Industries Inc., Gradiner, NY). The dried samples were ground to pass a 1-mm screen in a Wiley mill and then a 0.5-mm screen in a Tecator Cyclotec 1093 sample mill (Tecator, Hogänäs, Sweden). Fecal samples were thawed at room temperature for approximately 24 h. For each animal on each day, 20 g of wet material were weighed for both a.m. and p.m. samples into a beaker and covered with cheesecloth. Ten grams of both a.m. and p.m. samples for each animal on each day were combined in a beaker as a composite sample for the given day. Weighed fecal samples were then freeze-dried, ground to pass a 0.5-mm screen in a Tecator Cyclotec 1093 cyclone mill, and thoroughly mixed with a spoon. Freeze-dried, ground samples were analyzed for alkanes as described by Mayes et al. (1986). Briefly, 0.3 and 0.1 g of forage and feces, respectively,

Estimated forage intake and alkaloid detection in grazing steers

were placed along with 10 mg of internal standard (C34, n-tetratriacontane) in 20-mL Pyrex screw-capped culture tubes. The samples were saponified with 7 mL of a 10% ethanolic KOH solution at 90°C for 3 h in a water bath. Contents were vortexed every 30 min. After cooling to room temperature, 7 mL of deionized water and 7 mL of heptane were added and the contents were vortexed for approximately 15 s. The organic extract was removed and applied to a filtration column made of 5-mL disposable pipettes containing silica gel with a glass wool stopper. The extract was eluted with approximately 10 mL of heptane into 20-mL scintillation vials. The eluent was then dehydrated in a N evaporator unit (Organonmation Associates Inc., Berlin, MA) and redissolved in 1 mL of heptane. A 0.5-µL injection of each sample was applied to the capillary column (Rtx-1, 30 m long, 0.52 mm i.d., and 1.5 µm of fused silica film thickness; Restek Inc., Bellefonte, PA) of a gas chromatograph (Agilent Technologies 6890, Santa Clara, CA) equipped with a flame-ionization detector, integrator, and a 7683 autosampler. The oven temperature was programmed to hold at 240°C for 4 min, increase to 288°C at 3°C/min, and then increase to 298°C at 2°C/min. Helium was used as the carrier gas with a flow rate of 9.0 to 9.25 mL/min. Specific alkanes (nonacosane, C29; hentriacontane, C31; dotriacontane, C32; hentriacontane, C33; and hexatriacontane, C36) were identified by their retention times relative to known standards. Alkanes were quantified by peak areas compared with reference to C34 as an internal standard. Daily DMI was calculated using the following equation (Mayes et al., 1986): ö æ Fi DMI = ççç ´ RRj÷÷÷ ÷ø è Fj

ö æ ççHi - Fi ´ Hj÷÷, ÷÷ çè Fj ø

where DMI is daily herbage intake (kg DM/d), RRj is daily release rate of even-chained alkane (C32), Fi and Hi are the fecal and herbage concentrations of odd-chained alkane (C33),

Fj and Hj are the fecal and herbage concentration of even-chained alkane (C32). The fecal recovery (99.1%) of dosed alkane was validated by Scaglia et al. (2005) under similar experimental conditions. Alkaloid Analysis. Samples were analyzed for ergovaline (EV) and LSA as described by Hill et al. (1993) with slight modification (G. E. Rottinghaus, unpublished data). Briefly, 5 g of ground forage was weighed into polypropylene screw-capped bottles along with 100 mL of chloroform and 5 mL of 0.1 M sodium hydroxide. Samples were mixed overnight (approximately 18 h) on a rotator shaker. After mixing, 2 g sodium sulfate was added and samples were mixed for an additional 30 min. Twenty milliliters of extract was then filtered through Whatman PS-1 filter paper (Whatman Inc., Florham Park, NJ). Ten milliliters of extract was applied to an ergosil cleanup column under vacuum. Cleanup columns were prepared by placing a 12.7-mm biological disk (Whatman Inc.) in the bottom of a 6-mL disposable syringe barrel followed by 1) 1 mL ergosil (Analtech Inc., Newark, DE); 2) a 12.7-mm biological disk; 3) 1 mL ground sodium sulfate; and 4) a 12.7-mm biological disk. Pigments were removed by washing the columns with 1.8 mL acetone:chloroform (8:2) followed by 3 mL of petroleum ether under vacuum. Ergot alkaloids were then eluted with methanol to a final volume of 2 mL. The methanol eluent was passed through a small Romer Mycosep 224 column (Romer Labs Inc., Union, MO) to remove the remaining pigmentation before HPLC analysis. Ergovaline and LSA were determined by HPLC with fluorescence detection. Standards for each alkaloid (50 and 100 ppb) were prepared in methanol. Methanolic samples were loaded onto an autosampler (Perkin-Elmer ISS200) and 20 µL was injected into a Perkin-Elmer (LC-250) HPLC pump with a 100 × 4.6 mm (3-µm particle size) Luna C18 analytical column (Phenomenex, Torrance, CA) and detected with a fluorescence detector (excitation 250 nm, emission 420 nm;

