J. Anim Sci. 2007. 85:2932-2940. doi:10.2527/jas.2007-0303
© 2007 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Influence of endophyte consumption and heat stress on intravaginal temperatures, plasma lipid oxidation, blood selenium, and glutathione redox of mononuclear cells in heifers grazing tall fescue1
N. C. Burke*,
G. Scaglia
,
K. E. Saker*,
D. J. Blodgett
and
W. S. Swecker, Jr*,2
* Department of Large Animal Clinical Sciences, and
and
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061; and and
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061
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Abstract
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A grazing experiment was conducted to assess the effects of wild-type endophyte-infected (E+) tall fescue consumption and elevated ambient temperatures on intravaginal temperatures, plasma lipid peroxidation, and glutathione redox of peripheral blood mononuclear cells. Angus heifers (n = 34) were allotted by BW to 4 blocks consisting of E+ and endophyte-free (E–) fescue pastures. Monthly, in June, July, and August, temperature loggers were fixed into blank controlled internal drug releasers and inserted into a subsample of heifers (n = 16) for 2 d. After 48 h, heifers were weighed, and blood (30 mL) was collected via jugular venipuncture. Peripheral blood mononuclear cells were isolated for analysis of glutathione peroxidase activity, glutathione reductase activity, and reduced:oxidized glutathione. Plasma malondialdehyde was evaluated as a marker of lipid peroxidation, and whole blood Se concentration was determined. Serum prolactin was assayed after the grazing period. Heifer ADG was greatest in August and least in July (P < 0.001). In August, heifers grazing E+ fescue exhibited greater (P < 0.05) afternoon intravaginal temperatures and temperature fluctuations than heifers grazing E– fescue. In July and August, all heifers had greater afternoon temperatures (P < 0.02) and less reduced:oxidized glutathione (P < 0.0001) than in June. Glutathione reductase activity of all heifers was greater in June (P = 0.03) than in July. Similarly, all heifers exhibited decreased glutathione peroxidase activity (P < 0.0008) in July, whereas whole blood Se was reduced (P < 0.0001) in July and August. No treatment or date effects were detected for malondialdehyde, but serum prolactin was reduced at the end of the grazing period (P = 0.008) in heifers stocked on E+ fescue. Using these markers, differences in oxidative stress were not detected between heifers consuming E+ fescue and those consuming E– fescue. Date effects indicating altered glutathione redox and enzyme activity may have been related to heat stress and nutritional limitations.
Key Words: cattle • endophyte • fescue • glutathione • heat stress • oxidative stress
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INTRODUCTION
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Tall fescue (Festuca arundinacea Schreb.), the predominant forage for grazing animals in southwest Virginia, is commonly infected with the fungal endophyte Neotyphodium coenophialum [(Morgan-Jones and Gams) Glenn, Bacon, and Hanlin; Glenn et al., 1996
]. Although this fungus acts in a mutualistic way to improve plant hardiness, it also produces alkaloids that are detrimental to the performance of grazers (Stuedemann and Hoveland, 1988; Strickland et al., 1993; Omacini et al., 2005). Abnormalities are particularly notable during periods of high temperature-humidity indices, and cattle exposed to elevated ambient temperatures and endophyte alkaloids are predisposed to hyperthermia (Oliver, 1997). In addition, the low-molecular weight antioxidant, glutathione, may be compromised by fescue toxicosis.
Lakritz et al. (2002) reported a decrease in reduced glutathione (GSH) and an increase in oxidized glutathione (GSSG) in the blood of heat-stressed cattle consuming wild-type endophyte-infected fescue (E+). Furthermore, disrupted redox status could promote the accumulation of by-products from oxidant-induced tissue damage. Saker et al. (2004) observed increased lipid hydroperoxides in the plasma of wether lambs fed E+ fescue hay after a period of heat stress and found that erythrocyte glutathione peroxidase (GSH-Px) activity increased linearly during this time. Because immune cell function is impaired in cattle grazing E+ fescue (Saker et al., 1998, 2001), the status of the glutathione redox system in immune cells of cattle stocked on E+ fescue may be of interest. An experiment was conducted to test the hypothesis that fescue toxicosis is exacerbated by an imbalance in the glutathione redox system of leukocytes. The objectives were to evaluate intravaginal temperatures, plasma lipid oxidation, and redox balance of glutathione in leukocytes from cattle grazing E+ fescue in typical conditions such as those encountered during summertime in Virginia.
