Pig fetal skeletal muscle development is associated with genome-wide DNA hypomethylation and corresponding alterations in transcript and microRNA expression.

Fetal myogenesis represents a critical period of porcine skeletal muscle development and requires coordinated expression of thousands of genes. Epigenetic mechanisms, including DNA methylation, drive transcriptional regulation during development; however, these processes are understudied in developing porcine tissues. We performed bisulfite sequencing to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-days gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45 739 differentially methylated regions (DMRs) between stages, and the majority (N = 34 232) were hypomethylated at 70 versus 41 dg. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and expression. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We show that developmental DMRs are enriched for GWAS SNPs for muscle- and meat-related traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics of porcine myogenesis and reveal putative cis-regulatory elements governed by epigenetic processes.

Genome-wide association analyses of lesion counts in group-housed pigs.

Aggression in group-housed pigs is a welfare concern and can negatively affect production. Skin lesions are reliable indicators of aggression and are moderately heritable, suggesting that selective breeding may reduce aggression. To further understand the genetic control of behavioral traits, such as the aggressive response to regrouping, associated single nucleotide polymorphisms (SNPs) can be identified within the genome, and the region in which these SNPs are located can be related to known genes. To investigate SNPs associated with aggression, 1093 purebred Yorkshire pigs were strategically remixed into new groups of familiar and unfamiliar animals at three life stages and lesion counts were recorded. Genomic best linear unbiased prediction (GBLUP) models were fitted for each trait. The genetic additive effect was obtained from a genetic relationship matrix constructed from the 50 924 SNPs. SNP effects and their variances were estimated from the GBLUP objects. SNPs that were associated with a significant portion of the trait variance were identified for lesions to the anterior (three SNPs, FDR <5%) and central (one SNP, FDR <5%) portions of the body in grow-finish pigs. These SNPs were located on chromosome 11, suggesting that chromosome 11 contains a region explaining variation in lesion counts that should be further explored to identify genes underlying biological control of aggression.

Estimation of genetic parameters for lesion scores and growth traits in group-housed pigs.

Pigs housed in groups are remixed with unfamiliar individuals, which can trigger aggressive interactions, potentially compromising animal welfare. Skin lesions are a reliable indicator trait of aggression and are moderately heritable, suggesting that aggression may be reduced through selection. This study estimated genetic parameters of skin lesions of pigs at multiple life stages, explored genetic correlations of skin lesions between age groups and body location, and studied the relationship between skin lesions and production traits of commercial importance. A population of 1,079 Yorkshire pigs was strategically remixed into new groups of familiar and unfamiliar animals at 3 life stages (weaning, grow-finish, and mature gilts). Skin lesions (fresh, bright red cuts) were counted immediately prior to mixing and 24 h and 3 wk after mixing across 3 body regions: anterior, central, and caudal. Weights were recorded prior to each mixing event. Prior to slaughter, backfat thickness and loin muscle area were determined using ultrasound. Univariate analyses were performed to obtain heritability estimates of lesion scores. Bivariate analyses were performed with response variables being skin lesions, weight gain per life stage, backfat thickness, or loin muscle area, depending on the relationship of interest, to obtain correlations. Lesion score heritabilities ranged from 0.10 to 0.40 and were significant ( < 0.05). Heritability was highest for lesions on the anterior region of the body for 24 h and 3 wk after mixing. Lesions to the central and caudal areas showed the highest genetic correlation at each stage of production, whereas those to the anterior and caudal regions had the lowest correlation. The highest genetic correlation was found between the mature gilt and grow-finish stages, whereas the weaning and mature gilt stages had the lowest correlations. Genetic correlations between lesions and production traits were not significantly different from 0 for weight gain and backfat thickness, but loin muscle area was negatively correlated with lesions ( = 1.17 × 10, = 2.30 × 10, and = 6.08 × 10 for anterior, central, and caudal lesions, respectively). These results are promising for the industry because they suggest that pigs selected for reduced lesions will show increased loin muscle area without negative effects on growth. Alternatively, selection for these production traits would not increase lesions.

Genome-wide association study in an F2 Duroc x Pietrain resource population for economically important meat quality and carcass traits.

