Proximate composition

A food processor (KitchenAid, St. Joseph, MI) was used to finely mince raw steak (∼200 g) containing only the LT muscle from the 12th rib to analyze protein, fat, and moisture content in duplicate. Total fat and moisture content were determined using AOAC methods (AOAC International, 2013). Fat was evaluated with the CEM ORACLE System (AOAC International, 2013; CEM Corporation Matthews, NC), and moisture was evaluated with the CEM SMART 6 System (AOAC International, 2013; CEM Corporation). Protein content was measured with the CEM Sprint Rapid Protein Analyzer (AOAC International, 2011; CEM Corporation).

Whole muscle protein extraction

Frozen meat containing only the LT (200 g) was homogenized and powdered in liquid nitrogen. Samples from each postmortem timepoint (0.5 g) were homogenized, and muscle proteins were solubilized using 10 mM sodium phosphate (pH 7.0) and 2% sodium dodecyl sulfate (SDS) (wt/vol) as described by Carlson et al. (2017b).

Sarcoplasmic protein extraction

Frozen meat samples containing only the LT (200 g) were homogenized and powdered in liquid nitrogen. Samples from each postmortem timepoint (3 g) were homogenized, and sarcoplasmic proteins were extracted (4°C; 50 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid [pH 8.0]) as described by Carlson et al. (2017a).

Gel electrophoresis running conditions

The samples from the whole muscle preparation were used to evaluate degradation of desmin and troponin-T (1 h and 1, 3, 7, and 14 d postmortem) and calpain-1 autolysis (1 h and 1, 3, and 7 d postmortem) using one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) as described by Carlson et al. (2017a, 2017b). Desmin degradation products that were soluble in the sarcoplasmic fraction (1 h and 1, 3, 7, and 14 d postmortem) and sarcoplasmic calpain-1 autolysis (1 h and 1, 3, and 7 d postmortem) were determined using one-dimensional SDS-PAGE as described by Carlson et al. (2017a, 2017b). A reference sample, composed of 4 samples at 1 d postmortem and 4 samples from the same animals at 7 d postmortem, was created. This day 1/day 7 mixed reference sample was loaded on each gel for all western blot analysis. SE 260 Hoefer Mighty Small II electrophoresis units (Hoefer, Inc., Holliston MA) were used to run 15% SDS-PAGE gels (10 cm × 10 cm; acrylamide: N,N’-bis-methylene acrylamide = 100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] tetramethylenediamine, 0.05% [wt/vol] ammonium persulfate, 0.5 M Tris-HCl [pH 8.8]) for desmin and troponin-T analysis and 10% SDS-PAGE gels (10 cm × 10 cm; acrylamide: N,N’-bis-methylene acrylamide = 100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] tetramethylenediamine, 0.05% [wt/vol] ammonium persulfate, 0.5 M Tris-HCl [pH 8.8]) for calpain-1 autolysis. Protein samples (40 μg) were loaded into each well for whole muscle extract desmin and troponin-T analysis along with whole muscle extract and soluble sarcoplasmic calpain-1 autolysis. For analysis of soluble desmin degradation products in the sarcoplasmic fraction, 50 μg of protein was loaded into each well.

