Comparison of carcass traits, meat quality and expressions of MyHCs in muscles between Mashen and Large White pigs

Abstract The aim of this study was to evaluate the differences in carcase traits, meat quality and muscle characteristics (mean fibre diameter and MyHCs levels of expression) between Mashen and Large White pigs. A total of 60 pigs (30 Mashen pigs and 30 Large White pigs, half male and half female in each breed) were used in this experiment. Results showed that Mashen pigs exhibited lower slaughter weight, reduced back-fat thickness, reduced loin eye area and lower lean meat ratio than Large White pigs (p < .05). Meat from Large White pigs had higher pH1, greater drip losses, higher Warner-Bratzler shear force values, higher lightness, higher yellowness, and greater mean diameter of muscle fibres. Also, meat from Large white pigs had lower cooked yield, lower redness, and reduced intramuscular fat content than meat from Mashen pigs (p < .05). The contents of 17 amino acids, total amino acids, essential amino acids and umami amino acids in the longissimus dorsi of Mashen pigs were higher than those in Large White pigs (p < .05). Large White pigs had higher monounsaturated fatty acids and lower polyunsaturated fatty acids than Mashen pigs (p < .05). Meanwhile, the expression of MyHCI was significantly higher and the expression of MyHCIIb was lower in Mashen than in Large White pigs (p < .05). The higher MyHCI and lower MyHCIIb found in muscles from Mashen pigs might partially explain meat quality differences found between the two breeds in the present study. These results provide valuable information for meat quality differences between Mashen and Large White pigs. Highlights Carcase traits and meat quality between Large White and Mashen pigs exhibited significant differences. The total amino acids, essential amino acids and umami amino acids in Mashen pigs were higher than those in Large White pigs. Large White pigs had higher monounsaturated fatty acids and lower polyunsaturated fatty acids than Mashen pigs. The expression of MyHCI was significantly higher and the expression of MyHCIIb was lower in Mashen than in Large White pigs.


Introduction
Meat quality is a very important issue for consumers and it is also vital for the meat industry (Şirin et al. 2017). With the continuous improvement of living standards, the demand of consumers for high quality meat is constantly increasing. Consumers' requirements for meat include to be delicious, nutritious, safe and healthy ). There are many factors influencing meat quality such as breed, genotype, sex, age, nutrition and slaughter conditions. Skeletal muscle fibre composition is one of the important factors influencing meat quality (Gil et al. 2003;Klosowska and Fiedler 2003;Choi et al. 2007). Myosin heavy chain (MyHC) is transcriptionally regulated in pig skeletal muscle (Park et al. 2009). MyHCs gene expression was commonly used to define muscle fibre type composition in pigs, which was more precise and reliable than traditional methodologies (Gunawan et al. 2007;Choi and Kim 2009;Park et al. 2009). Four MyHC isoforms were found to be expressed in the skeletal muscle of adult pigs: type I, IIa, IIx, IIb: type I, IIa, IIx, IIb (Lefaucheur et al. 2002). MyHC defines the contractile nature of fibre types: MyHC type I is the slower contractile type, followed by types IIA, IIX and IIB in increasing contractile speed order. Those contractile fibre types are usually associated with different metabolic activities. Type I fibres have greater oxidative capacity to support sustained contraction, whereas type IIb fibres are predominantly glycolytic and rapidly use glycogen for short bursts of activity. The IIa and IIx fibres are intermediate to type I and IIb fibres (Chang et al. 2003). Muscle fibre type composition has different effects on post-mortem change of meat quality. (Ozawa et al. 2000;, and abundance of MyHC IIb fibres is associated with less favourable meat quality, both in terms of pH, drip loss, grain, colour, yield force and work done (Chang et al. 2003). Meanwhile, the presence of type I fibre was positively related to good meat quality (Wimmers et al. 2008). An increase in the proportion of muscle fibre type I leaded to a decrease of lightness and an improvement of water-holding capacity in pork (Gil et al. 2003;Choi et al. 2010) and with improved tenderness and juiciness in beef (Maltin et al. 1998). Previous studies have found that type IIb fibres were closely related to toughness, paleness, higher protein denaturation and lower waterholding capacity in porcine longissimus muscle (Karlsson et al. 1993;Larzul et al. 1997;Kauffman et al. 1998;Renand et al. 2001;Ryu et al. 2008;Choi et al. 2010).
The Mashen pig, one of the most important indigenous breeds in northern China, exhibits high adaptability, good meat quality, slow growth rate and low feed conversion ratio. On the other hand, the Large White pig is a common breed for its high growth rate, feed conversion ratio and lean meat percentage (Zhang et al. 2001;Yang et al. 2005;Zhao et al. 2015). Therefore, these two pig breeds can serve as an ideal comparison for studying meat quality. Up to now, little was known about muscle fibre composition and postmortem muscle metabolism in Mashen pigs, nor the breed differences between Mashen and Large White pigs. In the present study, the differences in muscle fibre composition, carcase traits and meat quality between Mashen and Large White pigs were compared. The aim of this study was to evaluate the differences in carcase traits, meat quality and muscle characteristics (IMFC, mean fibre diameter and MyHCs levels of expression) between Mashen and Large White pigs.

