Animal treatment and sampling
One hundred male broilers (from Arbor Acres) were raised under similar conditions (25 °C ± 1 °C, RH 58% ~ 62%) for the first six weeks. Twenty-four broilers with similar weight (2.32 ± 0.02 kg) were selected from those and eight broilers each (n = 8) were randomly assigned to either (1) 36 °C heat exposure, (2) 38 °C heat exposure, or (3) 40 °C heat exposure for 90 minutes. All 24 broilers were moved to a preparation room (25 °C) and caged individually for 24 hours before the SHE for adaption. After the adaptation period, the broilers were moved into a heat exposure room for applying the treatments for 90 min. All the cage positions in the heat exposure room were the same as in the preparation room, and water was offered throughout the treatment.
After 90 minutes of exposure, each broiler was moved out and then slaughtered and bled within five minutes. After exsanguination, boneless pectoralis major (PM) muscle from both sides of the bird was removed manually by knife cutting immediately, without scalding or defeathering, and stored at 4 °C for aging and analysis. Two grams of sample was collected from PM muscle from each side at 0.1 h, 1 h, 2 h, and 4 h postmortem for enzyme analysis or pH measurement. An additional sample from right breast muscle was collected at 24 h postmortem for the ultimate pH measurement. The collected samples were placed into 5 mL antifreeze tubes, frozen in liquid nitrogen, and stored at -80 °C until analysis.
Muscle pH value
The pH values of the right breast muscles were measured, according to Wang et al. [31]. One-gram muscle sample was homogenized in 9 mL of 5 mM iodoacetate solution for 60 s by a blender (IKA, T18, Staufen, Germany) at medium speed. The pH was measured by a pH meter (SevenGo, Mettler Toledo, Zurich, Switzerland). The pH value was calculated from the average of the three repetitions.
Enzyme activity measurement
The activity of enzyme glycogen phosphorylase a (GPa) was measured because GPa is typically more active than glycogen phosphorylase b, and the latter form can be converted to GPa by phosphorylation [32] or calcium activation [33]. Glycogen phosphorylase a (GPa) activity was estimated spectrophotometrically using a glycogen phosphorylase assay kit (GPA-2-Y, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). Approximately 100 mg sample was homogenized in 1 mL extraction solution. After centrifugation (8,000 × g at 4 °C, 10 min), 10 µL of the supernatant was mixed with 10 µL distilled water and 180 µL reaction mixture in a microvolume quartz cuvette and held at 37 °C. The absorbance values at 340 nm were recorded after 5 min (A1) and 10 min (A2). Protein concentration (Cpr) was determined using a BCA protein assay kit (BCAP-2-W, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). The activity of GPa was calculated as (643 × A)/Cpr where A = A2 – A1.
Hexokinase (HK) activity was determined spectrophotometrically with a hexokinase assay kit (HK-2-Y, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). Approximately 100 mg of sample was homogenized in 1 mL extraction solution. After centrifugation (8,000 × g at 4 °C, 10 min), 30 µL of the supernatant was mixed with 1008 µL reaction mixture in a quartz cuvette and held at 37 °C. The absorbance values at 340 nm were recorded after 20 s (A1) and 5 min 20 s (A2). Protein concentration (Cpr) was determined using a BCA protein assay kit (BCAP-2-W, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). The activity of HK was calculated as (1113 × A)/Cpr, where A = A1 – A2.
Phosphofructokinase-1 (PFK1) activity was determined spectrophotometrically with a phosphofructokinase-1 assay kit (PFK-2-Y, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). Approximately 100 mg of sample was homogenized in 1 mL extraction solution. After centrifugation (8,000 × g at 4 °C, 10 min), 30 µL of the supernatant was mixed with 810 µL reaction mixture in a quartz cuvette and held at 37 °C. The absorbance values at 340 nm were recorded after 20 s (A1) and 10 min 20 s (A2). Protein concentration (Cpr) was determined using a BCA protein assay kit (BCAP-2-W, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). The activity of PFK1 was calculated as (450 × A)/Cpr, where A = A1 – A2.
