Pre-slaughter stress due to transportation, fighting between animals, or lairage time can have a negative impact on meat quality. However, it is unknown if post-natal early-life stress influences carcass merit or pork quality. Given that weaning is a major stressor, this study aimed to examine if gilt piglets exhibiting divergent stress response patterns (resilient or vulnerable) at weaning had differences in carcass or meat quality traits as well as underlying molecular differences in cortisol receptors (NR3C1, NR3C2). Stress resilient gilts exhibited significantly lower live weights at slaughter (P = 0.045) and smaller loin muscle area (P = 0.008) than stress vulnerable gilts. Additionally, several associations were observed between tissue-specific transcript abundance and meat quality traits. Lower NR3C1 transcript abundance in subcutaneous adipose was significantly associated with higher marbling score (P = 0.028), higher 45 min pH (P = 0.012), greater pH decline (P = 0.003), higher subjective color score (P = 0.027), and higher Minolta L & lowast; values (P = 0.049). In conclusion, this study indicates weaning stress is not associated with substantive differences in carcass merit or meat quality, and revealed insights into potential molecular mechanisms by which stress affects meat quality through adipose-specific expression of the glucocorticoid and mineralocorticoid receptors.

Exposure to social stress during gestation has been demonstrated to influence offspring health, growth, and behavior. This study aimed to characterize differences in weaned pigs previously selected from sows classified as exhibiting resilience or vulnerability to gestational social stress using salivary cortisol. In short, purebred Yorkshire litters born to first or second parity sows were selected based on the sow’s salivary cortisol pattern at an acute mixing event at 30d of gestation. Saliva was collected at 1200h each day at -1, 0, +1, +2, +3, +7d relative to social mixing. Salivary cortisol pattern was used to identify 8 females whose salivary cortisol concentrations returned to baseline as stress resilient (SR) and 8 females whose salivary cortisol concentrations remained increased as stress vulnerable (SV). We previously observed reduced average daily gain (ADG) leading to reduced weaning weight in the piglets born to SV sows. To investigate if differences exist post-weaning, we characterized the stress response to weaning, aggression at two mixing events using skin lesions as a proxy, and growth performance up to 12wk of the pigs in these 16 litters (n=85 SR, n=73 SV, n=158 total). Stress response at weaning (26 +/- 3d) was assessed by measuring serum cortisol at -2, 0, +4d surrounding weaning. Skin lesions were counted at -1, +1, +5d surrounding social mixing at weaning and again at 8wk when pigs were transitioned to grow-finish rooms. Growth performance was assessed using body weight at 8wk and 12wk, as well as ADG calculated during the nursery stage (4wk-8wk) and early grow-finish stage (8wk-12wk). Variables were treated as repeated measures and analyzed using a Gaussian linear mixed model. The base model included dam stress group, day, the interaction between stress group and day, and piglet sex as fixed effects; additionally, each model included individual animal, litter, sire, and farrowing group as random effects. Piglets born to SR sows had a greater response to weaning demonstrated by significantly higher cortisol on the day of weaning compared to piglets born to SV sows (SR: 183 +/- 22nmol/L, SV: 140 +/- 23nmol/L, P< 0.001). Piglets did not differ in cortisol concentration pre-weaning (P=0.439) or 4d post-weaning (P=0.764). At 8wk, pigs born to SR sows did not differ in body weight from those born to SV sows (P=0.843), but they tended to weigh more at 12wk (SR: 43.5 +/- 0.9kg, SV: 41.8 +/- 0.9kg, P=0.085). Pigs did not differ in ADG during the nursery or early grow-finish stages. No significant differences in skin lesions were detected at weaning or 8wk social mixing events. These results suggest that exposure to prenatal social stress and maternal resilience may alter acute stress response but not post-weaning performance.