Meat quality is essential for consumer acceptance, it ultimately impacts pork production profitability and it is subject to genetic control. The objective of this study was to map genomic regions associated with economically important meat quality and carcass traits. We performed a genome-wide association (GWA) analysis to map regions associated with 38 meat quality and carcass traits recorded for 948 F2 pigs from the Michigan State University Duroc × Pietrain resource population. The F0, F1, and 336 F2 pigs were genotyped with the Illumina Porcine SNP60 BeadChip, while the remaining F2 pigs were genotyped with the GeneSeek Genomic Profiler for Porcine Low Desnisty (LD) chip, and imputed with high accuracy ( = 0.97). Altogether the genomic dataset comprised 1,019 animals and 44,911 SNP. A Gaussian linear mixed model was fitted to estimate the breeding values and the variance components. A linear transformation was performed to estimate the marker effects and variances. Type I error rate was controlled at a False Discovery Rate of 5%. Seven putative QTL found in this study were previously reported in other studies. Two novel QTL associated with tenderness (TEN) were located on SSC3 [135.6:137.5Mb; False Discovery rate (FDR) < 0.03] and SSC5 (67.3:69.1Mb; FDR < 0.02). The QTL region identified on SSC15 includes Protein Kinase AMP-activated É£ 3-subunit gene (), which has been associated with 24-h pH (pH24), drip loss (DL) and cook yield (CY). Also, novel candidate genes were identified for TEN in the region on SSC5 [A Kinase (PRKA) Anchor Protein 3 (], and for tenth rib backfat thickness (BF10) [Carnitine O-Acetyltransferase ()] on SSC1. The association of gene polymorphisms with pork quality traits has been reported for several pig populations. However, there are no SNP for this gene on the chip used, thus we genotyped the animals for 2 non-synonymous variants ( and ). We then performed a GWA conditioning on the genotype of both SNP and was associated with pH24, DL, protein content (PRO) and CY ( < 0.004) and T30N with Juiciness, TEN, shear force, pH24, PRO, and CY < 0.04). Finally, we performed a GWA conditioning on the genotype of the SNP peak detected in this study, and T30N remained associated only with PRO ( < 0.02). Therefore, in this study we identified 2 novel QTL regions, suggest 2 novel candidate genes, and conclude that other SNP in PRKAG3 or nearby gene(s) explain the observed associations on SSC15 in this population.

Estimates of the actual relationship between half-sibs in a pig population.

Genomic relationships based on markers capture the actual instead of the expected (based on pedigree) proportion of genome shared identical by descent (IBD). Several methods exist to estimate genomic relationships. In this research, we compare four such methods that were tested looking at the empirical distribution of the estimated relationships across 6704 pairs of half-sibs from a cross-bred pig population. The first method based on multiple marker linkage analysis displayed a mean and standard deviation (SD) in close agreement with the expected ones and was robust to changes in the minor allele frequencies (MAF). A single marker method that accounts for linkage disequilibrium (LD) and inbreeding came second, showing more sensitivity to changes in the MAF. Another single marker method that considers neither inbreeding nor LD showed the smallest empirical SD and was the most sensible to changes in MAF. A higher mean and SD were displayed by VanRaden's method, which was not sensitive to changes in MAF. Therefore, the method based on multiple marker linkage analysis and the single marker method that considers LD and inbreeding performed closer to theoretical values and were consistent with the estimates reported in literature for human half-sibs.

Impact of improving dietary amino acid balance for lactating sows on efficiency of dietary amino acid utilization and transcript abundance of genes encoding lysine transporters in mammary tissue.

