Clustering of multi-tissue transcriptomes in gilts with normal cyclicity or delayed puberty reveals genes related to pubertal development†.

In gilts, puberty is marked by standing estrus in the presence of a boar. Delayed puberty (DP; failure to display pubertal estrus) is a major reason for gilt removal. To investigate the physiological determinants underlying DP in gilts, transcriptomic data from tissues relevant to estrus and puberty, such as mediobasal hypothalamus, anterior pituitary gland, ovarian cortex, olfactory bulb, amygdala, and hippocampus, were obtained from age-matched DP (n = 8) and cyclic control gilts at follicular phase (n = 8) and luteal phase (n = 8) of the estrous cycle. A gene expression module analysis via three-way gene × individual × tissue clustering using tensor decomposition identified pituitary and ovary gene modules contributing to regulation of pubertal development. Analysis of gene expression in the hypothalamic-pituitary-ovary axis identified reduced expression of hypothalamic genes critical for stimulating gonadotropin secretion (KISS1 and TAC3) and reduced expression of LHB in the anterior pituitary of DP gilts compared with their cyclic counterparts. Consequently, luteinizing hormone-induced genes in the ovary important for folliculogenesis (OXTR, RUNX2, and PTX3) were less expressed in DP gilts. Other intrafollicular genes (AHR, PTGS2, PTGFR, and IGFBP7) and genes in the steroidogenesis pathways (STAR and CYP11A1) necessary to complete the ovulatory cascade were also less expressed in DP gilts. This is the first clustering of multi-tissue expression data from DP and cyclic gilts to identify genes differentially expressed in gilts of similar ages but at different levels of sexual development. A critical lack of gonadotropin support and reduced ovarian responsiveness underlie DP in gilts.

Functional genomics of reproduction in pigs: Are we there yet?

Reproductive failure is the main reason for culling females in swine herds and is both a financial and sustainability issue. Because reproductive traits are complex and lowly to moderately heritable, genomic selection within populations can achieve substantial genetic gain in reproductive efficiency. A better understanding of the physiological components affecting the expression of these traits will facilitate greater understanding of the genes affecting reproductive traits and is necessary to improve and optimize management strategies to maximize reproductive success of gilts and sows. Large-scale genotyping with single-nucleotide polymorphism (SNP) arrays are used for genome-wide association studies (GWAS) and have facilitated identification of positional candidate genes. Transcriptomic data can be used to weight SNP for GWAS and could lead to previously unidentified candidate genes. Resequencing and fine mapping of candidate genes are necessary to identify putative functional variants and some of these have been incorporated into new genotyping arrays. Sequence imputation and genotype by sequence are newer strategies that could reveal novel functional mutations. In this study, these approaches are discussed. Advantages and limitations are highlighted where additional research is needed.

Potential functional variants in AHR signaling pathways are associated with age at puberty in swine.

Puberty in female pigs is defined as age at first estrus and gilts that have an earlier age at puberty are more likely to have greater lifetime productivity. Because age at puberty is predictive for sow longevity and lifetime productivity, but not routinely measured in commercial herds, it would be beneficial to use genomic or marker-assisted selection to improve these traits. A GWAS at the US Meat Animal Research Center (USMARC) identified several loci associated with age at puberty in pigs. Candidate genes in these regions were scanned for potential functional variants using sequence information from the USMARC swine population founder animals and public databases. In total, 135 variants (SNP and insertion/deletions) in 39 genes were genotyped in 1284 phenotyped animals from a validation population sired by Landrace and Yorkshire industry semen using the Agena MassArray system. Twelve variants in eight genes were associated with age at puberty (P < 0.005) with estimated additive SNP effects ranging from 1.6 to 5.3 days. Nine of these variants were non-synonymous coding changes in AHR, CYP1A2, OR2M4, SDCCAG8, TBC1D1 and ZNF608, two variants were deletions of one and four codons in aryl hydrocarbon receptor, AHR, and the most significant SNP was near an acceptor splice site in the acetyl-CoA carboxylase alpha, ACACA. Several of the loci identified have a physiological and a genetic role in sexual maturation in humans and other animals and are involved in AHR-mediated pathways. Further functional validation of these variants could identify causative mutations that influence age at puberty in gilts and possibly sow lifetime productivity.