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MATERIALS AND METHODS
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All animal handling procedures were approved by the Virginia Polytechnic Institute and State University Animal Care and Use Committee.
Experimental Design
During the summer of 2006, an experiment was conducted at Virginia Polytechnic Institute and State University’s Kentland Farm (81°5' west longitude; 37°25' north latitude). Crossbred Angus heifers (n = 34; BW = 329 ± 13.7 kg) were purchased from the same origin and stocked for 3 wk on naturalized pasture composed predominantly of Kentucky bluegrass (Poa pratensis L.), orchardgrass (Dactylis glomerata L.), tall fescue, red clover (Trifolium pratense L.), and white clover (Trifolium repens L.). On May 5, 2006, heifers were allotted by BW to 4 pasture blocks. Each block contained two 1.1-ha Kentucky 31 tall fescue paddocks, 1 composed of endophyte-free fescue (E–), the other consisting of E+ fescue. The E+ and E– fescue stands were originally established in September of 2002 and showed an original stand persistence of 74 and 63%, respectively, in 2005 (Rotz, 2006). Routine forage analysis in 2005 and 2006 revealed mean total alkaloid concentrations of 1,565 ± 552 and 135 ± 49 ppb for E+ and E– pastures, respectively (Agrinostics Ltd. Co., Watkinsville, GA).
Each paddock was subdivided into 6 sections with polywire (O’Brien Plastics Ltd., Auckland, New Zealand) so that heifers could be allowed to strip-graze the paddock over time. Water and a Se-free trace mineral salt mixture (Cargill Inc., Minneapolis, MN) were provided ad libitum, and heifers were monitored daily. On June 28, 2006, temperature-recording data loggers (ACR Systems Inc., Surrey, Canada) were fixed into blank controlled internal drug releasers (InterAg, Hamilton, New Zealand) and inserted vaginally into a randomly selected subset of heifers (n = 16; 8 E+ and 8 E–) at 0800. Heifers were weighed and then returned to their assigned paddocks. After 48 h, data loggers were removed, and blood (30 mL) was collected from heifers via jugular venipuncture into evacuated tubes containing either heparin or EDTA (Becton Dickinson, Franklin Lakes, NJ). Blood was placed on ice for transport to the laboratory (21 km from the site), where it was processed immediately. The sampling protocol was followed 21 d later on July 19, 2006, and 56 d later on August 23, 2006. An additional evacuated tube of blood was also collected in August for analysis of serum prolactin.
Environmental Conditions and Heifer Temperatures
Weather data were collected throughout the grazing period from a WeatherWatch 2000 weather station (Campbell Scientific Inc., Logan, UT) located on-site at Kentland Farm, and temperature-humidity indices (THI) were calculated according to Amundson et al. (2006). Heifer temperature data were recorded every 15 min, and recordings were subsequently extracted from data loggers using software provided with loggers (ACR Systems Inc.). For each sampling date, assessment of intravaginal temperatures was carried out by comparing morning and evening average temperatures recorded by data loggers over the 2 d for which they were in place. This was done to appreciate day and night temperature fluctuations that we expected to occur in heat-stressed cattle consuming ergot alkaloids (Bourke, 2003). All intravaginal temperatures recorded from 0400 to 0800 on both days preceding sampling were averaged to represent daily minimum temperatures, whereas all intravaginal temperatures recorded from 1600 to 2000 on both days preceding sampling were averaged to assess daily maximum temperatures (Tmax). The daily intravaginal temperature fluctuation (
T) was defined as the difference between afternoon average and morning average.
