Research Note: Expression levels of collagen-related genes in PSE conditions and white striping features of broiler pectoralis major muscle.

The objective of this study was to compare the meat quality characteristics and collagen-related gene expression levels in the broiler pectoralis major (PM) muscle among the meat quality groups, including normal, pale, soft, and exudative (PSE), and white striping (WS) groups. The group was classified by their WS degree (moderate or severe striping) and quality traits including pH and lightness values at 24 h postmortem (normal group: pH24 h ≥5.7, 48≤L*≤53, without WS features; PSE group: pH24 h <5.7, L* >53, without WS features; WS group: pH24 h ≥5.7, 48≤L*≤53, with moderate or severe striping). The WS group revealed no differences in all measured meat quality traits compared to the normal group (P > 0.05). PM muscles exhibiting PSE conditions without WS indicated lower pH15 min and pH24 h values (P < 0.05). Whereas, lower lightness and cooking loss values were observed in the normal and WS groups compared to the PSE group (P < 0.05). No significant difference was observed in the level of type I collagen among the groups (P > 0.05), whereas a higher type III collagen level was observed in the WS group than in the other groups (P < 0.05). Additionally, the WS group showed a higher type IV collagen level compared to the normal group (P < 0.05) and a level not different from that of the PSE group (P > 0.05). In contrast, the expression levels of matrix metalloproteinase (MMP) 2, involved in type IV collagen degradation, and angiopoietin-like protein 7, associated with collagen accumulation, were higher in the WS group compared to the normal group (P < 0.05). However, no difference was detected in the MMP1 level among the all groups (P > 0.05). These results suggest that the occurrence of WS features in broiler PM muscle, unlike PSE and normal conditions, can be influenced by the expression levels of collagen-related genes associated with abnormalities in extracellular matrix components.

GWAS and Post-GWAS High-Resolution Mapping Analyses Identify Strong Novel Candidate Genes Influencing the Fatty Acid Composition of the Longissimus dorsi Muscle in Pigs.

Fatty acid (FA) composition is one of the most important parameters for the assessment of meat quality in pigs. The FA composition in pork can also affect human health. Our aim was to identify quantitative trait loci (QTLs) and positional candidate genes affecting the FA profile of the longissimus dorsi muscle in a large F2 intercross between Landrace and Korean native pigs comprising 1105 F2 progeny by genome-wide association studies (GWAS) and post-GWAS high-resolution mapping analyses. We performed GWAS using the PorcineSNP60K BeadChip and a linear mixed model. Four genome-wide significant QTL regions in SSC8, SSC12, SSC14, and SSC16 were detected (p < 2.53 × 10-7). Several co-localizations of QTLs in SSC12 for oleic acid, linoleic acid, arachidonic acid, monounsaturated FAs, polyunsaturated FAs, and the polyunsaturated/saturated FA ratio were observed. To refine the QTL region in SSC12, a linkage and linkage disequilibrium analysis was applied and could narrow down the critical region to a 0.749 Mb region. Of the genes in this region, GAS7, MYH2, and MYH3 were identified as strong novel candidate genes based on further conditional association analyses. These findings provide a novel insight into the genetic basis of FA composition in pork and could contribute to the improvement of pork quality.

Author Correction: Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Erratum to: Comparisons of Beef Fatty Acid and Amino Acid Characteristics between Jeju Black Cattle, Hanwoo, and Wagyu Breeds.

[This corrects the article DOI: 10.5851/kosfa.2019.e33.].

Evaluation of Myosin Heavy Chain Isoforms in Biopsied Longissimus Thoracis Muscle for Estimation of Meat Quality Traits in Live Pigs.

Estimating meat quality prior to slaughter will be beneficial for the rapid identification of specific traits or poor quality pork compared to a conventional assessment at postmortem. In this study, we identified and quantified myosin heavy chain (MHC) isoforms from a biopsied longissimus thoracis muscle of pigs, and determined their correlation with postmortem muscle fiber characteristics and meat quality. MHC slow and fast isoforms proportions from biopsied samples correlated with postmortem percentage of type I and type IIB muscle fibers, respectively (p < 0.05). The percentage of the biopsied MHC slow isoform showed a positive correlation with pH at 45 min postmortem, and negative correlations with filter-paper fluid uptake and drip loss in pork (p < 0.05). Furthermore, clustering the pigs into three groups based on the biopsied MHC isoform proportions was not only significantly associated with muscle fiber number and proportions of muscle fiber area, but also correlated with pH at 45 min postmortem and the National Pork Producers Council color score (p < 0.05). Collectively, our findings indicate that the biopsied MHC isoforms serve as parameter for estimating meat quality, with the association between the higher proportion of MHC slow isoforms and pH at 45 min postmortem in particular being indicative of better pork quality.

Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

The Jeju horse, indigenous to the Jeju Island in Korea may have originated from Mongolian horses. Adaptations to the local harsh environment have conferred Jeju horse with unique traits such as small-sized body, stocky head, and shorter limbs. These characteristics have not been studied previously at the genomic level. Therefore, we sequenced and compared the genome of 41 horses belonging to 6 breeds. We identified numerous breed-specific non-synonymous SNPs and loss-of-function mutants. Demographic and admixture analyses showed that, though Jeju horse is genetically the closest to the Mongolian breeds, its genetic ancestry is independent of that of the Mongolian breeds. Genome wide selection signature analysis revealed that genes such as LCORL, MSTN, HMGA2, ZFAT, LASP1, PDK4, and ACTN2, were positively selected in the Jeju horse. RNAseq analysis showed that several of these genes were also differentially expressed in Jeju horse compared to Thoroughbred horse. Comparative muscle fiber analysis showed that, the type I muscle fibre content was substantially higher in Jeju horse compared to Thoroughbred horse. Our results provide insights about the selection of complex phenotypic traits in the small-sized Jeju horse and the novel SNPs identified will aid in designing high-density SNP chip for studying other native horse breeds.

A functional regulatory variant of MYH3 influences muscle fiber-type composition and intramuscular fat content in pigs.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.

Comparisons of Beef Fatty Acid and Amino Acid Characteristics between Jeju Black Cattle, Hanwoo, and Wagyu Breeds.

Jeju black cattle are known as one of Korea's traditional cattle. However, Hanwoo is more well-known to Korean meat consumers as representative beef cattle. Despite the popularity of these two breeds, comparison of the nutritional characteristics between Jeju black cattle and Hanwoo have not been studied. Here, we compared the fatty acid and amino acid characteristics between two Korean traditional cattle and Wagyu breeds. A total of 62 cattle were used in this study. The Jeju black cattle beef had significantly higher unsaturated fatty acids than Hanwoo (p<0.05). Savory fatty acids, including oleic acid were also higher than in Hanwoo cattle (p<0.05). The negative flavor fatty acids, such as palmitic acid were significantly lower than in Hanwoo (p<0.001). On the other hand, linoleic acid which imparts a negative flavor was higher than Hanwoo (p<0.05). Amino acids, including alanine and glutamine, usually representative of the umami taste were present in significantly higher proportions in Jeju black cattle (p<0.05). In addition, bitter tasting amino acids, including valine, leucine, isoleucine, and methionine were lower in Jeju black cattle beef than in Hanwoo (p<0.05, p<0.001, p<0.001, and p<0.001 each). Taken together, our results suggest that Jeju black cattle beef had higher savory flavor and umami taste which affected consumers preference for the meat.

Effects of a Novel p.A41P Mutation in the Swine Myogenic factor 5(MYF5) Gene on Protein Stabilizing, Muscle Fiber Characteristics and Meat Quality.

Myogenic factor 5 (MYF5) plays an important role in regulating skeletal muscle fiber characteristics, consequently affecting meat production and quality. We identified a novel p.A41P mutation in exon1 of the porcine MYF5 gene by direct sequencing. The mutation was predicted to be destabilizing in protein structure based on the resultant amino acid substitution. We estimated the significant substitution effect of p.A41P on the energy stabilization of Myf5 protein structure. Then, we demonstrated that the mutation in Yorkshire population significantly affected muscle fiber type I composition (p<0.05), loin-eye area of lean meat content (p<0.05) and filter-fluid uptake of meat quality (p<0.01). Furthermore, dominant effects significantly influenced total muscle fiber number (p<0.05). This study suggests that the novel p.A41P mutation in porcine MYF5 may be a valuable genetic marker to affect the muscle fiber characteristics and consequently improve meat production quality and quantity.

Estimation of pork quality in live pigs using biopsied muscle fibre number composition.