Lactating multiparous Yorkshire sows ( = 64) were used in 2 experiments to test the hypothesis that reducing dietary CP intake and improving AA balance through crystalline AA (CAA) supplementation improves apparent dietary AA utilization efficiency for milk production and increases transcript abundance of genes encoding Lys transporter proteins in mammary tissue. In Exp. 1, 40 sows were assigned to 1 of 4 diets: 1) high CP (HCP; 16.0% CP, as-fed basis; analyzed concentration), 2) medium-high CP (MHCP; 15.7% CP), 3) medium-low CP (MLCP; 14.3% CP), and 4) low CP (LCP; 13.2% CP). The HCP diet was formulated using soybean meal and corn as the only Lys sources. The reduced-CP diets contained CAA to meet estimated requirements for essential AA that became progressively limiting with reduction in CP concentration, that is, Lys, Ile, Met + Cys, Thr, Trp, and Val. Dietary standardized ileal digestible (SID) Lys concentration was 80% of the estimated requirement. In Exp. 2, 24 sows were assigned to the HCP or LCP diets. In Exp. 1, blood samples were postprandially collected 15 h on d 3, 7, 14, and 18 of lactation and utilization efficiency of dietary AA for milk production was calculated during early (d 3 to 7) and peak (d 14 to 18) lactation. Efficiency values were estimated from daily SID AA intakes and milk AA yield, with corrections for maternal AA requirement for maintenance and AA contribution from body protein losses. In Exp. 2, mammary tissue was biopsied on d 4 and 14 of lactation to determine the mRNA abundance of genes encoding Lys transporter proteins. In peak lactation, Lys, Thr, Trp, and Val utilization efficiency increased with decreasing dietary CP (linear for Trp and Val, < 0.05; in sows fed the MHCP diet vs. sows fed the HCP diet for Lys and Thr, < 0.05). Total essential and nonessential 15-h postprandial serum AA concentrations increased with decreasing dietary CP (linear, = 0.09 and < 0.05, respectively), suggesting increased maternal body protein mobilization. Transcript abundance of several genes involved in Lys transport in mammary tissue did not differ between sows fed the LCP and HCP diets. Feeding lactating sows low-CP diets supplemented with CAA increases the efficiency of utilizing dietary Lys, Thr, Trp, and Val for milk protein production but is unrelated to abundance in mRNA of genes encoding Lys transport proteins in the mammary gland. Dietary Lys utilization for milk protein production in lactating sows appears to be optimized when crystalline Lys is included at a minimum of 0.10% in a diet containing 15.70% CP.

A comparison of methods to estimate genomic relationships using pedigree and markers in livestock populations.

Accurate prediction of breeding values depends on capturing the variability in genome sharing of relatives with the same pedigree relationship. Here, we compare two approaches to set up genomic relationship matrices for precision of genomic relationships (GR) and accuracy of estimated breeding values (GEBV). Real and simulated data (pigs, 60k SNP) were analysed, and GR were estimated using two approaches: (i) identity by state, corrected with either the observed (GVR-O ) or the base population (GVR-B ) allele frequencies and (ii) identity by descent using linkage analysis (GIBD-L ). Estimators were evaluated for precision and empirical bias with respect to true pedigree IBD GR. All three estimators had very low bias. GIBD-L displayed the lowest sampling error and the highest correlation with true genome-shared values. GVR-B approximated GIBD-L 's correlation and had lower error than GVR-O . Accuracy of GEBV for selection candidates was significantly higher when GIBD-L was used and identical between GVR-O and GVR-B . In real data, GIBD-L 's sampling standard deviation was the closest to the theoretical value for each pedigree relationship. Use of pedigree to calculate GR improved the precision of estimates and the accuracy of GEBV.

Refining genomewide association for growth and fat deposition traits in an F pig population.

The identification of genomic regions that affect additive genetic variation and contain genes involved in controlling growth and fat deposition has enormous impact in the farm animal industry (e.g., carcass merit and meat quality). Therefore, a genomewide association study was implemented in an F pig population using a 60,000 SNP marker panel for traits related to growth and fat deposition. Estimated genomic EBV were linearly transformed to calculate SNP effects and to identify genomic positions possibly associated with the genetic variability of each trait. Genomic segments were then defined considering the markers included in a region 1 Mb up- and downstream from the SNP with the smallest -value and a false discovery rate < 0.05 for each trait. The significance for each 2-Mb segment was tested using the Bonferroni correction. Significant SNP were detected on SSC2, SSC3, SSC5, and SSC6, but 2-Mb segment significant effects were observed on SSC3 for weight at birth (wt_birth) and on SSC6 for 10th-rib backfat and last-rib backfat measured by ultrasound at different ages. Furthermore, a 6-Mb segment on SSC6 was also considered because the 2-Mb segments for 10 different fat deposition traits were overlapped. Although the segment effects for each trait remain significant, the proportion of additive variance explained by this larger segment was slightly smaller in some traits. In general, the results confirm the presence of genetic variability for wt_birth on SSC3 (18.0-20.2 Mb) and for fat deposition traits on SSC6 (133.8-136.0 Mb). Within these regions, fibrosin () and myosin light chain, phosphorylatable, fast skeletal muscle () genes could be considered as candidates for the wt_birth signal on SSC3, and the SERPINE1 mRNAbinding protein 1 gene () may be a candidate for the fat deposition trait signals on SSC6.

