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Following the successful creation of a biobank from two adult Thoroughbred mares, this study aimed to recapitulate sample collection in two adult Thoroughbred stallions as part of the Functional Annotation of the Animal Genome (FAANG) initiative. Both stallions underwent thorough physical, lameness, neurologic, and ophthalmic (including electroretinography) examinations prior to humane euthanasia. Epididymal sperm was recovered from both stallions immediately postmortem and cryopreserved. Aseptically collected full thickness skin biopsies were used to isolate, culture and cryopreserve dermal fibroblasts. Serum, plasma, cerebrospinal fluid, urine, and gastrointestinal content from various locations were collected and cryopreserved. Under guidance of a board-certified veterinary anatomic pathologist, 102 representative tissue samples were collected from both horses. Whole tissue samples were flash-frozen and prioritized tissues had nuclei isolated and cryopreserved. Spatially contemporaneous samples of each tissue were submitted for histologic examination. Antemortem and gross pathologic examination revealed mild abnormalities in both stallions. One stallion (ECA_UCD_AH3) had unilateral thoracic limb lameness and bilateral chorioretinal scars. The second stallion (ECA_UCD_AH4) had subtle symmetrical pelvic limb ataxia, symmetrical prostatomegally, and moderate gastrointestinal nematodiasis. DNA from each was whole-genome sequenced and genotyped using the GGP Equine 70K SNP array. The genomic resources and banked biological samples from these animals augments the existing resource available to the equine genomics community. Importantly we may now improve the resolution of tissue-specific gene regulation as affected by sex, as well as add sex-specific tissues and gametes.

Introduction: Pubertal attainment is critical to reproductive longevity in heifers. Previously, four heifer pubertal classifications were identified according to attainment of blood plasma progesterone concentrations > 1 ng/ml: 1) Early; 2) Typical; 3) Start-Stop; and 4) Non-Cycling. Early and Typical heifers initiated and maintained cyclicity, Start-Stop started and then stopped cyclicity and Non-Cycling never initiated cyclicity. Start-Stop heifers segregated into Start-Stop-Discontinuous (SSD) or Start-Stop-Start (SSS), with SSD having similar phenotypes to Non-Cycling and SSS to Typical heifers. We hypothesized that these pubertal classifications are heritable, and loci associated with pubertal classifications could be identified by genome wide association studies (GWAS). Methods: Heifers (n = 532; 2017 - 2022) genotyped on the Illumina Bovine SNP50 v2 or GGP Bovine 100K SNP panels were used for variant component estimation and GWAS. Heritability was estimated using a univariate Bayesian animal model. Results: When considering pubertal classifications: Early, Typical, SSS, SSD, and Non-Cycling, pubertal class was moderately heritable (0.38 ± 0.08). However, when heifers who initiated and maintained cyclicity were compared to those that did not cycle (Early+Typical vs. SSD+Non-Cycling) heritability was greater (0.59 ± 0.19). A GWAS did not identify single nucleotide polymorphisms (SNPs) significantly associated with pubertal classifications, indicating puberty is a polygenic trait. A candidate gene approach was used, which fitted SNPs within or nearby a set of 71 candidate genes previously associated with puberty, PCOS, cyclicity, regulation of hormone secretion, signal transduction, and methylation. Eight genes/regions were associated with pubertal classifications, and twentytwo genes/regions were associated with whether puberty was attained during the trial. Additionally, whole genome sequencing (WGS) data on 33 heifers were aligned to the reference genome (ARS-UCD1.2) to identify variants in FSHR, a gene critical to pubertal attainment. Fisher's exact test determined if FSHR SNPs segregated by pubertal classification. Two FSHR SNPs that were not on the bovine SNP panel were selected for additional genotyping and analysis, and one was associated with pubertal classifications and whether they cycled during the trial. Discussion: In summary, these pubertal classifications are moderately to highly heritable and polygenic. Consequently, genomic tools to inform selection/management of replacement heifers would be useful if informed by SNPs associated with cyclicity and early pubertal attainment. [ABSTRACT FROM AUTHOR]

