Puberty classifications in beef heifers are moderately to highly heritable and associated with candidate genes related to cyclicity and timing of puberty.

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 twenty-two 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.

An autosomal recessive variant in PYGM causes myophosphorylase deficiency in Red Angus composite cattle.

BACKGROUND: Between 2020 and 2022, eight calves in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) displayed exercise intolerance during forced activity. In some cases, the calves collapsed and did not recover. Available sire pedigrees contained a paternal ancestor within 2-4 generations in all affected calves. Pedigrees of the calves' dams were unavailable, however, the cows were ranch-raised and retained from prior breeding seasons, where bulls used for breeding occasionally had a common ancestor. Therefore, it was hypothesized that a de novo autosomal recessive variant was causative of exercise intolerance in these calves. RESULTS: A genome-wide association analysis utilizing SNP data from 6 affected calves and 715 herd mates, followed by whole-genome sequencing of 2 affected calves led to the identification of a variant in the gene PYGM (BTA29:g.42989581G > A). The variant, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon (p.Arg650*). The protein product of PYGM, myophosphorylase, breaks down glycogen in skeletal muscle. Glycogen concentrations were fluorometrically assayed as glucose residues demonstrating significantly elevated glycogen concentrations in affected calves compared to cattle carrying the variant and to wild-type controls. The absence of the PYGM protein product in skeletal muscle was confirmed by immunohistochemistry and label-free quantitative proteomics analysis; muscle degeneration was confirmed in biopsy and necropsy samples. Elevated skeletal muscle glycogen persisted after harvest, resulting in a high pH and dark-cutting beef, which is negatively perceived by consumers and results in an economic loss to the industry. Carriers of the variant did not exhibit differences in meat quality or any measures of animal well-being. CONCLUSIONS: Myophosphorylase deficiency poses welfare concerns for affected animals and negatively impacts the final product. The association of the recessive genotype with dark-cutting beef further demonstrates the importance of genetics to not only animal health but to the quality of their product. Although cattle heterozygous for the variant may not immediately affect the beef industry, identifying carriers will enable selection and breeding strategies to prevent the production of affected calves.

A recessive CLN3 variant is responsible for delayed-onset retinal degeneration in Hereford cattle.

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.

Short Communication: Beta-adrenergic agonists alter oxidative phosphorylation in primary myoblasts.

Beta-adrenergic agonists (ß-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which ß-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no ß-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by ß-AA treatment in ovine or porcine cells. These findings indicate that ß-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to ß-AA incubation also demonstrates differing physiological responses to ß-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.

Beta-adrenergic agonists (ß-AAs) are supplemented to pigs and cattle to improve growth performance, carcass weight, and loin muscle area. Little is known about the mechanism taking place within individual cells by which ß-AAs achieve this outcome. Previous work reported that ß-AA supplementation improves the efficiency in which cells use glucose as an energy source and alters the expression of genes related to mitochondrial function, a key component of cellular energy production. To further our understanding of the impact of ß-AA supplementation on these cellular functions, our objective was to identify the influence of two ß-AAs used in livestock production, ractopamine HCl and zilpaterol HCl, on the mitochondrial respiratory activity of cells collected from the loin muscle and grown in culture. We isolated cells from cattle, pig, and sheep muscle and measured the oxygen consumption of the cells after treatment with ractopamine HCl, zilpaterol HCl, or with no supplement. We found that both ractopamine HCl and zilpaterol HCl enhance the efficiency of cellular energy production during a state of cellular stress in bovine muscle cells. There was no appreciable effect of the supplement on the energy production of pig or sheep cells. These data indicate that ß-AA supplementation in cattle may increase the muscle cell energy production capacity compared with non-supplemented cells. This study also demonstrates that the efficiency of cell energy production is one plausible mechanism underlying species differences in the response to ß-AA supplementation.

Transcriptome analyses indicate that heat stress-induced inflammation in white adipose tissue and oxidative stress in skeletal muscle is partially moderated by zilpaterol supplementation in beef cattle.

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. ß-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass, and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index [THI] = 68) or HS (THI = 73-85) conditions and were not supplemented or supplemented with ZH (8.33 mg/kg/d) for 21 d in a 2 × 2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25A4, and S100A12) were DE due to ZH on day 3, and 2 (RRAD, ALB) were DE due to the interaction of HS and ZH on day 10 (Padj < 0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on day 10, and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, and TMCO6) were DE due to environment on day 21 (Padj < 0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw < 0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P < 0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P < 0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS.

