The Expression of Myoz1 and ApoB is Positively Correlated with Meat Quality of Broiler Chicken

Broiler chicken (Gallus gallus) is a source of animal protein with a high nutritional content. The purpose of this study was to evaluate the quality of broiler chicken meat (Gallus gallus) by analyzing its nutritional value, genetic profile, and protein level. The chicken meat samples were obtained from four different districts in Malang city, Indonesia. We analysed the proximate composition of chicken meat to detect its nutrition content. Furthermore, we have examined the sequence of the Myoz1 gene as well as the level of ApoB proteins in the same meat. The nutritional analysis of chicken meat showed that in the four locations different levels of protein, ash, water, and lipids were observed. The Myoz1 gene of femur chicken broilers from the second and third districts has five and twenty-one substitution mutations, respectively. The ApoB expression level in locations 2 and 3 was higher than that in the other districts. In conclusion, Myoz1 and ApoB levels were correlated with the nutritional content and quality of broiler chicken meat.


Introduction
Indonesian people have increased awareness of the importance of animal protein for the growth of body tissues. Indonesia, especially the Malang area, is the area with the largest number in the broiler industry sector. Te main advantage of chicken meat is that it has a high nutritional value or content so that it is able to meet the nutritional needs of the organism. In addition, broiler chickens display a rapid body weight gain, the meat is tender, and the efciency of feed is relatively high which means that the most of the feed can be easily converted into meat and is easily available in traditional and modern markets.
Tere are many intrinsic and extrinsic factors that afect meat quality, including muscle properties, such as muscle diameter [1], muscle growth [2], and muscle fber type [3]. Muscle types can contribute to diferent metabolic rates in chickens due to diferent properties regarding oxidative capabilities, which have a large impact on feed conversion ratios. At the same time, the type of muscle fber is also associated with the stability of meat color, tenderness, and water retention capacity. In the previous decade, both layer and broiler industries have developed rapidly, and broiler farming's target has shifted from increasing quantity to increasing quality as meat quality has become one of the most important goals for satisfying consumer preferences [4]. One of the selection marker genes that determine meat quality is Myoz1 [5]. Tere are three members of the myozenin (Myoz) family, including myozenin 1 (Myoz1), myozenin 2 (Myoz2), and myozenin 3 (Myoz3) that encode the proteins calsarcin-2 (FATZ1), calsarcin-1 (FATZ2), and calsarcin-3 (FATZ3). Myoz1 is also reported as a candidate gene for determining heart failure and hypertrophy. Myoz1 expression increased in rats that had frequent exercise due to changes in muscle fber type. In addition, the Myoz1 gene expression was also assessed in two diferent chicken variants, and it was found that the Myoz1 gene was not expressed in fast-twitch fber chickens and that its expression was associated with tissue, sex, and age [6].
Te fat content of chicken meat also contributes to the determination of meat quality since excessive fat can afect meat favour. According to Fisher and Ginsberg [7], lipid accumulation in chicken broiler liver exposed to chronic heat stress can be determined from the ApoB threshold value. Apolipoprotein B (ApoB) is one of the hydrophobic proteins that has the highest molecular weight. Tis is the main structural protein of the triglyceride-rich low-density lipoprotein (LDL) receptor which plays important roles in transport and metabolism of lipids and energy [8,9]. It has been reported that the level of ApoB is associated with body weight, abdominal fat, growth-related traits, and the meat quality of poultry [10,11]. Te broiler chicken contains 4536 ApoB amino acid residues which afect the growth and development of fat and have an impact on energy absorption and reproduction occupation. Te aim of this study is to determine meat proximate analysis, genes profle (Myoz1), and protein expression level (ApoB) in samples from different district in Malang city, as well as to analyze the relation determining the possible candidate as selection markers in meat chicken quality.

Meat Samples.
Te samples of femur broiler chicken were obtained from 4 subdistricts in the Malang city. Te samples are grouped into BR1Fem for chicken meat obtained from district 1, BR2Fem for chicken meat obtained from district 2, BR3Fem for chicken meat obtained from district 3, and BR4Fem for chicken meat obtained from district 4. Tree meat samples were isolated from each district.

DNA Extraction and PCR.
Genomic DNA was isolated from the femur muscle by the standard method according to [12] with some modifcations. Te DNA concentration and purity were determined by a NanoDrop spectrophotometer, and the quality of the DNA was analysed using agarose gel 1%. Te PCR mix solution composition was referred to [12]. Te Myoz1 gene primer at exon 3 were F: 5′-AGGACCAAA CCCTGCAAATG-3′; R: 5′-CCCTAAGAGTAAGACTGG CACAAG-3′ [5] with the following procedure: hot start at 95°C for 10 min; 35 cycles at 95°C for 30 s; annealing at 60°C for 30 s, and extension at 72°C for 40 s; and then postextension at 72°C for 10 min. Te quality of the PCR product was analysed using agarose gel 1.5% and visualized using ChemiDoc gel imaging (Bio-Rad).

DNA Sequencing and In Silico Analysis. Te PCR
product was purifed using Wizard ® SV Gel and the PCR Clean-Up System (Promega). Te product was sequenced by an ABI-Prims 3730xl DNA Sequencer (Köln, Germany), and the sequencing results were aligned with BioEdit software.

