Introduction
Stress is defined as any event invoking a physical, psychological, or emotional response (McEwen and Sapolsky, 2006). In the short term, stress contributes to the fight or flight response and is beneficial to the survival of the animal. However, stress in the long term contributes to an extended use of the body’s energy stores designed for short-term purposes. Regardless of the duration, however, lipids and proteins are oxidized in vivo, resulting in oxidative stress. Increased stress antemortem has further proven to be detrimental to meat quality, including prevalence of dark-cutting beef, decreased hot-carcass weights, and decreased carcass quality postmortem (McKenna et al., 2002; Gagaoua et al., 2021). Despite the recognition of stress implications on carcass composition, few studies have further evaluated beef quality during aging and retail display.
Very little research is available relating in vivo oxidative stress to meat quality, specifically as it relates to lipid stability. Nonetheless, studies have still recognized the importance of maintaining visual appeal at a retail level, which should encourage improvement of beef lipid stability. Discoloration of beef during retail display costs the industry approximately $3.73 billion annually in discarded products (Ramanathan et al., 2022). Based on data gathered from prior studies on the effect of antemortem stress on the production of lipid-damaging reactive oxygen species (ROS; Bekhit et al., 2013; Ponnampalam et al., 2017; Deters and Hansen, 2020), it could be theorized that damaged lipids in vivo translate to unstable lipids postmortem. Therefore, it is essential to understand the impact of antemortem stress events and their contribution to retail instability via oxidative stress. Because of this, the objective of this study was to evaluate the effect of various antemortem stressors on live animal oxidative stress responses to correlate these with postmortem meat quality in a model calf study.
Materials and Methods
Animal Management and Treatment
All procedures were approved by the Guide for the Care and Use of Agricultural Animals in Research in Training and approved by the Institutional Animal Care and Use Committee at the Livestock Issues Research Unit (LIRU; protocol number LIRU IACUC 2022_05).
Holstein steer calves (n = 40; 110 ± 11.8 kg body weight [BW]) were purchased from a calf ranch and transported to the US Department of Agriculture Agricultural Research Services (USDA-ARS) LIRU. Calf management followed procedures described by Burdick Sanchez et al. (2025). In brief, calves were housed in individual pens (1.2 × 1.8 m) in an environmentally controlled room with ad libitum access to water and a starter ration. On arrival (day −5), calves were balanced by BW and randomly assigned to 1 of 4 treatment groups (n = 10/treatment): 1) control (CON; no treatment application), 2) transport (TRANS; transported in a livestock trailer for 4 h), 3) lipopolysaccharide (LPS; intravenous administration of 0.10 μg /kg BW LPS from Escherichia coli O111:B4), and 4) vaccine (Mannheimia haemolytica toxoid vaccine administered, subcutaneous; OneShot, Zoetis, Parsippany, NJ). Prior to application of treatments, on day −1, calves were restrained via halter in a calf chute for fitting of indwelling rectal temperature (RT) recording devices (modified based on procedure by Reuter et al., 2010), measuring RT continuously at 5-min intervals, as well as jugular vein catheters (Carroll et al., 2009). Whole blood samples were collected at −1, −0.5, 0, 1, 2, 2, 3, and 4 h relative to treatment administration at 0 h. For the purpose of this study, whole blood plasma was collected in blood collection tubes containing lithium-heparin and centrifuged immediately at 1500 × g for 20 min at 4°C. Isolated plasma was allocated to microcentrifuge tubes and stored at −80°C for further analysis.
Transportation Management
All transportation procedures were conducted following methods described in depth by Burdick Sanchez et al. (2025). In brief, immediately after collection of the 0-h sample, calves assigned to transportation were loaded onto a 10.9-m gooseneck livestock trailer fitted with 4 standard dairy calf pens (1.2 × 1.8 m), where 2 to 3 calves were placed in each pen within the trailer. Calf pens were used during transportation to prevent injury during transport, while also facilitating the collection of blood at each time point. Transportation occurred in 1-h increments for a total of 4 h, with a transportation route consisting of paved and unpaved roads. The floor of the trailer was fitted with rubber mats for calf stability, as well as windowed siding to allow for airflow during the transport period. Average temperatures within the trailer were determined to be 34.5°C. At each collection point, the trailer would come to a complete stop, returning to the USDA-ARS LIRU each time to allow for blood sample collection. Jugular catheters for TRANS calves were coiled and secured to the top of the neck with vet wrap for protection after blood collection. During transportation, calves did not have access to feed or water but were allowed ad libitum access to water following the completion of the final blood collection and removal from the trailers at 4 h.