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

A high-quality annotated transcriptome of swine peripheral blood.

BACKGROUND: High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes and/or transcriptomes. However, neither the reference genome nor the peripheral blood transcriptome of the pig have been sufficiently assembled and annotated to support such profiling assays in this emerging biomedical model organism. We aimed to assemble published and novel RNA-seq data to provide a comprehensive, well-annotated blood transcriptome for pigs by integrating a de novo assembly with a genome-guided assembly. RESULTS: A de novo and a genome-guided transcriptome of porcine whole peripheral blood was assembled with ~162 million pairs of paired-end and ~183 million single-end, trimmed and normalized Illumina RNA-seq reads (~6 billion initial reads from 146 RNA-seq libraries) from five independent studies by using the Trinity and Cufflinks software, respectively. We then removed putative transcripts (PTs) of low confidence from both assemblies and merged the remaining PTs into an integrated transcriptome consisting of 132,928 PTs, with 126,225 (~95%) PTs from the de novo assembly and more than 91% of PTs spliced. In the integrated transcriptome, ~90% and 63% of PTs had significant sequence similarity to sequences in the NCBI NT and NR databases, respectively; 68,754 (~52%) PTs were annotated with 15,965 unique gene ontology (GO) terms; and 7618 PTs annotated with Enzyme Commission codes were assigned to 134 pathways curated by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Full exon-intron junctions of 17,528 PTs were validated by PacBio IsoSeq full-length cDNA reads from 3 other porcine tissues, NCBI pig RefSeq mRNAs and transcripts from Ensembl Sscrofa10.2 annotation. Completeness of the 5' termini of 37,569 PTs was validated by public cap analysis of gene expression (CAGE) data. By comparison to the Ensembl transcripts, we found that (1) the deduced precursors of 54,402 PTs shared at least one intron or exon with those of 18,437 Ensembl transcripts; (2) 12,262 PTs had both longer 5' and 3' termini than their maximally overlapping Ensembl transcripts; and (3) 41,838 spliced PTs were totally missing from the Sscrofa10.2 annotation. Similar results were obtained when the PTs were compared to the pig NCBI RefSeq mRNA collection. CONCLUSIONS: We built, validated and annotated a comprehensive porcine blood transcriptome with significant improvement over the annotation of Ensembl Sscrofa10.2 and the pig NCBI RefSeq mRNAs, and laid a foundation for blood-based high throughput transcriptomic assays in pigs and for advancing annotation of the pig genome.

Genetic analysis of teat number in pigs reveals some developmental pathways independent of vertebra number and several loci which only affect a specific side.