Transferring conditions and western blot analysis

After running, the SDS-PAGE gels were transferred to polyvinylidene difluoride membranes with pore sizes of 0.2 μm (Immobilon-PSQ, 26.5 by 3.75 M RL, VCAT#ISEQ00010, Millipore Corporation, Billerica, MA) as described by Carlson et al. (2017b). Western blot analysis was conducted as described by Carlson et al. (2017a, 2017b). The following primary antibody concentrations were made by diluting with phosphate-buffered saline (PBS)-Tween and added to separate blots: desmin (1:40,000) using polyclonal rabbit anti-desmin antibody produced at Iowa State University (Huff-Lonergan et al., 1996; Carlson et al., 2017a, 2017b), troponin-T (1:10,000) using monoclonal mouse anti-troponin-T primary antibody (T6277, JLT-12; Sigma-Aldrich, St. Louis, MO), and calpain-1 (1:5,000) using monoclonal mouse anti-calpain-1 (MA3-940; Thermo-Scientific, Rockford, IL). All blots were incubated for 1 h at room temperature (∼22°C) with primary antibodies. Desmin and troponin-T secondary antibodies were diluted with PBS-Tween, and calpain-1 secondary antibodies were incubated in PBS-Tween mixed with 5% nonfat dry milk. Secondary antibodies were added to separate blots following washes at the following concentrations: desmin (1:20,000): goat anti-rabbit horseradish peroxidase (HRP) antibody (32430; Thermo-Scientific); troponin-T (1:5,000): goat anti-mouse HRP antibody (32430; Pierce); and calpain-1 (1:10,000): goat anti-mouse HRP antibody (A2554; Sigma Aldrich, St. Louis, MO). All blots were incubated for 1 h at room temperature (∼22°C) with secondary antibodies. Following incubation with secondary antibodies, desmin and troponin-T blots were washed with PBS-Tween 3 times for 10 min, and calpain-1 blots were washed 5 times for 10 min. Blots were analyzed as described by Carlson et al. (2017a, 2017b). Using the internal reference on each blot, the intensities of the 38-kDa desmin degradation product and a 30-kDa troponin-T degradation product were quantified as a comparative ratio of the sample protein band to the internal reference protein band on each gel. Calpain-1 autolysis was analyzed as a percentage of the 80-, 78-, or 76-kDa bands within each sample. All western blots were completed in at least duplicate with a coefficient of variance value of the band of interest of 20% or less.

Statistics

Data were analyzed using the Mixed procedure of SAS version 9.4 (SAS Institute Inc., Cary, NC) as a 2 × 2 factorial with fixed effects of Zn and RH and the interaction. Harvest group was included as a fixed effect for all analyses. Whole muscle extract desmin and troponin-T degradation products, soluble sarcoplasmic desmin degradation products, and whole muscle extract and soluble sarcoplasmic calpain-1 data were analyzed using the Mixed procedure of SAS (version 9.4; SAS Institute) as a 2 × 2 factorial with fixed effects of Zn and RH and the interaction. Gel was used as a random effect in the model. Pearson correlations were generated using PROC CORR using the following criteria: lowly correlated (r ≤ 0.35), moderately correlated (0.36 ≤ r ≤ 0.67), and highly correlated (r ≥ 0.68; Taylor, 1990). Normality was confirmed using the Shapiro-Wilk test. Cook’s D was used to determine outliers at Cook’s D ≥ 0.5. Significance levels for all analyses were set at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. Least-squares means and standard errors were reported for all measured attributes. Least-squares means were separated using SAS’s PDIFF procedure (version 9.4; SAS Institute Inc., Cary, NC).

Postmortem pH decline and Warner-Bratzler shear force values

The rate of pH decline and muscle temperature have been shown to interact and impact ultimate pH and tenderness development in beef (Hwang and Thompson, 2001a; Hopkins et al., 2014; Warner et al., 2014). This very complex biological reaction is still not fully defined. Early studies of this subject demonstrated differences in the rate of pH decline of different beef muscles even when the temperature was kept at a constant state (Bendall, 1978). This variation in rate of pH decline was originally thought to simply be a mechanism of excess calcium and adenosine triphosphate turnover in the muscle system but is now deemed far more complex. Several studies have demonstrated that an intermediate rate of pH decline results in the most tender beef products after aging (Marsh et al., 1987; Hwang and Thompson, 2001a). It has also been demonstrated that a rapid rate of pH decline early postmortem, typically due to electrical stimulation, can result in more tender beef products at 1 d postmortem (Hwang and Thompson, 2001b). These studies further demonstrate the complex relationship between early postmortem pH decline and tenderness development. In this study, postmortem pH, temperature, and WBSF values were not affected by the interaction of Zn and RH, so the main effects will be discussed for the remainder of this section.