The pork industry has recognized a need to develop more robust pigs, and one method to achieve this is through the selection of resilient animals. Resilience can be measured using a proxy variable over time. Our previous work has shown serum cortisol to be a promising potential proxy for measuring resilience in crossbred gilts at weaning. In this study, we replicate and expand on our previous work using serum cortisol response to classify resilience of pigs to weaning stress and investigate differences in growth and behavior in purebred Yorkshire gilts. One stress resilient (SR) gilt and one stress vulnerable (SV) gilt was selected from each of 13 litters at weaning (n = 26). Body weights were measured and average daily gain calculated for suckling stage (birth-4 wk-of-age), weaning at 4 wk-of-age, nursery stage (4-8 wk-of-age), transition to grow-finish at 8 wk-of age, early grow-finish (8-12 wk-of-age), and mid grow-finish (12-16 wk-of-age). Skin lesions were recorded before and after weaning as well as at a mixing event at 8 wk-of-age. To assess behavior, we conducted a dyadic contest at 7 wk-of-age, handling tests at 8 wk and 12 wk-of-age, and a novel object test at 12 wk-of-age. We detected no significant differences in overall growth between SR and SV gilts. However, during the first week post-weaning SR gilts gained twice as much per day as SV gilts (SR: 152 ± 31 g/d, SV: 79 ± 31 g/d, P = 0.06). SR gilts also tended to gain more per day during the first week in grow-finish (P = 0.09). This higher performance relative to SV gilts immediately following change may suggest SR gilts adapt faster to a new environment. No significant differences were detected between SR and SV gilts in skin lesions or behavior in the dyadic contest, handling tests, or novel object test. These results are inconsistent with our previous findings from commercial crossbreed pigs and indicate that classification based on serum cortisol pattern is associated with limited differences in Yorkshire gilts.

Abstract Social stress during gestation has been demonstrated to influence piglet health, growth, and behavior. This study aimed to classify pigs exhibiting resilience or vulnerability to gestational social stress using salivary cortisol and characterize differences in their piglets during lactation. At 30 d of gestation, 40 gilts and first parity purebred Yorkshire pigs were socially mixed in group pens (n = 8/pen). Saliva was collected at 1200 h each day at -1, 0, +1, +2, +3, +7d relative to social mixing. Salivary cortisol pattern was used to identify 6 females whose salivary cortisol concentrations returned to baseline as stress resilient (SR) and 6 females whose salivary cortisol concentrations remained increased as stress vulnerable (SV). After farrowing, all piglets of the 12 SR and SV sows had birth weight, weaning weight, and pre-weaning average daily gain (ADG) recorded. At approximately 3 wk of age (18 ± 2d), piglets underwent a novel object test (NOT) by lowering a novel object into each farrowing crate and capturing continuous overhead video recording for 10 min. Videos were manually decoded for each piglet to identify three behavioral measures: latency to touch the object, total time spent interacting with the object, and number of object touches. At 1 d pre-weaning (24 ± 4 d) total skin lesions were recorded for each piglet. All data were analyzed using linear mixed models correcting for sow parity (1 or 2), sex, body weight, age, and litter. Birth weights did not differ between those born to SR or SV sows (SR: 1.3 ± 0.1 kg, SV: 1.3 ± 0.1kg, P = 0.668). However, suckling piglets born to SR sows had significantly greater ADG compared with those born to SV sows (SR: 183 ± 4 g/d, SV: 168 ± 4 g/d, P = 0.010) resulting in significantly greater weaning weights (SR: 5.7 ± 0.1kg, SV: 5.5 ± 0.1kg, P = 6.7e-09). We detected no differences in any of the NOT measures. At the end of lactation, offspring born to SR sows tended to have fewer skin lesions than those born to SV sows (SR: 0.73 ± 0.12, SV: 1.32 ± 0.12, P = 0.065). These results suggest that maternal resilience to gestational social stress is associated with improved suckling pig growth performance and reduced intra-litter aggression.