Animals
Mashen pigs (n ¼ 30; castrated males, n ¼ 15; females, n ¼ 15; 72.5 ± 0.6 kg) and Large White pigs (n ¼ 30; castrated males, n ¼ 15; females, n ¼ 15; 119.5 ± 1.0 kg) under the same feeding management conditions (The composition of diets were shown in Table S1) were selected at 180-days old from the Datong Pig Farm (Shanxi, China). The selected pigs all were weaned at 28-days old and males were castrated when weaning. Among them, eight Mashen pigs (4 castrated males and 4 females, 72.8 ± 0.6 kg) and eight Large White pigs (4 castrated males and 4 females, 119.9 ± 1.0 kg) were selected randomly to determain the amino acids composition, fatty acids composition and levels of MyHCs relative expression. Carcase traits and other meat quality measurements were conducted in all thirty pigs per breed. All of the animal procedures were conducted per the Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments (http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm). The methods were performed in accordance with the Good Experimental Practices adopted by the College of Animal Science and Veterinary Medicine, Shanxi Agricultural University (Shanxi, China). Moreover, the local animal welfare laws, guidelines, and policies were strictly followed for the feed and use of experimental animals. The experiment was approved by the Animal Ethics Committee of Shanxi Agricultural University.

Carcase traits
Electric shock was used to stun pigs, which then were exsanguinated. After removing hair, head, hooves and internal organs, the carcases were weighed, and the carcase yields were calculated. In addition, the carcase length was measured from the midpoint of the anterior margin of the pubic symphysis to the midpoint of the anterior margin of the first cervical vertebra. The carcase bone, meat, skin and fat of the left side of carcases were separated and weighed to calculate lean meat ratio. The loin eye area was measured at the last rib level and calculated based on the loin eye height and width with 0.7 as the coefficient (Dai et al. 2009). The fat thickness of the first and last ribs, and the last lumbar vertebra were measured using a vernier calliper (Jiang et al. 2012). The average of those three measurements was used as the back-fat thickness.

Meat quality
Muscle pH (pH 1 ) was measured at 45 min post-mortem in the longissimus dorsi (between the 13th and 14th rib) using pear-type portable pH metre (IQ-150 pH metre and PH77-SS probe, IQ Scientific Instruments, USA).
The longissimus dorsi samples at 10th to 11th thoracic were taken within 1-2 h after death and pieced to 4 cm Â 4 cm Â 4 cm size and weighted. Then the meat cubes were placed in inflated plastic bags and hung for 48 h at 4 C (Honikel 1998). After that, meat cubes were reweighted. The drip loss is the percentage of two weight changes.
About 100 g psoas major sample was taken at 24 h post-mortem. After stripped of the outer membrane and the attached fat, the meat was placed in an aluminium pot and steamed for 30 min. After steaming, the sample was hung for 30 min before re-weighed. The difference of two weights was used to calculate the cooked meat percentage.
At 24 h post-mortem, longissimus dorsi muscle samples at 3rd to 6th lumbar vertebrae were taken and cut into 5 cm Â2 cm Â2 cm pieces along the fibres, which were used for the measurement of the Warner-Bratzler shear force (WBSF). Then those samples were packed in plastic bags and boiled about 30 min (The centre of the meat should attain 80 C). After that, they were cooled to room temperature rapidly. The digital display muscle tenderness metre (C-LM3B, Northeast agricultural university, Heilongjiang, China) was used to measure the WBSF. Six repetitions per sample.
Muscle colour of longissimus dorsi were measured by Chroma metre (CR-300, Minolta Camera, Tokyo, Japan) at the last rib level. Muscle colour was determined on the exposed cut surface of the longissimus muscle at 24 h post-mortem. Each measurement was performed in six replicates, taking the mean value as the assay result. The results were expressed as CIE (Commission International del'Eclairage) Lightness, redness and yellowness values.
Intramuscular fat (IMF) percentage was determined in the Longissimus lumborum (between the 3rd and 4th lumbar vertebrae) by ether extraction in a Soxhlet apparatus after acid hydrolysis.
Approximately 80-100 g of longissimus dorsi tissue between the 3rd and 4th lumbar vertebrae were taken at 24 h post-mortem. About 2 mg of extracted lipid was redissolved in 2 mL of n-hexane and 1 mL of KOH (0.4 M) for saponification and methylation. The fatty acid methyl esters were analysed with a gas chromatographer (ThermoFisher Trace 1310 ISQ, Thermo Corporation, USA) equipped with a capillary column (30 m Â 0.25 mm Â 0.25 mm film thickness). Nitrogen was used as carrier gas, the oven temperature was initially held at 80 C. The temperature was rose at a rate of 10 C min À1 to 200 C and then continue got up to 250 C (5 C min À1 ). With the rate of 2 C min À1 , the temperature finally was up to 270 C. The ion source and transmission line temperatures were maintained at 280 C. Each fatty acid was identified by comparing their retention times with those of authenticated standards and quantified as a percentage of total fatty acids.
Approximate 100 mg samples of longissimus dorsi tissue at the 3rd to 4th lumbar vertebrae were dissolved in a solution of water and methanol (1:1) at 4 C for 10 min and centrifuged at 10,000 Â g for 10 min. The supernatant was filtered through glass wool. The 500 lL of the supernatant was used for amino acids analysis using liquid chromatograph (ThermoFisher U3000, Thermo Corporation, USA). Chromatographic conditions: flow rate, 1.0 mL per minute; column temperature, 40 C; wavelength, 254 nm.