A trend (Figure 2; P = 0.06) for lower pH at 6 h postmortem was observed from steers supplemented with supranutritional Zn, specifically in the Zn-only (SUPZN-NO [pH 5.49]) treatment. Conversely, a higher (Figure 2; P = 0.04) pH was observed in muscle from steers supplemented with RH compared with steers not supplemented with RH, specifically in CON-RAC treatment (pH 5.86), at 6 h postmortem. These 2 effects offset one another in the combination treatment—SUPZN-RAC (pH 5.70)—showing greater similarity to CON-NO (pH 5.68). These effects were not influenced by differences in muscle temperature at any timepoint (Figure 3). This difference in the rate of pH decline followed a similar trend in the observed differences in WBSF values at 1 d postmortem. Supplementation of RH resulted in greater (Figure 4; P < 0.01) WBSF values at 1 d postmortem, whereas supranutritional Zn supplementation trended (Figure 4; P = 0.06) for a lower WBSF value at 1 d postmortem. Specifically, supplementation of supranutritional Zn alone (SUPZN-NO; 5.40 kg) resulted in a 2.2-kg-lower WBSF value at 1 d postmortem than supplementation of RH alone (CON-RAC; 7.64 kg). Compared with the control treatment (6.72 kg; CON-NO), SUPZN-NO demonstrated a 1.3-kg-lesser WBSF value at 1 d postmortem. Supplementation of RH has consistently resulted in greater WBSF values at 3–14 d postmortem in the longissimus lumborum of beef compared with cattle not supplemented with RH (Avendaño-Reyes et al., 2006; Scramlin et al., 2010; Boler et al., 2012; Bohrer et al., 2014; Lean et al., 2014). The consistency of greater WBSF values in steaks from cattle supplemented with RH remains unless steaks are aged for extended periods (≥14 d; Scramlin et al., 2010; Boler et al., 2012; Bohrer et al., 2014). This delay in onset of or slower rate of tenderization could provide the opportunity to investigate and understand biological factors that influence a delayed or hastened rate of tenderization, even if tenderness differences do not persist later postmortem (≥14 d). An understanding of this biological difference in rate of tenderness development could provide for application of alternative merchandizing strategies of beef products.

Figure 2.

Effect of supranutritional zinc (Zn) and ractopamine hydrochloride (RH) supplementation on pH values of beef longissimus thoracis muscle at different postmortem timepoints.^1,2,3

¹CON = no supplemental Zn (analyzed 36 mg Zn/kg dry matter [DM]); SUPZN = CON + 60 mg Zn/kg DM (from ZnSO4) + 60 mg Zn/kg DM (from Zn–amino acid complex; Availa-Zn; Zinpro Corporation, Eden Prairie, MN). Fed for the entire 89-d trial.

²NO = no supplemental RH; RAC = 300 mg RH per head per day (Actogain45; Zoetis, Parsippany, NJ) starting 28 d prior to harvest.

³Means and standard errors of the mean are reported.

Figure 3.

Effect of supranutritional zinc (Zn) and ractopamine hydrochloride (RH) supplementation on temperature values of beef longissimus thoracis muscle at different postmortem timepoints.^1,2,3

¹CON = no supplemental Zn (analyzed 36 mg Zn/kg dry matter [DM]); SUPZN = CON + 60 mg Zn/kg DM (from ZnSO4) + 60 mg Zn/kg DM (from Zn–amino acid complex; Availa-Zn; Zinpro Corporation, Eden Prairie, MN). Fed for the entire 89-d trial.

²NO = no supplemental RH; RAC = 300 mg RH per head per day (Actogain45; Zoetis, Parsippany, NJ) starting 28 d prior to harvest.

³Means and standard errors of the mean are reported.

Figure 4.

Effect of supranutritional zinc (Zn) and ractopamine hydrochloride (RH) supplementation on Warner-Bratzler shear force (WBS) values of beef longissimus thoracis steaks at different postmortem timepoints.^1,2,3.4

¹CON = no supplemental Zn (analyzed 36 mg Zn/kg dry matter [DM]); SUPZN = CON + 60 mg Zn/kg DM (from ZnSO4) + 60 mg Zn/kg DM (from Zn–amino acid complex; Availa-Zn; Zinpro Corporation, Eden Prairie, MN). Fed for the entire 89-d trial.

²NO = no supplemental RH; RAC = 300 mg RH per head per day (Actogain45; Zoetis, Parsippany, NJ) starting 28 d prior to harvest.

³WBS values were averaged across 2 adjacent steaks.

⁴Means and standard errors of the mean are reported.

The differences between the treatments in pH decline at 6 h postmortem and WBSF values at 1 d postmortem are strikingly similar in the observed figures (Figures 2 and 4). Correlation coefficients found in Table 1 demonstrate a moderately strong positive correlation of pH values at 3 h (r = 0.50; P = 0.03) and 6 h (r = 0.55; P = 0.01) postmortem with WBSF values at 1 d postmortem. This relationship is consistent with Hwang and Thompson (2001a) observations, demonstrating a lower early postmortem WBSF value in beef muscles with the most rapid rate of pH decline. These data further underscore the need to investigate the biochemistry underlying the relationship of early postmortem pH decline and metabolism with tenderness development.

