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Duration of restraint and isolation stress as a model to study the dark-cutting condition in cattle^1,2

Department of Animal Sciences, University of Arkansas, Fayetteville 72701

	Abstract

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Holstein steer calves (n = 32; 156 ± 33.2 kg average BW) were used to evaluate the duration of restraint and isolation stress (RIS) on endocrine and blood metabolite status and the incidence of dark-cutting LM. Calves were blocked by BW and assigned randomly within blocks to one of four stressor treatments: unstressed controls (NS) or a single bout of RIS for 2, 4, or 6 h. Venous blood was collected via indwelling jugular catheters at 40, 20, and 0 min before stressor application and at 20-min intervals during RIS. Unstressed calves remained in their home stanchions and, except for blood sampling, were subjected to minimal handling and stress. Serum cortisol and plasma lactate concentrations were increased (P <0.01) during the first 20 min after RIS application, and remained elevated throughout the 6 h of RIS. Plasma concentrations of glucose and insulin were greater (P <0.05) in RIS calves than in NS calves after 80 and 100 min of stressor application, respectively; however, RIS did not (P >0.80) affect plasma NEFA concentrations. Calves were slaughtered within 20 min of completion of RIS, and muscle samples were excised from right-side LM at 0, 0.75, 1.5, 3, 6, 12, 24, and 48 h after exsanguination for quantifying LM pH, and glycogen and lactate concentrations. The pH of the LM from calves subjected to 6 h of RIS exceeded 6.0, and was greater (P <0.05) at 24 and 48 h postmortem than the pH of NS calves or calves subjected to 2 or 4 h RIS. Muscle glycogen concentrations did not differ (P = 0.16; 25.58, 10.41, 13.80, and 14.41 µmol/g of wet tissue weight for NS and 2-, 4-, and 6-h RIS, respectively), and LM lactate concentrations tended to be lower (P = 0.08) in calves subjected to 6 h of RIS. At 48 h after exsanguination, the LM from calves subjected to 6 h of RIS had more (P <0.05) bound and less (P <0.05) free moisture than did the LM from NS calves or calves subjected to 2 or 4 h of RIS. Additionally, the LM from RIS calves was darker (lower L* values; P <0.05) than the LM of NS calves. Visual color scores for the LM were greatest (P < 0.05) for calves subjected to 6 h of RIS and least (P <0.05) for NS calves. Subjecting lightweight Holstein calves to 6, 4, and 2 h of RIS resulted in six (75%), two (25%), and two (25%) carcasses characteristic of the dark-cutting condition, respectively. There were no dark-cutting carcasses produced from NS calves. Thus, RIS may be a reliable animal model with which to study the formation of the dark-cutting condition.

Key Words: Cattle • Cortisol • Dark-Cutting Condition • Meat Quality • Postmortem Metabolism • Stress

	Introduction

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Dark-cutting meat is a persistent quality defect characterized by elevated muscle pH (6.0); high water-holding capacity; dry, firm, and "sticky" lean; and a dark-red to almost black lean color. It is widely accepted that decreased muscle glycogen reserves before slaughter are responsible for the formation of dark-cutting meat. Excessive antemortem glycogenolysis may arise from any change in the physical and/or psychological well being of an animal. More importantly, the dark-cutting condition (DCC) costs the U.S. beef industry between $132 and $170 million annually (Smith et al., 1992, 1995).

The inability to consistently produce dark-cutting carcasses impedes replication of experimental results, and hinders the ability to test possible management practices and/or treatments to decrease/eliminate the DCC. In an attempt to develop an animal model to study the DCC under controlled experimental conditions, sheep were used to test the effects of a physical stressor (treadmill exercise; Apple et al., 1994) and a psychological stressor (restraint and isolation; Apple et al., 1993a,b) on producing dark-cutting meat. Although plasma indicators of stress were evident in exercised sheep, no dark-cutting lamb carcasses were produced (Apple et al., 1994). After some modifications, Apple et al. (1995) found that exposing sheep to a single 6-h bout of restraint and isolation stress (RIS) effectively increased circulating stress hormone concentrations, dramatically decreased glycogen reserves before slaughter, and produced 100% dark-cutting carcasses. Nonetheless, questions arose concerning the applicability of this animal model to cattle; therefore, the primary objective of this experiment was to evaluate RIS as a repeatable, reliable animal model to study the DCC using lightweight, Holstein calves.