IntroductionAs pigs are exposed to multiple stressors in production systems, we must understand their ability to be resilient to a range of environmental challenges to maintain production and welfare. Stress-resilience (SR) is the capacity to cope with and recover from stressors while maintaining healthy emotional functioning. In contrast, stress-vulnerability (SV) contributes to and predicts the onset and persistence of mood disorders and pathological processes following exposure to stress.Methods52 focal gilts were identified through a physiological marker (cortisol) in a previous study to compare resilience to weaning stress and behavioral responses at weaning. Within pigs’ home pen, we observed agonistic behavior, non-agonistic social behavior, and daily maintenance behaviors. Behavior was observed over two 4-hour periods (6 AM to 10 AM): one (D1) and four days post-weaning (D4).ResultsOn D1, SV pigs displayed a higher average frequency of non-injurious contact behavior (P = 0.0198) compared to SR pigs, while SR pigs exhibited a significantly longer average duration of lying behavior (P = 0.018) compared to SV. On D4, SV pigs exhibited a significantly longer duration of fighting behavior (P = 0.025) on average compared to SR pigs. Additionally, a significant effect of time on behavioral adaptation patterns was observed. On D1 post-weaning, pigs spent more time fighting (P < 0.001) and exploring (P < 0.001) and showed more frequent non-injurious contact (P = 0.029) and drinking behaviors (P < 0.001) compared to D4. Conversely, on D4, pigs spent more time feeding (P = 0.005) and lying (P < 0.001) compared to D1.DiscussionOur findings imply that non-injurious contact and lying behaviors immediately after weaning and fighting behavior several days later may be promising indicators of pigs’ ability to be resilient to the stress associated with weaning. However, to better understand how pigs change their behavior in response to the stress of weaning, we need standard approaches for measuring their behavior and evaluating the degree of change. Understanding behavioral variation between SR and SV pigs can facilitate the development of resilience indexes that could be helpful in breeding programs, facilitating the selection of resilient pigs that overcome challenges associated with weaning.

Resilience is the capacity of animals to return quickly to their pre-stress status following a disturbance, including social, physical, and/or disease challenges. In modern farming, where there are many stressors, resilience is crucial for pigs because it affects their welfare and production. The goal of the study was to assess whether behavioral response to an auditory stimulus during a startle test (acute stress) differed between pigs designated at weaning (27 +/- 2d of age) as stress-resilient (SR) or stress-vulnerable (SV). Blood samples were collected from female piglets (n = 170) from 26 litters surrounding weaning at multiple time points. Using serum cortisol levels from these samples, two female pigs from each litter (n = 52) were classified as either SR (n = 26) or SV (n = 26) and used for the startle test. The startle test was conducted when pigs were 7wk-of-age while they were housed in the nursery room and recordings were captured utilizing cameras positioned on the ceiling. We assessed startle magnitude score (SM) and time to resume home pen behavior as indicators of the pigs’ behavioral responses. In addition to stress resilience designation, pigs’ relaxed-tense score (RT), orientation to stimulus (OS), and pen position were also observed which may affect startle response. Our data suggest no relationship between behavioral response to an auditory startle test and resilience or vulnerability to weaning stress. The difference in the type of stressors needs to be considered as an acute, simple auditory stimulus was used for the startle test and a longer-lasting, multi-modal weaning stress was used for initial resilience designation. However, the startle test could be a relatively easy way to assess the fearfulness of pigs on farms, as it requires no training of pigs and can be conducted in the home pen, but further methodological improvement is required. Though our results did not support a clear connection between a behavioral fear response and physiological stress resilience, examining the resilience of pigs during their early developmental phases using behavioral markers is worthwhile. Early detection of non-resilient animals could offer the opportunity to implement more effective management strategies beginning earlier in life when pigs might be more vulnerable to stressors or the effects more long-lasting, thereby improving the welfare of pigs. Furthermore, it facilitates the selection of robust animals for future breeding. Further research is required to identify behavioral traits clearly indicative of stress resilience.