Consistent evidence of the relationship of pH to tenderness supports the hypothesis that pH has an influence on biochemical factors that influence tenderness. Wu et al. (2014) classified longissimus dorsi muscle from bull carcasses into high (≥6.2), intermediate (5.80–6.19), and low (≤5.79) ultimate pH classifications based on 48-h pH measurements. Beef with highest ultimate pH resulted in the lowest WBSF value at each day of aging (1, 2, 7, 14, 21, and 28 d postmortem) (Wu et al., 2014). The intermediate ultimate pH classification resulted in the greatest WBSF values across all postmortem aging timepoints, and the lowest ultimate pH classification was in between the 2 classifications (Wu et al., 2014). In the current study, a trend for a moderately strong positive correlation was determined between the pH at 24-h-postmortem and 14-d-postmortem WBSF values (r = 0.39; P = 0.09). The 24-h pH values ranged from 5.38 to 5.62 in this study; thus, we did not have the extremes of ultimate pH values that Wu et al. (2014) suggested could strongly influence product quality.

As stated earlier, an intermediate rate of pH decline results in the most tender beef products (Marsh et al., 1987; Hwang and Thompson, 2001b). A moderately strong negative correlation was observed between 6-h pH and 7-d WBSF values (r = −0.44; P = 0.05), demonstrating a less extreme rate of pH decline resulting in a more tender product when given proper aging time (≥7 d).

The rate of pH and temperature decline are continuously interacting and influencing each other, thus collaborating to impact tenderness development (Hopkins et al., 2014; Warner et al., 2014). In this study, no differences in muscle temperature during harvest were observed among treatments or their interaction at any postmortem timepoint (Figure 3). A trend for a moderately strong negative correlation of pH at 6 h postmortem with temperature at 1 h postmortem was determined (Table 1; r = −0.40; P = 0.08). This correlation supports the hypothesis that a higher initial temperature can increase the rate of postmortem glycolysis, thus causing a more rapid pH decline (Kim et al., 2014). A moderately strong negative correlation was observed between temperature at 3 h (r = −0.50; P = 0.02), 6 h (r = −0.42; P = 0.06), and 24 h (r = −0.63; P < 0.01) postmortem and WBSF values at 1 d postmortem. Similarly, temperature at 3 h (r = −0.55; P = 0.01), 6 h (r = −0.52; P = 0.02), and 24 h (r = −0.53; P = 0.02) postmortem showed a moderately strong negative correlation with WBSF values at 3 d postmortem. This relationship between prerigor temperatures and early postmortem (1–3 d) tenderness development has been previously demonstrated (Hwang et al., 2004; Kim et al., 2014). Hwang et al. (2004) showed a lower WBSF value at 24 h postmortem in beef longissimus dorsi muscle that was held at a higher prerigor temperature after harvest. This difference in WBSF value was attributed to postmortem proteolysis variations. These correlations from this small data set are consistent with previous reports of the strong relationship between postmortem pH decline, temperature decline, and tenderness development in beef steaks from the LT.

Carcass characteristics and proximate analysis

The use of RH to improve growth performance and carcass lean yield has been extensively studied (Boler et al., 2012; Bohrer et al., 2014; Bittner et al., 2016, 2017; Genther-Schroeder et al., 2016b; Hagenmaier et al., 2017). However, many results related to these characteristics are inconsistent. Live weight, HCW, dressing percentage, and REA typically improve due to RH supplementation (Avendaño-Reyes et al., 2006; Boler et al., 2012; Bohrer et al., 2014). However, in this study, carcass characteristics were not affected by supplementation of Zn, RH, or the interaction (Table 2; P ≥ 0.17). Several previous studies have also demonstrated no difference in these lean yield characteristics of carcasses from cattle supplemented with RH (Avendaño-Reyes et al., 2006; Samuelson et al., 2016; Trotta et al., 2019).

No difference in 12th rib backfat, KPH, YG, or marbling score was observed in this study due to supplementation of Zn, RH, or their interaction (Table 2; P ≥ 0.17). Several studies have observed lesser fat thickness and KPH along with lower marbling scores and YG in carcasses from cattle supplemented with RH (Bohrer et al., 2014; Bittner et al., 2016; Trotta et al., 2019). Conversely, studies have shown no difference in carcass characteristics from cattle supplemented with RH (Samuelson et al., 2016; Trotta et al., 2019). These inconsistencies are thought to be due to several factors, including diet, background, dosage, length of time of dosage, the power of the study, and genetic variations.

