Porcine meat is the most consumed red meat worldwide. Pigs are also vital tools in biological and medical research. However, xenoreactivity between porcine's N-glycolylneuraminic acid (Neu5Gc) and human anti-Neu5Gc antibodies poses a significant challenge. On the one hand, dietary Neu5Gc intake has been connected to particular human disorders. On the other hand, some pathogens connected to pig diseases have a preference for Neu5Gc. The Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) catalyses the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. In this study, we predicted the tertiary structure of CMAH, performed molecular docking, and analysed the protein-native ligand complex. We performed a virtual screening from a drug library of 5M compounds and selected the two top inhibitors with Vina scores of -9.9 kcal/mol for inhibitor 1 and -9.4 kcal/mol for inhibitor 2. We further analysed their pharmacokinetic and pharmacophoric properties. We conducted stability analyses of the complexes with molecular dynamic simulations of 200 ns and binding free energy calculations. The overall analyses revealed the inhibitors' stable binding, which was further validated by the MMGBSA studies. In conclusion, this result may pave the way for future studies to determine how to inhibit CMAH activities. Further in vitro studies can provide in-depth insight into these compounds' therapeutic potential.

The sugar molecule N-glycolylneuraminic acid (Neu5Gc) is one of the most common sialic acids discovered in mammals. Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) catalyses the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc, and it is encoded by the CMAH gene. On the one hand, food metabolic incorporation of Neu5Gc has been linked to specific human diseases. On the other hand, Neu5Gc has been shown to be highly preferred by some pathogens linked to certain bovine diseases. We used various computational techniques to perform an in silico functional analysis of five non-synonymous single-nucleotide polymorphisms (nsSNPs) of the bovine CMAH (bCMAH) gene identified from the 1000 Bull Genomes sequence data. The c.1271C>T (P424L) nsSNP was predicted to be pathogenic based on the consensus result from different computational tools. The nsSNP was also predicted to be critical based on sequence conservation, stability, and post-translational modification site analysis. According to the molecular dynamic simulation and stability analysis, all variations promoted stability of the bCMAH protein, but mutation A210S significantly promoted CMAH stability. In conclusion, c.1271C>T (P424L) is expected to be the most harmful nsSNP among the five detected nsSNPs based on the overall studies. This research could pave the way for more research associating pathogenic nsSNPs in the bCMAH gene with diseases.

Understanding the genomic control of tissue-specific gene expression and regulation can help to inform the application of genomic technologies in farm animal breeding programs. The fine mapping of promoters [transcription start sites (TSS)] and enhancers (divergent amplifying segments of the genome local to TSS) in different populations of cattle across a wide diversity of tissues provides information to locate and understand the genomic drivers of breed- and tissue-specific characteristics. To this aim, we used Cap Analysis Gene Expression (CAGE) sequencing, of 24 different tissues from 3 populations of cattle, to define TSS and their coexpressed short-range enhancers (<1 kb) in the ARS-UCD1.2_Btau5.0.1Y reference genome (1000bulls run9) and analyzed tissue and population specificity of expressed promoters. We identified 51,295 TSS and 2,328 TSS-Enhancer regions shared across the 3 populations (dairy, beef-dairy cross, and Canadian Kinsella composite cattle from 2 individuals, 1 of each sex, per population). Cross-species comparative analysis of CAGE data from 7 other species, including sheep, revealed a set of TSS and TSS-Enhancers that were specific to cattle. The CAGE data set will be combined with other transcriptomic information for the same tissues to create a new high-resolution map of transcript diversity across tissues and populations in cattle for the BovReg project. Here we provide the CAGE data set and annotation tracks for TSS and TSS-Enhancers in the cattle genome. This new annotation information will improve our understanding of the drivers of gene expression and regulation in cattle and help to inform the application of genomic technologies in breeding programs.

Since domestication, a wide variety of phenotypes including coat color variation has developed in livestock. This variation is mostly based on selective breeding. During the beginning of selective breeding, potential negative consequences did not become immediately evident due to low frequencies of homozygous animals and have been occasionally neglected. However, numerous studies of coat color genetics have been carried out over more than a century and, meanwhile, pleiotropic effects for several coat color genes, including disorders of even lethal impact, were described. Similar coat color phenotypes can often be found across species, caused either by conserved genes or by different genes. Even in the same species, more than one gene could cause the same or similar coat color phenotype. The roan coat color in livestock species is characterized by a mixture of white and colored hair in cattle, pig, sheep, goat, alpaca, and horse. So far, the genetic background of this phenotype is not fully understood, but KIT and its ligand KITLG ( MGF ) are major candidate genes in livestock species. For some of these species, pleiotropic effects such as subfertility in homozygous roan cattle or homozygous embryonic lethality in certain horse breeds have been described. This review aims to point out the similarities and differences of the roan phenotype across the following livestock species: cattle, pig, sheep, goat, alpaca, and horse; and provides the current state of knowledge on genetic background and pleiotropic effects.