Heat stress (HS) negatively impacts livestock health and carcass quality. Supplementation of livestock with ß-adrenergic agonists (ß-AA) increases muscle mass and decreases fat deposition. The purpose of this study was to understand how HS and zilpaterol hydrochloride (ZH), a ß-AA, alter gene expression in muscle and in adipose of cattle. Twenty-four steers were assigned to thermoneutral (TN) or HS conditions and were not supplemented (NS) or supplemented with ZH for 21 d. RNA was isolated from muscle and adipose collected on days 3, 10, and 21 to identify changes in gene expression. Several individual loci were differentially expressed (DE) due to HS or ZH in both tissues while the interaction of HS and ZH altered expression in adipose. A less stringent definition of DE used to explore biological pathways predicted that both treatments alter metabolism. Pathway analyses also supported that HS increased inflammation in adipose, but that these inflammatory pathways were downregulated by ZH. HS also was predicted to induce oxidative stress in muscle although ZH moderated this response. This study provides information on how HS and ß-AA act independently and interact to alter physiology, lending insight useful for the development of management and mitigation strategies for stress.

Mandibulofacial Dysostosis Attributed to a Recessive Mutation of CYP26C1 in Hereford Cattle.

In spring 2020, six Hereford calves presented with congenital facial deformities attributed to a condition we termed mandibulofacial dysostosis (MD). Affected calves shared hallmark features of a variably shortened and/or asymmetric lower mandible and bilateral skin tags present 2-10 cm caudal to the commissure of the lips. Pedigree analysis revealed a single common ancestor shared by the sire and dam of each affected calf. Whole-genome sequencing (WGS) of 20 animals led to the discovery of a variant (Chr26 g. 14404993T>C) in Exon 3 of CYP26C1 associated with MD. This missense mutation (p.L188P), is located in an α helix of the protein, which the identified amino acid substitution is predicted to break. The implication of this mutation was further validated through genotyping 2 additional affected calves, 760 other Herefords, and by evaluation of available WGS data from over 2500 other individuals. Only the affected individuals were homozygous for the variant and all heterozygotes had at least one pedigree tie to the suspect founder. CYP26C1 plays a vital role in tissue-specific regulation of retinoic acid (RA) during embryonic development. Dysregulation of RA can result in teratogenesis by altering the endothelin-1 signaling pathway affecting the expression of Dlx genes, critical to mandibulofacial development. We postulate that this recessive missense mutation in CYP26C1 impacts the catalytic activity of the encoded enzyme, leading to excess RA resulting in the observed MD phenotype.

KDR-LacZ-expressing cells are involved in ovarian and testis-specific vascular development, suggesting a role for VEGFA in the regulation of this vasculature.

Our objectives were to evaluate kinase insert domain protein receptor (KDR)-ß-galactosidase (LacZ) expression as a marker for vascular development during gonadal morphogenesis and to determine whether any novel non-angiogenic KDR-LacZ expression was present in mouse testes or ovaries. Gonads were collected from mice expressing LacZ driven by the Kdr promoter (KDR-LacZ) from embryonic day 11 (E11) through postnatal day 60 (P60). At E11.5, mesonephric cells expressing KDR-LacZ seemed to migrate into the developing testis and surrounded developing seminiferous cords. Cells expressing KDR-LacZ appeared in the ovary with no apparent migration from the adjacent mesonephros, suggesting a different origin of endothelial cells. Testis organ cultures from E11 mice were treated with 8 µM VEGFR-TKI, a vascular endothelial growth factor A signal transduction inhibitor; subsequently, the amount of KDR-LacZ staining was reduced by 66%-99% (P<0.002), and the ability of KDR-expressing cells to form a densely organized vascular network was inhibited. Novel non-angiogenic KDR-LacZ staining was detected in the testis on specific subsets of germ cells at E16, E17, P4, P20, P30, and P60. In ovaries, staining was present on oocytes within oocyte cysts at E17 and within late secondary follicles of postnatal mice. Thus, KDR is an excellent marker for analyzing vascular development in the gonads. Inhibition of VEGFA signal transduction prevents the development of testis-specific vasculature. Furthermore, non-vascular KDR-LacZ staining suggests that KDR directly affects both spermatogenesis and somatic-oocyte interactions during gametogenesis.

Developments on standoff detection of explosive materials by differential reflectometry.

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.