Muscle characteristics
Within 45 min post-mortem, longissimus dorsi samples at the last rib were taken for histochemical analysis. Muscle samples at a size of 0.5 cm Â 0.5 cm Â 0.2 cm were obtained and immediately put into 4% paraformaldehyde solution for paraffin embedding, which were then cross-sectioned for HE staining. The microscopic images were captured at 100 times magnification (OLYMPUS microscope, Olympus Corporation, Japan). Muscle fibre diameters were measured on $100 fibres based on the smallest diameter. The diameter value was expressed as the mean value from all measurements. In addition, muscle fibre density was calculated by dividing the average number of muscle fibres in 10 randomly selected views by the view area (0.59 mm 2 ), which was converted to the number of muscle fibre roots per mm 2 .
The longissimus dorsi muscles between the 11th and 12th rib of Mashen pigs and Large White pigs were taken shortly after exsanguination, immediately snapped in liquid nitrogen, and stored at À80 C for subsequent use. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the mRNA expression patterns of MyHCI, MyHCIIa, MyHCIIx and MyHCIIb. The primers of MyHCs used in present research referred to the study of Hu et al (Hu et al. 2008). 18S rRNA was used as the internal control. Details of primers are shown in Table 1. qPCR was performed using a SYBR V R PrimeScriptTMII RT-PCR Kit (Takara, China) in conjunction with an ABI-7500 realtime PCR system (Applied Biosystems, USA). The reaction conditions were as follows: predenaturation at 95 C for 30 s; 45 cycles of 95 C for 30 s and 60 C for 34 s; and one cycle of 95 C for 30 s, 60 C for 1 min, 95 C for 30 s. To ensure robustness, each sample was analysed in triplicate. The relative expressions were quantified using the 2 ÀDDCt method.

Statistical methods
Carcase traits, meat quality and muscle characteristics between Mashen and Large White pigs were analysed by one-way ANOVA in SPSS22.0 software. p < .05 indicates the difference is significant. Data were expressed as "means ± standard error of means (SEM)". We are preprocessing the test data to analyse the effects of breed and sex. By building model found that gender had no significant influence on carcase traits and meat quality and muscle characteristics.

Carcase traits of Mashen and large white pigs
The carcase traits in Mashen and Large White pigs are shown in Table 2. Compared with Large White pigs, the slaughter weight of the Mashen pigs were lower. When slaughtered at 180 days of age, the weight of the Mashen pigs were 72.5 kg, while that of Large White pigs were 119.5 kg. Mashen pigs had significantly lower back fat thickness, loin eye area and lean meat yield compared to Large White pigs (30.0 vs. 33.7, 25.6 vs. 51.6, and 48.8% vs. 63.5%, respectively, p < .05).