Resilience is a key component of an animal's robustness in that failure to adapt to perturbations can result in inappropriate, detrimental responses to the production environment. Methodology used to identify pigs resilient to weaning stress could be applied in identification of pigs resilient to social stress and enable future selection and breeding of pigs, particularly gilts and sows, better suited to a group-housing environment allowing them to achieve their production potential. In this study, we introduced a novel phenotypic classification of resilience to weaning stress in gilt piglets using serum cortisol response pattern and assessed them for persistent behavioral differences during the grow-finish stage of production. At weaning (avg. 28d), 52 focal gilt piglets from 26 litters were designated as stress resilient (SR, n = 26) or stress vulnerable (SV, n = 26). At 8wk-of-age focal gilts were moved from nursery pens to grow-finish pens and mixed into new social cohorts. Skin wounds were counted 1d pre-, 1d post-, and 4d post-mixing. We found that SR gilts engaged in more agonistic interactions than SV gilts 1d post-mixing (p = 0.042), but this difference was not observed pre-mixing or 4d post-mixing. At 12wk-of-age focal gilts underwent a handling test and a novel object test (NOT), with tests spaced at least 2 days apart. No differences were observed in handling test scores or NOT latency measures. However, during NOT, SR gilts touched the object more (p = 0.029) and vocalized less (p = 0.037) compared to SV gilts. Overall, resilience to weaning stress was associated with behavioral differences in finishing gilts at mixing and when faced with novel situations.

Genetic control of carcass and meat quality traits is of great interest to the pork industry, and Hypothalamic-Pituitary-Adrenal (HPA) activation has been reported to have an inverse relationship with lean carcass growth traits in swine. The objective of this study was to determine if transcript abundance of the glucocorticoid receptor (NR3C1) in liver is associated with carcass trait variation. The liver is a key player in energy partitioning towards muscle or adipose accretion, and hepatic glucocorticoid receptors that bind cortisol are known to play a role in glucose metabolism. We hypothesized that NR3C1 expression levels are associated with differences in pork quality and carcass traits. We collected liver tissue from 18 gilts at slaughter (avg. BW =127 kg, age at slaughter = 164 d). Additionally, we collected measurements of carcass traits including dressing percentage, loin muscle area, 10th-rib backfat depth, subjective color and marbling scores, Minolta Colorimeter measures of L*, a*, b*, 45-min and ultimate pH, and driploss (Table 1). We extracted RNA from liver samples using a Qiagen miRNeasy kit and performed qRT-PCR using a TaqMan assay to quantify NR3C1 transcript abundance, with ACTB and HPRT1 as reference genes. Pearson correlation analysis was conducted between the carcass traits and NR3C1 expression. While liver NR3C1 transcript abundance was not significantly correlated with a majority of these traits, it was found to have a significant positive correlation with dressing percentage (r = 0.606, t = 3.05, P = 0.008). In conclusion, transcript abundance of NR3C1 in liver does not appear have a relationship with carcass merit and meat quality, but does have a positive relationship with dressing percentage. Further investigation into tissue-specific expression of NR3C1 may reveal the molecular mechanisms underlying the reported associations between HPA activation and differences in pork quality and carcass traits.

Acute stress before slaughter has been demonstrated to have a detrimental impact on pork quality, but little is known about whether early life stress has a similar effect. Our objective was to identify if stress resiliency or vulnerability at weaning had an impact on long-term performance resulting in differences in growth, carcass, or meat quality traits. At weaning, blood samples were collected from all gilt piglets from 26 crossbred litters (n = 170) at -1 d, 0 d, and +4 d pre- and post-weaning. For each litter, serum cortisol was used to identify the gilt most capable of returning to baseline levels by +4 d as stress resilient (SR, n = 26) and the gilt least capable as stress vulnerable (SV, n = 26). The SR and SV gilts were processed at the Michigan State University Meat Laboratory and carcass and meat quality data were collected. Carcass traits included finished live weight, hot carcass weight, dressing percentage, loin muscle area, and 10th-rib backfat depth. Meat quality traits included 45-min and ultimate pH, marbling score, subjective color score, Minolta colorimeter L*, a* and b*, and drip loss. Data were analyzed using Gaussian linear models. The base model included stress group and farrowing group as fixed effects and age at slaughter as a covariate. The SR gilts showed decreased ADG at the grow-finish stage resulting in lighter finished weight (P = 0.089 and P = 0.070, respectively). The SR gilts also had a lighter hot carcass weight (P = 0.05); however, no differences were observed in dressing percentage ( P= 0.924). the SR and SV gilts did not differ in backfat depth, but SR gilts had smaller loin muscle area (P = 0.019). No differences were observed in any of the meat quality measures. In summary, we found resiliency to weaning stress to have a minimal impact on carcass merit or meat quality. Future studies will aim to determine if the observed difference in loin muscle area is associated with gene expression differences.