Meta-analysis of genome-wide association from genomic prediction models.

Genome-wide association (GWA) studies based on GBLUP models are a common practice in animal breeding. However, effect sizes of GWA tests are small, requiring larger sample sizes to enhance power of detection of rare variants. Because of difficulties in increasing sample size in animal populations, one alternative is to implement a meta-analysis (MA), combining information and results from independent GWA studies. Although this methodology has been used widely in human genetics, implementation in animal breeding has been limited. Thus, we present methods to implement a MA of GWA, describing the proper approach to compute weights derived from multiple genomic evaluations based on animal-centric GBLUP models. Application to real datasets shows that MA increases power of detection of associations in comparison with population-level GWA, allowing for population structure and heterogeneity of variance components across populations to be accounted for. Another advantage of MA is that it does not require access to genotype data that is required for a joint analysis. Scripts related to the implementation of this approach, which consider the strength of association as well as the sign, are distributed and thus account for heterogeneity in association phase between QTL and SNPs. Thus, MA of GWA is an attractive alternative to summarizing results from multiple genomic studies, avoiding restrictions with genotype data sharing, definition of fixed effects and different scales of measurement of evaluated traits.

Implementing meta-analysis from genome-wide association studies for pork quality traits.

Pork quality plays an important role in the meat processing industry. Thus, different methodologies have been implemented to elucidate the genetic architecture of traits affecting meat quality. One of the most common and widely used approaches is to perform genome-wide association (GWA) studies. However, a limitation of many GWA in animal breeding is the limited power due to small sample sizes in animal populations. One alternative is to implement a meta-analysis of GWA (MA-GWA) combining results from independent association studies. The objective of this study was to identify significant genomic regions associated with meat quality traits by performing MA-GWA for 8 different traits in 3 independent pig populations. Results from MA-GWA were used to search for genes possibly associated with the set of evaluated traits. Data from 3 pig data sets (U.S. Meat Animal Research Center, commercial, and Michigan State University Pig Resource Population) were used. A MA was implemented by combining -scores derived for each SNP in every population and then weighting them using the inverse of estimated variance of SNP effects. A search for annotated genes retrieved genes previously reported as candidates for shear force (calpain-1 catalytic subunit [] and calpastatin []), as well as for ultimate pH, purge loss, and cook loss (protein kinase, AMP-activated, γ 3 noncatalytic subunit []). In addition, novel candidate genes were identified for intramuscular fat and cook loss (acyl-CoA synthetase family member 3 mitochondrial []) and for the objective measure of muscle redness, CIE a* (glycogen synthase 1, muscle [] and ferritin, light polypeptide []). Thus, implementation of MA-GWA allowed integration of results for economically relevant traits and identified novel genes to be tested as candidates for meat quality traits in pig populations.

Searching for causal networks involving latent variables in complex traits: Application to growth, carcass, and meat quality traits in pigs.

Structural equation models (SEQM) can be used to model causal relationships between multiple variables in multivariate systems. Among the strengths of SEQM is its ability to consider causal links between latent variables. The use of latent variables allows modeling complex phenomena while reducing at the same time the dimensionality of the data. One relevant aspect in the quantitative genetics context is the possibility of correlated genetic effects influencing sets of variables under study. Under this scenario, if one aims at inferring causality among latent variables, genetic covariances act as confounders if ignored. Here we describe a methodology for assessing causal networks involving latent variables underlying complex phenotypic traits. The first step of the method consists of the construction of latent variables defined on the basis of prior knowledge and biological interest. These latent variables are jointly evaluated using confirmatory factor analysis. The estimated factor scores are then used as phenotypes for fitting a multivariate mixed model to obtain the covariance matrix of latent variables conditional on the genetic effects. Finally, causal relationships between the adjusted latent variables are evaluated using different SEQM with alternative causal specifications. We have applied this method to a data set with pigs for which several phenotypes were recorded over time. Five different latent variables were evaluated to explore causal links between growth, carcass, and meat quality traits. The measurement model, which included 5 latent variables capturing the information conveyed by 19 different phenotypic traits, showed an acceptable fit to data (e.g., χ/df = 1.3, root-mean-square error of approximation = 0.028, standardized root-mean-square residual = 0.041). Causal links between latent variables were explored after removing genetic confounders. Interestingly, we found that both growth (-0.160) and carcass traits (-0.500) have a significant negative causal effect on quality traits (-value ≤ 0.001). This result may have important implications for strategies for pig production improvement. More generally, the proposed method allows further learning regarding phenotypic causal structures underlying complex traits in farm species.