Antemortem Plasma Measures
Thiobarbituric reactive substances of plasma. Thiobarbituric reactive substances (TBARS) were evaluated on collected plasma samples using a quantitative assay kit (OxiSelect, Cell BioLabs, Inc.). A malondialdehyde (MDA) equivalent standard was used for comparisons for quantifying concentrations of MDA and levels of oxidation in unknown samples. Plasma samples were thawed and evaluated for reactive substances according to direct TBARS procedures described in the kit protocol. Standards and samples were evaluated in duplicate on 96-well plate and measured via spectrophotometer at 532 nm. Intra assay coefficients of variation (CV) were accepted at 5% or less.
Total antioxidant capacity. Total antioxidant capacity (TAC) was evaluated on collected plasma using a quantitative assay kit (OxiSelect, Cell BioLabs Inc.). A uric acid standard was used for comparison of antioxidant capacity following the addition of reaction reagent and copper. Plasma samples were thawed and evaluated for TAC according to direct procedures described in the kit protocol. Standards and samples were evaluated in duplicate on 96-well plates and measured via spectrophotometer at 490 nm at the end of the reaction. Intra assay CV were accepted at 5% or less.
Reactive oxygen species. ROS of plasma were determined using a quantitative fluorometric assay kit (OxiSelect, Cell BioLabs, Inc.). A hydrogen peroxide (H2O2) standard was used for comparison of in quantifying concentrations of H2O2 as the primary ROS in unknown samples. Collected plasma samples were thawed and evaluated for H2O2 concentrations according to direct procedures described by the assay kit protocol. Samples were evaluated in quadruples, and free radical content was determined by comparison to a 2’,7-dichlorodihydrofluorescein and H2O2 standard curves fluorometrically. Samples were read on 96-well plates via standard fluorescence plate reader at 480 nm excitation per 530 nm emission. Intra assay CV were accepted at 20% or less.
Oxidative stress index. The ratio of ROS to TAC was used to calculate the oxidative stress index (OSi; Abuelo et al., 2013), determining a ratio of pro-oxidants to antioxidants.
Necropsy and Postmortem Collections
Following blood collections at 4 h, all animals were returned to their original pens and allowed to rest with water for 2 h postchallenge (hours 5 and 6). At 6 h posttreatment application, calves were immobilized and humanely euthanized by captive bolt and exsanguinated. From each calf, the entire Longissimus dorsi (LD) was collected immediately postmortem, so as to exclude bones and accessory muscles, and vacuum packaged (Koch Ultravac; UltraSource LLC., Kansas City, MO) before being transported to the G.W. Davis Meat Laboratory at Texas Tech University, where loins were allowed to undergo rigor mortis and aged for 7 d postmortem under vacuum and refrigeration (2–4°C).
Fabrication and Retail Display
At 7 d postmortem, loins were fabricated anterior to posterior into 2.54-cm thick steaks. Steaks (n = 5) from each loin were randomly assigned to a retail display period of 0, 3, 6, and 9 d, with each face steak assigned to analysis of metmyoglobin reducing activity (MRA). Steaks assigned to 3, 6, and 9 d of display were individually placed on black expanded polystyrene trays and overwrapped with polyvinyl chloride film using a Minipack-torre, Minispenser (Dalmine, Italy). Steaks assigned to 0 d of aging, face steaks, and extra steaks were individually vacuum packaged and frozen at −20°C until subsequent analysis. All steaks assigned to retail display were stored under continuous fluorescent lighting in 2 coffin-style cases within temperatures of 2°C to 4°C. Packages were rotated every 12 h within their original cases. Immediately following retail display, steaks were removed from overwrap packaging, transferred to vacuum packaging, and frozen at −20°C until oxidative analyses.
Instrumental and Trained Color Evaluation
The L*, a*, and b* values were (n = 3) recorded every 12 h during the entire display period on steaks receiving the 9-d aging treatment. A Hunter MiniScan EZ 4500 (Hunter Associates Laboratory, Inc., Reston, VA) utilizing an area of 45° per 0° directional viewing geometry, with a 31.8-mm port and 25-mm viewed area used for instrumental color data collection. The spectral range of the colorimeter determined values between 400 nm and 700 nm and were used to calculate the percentage of oxymyoglobin and metmyoglobin (MMb) production (King et al., 2023).
