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.

Daily Injection of the ß2 Adrenergic Agonist Clenbuterol Improved Muscle Glucose Metabolism, Glucose-Stimulated Insulin Secretion, and Hyperlipidemia in Juvenile Lambs Following Heat-Stress-Induced Intrauterine Growth Restriction.

Stress-induced fetal programming diminishes ß2 adrenergic tone, which coincides with intrauterine growth restriction (IUGR) and lifelong metabolic dysfunction. We determined if stimulating ß2 adrenergic activity in IUGR-born lambs would improve metabolic outcomes. IUGR lambs that received daily injections of saline or the ß2 agonist clenbuterol from birth to 60 days were compared with controls from pair-fed thermoneutral pregnancies. As juveniles, IUGR lambs exhibited systemic inflammation and robust metabolic dysfunction, including greater (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, increased (p < 0.05) intramuscular glycogen, reduced (p < 0.05) circulating IGF-1, hindlimb blood flow, glucose-stimulated insulin secretion, and muscle glucose oxidation. Daily clenbuterol fully recovered (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, hindlimb blood flow, muscle glucose oxidation, and intramuscular glycogen. Glucose-stimulated insulin secretion was partially recovered (p < 0.05) in clenbuterol-treated IUGR lambs, but circulating IGF-1 was not improved. Circulating triglycerides and HDL cholesterol were elevated (p < 0.05) in clenbuterol-treated IUGR lambs, despite being normal in untreated IUGR lambs. We conclude that deficient ß2 adrenergic regulation is a primary mechanism for several components of metabolic dysfunction in IUGR-born offspring and thus represents a potential therapeutic target for improving metabolic outcomes. Moreover, benefits from the ß2 agonist were likely complemented by its suppression of IUGR-associated inflammation.

A de novo mutation in CACNA1A is associated with autosomal dominant bovine familial convulsions and ataxia in Angus cattle.

Bovine familial convulsions and ataxia (BFCA) is considered an autosomal dominant syndrome with incomplete penetrance. Nine Angus calves from the same herd were diagnosed with BFCA within days of birth. Necropsy revealed cerebellar and spinal cord lesions associated with the condition. Parentage testing confirmed that all affected calves had a common sire. The sire was then bred to 36 cows across two herds using artificial insemination, producing an additional 14 affected calves. The objective of this investigation was to identify hypothesized dominant genetic variation underlying the condition. Whole-genome sequencing was performed on the sire, six affected and seven unaffected paternal half-sibling calves and combined with data from 135 unrelated controls. The sire and five of the six affected calves were heterozygous for a nonsense variant (Chr7 g.12367906C>T, c.5073C>T, p.Arg1681*) in CACNA1A. The other affected calves (N = 8) were heterozygous for the variant but it was absent in the other unaffected calves (N = 7) and parents of the sire. This variant was also absent in sequence data from over 6500 other cattle obtained via public repositories and collaborator projects. The variant in CACNA1A is expressed in the cerebellum of the ataxic calves as detected in the transcriptome and was not differentially expressed compared with controls. The CACNA1A protein is part of a highly expressed cerebellar calcium voltage gated channel. The nonsense variant is proposed to cause haploinsufficiency, preventing proper transmission of neuronal signals through the channel and resulting in BFCA.

A Comprehensive Allele Specific Expression Resource for the Equine Transcriptome.

Background: Allele-specific expression (ASE) analysis provides a nuanced view of cis-regulatory mechanisms affecting gene expression. Results: In this work, we introduce and highlight the significance of an equine ASE analysis, containing integrated long- and short-read RNA sequencing data, along with insight from histone modification data, from four healthy Thoroughbreds (2 mares and 2 stallions) across 9 tissues. Conclusions: This valuable publicly accessible resource is poised to facilitate investigations into regulatory variation in equine tissues and foster a deeper understanding of the impact of allelic imbalance in equine health and disease at the molecular level.

Daily injection of the ß2 adrenergic agonist clenbuterol improved poor muscle growth and body composition in lambs following heat stress-induced intrauterine growth restriction.