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Hitachi F-1200; Hitachi High Technologies America Inc., San Jose, CA). The mobile phase (30% acetonitrile in a 200 mg/L solution of ammonium carbonate in distilled water) was pumped at a rate of 1 mL/min.

Statistical Analysis Dry matter intake and alkaloid concentration data were analyzed as a randomized complete block design with forage as treatment by using repeated measures ANOVA in PROC MIXED (SAS Inst. Inc., Cary, NC). For alkaloid concentration, the model consisted of year, block, period, and period × treatment, whereas for DMI it also included treatment. Treatment was included as a fixed effect and year as the repeated variable for across-year analysis. Data representing sampling methods for DMI determination were analyzed for period one of 2004 with treatment and block in the model by using PROC GLM (SAS Inst. Inc.). All means reported are least squares means. The compound symmetry covariance structure provided the best fit data for analyses. Standard errors were calculated in SAS with the estimation of similar variances between treatments on any day. Differences were determined for the repeated measures by using PDIFF-adjusted P-values.

RESULTS AND DISCUSSION Estimation of Dry Matter Intake Effect of Sampling Time on Dry Matter Intake Estimation. No differences (P = 0.59) in DMI estimation attributable to sampling time of fecal matter were detected using a.m.-collected (22.6 g/kg BW) and p.m.-collected (22.8 g/kg BW) samples compared with daily composite samples (22.6 g/kg BW). Diurnal variation of marker concentration in feces when markers are dosed once daily is a concern in DMI estimation. However, increasing the number of daily doses to decrease diurnal variation is impractical because of

Stewart et al.

582 increased labor and effect on animal grazing behavior (Burns et al., 1994). The development of the CRD was intended to decrease this variation by releasing a consistent concentration of marker and decreased labor and handling time of animals (Dove and Mayes, 1996). However, this method still raises concerns involving marker release rate and recovery in feces (Burns et al., 1994). When using dosed, artificial alkanes, diurnal variation in recovery rate may occur because of the nature of natural, oddchained alkanes to associate with the particulate phase and artificial, evenchained alkanes to associate with the liquid phase (Dove and Mayes, 1996). The use of alkanes adjacent in chain lengths has reduced the errors associated with incomplete recovery rate of alkanes (Dove and Mayes, 1991, 1996; Mayes and Dove, 2000) The current research supports these concepts, showing no differences in DMI estimation when using a CRD and sampling feces at different points over a 24-h period. Effect of Forage Sampling Method on Dry Matter Intake Estimation. Intake estimated from whole-plant samples tended to be lower (P = 0.06) as compared with hand-picked samples (17.8 vs. 19.1 g/kg BW). The diet of the grazing animal will differ from clipped samples harvested at ground level (Weir, 1959; Coleman and Barth, 1973; De Vries, 1995). Dubbs et al. (2003) reported that forage samples collected from esophageally fistulated steers grazing tall fescue were lesser in NDF and ADF and greater in CP compared with samples clipped by hand to ground level. The use of esophageally fistulated animals is not always practical in grazing research; thus, a procedure is used of collecting forage samples that closely replicate what the animal selects (Dubbs et al., 2003). De Vries (1995) reported that under range conditions, hand-plucked samples collected to closely mimic the selection of the grazing animals were similar in nutritive value to samples collected from esophageally fistulated steers. When herbage allowance is not

Table 2. Average daily estimated DMI of steers grazing ‘Kentucky-31’ endophyte infected (E+) and endophyte free (E−), and QuantumAR542 (Q) non-ergot alkaloidproducing endophyte-infected tall fescues Period and treatment1 Period 1   E–  Q   E+ Period 2   E–  Q   E+ Period 3   E–  Q   E+ Pooled SE

DMI, g/kg BW 2004

2005

27.6a 19.9b 22.6b

24.6a 23.4a 19.0b

23.2a 14.3b 15.9b

20.6 19.1 20.8

18.1a 24.9a 12.9b 21.8b 13.3b 23.6ab 1.01 0. 77

a,b

Within a period and year, means without a common superscript letter differ, P < 0.05.