Trained color evaluators were responsible for determining overall redness and percent of discoloration in samples displayed for 9 d, following the methods described by King et al. (2023). Evaluators were screened on normal acuity and ability to discriminate color differences using the Farnsworth-Munsell 100-Hue test. Successful evaluators showed scores of 50 or less as per guidelines of King et al. (2023). Evaluators were further trained and tested on samples for approximately 14 h to objectively evaluate samples for meat-color stability of displayed whole muscle and surface discoloration as a reference. Samples were quantified on an unstructured line scale from 0 to 100, with “0” representing extremely pale samples or no discoloration, while “100” represented extremely dark red or completely discolored samples. Ballots were provided to evaluators via electronic tablets (iPad, Apple Inc., Cupertino, CA) using electronic surveys (Qualtrics, Provo, UT). Evaluators were asked to evaluate samples every 12 h (± 1 h) for the entire duration of the 9-d display period.
Metmyoglobin Reducing Activity
MRA was determined by following methods described by Ramanathan et al. (2019). In brief, samples from face steaks were submerged in a 0.3% weight/volume solution of sodium nitrite (Sigma Aldrich, St. Louis, MO) for 20 min at 30°C. After 30 min, steaks were removed from the solution and blotted to remove visible solution. MMb content on the surface was determined by Hunter MiniScan. Ratios of MRA were determined as K/S572 ÷ K/S525, where higher ratios were indicative of a greater MRA.
pH
Powdered homogenate from day 0 steaks were evaluated for pH in a 1 to 10 ratio to deionized water. Two grams of powder (± 0.1 g) were weighed into 50-mL centrifuge tubes and combined with 20 mL of deionized water. Samples were blended for 30 s by Polytron. The pH of the muscle homogenate was obtained by an Accumet combination glass electrode connected to an Accumet 50 pH meter. The electrode was standardized using pH 4 and 7 buffer prior to use.
Thiobarbituric Reactive Substances of Steaks
TBARS of steaks were determined using modified methods of Buege and Aust (1978), described by Luque et al. (2011). In brief, following retail display, all steaks from days 0, 3, 6, and 9 were cubed (approximately steak thickness × 1 cm), flash frozen in liquid nitrogen, and homogenized (NutriBullet, NutriBullet, LLC, Los Angeles, CA). In duplicate, 5 g (± 0.1 g) of each sample were weighed into 50-mL centrifuge tubes. Remaining homogenate was stored at −80°C. Fifteen milliliters of cold, deionized water were added to each tube and vortexed for 30 s. After briefly vortexing, samples were blended by Polytron (Homogenizer 850; Fisher Scientific, Waltham, MA) for approximately 30 s. Samples were then centrifuged for 10 min at 3000 rpm. After centrifugation, 2 mL of clear supernatants from each tube were transferred over to a 15-mL centrifuge tube. To each tube, 4 mL of TCA/TBA reagent and 100 μL butylated hydroxyanisole were added before vortexing again for approximately 1 min. Sample tubes were placed in a water bath (100°C) for 15 min. After heating, tubes were allowed to cool in ice water for 10 min before centrifuging again at 3000 rpm for 10 min. Once centrifugation was complete, 200 μL of supernatant, ranging in color from clear to pink, were transferred to a 96-well plate. Plates were analyzed via spectrophotometer (Epoch; BioTek Instruments Inc., Winooski, VT) at 531 nm and calculated according to a standard curve. Intra assay CV were accepted at 5% or less.
Statistical Analysis
Data accumulated for trained color panels, instrumental color data, steak TBARS, plasma TBARS, TAC, and ROS were averaged for each sample and analyzed as a repeated measure using the GLIMMIX procedure of SAS (Version 9.4; SAS Institute, Cary, NC). Stressor type and time or day of retail display served as main effects, with calf serving as the repeated measure. The covariance structure with the lowest Akaike Information Criterion was used. Significance level was set at a P value of .05 or less, and tendencies were considered when α was greater than 0.05 but less than 0.10. The Kenward-Roger adjustment was used to estimate denominator degrees of freedom.