Sample Processing and Analysis
Blood with EDTA was centrifuged for 5 min at 2,500 x g, and the buffy coat was removed and dispersed into 12 mL of lysis buffer (pH = 7.2, 150 mM NH4Cl, 10 mM NaHCO3, 10 mM EDTA) to remove residual erythrocytes. The bovine buffy coat consists almost entirely of mononuclear cells (Carlson and Kaneko, 1973), as confirmed by our own cell differentials (data not shown). Blood smears performed on buffy coat leukocytes resuspended in autologous plasma revealed 97% peripheral blood mononuclear cells (PBMC). Lysis buffer containing PBMC was incubated for 10 min at room temperature and then centrifuged at 470 x g for 10 min to pellet the cells. Cells were washed in 10 mL of Hank’s Balanced Salt Solution (Gibco Industries Inc., Langley, OK) and repelleted by centrifugation at 470 x g. Pellets were reconstituted into 1 mL of distilled water, aliquoted into storage vials, and frozen at –70°C until assay for glutathione reductase (GR) and GSH-Px activity.
For analysis of GSH:GSSG in PBMC isolates, blood was handled via a slight modification of the procedures described above. To assess GSSG apart from GSH, 100µL of the isolate was added to 10 µL of the thiol-scavenging reagent, 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (Oxis International, Portland, OR), before freezing and storage. Heparinized blood was centrifuged for 5 min at 3,000 x g and 4°C, and the plasma was separated into vials for storage at –70°C until analysis of malondialdehyde (MDA) concentration. Assessment of PBMC GR, GSH-Px, and GSH:GSSG, as well as plasma MDA, was carried out with colorimetric assay kits (OXIS International, Portland, OR). Glutathione peroxidase activity was measured using the coupled test procedure (Flohe, 1989), in which the action of GSH-Px is coupled to that of GR, and the change in absorbance at 340 nm measured as NADPH was oxidized in the presence of the substrate tert-butyl hydroperoxide (Oxis International). Glutathione reductase activity was determined spectrophotometrically after the oxidation of NADPH at 340 nm in the presence of excess GSSG (Carlberg and Mannervik, 1985). Enzyme activity was expressed in milliunits and was defined as the amount of enzyme catalyzing the reduction of 1 nmol of tert-butyl hydroperoxide per minute at pH 7.6 and 25°C for GSH-Px and as the amount of enzyme catalyzing the reduction of 1 nmol of GSSG per minute at pH 7.6 and 25°C for GR. All enzyme measurements were adjusted based on the total protein content of the PBMC isolate (BioRad Laboratories, Hercules, CA), which was quantified using the Coomassie Blue method of Bradford (1976). Determination of the GSH:GSSG ratio was accomplished by employing a spectrophotometric methodology based on the enzymatic techniques developed by Tietze (1969). Malondialdehyde was evaluated as a marker of lipid peroxidation by recording formation of a spectrophotometrically detectable carbocyanine dye, which results from the reaction of MDA with N-methyl-2-phenylindole at 45°C (Gerard-Monnier et al., 1998). Whole blood Se concentration was determined using graphite furnace atomic absorption spectrophotometry on a Perkin Elmer AAnalyst 800 (PerkinElmer Life and Analytical Science Inc., Wellesley, MA). Blood was diluted 10-fold in distilled water and, after addition of a Pd/Mg nitrate matrix modifier (Van Cauwenbergh et al., 1990), and absorption was measured at 196 nm. Serum prolactin was assayed by RIA (Miller et al., 1999). Sensitivity was 1.04 ng/mL. Intra- and interassay CV were 10.5 and 9.1%, respectively.
Statistical Analysis
Data were analyzed using repeated measures, using PROC MIXED with paddock within block as the experimental unit and block as a random effect (SAS Inst. Inc., Cary, NC). Fescue type, date, and the fescue type x date interaction were the main effects. The arh (1) covariance structure was the best fit for data based on Akaike’s information criterion. Tukey’s test was applied post hoc to test significant fescue type x date interactions. For gain, initial BW was added to the model as a covariate. Significance was determined at P < 0.05 and a trend at P < 0.10.
