Muscle fiber characteristics and postmortem quality of longissimus thoracis, psoas major and semitendinosus from Chinese Simmental bulls

Abstract Using Chinese Simmental cattle semitendinosus, psoas major, and longissimus thoracis samples, we assessed muscle fiber characteristics and postmortem quality. The type I, IIA, and IIB fiber diameters were greater in semitendinosus and longissimus thoracis relative to psoas major, with psoas major, semitendinosus, and longissimus thoracis having the highest respective percentages of type I, IIB, and IIA fibers. Psoas major had the highest R 248 and R 250 values and lowest R 258 values at 1‐ and 6‐hr postmortem. Psoas major had the lowest Warner–Bratzler shear force (WBSF), hardness, and chewiness values. The trends of WBSF, hardness, and chewiness changes decreased with increasing aging time. Semitendinosus had higher changes in WBSF than psoas major, and the number % type I fibers was correlated negatively with % changes of WBSF. Therefore, muscles with a high proportion of type IIB fibers and a low proportion of type I had lower tenderness and higher tenderization rate. Further research should be done to seek the optimal composition of muscle fiber type in order to improve beef quality, as muscle fiber type has opposite effect of tenderness background and tenderization rate.

Muscle fiber types differ according to their metabolism and contractile response. Muscle fiber type can be divided into type I, IIA, and IIB through the histochemical techniques (ATPase staining method; Brooke & Kaiser, 1970). Four fiber types (I, IIA, IIB, and IIX) can be classified from the detection of myosin heavy chain isoforms (MyHC I, IIa, IIb, and IIx) using immunohistochemical techniques and electrophoretic separation method. However, many studies found that IIB fiber (expressing IIB myosin heavy chain) was not expressed in trunk and limb muscles of bovine .
Muscle fiber characteristics are key factors significantly affecting meat qualities, such as pH decline, tenderness, water-holding capacity, color parameters (Hwang et al., 2010;Kirchofer, Calkins, & Gwartney, 2002;Van Bibber-Krueger et al., 2020), connective tissue component, and intramuscular fat content . Muscle fiber types significantly affect glycogen and mitochondria contents, determined the glycolytic rate, then influenced myofibrillar protein degradation, enzymatic activity of glycolytic enzymes and calcium-dependent proteolysis system, and finally affected the ultimate meat quality (Choi & Kim, 2009;. Studies showed that total collagen, insoluble collagen, and chemical cross-links were positively and negatively correlated with type IIA and IIB + X muscle fibers, insoluble collagen, and sensory tenderness was correlated with type I muscle fibers in cattle muscle, regardless of muscle and animal type . Type IIB muscle fibers have higher calpain/calpastatin ratios compared to type I fibers in porcine muscle; myosin heavy chain (MHC) slow isoform contents are positively correlated with calpastatin activity (Choi & Kim, 2009;Ouali & Talmant, 1990). Muscle proteins, for example, desmin and troponin T, of different muscle fiber types have different degradation rates (Christensen, Henckel, & Purslow, 2004;Muroya, Ertbjerg, Pomponio, & Christensen, 2010). Therefore, muscle fiber type influences glycolytic and protein degradation rates, thereby affecting postmortem meat quality. Several studies have focused on how characteristics of muscle fibers impact meat quality (Chang et al., 2003;Hwang et al., 2010;Renand, Picard, Touraille, Berge, & Lepetit, 2001;Zhang et al., 2014); however, there is little information on the effect of muscle fiber characteristics on the postmortem meat quality of Chinese Simmental cattle, especially on beef tenderization rate. This study thus aimed to evaluate how the characteristics of muscle fibers impacted the postmortem meat quality of longissimus thoracis (LT), psoas major (PM), and semitendinosus (ST) from Chinese Simmental bulls in an effort to better understand how these characteristics can be manipulated to modulate postmortem beef quality.