Data accumulated for MRA and pH were averaged for each sample and analyzed as a complete randomized design using the GLIMMIX procedure of SAS (Version 9.4; SAS Institute, Cary, NC). Stressor type served as a main effect, with individual steak serving as the experimental unit. Probability values (P values) less than or equal to α equals 0.05 were considered significant, while tendencies were considered when α was greater than 0.05 but less than 0.10. The Kenward-Roger adjustment was used to estimate denominator degrees of freedom.
Results
Thiobarbituric Reactive Substances of Plasma
An interaction occurred between treatment and time (P < .001; Figure 1) for antemortem plasma TBARS concentrations. CON and vaccine calves tended to start the study with less initial concentrations (−1 h) of MDA than TRANS calves (P < .05) compared to LPS, which were similar initially (P > .05). However, at −0.5 h, all treatment groups were similar (P > .05). At hour 0, plasma MDA concentrations in LPS calves increased (P < .05), while concentrations were the least in CON and vaccine calves (P < .05). TRANS calf plasma MDA concentrations were intermediate at 0 h (P > .05). At hour 1, MDA concentrations did not differ across treatment groups (P > .05). By 2 h, plasma MDA concentrations increased again in LPS calves (P < .05), reaching peak values. Meanwhile, plasma MDA concentrations in TRANS, vaccine, and CON calves were similar (P > .05) and less than in LPS calves (P < .05). At 3 h, LPS calf plasma MDA concentrations decreased (P < .05) and were similar to TRANS calves (P > .05). Concentrations in CON calves also decreased at 3 h (P < .05) but were similar to TRANS and LPS calves (P > .05). Vaccine calf concentrations increased at 3 h compared to TRANS and CON calves (P < .05), exhibiting the greatest concentration. At 4 h, a sharp decrease in MDA concentrations were observed in TRANS, vaccine, and CON calves (P < .05), but concentrations were similar to each other (P > .05). MDA concentrations in LPS calves did not differ by 4 h (P > .05) but remained elevated compared to the other treatment groups (P < .05).
Interaction between challenge time (−1, 0.5, 0, 1, 2, 3, 4) and treatment type on malondialdehyde (μM) in plasma antemortem (P < .001). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, body weight; CON, control; LPS, lipopolysaccharide; MDA, malondialdehyde concentrations; SEM, standard error of the mean; TRANS, transport.
Total Antioxidant Capacity
An interaction occurred between treatment and time (P < .001; Figure 2) for copper reducing equivalents (CRE) in antemortem plasma. At −1 h, LPS, vaccine, and CON calves began with similar (P > .05) concentrations, while TRANS calves exhibited the greatest concentration of CRE at −1 h (P < .05). Concentrations of CRE in TRANS calves decreased at −0.5 h but were still greater than the other treatment groups (P < .05). At hour 0, vaccine and LPS calves had the least concentrations of CRE, while TRANS calves still had the greatest (P < .05). CON calves exhibited intermediate concentrations of CRE (P < .05) but were similar to LPS calves (P > .05). All treatment groups showed increased concentrations of CRE at hour 1, where LPS and vaccine calves were greater (P < .05) than CON and TRANS calves, which were similar (P > .05). Concentrations for all treatment groups were similar at 2 h (P > .05). TRANS calves exhibited drastically elevated concentrations of CRE at 3 h (P < .05). Similarly, CRE concentrations in CON calves also increased (P < .05) but to a lesser extent. However, in vaccine and LPS calves, the concentration had decreased (P < .05), but they were similar to each other (P > .05). By 4 h postchallenge, concentrations decreased in both vaccine and TRANS calves (P < .05) to a point where there were similar to concentrations found in vaccine and LPS calves (P > .05).
Interaction between challenge time (−1, 0.5, 0, 1, 2, 3, and 4 h) and treatment type on copper reducing equivalents, contributing to total antioxidant capacity in plasma antemortem (P < .001). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, boy weight; CON, control; CRE, copper reducing equivalent; LPS, lipopolysaccharide; SEM, standard error of the mean; TRANS, transport.