BACKGROUND: Number of functional teats is an important trait in commercial swine production. As litter size increases, the number of teats must also increase to supply nutrition to all piglets. Therefore, a genome-wide association analysis was conducted to identify genomic regions that affect this trait in a commercial swine population. Genotypic data from the Illumina Porcine SNP60v1 BeadChip were available for 2951 animals with total teat number (TTN) records. A subset of these animals (n = 1828) had number of teats on each side recorded. From this information, the following traits were derived: number of teats on the left (LTN) and right side (RTN), maximum number of teats on a side (MAX), difference between LTN and RTN (L - R) and absolute value of L - R (DIF). Bayes C option of GENSEL (version 4.61) and 1-Mb windows were implemented. Identified regions that explained more than 1.5% of the genomic variation were tested in a larger group of animals (n = 5453) to estimate additive genetic effects. RESULTS: Marker heritabilities were highest for TTN (0.233), intermediate for individual side counts (0.088 to 0.115) and virtually nil for difference traits (0.002 for L - R and 0.006 for DIF). Each copy of the VRTN mutant allele increased teat count by 0.35 (TTN), 0.16 (LTN and RTN) and 0.19 (MAX). 15, 18, 13 and 18 one-Mb windows were detected that explained more than 1.0% of the genomic variation for TTN, LTN, RTN, and MAX, respectively. These regions cumulatively accounted for over 50% of the genomic variation of LTN, RTN and MAX, but only 30% of that of TTN. Sus scrofa chromosome SSC10:52 Mb was associated with all four count traits, while SSC10:60 and SSC14:54 Mb were associated with three count traits. Thirty-three SNPs accounted for nearly 39% of the additive genetic variation in the validation dataset. No effect of piglet sex or percentage of males in litter was detected, but birth weight was positively correlated with TTN. CONCLUSIONS: Teat number is a heritable trait and use of genetic markers would expedite selection progress. Exploiting genetic variation associated with teat counts on each side would enhance selection focused on total teat counts. These results confirm QTL on SSC4, seven and ten and identify a novel QTL on SSC14.

Polymorphism in the intron 20 of porcine O-linked N-acetylglucosamine transferase.

OBJECTIVE: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone level and testes size. The objective of this study was to find the variations of OGT between European and Chinese pigs. METHODS: Pigs were tested initially for polymorphism in OGT among European and Chinese pigs by polymerase chain reaction and sequencing at the U.S. Meat Animal Research Center (USMARC). The polymorphism was also determined in an independent population of pigs including European and Chinese Meishan (ME) breeds at the National Institute of Animal Science (NIAS, RDA, Korea). RESULTS: The intron 20 of OGT from European and Chinese pigs was 514 and 233 bp, respectively, in the pigs tested initially. They included 1 White composite (WC) boar and 7 sows (2 Minzu×WC, 2 Duroc [DU]×WC, 2 ME×WC, 1 Fengzing×WC) at USMARC. The 281-bp difference was due to an inserted 276-bp element and GACTT in European pigs. When additional WC and ME boars, the grandparents that were used to generate the 1/2ME×1/2WC parents, and the 84 boars of 16 litters from mating of 1/2ME×1/2WC parents were analyzed, the breeds of origin of X chromosome quantitative trait locus (QTL) were confirmed. The polymorphism was determined in an independent population of pigs including DU, Landrace, Yorkshire, and ME breeds at NIAS. OGT was placed at position 67 cM on the chromosome X of the USMARC swine linkage map. CONCLUSION: There was complete concordance with the insertion in European pigs at USMARC and NIAS. This polymorphism could be a useful marker to identify the breed of origin of X chromosome QTL in pigs produced by crossbreeding Chinese and European pigs.

Genome-wide association and identification of candidate genes for age at puberty in swine.

BACKGROUND: Reproductive efficiency has a great impact on the economic success of pork production. Gilts comprise a significant portion of breeding females and gilts that reach puberty earlier tend to stay in the herd longer and be more productive. About 10 to 30% of gilts never farrow a litter and the most common reasons for removal are anestrus and failure to conceive. Puberty in pigs is usually defined as the female's first estrus in the presence of boar stimulation. Genetic markers associated with age at puberty will allow for selection on age at puberty and traits correlated with sow lifetime productivity. RESULTS: Gilts (n = 759) with estrus detection measurements ranging from 140-240 days were genotyped using the Illumina PorcineSNP60 BeadChip and SNP were tested for significant effects with a Bayesian approach using GENSEL software. Of the available 8111 five-marker windows, 27 were found to be statistically significant with a comparison-wise error of P < 0.01. Ten QTL were highly significant at P < 0.005 level. Two QTL, one on SSC12 at 15 Mb and the other on SSC7 at 75 Mb, explained 16.87% of the total genetic variance. The most compelling candidate genes in these two regions included the growth hormone gene (GH1) on SSC12 and PRKD1 on SSC7. Several loci confirmed associations previously identified for age at puberty in the pig and loci for age at menarche in humans. CONCLUSIONS: Several of the loci identified in this study have a physiological role for the onset of puberty and a genetic basis for sexual maturation in humans. Understanding the genes involved in regulation of the onset of puberty would allow for the improvement of reproductive efficiency in swine. Because age at puberty is a predictive factor for sow longevity and lifetime productivity, but not routinely measured or selected for in commercial herds, it would be beneficial to be able to use genomic or marker-assisted selection to improve these traits.

Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle.

The purpose of this investigation was to determine the extent to which dystrophin insufficiency caused histomorphological changes in a novel pig model of Becker muscular dystrophy. In our procedures, we used a combination of biochemical approaches, including quantitative PCR and Western blots, along with a histological analysis using standard and immunohistological measures. We found that 8-wk-old male affected pigs had a 70% reduction in dystrophin protein abundance in the diaphragm, psoas major, and longissimus lumborum and a 5-fold increase in serum creatine kinase activity compared with healthy male littermates. Dystrophin insufficiency in the diaphragm and the longissimus resulted in muscle histopathology with disorganized fibrosis that often colocalized with fatty infiltration but not the psoas. Affected animals also had an 80-85% reduction in α-sarcoglycan localization in these muscles, indicating compromised assembly of the dystrophin glycoprotein complex. Controls used in this study were 4 healthy male littermates, as they are most closely related to the affected animals. We concluded that pigs with insufficient dystrophin protein expression have a phenotype consistent with human dystrophinopathy patients. Given that and their similarity in body size and physiology to humans, we further conclude that this pig line is an appropriate translational model for dystrophinopathies.

A study of vertebra number in pigs confirms the association of vertnin and reveals additional QTL.

BACKGROUND: Formation of the vertebral column is a critical developmental stage in mammals. The strict control of this process has resulted in little variation in number of vertebrae across mammalian species and no variation within most mammalian species. The pig is quite unique as considerable variation exists in number of thoracic vertebrae as well as number of lumbar vertebrae. At least two genes have been identified that affect number of vertebrae in pigs yet considerable genetic variation still exists. Therefore, a genome-wide association (GWA) analysis was conducted to identify additional genomic regions that affect this trait. RESULTS: A total of 1883 animals were phenotyped for the number of ribs and thoracolumbar vertebrae as well as successfully genotyped with the Illumina Porcine SNP60 BeadChip. After data editing, 41,148 SNP markers were included in the GWA analysis. These animals were also phenotyped for kyphosis. Fifty-three 1 Mb windows each explained at least 1.0 % of the genomic variation for vertebrae counts while 16 regions were significant for kyphosis. Vertnin genotype significantly affected vertebral counts as well. The region with the largest effect for number of lumbar vertebrae and thoracolumbar vertebrae were located over the Hox B gene cluster and the largest association for thoracic vertebrae number was over the Hox A gene cluster. Genetic markers in significant regions accounted for approximately 50% of the genomic variation. Less genomic variation for kyphosis was described by QTL regions and no region was associated with kyphosis and vertebra counts. CONCLUSIONS: The importance of the Hox gene families in vertebral development was highlighted as significant associations were detected over the A, B and C families. Further evaluation of these regions and characterization of variants within these genes will expand our knowledge on vertebral development using natural genetic variants segregating in commercial swine.

Incorporation of genetic technologies associated with applied reproductive technologies to enhance world food production.