| Materials
Ten Chinese Simmental cattle (26 month of age, male, carcass weight: 378 ± 30 kg), reared in a grass-based system supplemented with concentrates, were chosen from a local feedlot and transported to a modern beef slaughterhouse (Beijing Yuxiangyuan livestock co., Ltd).
After a 12-hr fasting and freedom drinking water, animals were randomly slaughtered via Halal approaches. At 30 min following slaughter, LT, PM, and ST were excised from the right carcasses, separated to produce five steaks of 6 cm in thickness, vacuum-packed, and kept for 1, 3, 7, 14, and 21 days at 4°C. Roughly samples (1 × 1×1 cm cubes) utilized for histochemical analyses were isolated from the centers of LT (sixth rib), PM, and ST muscles within 1-hr postslaughter, snap-frozen in liquid nitrogen, and then stored at −80°C (Oury, Dumont, Jurie, Hocquette, & Picard, 2010). In order to cover a wide range of tenderness and muscle fibers composition, these three major muscles were chosen due to their different beef qualities and muscle fiber characteristics (Belew et al., 2003;Kirchofer et al., 2002).

| Histochemical analysis
Histochemical analyses were performed with a modified version of a protocol as reported by Brooke and Kaiser (1970). Transverse serial sections (8 μm) were generated with a cryostat (CM1950, Leica Microsystems Nussloch GmbH) and stained for myosin ATPase after preincubation with alkaline (pH 9.4) and acid (pH 4.6). Images of all sections were captured by orthopedic biological microscope (Olympus BX61, Olympus Corporation). About 300 fibers of each sample were analyzed in terms of size and fiber type, that is, I, IIA, or IIB. In this paper, IIB fiber, which was classified by ATPase staining method, was corresponding to IIX fiber (expressing IIX myosin heavy chain isoform). Fiber number and area percentages, as well as fiber diameters (FNP, FAP, and FD, respectively), were established with Image-Pro Plus 6.0 software (Media Cybernetics). FNP refers to the ratio of counted fiber number of each fiber type to the total counted fiber number. FAP was the ratio of total cross-sectional area of each fiber type to total fiber area (Hwang, Joo, Bakhsh, Ismail, & Joo, 2017;Joo, Lee, Hwang, & Joo, 2017).

| pH
pH was measured at 1, 6, 12, and 24 hr and 3, 7, 14, and 21 days postmortem in the center of the steak of three muscles (Lu, Zhang, Zhu, Luo, & Hopkins, 2018). IQ pH meter (IQ Scientific Instruments, Inc) equipped with a combination electrode was used, and the pH meter was calibrated with pH 4.0 and 7.0 buffers.

| Tenderness
Warner-Bratzler shear force (WBSF) was assessed according to Li et al. (2006). Steaks were cooked at 80°C in a water bath to 70°C internal temperature. Temperature was monitored with a testo 205 (Testo Instrument Company). Six beef cubes (1 × 1 × 3 cm), having the longest axis (3 cm) parallel to the fiber direction, were obtained, and WBSF was determined by texture analyzer (TA.XT Plus, Stable Micro System, Ltd) using the HDP/ BSW blade.

| Pressing loss and cooking loss
Pressing and cooking loss were established based on approaches reported by Li, Zhang, et al. (2012)) and Li, Liu, et al. (2012). Cooking loss was measured from steaks prepared for WBSF measurements and given as percentage weight loss prior to and following completion of cooking.

| Color
Meat color was analyzed after exposing freshly cut surface to atmospheric air at 4°C for 30 min. Meat color was taken with a CR-400 chroma meter (Minolta Co., Osaka, Japan) using illuminant D65 for L* (light), a* (red), and b* (yellow) (Cho et al., 2010). The instrument was precalibrated on a white plate according to the guidelines of the manufacturer.

| Electron microscopy
Samples were prefixed in 2.5% glutaraldehyde and then fixed with 1% OsO4, followed by ethanol dehydration and spur resin embedding. A Leica UC6 ultramicrotome was used to prepare sections that were stained using uranyl acetate as well as lead citrate (Lang et al., 2016). And the images were taken using a transmission electron microscope (Hitachi H-7500). The sarcomere length was assessed with Image-Pro Plus 6.0 software (Media Cybernetics).