A multi-dimensional transcriptome map was generated using a diverse catalogue of tissue samples collected from six individuals of both sexes, different ages, kept in different environments and from three divergent breeds/crosses. More than 15k genes exhibit at least one potentially novel transcript in comparison with existing annotations. Tissue-specific miRNAs were detected contributing to the understanding of vital body functions in bovine. PIWI-interacting RNAs (piRNAs) were detected in ovary and testis only and, the repertoire of piRNA clusters in the male germline was expanded. A regulatory landscape was also generated including open chromatin regions, putative active promoters and transcription start sites, active enhancers, repressive states and insulators. This constitutes a key repository dataset for biology-driven genomic prediction that will help the scientific community and industry stakeholders to apply genomics to address the challenges facing cattle production.

Recently, expressed genomic information e.g. expression quantitative loci (eQTL), have been utilised in both dairy and beef breeding to link genotype to phenotype. The fine mapping of promoters (transcription start sites; TSS) and enhancers (divergent amplifying segments local to TSS) in different populations of cattle across multiple tissues can help to unravel breed- and tissue-specific phenotypes. This study provides a high-resolution analysis of TSS complexity including drivers of transcription using Cap Analysis Gene Expression (CAGE) sequencing in 3 populations of cattle (Holstein, Charolais × Holstein and Kinsella beef composite; KC). We identified 55,139 TSS and 2,543 TSS-Enhancer regions shared by 3 populations along with population-specific sets. Comparative analysis revealed a cattle-specific set of TSS. Improving our understanding of the genomic architecture of gene expression and regulation in this way can help to inform the application of genomic technologies in breeding programmes for cattle.

Maternal lineages are considered an important factor in breeding. Mitochondrial DNA (mtDNA) is maternally inherited and plays an important role in energy metabolism. It has already been associated with energy consumption and performances, e.g., stamina in humans and racehorses. For now, corresponding studies are lacking for sport performance of warmblood breeds. MtDNA sequences were available for 271 Holstein mares from 75 maternal lineages. As all mares within a lineage showed identical haplotypes regarding the non-synonymous variants, we expanded our data set by also including non-sequenced mares and assigning them to the lineage-specific haplotype. This sample consisting of 6334 to 16,447 mares was used to perform mitochondrial association analyses using breeding values (EBVs) estimated on behalf of the Fédération Équestre Nationale (FN) and on behalf of the Holstein Breeding Association (HOL). The association analyses revealed 20 mitochondrial SNPs (mtSNPs) significantly associated with FN-EBVs and partly overlapping 20 mtSNPs associated with HOL-EBVs. The results indicated that mtDNA contributes to performance differences between maternal lineages. Certain mitochondrial haplogroups were associated with special talents for dressage or show jumping. The findings encourage to set up innovative genetic evaluation models that also consider information on maternal lineages.

The insertion of an endogenous retroviral long terminal repeat (LTR) sequence into the bovine apolipoprotein B (APOB) gene is causal to the inherited genetic defect cholesterol deficiency (CD) observed in neonatal and young calves. Affected calves suffer from developmental abnormalities, symptoms of incurable diarrhoea and often die within weeks to a few months after birth. Neither the detailed effects of the LTR insertion on APOB expression profile nor the specific mode of inheritance nor detailed phenotypic consequences of the mutation are undisputed. In our study, we analysed German Holstein dairy heifers at the peak of hepatic metabolic load and exposed to an additional pathogen challenge for clinical, metabolic and hepatic transcriptome differences between wild type (CDF) and heterozygote carriers of the mutation (CDC). Our data revealed that a divergent allele-biased expression pattern of the APOB gene in heterozygous CDC animals leads to a tenfold higher expression of exons upstream and a decreased expression of exons downstream of the LTR insertion compared to expression levels of CDF animals. This expression pattern could be a result of enhancer activity induced by the LTR insertion, in addition to a previously reported artificial polyadenylation signal. Thus, our data support a regulatory potential of mobile element insertions. With regard to the phenotype generated by the LTR insertion, heterozygote CDC carriers display significantly differential hepatic expression of genes involved in cholesterol biosynthesis and lipid metabolism. Phenotypically, CDC carriers show a significantly affected lipomobilization compared to wild type animals. These results reject a completely recessive mode of inheritance for the CD defect, which should be considered for selection decisions in the affected population. Exemplarily, our results illustrate the regulatory impact of mobile element insertions not only on specific host target gene expression but also on global transcriptome profiles with subsequent biological, functional and phenotypic consequences in a natural in-vivo model of a non-model mammalian organism.