Animal breeding and reproductive physiology have been closely related throughout the history of animal production science. Artificial insemination provides the best method of increasing the influence of sires with superior genetics to improve production traits. Multiple ovulation embryo transfer (MOET) provides some ability to increase the genetic influence of the maternal line as well. The addition of genetic technologies to this paradigm allows for improved methods of selecting sires and dams carrying the best genes for production and yield of edible products and resistance to diseases and parasites. However, decreasing the number of influential parents within a population also increases the risk of propagating a recessive gene that could negatively impact the species (Reprod Domest Anim 44:792-796, 2009; BMC Genomics 11:337, 2010). Furthermore, antagonistic genotypic relationships between production traits and fertility (Anim Prod Sci 49:399-412, 2009; Anim Genet 43:442-446, 2012) suggest that care must be taken to ensure that increasing the frequency of genes with a positive influence on production does not negatively impact the fertility of the replacement females entering the herd.

Analysis of copy number variations among diverse cattle breeds.

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

Relationships of genomic estimated breeding values for age at puberty, birth weight, and growth during development in normal cyclic and acyclic gilts.

Managing replacement gilts to reach optimal body weight and growth rate for boar stimulation and first breeding is a key component for sow reproductive longevity and producer profitability. Failure to display pubertal estrus remains a major reason that gilts are culled from the herd. Puberty is metabolically gated so evaluating phenotypic and genetic relationships between birth weight and growth traits with age at puberty and acyclicity can provide valuable insight for efficient gilt development. Data on a litter of origin of the gilt, average daily gain at different stages of development, and age at puberty were available for age-matched cyclic (n = 4,861) and acyclic gilts (prepubertal anestrus, n = 578; behavioral anestrus, n = 428). Genomic estimated breeding values were predicted for each trait using genomic best linear unbiased prediction. Primiparous sows produced more acyclic gilts than multiparous sows (P < 0.05). Accounting for effects of parity and litter size, prepubertal anestrus gilts were heavier at birth and behaviorally anestrus gilts grew faster during the finisher period compared to cyclic gilts (P < 0.05), reflecting possible prenatal programming that negatively affects optimal pubertal development and antagonistic effects between adolescent growth and expression of estrus of gilts from first parity sows. Regression of phenotypic age at puberty with lifetime growth rate (birth to selection) showed a negative linear relationship whereas genomic estimated breeding values showed a negative quadratic relationship indicating that gilts with the least and greatest growth are less optimal as replacements. The slopes of these relationships are small with low negative phenotypic (r = -0.06) and genetic correlations (r = -0.13). The addition of data from acyclic gilts did not substantially change the estimates for genetic relationships between growth and pubertal onset. Although this study identified differences in birth weight and growth rate between cyclic and acyclic gilts the genetic relationships are weak, suggesting that genetic selection for these traits can be achieved separately. Avoiding the smallest and largest gilts in a cohort born to first parity sows could result in gilts with optimal development and reduce the proportion of replacement gilts that are acyclic.

Failure to display pubertal estrus is major reason replacement gilts are culled from the herd. Two types of prebreeding estrus failure are delay in attaining puberty due to sexual immaturity known as prepubertal anestrus (PPA) and silent ovulation without signs of estrus known as behavioral anestrus (BA). For efficient gilt development, it is important to understand what contributes to these acyclic phenotypes. Comparison of birth weight and growth rate in age-matched cyclic, PPA, and BA gilts showed that PPA gilts were heavier at birth and BA gilts grew faster during the finisher period, reflecting negative effects of larger birth weight and faster growth on sexual maturity and behavioral estrus. The genetic relationship between growth and puberty onset indicated that gilts with the least and greatest growth rates are less optimal as replacements. A selection criterion to avoid the smallest and largest gilts in a cohort could result in gilts with optimal development for boar stimulation and reduce the proportion of acyclic gilts. This management strategy would be most effective if targeted to first parity sows.

gBLUP-GWAS identifies candidate genes, signaling pathways, and putative functional polymorphisms for age at puberty in gilts.