	Materials and Methods

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Animals and Stressor Treatment
Before the initiation of this experiment, animal treatment and all experimental protocols were approved by the University of Arkansas Interdepartmental Animal Care and Use Committee (Project No. 01028). Thirty-two Holstein steer calves (156 ± 33.2 kg average BW) were purchased from a local backgrounder, and blocked by weight into four blocks (eight calves per block). Within blocks, calves were assigned randomly to one of four stressor treatments: 1) unstressed controls; 2) subjected to a single 2-h bout of RIS; 3) subjected to a single 4-h bout of RIS; and 4) subjected to a single 6-h bout of RIS. Calves had ad libitum access to a concentrate diet (Table 1) and water for a minimum of 4 wk. Seven days before a stressor treatment, each block of calves was moved from the University of Arkansas Beef Cattle Research Unit to the University of Arkansas Calf Research Facility, stanchioned in 1.65 m x 0.64 m metabolic crates with rubber-coated woven-wire floors (raised 0.38 m off the ground), and individually fed twice daily the same concentrate diet at a rate of 2.5% of their individual BW. Body weights were the average of weights obtained on two consecutive days before the calves were moved. Calves had ad libitum access to water via automated bowl-waterers attached to each crate.

Restraint and isolation stressor treatment consisted of moving calves from their home stanchions approximately 50 m to another barn where they were isolated from visual and tactile contact with other calves. Restraint was achieved by placing calves in right lateral recumbency and binding both forelimbs and both hind-limbs together with a nonadhesive tape (Vetrap bandaging tape, 3M Animal Care Products, St. Paul, MN). Finally, both sets of limbs were bound together with elastic adhesive tape (Elastikon, Johnson & Johnson Products, New Brunswick, NJ). To minimize physical discomfort during stressor application, restrained calves were placed on 12.7 cm of dense carpet padding. With the exception of blood sampling, unstressed controls remained in their home stanchions and were subjected to minimal handling and stress. Upon completion of the specified duration of RIS, calves were transported approximately 275 m to the University of Arkansas Red Meat Research Abattoir, and slaughtered within 20 min of completion of RIS according to industry-accepted procedures. Unstressed controls were transported and slaughtered immediately after the calves that were subjected to 6 h of RIS.

Blood Collection and Assays
On the morning of each day of stressor application, samples of venous blood were collected 40, 20, and 0 min before RIS application. The RIS calves (two per duration) were then moved and subjected to restraint and isolation as described previously. After initiation of stressor treatment, blood samples were collected at 20-min intervals for the calf’s assigned duration. Aliquots of blood samples were placed into three tubes: one plain glass tube used for serum contisol; one tube containing sodium fluoride and potassium oxalate used for plasma glucose and lactate; and one tube containing EDTA used for plasma NEFA. Blood samples were kept on ice until transportation to the laboratory, where samples were centrifuged at 1,200 x g for 30 min, and the serum or plasma was stored at –20°C until analyzed. Serum cortisol and plasma insulin concentrations were measured using direct RIA, in which the appropriate antibody was coated to plastic tubes (Diagnostic Products Corp., Los Angeles, CA). The intra- and interassay CV for serum cortisol were 5.11 and 11.5%, respectively, whereas the intra- and interassay CV for plasma insulin were 13.1 and 6.20%, respectively. Glucose and lactate were measured in plasma fractions using commercially available diagnostic kits (Sigma Chemical, St. Louis, MO), whereas plasma NEFA concentrations were quantified with a commercially available enzymatic kit (Wako Chemicals USA, Dallas, TX).

Animal Slaughter and Muscle Data Collection
Calves were rendered unconscious and insensitive to pain via nonpenetrating captive bolt, and were exsanguinated. Immediately following exsanguination, two 1.27-cm-diameter cores were removed from the LM perpendicular to the length of the LM on the right side at the level of the 7th thoracic vertebrae. Subsequently, LM samples were removed at 0.75, 1.5, 3, 6, 12, 24, and 48 h after exsanguination at 5-cm distances caudally from the previous sample. Approximately 2 g of LM was removed from one core and used for pH determinations, whereas the remainder of the core, as well as the second core, was immediately frozen in liquid N₂ and stored at –20°C for determination of LM glycogen (Lo et al., 1970) and lactate (McGinnis et al., 1989) concentrations.

Samples for pH were homogenized in 20 mL of 5 mM sodium iodoacetate in 150 mM of KCl (Bendall, 1973). The pH of the homogenate was measured with a temperature-compensating combination electrode (model 300731.1, Denver Instrument Co., Arvada, CO) attached to a pH/ion/FET-meter (model AP25, Denver Instrument Co.).