Thirteen American Hereford cattle were reported blind with presumed onset when ~12-mo-old. All blind cattle shared a common ancestor through both the maternal and paternal pedigrees, suggesting a recessive genetic origin. Given the pedigree relationships and novel phenotype, we characterized the ophthalmo-pathologic changes associated with blindness and identified the responsible gene variant. Ophthalmologic examinations of 5 blind cattle revealed retinal degeneration. Histologically, 2 blind cattle had loss of the retinal photoreceptor layer. Whole-genome sequencing (WGS) of 7 blind cattle and 9 unaffected relatives revealed a 1-bp frameshift deletion in ceroid lipofuscinosis neuronal 3 (CLN3 ; chr25 g.26043843del) for which the blind cattle were homozygous and their parents heterozygous. The identified variant in exon 16 of 17 is predicted to truncate the encoded protein (p. Pro369Argfs*8) battenin, which is involved in lysosomal function necessary for photoreceptor layer maintenance. Of 462 cattle genotyped, only blind cattle were homozygous for the deletion. A query of WGS data of > 5,800 animals further revealed that the variant was only observed in related Hereford cattle. Mutations in CLN3 are associated with human juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which results in early-onset retinal degeneration and lesions similar to those observed in our cases. Our data support the frameshift variant of CLN3 as causative of blindness in these Hereford cattle, and provide additional evidence of the role of this gene in retinal lesions, possibly as a model for human non-syndromic JNCL. [ABSTRACT FROM AUTHOR]

The mitochondrion is a maternally inherited organelle that produces approximately 90% of cellular energy. The mitochondrial genome (mtDNA) consists of 13 genes that are critical components of oxidative phosphorylation. Identifying and understanding variation within this genome could elucidate differences in metabolic and physiological traits, specifically growth efficiency in cattle. Our aim is to identify how variation in the mitochondrial genome influences efficiency in beef cattle. Low-pass sequencing data (average 0.5X coverage of the autosomes) were collected (Neogen) from 49 animals within the UNL herd (composite Simmental, Red Angus, and Gelbvieh). The data were trimmed and mapped (BWA-MEM) to the ARS-UCD1.2 cattle genome. After variant impacts were predicted (Ensembl VEP) haplotype networks were created to visualize variation among samples. Over 60X coverage was achieved across the mitochondrial genome, on average. Excluding the D-loop, 62 variants were identified in the 49 animals: 2 tRNA, 6 rRNA, and 54 coding single nucleotide polymorphisms (SNPs). These 62 variants were encompassed in 23 unique haplotypes. Of the coding variants, 13 were nonsynonymous and found in the following genes encoding components of oxidative phosphorylation: ND1 (n = 3), ND2 (n = 1), ND3 (n = 1), ND4 (n = 1), ND5 (n = 1), ND6 (n = 3), COX3 (n = 2), and CYTB (n = 1). Nine unique haplotypes were present considering only nonsynonymous variants. Nonsynonymous mutations were identified in complex I genes (ND1-6), in the complex IV gene COX3, and in CYTB, which is involved in multiple processes including the electron transport chain. These data, from a closed herd at UNL, support that there is a great deal of mtDNA variation present among beef cattle for which to analyze the impact of mitochondrial genotype on animal efficiency. Performance data demonstrate variation in average daily gain (ADG) and residual feed intake (RFI) in this herd, with a mean ADG of 1.95 kg/d (SD = 0.23 kg/d) and a mean RFI of -0.11 kg (SD = 0.57 kg). Data on individual feed intake, growth rates, and carcass performance values will continue to be collected on all animals sequenced. Based upon these data we hypothesize that variation in the mitochondrial genome will be associated with altered complex I and IV activity. For a more comprehensive understanding of this variation, the sample size will be expanded to over 1,500 animals in the next 2 yr. Overall, from our initial analysis of the mitochondrial genomes from 49 animals, we have identified 62 variants that we anticipate will explain variation in cellular and growth efficiency. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of acute heat stress on the skeletal muscle epigenome using reduced representation bisulfite sequencing (RRBS). March-born crossbred (5/8 Red Angus, 3/8 Simmental) steers (n = 14; average body weight = 308.7 kg) were exposed to heat stress (HS) conditions of 35°C + 35% relative humidity (Temperature Humidity Index = 81) for 12h/d for 2 d (48 h). Longissimus dorsi samples were collected via biopsy and flash-frozen in liquid nitrogen immediately following the HS period at 48 h and from the contralateral longissimus dorsi 5 d later. DNA was isolated and underwent RRBS using 150 bp paired-end reads at an achieved average depth of 15.3 million reads/ sample. Reads were trimmed and mapped to a bisulfiteconverted bovine genome (ARSUCD1.2). Methylation calls were extracted and used to identify differentially methylated regions (DMRs) with methylkit in R comparing samples collected 5 d post-HS to those taken immediately post-48 h of HS. Differentially methylated regions (1,000 bp) had q < 0.05 and at least a 15% difference in methylation. There were 1,015 DMRs in genes. Hypomethylated DMRs 5 d post-HS included IGF1R, IGF2, IGF2BP1, IGFBP4, IGF2R, IKBKB, and IL34. Hypermethylated DMRs 5 d post-HS included FGF5, FGFR1, and FGFR3. There were 81 DMRs in putative promoter regions (2000 bp 5’ of a gene). MYLK2, SIGIRR, and 25 other genes had hypomethylated promoter regions. Genes that contained DMRs were used as input for DAVID to explore KEGG pathways. Enriched pathways (P < 0.1) included multiple hormone pathways, MAPK, PI3K-Akt, HIF-1, and VEGF. Hypomethylation of IGF-related genes and hypermethylation of FGF-related genes suggests impaired growth capacity post-HS, as does the enrichment of MAPK and PI3K-Akt pathways. Hypomethylation of SIGIRR’s promoter region, IL34 and IKBKB, indicates enhanced anti-inflammatory activity given their known regulation of inflammatory cytokine production. This, coupled with enrichment of the HIF-1 and VEGF pathways, suggests regulatory dampening of HS-induced oxidative stress. These findings reveal key regulatory changes in muscle growth, inflammatory response, and oxidative stress activity for cattle recovering from acute HS. Previous cattle skeletal muscle RNA studies predicted that HS altered metabolism, inflammation, and oxidative stress pathways, similar to the methylation results of this study. Overall, evidence suggests growth dysregulation, which may help explain observed HS-induced poor growth due to HS, as well as subsequent anti-inflammatory activity during recovery from HS. [ABSTRACT FROM AUTHOR]