Successful development of replacement gilts determines their reproductive longevity and lifetime productivity. Selection for reproductive longevity is challenging due to low heritability and expression late in life. In pigs, age at puberty is the earliest known indicator for reproductive longevity and gilts that reach puberty earlier have a greater probability of producing more lifetime litters. Failure of gilts to reach puberty and display a pubertal estrus is a major reason for early removal of replacement gilts. To identify genomic sources of variation in age at puberty for improving genetic selection for early age at puberty and related traits, gilts (n = 4,986) from a multigeneration population representing commercially available maternal genetic lines were used for a genomic best linear unbiased prediction-based genome-wide association. Twenty-one genome-wide significant single nucleotide polymorphisms (SNP) located on Sus scrofa chromosomes (SSC) 1, 2, 9, and 14 were identified with additive effects ranging from -1.61 to 1.92 d (P < 0.0001 to 0.0671). Novel candidate genes and signaling pathways were identified for age at puberty. The locus on SSC9 (83.7 to 86.7 Mb) was characterized by long range linkage disequilibrium and harbors the AHR transcription factor gene. A second candidate gene on SSC2 (82.7 Mb), ANKRA2, is a corepressor for AHR, suggesting a possible involvement of AHR signaling in regulating pubertal onset in pigs. Putative functional SNP associated with age at puberty in the AHR and ANKRA2 genes were identified. Combined analysis of these SNP showed that an increase in the number of favorable alleles reduced pubertal age by 5.84 ± 1.65 d (P < 0.001). Candidate genes for age at puberty showed pleiotropic effects with other fertility functions such as gonadotropin secretion (FOXD1), follicular development (BMP4), pregnancy (LIF), and litter size (MEF2C). Several candidate genes and signaling pathways identified in this study play a physiological role in the hypothalamic-pituitary-gonadal axis and mechanisms permitting puberty onset. Variants located in or near these genes require further characterization to identify their impact on pubertal onset in gilts. Because age at puberty is an indicator of future reproductive success, these SNP are expected to improve genomic predictions for component traits of sow fertility and lifetime productivity expressed later in life.

Selecting for replacement gilts is challenging because sow reproductive traits are lowly heritable and expressed late in life. Age at puberty is the earliest indicator of future reproductive success of gilts. Genetic selection for early onset of puberty could be feasible with the availability of molecular genetic predictors for age at puberty. To identify genomic sources associated with variation in age at puberty in gilts, a large-scale genome-wide association study was conducted at the U.S Meat Animal Research Center, Clay Center, Nebraska. Novel genomic associations for age at puberty were identified. Several candidate genes identified for age at puberty are involved in signaling pathways that regulate ovarian functions and pubertal onset. Potential causative genetic variants for age at puberty were identified within the candidate genes. These novel SNP are important new markers for use in genomic selection of replacement gilts with early puberty and provide critical new insight into biological mechanisms important for pubertal development in gilts.

A defect in dystrophin causes a novel porcine stress syndrome.

BACKGROUND: Losses of slaughter-weight pigs due to transport stress are both welfare and economic concerns to pork producers. Historically, the HAL-1843 mutation in ryanodine receptor 1 was considered responsible for most of the losses; however, DNA testing has effectively eliminated this mutation from commercial herds. We identified two sibling barrows in the USMARC swine herd that died from apparent symptoms of a stress syndrome after transport at 12 weeks of age. The symptoms included open-mouth breathing, skin discoloration, vocalization and loss of mobility. RESULTS: We repeated the original mating along with sire-daughter matings to produce additional offspring. At 8 weeks of age, heart rate and electrocardiographs (ECG) were monitored during isoflurane anesthesia challenge (3% for 3 min). Four males from the original sire-dam mating and two males from a sire-daughter mating died after one minute of anesthesia. Animals from additional litters were identified as having a stress response, sometimes resulting in death, during regular processing and weighing. Affected animals had elevated plasma creatine phosphokinase (CPK) levels before and immediately after isoflurane challenge and cardiac arrhythmias. A pedigree containing 250 pigs, including 49 affected animals, was genotyped with the Illumina PorcineSNP60 Beadchip and only one chromosomal region, SSCX at 25.1-27.7 Mb over the dystrophin gene (DMD), was significantly associated with the syndrome. An arginine to tryptophan (R1958W) polymorphism in exon 41 of DMD was the most significant marker associated with stress susceptibility. Immunoblots of affected heart and skeletal muscle showed a dramatic reduction of dystrophin protein and histopathology of affected hearts indicated muscle fiber degeneration. CONCLUSIONS: A novel stress syndrome was characterized in pigs and the causative genetic factor most likely resides within DMD that results in less dystrophin protein and cardiac abnormalities that can lead to death under stressful conditions. The identification of predictive markers will allow us to determine the prevalence of this disease in commercial swine populations. This defect also provides a unique biomedical model for human cardiomyopathy associated with muscular dystrophy that may be superior to those available because of the similarities in anatomy and physiology and allow advances in gene therapies for human disease.