When quantifying LM lactate concentrations, approximately 1 g of frozen LM was homogenized in 2.0 mL of 6% (vol/vol) perchloric acid for 30 s with a homogenizer (model PRO250, PRO Scientific, Inc., Monroe, CT) set on high. Additional perchloric acid was added to yield a final volume of 19.3 mL/g of tissue, and was homogenized for 1 min. The homogenate was filtered under vacuum, and a 4-mL aliquot of filtrate was neutralized with 0.2 mL of 5 M potassium carbonate and centrifuged at 1,500 x g for 10 min. The resulting supernatant was aspirated into plastic tubes, capped, and frozen in liquid N₂ to be used for determination of LM lactate concentrations with a commercially available kit (Sigma Chemical) according to the procedure of McGinnis et al. (1989).

For muscle glycogen levels, 30 to 60 mg of frozen LM and 0.5 mL of 30% KOH were boiled in test tubes for approximately 25 min in a 100°C water bath. Samples were then cooled on ice, and 0.7 mL of 95% ethanol was added to each tube, placed on ice for 30 min, and then centrifuged at 2,000 x g for 20 min. Supernatant fluid was aspirated and discarded, and 3 mL of distilled water were added to the precipitate and vortexed, after which 1 mL of 5% phenol and 5 mL of concentrated H₂SO₄ were added to a 1-mL aliquot of sample, vortexed, cooled, and the absorbance of sample was read at 490 nm according to the procedure of Lo et al. (1970). Standard curves were run with each assay to determine LM glycogen concentrations, which were reported as micromoles per gram of wet tissue weight.

Carcass Data Collection
Carcasses were chilled conventionally at 1°C for 48 h, after which left sides of each carcass were ribbed at the 12th-/13th-rib juncture, and wholesale ribs were removed from the left-side forequarters. Beginning at the posterior end of each rib, three 2.54-cm-thick LM chops were cut. Two chops were used for visual and instrumental color measurements, whereas the third chop was designated for moisture determinations. Visual color of the LM chops was evaluated using the six-point Japanese color standards for pork (Nakai et al., 1975) by a three-person panel after a 30-min bloom period at 2°C. The Japanese pork color scoring system consists of six plastic disks with meat-like texture and appearance developed from objective colorimetry (1 = pale gray to 6 = dark purple) and has been widely used as the standards for visual appraisal of veal color (E. D. Mills, Pennsylvania State University, University Park, personal communication,). Instrumental color (L*, a*, and b* values) of LM chops was determined from a mean of eight random readings (four per LM chop) using a Hunter MiniScan XE (model 45/0-L, Hunter Associates Laboratory, Reston, VA) using illuminant C and a 10° standard observer. Immediately after color data collection, both 2.54-cm-thick chops were wrapped in freezer paper and frozen at –20°C before cooking and Warner-Bratzler shear force (WBSF) determinations.

LM Moisture and Water-Holding Capacity Determinations
Moisture content of the LM was determined using the freeze-drying procedure of Apple et al. (2001). Briefly, duplicate 5-g samples of LM were weighed, placed in 30-mL beakers, and reweighed. Then, beakers containing samples of LM were dried for 60 h in a Labconco freeze-dryer (model 4.5, Labconco Corp., Kansas City, MO), with a temperature of –50°C and a vacuum of less than 10 µm Hg. The difference between initial and dried beaker weights was divided by the initial sample weight to calculate LM moisture percent.

Additionally, LM water-holding capacity was measured with the compensating planar planimeter method of Urbin et al. (1962), where approximately 500 mg of minced LM and Whatman No. 1 filter paper (Maidstone, U.K.) were weighed. Then, the sample was placed on the filter paper, placed between two Plexiglas sheets, and pressed for 1 min at 35.2 kg/cm² of force in a Carver press (Fred S. Carver, Inc., Summit, NJ). Plexiglas sheets were separated, and the inner (meat film area) and outer (total surface area) moisture edges were carefully traced, and areas of each circle were measured using a compensating planimeter. The percentage of free moisture/water was calculated as follows: [(total surface area – meat film area) x 61.1]/total moisture content of the LM sample, with the result multiplied by 100. The percentage of bound moisture/water was calculated as 100 – percentage of free moisture/water.