| Statistical analysis
SAS v9.1 was used for statistical assessment. ANOVA and Duncan's multiple range test were utilized for comparing the mean values of muscle characteristics and meat quality at a 5% level of significance. Furthermore, percentage changes from days 1-21 postmortem was determined for WBSF as follows: % change WBSF = (WBSF 1d -WBSF 21d )/WBSF 1d × 100%, and was defined as tenderization rate Li, Liu, et al., 2012;Li, Zhang, et al., 2012). Spearman correlation coefficients were used to describe the relationship between WBSF and muscle fiber characteristics.

| Characteristics of muscle fibers
Muscle type had significant effects on muscle fiber diameter and composition (p < .05), as shown in Table 1 and Figure 1. In order of decreasing FD, the muscle fiber types were IIB > IIA > I. FD of type I, IIA, and IIB fibers in ST and LT was bigger relative to PM (p < .05), and this was in line with the study by Kim, Yang, and Jeong (2016) and . FNP of type I was the highest in PM and the lowest in ST (p < .05). FNP of type IIA was higher in LT than PM (p < .05); no differences were obtained in FNP of type IIA between LT and ST or between PM and ST (p > .05). FNP of type IIB was the highest in ST and the lowest in PM (p < .05). LT had an increased FAP of type IIA than PM and ST (p < .05). PM had a higher FAP of type I, but had a lower FAP of type IIB (p < .05). ST had a lower FAP of type I and IIA, but had a higher FAP of type IIB (p < .05).

| R values
R values, which reflect the muscle metabolism, are related to the ratio of the relative concentrations of the adenine nucleotides to inosine monophosphate and inosine (Calkins, Dutson, Smith, & Carpenter, 1982;Ryu & Kim, 2005). As shown in

| pH
The pH decline of LT, PM, and ST during postmortem aging was determined ( Figure 2). During the first 24-hr postmortem, pH decreased and then reached a plateau at 24 hr. At 1 hr, pH values of PM were statistically lower than that of LT and ST (p < .05). At 6-hr postmortem, ST had higher pH values than LT (p < .05), in line with the former study by Wang et al. (2018). From 12-hr to 21-day postmortem, pH values among LT, PM, and ST did not differ significantly (p > .05). The result suggested that PM had a higher pH decline rate following slaughter, and this was in agreement with the R values and the literature that red muscles had higher rate and extent of pH decline than meat with more percentages of fast-twitch glycolytic fibers ).

| Tenderness
The WBSF changes during postmortem aging time are shown in  Table 3). The relationship between myofiber characteristics and tenderization rate was analyzed ( Our study shows that PM muscle had lower WBSF, hardness, and chewiness values than LT and ST muscle, and this was consistent in line with work from Kim et al. (2016) and Rodriguez et al. (2014). The results may be partly explained muscle fiber composition and fiber size of these three major muscles. Some former studies had shown that tenderness was higher for muscle with high frequency of type I and lower frequency of type IIB fibers. Muscles with larger fiber area had greater hardness than muscle with smaller fiber size, particularly type IIB fiber (Renand et al., 2001;Żochowska et al., 2005). And this was consistent with our study that PM had higher percentage of type I fibers and also small fiber size. However, some studies have reported that muscles with increased type I fiber frequencies and a low proportion of type IIB fibers have higher WBSF values (Chriki et al., 2013;Ozawa et al., 2000;Ryu & Kim, 2005). The contradictory results may be explained by breeds, muscle type, and muscle location used in the experiments (Jurie et al., 2007;Maltin, Balcerzak, Tilley, & Delday, 2003;Oury et al., 2010;Van Bibber-Krueger et al., 2020).
In this study, we first described the relationship between muscle fiber type and % changes of WBSF. PM had fewer changes in WBSF, ST had higher changes in WBSF, and FNP of type I was negatively correlated with % changes of WBSF. And this was consistent with the former study that type I fibers have lower calpain/calpastatin ratios than type IIB fibers (Ouali & Talmant, 1990). Moreover, muscle fiber types have different susceptibility to protein denaturation, especially desmin and troponin T (Bowker, Swartz, Grant, & Gerrard, 2005;Christensen et al., 2004). Type IIB fibers have thinner Z-band than type I fibers; proteins comprising Z-bands in type IIB fibers are more vulnerable to rapid postmortem proteolytic breakdown compared with those of type I fibers (Muroya et al., 2010).