1

In 2004, period 1 = May 26 to June 1; period 2 = July 7 to 13; period 3 = September 1 to 7. In 2005, period 1 = June 13 to 19; period 2 = August 1 to 7; period 3 = September 20 to 26.

limiting, grazing animals select forage of greater nutritive value (Parsons et al., 1994). Therefore, when DMI of grazing animals is to be measured, it is apparent that sampling method affects DMI estimation. Estimation of Daily Dry Matter Intake. A year effect was detected (P < 0.01) on DMI; estimated DMI was less in 2004 compared with 2005 (19.1 vs. 22.2 g/kg BW). In addition, there was a treatment × period interaction in 2004 and 2005 (P < 0.01). Therefore, data are presented by year and period. In each of the 3 periods of the 2004 grazing season, DMI of steers grazing E– was greater than that of steers grazing Quantum or E+ (Table 2). Dry matter intake of steers grazing E– and E+ followed similar seasonal trends, decreasing from May to July

and again in September, whereas DMI of steers grazing Quantum decreased from May to July and remained similar in September. Across treatments from May to June (period 1) to July (period 2) of 2004, HA, NDF, and ADF decreased and CP increased (Table 3). These changes in forage characteristics are likely due to stage of development. In May to June, steers were grazing paddocks where stems with reproductive structures were present. The presence of stems in this stage of development is associated with increased fiber and reduced CP (Nelson and Moser, 1995). In early July, steers were grazing paddocks in which hay was previously harvested, which removed mature and stemmy material. This resulted in forage of greater nutritive value, but reduced herbage mass. Vazquez and Smith (2000) evaluated forage variables from 27 previously published studies and found that DMI was negatively correlated (r = −0.31) with diet NDF concentration. Average THI indicated longer periods of mild stress in July (Table 4). Seman et al. (1997) reported a negative correlation between solar radiation and grazing time of steers grazing both E+ and E– tall fescues. The increased heat stress during July may explain the decrease in DMI of steers grazing all fescue types despite the improved forage nutritive value. From July to September, DMI of steers grazing all fescue types decreased even though HA increased and nutritive value improved (Table 3). In addition, THI indicated lower heat stress conditions (Table 4). The decrease in DMI during this period, even though environmental conditions and forage nutritive value improved, was unexpected. In 2005, there was a treatment × period interaction for DMI (Table 2). In June, estimated DMI of steers grazing E– and Quantum was greater than that of steers grazing E+. In August, there was no difference in DMI of steers grazing E–, Quantum, and E+. In September, DMI of steers grazing E– was greater compared

Estimated forage intake and alkaloid detection in grazing steers

583

Table 3. Herbage allowance (HA) and nutritive value of ‘Kentucky-31’ endophyte infected (E+) and endophyte free (E−), and Quantum-AR542 (Q) non-ergot alkaloid-producing endophyte-infected tall fescues 2004 Period and treatment1 Period 1   E–  Q   E+   Average Period 2   E–  Q   E+   Average Period 3   E–  Q   E+   Average Pooled SE a–c d,e

2005

HA,2 kg/ kg BW

CP,2 % of DM

NDF,2 % of DM

ADF,2 % of DM

HA,3 kg/kg BW

7.9 7.2 6.1 7.1a

8.8 8.7 8.2 8.5c

69.1 68.9 71.2 69.7a

39.8 38.7 41.7 40.1a

3.7 4.2 6.4 4.8a

2.8 2.3 3.6 2.9c

12.1 13.4 12.4 12.6b

59.9 60.4 60.7 60.3b

31.4 31.9 32.7 32.0b

4.2e 3.4e 7.1d 5.0b 1.3

17.6 17.8 17.7 17.7a 0. 7

59.4 60.1 59.8 59.8b 1.2

28.3 28.5 28.9 28.6c 0.8

CP,2 % of DM

NDF,2 % of DM

ADF,2 % of DM

11.4 11.1 10.4 11.0a

67.1 66.7 64.1 66.0a

33.3 33.6 34.1 33.9a

5.7 4.9 5.4 5.3a

11.0 12.3 10.8 11.3a

66.8 65.6 64.9 65.8a

35.1 33.6 36.5 35.1a

2.3 2.8 2.9 2.7b 1.0

15.4 15.9 14.3 15.2b 0.50

54.5 56.3 52.6 54.5b 1.5

26.9 28.2 29.0 28.0b 0.50

Within a column, period means without a common superscript letter differ, P < 0.05.