Warner-Bratzler Shear Force and Cooking Loss Determinations
Chops were thawed for 16 h at 2°C, deboned, weighed, and then cooked to an internal temperature of 71°C in a commercial convection oven (Zephaire E, Blodgett Oven Co., Burlington, VT) preheated to 165°C. Internal temperature was monitored with Teflon-coated thermocouple wires (Type T; Omega Engineering, Inc., Stamford, CT) placed in the geometric center of each chop and attached to a multichannel data logger (model 245A, VAS Engineering, Inc., San Diego, CA). Chops were turned once during cooking when the internal temperature reached 35°C. Immediately after removal from the oven, chops were blotted dry on paper towels, weighed, and the difference between pre- and post-cooked weights was used to calculate cooking loss percentage. Chops were chilled at 4°C for 24 h, and at least six good, 1.27-cm-diameter cores were removed parallel to the muscle fiber orientation, and sheared through the center with a WBSF device attached to an Instron Universal Testing Machine (model 4466, Instron Corp., Canton, MA), equipped with a 55-kg tension/compression load cell, and a crosshead speed set at 250 mm/min. Shear force values of the six cores from each chop were averaged for statistical analyses.

Statistical Analyses
All data were analyzed as a randomized complete block design with individual calf as the experimental unit. Data from one calf (unstressed control) were removed because at slaughter, it was discovered that the calf had a metabolic disorder and internal hemorrhaging. Blood data (pooled across all RIS durations), as well as postmortem muscle data, were analyzed as repeated measures using the PROC MIXED with MIVQUE(0) procedure of SAS (SAS Inst., Inc., Cary, NC), with sampling time as the repeated variable and calf as the subject. Fixed effects included in the model were stressor treatment, sampling time, and the treatment x time interaction, whereas block and the block x treatment x calf interaction were included in the model as random effects. The ANOVA for all quality data also was generated with PROC MIXED, with stressor treatment as the lone fixed effect and block as the random effect in the statistical model. Least squares means were calculated for all main and interactive effects, and were separated statistically using the PDIFF option of SAS when the F-test was significant at P 0.05.

	Results

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Within 20 min of stressor application, serum cortisol concentrations were elevated (P <0.001) in RIS calves compared with their unstressed counterparts, and remained elevated throughout stressor treatment (treatment x time interaction; P <0.001; Figure 1). Additionally, plasma glucose (Figure 2) and lactate (Figure 3) concentrations were higher (P <0.01) in calves subjected to RIS than in unstressed controls. Plasma glucose concentrations were greater (P <0.01) in RIS than in unstressed calves after 80 min of RIS (treatment x time interaction; P <0.001; Figure 2). Conversely, plasma lactate concentrations rose immediately after stressor application, and were higher (P <0.01) in RIS calves than in control calves from 20 to 360 min after stressor application (treatment x time interaction; P <0.001; Figure 3). Plasma insulin concentrations did not differ (P >0.15) during the first 80 min after stressor application, but from 100 to 340 min of RIS, stressed calves had greater (P <0.05) insulin concentrations than unstressed calves (treatment x time interaction; P <0.001; Figure 4). At the end of RIS (360-min sample), circulating insulin concentrations did not (P = 0.11) differ between RIS and control calves. Finally, plasma NEFA concentrations did not differ (main effect; P >0.80) between RIS and unstressed calves (Figure 5).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of restraint and isolation stress (RIS) on serum cortisol concentrations (treatment x time; P <0.001). Data for unstressed controls and RIS calves from 280 to 360 min represent the least squares means (±SE) of seven and eight calves, respectively. Data for RIS calves from –40 to 120 min represent the least squares means (±SE) of 24 RIS calves, and data from 140 to 240 min represent 16 calves. Points with an asterisk (*) differ (P <0.001) within a specific time.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of restraint and isolation stress (RIS) on plasma glucose concentrations (treatment x time; P <0.001). Data for unstressed controls and RIS calves from 280 to 360 min represent the least squares means (± SE) of seven and eight calves, respectively. Data for RIS calves from –40 to 120 min represent the least squares means (±SE) of 24 RIS calves, and data from 140 to 240 min represent 16 calves. Points with an asterisk (*) differ (P <0.01) within a specific time.

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of restraint and isolation stress (RIS) on plasma lactate concentrations (treatment x time; P <0.001). Data for unstressed controls and RIS calves from 280 to 360 min represent the least squares means (±SE) of seven and eight calves, respectively. Data for RIS calves from –40 to 120 min represent the least squares means (±SE) of 24 RIS calves, and data from 140 to 240 min represent 16 calves. Points with an asterisk (*) differ (P <0.01) within a specific time.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of restraint and isolation stress (RIS) on plasma insulin concentrations (treatment xtime; P <0.001). Data for unstressed controls and RIS calves from 280 to 360 min represent the least squares means (±SE) of seven and eight calves, respectively. Data for RIS calves from –40 to 120 min represent the least squares means (±SE) of 24 RIS calves, and data from 140 to 240 min represent 16 calves. Points with an asterisk (*) differ (P <0.05) within a specific time.