Between 2020 and 2022, eight calves (6 heifers, 2 bulls) in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) experienced exercise intolerance during extended physical activity. These calves exhibited an inability to keep pace with the rest of the herd, with some eventually succumbing to physical collapse from which they did not recover. Increased creatine kinase, an indication of muscle degeneration, was confirmed in biopsy and necropsy samples. Available sire pedigrees revealed a common paternal ancestor within 2 to 4 generations in all the affected calves. Dam pedigrees were unavailable; however, the herd retained replacement females that occasionally shared common ancestors with breeding bulls. Thus, a de novo autosomal recessive mutation was hypothesized as the cause of exercise intolerance in these calves. Genomic data (100K SNP) from 6 affected calves and 715 herd mates were initially used for a whole genome-wide association study, pinpointing a significant region on chromosome 29. Whole-genome sequencing of 2 affected calves combined with 145 other sequenced cattle led to the identification of a nonsense mutation in the PYGM gene, also located on chromosome 29. The mutation, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon. The protein product of PYGM, myophosphorylase, has a crucial role in metabolizing glycogen stored within skeletal muscle to generate energy for muscle cells. As a result, the recessive genotype led to greater (P < 0.05) glycogen concentrations in affected calves (n = 2) compared with heterozygous animals (n = 3) and wild-type controls (n = 2). Immunohistochemistry and label-free quantitative proteomics analysis confirmed the absence of the PYGM protein product in skeletal muscle. Myophosphorylase is needed for efficient glycogen utilization, which is crucial for producing high-quality beef. In the 2 affected calves harvested, increased skeletal muscle glycogen persisted postmortem, resulting in increased pH and dark-cutting beef, which is associated with a negative consumer perception and economic losses to the industry. In addition to identifying the cause of exercise intolerance and welfare concerns in these cattle, this study is among the first to identify a specific genetic mutation associated with dark-cutting beef. Carriers of the mutation did not display any discernible differences in meat quality or measures of animal well-being. While cattle heterozygous for the mutation may not immediately impact the beef industry, identifying carriers is paramount for implementing proactive selection and breeding strategies to prevent the production of affected calves. [ABSTRACT FROM AUTHOR]

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide-major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I-restricted [CD8.sup.+] T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogenderived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key-like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease., Introduction T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell [...]

Differential reflectometry (DR) is an effective tool to supplement existing explosives detection systems thus making the combined unit more effective than one tool alone. It is an optical technique in which the light beam (UV) emanates from an extended distance onto the substance under investigation, thus rendering it to be a standoff method. DR allows the measurement of the energies that electrons absorb from photons as they are raised into higher, allowed energy states. These electron transitions serve as a 'fingerprint' for identifying substances. The device can be made portable; it is fast, safe for the public, does not require human involvement, is cost effective, and most of all, does not require ingestion of a suspicious substance into an instrument. Various embodiments are presented. OCIS codes: 120.0280, 260.7190, 260.7190.