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RESULTS AND DISCUSSION
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One goal of the current experiment was to accurately assess heifer body temperatures at rest. Rectal temperatures collected while cattle are in the squeeze chute may be affected by confounding variables (e.g., exertion required in walking from paddocks to handling facilities, handling-induced stress, barn environment, and waiting time). For this reason, indwelling data loggers were used to measure intravaginal temperatures over the entire 48-h period preceding blood collection. Because we were concerned that heat stress may be a function of both body heating and body cooling, we chose to analyze average intravaginal temperatures taken from periods when heifers were hottest (1600 to 2000) and coolest (0400 to 0800). Heifers consuming E+ fescue tended (P = 0.075) to have greater Tmax than heifers consuming E– fescue. Heifers had greater Tmax in the hotter months, July (P < 0.0001) and August (P = 0.01), than in June (Table 1). Severity of heat events is often defined by their respective THI. Livestock managers are advised to be on alert when THI range from 75 to 78, whereas THI from 79 to 84 are classified as dangerous conditions for cattle (Hubbard et al., 1999). Although heifers were exposed to THI in the alert zone during all 3 mo, ambient conditions were most severe during July and August, with July THI reaching the danger threshold (Figure 1). The normal range for bovine core temperatures is 38.0 to 39.0°C (Jackson and Cockcroft, 2002). With the exception of the heifers stocked on E– fescue in August, average Tmax exceeded these ranges in July and August. Bourke (2003) suggested that 39.5°C is the critical rectal temperature at which hyperthermia can be said to exist in cattle. Although the mean Tmax in our heifers never surpassed this value, the observation that average afternoon body temperatures in late summer exceeded the reference range indicated that the animals were heat-stressed. During August, there tended to be a fescue x date interaction (P = 0.095), with E+ heifers having greater Tmax than E– heifers (39.4 ± 0.16°C vs. 38.8 ± 0.16°C). Differences in morning heifer temperatures were not detected at any sampling dates (P = 0.430).
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Table 1. Least squares means for intravaginal temperature1 of heifers grazing endophyte-infected (E+) or endophyte-free (E–) tall fescue in June, July, and August
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Date and a fescue type x date interaction were detected for T (Table 1
), with the greatest T in July, least T in June, and August T being intermediate. Ambient temperature followed a similar pattern. In August, E+ heifers exhibited greater (P = 0.046) T than E– heifers (1.41 ± 0.09°C vs. 1.14 ± 0.09°C). Morning heifer temperatures were numerically not different, so the interaction was associated with daytime temperature elevations in Tmax. Differences in nighttime and daytime THI were also greatest during August (Figure 1).
A date effect on ADG was detected (Table 2). Daily gain was greatest (P < 0.0001) in all heifers during the month of August, whereas weight loss occurred during July. Cattle gains on E+ fescue are generally negatively influenced by the presence of the endophyte (Stuedemann and Hoveland, 1988; Strickland et al., 1993; Oliver, 1997). However, there was no effect of fescue type on heifer ADG. Elevated ambient temperatures during the current experiment, along with summertime declines in forage availability such as those described by Rotz (2006), may explain the observed BW losses.
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Table 2. Least squares means for ADG, reduced to oxidized glutathione ratio, enzyme activity, malondialdehyde, and blood Se of heifers grazing endophyte-infected (E+) or endophyte-free (E–) tall fescue in June, July, and August
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A decrease in PBMC GSH:GSSG was observed after the first sampling date in heifers grazing E+ and E– fescue (Table 2). As the predominant low-molecular weight thiol in animal cells, GSH serves as a crucial antioxidant to offset environmentally derived oxidative insult. Oxidation of GSH occurs either directly or through enzymatic means, as free radicals and other reactive oxygen species are scavenged (Wu et al., 2004). The GSH:GSSG in heifer PBMC was less (P < 0.0001) in both July and August than at the initial sampling time (Table 2). A lower ratio, which is commonly used as an indicator of cellular redox, indicates either an increased oxidative load on the cells or decreased anti-oxidant defenses. Failure to detect any fescue effects in conjunction with the date effects may indicate that the elevated THI during these months were sufficient to create this outcome