The Longissimus thoracic et lumborum (LM), Triceps brachii (TB), Semimembranosus (SM) and Psoas major (PM) were collected from each carcass approximately 26 h postmortem. All cuts were vacuum packed and transported on ice to the Iowa State University sensory evaluation lab. Fresh pork was aged (PM 7 d, SM 8 d, LM 10 d, and TB 13 d) under refrigeration (4°C) and used to determine treatment effect on ultimate pH, purge loss, cook loss, star probe, Warner-Bratzler shear force (WBS), and sensory quality. Purge measurements were collected following aging. Samples were removed from their vacuum bag and weighed. The weight of the bag was then subtracted from the weight of purge remaining in the bag and purge was determined as a percentage of the original muscle weight [(initial weight – final weight)/initial weight] × 100. Ultimate pH was recorded on whole cuts and 2.54 cm thick chops (LM and SM) were prepared. Hunter L, a, and b values were collected using a Minolta Chroma Meter fitted with a 50 mm aperture and a D65 light source with a 2° observer. Color scores (National Pork Board, 1999; standard 6-point scale, 1 = pale pinkish gray to white; 6 = dark purplish red) marbling scores (National Pork Board, 1999; standard 10-point scale, 1 = 1.0% intramuscular fat; 10 = 10.0% intramuscular fat) were assigned to chops (SM and LM).

Raw weights were collected on chops (LM and SM) and roasts (PM and TB) for cook loss determinations. Chops were cooked in clamshell grills to an internal temperature of 68°C (Santos et al., 2018). Roasts (PM and TB) were cooked in a rotary hearth oven at 176°C to an internal temperature of 68°C. Cooked weight was then collected and samples were divided into 1.27 × 1.27 × 2.54 for trained sensory analysis (n = 4; Carlson et al., 2017a). Two additional chops or roasts were cooked using the same procedures as listed above. These chops were allowed to cool to 22°C. An Instron Universal Testing Machine (Instron Industrial Products, Grove City, PA) fitted with Warner-Bratzler shear force head was used to determine Warner-Bratzler shear force values (Schulte et al., 2019). A 5-pointed star probe was fitted to the Instron and compressed the sample to 80% of its height to determine star probe value (Lonergan et al., 2007; Schulte et al., 2019).

Sensory analysis was conducted as described by Carlson et al. (2017a) to determine tenderness, chewiness, juiciness, flavor, and off-flavor. Trained panelists (n = 4) evaluated samples on a 10-point anchored scale. A lesser value represents a lesser degree of a trait and greater values represents a greater degree of the trait (1 = tough, 10 = tender; 1 = not chewy, 10 = chewy; 1 = dry, 10 = juicy; 1 = no pork flavor, 10 = intense pork flavor; 1 = no off-flavor, 10 = intense off-flavor). Panelists were required to complete a 6-mo training period. During this period, they were provided with samples that exhibited extremes of the sensory attributes measured in each cut included in the study. Panelists were served 1.27 × 1.27 × 2.54 cm cubes of each sample immediately following cooking. Unsalted crackers and ice water were provided between samples.

Samples aged 2 and 10 d postmortem were frozen in liquid nitrogen and then powdered using a Waring blender (Waring Commercial, New Hartford CT). Whole muscle protein was extracted from frozen powdered samples as described by Carlson et al. (2017a, 2017b) using a solubilizing buffer (2% wt/vol SDS, 10mM sodium phosphate, pH 7.0). Protein concentration was determined using a Lowry protein assay with premixed reagents (DC protein assay, Bio-Rad Laboratories, Hercules, CA). Protein solutions were adjusted to 4 mg/mL in Wang’s tracking dye (3 mM EDTA, 3% [wt/vol] SDS, 30% [vol/vol] glycerol, 0.01% [wt/vol] pyronin-Y, 30 mM Tris HCl, pH 8.0), vortexed, heated (approximately 50°C for 15 min), and then stored at –80°C.