Weaning is an acute early-life stressor and therefore, an excellent model for examining lasting impacts of stress resilience. Our objective was to identify gilts exhibiting resilience or vulnerability to weaning stress and characterize long-term impact on behavior. At weaning, blood samples were collected from all gilt piglets of 17 crossbred litters (n=112) at -1d, 0d, and +4d pre- and post-weaning. Serum cortisol concentrations were quantified using a commercial porcine-specific ELISA kit. For each litter, serum cortisol was used to identify the gilt most capable to return to baseline concentrations by +4d as stress resilient (SR, n=17) and the gilt least capable as stress vulnerable (SV, n=17). SR and SV gilts were followed and mixed into new social cohorts at approximately 8 wk-of-age. Skin wounds counts were recorded at -1d, +1d, and +4d surrounding the mix event. At approximately 12 wk-of-age, SR and SV gilts underwent a novel object test (NOT) to assess reactivity to a novel situation. Measures collected during the NOT included latency to cross 1m line from object, latency to cross 0.5m line from object, latency to touch object, total vocalizations, and percent of vocalizations that were high pitched. A paired t-test was conducted to analyze differences between SR and SV gilts. SR gilts had significantly more skin wounds at 1d post-mixing than SV gilts (P=0.007). No significant differences in vocalizations or latency to cross 1m line from object were found between the two groups. However, SR gilts had significantly shorter latency to cross the 0.5m line (P=0.010) and marginally shorter latency to touch the novel object (P=0.088). We found behavioral differences associated with resilience to weaning stress. Gilts resilient to weaning stress engaged in more agonistic behavior at mixing and were bolder when faced with a novel situation.

Background Genetics studies in the porcine immune system have enhanced selection practices for disease resistance phenotypes and increased the efficacy of porcine models in biomedical research; however limited functional annotation of the porcine immunome has hindered progress on both fronts. Among epigenetic mechanisms that regulate gene expression, DNA methylation is the most ubiquitous modification made to the DNA molecule and influences transcription factor binding as well as gene and phenotype expression. Human and mouse DNA methylation studies have improved mapping of regulatory elements in these species, but comparable studies in the pig have been limited in scope. Results We performed whole-genome bisulfite sequencing to assess DNA methylation patterns in nine pig immune cell populations: CD21 + and CD21 − B cells, four T cell fractions (CD4 + , CD8 + , CD8 + CD4 + , and SWC6γδ + ), natural killer and myeloid cells, and neutrophils. We identified 54,391 cell differentially methylated regions (cDMRs), and clustering by cDMR methylation rate grouped samples by cell lineage. 32,737 cDMRs were classified as cell lowly methylated regions (cLMRs) in at least one cell type, and cLMRs were broadly enriched in genes and regions of intermediate CpG density. We observed strong correlations between differential methylation and expression across immune cell populations, with cell-specific low methylation disproportionately impacting genes exhibiting enriched gene expression in the same cell type. Motif analysis of cLMRs revealed cell type-specific enrichment of transcription factor binding motifs, indicating that cell-specific methylation patterns may influence accessibility by trans-acting factors. Lastly, cDMRs were enriched for immune capacity GWAS SNPs, and many such overlaps occurred within genes known to influence immune cell development and function ( CD8B, NDRG1 ). Conclusion Our DNA methylation data improve functional annotation of the porcine genome through characterization of epigenomic regulatory patterns that contribute to immune cell identity and function, and increase the potential for identifying mechanistic links between genotype and phenotype.