regardless of endophyte toxins. To the best of the authors’ knowledge, this is the first report of glutathione redox balance in PBMC from animals grazing E+ fescue. Lowered GSH:GSSG could result from depletion of GSH, increased generation of GSSG, or both. Lakritz et al. (2002) indicated that heat stress induced an increase in GSSG in bovine whole blood, independent of endophyte consumption. Hyperthermia alone can increase the reactivity of oxygen-based radicals (Issels et al., 1986). Settivari et al. (2006) observed that rats consuming E+ fescue seeds downregulated transcription of genes responsible for GSH production in vivo. Support for the notion that glutathione production is compromised is supported by the finding that heat stress in combination with E+ fescue consumption depletes total glutathione (GSH + GSSG) concentration in whole blood (Lakritz et al., 2002). Similarly, application of heat stress to cultured bovine lymphocytes reduces the concentration of total intracellular glutathione (Paula-Lopes et al., 2003). Unfortunately, our experimental methods did not allow the determination of the total amount of glutathione (GSH + GSSG). However, GSH:GSSG comparisons can be made to ratios extrapolated from previous work. Whole blood GSH:GSSG ratios ranged from 162 in cows in thermoneutral conditions to 73 in cows exposed to heat stress and E+ fescue (Lakritz et al., 2002). This same ratio in blood of dairy cows of normal vs. high BCS was reported to be 0.66 and 1.9, respectively (O’Boyle et al., 2006). In the latter study, the authors cited extreme variability in GSH:GSSG, which may have been related to a delay in postsampling addition of the thiol scavenger. In the current study, GSH:GSSG was determined in isolated PBMC. Although we are not aware of any reports on GSH:GSSG in bovine immune cells, values from the current study are within the range of those cited in canine (15 to 80) PBMC (Zickler et al., 2002), murine (25 to 142) peritoneal leukocytes (Alvarez et al., 2006), and human (18.4 to 22.2) neutrophils (Tauler et al., 2002).
Similar to the GSH:GSSG ratio, GR activity was influenced by date (Table 2). Activity of GR was less (P = 0.034) at the July sampling than in June, whereas GR activity at the August sampling only tended (P = 0.053) to be lower than that in June (Table 2). The decrease of GR activity is related to the decrease in GSH:GSSG, because GR is important in maintaining normal physiological ratios of GSH:GSSG (Griffith, 1999). Several authors have reported the possibility of an adaptive upregulation of GR with the onset of oxidant-related stress (Schirmer et al., 1989; Townsend et al., 2003). Current observations regarding GSH:GSSG strongly support the presence of thermally induced oxidative stress in bovine PBMC, yet contrary to an adaptive response, a reduction in GR activity was observed. We are unaware of any previous literature regarding GR activity in response to endophyte consumption.
Several speculations are offered in support of the present data. As evidenced by the negative BW gains observed between the June and July sampling dates, forage availability may have become limiting. In addition, heat stress is known to decrease feed intake and to reduce the length of daily grazing time (VanSoest, 1982; Trout et al., 1998). Thus, activity of GR may have been nutritionally related. Food-deprived rats have less activity of erythrocyte GR than rats fed ad libitum (Wohaieb and Godin, 1987). Glutathione reductase is an enzyme that depends on flavin adenine dinucleotide as a prosthetic group (Schirmer et al., 1989) and is dependent upon an adequate supply of riboflavin. In humans, altered riboflavin status can account for dissipation of GR activity (Tessier et al., 1995; Tauler et al., 2006a). Rumen microflora should synthesize adequate riboflavin to render such a hypothesis unlikely in cattle (Santschi et al., 2005; Schwab et al., 2006). An alternative explanation for reduced GR activity in July could be a possible depletion of total glutathione. Total glutathione availability is compromised by heat stress (Paula-Lopes et al., 2003) or reduced energy intake (Tateishi, 1990; Lu, 2000; Wu et al., 2004). Depletion of total glutathione could cause a decrease in the overall concentration of GSSG in spite of increased GSH:GSSG. Oxidized glutathione plays a role in protecting GR against redox inactivation, and a decrease in GSSG concentration can reduce GR activity in vitro (Mata et al., 1985; Lopez-Barea et al., 1990). This conjecture would not be supported by the findings of Lakritz et al. (2002), which indicated increases in bovine blood GSSG despite exposure to thermal stress. Still, heifers used in the current experiment were subjected to heat stress and, as previously suggested, may have had limited intake in July, so the possibility that GR was redox inactivated cannot be ruled out.