Background: Intrauterine growth restriction (IUGR) is associated with reduced ß2 adrenergic sensitivity, which contributes to poor postnatal muscle growth. The objective of this study was to determine if stimulating ß2 adrenergic activity postnatal would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Methods: Time-mated ewes were housed at 40°C from day 40 to 95 of gestation to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of 0.8 µg/kg clenbuterol HCl (IUGR+CLEN; n = 11) or saline placebo (IUGR; n = 12). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Biometrics were assessed weekly and body composition was estimated by ultrasound and bioelectrical impedance analysis (BIA). Lambs were necropsied at 60 days of age. Results: Bodyweights were lighter (p ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, day 30, and day 60. Average daily gain was less (p ≤ 0.05) for IUGR lambs than controls and was intermediate for IUGR+CLEN lambs. At day 58, BIA-estimated whole-body fat-free mass and ultrasound-estimated loin eye area were less (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. At necropsy, loin eye area and flexor digitorum superficialis muscles were smaller (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Longissimus dorsi protein content was less (p ≤ 0.05) and fat-to-protein ratio was greater (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (p ≤ 0.05) ß2 adrenoreceptor content, fewer (p ≤ 0.05) proliferating myoblasts, tended to have fewer (p = 0.08) differentiated myoblasts, and had smaller (p ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size were recovered (p ≤ 0.05) in IUGR+CLEN lambs compared to IUGR lambs, but ß2 adrenoreceptor content and differentiated myoblasts were not recovered. Semitendinosus lipid droplets were smaller (p ≤ 0.05) in size for IUGR lambs than for controls and were further reduced (p ≤ 0.05) in size for IUGR+CLEN lambs. Conclusion: These findings show that clenbuterol improved IUGR deficits in muscle growth and some metabolic parameters even without recovering the deficit in ß2 adrenoreceptor content. We conclude that IUGR muscle remained responsive to ß2 adrenergic stimulation postnatal, which may be a strategic target for improving muscle growth and body composition in IUGR-born offspring.

The localization of centromere protein A is conserved among tissues.

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.

Imputed genomes of historical horses provide insights into modern breeding.

Historical genomes can provide important insights into recent genomic changes in horses, especially the development of modern breeds. In this study, we characterized 8.7 million genomic variants from a panel of 430 horses from 73 breeds, including newly sequenced genomes from 20 Clydesdales and 10 Shire horses. We used this modern genomic variation to impute the genomes of four historically important horses, consisting of publicly available genomes from 2 Przewalski's horses, 1 Thoroughbred, and a newly sequenced Clydesdale. Using these historical genomes, we identified modern horses with higher genetic similarity to those in the past and unveiled increased inbreeding in recent times. We genotyped variants associated with appearance and behavior to uncover previously unknown characteristics of these important historical horses. Overall, we provide insights into the history of Thoroughbred and Clydesdale breeds and highlight genomic changes in the endangered Przewalski's horse following a century of captive breeding.

Genetic diversity and signatures of selection in four indigenous horse breeds of Iran.

Indigenous Iranian horse breeds were evolutionarily affected by natural and artificial selection in distinct phylogeographic clades, which shaped their genomes in several unique ways. The aims of this study were to evaluate the genetic diversity and genomewide selection signatures in four indigenous Iranian horse breeds. We evaluated 169 horses from Caspian (n = 21), Turkmen (n = 29), Kurdish (n = 67), and Persian Arabian (n = 52) populations, using genomewide genotyping data. The contemporary effective population sizes were 59, 98, 102, and 113 for Turkmen, Caspian, Persian Arabian, and Kurdish breeds, respectively. By analysis of the population genetic structure, we classified the north breeds (Caspian and Turkmen) and west/southwest breeds (Persian Arabian and Kurdish) into two phylogeographic clades reflecting their geographic origin. Using the de-correlated composite of multiple selection signal statistics based on pairwise comparisons, we detected a different number of significant SNPs under putative selection from 13 to 28 for the six pairwise comparisons (FDR < 0.05). The identified SNPs under putative selection coincided with genes previously associated with known QTLs for morphological, adaptation, and fitness traits. Our results showed HMGA2 and LLPH as strong candidate genes for height variation between Caspian horses with a small size and the other studied breeds with a medium size. Using the results of studies on human height retrieved from the GWAS catalog, we suggested 38 new putative candidate genes under selection. These results provide a genomewide map of selection signatures in the studied breeds, which represent valuable information for formulating genetic conservation and improved breeding strategies for the breeds.