Reactive Oxygen Species
An interaction between treatment and time occurred for ROS concentrations in plasma (P < .001; Figure 3). Calves in the TRANS, LPS, and vaccine treatment groups had greater concentrations of ROS and were similar to each other at −1 h (P > .05) compared to CON calves at −1 h (P < .05). However, at −0.5 h, concentrations increased in CON calves so that all treatment groups had similar concentrations (P > .05). Concentrations decreased substantially in all treatment groups at 0 h (P < .05), where CON, LPS, and TRANS calves were the lowest and similar to each other (P > .05), while vaccine calves had greater concentrations (P < .05). By 1 h posttreatment application, LPS and CON calves showed the greatest increase in ROS compared to 0 h (P < .05), while TRANS calves also increased (P < .05), although less substantially, and vaccine-treated calves remained similar to the previous hour (P > .05) Values remained consistent for all treatment groups from 1 h to 2 h (P > .05) but decreased again for vaccine and CON calves (P < .05). TRANS and LPS calves did not change at 3 h (P > .05). At 4 h posttreatment, TRANS calves had the least concentration of ROS (P < .05) but were still similar in concentration to vaccine calves (P > .05).
Interaction between challenge time (−1, 0.5, 0, 1, 2, 3, and 4 h) and treatment type on reactive oxygen species in plasma antemortem (P < .001). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, body weight; CON, control; H2O2, hydrogen peroxide; LPS, lipopolysaccharide; SEM, standard error of the mean; TRANS, transport.
Oxidative Stress Index
A treatment type and time interaction occurred for OSi (P < .001; Figure 4). At −1 h, CON and TRANS calves had similar OSi values (P > .05) that were less (P < .05) compared to LPS and vaccine calves, whose OSi values were similar to each other (P > .05). CON, TRANS, and LPS calves had increased OSi values at −0.5 h (P < .05), while vaccine calves did not change (P > .05). A substantial reduction in OSi values occurred for all treatment groups (P < .05) at 0 h; however, vaccine calves had greater OSi values than the other treatment groups (P < .05). Index values in LPS and CON calves were elevated at 1 h (P < .05), while TRANS OSi values increased slightly (P < .05) and were similar to values in vaccine calves (P > .05), whose OSi values remained constant from 0 h to h (P > .05). Index values did not change in LPS calves from 1 h to 3 h (P > .05) or in CON and TRANS calves from 1 to 2 h (P > .05). The OSi values in CON and LPS calves all decreased at 3 h postchallenge (P < .05). A decrease in OSi values occurred for LPS calves at 4 h (P < .05). Values for TRANS calves did not change (P > .05).
Interaction between challenge time (−1, 0.5, 0, 1, 2, 3, and 4 h) and treatment type on oxidative stress index in plasma antemortem (P < .001). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, body weight; CON, control; LPS, lipopolysaccharide; OSi, oxidative stress index; SEM, standard error of the mean; TRANS, transport.
Instrumental Color Evaluation
There were no interactions between calf treatment and hours of display for any instrumental values and calculations (P ≥ .551). However, instrumental values for L* and b* were affected by calf treatment (P ≤ .006; Table 1). Percentage of calculated MMb was also impacted by calf treatment (P < .001; Table 1). Tendencies occurred for a* values as an effect of calf treatment (P = .088; Table 1).
Treatment effect on instrumental L*, a*, and b* values and percentage of metmyoglobin
| Treatment1 | Instrumental Value | |||
|---|---|---|---|---|
| L* | a* | b* | MMb2 | |
| LPS | 52.89a | 18.66 | 17.97b | 38.47a |
| TRANS | 50.49c | 19.34 | 18.54a | 38.88a |
| Vaccine | 51.67b | 19.75 | 18.45a | 37.12b |
| CON | 51.25bc | 19.71 | 17.97b | 36.82b |
| SEM | 0.36 | 0.34 | 0.15 | 0.42 |
| P value | <.001 | .088 | .005 | <.001 |
Abbreviations: BW, body weight; CON, control; LPS, lipopolysaccharide; MMb, metmyoglobin; SEM, standard error of the mean; TRANS, transport.
LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups.
MMb percentage.
Means within columns lacking common superscripts differ.
Instrumental L* values, or lightness, were greatest in steaks from calves treated with LPS (P < .05), while steaks from TRANS calves had the least L* values (P < .05). Vaccine-treated calves showed intermediate brightness values, while CON steaks were similar (P > .05) to vaccine and TRANS calves. Blueness, determined by b* values, was greatest in TRANS and vaccine-treated steaks (P < .05), while LPS and CON calf steaks were the least (P < .05). Calculated MMb percentages were greatest in LPS and TRANS calf steaks (P < .05), while vaccine and CON calf steaks showed the least MMb (P < .05). Redness, determined by a* values, showed a tendency (P = .08) for greater a* values in vaccine-treated and CON calf steaks (P > .05) and the least values in LPS-treated calf steaks (P < .05), while TRANS treated calves were similar (P > .05) to LPS-treated calf steaks.