Meat quality characteristics of Mashen and Large White pigs
As shown in Table 3, meat quality characteristics between two breeds were significantly different (p < .05). Compared with Large White pigs, Mashen pigs had lower pH 1 , drip loss, WBSF, lightness, yellowness and higher cooked yield, redness and IMF.
The content of amino acids (except lysine) in Mashen pigs was significantly higher than that in Large White pigs (Table 4) (p < .05). In addition, the sum of amino acids, umami amino acid and essential amino acids of Mashen pigs were significantly higher than those of Large White pigs (Table 4) (p < .05). Among   amino acids, the content of Glutamic acid (Glu) was the highest (Mashen pig, 4.40 g/100g; Large White pig, 3.75 g/100g) (Table 4) (p < .05). As shown in Table 5, the content of monounsaturated fatty acids in Mashen pigs was significantly lower than that in Large White pigs. On the contrary, polyunsaturated fatty acids were more abundant in Mashen pigs (p < .05).

Muscle characteristics of Mashen and Large White pigs
As shown in Table 6, compared with Large White pigs, Mashen pigs had lower diameter and higher density of muscle fibres.
The mRNA level of MyHCI was the lowest and MyHCIIb was the highest between the 4 MyHC isoforms compared within each pig breed. The mRNA expression of MyHCI and MyHCIIa in Mashen pigs was higher than that in Large White pigs (p < .05). On the other hand, the expression of MyHCIIb in Large White pigs was higher than that in Mashen pigs (p < .05). Regarding MyHCIIX mRNA levels of expression, no significant differences were observed between the two studied breeds (p>.05).