White coat color of a Black Angus calf attributed to an occurrence of the delR217 variant of MITF.

A white calf, with minimal pigmented markings, was born to two registered Black Angus parents. Given the possibility of an unknown recessive or de novo dominant mutation, whole-genome sequencing was conducted on the trio of individuals. A 3-bp in-frame deletion in MITF was identified; this mutation was unique to the calf but identical to the delR217 variant reported in both humans and murine models of Waardenburg syndrome type 2A and Tietz syndrome. Given the coat color phenotype and identity of the mutation, our data support that this calf represents the first instance of this recurring MITF mutation in cattle.

Functional annotation of the animal genomes: An integrated annotation resource for the horse.

The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

Type 2 polysaccharide storage myopathy in Quarter Horses is a novel glycogen storage disease causing exertional rhabdomyolysis.

BACKGROUND: Both type 1 (PSSM1) and type 2 polysaccharide storage myopathy (PSSM2) are characterised by aggregates of abnormal polysaccharide in skeletal muscle. Whereas the genetic basis for PSSM1 is known (R309H GYS1), the cause of PSSM2 in Quarter Horses (PSSM2-QH) is unknown and glycogen concentrations not defined. OBJECTIVES: To characterise the histopathological and biochemical features of PSSM2-QH and determine if an associated monogenic variant exists in genes known to cause glycogenosis. STUDY DESIGN: Retrospective case control. METHODS: Sixty-four PSSM2-QH, 30 PSSM1-QH and 185 control-QH were identified from a biopsy repository and clinical data, histopathology scores (0-3), glycogen concentrations and selected glycolytic enzyme activities compared. Coding sequences of 12 genes associated with muscle glycogenoses were identified from whole genome sequences and compared between seven PSSM2-QH and five control-QH. RESULTS: Exertional rhabdomyolysis in PSSM2-QH occurred predominantly in barrel racing and working cow/roping performance types and improved with regular exercise and a low starch/fat-supplemented diet. Histopathological scores, including the amount of amylase-resistant polysaccharide (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, control-QH 0 ± 0, p < 0.001), and glycogen concentrations (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, control-QH 80 ± 27 mmol/kg, p < 0.0001) were intermediate in PSSM2-QH with significant differences among groups. In PSSM2-QH, abnormal polysaccharide had a less filamentous ultrastructure than PSSM1-QH and phosphorylase and phosphofructokinase activities were normal. Seventeen of 30 PSSM2-QH with available pedigrees descended from one of three stallions within four generations. Of the 29 predicted high or moderate impact genetic variants identified in candidate genes, none were present in only PSSM2-QH and absent in control-QH. MAIN LIMITATIONS: Analyses of PSSM2-QH and PSSM1-QH were performed on shipped samples, controls on frozen samples. CONCLUSIONS: PSSM2-QH is a novel glycogen storage disorder that is not the result of a mutation in genes currently known to cause muscle glycogenoses in other species.

