The selection of high feed efficiency (FE) animals impacts sustainability andprofitability of beef and dairy cattle production systems. An approach toinvestigate the mechanisms of FE involves analyzing gene expression profile inliver. This study used residual feed intake as a metric of FE to select 10 Gir xHolstein crossbred cows (Girolando F1) divided into high (HE) and low (LE) FEgroups. Hepatic biopsies were used for differential gene expressioninvestigation using RNA-seq analyses which revealed 20,787 known genes mappedaccordingly to the bovine reference genome. The comparison of HE and LE revealed149 significantly differentially expressed genes (DEG), 41 up-regulated, and 108down-regulated in the LE group. Among DEG, some stood out as potential candidategenes, including DLK1, CACNG4, SLC2A12, SLC26A4, DUOX2, and DUOXA2. Functionalenrichment analyses showed pathways that potentially influence FE, such as thenegative regulation of leukocyte migration, regulation of calcium channelactivity, negative regulation of cell migration and adhesion, extracellularmatrix (ECM) organization, and thyroid hormone synthesis. ECM composition andimmune system roles were also highlighted. These results could helpunderstanding the mechanisms related to FE in dairy cattle and the developmentof selection strategies to improve this trait.

Simple SummaryHanwoo cattle are valued for their excellent meat quality and distinctive marbling; however, the genetic reasons behind the development of fat and muscle remain unclear. In this study, we analyzed gene expression in five tissues to understand how certain genes interact to shape these traits. We identified several groups of genes that were more active either in fat or in muscle. In fat tissue, many genes were related to lipid metabolism and tissue structure, whereas in muscle tissue, genes were primarily involved in energy production and mitochondrial function. Some genes, such as AGPAT5 and ARPC5, seemed to play key roles in linking these biological processes. These results give us a clearer picture of how gene networks contribute to the balance between fat and muscle in Hanwoo cattle. The findings may help improve breeding programs by enhancing marbling and reducing excess fat, ultimately supporting better beef quality and sustainable production.This study aims to characterize tissue-specific expression patterns in Hanwoo steers by identifying co-expression modules, functional pathways, and hub genes related to fat and muscle traits using Weighted Gene Co-expression Network analysis (WGCNA). RNA-Seq data were generated from three muscle tissues (longissimus muscle, tenderloin, and rump) and two fat tissues (back fat and abdominal fat) collected from six 30-month-old Hanwoo steers. Quality control of raw sequencing reads was performed using FastQC, and trimmed reads were aligned to the bovine reference genome (ARS-UCD1.3) using HISAT2. We also identified a gene co-expression network via WGCNA using normalized gene expression values. Modules were defined based on topological overlap and correlated with tissue-specific expression patterns. Modules with a significant association (p < 0.05) were used for functional enrichment based on Gene Ontology (GO) and KEGG pathways, as well as Protein–Protein Interaction Network analysis. A total of seven co-expression modules were identified by WGCNA and labeled in distinct colors (yellow, blue, red, brown, turquoise, green, black). Among them, the yellow and blue modules were positively associated with back fat, while the turquoise and green modules showed a negative correlation with abdominal fat. Additionally, the turquoise or green module was positively correlated with longissimus and rump tissues, indicating distinct gene expression patterns between fat and muscle. This study identified key co-expression modules and hub genes associated with muscle and fat metabolism. Notably, ARPC5 (blue module) was involved in lipid metabolism and energy storage, whereas AGPAT5 (turquoise module) was linked to maintaining muscle cell structure and function. These findings reveal biological mechanisms for tissue-specific gene regulation, providing targets for enhancing meat quality in Hanwoo.

Abstract This study had the objective of evaluating the feasibility of genomic prediction from biopsied bovine embryos. A total of 93 embryos were produced, derived from 28 donor cows and fertilized in vitro with semen from two bulls, all animals were of the Nellore breed. Embryo biopsies were performed, followed by DNA extraction and amplification, in addition to genotyping with the Illumina Bovine 50K v2 chip. FImpute software was used to check for Mendelian inconsistencies, correct genotyping errors and impute missing genotypes. Quality control of the genotype file was performed by eliminating non-autosomal SNPs and with the same position on the chromosome, based on the bovine reference genome ARS-UCD1.2. After quality control, analyses were performed using 34,900 SNPs from the 50K chip and 615,397 SNPs from the HD chip. The quality of genotyping was assessed by the call rate, which presented an average of 93%, ranging from 75.1% to 97.9%. It is worth noting that approximately 80% of the samples presented a call rate above 90%. After imputing the genotypes for the high-density panel, the call rate of the samples reached 100%, correcting all Mendelian inconsistencies. Heterozygosity increased from 11.7% in observed genotypes to 22.5% in imputed genotypes. The determination of the sex of the embryos was performed based on the heterozygosity of the X chromosome, identifying 41 males and 19 females, while 33 embryos did not show conclusive heterozygosity. The embryos were transferred, and the pregnancy rate of the recipient cows was 31%, similar to the control group of non-biopsied embryos, at 24%. All embryos had their genomic genetic values estimated, in addition to the selection index calculated according to the methodology used by the genetic breeding program of which the ranch participates. The average selection index calculated for all embryos was 11.25, with a minimum of 4.54 and a maximum of 20.06. Thus, the study showed that genotyping and genetic prediction from biopsied embryos can be feasible in Nellore cattle breeding programs. However, challenges related to DNA extraction and amplification still affect the quality of genotypes, making it essential to use techniques such as imputation to improve the accuracy of results.