Trained Color Evaluation
An interaction occurred between calf treatment and hours of retail display for redness (P < .001; Figure 5) and discoloration (P = .011; Figure 6) determined by trained evaluators. For redness, steaks from CON calves were the darkest red at 0 h (P < .05), while LPS calf steaks were the palest red (P < .05). Steaks from TRANS and vaccine calves were intermediate and similar (P > .05). Redness decreased for all steaks throughout the display period (P < .05), where CON calf steaks were the reddest compared to all other treatments until 120 h, at which point CON and vaccine calf steaks were similar (P > .05). By the end of retail display, all calves were similar in redness (P > .05) with the exception of LPS and TRANS calves (P < .05), where TRANS calves were paler red.
Interaction between hours of display (9 d of display with measurements every 12 h) and treatment type on redness values in steaks determined by trained color evaluators (P < .001). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, body weight; CON, control; LPS, lipopolysaccharide; SEM, standard error of the mean; TRANS, transport.
Interaction between hours of display (9 d of display with measurements every 12 h) and treatment type on percent discoloration in steaks determined by trained color evaluation (P = .011). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: BW, body weight; CON, control; LPS, lipopolysaccharide; SEM, standard error of the mean; TRANS, transport.
Discoloration did not occur visually for any steaks until after 60 h of display. By 72 h of display, all steaks had discolored; however, CON calf steaks showed the greatest percentage of discoloration compared to TRANS and vaccine calves (P < .05) but were similar to LPS calves (P > .05) Discoloration increased throughout the remainder of the display period (P < .05). By 120 h, CON steaks were the least discolored compared to TRANS and LPS calves (P < .05) but similar to steak from vaccine calves (P > .05). At 204 h, the end of retail display, all steaks were similar in discoloration (P > .05).
Metmyoglobin Reducing Activity and pH
There was no effect of calf treatment on MRA in the samples evaluated (P = .156; Data not shown). A treatment effect occurred for pH values on steaks from treated calves (P = .019; Data not shown). The pH of steaks from CON calves (5.63 ± 0.018; P < .05) was higher than LPS and vaccine calf steaks but similar to TRANS calves (5.61 ± 0.018; P > .05). Vaccine (5.56 ± 0.018) and LPS calves (5.56 ± 0.018) had the lowest pH and were similar to each other (P > .05).
Thiobarbituric Reactive Substances of Steaks
An interaction occurred between treatment and days of display (P = .014; Figure 7) for TBARS concentrations in displayed steaks. In general, MDA concentrations increased for all treatments throughout the display period (P < .05). On day 0, steaks from CON and LPS calves were similar (P > .05) and exhibited the least concentration of MDA compared to TRANS and vaccine-treated steaks, which were greater in concentration but similar to each other (P > .05). At day 3 of retail display, CON, LPS, and vaccine calf steaks were similar (P > .05), while steaks from TRANS calves showed the greatest concentrations of MDA (P < 0.05). At 6 h of display, steaks from all treatment groups contained similar concentrations of MDA (P > .05). After 9 d of display, concentrations of MDA were greatest in TRANS calf steaks (P < .05), while CON, TRANS, and vaccine calf steaks were similar (P > .05).
Interaction between days of display (0, 3, 6, and 9 d) and treatment type on malondialdehyde (mg/kg of sample) concentrations in steaks (P = .014; SEM = 0.06). Treatment types include LPS (0.10 μg/kg BW), 4 h TRANS, Mannheimia haemolytica toxoid vaccine, and CON groups. Abbreviations: CON, control; LPS, lipopolysaccharide; MDA, malondialdehyde; SEM, standard error of the mean; TRANS, transport.