Activity of PBMC GSH-Px followed a pattern similar to that of GR. Like GR, GSH-Px activity was less during the month of July compared with June (P < 0.001) and August (P < 0.001; Table 2). Again, present results are contrary to the suggestion that GSH-Px is an enzyme that is induced by oxidant-applied stress (Townsend et al., 2003; Surai, 2006a; Tauler et al., 2006b). Saker et al. (2004) previously observed increases in erythrocyte GSH-Px activity in heat-stressed wethers consuming E+ fescue hay and suggested an endogenously regulated protective response by this antioxidant enzyme. Similarly, after an initial decline, white blood cell GSH-Px activity in the wethers increased in response to heat stress. Bernabucci et al. (2002) found that plasma GSH-Px activity was not different between transition dairy cows exposed to elevated summertime THI and those in temperate conditions. In contrast, heat-stressed cows had erythrocyte GSH-Px activity that exceeded that of the cows transitioning during cooler periods. Conversely, our data exhibited a decrease in PBMC GSH-Px activity in spite of elevated THI. Like GR, the reduced GSH-Px activity may be nutritional in nature. Because of the essentiality of Se in GSH-Px, maintenance of enzyme activity is tightly linked to Se status (Rotruck et al., 1973).
The Se status of heifers was affected by sampling date (Table 2). Fescue type had no effect on whole blood Se (P = 0.86), but Se concentrations were lower (P < 0.001) in both July and August than in June. This alteration in Se availability is possibly related to the decrease in GSH-Px activity that was observed in July. Cattle are Se-deficient when the concentration in whole blood is less than 80 ppb (Puls, 1988), and the correlation between subadequate GSH-Px activity and Se status is greater in Se-deficient cattle than in Se-adequate cattle (Surai, 2006b). Heifers were given ad libitum access to a mineral mixture without Se. Thus, reduced forage DMI or prolonged consumption of low Se forage could have caused the observed decline in Se status. The apparent recovery in GSH-Px activity during the month of August despite a still low Se status is difficult to explain but may relate to utilization of body Se reserves. According to Surai (2006a), when Se availability is adequate, Se-containing AA are nonspecifically incorporated into many body proteins, such as those making up skeletal muscle. However, when Se becomes limiting, stress conditions may increase selenoprotein requirements despite decreased Se bioavailability. Proteosomes that degrade body proteins are suggested to be activated during such conditions, thereby releasing Se-containing AA for additional synthesis of selenoproteins like GSH-Px (Surai, 2006a). Proteosome activity may be partially regulated by redox balance, so it is possible that this mechanism could account for the rebound in GSH-Px activity that was observed in the heifers during August. Alternatively, the increase in GSH-Px activity observed at the last sampling could represent an adaptive increase to oxidative stress experienced over the previous 2 mo, with the lag in response due to the life span of the PBMC. Whole blood Se and erythrocyte GSH-Px are highly correlated in cattle, but plasma Se and plasma GSH-Px are not (Scholz and Hutchinson, 1979). The relationship between plasma Se and PBMC GSH-Px activity is not well defined. Association of these 2 variables may be weakened by the fact that plasma Se is a short-term marker of Se status, whereas PBMC GSH-Px, because of the life span of the cell, is a longer-lived response variable.
No treatment or date effects were detected for the concentration of plasma MDA (Table 2). Malondialdehyde is a by-product of lipid peroxidation, and peripheral accumulation indicates oxidative damage of tissues. Regardless of the lowered GSH:GSSG in PBMC, no evidence of oxidative insult could be found using this whole-body biomarker of oxidative stress. Realini et al. (2005) previously showed that tissue from cattle grazing E+ fescue is not at increased risk of postmortem lipid oxidation. However, it was expected that some consequence of E+ consumption on plasma lipid peroxidation might be observed in the current experiment. Plasma lipid hydroperoxides increased in wether lambs fed E+ hay during a period of heat stress (Saker et al., 2004), but this effect could have been due to the heat, rather than the presence of the endophyte. Bernabucci et al. (2002) noted that transition dairy cows exposed to a hot environment displayed greater erythrocyte MDA than similar cows exposed to a thermoneutral environment. Interestingly, a difference in plasma MDA concentrations in the same cows was not detected. Similarly, Trout et al. (1998) found that dairy cattle exposed to heat stress via controlled chambers exhibited no evidence of increased MDA concentration in muscle tissue. Likewise, there was no effect of elevated ambient temperatures on lipid oxidation markers in the plasma in the current study.