Humans frequently interact with pigs, whose meat is also one of the primary sources of animal protein. They are one of the main species at the center of sialic acid (Sia) research. Sias are sugars at terminals of glycoconjugates, are expressed at the cell surfaces of mammals, and are important in cellular interactions. N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) are notable Sias in mammals. Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) encodes the CMAH enzyme that biosynthesizes Neu5Gc. Although humans cannot endogenously synthesize Neu5Gc due to the inactivation of this gene by a mutation, Neu5Gc can be metabolically incorporated into human tissues from red meat consumption. Interactions between Neu5Gc and human anti-Neu5Gc antibodies have been associated with certain diseases and disorders. In this review, we summarized the sialic acid metabolic pathway, its regulation and link to viral infections, as well as the importance of the pig as a model organism in Sia research, making it a possible source of Neu5Gc antigens affecting human health. Future research in solving the structures of crucial enzymes involved in Sia metabolism, as well as their regulation and interactions with other enzymes, especially CMAH, could help to understand their function and reduce the amount of Neu5Gc.

Bovine mammary function at molecular level is often studied using mammary tissue or primary bovine mammary epithelial cells (pbMECs). However, bulk tissue and primary cells are heterogeneous with respect to cell populations, adding further transcriptional variation in addition to genetic background. Thus, understanding of the variation in gene expression profiles of cell populations and their effect on function are limited. To investigate the mononuclear cell composition in bovine milk, we analyzed a single-cell suspension from a milk sample. Additionally, we harvested cultured pbMECs to characterize gene expression in a homogeneous cell population. Using the Drop-seq technology, we generated single-cell RNA datasets of somatic milk cells and pbMECs. The final datasets after quality control filtering contained 7,119 and 10,549 cells, respectively. The pbMECs formed 14 indefinite clusters displaying intrapopulation heterogeneity, whereas the milk cells formed 14 more distinct clusters. Our datasets constitute a molecular cell atlas that provides a basis for future studies of milk cell composition and gene expression, and could serve as reference datasets for milk cell analysis.

Maternal lineages are important for the breeding decision in the Holstein horse breed. To investigate the genetic diversity of the maternal lineages and the relationships between founder mares, the maternal inherited mitochondrial genome (except the repetitive part of the non-coding region) of 271 mares representing 75 lineages was sequenced. The sequencing predominantly revealed complete homology in the nucleotide sequences between mares from one lineage with exceptions in 13 lineages, where differences in one to three positions are probably caused by de novo mutations or alternate fixation of heteroplasmy. We found 78 distinct haplotypes that have not yet been described in other breeds. Six of these occurred in two or three different lineages indicating a common ancestry. Haplotypes can be divided into eight clusters with all mares from one lineage belonging to the same cluster. Within a cluster, the average number of pairwise differences ranged from zero to 16.49 suggesting close maternal relationships between these mares. The results showed that the current breeding population originated from at least eight ancestral founder mares.

Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.

Background Tetradysmelia is a rare genetic disorder that is characterized by an extremely severe reduction of all limb parts distal of the scapula and pelvic girdle. We studied a Holstein Friesian backcross family with 24 offspring, among which six calves displayed autosomal recessive tetradysmelia. In order to identify the genetic basis of the disorder, we genotyped three affected calves, five dams and nine unaffected siblings using a Bovine Illumina 50 k BeadChip and sequenced the whole genome of the sire. Results Pathological examination of four tetradysmelia cases revealed a uniform and severe dysmelia of all limbs. Applying a homozygosity mapping approach, we identified a homozygous region of 10.54 Mb on chromosome 14 ( Bos taurus BTA14). Only calves that were diagnosed with tetradysmelia shared a distinct homozygous haplotype for this region. We sequenced the whole genome of the cases’ sire and searched for heterozygous single nucleotide polymorphisms (SNPs) and small variants on BTA14 that were uniquely present in the sire and absent from 3102 control whole-genome sequences of the 1000 Bull Genomes Project, but none were identified in the 10.54-Mb candidate region on BTA14. Therefore, we subsequently performed a more comprehensive analysis by also considering structural variants and detected a 50-kb deletion in the targeted chromosomal region that was in the heterozygous state in the cases’ sire. Using PCR, we confirmed that this detected deletion segregated perfectly within the family with tetradysmelia. The deletion spanned three exons of the bovine R-spondin 2 ( RSPO2 ) gene, which encode three domains of the respective protein. R-spondin 2 is a secreted ligand of leucine-rich repeats containing G protein-coupled receptors that enhance Wnt signalling and is involved in a broad range of developmental processes during embryogenesis. Conclusions We identified a 50-kb deletion on BTA14 that disrupts the coding sequence of the RSPO2 gene and is associated with bovine tetradysmelia. To our knowledge, this is the first reported candidate causal mutation for tetradysmelia in a large animal model. Since signalling pathways involved in limb development are conserved across species, the observed inherited defect may serve as a model to further elucidate fundamental pathways of limb development.