Gene expression in the amygdala and hippocampus of cyclic and acyclic gilts.

Age at first estrus is the earliest phenotypic indicator of future reproductive success of gilts. Prebreeding anestrus is a major reason for reproductive failure leading to culling of replacement gilts. The two types of prebreeding anestrus are delay in attaining puberty (prepubertal anestrus, PPA) and silent ovulation (behavioral anestrus, BA). Neural tissues such as amygdala and hippocampus play a major role in regulating sexual behavior, social interactions, and receptivity to males. Differences in gene expression in the amygdala and hippocampus of gilts were analyzed in three comparisons: 1) PPA cases and cyclic controls at follicular phase of estrous cycle, 2) BA cases and cyclic controls at luteal phase of estrous cycle, and 3) gilts at different stages of the ovarian cycle (cyclic gilts at follicular phase and luteal phase of estrous cycle) to gain functional understanding of how these rarely studied tissues may differ between pubertal phenotypes and different stages of the estrous cycle of gilts. Differentially expressed genes (DEG) between PPA and BA cases and their respective cyclic controls were involved in neurological and behavioral disorders as well as nervous system functions that could directly or indirectly involved in development of behaviors related to estrus. The comparison between cyclic follicular and luteal phase control gilts identified the greatest number of DEG in the hippocampus and amygdala. These DEG were involved in adult neurogenesis and neural synapse (e.g., GABAergic, dopamine, cholinergic), suggesting that these tissues undergo structural changes and synaptic plasticity in gilts. This is the first report to demonstrate that the stage of estrous cycle is associated with dynamic changes in gene expression within porcine hippocampus and amygdala and indicates a role of gonadal steroids in regulating their biology.

Dam parity structure and body condition during lactation influence piglet growth and gilt sexual maturation through pre-finishing.