Here, we newly provided the parameters for estimating meat quality in live pigs using the muscle biopsy. The biopsied longissimus thoracis muscle was used to identify the muscle fibre characteristics (MFCs). Of the various MFCs in biopsied muscle, muscle fibre number (MFN) composition showed the greatest correlation with the MFCs in postmortem muscle (P<0.001). Moreover, the pigs cluster groups, based on the biopsied MFN composition, demonstrated statistically significant differences in meat quality traits such as muscle pH, drip loss, and meat colour (P<0.05). Therefore, we conclude that the MFN parameters in live pigs are closely related to the postmortem muscle metabolic rate and ultimately with the quality of meat. We suggest that the higher type I and lower type IIB MFN in biopsied muscle will result in better pork quality.

Effects of mushroom extract on textural properties and muscle protein degradation of bovine longissimus dorsi muscle.

We investigated the effects of Sarcodon aspratus, Agaricus bisporus, and Lentinula edodes aqueous extracts on the tenderization of bovine longissimus dorsi muscle. Meat quality and muscle protein degradation were examined as well. Beef chunks were marinated in distilled water (control), 5% S. aspratus (SA), 5% A. bisporus (AB), or 5% L. edodes (LE) extracts. SA was shown to have a higher enzymatic activity (p < 0.001) and water-holding capacity than LE (p < 0.01). SA and AB extracts exhibited lower shear force values compared with the control (p < 0.05). SA, AB, and LE showed superior muscle proteolytic effects compared with the control. SA demonstrated the ability to degrade myosin heavy chains and actin, which was not observed after AB and LE extract treatments. This suggests that SA extract may affect tenderization. Taken together, our results show that aqueous extract of S. aspratus affects the tenderness of the bovine longissimus dorsi muscle.

Effects of Morphological Characteristics of Muscle Fibers on Porcine Growth Performance and Pork Quality.

The aim of this study was to investigate the effects of morphological characteristics of porcine muscle fibers on growth performance, muscle fiber characteristics, and pork quality taken from the longissimus dorsi muscle. A total of 239 crossbred pigs (164 castrated males and 75 females) were used in this study. Experimental pigs were categorized by the total number of muscle fiber (TNF: High and Low) and cross sectional area of muscle fiber (CSAF: Large, Middle, and Small). Their combinations were classified into six groups (High-Large, HL; High-Middle, HM; High-Small, HS; Low-Large, LL; Low-Middle, LM; Low-Small, LS). The TNF and CSAF were significantly (p<0.05) correlated with growth rate and carcass productivity, while the only of the type I number had no meaningful relationships excluding the correlation with loin area (p<0.001). The proportion of type I area was positively correlated with pH45 min while the proportion of type IIB area was negatively correlated with pH45 min and pH24 h (p<0.05). Drip loss and protein denaturation had strong relationships with the proportion of type IIB number or area. The HL group exhibited the greatest growth performance. In addition, the HL group had significantly greater values in protein solubility than the other groups. In conclusion, this study suggest that high TNF combined to large CSAF improve the ultimate lean meat productivity and assure normal meat quality simultaneously with increased both proportion of number and area of type I, type IIA muscle fibers and lowered proportion of number and area of type IIB.

Genome-wide Association Study (GWAS) and Its Application for Improving the Genomic Estimated Breeding Values (GEBV) of the Berkshire Pork Quality Traits.

The missing heritability has been a major problem in the analysis of best linear unbiased prediction (BLUP). We introduced the traditional genome-wide association study (GWAS) into the BLUP to improve the heritability estimation. We analyzed eight pork quality traits of the Berkshire breeds using GWAS and BLUP. GWAS detects the putative quantitative trait loci regions given traits. The single nucleotide polymorphisms (SNPs) were obtained using GWAS results with p value <0.01. BLUP analyzed with significant SNPs was much more accurate than that with total genotyped SNPs in terms of narrow-sense heritability. It implies that genomic estimated breeding values (GEBVs) of pork quality traits can be calculated by BLUP via GWAS. The GWAS model was the linear regression using PLINK and BLUP model was the G-BLUP and SNP-GBLUP. The SNP-GBLUP uses SNP-SNP relationship matrix. The BLUP analysis using preprocessing of GWAS can be one of the possible alternatives of solving the missing heritability problem and it can provide alternative BLUP method which can find more accurate GEBVs.

Genome-wide detection and characterization of positive selection in Korean Native Black Pig from Jeju Island.