CONTEXTO: Ambos os tipos 1 e 2 de miopatia por acúmulo de polissacarídeo (PSSM) são caracterizados por agregados de polissacarídeos anormais no músculo esquelético. Enquanto a base genética do PSSM 1 é conhecida (R309H GYS1), a causa do PSSM2 em cavalos Quarto de Milha (PSSM2-QH) é desconhecida, e a concentração de glicogênio não é definida. OBJETIVOS: Identificar as características histopatológicas e bioquímicas do PSSM-QH e determinar se há uma variante monogênica em genes conhecidos por causar glicogenose. DELINEAMENTO DO ESTUDO: Caso controlado retrospectivo. METODOLOGIA: 64 PSSM2-QH, 30 PSSM1-QH e 185 QH controles foram identificados em um arquivo de dados. Informação clínica, achados histológicos (escala 0-3), concentração de glicogênio e atividade enzimática de algumas enzimas glicolíticas foram comparadas. Sequências codificadas de 12 genes associados com glicogenose muscular foram identificados nas sequências genômicas completas, e comparadas entre 7 PSSM2-QH e 5 QH controles. RESULTADOS: Rabdomiólise por exercício em PSSM2-QH ocorreu predominantemente em cavalos de corrida de tambor e cavalos de team roping/trabalho com gado, e melhorou com exercício regular e uma dieta com baixo amido e alta gordura. A escala histopatológica, incluindo a quantidade de polissacarídeos resistentes à amilase (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, controle-QH 0 ± 0, P < 0.001), e concentrações de glicogênio (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, controle-QH 80 ± 27 mmol/kg, P < 0.0001) foram intermediárias em PSSM2-QH com diferença significante entre grupos. Em PSSM2-QH, polissacarídeo anormal teve uma ultraestrutura menos filamentosa do que PSSM1-QH e as atividades de fosforilase e fosfofrutoquinase foram normais. Dezessete dos 30 PSSM2-QH com pedigree disponível descendiam de 1 de 3 garanhões dentro de 4 gerações. Das 29 variações genéticas preditas a terem impacto moderado ou alto como genes candidatos, nenhuma estava presente apenas em PSSM2-QH e ausente no grupo controle-QH. PRINCIPAIS LIMITAÇÕES: As análises feitas nas amostras de PSSM2-QH e PSSM1-QH foram realizadas em amostras enviadas por correio, e as amostras dos animais controles eram amostras congeladas. CONCLUSÕES: PSSM2-QH é uma nova doença por acúmulo de glicogênio que não é o resultado de uma mutação nos genes conhecidos por causarem glicogenose muscular em outras espécies.

Absence of myofibrillar myopathy in Quarter Horses with a histopathological diagnosis of type 2 polysaccharide storage myopathy and lack of association with commercial genetic tests.

BACKGROUND: Genetic tests for variants in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 or P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) genes are offered commercially to diagnose myofibrillar myopathy (MFM) and type 2 polysaccharide storage myopathy (PSSM2) in Quarter Horses (QH). OBJECTIVES: To determine if PSSM2-QH has histopathological features of MFM. To compare genotype and allele frequencies of variants P2, P3, P4 between control-QH and PSSM2-QH diagnosed by histopathology. STUDY DESIGN: Retrospective cross-sectional. METHODS: The study includes a total of 229 healthy control-QH, 163 PSSM2-QH GYS1 mutation negative. Desmin stains of gluteal/semimembranosus muscle were evaluated. Purported disease alleles P2, P3a, P3b, P4 were genotyped by pyrosequencing. Genotype, allele frequency and total number of variant alleles or loci were compared between phenotypes using additive/genotypic and dominant models and quantitative effects evaluated by multivariable logistic regression. RESULTS: Histopathological features of MFM were absent in all QH. A P variant allele at any locus was not associated (P > .05) with a histopathological diagnosis of PSSM2 and one or more P variants were common in control-QH (57%) and PSSM2-QH (61%). Allele frequencies (control/PSSM2) were: 0.24/0.21 (P2), 0.07/0.12 (P3a), 0.07/0.11 (P3b) and 0.06/0.08 (P4). P3a and P3b loci were not independent (r2  = 0.894); and not associated with PSSM2 histopathology comparing the haplotype of both P3a and P3b variants to other haplotypes. A receiver operator curve did not accurately predict the PSSM2 phenotype (AUC = 0.67, 95% CI 0.62-0.72), and there was no difference in the total number of variant loci or total variant allele count between control-QH and PSSM2-QH. MAIN LIMITATIONS: P3a and P3b were not in complete linkage disequilibrium. CONCLUSIONS: The P2, P3 and P4 variants in genes associated with human MFM were not associated with PSSM2 in 392 QH. Their use would improperly diagnose PSSM2/MFM in 57% of healthy QH and fail to diagnose PSSM2 in 40% of QH with histopathological evidence of PSSM2.