Roan (Rn) horses show a typical seasonal change of color. Their body is covered with colored and white hair. We performed a descriptive statistical analysis of breeding records of Icelandic horses to challenge the hypothesis of roan being lethal in utero under homozygous condition. The roan to non-roan ratio of foals from roan × roan matings revealed homozygous roan Icelandic horses to be viable. Even though roan is known to be inherited in a dominant mode and epistatic to other coat colors, the causative mutation is still unknown. Nevertheless, an association between roan phenotype and the KIT gene was shown for different horse breeds. In the present study, we identified KIT variants by Sanger sequencing, and show that KIT is also associated with roan in the Icelandic horse breed.

Purpose: To define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15. Methods: A genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses. Results: GWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype. Conclusions: The study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

Aberrant photoreceptor function or morphogenesis leads to blinding retinal degenerative diseases, the majority of which have a genetic aetiology. A variant in PRCD previously identified in Portuguese Water Dogs (PWDs) underlies prcd (progressive rod-cone degeneration), an autosomal recessive progressive retinal atrophy (PRA) with a late onset at 3–6 years of age or older. Herein, we have identified a new form of early-onset PRA (EOPRA) in the same breed. Pedigree analysis suggested an autosomal recessive inheritance. Four PWD full-siblings affected with EOPRA diagnosed at 2–3 years of age were genotyped (173,661 SNPs) along with 2 unaffected siblings, 2 unaffected parents, and 15 unrelated control PWDs. GWAS, linkage analysis and homozygosity mapping defined a 26-Mb candidate region in canine chromosome 20. Whole-genome sequencing in one affected dog and its obligatory carrier parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT (CanFam3.1); c.2262_c.2263insA) in CCDC66 predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Screening of an extended PWD population confirmed perfect co-segregation of this genetic variant with the disease. Western blot analysis of COS-1 cells transfected with recombinant mutant CCDC66 expression constructs showed the mutant transcript translated into a truncated protein. Furthermore, in vitro studies suggest that the mutant CCDC66 is mislocalized to the nucleus relative to wild type CCDC66. CCDC66 variants have been associated with inherited retinal degenerations (RDs) including canine and murine ciliopathies. As genetic variants affecting the primary cilium can cause ciliopathies in which RD may be either the sole clinical manifestation or part of a syndrome, our findings further support a role for CCDC66 in retinal function and viability, potentially through its ciliary function.

OBJECTIVE:To conduct a genetic and candidate gene association study with samples from phenotype-ascertained dogs to identify putative disease-associated gene/mutation for optic nerve hypoplasia (ONH) in the miniature poodle.ANIMALS STUDIED:A total of 43 miniature poodles from the United States and Europe, nine affected bilaterally with ONH, were included in the study. Pedigree information was recorded.PROCEDURES:A pedigree including all animals studied was assembled. Twenty-one genes typically expressed in ganglion cells or that are associated with ocular malformations and have a critical function in eye and neural retina development were selected. Exons and exon-intron boundaries of eight genes were sequenced in four ONH cases and four controls. Furthermore, cases and controls were genotyped with the Illumina CanineHD BeadChip to obtain genotypes for 13 additional candidate genes for haplotype association.RESULTS:The assembled pedigree connected all ONH-affected dogs to a possible common founder. Identified variants and haplotypes of the tested candidate genes did not segregate with the phenotype using Identity by Descent approach assuming autosomal recessive inheritance with variable but yet unknown penetrance.CONCLUSIONS:Pedigree analysis did not reveal the inheritance pattern. There is no evidence of association of the evaluated candidate genes with ONH; therefore, the screened candidate genes can provisionally be ruled out as causally associated with the disease.