BACKGROUND: In the 1980s, Korean native black pigs from Jeju Island (Jeju black pigs) served as representative sample of Korean native black pigs, and efforts were made to help the species rebound from the brink of extinction, which occurred as a result of the introduction of Western pig breeds. Geographical separation of Jeju Island from the Korean peninsula has allowed Jeju black pigs not only to acquire unique characteristics but also to retain merits of rare Korean native black pigs. RESULTS: To further analyze the Jeju black pig genome, we performed whole-genome re-sequencing (average read depth of 14×) of 8 Jeju black pig and 6 Korean pigs (which live on the Korean peninsula) to compare and identify putative signatures of positive selection in Jeju black pig, the true and pure Korean native black pigs. The candidate genes potentially under positive selection in Jeju black pig support previous reports of high marbling score, rare occurrence of pale, soft, exudative (PSE) meat, but low growth rate and carcass weight compared to Western breeds. CONCLUSIONS: Several candidate genes potentially under positive selection were involved in fatty acid transport and may have contributed to the unique characteristics of meat quality in JBP. Jeju black pigs can offer a unique opportunity to investigate the true genetic resource of once endangered Korean native black pigs. Further genome-wide analyses of Jeju black pigs on a larger population scale are required in order to define a conservation strategy and improvement of native pig resources.

Tenderization of Bovine Longissimus Dorsi Muscle using Aqueous Extract from Sarcodon aspratus.

The aim of this study was to investigate the effects of aqueous extract from Sarcodon aspratus on tenderization of the bovine longissimus dorsi muscles in comparison with commercial proteolytic enzymes. Furthermore, meat quality and muscle protein degradation were examined. We marinated meat with 2% Sarcodon aspratus extract, 2% kiwi extract, and 0.2% papain. Beef chunks (3×3×3 cm(3)) were marinated with distilled water (control), Sarcodon aspratus extract (T1), kiwi extract (T2) or papain (T3) for 48 h at 4℃. There were no significant differences in muscle pH and lightness between control and treated samples. T1 had the lowest redness (p<0.01), and higher cooking loss and water holding capacity than control and T2 (p<0.05). T1 and T3 exhibited lower shear force values than control (p<0.05). Total protein solubility did not differ significantly between T1 and control, but T1 had less myofibrillar protein solubility than control and T2 (p<0.001). The degradation of myosin heavy chain in T1 and T3 was observed. This degradation of myofibrillar protein suggests that Sarcodon aspratus extract could influence tenderization. These results show that aqueous extract of Sarcodon aspratus extract actively affect the tenderness of the bovine longissimus dorsi muscle.

The influences of weaning age and weight on carcass traits and meat quality of pigs.

The purpose of this study was to establish the effects of weaning age and weight on pigs, and their interaction with carcass traits and meat quality. A total of 468 piglets were obtained from 57 sows and four boars and grouped by age at weaning (D21, 18-24 days; D28, 25-32 days). Each weaning group was subdivided into three weight groups (L, M and H) according to weaning weight. The D28 group had heavier carcass weight, redness and yellowness, but had lower marbling scores and less drip loss than the D21 group (P < 0.05). The pigs with a light weight at weaning had higher carcass weights and lower yellowness than did pigs with a medium or heavy weight at weaning (P < 0.05). Weaning age was found to have a negative correlation with drip loss, while weaning weight was negatively correlated with carcass weight and drip loss (P < 0.05). We concluded that carcass and meat quality traits in pigs were significantly related to their age and weight at weaning. Therefore, we find that piglet weaning age and weight are no less important than post-weaning growth performance and behavior, with regard to carcass traits and meat quality.

Genetic traceability of black pig meats using microsatellite markers.

Pork from Jeju black pig (population J) and Berkshire (population B) has a unique market share in Korea because of their high meat quality. Due to the high demand of this pork, traceability of the pork to its origin is becoming an important part of the consumer demand. To examine the feasibility of such a system, we aim to provide basic genetic information of the two black pig populations and assess the possibility of genetically distinguishing between the two breeds. Muscle samples were collected from slaughter houses in Jeju Island and Namwon, Chonbuk province, Korea, for populations J and B, respectively. In total 800 Jeju black pigs and 351 Berkshires were genotyped at thirteen microsatellite (MS) markers. Analyses on the genetic diversity of the two populations were carried out in the programs MS toolkit and FSTAT. The population structure of the two breeds was determined by a Bayesian clustering method implemented in structure and by a phylogenetic analysis in Phylip. Population J exhibited higher mean number of alleles, expected heterozygosity and observed heterozygosity value, and polymorphism information content, compared to population B. The FIS values of population J and population B were 0.03 and -0.005, respectively, indicating that little or no inbreeding has occurred. In addition, genetic structure analysis revealed the possibility of gene flow from population B to population J. The expected probability of identify value of the 13 MS markers was 9.87×10(-14) in population J, 3.17×10(-9) in population B, and 1.03×10(-12) in the two populations. The results of this study are useful in distinguishing between the two black pig breeds and can be used as a foundation for further development of DNA markers.