SDS-PAGE and Western Blot were all conducted as described by Carlson et al. (2017a, 2017b). Briefly, desmin degradation on all samples (PM, TB, SMS, SMD, and LM) aged 2 and 10 d postmortem was determined using 10% polyacrylamide separating gels (acrylamide/bisacrylamide = 100:1 [wt/wt], 0.1% [wt/vol] SDS, 0.05% [vol/vol] tetramethylenediamine [TEMED], 0.05% [wt/vol] ammonium persulfate [AMPER], and 0.5 M Tris HCl pH 8.8). Troponin-T degradation was determined on LM samples aged 2 and 10 d using 15% polyacrylamide gels. Autolysis of calpain-1 in LM samples aged 2 d was determined using 8% polyacrylamide. For desmin degradation and calpain-1 autolysis analysis, 40 µg of sample was loaded in each lane, while 20 µg of sample was used in troponin-T degradation determination. Gels (10 cm wide × 8 cm tall) were run in SE 260 Hoefer Mighty Small II (Hoefer, Inc., Holliston, MA) electrophoresis units. The running buffer was a solution composed of 25 mM Tris, 192 mM glycine, 2 mM EDTA, and 0.1% [wt/vol] SDS. Desmin gels were run for approximately 375 V-hours, troponin-T gels were run for approximately 360 V-hours and µ-calpain gels were run for approximately 350 V-hours. Proteins from gels were transferred to polyvinylidene difluoride (PVDF) membrane. Blots were blocked for 1 h at 22°C using 5% non-fat dry milk [wt/vol] in PBS-Tween (80 mM Na₂HPO₄, anhydrous, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% [vol/vol] polyoxyethylene sorbitan monolaurate [Tween-20]. Blocking solution was then discarded and primary antibodies were added to PBS-Tween in the following dilutions, desmin- 1:40,000 using rabbit anti-desmin polyclonal antibody produced in house, troponin-T- 1:40,000 using monoclonal mouse anti-troponin-T (T6277, JLT-12, Sigma, Saint Louis, MO) and µ-calpain- 1:5,000 using monoclonal mouse anti-µ-calpain (MA3–940, Thermo Scientific, Rockford, IL). Blots were incubated in 10 mL of diluted antibody solution overnight at 4°C. Blots were then washed 3 times in 10 min intervals using 10 mL of PBS-Tween before a 1 h incubation in secondary antibody (22°C). Dilutions for secondary antibodies in PBS-Tween are as follows: desmin- 1:20,000 using goat anti-rabbit antibody (31460, Thermo Scientific), troponin-T- 1:30,000 using goat anti-mouse antibody (A2554, Sigma), and µ-calpain- 1:10,000 using goat anti-mouse antibody (A2554, Sigma). Following secondary antibody incubation, blots were again washed 3 times in 10 min intervals using 10 ml of PBS-Tween before detection using a chemiluminescent detection kit (ECL Prime, GE Healthcare, Piscataway, NJ).

Densities of immunoreactive protein bands were quantified using ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and Alpha EaseFC (v. 2.03; Alpha Innotech). Desmin degradation was indicated by a decrease in the intensity of the approximate 55 kDa band and compared against a reference collected from a pork LM aged 7 d postmortem, troponin-T degradation was indicated by an increase in intensity of the approximate 30 kDa band and compared against a reference collected from a pork LM aged 7 d postmortem, and 2 d µ-calpain autolysis was indicated by an increase in the approximate 76 kDa band and compared to LM reference aged 12 h postmortem.

Calpastatin activity was determined on LM samples at 2 d postmortem following procedures of Cruzen et al. (2014). Fresh muscle was finely minced and 10 g was homogenized in 30ml extraction buffer (100 mM Tris-HCl pH 8.3, 10 mM mercaptoethanol, 10mM EDTA, 100mg/L ovomucoid) using a Polytron PT 3100 (Lucerne, Switzerland). The solution was then centrifuged at 25,000 × g for 20 min and filtered through cheesecloth. Samples were then dialyzed for at least 6 h against 40ml of 40mM Tris-HCl pH 7.4, 1mM EDTA. Following dialysis, samples were heated for 20 min in a 95°C water bath and immediately chilled in ice cold water for 15 min. Samples including proteins in suspension and solution were transferred to 30 mL centrifuge tubes, centrifuged at 25,000 × g for 20 min, filtered through cheesecloth and volumes recorded for calpastatin activity calculation. Calpastatin activity was using partially purified lung m-calpain (Koohmaraie, 1990; de Oliveira et al., 2019).

One LM chop was collected 30 h postmortem, frozen and stored until analysis. Powdered frozen muscle tissue was suspended in a sucrose buffer (20 mM Sucrose, 20 mM potassium chloride, 5 mM sodium iodoacetate; pH 7.0) on the slide. Approximately 50 ML of the homogenate was placed on a slide with a cover slip. The beam of a helium-neon laser (γ = 0.6328 nm; Melles Griot, Carlsbad, CA) was passed through the homogenate and the distance between first-order diffraction bands was recorded. Sarcomere length was calculated using the equation prescribed by Cross et al. (1981). Five replicates were conducted for each sample.

Data were analyzed using a mixed linear model (PROC MIXED, SAS Enterprise Guide 5.1, SAS Inst. Inc., Cary, NC). The model included fixed effects of Treatment (TRT), Harvest day, and TRT*Harvest day. Carcasses (n = 40) served as random effects. To determine the effect of carcass composition, covariates of HCW, FFL, and the quadratic of each were added to the model. Least square means and standard errors were reported for each trait. A model using fixed effects of TRT, Harvest day, TRT*Harvest day, Location (SMS vs. SMD), and Location*TRT and carcass as a random effect was used to determine the effect of location within the SM. Significance level was determined at P ≤ 0.05.