The toxic potential of the paddocks utilized in the present experiment had been evaluated in previous grazing trials. In the year preceding the current study, E+ paddocks had ergovaline and lysergic acid amide concentrations of 330 and 424 µg/kg of DM, respectively (Stewart, 2006). Neither ergovaline nor lysergic acid amide were detectable in samples taken from E– paddocks. In 2004 (Boland, 2005) and 2005 (Stewart, 2006), immunoblot testing of tillers (Hiatt et al., 1997) revealed a rate of endophyte infection exceeding 80% in E+ paddocks, whereas endophyte was not detectable in E– paddocks. One consistent physiological response to fescue toxicosis in cattle is a reduction in serum prolactin (Porter and Thompson, 1992; Strickland et al., 1993). Indeed, this effect is so well characterized that it has been routinely used to document that livestock are being affected by E+ alkaloids (Oliver, 1997). In addition to the historical data provided above, we measured serum prolactin concentrations (intraassay CV 10.5%; interassay CV 9.1%) at the last sampling date to verify the distress produced by E+ fescue consumption. Serum prolactin concentrations were lower (P = 0.008) in heifers stocked on E+ fescue (20 ± 3.0 ng/mL) in August than in heifers stocked on E– fescue (167 ± 18.0 ng/mL). Rates of endophyte infection or alkaloid concentrations required to reduce serum prolactin have not been precisely defined. Watson et al. (2004) did report reduced prolactin in cattle grazing pastures with only 448 ppb of ergot alkaloids, whereas Parish et al. (2003) observed prolactin declines when cattle grazed E+ pastures ranging from 822 to 1208 ppb in alkaloid content. Wide ranges of serum prolactin were reported by these authors depending on season, location, animal sex, and age. In addition, alkaloid concentrations of pastures used in their studies were less than those of the E+ paddocks (1,565 ppb) in the present experiment. The magnitude of difference in serum prolactin from heifers grazing E+ vs. E– fescue in the current experiment is similar to the magnitude of difference reported in cattle grazing E+ vs. nonergot alkaloid-producing endophyte-infected fescue (Nihsen et al., 2004) and similar to the magnitude of change reported when cattle are switched from E– to E+ fescue pastures (Aiken et al., 2006).
Heifers grazing E+ fescue in the present experiment had lowered serum prolactin, indicating that they were experiencing the physiological effects of fescue toxicosis. Our data suggested no influence of E+ fescue consumption on glutathione redox in PBMC or accumulation of lipid peroxidation products in plasma. Using the selected biomarkers, we found no evidence that consumption of endophyte toxins promotes oxidative stress in cattle. Our experiment is the first to report GSH:GSSG and GR activity in immune cells from animals grazing E+ fescue under heat stress. Results indicated that cattle experiencing heat stress have altered immune cell redox, which may be exacerbated when nutrition becomes limiting. Forage availability and DMI were speculated to affect antioxidant enzyme activity, but the nature of this influence was not well characterized. Further research will clarify this relationship and the magnitude of its importance in heat-stressed cattle.
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Footnotes
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1 Research supported in part by Pasture-Based Beef Systems for Appalachia, a regional initiative funded by USDA-ARS. We are thankful to W. E. Beal for loan of the heifers and to A. O. Abaye for use of the fescue pastures. In addition, we appreciate the assistance of T. Shanklin, J. Martin, C. Sink, A. Lillie, H. Boland, R. Stewart, J. Rotz, and A. Lucas (Virginia Polytechnic Institute and State University). 
2 Corresponding author: cvmwss{at}vt.edu
Received for publication May 26, 2007.
Accepted for publication July 30, 2007.
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Abstract
INTRODUCTION