CONTEXTO: Testes genéticos para detecção das mutações MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 ou P3b; rs1142918816) e MYOZ3 (P4; rs1142544043) são oferecidos comercialmente para diagnosticar miopatia miofibrilar (MMF) e miopatia por acúmulo de polissacarídeo tipo 2 (PSSM2) em cavalos Quarto de Milha (QM). HIPÓTESES/OBJETIVOS: Determinar se PSSM2-QM tem características similares à MMF. Comparar o genótipo e a frequência dos alelos variantes P2, P3, e P4 entre cavalos QM controle, e PSSM2-QM diagnosticados por histologia. MÉTODOS: 229 cavalos QH saudáveis como controle, e 163 PSSM2-QM positivos na histologia e negativos para a mutação GYS1. METODOLOGIA: Amostras dos músculos glúteo/semimembranoso foram avaliadas após coloração com desmina. Os pretensos genes alelos P2, P3a, P3b e P4 foram genotipados por pirosequenciamento. Genótipo, frequência alélica, e número total de variância alélica ou loci foram comparados entre os fenótipos usado aditivo/genotípico e modelos dominantes e efeitos quantitativos através de regressão logística multivariável. RESULTADOS: Características histopatológicas de MMF não foram encontradas em nenhum QM. Uma variante alélica P em qualquer uma dos loci não foi associada (P > .05) com o diagnóstico histopatológicos de PSSM2 e uma ou mais variante P foram comuns em QM controles (57%) e PSSM2-QM (61%). Frequência alélica (controle/PSSM2) foram: 0.24/0.21 (P2), 0.07/0.12 (P3a), 0.07/0.11 (P3b), e 0.06/0.08 (P4). P3a e P3b loci não foram independentes (r2  = 0.894); e não foram associados com achados histopatológicos de PSSM quando comparando o haplótipo de ambas as variantes P3a e P3b com os outros haplótipos. A curva característica de operação do receptor não previu acuradamente o fenótipo PSSM2 (AUC = 0.67, 95% IC 0.62-0.72), e não houve diferença no número dotal de variantes no loci ou na contagem de variantes alélicas total entre QM controles e PSSM2-QM. PRINCIPAIS LIMITAÇÕES: P3a e P3b não estavam em desequilíbrio de ligação. CONCLUSÕES: As variantes P2, P3 e P4 em genes associados com MMF em humanos não foram associadas com PSSM em 392 QM. O seu uso diagnosticaria impropriamente PSSM2 e MMF em 57% dos cavalos saudáveis utilizados como controle e não diagnosticaria PSSM2 em 40% dos QM com evidência histológica de PSSM2.

Epidemiological Aspects of Equid Herpesvirus-Associated Myeloencephalopathy (EHM) Outbreaks.

Equid Herpesvirus Myeloencephalopathy (EHM) is a multifactorial disease following an EHV-1 infection in Equidae. We investigated a total of 589 horses on 13 premises in Europe in search of risk factors for the development of EHM. We found that fever (p < 0.001), increasing age (p = 0.032), and female sex (p = 0.042) were risk factors for EHM in a logistic mixed model. Some breeds had a decreased risk to develop EHM compared to others (Shetland and Welsh ponies; p = 0.017; p = 0.031), and fewer EHV-1-vaccinated horses were affected by EHM compared to unvaccinated horses (p = 0.02). Data evaluation was complex due to high variability between outbreaks with regards to construction and environment; viral characteristics and the virus's transmissibility were affected by operational management. This study confirms earlier suspected host-specific risk factors, and our data support the benefit of high vaccine coverage at high-traffic boarding facilities.

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.

Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways.

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) ß-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.

Myoblasts are stems cells whose functional capacity can limit muscle growth. However, stressful intrauterine conditions cause these cells to be intrinsically dysfunctional. This restricts muscle growth capacity, leading to intrauterine growth restriction (IUGR) of the fetus, low birth weight, and less muscle mass after birth. Consequently, meat yield is reduced in IUGR-born food animals and glucose homeostasis is impaired in IUGR-born humans, which contributes to metabolic dysfunction. Intrinsic dysfunction of IUGR myoblasts has been previously observed, but the fetal programming changes (i.e., permanent changes in the development of cellular mechanisms that explains different functional outcomes) have not been identified. This study shows that one mechanism is the enhancement of signaling pathways for TNF-α and other inflammatory cytokines. These cytokines have roles in stress responses and regulation of muscle growth. Programmed enhancement of these pathways means that IUGR myoblasts are more responsive to even normal amounts of circulating cytokines. Unfortunately, the primary response of myoblasts to cytokines is slower differentiation (i.e., cellular transformation necessary for muscle growth). Programmed enhancement of this response directly impedes myoblast-dependent muscle growth, and the deficit is lifelong. However, identifying this mechanism is a fundamental step for developing strategies to improve muscle growth in low birth weight offspring.

