Sample collection and preparation

Beef strip loins [IMPS #180; n = 117; 39/marbling score group (Top Choice, Low Choice, and Select)] of 3 marbling texture treatments (fine, medium, and coarse) were selected through visual appraisal using the USDA-AMS-LS-SB-02 marbling texture reference card. This reference card provides a visual reference for fine, medium, and coarse marbling based on marbling spec size. All strip loins were graded and selected by trained Kansas State University (KSU) research personnel to fit each of the various marbling score and texture treatments. To be selected for the study, greater than 75% of the marbling in the ribeye was required to meet the USDA visual standard for the designated marbling texture treatment. After selection, strip loins were transported under refrigeration (2°C) to the KSU Meat Laboratory for steak cutting and processing. Strip loins were fabricated into 2.54-cm thick steaks from anterior to posterior using a manual electric slicer (Model KAMS-14; Pro-Cut, Houston, TX) 72 h postmortem. The anterior most “face steak” was removed and the next steak was used for histology analysis.

Histochemistry and immunohistochemistry

Within each histology steak, 3 marbling flecks that best represented the marbling texture treatment were selected for extraction from the medial, central, and lateral portion of the steak to provide the best representation of the entire steak in the analyses. A 1.27 cm × 1.27 cm × 2.54 cm area around the fleck was extracted and embedded in optimum cutting temperature tissue freezing medium (Tissue Tek OCT; VWR; Radnor, PA), frozen in liquid nitrogen cooled isopentane, and stored at -80°C until subsequent analysis. After sample embedding, the remaining portions of each steak were vacuum packaged in 3 mil standard barrier vacuum bags with oxygen transmission rates of 4.5 cm³ × 100 cm^–2 × 24 h^–1 at 23°C and 65% humidity (Prime Source Vacuum Pouches; Bunzl Processor Division, Koch Supplies, Kansas City, MO) using a Multivac C500 (Cryovac Inc., Elmwood, NJ) and aged for 21-d postmortem at 2 to 4°C.

The methods of Phelps et al. (2014) were used for cryosection collection and immunohistochemical analysis. Seven-micrometer cryosections were collected on positively charged glass microscope slides. For each marbling fleck, 1 cryosection was taken for immunofluorescent fiber type staining and a serial cryosection was taken for Masson’s trichrome staining for perimysial collagen and adipocyte staining. Samples were trimmed 2 mm from the location of the first cryosection and 2 serial cryosections were collected.

Immunohistochemical cryosections were incubated in a blocking solution of 10% horse serum and 0.2% Triton X-100 in phosphate buffer solution (PBS) for 30 m at room temperature. Slides were then incubated in the primary antibody solution of 1:500 rabbit ɑ-dystrophin (Thermo-Fisher Scientific, Waltman, MA), 1:10 supernatant mouse anti-MHC, slow IgG2b (BA-D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and 1:10 mouse anti-MHC all but type IIX, IgG1 (BF-35; Developmental Studies Hybridoma Bank). After the rinsing step of incubation of slides with PBS for 5 min, a solution of secondary antibodies was applied for 30 min. The secondary antibody solution included 1:100 Alexa-Fluor 488 goat anti-mouse IgG1 (Invitrogen, Grand Island, NY); 1:1000 Alexa Fluor 633 goat anti-mouse IgG2b (Invitrogen); 1:1000 Alexa-Fluor 594 goat anti-rabbit H&L (Invitrogen); and 1:1000 DAPI (Thermo-Fisher Scientific). After another washing step, slides were coverslipped for imaging.

A Masson’s trichrome staining kit was used (Sigma-Aldrich; St. Louis, MO) for staining histology cryosections. Slides were incubated in deionized water for 2 min, followed by a Bouin’s solution (Sigma-Aldrich; St. Louis, MO) incubation for 15 min at 56°C. Samples were then successively incubated in tap water, Weigert’s Hematoxylin solution, tap water again, deionized water, Beibrich Scarlet-Acid Fuchsin stain, a phosphotungstic and phosphomolybdic acid solution, and aniline blue solution for 5 min each. Following these solutions, slides were then incubated in a 1% acetic acid solution for 2 min, and then successively incubated in deionized water, 100% ethanol, and xylenes for 1 min each. Slides were coverslipped for imaging.

Cryosections were imaged at 100-fold magnification with a Nikon TI-U inverted microscope (Nikon; Lewisville, TX) fitted with an X-Cite 120XL epifluorescence illumination system (EXFO; Mississagua, Ontario, Canada), a DS-QiMC digital camera (for immunohistochemical imaging), and a DS-Fi1 digital camera (for histochemical imaging). Images were taken specifically near the marbling fleck in order to evaluate the impact of marbling texture on the specified traits.