Changes to the epigenome, including those to DNA methylation, have been proposed as mechanisms by which stress can induce long-term physiological changes in livestock species. Pig weaning is associated with dietary and social stress, both of which elicit an immune response and changes to the hypothalamic–pituitary–adrenal (HPA) axis. While differential methylation following stress has been assessed in model organisms, it remains poorly understood how the pig methylome is altered by stressors in production settings. We quantified changes in CpG methylation and transcript abundance in piglet peripheral blood mononuclear cells (PBMCs) following weaning and also assessed differential patterns in pigs exhibiting high and low stress response as measured by cortisol concentration and lesion scores. Blood was collected from nine gilt piglets 24 h before and after weaning, and whole-genome bisulfite sequencing (WGBS) and RNA-sequencing were performed on six and nine animals, respectively, at both time points. We identified 2,674 differentially methylated regions (DMRs) that were enriched within promoters of genes associated with lymphocyte stimulation and transcriptional regulation. Stress groups displayed unique differential methylation and expression patterns associated with activation and suppression of T cell immunity in low and high stress animals, respectively. Differential methylation was strongly associated with differential expression; specifically, upregulated genes were enriched among hypomethylated genes. We observed post-weaning hypermethylation of the glucocorticoid receptor (NR3C1) promoter and a significant decrease in NR3C1 expression (n = 9, p = 6.1 × 10–3). Our results indicate that weaning-associated stress elicits genome-wide methylation changes associated with differential gene expression, reduced T cell activation, and an altered HPA axis response.

Determining mechanisms regulating complex traits in pigs is essential to improve the production efficiency of this globally important protein source. MicroRNAs (miRNAs) are a class of non-coding RNAs known to post-transcriptionally regulate gene expression affecting numerous phenotypes, including those important to the pig industry. To facilitate a more comprehensive understanding of the regulatory mechanisms controlling growth, carcass composition, and meat quality phenotypes in pigs, we integrated miRNA and gene expression data from longissimus dorsi muscle samples with genotypic and phenotypic data from the same animals. We identified 23 miRNA expression Quantitative Trait Loci (miR-eQTL) at the genome-wide level and examined their potential effects on these important production phenotypes through miRNA target prediction, correlation, and colocalization analyses. One miR-eQTL miRNA, miR-874, has target genes that colocalize with phenotypic QTL for 12 production traits across the genome including backfat thickness, dressing percentage, muscle pH at 24 h post-mortem, and cook yield. The results of our study reveal genomic regions underlying variation in miRNA expression and identify miRNAs and genes for future validation of their regulatory effects on traits of economic importance to the global pig industry.

Economically important growth and meat quality traits in pigs are controlled by cascading molecular events occurring during development and continuing throughout the conversion of muscle to meat. However, little is known about the genes and molecular mechanisms involved in this process. Evaluating transcriptomic profiles of skeletal muscle during the initial steps leading to the conversion of muscle to meat can identify key regulators of polygenic phenotypes. In addition, mapping transcript abundance through genome-wide association analysis using high-density marker genotypes allows identification of genomic regions that control gene expression, referred to as expression quantitative trait loci (eQTL). In this study, we perform eQTL analyses to identify potential candidate genes and molecular markers regulating growth and meat quality traits in pigs.