Comparative transcriptomic analysis to identify differentially expressed genes in fat tissue of adult Berkshire and Jeju Native Pig using RNA-seq.

Pork is a major source of animal protein for humans. The subcutaneous, intermuscular and the intramuscular fat are the factors responsible for meat quality. RNA-seq is rapidly adopted for the profiling of the transcriptomes in the studies related to gene regulation. The discovery of differentially expressed genes (DEGs) between adult animals of Jeju Native Pig (JNP) and Berkshire breeds are of particular interest for the current study. RNA-seq was used to investigate the transcriptome profiling in the fat tissue. Sequence reads were obtained from Ilumina HiSeq2000 and mapped to the pig genome using Tophat2. Total 153 DEGs were identified and 71 among the annotated genes, have BLAST matches in the non- redundant database. Metabolic, immune response and protein binding are enriched pathways in the fat tissue. In our study, biological adhesion, cellular, developmental and multicellular organismal processes in fat were up-regulated in JNP as compare to Berkshire. Multicellular organismal process, developmental process, embryonic morphogenesis and skeletal system development were the most significantly enriched terms in fat of JNP and Berkshire breeds (p = 1.17E-04, 0.044, 3.47E-04 and 4.48E-04 respectively). COL10A1, COL11A2, PDK4 and PNPLA3 genes responsible for skeletal system morphogenesis and body growth were down regulated in JNP. This study is the first statistical analysis for the detection of DEGs from RNA-seq data generated from fat tissue sample. This analysis can be used as stepping stone to understand the difference in the genetic mechanisms that might influence the identification of novel transcripts, sequence polymorphisms, isoforms and noncoding RNAs.

The characteristics of myosin heavy chain-based fiber types in porcine longissimus dorsi muscle.

The aim of this study is to type fibers from porcine longissimus dorsi (LD) muscles according to their distribution of myosin heavy chain (MHC) isoforms as well as to investigate fiber characteristics. Four pure types, including types I, IIA, IIX, and IIB were labeled and two hybrid fiber types were subdivided into type IIAX and IIXB by immunohistochemistry using four monoclonal antibodies. Porcine LD muscles were found to have 92.79 (number) and 92.10% (area) of pure type composition, while the composition of hybrid fibers was 8.22 (number) and 9.71% (area). The activities of myosin ATPase increased in the following order: type I, IIAX, IIA = IIX = IIB and IIXB. The succinate dehydrogenase and glycerol-3-phosphate dehydrogenase activities were higher in fiber types I and IIB, respectively. The characteristics of hybrid fibers were observed that their characteristics did not lie between the properties for their respective pure phenotypes.

Relationship between pork quality and characteristics of muscle fibers classified by the distribution of myosin heavy chain isoforms.

A total of six fiber types, including four pure types (type I, IIA, IIX, and IIB) and two hybrid types (type IIAX and IIXB), were classified according to the expression of myosin heavy chain (MHC) isoforms by immunohistochemistry with MHC specific monoclonal antibodies. The comparison of the muscle fiber characteristics and pork quality between pork quality groups (DFD: dark, firm, and dry; PSE: pale, soft, and exudative; RFN: reddish pink, firm, and nonexudative; and RSE: reddish pink, soft, and exudative) classified by muscle pH, drip loss, and lightness was conducted and the relationship of myofiber characteristics to pork quality was investigated. The DFD group had the highest value of IIAX fiber density (P<0.05). The DFD group also showed the greatest fiber relative area of type I, IIA, and IIAX (P<0.05) whereas there were no significant differences in area composition for types I, IIA, and IIAX among the other groups including PSE, RFN, and RSE (P>0.05). The DFD group had the highest cross-sectional area (CSA) in types I, IIA, and IIX among the groups. The increase in density of type IIAX was related with the higher pH and the lower hue and drip loss. An increase in the fiber number composition of hybrid type IIXB increased the lightness and cooking loss and decreased sarcoplasmic protein solubility (SPS). Regarding fiber relative area, pure type I and IIA and hybrid type IIAX were greater in the DFD group and had lower lightness and drip loss. Hybrid type IIAX influences the desirability of the pork due to its association with low lightness and high pH and water-holding capacity (WHC). In contrast, type IIXB was related to poor quality pork, including pale color, low WHC in cooked meat, and low SPS.