Immunohistochemistry photomicrographs were analyzed for muscle fiber cross-sectional area and myosin heavy chain (MHC) type distribution using NIS-Elements Imaging Software (Basic Research 3.3; Nikon Instruments, Inc., Melville, NY). For each marbling fleck sample, a minimum of 3 photomicrographs and 300 fibers were analyzed per section. Fibers that stained positive for BA-D5 antibody were considered as MHC Type I fibers. Fibers that stained positive for the BF-35 antibody and negative for BA-D5 antibody were considered as MHC type IIA fibers. Any fibers that did not stain positive for both BF-35 and BA-D5 were considered as MHC Type IIX fibers. The ɑ-dystrophin ring around each fiber identified the fiber cross-sectional area (Ebarb et al., 2016).

Histochemistry photomicrographs were analyzed for adipocyte cross-sectional area and perimysial thickness using NIS-Elements Imaging Software (Basic Research 3.3; Nikon Instruments, Inc.). For each marbling fleck, a minimum of 200 adipocytes per steak were measured and perimysium thickness was measured every 10 micrometers with a minimum of 10 measurements per image (Figure 1).

Figure 1.

Representative photomicrographs of Masson’s trichrome staining of bovine longissimus lumborum intramuscular adipose and muscle tissue; a) Photomicrograph utilized for intramuscular adipocyte cross-sectional area analysis, b) Photomicrograph utilized for perimysium thickness measures (A = perimysium; B = marbling adipocyte; C = muscle cell). Scale bars = 100 µm.

Collagen solubility

A modified method described by Gonzalez et al. (2014) was used to determine hydroxyproline content of each sample. Briefly, following the 21-d aging period, steaks were diced and frozen in liquid nitrogen, then homogenized using a Waring blender (Waring Products Division; Hartford, CT) and stored at -80°C until further analysis. Three grams of ground sample in duplicate were mixed with 0.25 strength Ringer’s Solution. The tubes were then placed in a hot water bath at 77°C for 70 m and stirred every 10 m. Samples were then centrifuged at 20°C for 15 m at 5,200 × g. Soluble samples were then decanted into autoclave milk bottles through Fisher 09-795 filter paper (Thermo-Fisher Scientific). Soluble samples were re-centrifuged with 0.25 strength Ringer’s solution under the same conditions, and then re-decanted. After the sample was filtered, 25 mL of 12 N hydrochloric acid was added to the bottles and they were sealed. The remaining pellet was transferred into autoclave bottles with the filter paper used for decantation and labeled as the insoluble fraction. The centrifuge tubes were rinsed with 25 mL of 6 N hydrochloric acid and decanted into the bottles.

Following decantation, all samples were autoclaved for 18 h at 121°C at 18 to 21 psi. One gram of decolorizing charcoal was added to each sample bottle and mixed thoroughly. Following the addition of charcoal, all samples were filtered through Whatman #2 filter paper (Thermo-Fisher Scientific). Sample bottles were rinsed with deionized water to bring the soluble samples to 175 mL and insoluble samples to 300 mL. Each sample’s pH was adjusted to 6 using 12 N sodium hydroxide and various concentrations of hydrochloric acid. Soluble samples were diluted to 250 mL and insoluble samples were diluted to 500 mL in volumetric flasks. All samples were thoroughly mixed and filtered through Fisher 09-795 filter paper into 15 mL centrifuge tubes and frozen at -40°C until reading. Hydroxyproline determination was accomplished using procedures of Bergman and Loxley (1963). A BioTek Eon spectrophotometer (Biotek Instruments Inc., Winooski, VT) was used to read absorbance at 558 nm and oriented using a blank of distilled water and a standard curve created for each group of analyses. To determine total and fractional collagen contents, hydroxyproline content was multiplied by 7.52 for the soluble fraction and 7.25 for the insoluble fraction (Cross et al., 1973).

Transition temperature of collagen

The protocol of Light and Champion (1984) was used to extract the perimysial fraction of muscle. In short, 50 g of tissue was diced, homogenized in cold 0.05 M calcium chloride in a glass Waring blender (Waring Products Division; Hartford, CT), and filtered through a 1-mm² sieve. This process was repeated 3 times with tissue remaining on top of the screen. Following separation, samples were freeze dried for 24 h and 20 mg of the dried tissue were weighed into aluminum pans (Shimadzu Scientific Instruments, Kyoto, Japan). Peak thermal transition temperature was analyzed using a differential scanning calorimeter (Shimadzu Scientific Instruments), and analyzed using TA 60WS software (Shimadzu Scientific Instruments).

Statistical analysis

Statistical analysis was performed using the PROC GLIMMIX procedure of SAS (Version 9.4; SAS Inst. Inc., Cary, NC). Data were analyzed with strip loin as the experimental unit as a completely randomized design with a 3 × 3 factorial arrangement, with marbling score group, marbling texture and the marbling score group × texture interaction serving as fixed effects. The Kenward-Roger adjustment was used for all analyses. Additionally, effects were considered significant with an ɑ of 0.05.