Proximate results determined no difference in protein, moisture, or fat percentages (Table 2; P > 0.10) due to supplementation of Zn, RH, or their interaction.

Limited differences in quality characteristics existed. Data for these characteristics can be found in Supporting Information Table S1.

Calpain-1 autolysis and desmin and troponin-T postmortem protein degradation

The calpain proteolytic system, specifically calpain-1, is the primary contributor to postmortem protein degradation of myofibrillar, cytoskeletal, and intermediate filament proteins in postmortem muscle (Geesink et al., 2006; Koohmaraie and Geesink, 2006). Autolysis of calpain-1 (76-kDa product) negatively correlates with intact desmin, vinculin, and talin in pork (Bee et al., 2007). A variety of factors can influence calpain-1 activity, including pH decline (Melody et al., 2004), oxidative conditions (Rowe et al., 2004; Maddock Carlin et al., 2006), nitric oxide (Li et al., 2014; Liu et al., 2016, 2019; Zhang et al., 2018; Hou et al., 2020), and calpastatin activity (Pringle et al., 1993; Rowe et al., 2004). Zinc infused at high levels via the vascular system in carcasses immediately postmortem has been shown to limit calpain activity (Koohmaraie, 1992). Investigation into different fractions of muscle, such as the sarcoplasmic and myofibrillar fractions, has demonstrated that myofibrillar bound calpain-1 is nearly inactive proteolytically and arises from precipitation of calpain-1 onto the myofibril (Boehm et al., 1998). Calpain-1 autolysis in the whole muscle and in the sarcoplasmic fraction was not impacted by supplementation of Zn, RH, or their interaction (Table 3; P ≥ 0.06). The observed trends for greater 78-kDa and lesser 76-kDa calpain-1 (Table 3; P < 0.10) in the sarcoplasmic fraction of muscle from cattle supplemented with supranutritional Zn is interesting. In ovine muscle, Koohmaraie (1992) infused high levels of Zn chloride (1 mM) to examine the impact of Zn infusion on postmortem proteolysis and observed reduced calpain-1 activity (2.4% of maximal activity) in the Zn-treated muscle. In the study by Koohmaraie (1992), it was concluded that Zn reduced the rate of calpain-1 autolysis rather than completely inhibiting autolysis of calpain-1. This relationship between calpain-1 autolysis and Zn needs to be studied further under physiological conditions. Additionally, a faster rate of pH decline has been demonstrated to be related to a more rapid rate of calpain-1 autolysis in the psoas major of pork (Melody et al., 2004). Differences in the rate of pH decline could be impacting differences in calpain-1 activity and subsequent loss of activity earlier postmortem. Although activity was not measured in this study, several studies have demonstrated the relationship between autolysis and activity of calpain-1 in postmortem muscle (Boehm et al. 1998; Melody et al., 2004).

Postmortem protein degradation significantly impacts tenderness development (Wu et al., 2014). Desmin and troponin-T degradation have commonly been examined as indicators of postmortem proteolysis because of their known roles in structural integrity of skeletal muscle and contraction (Clark et al., 2002). Analysis of the whole muscle extract samples showed that supranutritional supplementation of Zn trended to have lesser whole muscle extract desmin degradation products at 3 and 7 d postmortem and lesser sarcoplasmic desmin degradation products at 3 and 14 d postmortem (Table 4; P < 0.10). Additionally, supranutritional Zn supplementation resulted in lesser troponin-T degradation at 3 and 14 d postmortem (Table 4; P ≤ 0.05). This may be related to the observed trends for greater 78-kDa and lesser 76-kDa calpain-1 (Table 3; P < 0.10) in the sarcoplasmic fraction at 7 d postmortem of samples supplemented with supranutritional Zn. Reduced calpain-1 activity or earlier loss of calpain-1 activity will result in less postmortem protein degradation (Rowe et al., 2004). Previously, Zn introduced to muscle at levels higher than seen physiologically has been shown to have an inhibitory effect on calpain-1 activity (Koohmaraie, 1992). Thus, our results provide evidence that the relationship between physiologically achievable Zn concentrations and calpain activity/autolysis needs to be characterized more fully. However, it must be kept in mind that, despite these observed trends of differences in calpain-1 autolysis at 7 d postmortem, this did not result in differences in WBSF values at 7 or 14 d postmortem between treatments.