Advancements in sequencing technology, improvements in genome annotation, and development of quantitative genetic models have been instrumental to the significant genetic gains achieved in pork production. Several quantitative trait loci (QTL) have been identified for growth, meat quality and carcass composition (GMC) phenotypes, however, the biological mechanisms underlying most QTL remain unknown. Functional genomic analysis can reveal insights on the genetic architecture of complex traits, and transcriptomic profiling of skeletal muscle during the conversion of muscle to meat can identify critical regulators of GMC phenotypes. Gene transcripts obtained with RNA-seq of longissimus muscle from 168 pigs were used to estimate gene expression variation subject to genetic control by mapping expression QTL (eQTL) and allele-specific expression (ASE). A total of 334 eQTL were mapped (FDR≤0.01) and joint association of eQTL with phenotypic QTL (pQTL) segregating in our population revealed 16 genes significantly associated with 21 pQTL for GMC phenotypes. ASE analysis facilitates the identification of cis-acting regulation of transcript abundance. We tested for ASE in 69,502 coding SNP (cSNP) and a total of 18,234 cSNP with significant ASE were identified (FDR≤0.01). A gene-wise conditional analysis fitting all ASE cSNP per gene for each phenotype identified 60 genes associated with GMC phenotypes. A comparison of eQTL with ASE cSNP showed an overlap of 136 genes. Pearson correlations of peak eQTL SNP with ASE cSNP was significant for 51% of these genes. The ASE analysis showed more precision in the identification of cis-acting effects than the genome-wide eQTL analysis; however, both approaches provide valuable information on the regulation of transcript abundance. For instance, we observed 24 genes associated with distant eQTL (trans effects) and exhibiting ASE. This study provides new information on the complex regulation of the pig longissimus muscle transcriptome and associations with measurable differences in economically important phenotypic traits.

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.

MicroRNAs (miRNAs) are a class of noncoding RNAs known to post-transcriptionally regulate gene expression through binding with target mRNAs, ultimately affecting a multitude of biological processes and phenotypes. It has been documented that miRNAs influence skeletal muscle development; however lack of miRNA annotation in pigs hinders understanding of molecular mechanisms underlying this process. We sought to identify novel miRNAs in fetal longissimus dorsi (LD) muscle and compare expression of these miRNAs at 41 days gestation (dg) and 70 dg (n=3 per stage), representing primary and secondary fetal myogenesis. Total RNA was isolated from LD samples of fetuses obtained from Yorkshire x Landrace gilts. Small-RNA sequencing was performed on the Illumina HiSeq 4000 platform, generating 30-60 million 1x50 reads per sample. High-quality reads were aligned to the S. scrofa reference genome (v11.1), and mapping and prediction of novel miRNAs was performed using miRDeep2. Predicted miRNAs with significant randfold p-values, miRDeep2 scores >7, and total read counts per million ≥1 for each sample were retained. Annotated human miRNAs with ≤2 mismatches with common and stage-specific novel miRNAs were found using miRBase. Differential expression analysis was performed on novel miRNAs using DESeq2. TargetScan was used to find conserved targets of human miRNAs with sequence identity to differentially expressed (DE) pig miRNAs. At 41 dg and 70 dg, 83 and 73 novel miRNAs were predicted in at least two samples, respectively. Of these, 59 were common to both stages. We identified 10 DE miRNAs (|log2 fold change|>1 and adj.p<0.05), nine of which were downregulated and one upregulated. One novel DE miRNA had 95% identity (1 mismatch) with a known pig miRNA (ssc-miR-26a). miR-26a has been found to play a major role in repression of myogenesis through the TGF-β/BMP signaling pathway in mice. In addition, six DE miRNAs had ≤2 mismatches with the known human miRNAs: miR-188-5p, miR-200ab-5p, miR-3194-5p, miR-33a-5p, miR-34b-5p, miR-93-5p. miR-34b has been shown to play a role in muscle cell differentiation during development in C2C12 mouse myoblast cells. Targets of DE miRNAs were enriched for Gene Ontology terms and KEGG pathways related to skeletal muscle development including axon guidance, regulation of actin cytoskeleton, Wnt signaling pathway, and cadherin binding involved in cell-cell adhesion. This study identified novel pig miRNAs with putative roles in myogenesis as supported by research in model organisms. Future efforts will analyze specific gene targets and their roles in skeletal muscle development.