Within a column and period, means without a common superscript letter differ, P < 0.10.

1

In 2004, period 1 = May 26 to June 1; period 2 = July 7 to 13; period 3 = September 1 to 7. In 2005, period 1 = June 13 to 19; period 2 = August 1 to 7; period 3 = September 20 to 26.

2

Period effect, P < 0.001.

3

Period effect, P < 0.10.

with those grazing Quantum with E+ intermediate. In 2005, HA and nutritive value (NDF, ADF, and CP) did not differ among treatments, but there was a difference (P < 0.05) attributable to period (Table 3). In September (period 3), DMI increased, whereas HA was the lowest of the season. Nutritive value also improved in September, suggesting that DMI followed trends of nutritive value and not HA. This contradicts Vallentine (2001) who indicated that herbage availability is a major factor influencing DMI of grazing animals. It appears that forage was not limiting DMI during any of the periods studied because of HA being greater than 2.0 kg/kg BW. Previous research has reported DMI of steers grazing tall fescue in similar grazing conditions. Scaglia et al. (2005) reported an estimated daily DMI (using a ratio of dosed C32 to forage C33 alkanes) of steers (330 ± 11

kg) grazing E+ tall fescue in May and June of 32.7 g/kg BW. This value was greater than any treatment in the current research and may be explained by greater CP and HA (17.5% and 9.32 kg/kg BW, respectively) and lesser NDF (55%) compared with the current research. At greater HA, animals can select for green leafy material and against stems (Heitschmidt and Stuth, 1991). Reduced performance of animals grazing E+ tall fescue has been related to reduced forage DMI; however, previous research results are conflicting. Stewart (2006) reported that the ADG of steers grazing E− was greater than those on E+ from April to September of 2004 (0.54 and 0.22 kg, respectively), whereas DMI was consistently greater for steers on E− during the 3 periods it was estimated (Table 2). However, in 2 of the 3 periods that DMI was estimated in 2005, there were no differences between E− and

Table 4. Temperature heat index (THI) during the periods of DMI estimation Year and period2 2004   Period 1   Period 2   Period 3 2005   Period 1   Period 2   Period 3

THI1 Minimum Maximum 16.0 16.9 15.7

22.5 25.5 22.3

13.7 17.6 15.5

23.3 26.5 24.1

1

Mild stress = 20.0 to 26.3; medium stress = 26.3 to 38.0; severe stress >38.0.

2

In 2004, period 1 = May 26 to June 1; period 2 = July 7 to 13; period 3 = September 1 to 7. In 2005, period 1 = June 13 to 19; period 2 = August 1 to 7; period 3 = September 20 to 26.

584 E+ (Table 2), and ADG were greater (P < 0.05) for E− compared with E+ (0.54 and 0.30 kg/d, respectively). These results may be explained by an interaction of environmental conditions and fescue toxicosis, where the toxicosis effects are increased as temperature increases as suggested by Peters et al. (1992). Temperature humidity index can be used as a measure of heat stress that accounts for relative humidity in addition to ambient temperature. This index indicates times of mild stress during all periods of DMI estimation (Table 4), and may partly explain the decrease DMI during these periods. However, DMI was not different among treatments in 2005 when THI indicated the periods of mild or medium stress. This would suggest an effect of fescue toxicosis on DMI independent of environmental conditions. Hannah et al. (1990) reported that increasing EV concentration in the diet of sheep by the inclusion of E+ tall fescue seed decreased (P < 0.10) ruminal and total tract OM, NDF, and cellulose digestibility. The reduced digestibility of these fractions may have led to decreased DMI because of reduced passage rate from the rumen. Goetsch et al. (1987) reported that passage rate of particulates from the rumen decreased as the proportion of E+ tall fescue hay in the diet increased. This may explain the differences observed in DMI of steers grazing E– and E+ in the present study. Seman et al. (1997) reported that time spent lying down and grazing of steers grazing E+ was reduced (P < 0.05) compared with E– tall fescues. Similarly, Boland (2005) reported that steers grazing E+ spent more (P < 0.05) time standing and idling compared with steers on E– and Quantum. This increased idling time may partly explain the difference in DMI of E– and E+. The use of non-ergot alkaloidproducing endophyte-infected fescue is a relatively new concept (Bouton et al., 2002); therefore, limited data were available when DMI of animals grazing these forages was estimated. The findings of the current research are