Western Blotting Analysis.
Expression of APOB and β actin was analysed at the protein level in chicken meat. Protein of femur meat samples was separated in polyacrylamide gradient gels and then blotted to polyvinylidene fuoride (PVDF) transfer membrane (UltraCruz ® PVDF Transfer membrane). Te nonspecifc protein blocking was applied to 5% skim milk in phosphate bufered saline (PBS) for an-hour's incubation at room temperature. Te blotted membranes were incubated with polyclonal rabbit anti-Apolipoprotein B Polyclonal Antibody (Bioss, USA) and mouse anti-β actin (SantaCruz-Biotech., USA). Te membranes were incubated with polyclonal AP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies (TermoFisher Scientifc, USA). Te membrane was exposed using BCIP/NBT Membrane Alkaline Phosphatase Substrate (Rockland, Limerick, PA, USA) and visualized by the ChemiDoc imaging system (Bio-Rad Laboratories Inc, USA). Te intensity of a specifc protein was measured by Quantity One software.
2.6. Statistical Analysis. Statistical analysis was conducted using the GraphPad Prism5 statistical package (Graphpad software, USA). Te one-way analysis of variance (ANOVA)

Proximate Composition.
In this study, we performed proximate analysis to examine the levels of protein, ash, water, and lipids in broiler chicken meat isolated from 4 diferent locations. Te result showed that broiler chicken meat at location 1 had a higher level of water (75.93 ± 0.04%) and ash content (1.35 ± 0.27%), and also a lower level of lipid (0.02%) than meat from other locations (Table 1). Importantly, we found a high level of protein in broiler chicken meat from location 1 (23.1 ± 0.04%) and location 3 (23.4 ± 0.16%).

Identifcation of Sequence Mutation of the Chicken Myoz1
Gene. To identify the presence of mutations in the Myoz1 gene of broiler chicken meat, we amplify the DNA sequence of Myoz1 and then subject it to sequencing analysis. Te amplifcation of the broiler Myoz1 gene was identifed using PCR, and the DNA band profle was observed at 379 bp (Figure 1(a)). Te results of the sequence alignment of four Myoz1 amplicons are shown in Figure 1

Te ApoB Expression Level of Broiler
Chicken. In this study, the level of ApoB protein was measured using western blotting analysis of total proteins isolated from broiler chicken meat. Te level of ApoB protein was signifcantly higher

Discussion
Te proximate analysis of broiler chicken meat obtained from 4 diferent locations showed some variations in the level of protein, ash, water, and lipid (Table 1). Te diferences between those parameters could be afected by the geographical location, diet, and hybrid used. Tese factors infuenced the levels of protein, fat, ash, and water of chicken meat, thereby increasing variation in the quality of broiler chicken meat.
Our results show that these four diferent locations provide meat of diferent quality, although they are still in the same city. Other factors that could afect the quality of chicken meat are rearing system, age, muscle pH, chemistry composition, muscle microstructure, postmortem aging, and processing methods [13]. Te quality of chicken meat is also related to its composition, especially muscle protein, intramuscular collagen, and intramuscular fat content which are strongly infuenced by breed, system maintenance, and the age of the chicken [13][14][15][16]. According to Dirong et al. [17], several methods can be applied to distinguish chickens from other species, including genomics, transcriptomics, proteomics, lipidomics, metabolomics, and glycomics, as well as the application of bioinformatics or chemometrics. It has been reported that Myoz1 is expressed in fast-twitch skeletal muscles of humans, mice, and chicken [5,6]. In this study, we analysed the sequence of the Myoz1 gene since its expression and single-nucleotide polymorphism are associated with the quality of chicken meat [5]. We found that chicken meat from the Myoz1 gene from locations 2 (BR2Fem) and 3 (BR3Fem) has substitution mutations. Previous studies have shown that mutations found in the chicken Myoz1 gene were associated with various traits including the weight of leg and breast muscle. Terefore, we hypothesized that mutations of this gene may afect the muscle weight and protein level of the chicken meat obtained from locations 2 (BR2Fem) and 3 (BR3Fem). It is known that Myoz1 defciency decreases performance due to reduced body weight and fast-twitch muscles, resulting in excessive CaN/NFAT activation [6]. Te NFAT activation can induce a muscle fber type switch toward a slow-twitch and oxidative phenotype, related to the increased expression of a subset of genes associated with type I myofbers such as myoglobin and troponin I slow [18,19]. We have previously reported that the substitution mutation of GAPDH gene sequences of chicken broilers can change amino acid sequences and the three-dimension structure of GAPDH protein [20]. Myoz family proteins are often used in the feld of animal husbandry. Since the discovery of these three members, the Myoz family has emerged as one of the gene families most involved in the type of muscle fber. In the pig species, all three members of the Myoz family were cloned and characterized [21], and the Myoz1 gene is a potential candidate as a marker to determine meat quality in animals. In addition, previous studies showed that Myoz1 was expressed in the liver, heart, breast muscle, and leg muscle of chicken, with a higher expression in both the leg and breast muscles than in the liver and heart [5]. Te results of this study indicate that chicken meat from two locations has diferent quality and may have come from diferent breeds.
In our study, the ApoB expression level of BR2Fem and BR3Fem was high, suggesting that the lipid transport in the chicken muscle was accelerated and lipid accumulation was reduced [7]. Tis result indicates that both meat from BR2Fem and BR3Fem has a high quality. Several proteins that play an important role in determining intramuscular fat and meat quality include adipose diferentiation related protein (ADFP), fatty acid transport protein 1 (FATP1), and apolipoprotein B (APOB) [11]. In conclusion, the meat qualities from 4 locations in the Malang area have a diferent composition which is infuenced by environmental parameters and genotype. Te Myoz1 gene of femur chicken broilers from the second districts and third districts has fve and twenty-one substitution mutations, respectively. Te ApoB expression level in locations 2 and 3 have a high level that correlated with lipid accumulation directly. Indeed, both Myoz1 and ApoB can be markers for determining the meat quality of chicken.

Data Availability
All data generated during this study are available from the corresponding author on request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.