View larger version (24K): [in this window] [in a new window]   Figure 5. Effect of restraint and isolation stress (RIS) on plasma NEFA concentrations (treatment xtime; P = 0.9213). Data for unstressed controls and RIS calves from 280 to 360 min represent the least squares means (±SE) of seven and eight calves, respectively. Data for RIS calves from –40 to 120 min represent the least squares means (±SE) of 24 RIS calves, and data from 140 to 240 min represent 16 calves.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effects of restraint and isolation stress (RIS) duration on LM pH (treatment xtime; P <0.001). Data represent the least squares means (±SE) of seven unstressed control calves and eight calves per RIS duration (2, 4, or 6 h). Within a specific time postmortem, points that do not have common letters differ, P <0.05.

Total LM moisture content did not differ (P = 0.48) among the treatment groups, but the LM of calves subjected to 6 h of RIS had a greater (P <0.05) percentage of bound and a lower (P <0.05) proportion of free moisture/water than did the other treatment groups (Table 2). Although noticeable differences existed in the water-holding capacity between RIS and unstressed calves, cooking loss percents did not (P = 0.26) differ among treatments. However, cooked LM from RIS calves, regardless of RIS duration, had lower (P <0.05) WBSF values than the LM from unstressed controls.

	Discussion

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The incidence of dark-cutting carcasses is quite variable, ranging from 5.0% of all steers and heifers slaughtered in 1990 (Lorenzen et al., 1993) to 2.7 and 2.3% in 1995 (Boleman et al., 1998) and 2000 (McKenna et al., 2002), respectively. Moreover, in a study evaluating slaughter data for a 1-yr period from four beef packing plants, Kreikemeier et al. (1998) found that the highest incidence of dark-cutting carcasses occurred during August, September, and October (1.1 to 1.4%), whereas the lowest incidence occurred between December and February (0.43 to 0.56%). Incidence of dark-cutting carcasses also varies among feed yards (0.05 to 0.64%; Scanga et al., 1998) and packing plants (0.28 to 1.24%; Kreikemeier et al., 1998). The manifestation of the DCC in feedlot heifers ranges from 0.38 (Scanga et al., 1998) to 4.7% (Lorenzen et al., 1993), whereas 70% of bull carcasses were classified as dark cutters (Bartos et al., 1988). In addition, Lorenzen et al. (1993) reported that 9.7, 4.7, and 4.4% of dairy, "native," and Bos indicus cattle slaughtered produced dark-cutting carcasses, respectively.

As early as 1944, scientists concluded that the elevated pH of dark-cutting meat was directly related to a deficiency of muscle glycogen before slaughter (Hall et al., 1944). Since that time, antemortem glycogenolysis has been readily accepted as the mechanism by which the DCC is elicited. Decreased glycogen reserves before slaughter and the formation of the DCC may arise from any change in the physical or psychological well being of an animal, including long-term (Price and Tennessen, 1981) or short-term (Kenny and Tarrant, 1987) transportation, animal handling (Warriss, 1990), food withdrawal (Warriss et al., 1987), commingling/mixing strange animals (Warriss et al., 1984; Schaefer et al., 1990), estrogenic implants (Scanga et al., 1998), and females in estrus (Kenny and Tarrant, 1988), as well as forced exercise (Chrystall et al., 1982) and shearing and preslaughter washing (Bray et al., 1989) in sheep. Thus, the considerable variation in genetics, environment, and management of animals, coupled with the inability to consistently produce dark-cutting carcasses under controlled conditions, has made it quite difficult to repeat previous experimental results, thereby hindering the ability to test possible management practices and/or treatments to decrease/eliminate the DCC in meat-producing animals.

Several attempts have been made to develop model-systems to study the DCC. Lister and Spencer (1983) infused sheep with methyl pyrazole carboxylic acid to inhibit lipolysis, and found that it effectively depleted muscle glycogen reserves and produced dark-cutting carcasses; however, this treatment did not alter plasma ACTH or cortisol levels typically associated with pre-slaughter stress. Research also was conducted on the use of epinephrine injections as a method to elicit the DCC in beef cattle, but the patterns of glycogen depletion and muscle pH elevation were considerably different between epinephrine-treated and mixed/commingled cattle (Tarrant and Sherington, 1980; Lacourt and Tarrant, 1985). Even though injections and/or infusions elicit the consequences of a stressor, these types of models do not provoke the central nervous system in the cognition, integration, and homeostatic responses typically associated with exposure to an acute or chronic stressor (Minton, 1994).