Energy demands during lactation greatly influence sow body condition and piglet performance. We hypothesized that primiparous sows or sows with reduced body condition would produce piglets with reduced growth and delayed sexual maturation. Eight weekly farrowing seasons were used to evaluate sow body condition (post-farrowing, PF and weaning, WN) and piglet growth from 157 dams. Body condition was measured at PF and WN using sow calipers (last rib and hip) and 10th rib ultrasound. Sows were categorized as thin, moderate, or fat by caliper (PF or WN). Individual pig weights were recorded on approximately 1, 10, WN, 45, 100, and 145 d of age. At 100 and 145 d of age, 10th-rib backfat and loin eye area were measured on 567 pigs and first estrus was monitored in 176 gilts reserved for breeding selection beginning at approximately 170 d of age. Sows had similar (P > 0.10) PF last rib caliper measurements but at WN, first parity sows had the smallest caliper measurements compared to other parities (P < 0.05). Parities 1, 2, and 3 sows had similar (P > 0.10) loin eye area at PF; however, at WN first parity sows had the smallest loin eye area (P < 0.05; 38.2 ± 0.63 cm2). Parity 1 sows had the greatest (P < 0.05) reduction of backfat and loin eye area over the lactation period (-2.9 ± 0.31 mm and -2.6 ± 0.49 cm2, respectively). At 1 d of age and WN, piglets from first parity sows weighed the least (P < 0.05) but were the heaviest (P < 0.05) at 100 and 145 d of age. Pigs from first parity litters had larger (P < 0.05) loin eye area at 100 and 145 d of age and greater backfat (P < 0.05) at 145 d of age. Fat sows at WN (last rib or hip) had the lightest (P < 0.05) piglets at 10 d of age and WN. However, at 45 d of age, piglets from fat sows (last rib or hip) were heavier (P < 0.05) than piglets from moderate and thin sows. Tenth rib backfat at 100 and 145 d of age tended (P < 0.10) to be less in pigs reared by thin sows (PF and WN hip). Tenth rib loin eye area was similar among pigs reared by fat, moderate, or thin sows. Gilts developed in litters from fourth parity sows had (P < 0.05) delayed age at puberty in contrast to gilts from first or third parity sows (200.9 ± 4.96 d vs. 189.0 ± 2.29 d and 187.5 ± 2.84 d, respectively). Although progeny body weights were typically less from first parity dams through 45 d of age, these progeny were similar or heavier at 100 and 145 d of age in contrast to progeny from other parities. Furthermore, gilt progeny from first parity dams did not have delayed pubertal attainment.

Young female swine have a greater challenge successfully producing and raising a litter of piglets as they are still maturing themselves and nursing is an extremely energy demanding event. Piglet growth during the nursing phase can have extended impact on growth and development later in life. Piglets raised by young first-time mothers were smaller at birth and weaning but grew to similar weight and body composition later in life as their contemporaries raised by older more mature mothers. Young female pigs raised by first-time mothers had similar or better sexual maturity than counterparts raised by mature mothers. These findings indicate that piglets reared by first time mothering dams will not have detrimental effects on maturity and reproductive parameters. Producers can confidently select females that were reared by first-time mothers for the breeding herd without sacrificing quality.

Genomic regions associated with kyphosis in swine.

BACKGROUND: A back curvature defect similar to kyphosis in humans has been observed in swine herds. The defect ranges from mild to severe curvature of the thoracic vertebrate in split carcasses and has an estimated heritability of 0.3. The objective of this study was to identify genomic regions that affect this trait. RESULTS: Single nucleotide polymorphism (SNP) associations performed with 198 SNPs and microsatellite markers in a Duroc-Landrace-Yorkshire resource population (U.S. Meat Animal Research Center, USMARC resource population) of swine provided regions of association with this trait on 15 chromosomes. Positional candidate genes, especially those involved in human skeletal development pathways, were selected for SNP identification. SNPs in 16 candidate genes were genotyped in an F2 population (n = 371) and the USMARC resource herd (n = 1,257) with kyphosis scores. SNPs in KCNN2 on SSC2, RYR1 and PLOD1 on SSC6 and MYST4 on SSC14 were significantly associated with kyphosis in the resource population of swine (P ≤ 0.05). SNPs in CER1 and CDH7 on SSC1, PSMA5 on SSC4, HOXC6 and HOXC8 on SSC5, ADAMTS18 on SSC6 and SOX9 on SSC12 were significantly associated with the kyphosis trait in the F2 population of swine (P ≤ 0.05). CONCLUSIONS: These data suggest that this kyphosis trait may be affected by several loci and that these may differ by population. Carcass value could be improved by effectively removing this undesirable trait from pig populations.

An improved pig reference genome sequence to enable pig genetics and genomics research.

BACKGROUND: The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. RESULTS: We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. CONCLUSIONS: These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.

MicroRNA transcriptome profiles during swine skeletal muscle development.

BACKGROUND: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. RESULTS: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. CONCLUSION: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.