A comparison of alternative random regression and reaction norm models for whole genome predictions.

Whole genome prediction (WGP) based on high density SNP marker panels is known to improve the accuracy of breeding value (BV) prediction in livestock. However, these accuracies can be compromised when genotype by environment interaction (G×E) exists but is not accounted for. Reaction norm (RN) and random regression (RR) models have proven to be useful in accounting for G×E in pre-WGP evaluations by modeling BV as linear or higher order functions of environmental or temporal covariates. We extend these RR/RN models based on several alternative specifications for SNP-specific intercepts and linear slopes on environmental covariates. One specification is based on bivariate normality (BVN) of SNP-specific intercepts and slopes, whereas 2 others, IW-BayesA and based on inverted Wishart (IW) extensions IW-BayesB, are, respectively, bivariate Student t extensions of currently popular models without (BayesA) or with (BayesB) variable selection. We highlight alternative specifications based on the square root free Cholesky decomposition (CD) of SNP-specific variance-covariance (VCV) matrices in an attempt to better differentially model environmentally sensitive from environmentally robust QTL. Two CD specifications were considered with (CD-BayesB) or without (CD-BayesA) any variable selection on intercept and slope effects. We compared each of the 5 models based on an RN simulation study. Six scenarios were considered based on differences in overall genetic correlations between SNP-specific intercept and slope effects as well as on heritabilities and numbers of environmentally robust versus sensitive QTL. In most scenarios, IW-BayesA had the greatest accuracy, whereas CD-BayesB exhibited the greatest accuracy in low complexity architectures (i.e., low number of QTL). In an RR application of a Duroc × Pietrain resource population at Michigan State University, 5,271 SNP markers and 928 F2 animals with known pedigree were analyzed for backfat thickness at wk 10, 13, 16, 19, and 22. SNP-based RR methods had a 2.5% greater (P < 0.0001) cross-validation accuracy for predicting phenotypes than the SNP-based conventional BayesA/BayesB and/or pedigree based RR BLUP; however, none of the proposed RR models had performances that were different from each other.

Estimation of U.S. Yorkshire breed composition using genomic data.

The Yorkshire breed uses white coat color as a selection requirement to indicate breed purity. In this study, genomic information from chromosomal regions, as well as the whole genome, was applied to estimate breed composition of purebred Yorkshire animals. Genotypes for approximately 60,000 SNP from the Illumina PorcineSNP60 BeadChip (60K) were available for reference animals for which the genetic background was known, study animals that included Yorkshire sires (Tes_York, n = 889), and known crossbred animals that had Yorkshire heritage (Tes_U, n = 12). Haplotypes of SNP flanking the KIT (Dominant white locus) and MC1R (Melanocortin receptor 1) genes were developed for reference animals for the Duroc, Hampshire, Landrace, Yorkshire, and Pietrain breeds. For the KIT region, haplotypes observed in Yorkshire reference animals were also found in 84 and 7% of the haplotypes in Landrace and Pietrain reference animals, respectively. They were not found in Duroc or Hampshire reference animals. The sensitivity and specificity of haplotype analysis was 0.93 and 0.75, respectively. In addition, whole genome SNP information was used in regression analyses to further differentiate breed composition. Using 60K, 90% of regression coefficients for Yorkshire, indicating relative Yorkshire composition, ranged from 0.791 to 1.073 and 0.524 to 1.06 in Tes_York and Tes_U, respectively. Regression coefficients for 90% of Hampshire ranged from -0.029 to 0.052 and -0.005 to 0.379 in Tes_York and Tes_U, respectively. Animals in Tes_U were likely of Yorkshire and Hampshire breed origin. The sensitivity and specificity of regression analysis was 0.96 and 0.58, respectively. Combining haplotype and regression analyses, 810 Tes_York animals were accepted as purebred Yorkshire. Genomic information can be used as a tool to describe an animal's breed composition and reduce the need for progeny testing for white coat color verification.

Deep transcriptome sequencing reveals differences in global gene expression between normal and pale, soft, and exudative turkey meat.