Discussion
The effect of stress on cattle has been well reviewed in literature over the years (Deters and Hansen, 2020; Shephard and Maloney, 2023; Smock et al., 2023), whether immune, transportation, or some other form of stress. There have been few studies, however, which have recognized the full extent that stress impacts meat quality. Instrumental color evaluation offers a quantitative value when analyzing the spectrum of light to dark (L*), green to red (a*), and yellow to blue (b*). Although differences were observed between treatments in the current study, biological significance may be lacking. A study evaluating gentle and aggressive handling of veal calves prior to harvest evaluated the L*, a*, and b* values of the Rectus abdominus and Semimembranosus 24 h postmortem and did not determine any differences between calves (Lensink et al., 2000). Lensink et al. (2000) suggest that veal color was either unaffected by the stressors presented, or stressed calves had an opportunity to adjust over a longer period of time than calves in the current study. Nonetheless, the current study evaluated the LD and color evaluations were conducted on aged and displayed samples. Furthermore, samples in the current study have a range of L*, a*, and b* values much greater than the typical values reported in veal calves (Denoyelle and Berny, 1999; Lensink et al., 2000; Lagoda et al., 2002). However, the calves in the current study were used as a model and not for the production of veal, receiving diets unlike those of the typical veal calf. Additionally, where this study evaluated the LD for color during display, the previous studies listed evaluated the Rectus abdominus, Semimembranosus, or the breast lean tissues. Lagoda et al. (2002) used special-fed calves (milk-based diet) that were Kosher slaughtered and evaluated the color of the breast meat up to 24 h postmortem via Minolta Chromameter. Similarly, Denoyelle and Berny (1999) used a Minolta Chromameter, and while the study evaluated a large sample of calves, the standard diet for calves in Europe at the time of the study, and still today, was a primarily milk-fed diet (EU Council, 1997). It may, instead, be more worth recognizing the potential for larger differences if the current study was conducted on larger, harvest-ready cattle. In general, meat from veal calves is considered “high quality” by consumers when pale or “white” in color compared to the darker red seen in fed-beef cattle (Ngapo and Gariépy, 2006). While the animals in the current study were used for proof of concept, it is necessary to remember that myoglobin content is drastically reduced in calves compared to animals that have reached the standard 9 mo to 30 mo of maturity seen in modern fed-beef production (Boccard et al., 1979; Tuma et al., 1962). Regardless, instrumental and trained color evaluations did prove to be similar, where LPS-treated calves tended to show fewer red steaks according to both instrumental a* and redness values determined by panelists. Herrera et al. (2021) evaluated the effect of LPS on lamb chops and noted the opposite, stating a* values were increased in lambs treated with LPS compared to CON lambs. Kadim et al. (2007) acknowledged L*, a*, and b* values were decreased in sheep receiving a 2-h transport treatment under high environmental temperatures (37.5°C). The study further recognized that transportation also increased pH and decreased tenderness, although the latter was not evaluated in the current study. As expected, discoloration increased in all steaks as retail display time increased due to normal degradation of meat quality.
Lipid oxidation measures, specifically TBARS, tend to increase during retail display based on the normal lipid degradation that occurs during aging. Lipid peroxidation further results in increased discoloration, impaired water-holding capacity, and formation of off odors (Xing et al., 2018). Unfortunately, few studies have determined whether antemortem stress may speed up the lipid oxidation process. Nonetheless, when combining the results of steak and antemortem TBARS concentrations, a better relationship between the two may be explained. Transit may often be a forgotten piece of the beef cattle stress puzzle because it is relied on so heavily throughout their entire lifespan. However, the current study is suggestive of its greater effects on lipid oxidation, agreeing with former studies (Marques et al., 2012; Deters and Hansen, 2020).
It may be worth noting that transportation within the current study served as a model, where the method of transportation described is common for calves early in life but is less common for fed-cattle being transported to larger packing facilities in the United States. Nonetheless, it has been documented that calves may show the greatest potential for adverse effects during transportation compared to larger, more mature animals. Eicher et al. (2006) suggests calves are more susceptible to increased stress during transportation because of incomplete development of the hypothalamus-pituitary-adrenal (HPA) axis. This could explain elevated effects exhibited by the calves in the current study. Calves receiving transportation in the current study were also handled more than those in other treatment groups, adding to the potential for increased stress in vivo compared to other treatment groups. While cortisol is a common stress indicator produced by the HPA axis, concentrations did not increase in transported calves (Sanchez et al., 2025), suggesting other markers, such as those evaluated for oxidative stress in the current study, may be more important when measuring the stress response over short-distance transportation. In broilers, acute preharvest environmental stress, including transportation, has contributed to meat-quality defects similar to pale, soft, and exudative (PSE) meat in pork (McKee and Sams, 1998; Sams, 1999). These studies have also noted defects such as decreased pH, water-holding capacity, and increased cook loss. Nonetheless, the effects observed in poultry did not agree with the results observed in the current study, although few studies have been conducted similarly in fed-beef poultry.