Discussion
In the present study, the muscle fibre diameters of Mashen pigs were smaller than Large White pigs, which might partially explain the lower drip loss, WBSF and higher cooked yield in Mashen pork. Previous studies suggested that muscle fibre diameter influences meat quality, and pork with greater muscle fibre diameters have higher levels of shear force, drip loss, cooking loss (Seideman and Crouse 1986;Rehfeldt et al. 2000;Bulotiene and Jukna 2008). We found that the IMF of Mashen pigs was 3.73% and Large White pigs was 2.51%. Consistently, it has been reported that the IMF of European pigs is 2% to 3%, while in Chinese indigenous pigs, the IMF is around 4% or even higher (Liu et al. 2016;Yang et al. 2016). In addition, Mashen pigs exhibited reduced loin eye area, reduced lean meat rate, lower drip loss, lower WBSF, and lower lightness values than Large White pigs. This is consistent with other studies evaluating meat quality of Chinese indigenous pig (Young 1995;Miao et al. 2009;Guo et al. 2011;Jiang et al. 2011;Wu et al. 2013;Liu et al. 2015). Because of genetic selection, the Large White pigs have higher growth rate and lean ratio when compared to less selected native pig breeds (Mashen pig). Consistently, Korean native pigs exhibited a higher IMF content and backfat thickness than European meat breeds ). The Lantang pig is native to the southern part of China, which had a higher IMF content and back-fat thickness than the commercial European pig Landrace, while the Landrace exhibited a higher loin eye area and ash content than Lantang pig (Dai et al. 2009). The Min   pig (such as Heilongjiang Province), a well-known Chinese fat-type breed, is famous for its high IMF, superior meat quality, and stress resistance. Compared with Min pig, the Large White pig has a faster growth rate and higher lean meat ratio (Liu et al. 2017).
Muscle fibre types were related to meat quality and carcase traits in various species, including cattle (Ozawa et al. 2000;Picard et al. 2006;Hwang et al. 2010), pigs (Larzul et al. 1997;Ryu and Kim 2006) and poultry (Dransfield and Sosnicki 1999;Kim et al. 2008). In adult pigs, skeletal muscles consist of four contractile fibre types (Type I, IIa, IIx, IIb), which are characterised by the expression of MyHC gene isoforms (Lefaucheur et al. 2002). The expression of MyHC genes was highly correlated with the relative amount of the corresponding protein and is used to classify the muscle fibre type composition (Gunawan et al. 2007). The positive correlation between MyHC mRNA expression and its corresponding protein level has also been shown by others (Cox and Buckingham 1992;Schiaffino and Reggiani 1996;Mckoy et al. 1998). Therefore, MyHCs gene expression was used to define muscle fibre type composition, which was more precise and reliable than traditional methodologies (Gunawan et al. 2007;Choi and Kim 2009;Park et al. 2009).
In the present study, both Mashen and Large White pigs, showed higher levels of expression of MyHCIIb than that of MyHCI. This is consistent with previous studies which generally show that the longissimus muscle of pigs has higher proportions of type IIb than type I fibres (Marita and Eero 2004;Ryu et al. 2008;Lee et al. 2012). The proportions of type IIb fibres were 69.7-90.3% in pig longissimus dorsi from various breeds, such as Berkshire, Yorkshire, Landrace, Duroc and their crossbred pigs (Marita and Eero 2004;Ryu et al. 2008;Lee et al. 2012). Importantly, the expression of MyHCI was higher in Mashen than in Large White pigs, whereas the expression of MyHCIIb was higher in Large White pigs. Each muscle fibre type has different biochemical and biophysical characteristics. The differentially expressed muscle fibre types may be the most important factors influencing meat quality, particularly IMF content and drip loss (Ozawa et al. 2000;Kim et al. 2008;Guo et al. 2011). Type I fibres contain neutral lipids, whereas only 26% and 1% of types IIa and IIb fibres contain lipids (Karlsson et al. 1999). The type I fibres contain abundant mitochondria and greater amount of intramyofibrilar lipid content (Lefaucheur et al. 2002), while pig muscle with a higher proportion of fast-twitch fibres had a lower intramuscular lipid content . Hence, the higher IMF in Mashen pigs than Large White pigs might be partially due to a higher proportion of type I and a lower proportion of type IIb in Mashen pigs. In addition, drip loss is influenced by the characteristics of muscle fibres, especially type IIb (Larzul et al. 1997;Ryu and Kim 2006). Meat from Large White pigs had a higher drip losses and lower pH 1 , which might be due to a higher proportion of type IIb fibres. Type IIb fibres are predominantly glycolytic, which contribute to the rapid metabolism of glycogen during the early post-mortem period, producing higher amounts of intramuscular lactate and faster pH decline rates, resulting in poorer meat quality (Schiaffino and Reggiani 1996;Pette and Staron 2001;Choi and Kim 2009). Consistently, meat with a higher percentage of type IIb fibres and lower percentage of type I fibres showed lower pH, paler colour, and higher drip loss (Ryu and Kim 2006;Choi et al. 2007;Choe et al. 2008;Choi et al. 2010). With the higher expression level of MyHCI and lower expression level of MyHCIIb, Mashen pigs had a higher redness and lower lightness, and lower WBSF. In agreement, tenderness is affected by fibre type, especially type IIb fibres. Muscles with larger type IIb fibres may be tougher or may have greater hardness (Karlsson et al. 1993;_ Zochowska et al. 2005). In addition, due to the presence of abundant mitochondria and myoglobin in type I fibres, higher percent of type I fibres were related to lower lightness and higher redness (Ryu and Kim 2006;Hwang et al. 2010;Kim et al. 2013).
The soluble amino acid content correlates with the flavour of meat (Yang et al. 1994;Shi et al. 2003; Chen  et al. 2004;Zhu et al. 2008). Six umami amino acids (Gly, Ile, Pro, Ser, Ala and Glu) are directly related to the flavour, of which Glu is the most important umami amino acid, (Hood and Allen 1973). Comparing the amino acid composition of Bamei, Landrace and their hybrid pigs, 17 amino acids were significantly different among these breeds, of which Bamei pigs had the highest content of umami amino acids (such as Glu) (Yang et al. 1994). In this study, the contents of Glu, Gly, Ile, Pro, Ser and Ala, were higher in Mashen pigs than that in Large White pigs, which may cause the Mashen pig has the better taste of pork.
In present study, the monounsaturated fatty acids content was lower in Mashen pigs than in Large White pigs, while the content of polyunsaturated fatty acids was higher in Mashen pigs. The content of saturated fatty acids is also related to muscle quality (Hood and Allen 1973;Cameron and Enser 1991). Unsaturated fatty acids are not only an important precursor to meat flavour, but also an indispensable nutrient for human body (Hallenstvedt et al. 2012). On the other hand, saturated fatty acids can cause atherosclerosis, elevated blood lipids, thrombotic disorders and other issues, while increased unsaturated fatty acids (n-3 polyunsaturated fatty acids) uptake can decrease blood cholesterol level and prevent arteriosclerosis (Bhavsar and Stonge 2016). In addition, a higher content of C18:3 was found in Mashen pork than Large White pork. Polyunsaturated fatty acids, especially the n-3 fatty acids, are beneficial to human health (Webb and O'Neill 2008;Wood et al. 2008). Therefore, Mashen pig meat products may have more nutritional value and may be more beneficial to human health.

Conclusions
The Mashen pigs had lower drip loss, WBSF, lightness, yellowness, pH 1 , diameter of muscle fibres and higher cooked yield, redness, IMF than Large White pigs. The content of MyHCI was higher and MyHCIIb was lower in Mashen pigs, which might relate to their meat quality. These data provide valuable information for meat quality difference of Mashen and Large White pigs.