Stewart et al.

inconsistent with those of Parish et al. (2003). These authors compared DMI of steers grazing E−, E+, and nonergot alkaloid-producing endophyte AR542 infected ‘Jesup’ fescues. In spring, Parish et al. (2003) reported DMI of steers grazing E– (14.6 g/kg BW) and the ‘Jesup’ tall fescue (14.1 g/kg BW) was greater (P < 0.10) than that of steers grazing E+ (10.2 g/kg BW) tall fescue. However, in the current research, DMI of E− was greater than Quantum in spring of 2004, but not 2005 (Table 2) In fall, Parish et al. (2003) reported DMI of steers grazing E– (14.0 g DM/kg BW) was greater (P < 0.10) than for E+ (9.3 g DM/kg BW), and non-ergot alkaloid-producing endophyte-infected ‘Jesup’ tall fescue (11.2 g DM/kg BW) was intermediate; in the current research, DMI of E− was greater than Quantum in both years (Table 2). The difference observed between estimated DMI of steers grazing E– and Quantum in the current experiment was unexpected. This difference in DMI may be attributed to differences in the potentially digestible fraction of these fescue types. Boland et al. (2007) determined the DM digestibility fractions of the forages in the present study using the nonlinear regression model described by Ørskov et al. (1980). The total potentially digestible fraction was less (P < 0.05) in Quantum (66.5%) compared with E– (72.4%) and E+ (79.5%). Additionally, the indigestible fraction of Quantum (4.0%) was greater compared with E– and E+ (2.2 and 2.5%, respectively). The reduced digestible fraction of Quantum likely caused a decrease in passage rate and may have limited DMI because of gut fill. Another explanation may involve differences in fescue variety. The fescue-endophyte combination used by Parish et al. (2003) involved the same endophyte strain (AR542) as the current research but infected in different fescue varieties. The E– and E+ fescue cultivar for this present study was ‘Kentucky-31’, whereas the non-ergot alkaloid producing cultivar used was ‘Quantum’. The difference in variety may involve differences in palatability

of forage of Quantum compared with E–.

Alkaloids in Forage In 2004 and 2005, EV and LSA were not detectable in forage samples of E– and Quantum. In 2004, EV concentrations in E+ forage were greatest (P = 0.04) in late spring when forage was in a reproductive stage (Table 5). This is consistent with the findings of Rottinghaus et al. (1991) in which EV concentration of forage was increased because of the presence of seed heads. Both the endophyte and EV concentrate in the seed head as tall fescue enters a reproductive stage in late spring because of the asexual nature of the endophyte (Siegel et al., 1987). Unlike EV, LSA concentrations were lowest (P = 0.01) during this time (Table 5). After seed heads were removed, EV concentrations decreased, whereas LSA increased. Concentrations of both EV and LSA remained similar in late summer. In 2005, forage alkaloids were determined after seed heads were removed. In late spring of 2005, EV was lower and LSA was greater compared with late spring of 2004 (Table 5). Ergovaline and LSA decreased slightly in mid-summer before increasing in late summer to early fall. The presence of the endophyte in tall fescue increases plant tolerance to adverse ambient stresses, including drought (Schardl et al., 2004). Ergovaline concentrations in tall fescue plants are increased under drought conditions (Arechavaleta et al., 1992). Therefore, dry conditions before forage sampling in September of 2005 (there was no rainfall in the 21 d before the sampling day) may explain the increase in forage alkaloid concentrations. Literature related to LSA levels in forage is not readily available. Lysergic acid amide is a chemical analog of lysergic acid that has been reported to occur naturally in E+ tall fescue because of the presence of alkaloidproducing endophytes (Petroski and Powell, 1991). Oliver et al. (1993) reported that LSA has vasoconstrictor activity and acts as a partial agonist