Another lesson from unmapped reads: in-depth analysis of RNA-Seq reads from various horse tissues.

In recent years, a vast amount of sequencing data has been generated and large improvements have been made to reference genome sequences. Despite these advances, significant portions of reads still do not map to reference genomes and these reads have been considered as junk or artificial sequences. Recent studies have shown that these reads can be useful, e.g., for refining reference genomes or detecting contaminating microorganisms present in the analyzed biological samples. A special case of this is RNA sequencing (RNA-Seq) reads that come from tissue transcriptomes. Unmapped reads from RNA-Seq have received much less attention than those from whole-genome sequencing. In particular, in the horse, an analysis of unmapped RNA reads has not been performed yet. Thus, in this study, we analyzed the unmapped reads originating from the RNA-Seq performed through the Functional Annotation of Animal Genomes (FAANG) project in the horse, using eight different tissues from two mares. We demonstrated that unmapped reads from RNA-Seq could be easily assembled into transcripts relating to many important genes present in the sequences of other mammals. Large portions of these transcripts did not have coding potential and, thus, can be considered as non-coding RNA. Moreover, reads that were not mapped to the reference genome but aligned to the entries in NCBI database of horse proteins were enriched for biological processes that largely correspond to the functions of organ from which RNA was isolated and thus are presumably true transcripts of genes associated with cell metabolism in those tissues. In addition, a portion of reads aligned to the common pathogenic or neutral microbiota, of which the most common was Brucella spp. These data suggest that unmapped reads can be an important target for in-depth analysis that may substantially enrich results of initial RNA-Seq experiments for various tissues and organs.

Assessing Population Structure and Genetic Diversity in US Suffolk Sheep to Define a Framework for Genomic Selection.

Long-term sustainability of breeds depends on having sufficient genetic diversity for adaptability to change, whether driven by climatic conditions or by priorities in breeding programs. Genetic diversity in Suffolk sheep in the United States was evaluated in four ways: 1) using genetic relationships from pedigree data [(n = 64 310 animals recorded in the US National Sheep Improvement Program (NSIP)]; 2) using molecular data (n = 304 Suffolk genotyped with the OvineHD BeadChip); 3) comparing Australian (n = 109) and Irish (n = 55) Suffolk sheep to those in the United States using molecular data; and 4) assessing genetic relationships (connectedness) among active Suffolk flocks (n = 18) in NSIP. By characterizing genetic diversity, a goal was to define the structure of a reference population for use for genomic selection strategies in this breed. Pedigree-based mean inbreeding level for the most recent year of available data was 5.5%. Ten animals defined 22.8% of the current gene pool. The effective population size (Ne) ranged from 27.5 to 244.2 based on pedigree and was 79.5 based on molecular data. Expected (HE) and observed (HO) heterozygosity were 0.317 and 0.306, respectively. Model-based population structure included 7 subpopulations. From Principal Component Analysis, countries separated into distinct populations. Within the US population, flocks formed genetically disconnected clusters. A decline in genetic diversity over time was observed from both pedigree and genomic-based derived measures with evidence of population substructure as measured by FST. Using these measures of genetic diversity, a framework for establishing a genomic reference population in US Suffolk sheep engaged in NSIP was proposed.

Prediction of histone post-translational modification patterns based on nascent transcription data.

The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.

DNA methylation aging and transcriptomic studies in horses.

Cytosine methylation patterns have not yet been thoroughly studied in horses. Here, we profile n = 333 samples from 42 horse tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). Using the blood and liver tissues from horses, we develop five epigenetic aging clocks: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. In addition, using blood methylation data from three additional equid species (plains zebra, Grevy's zebras and Somali asses), we develop another clock that applies across all equid species. Castration does not significantly impact the epigenetic aging rate of blood or liver samples from horses. Methylation and RNA data from the same tissues define the relationship between methylation and RNA expression across horse tissues. We expect that the multi-tissue atlas will become a valuable resource.

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.