Previous studies from our laboratory suggested that differential expression of genes between normal and pale, soft, and exudative (PSE) turkey is associated with development of the PSE syndrome. However, a detailed understanding of molecular mechanisms responsible for the development of this meat defect remains unclear. The objective of this study was to extend and complement our previous work by using deep transcriptome RNA sequence analysis to compare the respective transcriptome profiles and identify molecular mechanisms responsible for the etiology of PSE turkey meat. Turkey breasts (n = 43) were previously classified as normal or PSE using marinade uptake as an indicator of quality (high = normal; low = PSE). Total RNA from breast muscle samples with the highest (n = 4) and lowest (n = 4) marinade uptake were isolated and sequenced using the Illumina GA(IIX) platform. The results indicated differential expression of 494 loci (false discovery rate < 0.05). Changes in gene expression were confirmed using quantitative real-time PCR. Pathway analysis of differentially expressed genes suggested abnormalities of calcium homeostasis and signaling pathways regulating actin cytoskeleton structure as well as carbohydrate metabolism and energy production in PSE samples. Dysregulation of postmortem glucose oxidation in PSE turkey was suggested by both dramatic downregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA, the most downregulated gene, and a decrease in the protein product (P = 0.0007) as determined by immunoblot analysis. These results support the hypothesis that differential expression of several genes and their protein products contribute to development of PSE turkey.

Differential gene expression between normal and pale, soft, and exudative turkey meat.

In response to high consumer demand, turkeys have been intensively selected for rapid growth rate and breast muscle mass and conformation. The success in breeding selection has coincided with an increasing incidence of pale, soft, and exudative (PSE) meat defect, especially in response to heat stress. We hypothesized that the underlying mechanism responsible for the development of PSE meat arises from differences in expression of several critical genes. The objective of this study was to determine differential gene expression between normal and PSE turkey meat using a 6K turkey skeletal muscle long oligonucleotide microarray. Breast meat samples were collected from Randombred Control Line 2 turkeys at 22 wk of age, and classified as normal or PSE primarily based on marinade uptake (high = normal, low = PSE). Total RNA was isolated from meat samples with the highest (normal, n = 6) and the lowest (PSE, n = 6) marinade uptake. Microarray data confirmation was conducted using quantitative real-time PCR. Selection of differentially expressed genes for pathway analysis was performed using a combination of fold change (FC) ranking (FC < -1.66, FC >1.66) and false discovery rate (<0.35) as criteria. The calcium signaling pathway was highlighted as the top canonical pathway associated with differential gene expression between normal and PSE turkey. Dramatic downregulation of fast-twitch myosin heavy chain coupled with upregulation of slow-twitch myosin and troponin C suggested a switch of skeletal muscle isoforms, which may alter muscle fiber arrangement and formation of actin-myosin complexes. Changes in expression of genes in the actin cytoskeleton signaling pathway also suggest altered structures of actin filaments that may affect cell motility as well as strength and flexibility of muscle cells. Substantial downregulation of pyruvate dehydrogenase kinase, isozyme 4 was observed in PSE samples, suggesting altered regulation of the aerobic metabolic pathway in the birds that developed PSE meat defect.

Association of halothane sensitivity with growth and meat quality in pigs.

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire-Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (-0.40 v. -0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.

Versican, matrix Gla protein, and death-associated protein expression affect muscle satellite cell proliferation and differentiation.

Our previous transcriptional profiling study using a turkey skeletal muscle-specific oligonucleotide microarray revealed over 3,000 genes that were differentially expressed at 3 critical stages of muscle development: 18 d embryonic, 1 d posthatch, and 16 wk of age. The genes versican, matrix Gla protein (MGP), and death-associated protein (DAP) were selected to study for their potential effects on muscle satellite cell proliferation and differentiation, as their functions in other tissues are suggestive of possible key roles in the regulation of myogenesis and they are differentially expressed throughout muscle development in the turkey. Using small interfering RNA to knockdown the expression of these genes during proliferation and differentiation, each of the genes was found to differentially affect proliferation and differentiation. Versican and MGP predominantly affected proliferation with line effects, but later stages of differentiation were affected by the knockdown of versican and MGP. The underexpression of DAP inhibited myotube formation, which is a necessary stage in the development of muscle fibers. Without myotube development, muscle fiber formation will be inhibited or abolished. This is the first report that these genes with no previously documented functions with regard to muscle development play a critical role in muscle cell proliferation and differentiation.

Identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Differences in gene expression were compared between RNAs from lungs of high (HR) and low (LR) porcine reproductive and respiratory syndrome virus (PRRSV) burden pigs using the swine protein-annotated long oligonucleotide microarray, the Pigoligoarray. Pathway analyses were carried out to determine biological processes, pathways and networks that differ between the LR and HR responses. Differences existed between HR and LR pigs for 16 signalling pathways [P < 0.01/-log (P-value) >1.96]. Top canonical pathways included acute phase response signalling, crosstalk between dendritic cells and natural killer cells and tight junction signalling, with numerous immune response genes that were upregulated (SOCS1, SOD2, RBP4, HLA-B, HLA-G, PPP2R1A and TAP1) or downregulated (IL18, TF, C4BPA, C1QA, C1QB and TYROBP). One mechanism, regulation of complement activation, may have been blocked in HR (PRRSV-susceptible) pigs and could account for the poor clearance of PRRSV by infected macrophages. Multiple inhibiting signals may have prevented effective immune responses in susceptible HR pigs, although some protective genes were upregulated in these pigs. It is likely that in HR pigs, expression of genes associated with protection was delayed, so that the immune response was not stimulated early; thus, PRRSV infection prevented protective immune responses.

Evaluation of BTA1 and BTA5 QTL Regions for Growth and Carcass Traits in American and Korean Cattle.

Previously identified QTL regions on BTA1 and BTA5 were investigated to validate the QTL regions and to identify candidate genes for growth and carcass traits in commercial cattle populations from the USA and Korea. Initially, a total of 8 polymorphic microsatellite (MS) markers in the BTA1 and 5 QTL regions were used for Chi-square tests to compare the frequencies of individual alleles between high and low phenotypic groups for the US (Michigan Cattleman's Association/Michigan State University; MCA/MSU) cattle. For a subsequent study, 24 candidate genes containing missense mutations and located within the QTL regions based on bovine genome sequence data were analyzed for genotyping in the two commercial cattle populations. Re-sequencing analyses confirmed 18 public missense SNPs and identified 9 new SNPs. Seventeen of these SNPs were used for genotyping of the MCA/MSU cattle (n = 98) and Korean native cattle (n = 323). On BTA1, UPK1B, HRG, and MAGEF1 polymorphisms residing between BM1312 and BMS4048 were significantly associated with growth and carcass traits in one or both of the MCA/MSU and Korean populations. On BTA5, ABCD2, IL22 and SNRPF polymorphisms residing between BL4 and BR2936 were associated with marbling and backfat traits in one or both of the MCA/MSU and Korean cattle populations. These results suggested that BTA 1 and 5 QTL regions may be segregating in both Korean Hanwoo and USA commercial cattle populations and DNA markers tested in this study may contribute to the identification of positional candidate genes for marker-assisted selection programs.

Transcriptional profiling during foetal skeletal muscle development of Piau and Yorkshire-Landrace cross-bred pigs.

Skeletal muscle development is a complex process involving the coordinated expression of thousands of genes. The aim of this study was to identify differentially expressed genes in longissimus dorsi (LD) muscle of pigs at 40 and 70 days (d) of gestation (developmental stages encompassing primary and secondary fibre formation) in Yorkshire-Landrace (YL) cross-bred pigs and Piau pigs (a naturalized Brazilian breed), which are two breed types that differ in muscularity. Foetuses were obtained from gilts at each gestational age (n = 3 YL; n = 4 Piau), and transcriptional profiling was performed using the Pigoligoarray microarray containing 20 400 oligonucleotides. A total of 486 oligonucleotides were differentially expressed (fold change (FC) ≥ 1.5; false discovery rate (FDR) ≤ 0.05) between 40 and 70 d gestation in either YL or Piau pigs, and a total of 1300 oligonucleotides were differentially expressed (FC ≥ 1.5; FDR ≤ 0.05) between YL and Piau pigs at either age. Gene ontology annotation and pathway analyses determined functional classifications for differentially expressed genes and revealed breed type-specific developmental expression patterns. Thirteen genes were selected for confirmation by qRT-PCR analyses, and expression patterns for most of these genes were confirmed, providing further insight into the roles of these genes in pig muscle development. This study revealed both developmental and breed type-specific patterns of gene expression in foetal pig skeletal muscle, including genes not previously associated with myogenesis. This information will contribute to future pig genetic improvement efforts.