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.

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.

DNA methylation profile in beef cattle is influenced by additive genetics and age.

DNA methylation (DNAm) has been considered a promising indicator of biological age in mammals and could be useful to increase the accuracy of phenotypic prediction in livestock. The objectives of this study were to estimate the heritability and age effects of site-specific DNAm (DNAm level) and cumulative DNAm across all sites (DNAm load) in beef cattle. Blood samples were collected from cows ranging from 217 to 3,192 days (0.6 to 8.7 years) of age (n = 136). All animals were genotyped, and DNAm was obtained using the Infinium array HorvathMammalMethylChip40. Genetic parameters for DNAm were obtained from an animal model based on the genomic relationship matrix, including the fixed effects of age and breed composition. Heritability estimates of DNAm levels ranged from 0.18 to 0.72, with a similar average across all regions and chromosomes. Heritability estimate of DNAm load was 0.45. The average age effect on DNAm level varied among genomic regions. The DNAm level across the genome increased with age in the promoter and 5' UTR and decreased in the exonic, intronic, 3' UTR, and intergenic regions. In addition, DNAm level increased with age in regions enriched in CpG and decreased in regions deficient in CpG. Results suggest DNAm profiles are influenced by both genetics and the environmental effect of age in beef cattle.

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.

Fine mapping genetic variants associated with age at puberty and sow fertility using SowPro90 genotyping array.

Sow fertility traits, such as litter size and the number of lifetime parities produced (reproductive longevity), are economically important. Selection for these traits is difficult because they are lowly heritable and expressed late in life. Age at puberty (AP) is an early indicator of reproductive longevity. Here, we utilized a custom Affymetrix single-nucleotide polymorphisms (SNPs) array (SowPro90) enriched with positional candidate genetic variants for AP and a haplotype-based genome-wide association study to fine map the genetic sources associated with AP and other fertility traits in research (University of Nebraska-Lincoln [UNL]) and commercial sow populations. Five major quantitative trait loci (QTL) located on four Sus scrofa chromosomes (SSC2, SSC7, SSC14, and SSC18) were discovered for AP in the UNL population. Negative correlations (r = -0.96 to -0.10; P < 0.0001) were observed at each QTL between genomic estimated breeding values for AP and reproductive longevity measured as lifetime number of parities (LTNP). Some of the SNPs discovered in the major QTL regions for AP were located in candidate genes with fertility-associated gene ontologies (e.g., P2RX3, NR2F2, OAS1, and PTPN11). These SNPs showed significant (P < 0.05) or suggestive (P < 0.15) associations with AP, reproductive longevity, and litter size traits in the UNL population and litter size traits in the commercial sows. For example, in the UNL population, when the number of favorable alleles of an SNP located in the 3' untranslated region of PTPN11 (SSC14) increased, AP decreased (P < 0.0001), while LTNP increased (P < 0.10). Additionally, a suggestive difference in the observed NR2F2 isoforms usage was hypothesized to be the source of the QTL for puberty onset mapped on SSC7. It will be beneficial to further characterize these candidate SNPs and genes to understand their impact on protein sequence and function, gene expression, splicing process, and how these changes affect the phenotypic variation of fertility traits.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Evaluation of genotype quality parameters for SowPro90, a new genotyping array for swine1.

Understanding early predictors of sow fertility has the potential to improve genomic predictions. A custom SNP array (SowPro90 produced by Affymetrix) was developed to include genetic variants overlapping quantitative trait loci for age at puberty, one of the earliest indicators of sow fertility, as well as variants related to innate and adaptive immunity. The polymorphisms included in the custom genotyping array were identified using multiple genomic approaches including deep genomic and transcriptomic sequencing and genome-wide associations. Animals from research and commercial populations (n = 2,586) were genotyped for 103,476 SNPs included in SowPro90. To assess the quality of data generated, genotype concordance was evaluated between the SowPro90 and Porcine SNP60 BeadArray using a subset of common SNP (n = 44,708) and animals (n = 277). The mean genotype concordance rate per SNP was 98.4%. Differences in distribution of data quality were observed between the platforms indicating the need for platform specific thresholds for quality parameters. The optimal thresholds for SowPro90 (≥97% SNP and ≥93% sample call rate) were obtained by analyzing the data quality distribution and genotype concordance per SNP across platforms. At ≥97% SNP call rate, there were 42,151 SNPs (94.3%) retained with a mean genotype concordance of 98.6% across platforms. Similarly, ≥94% SNPs and ≥85% sample call rates were established as thresholds for Porcine SNP60 BeadArray. At ≥94% SNPs call rate, there were 41,043 SNPs (91.8%) retained with a mean genotype concordance of 98.6% across platforms. Final evaluation of SowPro90 array content (n = 103,476) at ≥97% SNPs and ≥93% sample call rates allowed retention of 89,040 SNPs (86%) for downstream analysis. The findings and strategy for quality control could be helpful in identifying consistent, high-quality genotypes for genomic evaluations, especially when integrating genotype data from different platforms.

A Missense Mutation in SLC45A2 Is Associated with Albinism in Several Small Long Haired Dog Breeds.

Homozygosity for a large deletion in the solute carrier family 45, member 2 (SLC45A2) gene causes oculocutaneous albinism (OCA) in the Doberman Pinscher breed. An albino Lhasa Apso did not have this g.27141_31223del (CanFam2) deletion in her SLC45A2 sequence. Therefore, SLC45A2 was investigated in this female Lhasa Apso to search for other possible variants that caused her albinism. The albino Lhasa Apso was homozygous for a nonsynonymous substitution in the seventh exon, a c.1478G>A base change that resulted in a glycine to aspartic acid substitution (p.G493D). This mutation was not found in a wolf, a coyote, or any of the 15 other Lhasa Apso dogs or 32 other dogs of breeds related to the Lhasa Apso. However, an albino Pekingese, 2 albino Pomeranians, and an albino mixed breed dog that was small and long haired were also homozygous for the 493D allele. The colored puppies of the albino Lhasa Apso and the colored dam of the 2 albino Pomeranians were heterozygous for this allele. However, an albino Pug was homozygous for the 493G allele and therefore although we suggest the 493D allele causes albinism when homozygous in several small, long haired dog breeds, it does not explain all albinism in dogs. A variant effect prediction for the albino Lhasa Apso confirms that p.G493D is a deleterious substitution, and a topology prediction for SLC45A2 suggests that the 11th transmembrane domain where the 493rd amino acid was located, has an altered structure.