Supplementation of RH resulted in lesser whole muscle extract desmin and troponin-T degradation products at 1 d postmortem (Table 4; P ≤ 0.05) compared with samples not supplemented with RH. Lesser protein degradation at 1 d postmortem in samples supplemented with RH compared with samples not supplemented with RH could explain the increased WBSF values at 1 d postmortem in samples supplemented with RH compared with samples not supplemented with RH. Along with a higher pH at 6 h postmortem, a strong relationship between rate of pH decline, day-1 WBSF values, and day-1-postmortem proteolysis of desmin and troponin-T of whole muscle extracts is demonstrated in samples supplemented with RH. Correlation analysis further demonstrates this relationship between pH decline and postmortem proteolysis (Table 5). Sarcoplasmic calpain-1 76-kDa autolysis product demonstrated a moderately strong negative relationship with pH values at 3 h (r = −0.58; P = 0.01) and 6 h (r = −0.61; P < 0.01) postmortem. This relationship is strengthened with the shown correlation of pH values at 6 h postmortem trending to have a moderately strong negative correlation with whole muscle desmin (r = −0.44; P = 0.06) and troponin-T (r = −0.39; P = 0.09) degradation products at 1 d postmortem.

Additionally, RH supplementation resulted in less desmin degradation product in the whole muscle extract at 3 and 7 d postmortem and less appearance of the soluble desmin degradation product in the sarcoplasmic fraction at 3 d postmortem (Table 4; P < 0.01). Less postmortem proteolysis in samples from RH-supplemented cattle could potentially also be due to greater calpastatin activity and inhibition of the calpains, resulting in less calpain-1 activity in muscle from cattle fed RH (Strydom et al., 2009).

The interaction of Zn and RH supplementation resulted in less whole muscle extract desmin degradation product at 1 h (Table 4; P = 0.04) and 14 d (Table 4; P < 0.01) postmortem compared with samples not supplemented with Zn or RH. Additionally, the interaction of Zn and RH supplementation resulted in less soluble sarcoplasmic desmin degradation products at 7 d (Table 4; P < 0.01) postmortem compared with samples not supplemented with Zn or RH. These observed differences in protein degradation further lend credence to the need to more fully understand the role of Zn and RAC fed in combination in muscle growth and early postmortem metabolism.

The relationship between postmortem protein degradation and instrumental tenderness values has been extensively studied (Huff-Lonergan et al., 1996; Hwang et al., 2004; Melody et al., 2004; England et al., 2012; Carlson et al., 2017b), and these data continue to demonstrate this strong relationship (Table 5). A moderately strong negative relationship was demonstrated between WBSF values at 3 d postmortem and the following: whole-muscle desmin degradation products at 1 h (r = −0.49; P = 0.03), 3 d (r = −0.41; P = 0.07), and 7 d (r = −0.46; P = 0.04) postmortem; sarcoplasmic calpain-1 autolysis at 7 d (r = −0.49; P = 0.03) postmortem; and whole-muscle troponin-T degradation products at 1 d (r = −0.40; P = 0.08), 3 d (r = −0.52; P = 0.02), and 14 d (r = −0.39; P = 0.09) postmortem. Furthermore, WBSF value at 7 d postmortem showed moderately and highly negative correlations with whole-muscle desmin degradation products at 7 d (r = −0.48; P = 0.03) and 14 d (r = −0.59; P = 0.01) postmortem, sarcoplasmic calpain-1 autolysis at 7 d (r = −0.69; P < 0.01) postmortem, and whole-muscle troponin-T degradation products at 7 d (r = −0.62; P < 0.01) and 14 d (r = −0.50; P = 0.02) postmortem. Warner-Bratzler shear force values at 14 d postmortem were moderately and highly negatively correlated with whole-muscle desmin degradation products at 3 d (r = −0.39; P = 0.09), 7 d (r = −0.60; P = 0.01), and 14 d (r = −0.55; P = 0.01) postmortem, sarcoplasmic calpain-1 autolysis at 7 d (r = −0.57; P = 0.01) postmortem, and troponin-T degradation products at 3 d (r = −0.45; P = 0.05), 7 d (r = −0.70; P < 0.01), and 14 d (r = −0.71; P < 0.01) postmortem. These results show the very strong relationship between tenderness development and postmortem protein degradation that has been identified by the literature.