Oxidative stress in livestock has been recognized as multifactorial, making it difficult to recognize the true cause of meat-quality deterioration. Nonetheless, Chirase et al. (2004) recognized transport stress increased plasma MDA. Increased concentrations of MDA were further correlated to episodes of bovine respiratory disease (BRD) and calf mortality. Within the current study, plasma MDA concentrations agree with research of Chirase et al., (2004) where concentrations were elevated in transport calves compared to other treatment groups. Similarly, MDA concentrations in LPS calves in the current study agreed with our work conducted in previously published models (Barker et al., 2024), where LPS caused an increase in plasma MDA during the acute phase response. Xing et al. (2017) further recognized in broilers that transport during high ambient temperatures, perhaps similar to the conditions of the current study conducted in midsummer in Texas, contributed to an overproduction of ROS, excessive oxidation, and PSE-like meat.
Other studies evaluating the health status and antioxidant capacity of calves during stressful life events (i.e., weaning and first transport) have suggested that TAC is drastically reduced during these periods. Even more important, these studies recognize that TAC does not appear to return to normal values for up to 28 d to 60 d postweaning (Chirase et al., 2004; Pregel et al., 2005; Mattioli et al., 2020). While the stress of weaning is extensive, it is worth noting that the results in this study do not reflect the substantial changes observed in other studies. Instead, calves in the current study showed elevated TAC, especially at 3 h posttreatment application. The current study would be suggestive of antemortem stress being greater in the form of an acute phase response via endotoxin and vaccination against disease. Nonetheless, an elevated TAC antemortem may have been utilized quickly during lairage or immediately postharvest, offering little use by the time the retail display period occurred.
Respiratory bursts are an essential function of the immune response that allows leukocytes to destroy pathogens. However, every respiratory burst results in a rapid increase in production of ROS (Górski et al., 2012). Pathogenic bacteria and viruses may elicit this response in the cells during infection. Toxoid vaccines inject a nontoxic bacterial toxin into the body, similarly, stimulating a strong humoral immune response (Backx and Freedman, 2009; Gupta and Pellett, 2023). Therefore, LPS and vaccine calves could have been expected to similar responses in the study due to the presence of an “invader” in the body. In industry setting, vaccine efficacy is dependent on the host’s ability to produce an immune response to the pathogen while showing effective clearance and prevention of damage (Skugor et al., 2009). The detection of pathogens as a result of vaccination can produce an immune response contributing to ROS production. In calf studies, plasma MDA, a secondary lipid oxidation product, was increased in calves that died from BRD (43% increase; Urban-Chmiel, 2006). A study evaluating vaccines in trout acknowledged an increase in ROS and oxidative biomarkers in vaccinated trout compared to unvaccinated trout (Tkachenko, et al., 2014). The study suggested that increases in ROS were due to an immune response against the injected vaccine.
Conclusion
Decreased antioxidant capacity during stress contributes to decreased ability to mitigate oxidative events in vivo. These data suggest that when stress is not managed in the live animal, meat products may be affected by increased rates of lipid oxidation and discoloration. In the current study, transportation events prior to harvest contribute the greatest to antemortem stress and postharvest deterioration based on TBARS values. Nonetheless, in vivo TAC concentrations were the greatest in transported calves, suggesting a disconnect between antioxidant capacity and its actual effect on meat quality. Therefore, further research should be done to investigate strategies minimizing oxidative stress prior to harvest to preserve meat-quality attributes. Additionally, there may be potential to use these oxidative stress markers to predict the color shelf life of the final meat product.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgments
This project was supported in part by the National Bio and Agro-Defense Facility National Laboratorian Training Program.
Author Contribution
Samantha N. Barker: conceptualization, investigation, methodology, visualization, writing—original draft, and writing—review and editing; Kesley B. Kohl: investigation, methodology, and writing—review and editing; Nicole C. Burdick Sanchez: conceptualization, data curation, formal analysis, investigation, methodology, supervision, validation, visualization, and writing—review and editing; P. Rand Broadway: conceptualization, data curation, formal analysis, investigation, methodology, supervision, validation, visualization, and writing—review and editing; Christy L. Bratcher: funding acquisition, investigation, project administration, writing—review and editing; and Jerrad F. Legako: conceptualization, data curation, formal analysis, investigation, methodology, program administration, supervision, validation, visualization, and writing—review and editing.
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