| Pressing loss and cooking loss
Muscle type did not impact cooking loss (p > .05, Figure 5), but it had significant effects on pressing loss (p < .05). At 1, 3, and 21 days after death, ST and LT exhibited less pressing loss than did PM (p < .05).
At 14-day postmortem, pressing loss in ST was lower than that in PM (p < .05); pressing loss did not differ between ST and LT (p > .05).
Therefore, PM had a lower water-holding capacity than LT and ST.
Aging time had significant effects on cooking loss and pressing loss (p < .01). Cooking loss of LT, PM, and ST increased from 1-to 3-day postmortem (p < .05), and the trends of cooking loss changes were not obvious from 3-to 21-day postmortem (p > .05). PM pressing loss during 21-day postmortem did not differ significantly (p > .05).
Pressing loss in LT and ST at 7-and 14-day postmortem were higher than that at 1-day postmortem (p < .05), but had no differences from that at 3-and 21-day postmortem (p > .05).
Results showed that PM had a lower water-holding capacity than ST and LT. In line with past results, there was a negative correlation between myofiber cross-sectional area and drip loss and thawing loss (Berri et al., 2007;Kim et al., 2008), and this was consistent with our study that the diameter of PM was lower in PM than this in LT and ST. However, some studies showed that early postmortem drip loss was greater when muscles contained fewer type I fibers and more type IIB fibers (Choe et al., 2008;Lefaucheur, 2010). These differences may be associated with muscle physiology, buffering capacity, and pH (Lee et al., 2010). And the fast pH decline causes autolysis of enzyme, denaturation of sarcoplasmic and myofibrillar protein, resulting in a decrease in the ability to hold water (Pomponio, Ertbjerg, Karlsson, Costa, & Lametsch, 2010;Scheffler & Gerrard, 2007).

| Color parameters
Muscle type had significant impacts on color parameters (p < .05, PM had higher a* values. Some studies reported that muscle fiber type was closely related with color parameters and muscles with lower percentage of type I fibers showed lower a* values and higher L* values (Choe et al., 2008;Kim et al., 2013), and this was consistent with our study.

| Electron microscopy
Electron microscopy of LT, PM, and ST was shown in Figure 7. At 1-hr postmortem, the sarcomere length of PM was longer greatly, due to the I band of PM was weak. At 1-day postmortem, myofibril structure was disrupted, I band breakage appeared in both PM and ST muscles, while M-lines in LT and ST became obscured even disappeared. At 3-day postmortem, boundaries between the A and I bands and the H region were visibly weaker among LT, PM, and ST muscles. As the aging time increased, myofibrils ruptured and fragmented. Results show that muscle type affects myofibrils degradation and the degradation of myofibril proteins in PM was faster than LT and ST. This was consistent with the former study that calpain can be activated earlier, due to increased sarcoplasmic Ca 2+ concentration in PM muscle (Melody et al., 2004). Calpains are widely considered as the major endogenous enzyme in postmortem proteolysis of myofibrillar proteins (Ding et al., 2018), and the earlier activation of μ-calpain may contribute to the higher rate of tenderization (Yan et al., 2018).

| CON CLUS ION
This study is the first to describe the relationship between muscle fibers composition and tenderization rate. Muscle type had significant effects on muscle fiber composition, R values, and overall qualities of meat, especially tenderness. Relative to LT and ST, PM muscle exhibited a higher rate of pH decline, R 248 and R 250 values. PM muscle, with type I fibers being more prevalent and type IIB fibers being present in lower proportions, had higher tenderness and lower tenderization rate. ST muscle, which has higher type IIB fiber proportions and reduced type I fiber proportions, had lower tenderness and faster tenderization rate. Therefore, muscle with high meat quality should have the optimization of muscle fiber type composition in order to balance the tenderness background and its tenderization rate. Further research should be done to seek the optimal composition of muscle fiber type, as muscle fiber type has opposite effect of tenderness background and tenderization rate.

ACK N OWLED G M ENTS
The authors gratefully acknowledge the financial support of National Natural Science Foundation of China (31701531), Hebei Natural Science Foundation (C2018201146), National Beef Cattle Industry and Technology System (CARS-37), and One Province One School Project of China.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest in this work.