Use of acute stressor models has yielded varying results in producing dark-cutting carcasses. Treadmill exercise to near exhaustion failed to alter antemortem glycogen reserves or to produce dark-cutting carcasses in sheep (Forrest et al., 1964; Apple et al., 1994), but slaughtering sheep within 1 h of forced swimming resulted in mean LM pH values greater than the characteristic 6.0 (Petersen, 1983). Subjecting sheep to three consecutive days of RIS effectively decreased glycogen content of the semitendinosus, and elevated the ultimate (24-h) pH of the semimembranosus, biceps femoris, and infraspinatus, but no dark-cutting carcasses were detected (Apple et al., 1993b); however, sheep in that trial had access to a high-concentrate diet and water after each bout of RIS, and were not slaughtered until 18 h after the last bout of RIS. Conversely, when sheep were subjected to a single, 6-h bout of RIS and slaughtered within 30 min of RIS completion, antemortem glycogen reserves were depleted before slaughter, mean ultimate pH was approximately 6.4, and all RIS carcasses were classified as dark-cutters (Apple et al., 1995).

The present study was designed to test the applicability of the RIS sheep model of Apple et al. (1995) to cattle. In the present study, serum cortisol concentrations were robustly increased in calves subjected to RIS, which was consistent with observed increases in circulating ACTH and cortisol in sheep subjected to RIS (Apple et al., 1993a; Minton et al., 1995). Interestingly, Apple et al. (1995) reported that plasma cortisol concentrations increased dramatically within the first 12 min after RIS application in sheep, and serum cortisol concentrations of RIS calves in the present study were increased substantially within the first 20 min of stressor application. Furthermore, cortisol responses to RIS in the current study were consistent with increased blood cortisol concentrations in cattle to social isolation (Munksgaard and Simonsen, 1996; Rushen et al., 1999) and physical restraint (Lay et al., 1992; Wohlt et al., 1994).

In the present experiment, subjection of Holstein calves to 6 h of RIS also elicited dramatic increases in circulating glucose (Figure 2), lactate (Figure 3), and insulin (Figure 4), but it did not alter plasma NEFA concentrations (Figure 5), which mirrored response curves of these energy metabolites and insulin in sheep exposed to a single bout of RIS (Apple et al., 1995). Increases in blood lactate have been shown in cattle in response to handling and transportation (Mitchell et al., 1988; Schaefer et al., 1992; Steiger et al., 1999), and may be indicative of enhanced muscle glycogenolysis and hepatic gluconeogenesis (Brooks, 1986; Wasserman et al., 1987), as well as enhanced absorption of ruminally produced lactate (Ballard et al., 1969).

Changes in blood glucose in response to a stressor, however, have been variable. Some studies have reported a hyperglycemic effect of stress (Kent and Ewbank, 1983, 1986), whereas others have demonstrated that stress lowers (Cole et al., 1988; Kegley et al., 1997) or fails to alter (Locatelli et al., 1989; Sartorelli et al., 1992) circulating glucose levels. Nonetheless, it is readily accepted that blood glucose concentrations increase rapidly within approximately 1 h after initiation of a stressor due to mobilization of existing energy reserves by glucocorticoids and catecholamines (Eigler et al., 1979), as well as inhibition of further storage through rapid insulin resistance (Sapolsky et al., 2000). Even though glucocorticoids are antagonistic to insulin with respect to blood glucose concentrations, appetite, gluconeogenesis, protein synthesis and degradation, lipolysis, and lipogenesis (Strack et al., 1995), elevated glucocorticoids increase blood insulin concentrations (Dallman et al., 1993), and sustained glucocorticoid secretion causes sustained insulin secretion (Sapolsky et al., 2000). Thus, the classical insulin resistance observed in Holstein steers (present experiment) and sheep (Apple et al., 1995) subjected to RIS, is due to the negative effects of glucocorticoids on glucose transport into muscle (Weinstein et al., 1995; Dimitriadis et al., 1997) and adipocytes (Carter-Su and Okamoto, 1987), as well as decreased insulin sensitivity (Willi et al., 2002). During long-distance transportation or repeated transportations, blood cortisol declines to near or below baseline levels in response to stressor habituation (Warriss et al., 1995; Lay et al., 1996), thereby decreasing the inhibitory actions of glucocorticoids on insulin-mediated glucose transport and reestablishment of baseline blood glucose concentrations (Sapolsky et al., 2000). Additionally, some of the aforementioned studies imposed food deprivation (>24 h) on cattle before transportation (Cole et al., 1988), thereby complicating the interpretation of the hypoglycemic response to transportation stress. Thus, results of the present study with calves, coupled with previously published information from sheep (Apple et al., 1995; Minton et al., 1995), demonstrate that RIS is an effective stressor model that evokes participation of the central nervous system and probably the autonomic nervous system (Apple et al., 1995), as well as the hypothalamic-pituitary-adrenal axis, in the calf’s stress responses.