Sarcomere length can influence water holding capacity and tenderness (Ertbjerg and Puolanne, 2017). In the current study, sarcomere length in the LM was not different across chilling treatments (Table 3) and therefore quality differences cannot be attributed to sarcomere length in the LM. It was hypothesized that rapid chilling would result in less observed protein degradation in the early postmortem period. However, chilling did not affect LM calpain-1 autolysis at 2 d postmortem (Table 3) nor affect intact desmin intensity as measured by the 55 kDa band in the LM (Table 3), PM, SM, or TB measured 2 or 10 d postmortem (Table 4). Furthermore, there was no difference in troponin-T degradation product as measured by the 30 kDa band in the LM aged 2 and 10 d postmortem (Table 3). Calpastatin activity at 2 d postmortem in the LM also was not influenced by chilling treatment (Table 4).

Least squares means of purge loss and cook loss are summarized in Table 5. Chilling regime did not have an effect on purge loss in cuts from the LM, SM or TB. These results are in contrast to other reports that reported rapid chilling resulted in less purge and drip loss (McFarlane and Unruh, 1996; Janiszewsi et al., 2018). The difference may be that the control methods in those investigations had a slower chilling rate.

Roasts from the PM of BC sides had greater drip loss than CC sides. Because of the PM’s exposed location in a dressed carcass, it is less insulated and it is likely that temperature decline will be most rapid in this muscle, and will experience a cold shortening effect that will decrease myofibrillar space for water storage (Honikel et al., 1986; Offer and Cousins, 1992). Offer and Cousins (1992) found that the formation of drip in the event of sarcomere shortening could be explained by the formation of “drip channels” allowing water to escape from muscle fiber to the endomysium and ultimately lost from muscle tissue. This could explain the increased purge loss in roasts from the PM of BC sides. Although sarcomere length was not measured in these samples, a discussion point is that more rapid chilling in the PM might have created a cold shortening effect, creating drip channels and increasing purge lost.

Increased purge in the PM could also be explained by formation of ice crystals on the surface of muscle tissue. Rapid chilling of pork, specifically by air chilling, has been found to lead to ice crystal formation throughout muscle structure (Ngapo et al., 1999; Hansen et al., 2003). When muscle thaws, there is a significant increase in purge loss due to ice formation and melting.

Linkage of myofibrils in the LM may have an effect on water loss during cooking due to sarcomere shortening. This greater contraction during chilling could be a result of lack of early postmortem degradation of intermediate filament proteins which link muscle fibers to the sarcolemma and other muscle fibers at the Z-line causing increased purge loss (Morrison et al., 1998; Kristensen and Purslow, 2001; Melody et al., 2004; Bee et al., 2007). Melody et al. (2004) found that degradation of desmin in the PM began as early as 45 min postmortem in conventionally chilled pork carcass. Pomponio et al. (2010) found that early postmortem pH decline influenced µ-calpain activity with muscle with faster postmortem pH decline having increased µ-calpain activity postmortem. No difference in desmin degradation was noted at 2 or 10 d in due to treatment in the current study, but no commentary can be provided for the contribution to rate of proteolysis before the 2 d postmortem.

Chops from the LM of BC sides had greater cook loss (Table 5) and WBS (Table 6) than chops from CC sides but demonstrated no significant difference in proteolysis, calpastatin activity or calpain autolysis (Table 3). Results from the current study are consistent with previous results (Shackelford et al., 2012) that also found an increased toughness in the LM as determined by slice shear force value but found no difference in proteolysis after 15 d aging. Increased WBS values were not reflected in star probe values, or for sensory tenderness or chewiness.

Roasts from the PM of BC sides were juicier, more tender and less chewy than roasts from CC sides (Table 7) but treatment did not affect WBS or star probe. Blast chilling had an effect in the SM with decreased Hunter a value indicating a lower intensity of red pigment, and a darker observed color score (Table 8). No treatment effect was found in cuts from the TB (Table 9).

Postmortem pH decline, protein degradation, and changing postmortem muscle structure impacts fresh pork quality. The current study confirms the influence of chilling on fresh pork quality varies with muscle metabolism and carcass location. Postmortem proteolysis and changes in muscle biochemistry are responsible for the development of fresh pork quality. Slowing the rate of postmortem pH decline in the LM has a direct effect on the rate and extent of normal changes in the conversion of muscle to meat. These changes include protein degradation and denaturation. Rapid chilling did result in greater WBS in LM chops, but did not affect star probe, sensory tenderness, or sarcomere length. In addition, no difference in proteolysis due to chilling protocol was detected in any muscle evaluated. The influence of chilling on early postmortem muscle modification may warrant further investigation.