Pyruvate kinase (PK) activity was determined spectrophotometrically with a pyruvate kinase kit (PK-2-Y, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). Approximately 100 mg of sample was homogenized in 1 mL extraction solution. After centrifugation (8,000 × g at 4 °C, 10 min), 50 µL of the supernatant was mixed with 950 µL reaction mixture in a quartz cuvette and held at 37 °C. The absorbance values at 340 nm were recorded after 20 s (A1) and 2 min 20 s (A2), respectively. Protein concentration (Cpr) was determined using a BCA protein assay kit (BCAP-2-W, Suzhou Comin Biotechnology Co. Ltd, Suzhou, China). The activity of PK was calculated as (2613 × A)/Cpr, where A = A1 – A2.
Immunoblot analysis for phosphorylation of AMPK
The determination of p-AMPK-α[Thr172] was modified based on previous methods [14, 34]. Frozen muscle samples (0.5 g) were homogenized (13,500 × g for 10 s) using a polytron homogenizer (IKA Works, Inc., Wilmington, NC, USA) in 5 mL pre-cooled buffer containing 20 mmol/L Tris-HCl (pH 7.4 at 4 °C), 2% sodium dodecyl sulfate (SDS), 5 mmol/L ethylenediamine tetraacetic acid (EDTA), 5 mmol/L ethyleneglycoltetracatic acid (EGTA), 1 mmol/L dithiothreitol (DTT), 100 mM sodium fluoride (NaF), 2 mmol/L sodium vanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin and 10 µg/mL pepstatin. Muscle homogenates were then centrifuged at 17,500 × g at 4 °C for 20 min, and the supernatants were collected into fresh tubes. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Sigma, Dorset, UK).
For electrophoresis, each homogenate was mixed with an equal amount of 2 × standard sample loading buffer and heated for 3 min in a dry heater (100 °C). Equal amounts of total protein (45 µg) were resolved by electrophoresis (Bio-Rad, Richmond, CA, USA) on 10% SDS-PAGE (polyacrylamide gel electrophoresis) before being transferred electrophoretically to a polyvinylidenefluoride (PVDF) membrane. The transfer was done with a transfer buffer containing 25 mmol/L Tris, 192 mmol/L glycine, 15% (v/v) methanol, 0.01% (w/v) SDS using 90 V for 90 min at 4 °C. After transfer, the membranes were incubated in 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBST; 0.8% NaCl (wt/vol), 0.02% KCl (wt/vol), 0.24% Tris (wt/vol), 0.05% Tween-20 (vol/vol)) for 1 h. Primary antibody, phospho-AMPK-α[Thr172] antibody (Cell Signaling Technology, Beverly, MA, USA) or monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Bio-world, St. Louis Park, MO, USA) was diluted in TBST. The PVDF membranes were incubated overnight at 4 °C with the primary antibody. The blots were washed and incubated for 1.5 h with a secondary antibody (Jackson, West Grove, PA, USA). After 30 min washing, membranes were visualized using ECL Western blotting reagents (Amersham Bioscience, Piscataway, NJ, USA) and exposed to film (MR; Kodak, Rochester, NY, USA). The band densities were quantified by using Imager Scanner II and Image Quantity One software (GE, CT, USA). Band density among different blots was normalized according to the density of the reference band.
Statistical analysis
A split-plot design was used to evaluate the effects of antemortem SHE temperature and postmortem time on muscle pH, glycolytic enzyme activity, and AMPK phosphorylation. Samples were assigned to temperature x postmortem time combinations. Data analysis was performed by R (version 3.6.1) using the lme4 package as a mixed model, where temperature, postmortem time, and their interactions were fixed effects, and the random effect in the model was the individual bird. Differences between least-square means (P < 0.05) were determined by Tukey’s multiple comparisons.