The leptin receptor (LEPR) is a type I cytokine receptor that binds the leptin protein known to regulate food intake and energy metabolism, and is a functional candidate gene for regulating economically important phenotypic traits in pigs. The objective of our research is to determine the effect of a single nucleotide polymorphism (SNP) in exon 14 of the LEPR gene on adipose tissue LEPR transcript abundance and carcass composition phenotypes in the Michigan State University Pig Resource Population. This population is a F2 cross between Duroc and Pietrain breeds. A total of 51 female pigs were selected based on LEPR exon 14 SNP genotypes (n=17 per CC, CT, and TT genotype). Total RNA was isolated from subcutaneous fat tissue samples and analyzed using real-time RT-PCR to obtain LEPR expression levels, with SDHA and ACTB as controls. A conditional analysis was performed using Genomic Best Linear Unbiased Prediction with the LEPR exon 14 genotypes as fixed effects, and LEPR gene expression and carcass composition phenotypes as response variables. A significant dominance effect was identified with the TT genotype showing significantly lower expression than the CC and CT genotypes (p=1.20e-02 and 4.05e-02, respectively). The presence of a C allele was associated with significantly higher LEPR expression than the T allele (p=8.61e-03). The heritability of LEPR gene expression was found to be moderate at 0.29. However, the proportion of variance explained by the LEPR SNP was 12% of the total gene expression variance. Conditional analysis for carcass composition phenotypes identified significant dominance effects for eight phenotypic traits including marbling, carcass loin muscle area, ham and loin weight and four backfat thickness measures (p≤0.05). The T allele was significantly associated with decreased backfat thickness and increased carcass loin muscle area, and ham and loin weight. A genome-wide association analysis identified two quantitative trait loci (QTL) for tenth-rib and last-lumbar backfat phenotypes on SSC6. The heritability of these traits were moderate at 0.43. The conditional analysis for these two backfat phenotypes completely removed the significance of the QTLs on SSC6, accounting for up to 8% of the phenotypic variance. This study highlights the LEPR exon 14 SNP as a candidate marker regulating variation in LEPR transcript abundance, and backfat and muscle phenotypes, with the C allele associated with increased LEPR gene expression and backfat thickness, and decreased muscle mass.

Characterizing the temporal expression patterns of genes throughout fetal myogenesis and postnatal skeletal muscle hypertrophy in pigs is critical to obtain a better understanding of the genes involved in these important processes. Previous transcriptome-wide expression profiling studies utilizing the 70-mer Pigoligoarray microarray revealed many genes exhibiting dynamic expression during fetal and postnatal development. The objective of this study was to confirm expression profiles for 12 genes in pig Longissimus dorsi (LD) skeletal muscle at seven developmental stages: 57, 70, and 105 days of gestation (dg), birth, and 1, 3, and 5 weeks postnatal. Total RNA was extracted from LD samples of male and female fetuses (n≈3 per sex per stage) obtained from Yorkshire x Landrace gilts. Expression profiles for each gene at each stage were determined using real-time RT-qPCR assays with TaqMan chemistry. Target genes were ATF4, ATXN10, BTC, CACYBP, CYTH2, DCN, DLK1, FST, MYOZ1, NRAP, USP13, and WRAP73, along with PPIA, HPRT1, and RPS18 for normalization. The relative expression of each gene (∆Cts) was used as the response variable in a linear model including fixed effects of sex, age, and sex by age interaction. Results of this linear model were analyzed with ANOVA to assess the significance of each effect. Tukey-Kramer adjustments were used to conduct pairwise comparisons between developmental stages for each gene. A significant effect of age was observed on the expression of all target genes except for ATXN10 (p-value range: 5.89e-14 to 0.007). Six genes (ATF4, BTC, CACYBP, MYOZ1, NRAP, and USP13) increased in expression from 57 dg to 5 wk postnatal, while DLK1 decreased over time. Four genes (CYTH2, DCN, FST, and WRAP73) exhibited variable expression throughout development. These expression patterns were consistent with previously observed patterns of expression obtained from microarray experiments. These genes have been shown in other species to be involved in multiple biological processes including: metabolic disease, lipid metabolism, and molecular transport (CACYBP, DLK1, USP13, and WRAP73); organ morphology, skeletal and muscular system development and function, and skeletal and muscular disorders (BTC, DCN, MYOZ1, and NRAP); and cell signaling, DNA replication, recombination and repair, and nucleic acid metabolism (CYTH2 and FST). Further research into temporal gene expression patterns will enhance our understanding of the regulation of muscle development in pigs.