Dark-cutting meat is characterized by an ultimate muscle pH value in excess of 6.0, a dark-red to almost black lean color, and a high muscle water-holding capacity (Tarrant, 1989). Postmortem pH, the first criterion, decreases in a curvilinear fashion from an at-slaughter value of approximately 7.1 to 7.2 to an ultimate (24-h) pH value between 5.5 and 5.7 because of lactic acid accumulation in the muscle (Greaser, 1986). Postmortem pH decline in the LM of unstressed calves followed the typical pH decline, from an initial (0-min postexsanguination) value of 7.10 to 24- and 48-h pH values of 5.79 and 5.64, respectively (Figure 6). Conversely, the range in initial pH values for the LM of RIS calves was 7.08 to 7.19, and, more importantly, ultimate (24- and 48-h) pH values in the LM of calves subjected to 6 h of RIS exceeded 6.0 (mean pH values of 6.34 and 6.30, respectively).

In the present study, LM muscle glycogen concentrations were highest (P = 0.16) in unstressed controls compared with calves subjected to RIS (Table 2); however, in support of the relationship between postmortem pH decline and lactic acid accumulation, LM lactate concentrations of calves subjected to 6 h of RIS were lower than those of calves subjected to 2 and 4 h of RIS or unstressed controls (Table 2). Muscle glycogen concentrations of calves in the present study contrasted the reductions in glycogen concentrations in the semitendinosus (Apple et al., 1993b) and LM (Apple et al., 1995) of sheep subjected to RIS; however, Apple et al. (1995) observed that LM lactate concentrations were considerably lower at all measuring times postmortem in sheep subjected to RIS than in unstressed sheep. More importantly, postmortem pH decline in the LM of calves subjected to RIS are unmistakably similar to those of Apple et al. (1995), who demonstrated that subjecting sheep to a single 6-h bout of RIS increased mean 24-h postmortem pH to approximately 6.4 in the LM. According to Page et al. (2001), 91.7% of beef carcasses classified as dark-cutters had ultimate LM pH values of 5.87 or higher, suggesting a lower critical LM pH value for defining the DCC. Interestingly, subjecting Holstein calves to shorter durations of RIS (2 and 4 h) resulted in mean pH values of 5.98 and 5.95 at 24 h postmortem, respectively, and 5.83 and 5.91 at 48 h postmortem, respectively.

Subjecting Holstein calves to RIS produced a darker (lower L* values), less yellow (lower b* values) LM that had a considerably higher water-holding capacity (less free and more bound water; Table 2). Apple et al. (1995) reported that subjecting sheep to a single 6-h bout of RIS caused a decrease in L*, a*, and b* values of the LM, whereas subjecting sheep to three consecutive days of RIS lowered a* and b* values of the LM, semitendinosus, biceps femoris, and semimembranosus chops (Apple et al., 1993b). Page et al. (2001) reported a negative relationship between a* and b* values with muscle pH, with higher muscle pH associated with decreased a* and b* values, which is consistent with results of the present study. Moreover, at high pH values, muscle proteins bind more water tightly, resulting in less free water and a firmer, more closed muscle fiber matrix (Greaser, 1986). The LM from calves subjected to RIS had less free and more bound water, and was visually a darker, more purplish color than that of unstressed calves (Table 2). Thus, the darker color of the high-pH LM was a result of more light absorbed by the LM and less light reflected off the free moisture (Ledward et al., 1992). Additionally, insufficient acid formation during postmortem glycolysis fails to inactivate mitochondrial respiration, allowing myoglobin to remain in a deoxygenated state (Egbert and Cornforth, 1986; Ledward et al., 1992), which would also contribute to the dark, purplish color of the LM of calves subjected to RIS.