Estimated forage intake and alkaloid detection in grazing steers

Table 5. Ergovaline (EV) and lysergic acid amide (LSA) levels in forage and ruminal fluid of steers grazing ‘Kentucky-31’ endophyte infected (E+) tall fescue1 Item Forage   2004    Period 1    Period 2    Period 3    SE   2005    Period 1    Period 2    Period 3    SE Ruminal fluid   2004    May 3    May 28    July 23    Aug 20    SE   2005    May 5    June 25    July 28    Sept 20    SE

EV, µg/kg LSA, µg/kg

551b 281a 244a 56.8

56a 217b 235b 11.0

300ab 247a 330b 12.5

283b 160a 424c 13.4

ND2 ND ND ND —

ND 18.5b 7.8a 10.0ab 2.8

ND ND ND ND —

5.3a 36.4c 12.0ab 16.8b 2.0

a-c

Within a column and year, means without a common superscript letter differ, P < 0.05. 1

In 2004, period 1 = May 26 to June 1; period 2 = July 7 to 13; period 3 = September 1 to 7. In 2005, period 1 = June 13 to 19; period 2 = August 1 to 7; period 3 = September 20 to 26.

2

ND = not detected.

at adrenergic receptors and as an antagonist at serotonergic receptors in the bovine lateral saphenous vein and dorsal metatarsal artery. However, little research has been conducted trying to relate this compound with fescue toxicosis. The data from the current research suggest that, although not specifically measured, LSA concentrations in reproductive tillers are low compared with other plant parts. Low concentrations were detected in whole-plant samples when these structures were present as

compared with high concentrations in forage regrowth. In addition, in drought conditions, LSA concentrations increased more than EV.

Alkaloids in Ruminal Fluid In 2004, LSA was not detected (Table 5) in the ruminal fluid of steers before the beginning of the grazing season (May 3, 2004). Before the experiment, all steers were backgrounded on an alkaloid-free diet for 60 d. During early spring, LSA was detected, decreased to a minimum in July, and increased in late summer. In 2005, LSA was detectable (Table 5) in samples of ruminal fluid taken before the beginning of the experiment (May 5, 2005). The pretreatment diet of these steers is unknown, but it is likely that some animals grazed E+ tall fescue before their purchase. In late spring, ruminal fluid LSA concentration was twice (P = 0.0087) that of samples at a similar time in 2004, decreased in mid-summer, and increased slightly in late summer. Ruminal fluid LSA concentration was greater (P = 0.009) during late spring to early summer of 2005 compared with 2004. Although forage alkaloid concentrations were similar, greater LSA in 2005 may have caused greater concentrations of LSA in ruminal fluid. Previous research has pointed to EV as the toxic alkaloid causing fescue toxicosis because of its high recovery from E+ tall fescue (Agee and Hill, 1994). However, because of the site and greater potential of transport, Hill et al. (2001) suggested that a simple ergot alkaloid, and not an ergopeptine alkaloid, is responsible for fescue toxicosis. We are not aware of any literature suggesting the appearance of LSA in ruminal fluid of animals grazing E+ tall fescue. Another simple ergoline alkaloid, lysergic acid was quantified in different matrices: forage, ruminal fluid, urine, and feces of steers consuming E+ tall fescue straw with 400 µg/kg EV (Lodge-Ivey et al., 2006). These authors reported that straw, ruminal fluid, urine, and feces contained 24.2, 13.3, 26.3, and 20.6 µg/kg, respectively. These find-

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ings, along with those of the current research, suggest that these simpler alkaloids are present in the rumen in their original form, as metabolites of larger alkaloids, or both, and are available for transport across gastric tissue.

IMPLICATIONS Dry matter intake estimates of steers grazing E–, Quantum, and E+ did not vary diurnally when using the dosed alkane C32 and natural alkane C33. Therefore, time of sampling does not appear to be a concern when estimating DMI with alkanes in fescue. Differences between clipped whole-plant and hand-plucked forage sampling indicate the importance of closely representing the diet of the grazing animal to correctly estimate DMI. Lesser estimated DMI contributed to decreased animal performance on E+ forages and indicate Quantum may not be an optimal fescue variety in this environment. This is the first report of the appearance of LSA in the ruminal fluid of animals grazing E+ tall fescue, and suggests that this compound may contribute to fescue toxicosis. Bioavailability of this compound in the rumen needs further investigation to develop strategies to decrease the occurrence of fescue toxicosis.

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