Cooked LM chops from calves subjected to RIS had lower WBSF values than unstressed controls (Table 2), which also was observed in the LM of sheep subjected to a single 6-h bout of RIS (Apple et al., 1995). However, current results conflict with those of Wulf et al. (1997, 2002), who reported that beef from dark-cutting carcasses had higher calpastatin activity and shear force values, as well as lower palatability ratings for tenderness, juiciness, and flavor. Purchas (1990) and Watanabe et al. (1996) reported a curvilinear relationship between ultimate muscle pH and meat tenderness, with maximal meat toughness occurring between muscle pH values of 5.8 to 6.0. Even though the mean 48-h pH of LM chops from calves subjected to 2 and 4 h of RIS fell within this range, WBSF values of these chops were still lower than those from the unstressed controls. Moreover, pH values of LM chops from calves subjected to 6 h of RIS were in excess of 6.0 and had the lowest WBSF values, which is consistent with the results of Marsh et al. (1980/1981), who reported that meat tenderness was improved in muscles with ultimate pH values in excess of 6.0. The discrepancies in published relationships between ultimate muscle pH and tenderness may be that cattle and sheep of marketable weights and ages were used to establish these relationships, and may not be applicable to calves similar in age and weight of those used in the present study.

Six dark-cutting carcasses were produced from the eight calves subjected to 6 h of RIS, whereas only four dark-cutting carcasses (two per treatment) were elicited when calves were subjected to either 2 or 4 h of RIS, and no unstressed calves produced carcasses characteristic of the DCC. It should be noted that the six dark-cutting carcasses from calves exposed to 6 h of RIS were obtained in the first, second, and fourth blocks of this experiment. However, a severe weather change occurred on the day of the third block, with temperatures approximately 4°C colder than the other three blocks. Space heaters were brought in to maintain calf comfort during RIS, as well as improving the environment of the student-volunteers involved in blood collection. Researchers were initially concerned that the sudden weather shift would cause all calves to produce dark-cutting carcasses (Kreikemeier et al., 1998; Scanga et al., 1998), but none of the RIS calves in the third block produced dark-cutting carcasses. Because it was warmer near the RIS calves in the third block, the students stayed with their assigned calves during the duration of RIS; thus, it was postulated that the gentle touching and interaction between the students and their calves actually resulted in a calming effect (Lensink et al., 2000a, 2001). In fact, isolated calves tend to allow humans to approach more closely than group-reared calves (Arave et al., 1992), resulting in an enhanced human-to-calf bond (Purcell and Arave, 1991). Moreover, positive human contact (gentle touching, petting, letting calves suck fingers, or talking in a soft voice) have been shown to decrease stress responses (Gonyou, 1994) and fear reactions (Lensink et al., 2001) of calves subjected to handling and transportation, resulting in greater muscle glycogen reserves before slaughter (Lensink et al., 2000b), lower ultimate LM pH values, and a lighter, more pale lean color (Lensink et al., 2001). Although physical and visual isolation of Holstein calves from their contemporaries is stressful (Le Neindre, 1993), the gentle, positive contact between student volunteers and calves that occurred with the third block in the present study resulted in less dramatic changes in blood cortisol levels and no dark-cutting carcasses. Thus, although this RIS model produced carcasses indicative of the DCC, results from the third block of this experiment imply that human contact must be limited to elicit effectively a typical stress response and, more importantly, to produce dark-cutting carcasses repeatedly.

	Implications

Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 
Subjecting calves to restraint and isolation stress effectively elicited dramatic elevations in cortisol and circulating energy reserves, propagated insulin resistance, and curtailed normal postmortem pH decline. Thus, results of the present study indicate that exposing Holstein calves to a single 6-h bout of restraint and isolation stress may be an effective, reliable animal model to study the dark-cutting condition in a controlled laboratory setting.

	Footnotes

 
¹ This project was funded by the Arkansas Beef Council and Arkansas beef producers through their $1.00 per animal check-off program.

² The authors thank P. Hornsby, G. Carte, and J. Sligar for animal care and management; W. J. Roberts, C. B. Boger, M. E. Davis, J. E. Turner, B. Sandelin, S. Gadberry, R. Panivivat, B. McGinley, and D. C. Brown for assistance with blood collection; J. Stephenson, J. Leach, J. Jimenez-Villarreal, R. Miller, and N. Simon for assistance with steer slaughter; K. S. Anschutz for assistance in the laboratory; and Z. B. Johnson for assistance with statistical analyses.

⁴ Current address: Morehead State Univ., Morehead, KY 40351.

³ Correspondence: B-103C AFLS Bldg. (phone: 479-575-4840; fax: 479-575-7294; e-mail: japple{at}uark.edu).

Received for publication October 13, 2004. Accepted for publication January 21, 2005.

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