Staphylococcus aureus is a major zoonotic bacterial pathogen that causes a broad spectrum of human and animal diseases, including skin infections, sepsis, endocarditis, and bovine mastitis. In the geographically isolated setting of New Zealand, the population structure of S. aureus exhibits a distinct genomic profile. Globally, bovine S. aureus isolates are primarily associated with sequence types (STs), ST97 and ST151, whereas in New Zealand, ST1 predominates, accounting for approximately 70% of bovine isolates. ST1 is also a clinically significant sequence type in humans. This study employed a comparative One Health approach to investigate genetic differences in 520 S. aureus ST1 isolated from bovine milk and human clinical samples. We aimed to explore genomic features associated with persistence and diversification across hosts, focusing on antimicrobial resistance (AMR), virulence, and mobile genetic elements. Comparative genomics revealed that human isolates carried a significantly higher burden of AMR genes, consistent with clinical selective pressure. In contrast, 83% of bovine isolates harbored a prophage (φSabovST1) similar to S. aureus prophage φSaov3. This prophage encodes bovine-adapted leukocidins (LukMF'), supporting host-specific adaptation. Phylogenetic analysis revealed long branches, suggesting insufficient sampling, highlighting the need for broader genomic surveillance to resolve evolutionary relationships and transmission dynamics of S. aureus ST1 in New Zealand. These findings highlight the complex history of host interactions, historical transmission events, and ongoing bacterial adaptation. Expanding sampling efforts across human, animal, and environmental reservoirs will provide deeper insights into strain diversity, elucidate transmission pathways, and inform strategies to mitigate zoonotic risks.IMPORTANCEThis study presents a comprehensive genomic analysis of S. aureus ST1, a lineage that is unusually dominant in both bovine and human populations in New Zealand. Leveraging New Zealand's geographical isolation, we provide critical insights into the persistence, diversification, and adaptation of S. aureus, offering valuable knowledge to advance disease prevention in both public and veterinary health and strengthening global biosecurity. The development of the first bovine ST1 reference genome serves as a valuable resource for future research, while the identification of a novel prophage (φSabovST1) carrying bovine-specific leukocidins underscores the role of mobile genetic elements in host specificity and virulence. Human isolates exhibited a higher prevalence of antimicrobial resistance genes. Phylogenetic analysis further revealed two main circulating clades of ST1 with interspersed host origins, highlighting the critical need for integrated One Health approaches to more effectively monitor and manage zoonotic pathogens across agricultural and public health systems.

Mastitis is an inflammatory reaction caused by bacterial infection of the teat, and a relationship between its onset and cattle major histocompatibility complex (BoLA) region has been reported. However, no comprehensive genetic analysis of mastitis caused by environmental streptococci has been reported. Here, we resequenced the BoLA region using a hybridisation capture target next-generation sequencing (NGS) method to identify disease susceptibility markers mapped to the BoLA region in environmental streptococcal mastitis. This study examined 75 cows with mastitis caused by environmental streptococci selected from 1641 cows with mastitis and 222 healthy cows without mastitis in Japan. Targeted sequences obtained from MiSeq NGS were aligned to the bovine reference genome (ARS-UCD1.2/bosTau9), and 2,920,355 variants were detected within the BoLA region of the 297 Holstein cattle. In an association study using 2264 variants after quality control, the top 20 variants with the lowest P values were selected and assigned to the 18 surrounding candidate genes, and a gene network analysis of these genes resulted in the narrowing down of five candidate genes POU5F1, IER3, GNL1, ABCF1, and PRR3. Multivariate effect analysis of all 6 SNPs associated with these 5 genes revealed that they were significantly correlated with mastitis, indicating that they were useful for classification of mastitis-resistant and mastitis-susceptible cattle. This is the first report to identify SNPs associated with environmental streptococcal mastitis with an NGS-based association study using targeted resequencing in the BoLA region, and understanding host factors may provide important clues for mastitis control.

Domestic cattle (Bos taurus) play a significant role in human society as they provide abundant food resources and contribute to the development of agriculture and traditional culture. Dengchuan cattle, a local breed from Yunnan, Southwest China, are known for their high-quality milk and are at risk of extinction due to crossbreeding. To preserve the superior genetic resources of Dengchuan cattle, this study conducted whole-genome sequencing of 54 Dengchuan cattle using blood DNA samples, generating approximately 3.56 TB of clean data with an average sequencing depth of 32.78X. The sequencing data were aligned to the bovine reference genome (ARS-UCD2.0), achieving an average alignment rate of 99.85%. A total of 9,950,420 SNPs and 2,476,207 indels were detected using variant calling workflow. These data were utilized to characterize genomic profile of this unique cattle breed. The data generated in this study can be incorporated into the global cattle genomic diversity database, providing valuable information for comparative studies on cattle.

Abstract Short interspersed nuclear elements (SINEs) constitute a class of nonautonomous retrotransposons endowed with the capability to multiply and spread within a genome. SINEs serve as reliable indicators for assessing genetic variability in dairy populations, aiding in precise determination of breed composition. However, SINEs within the bovine genome remain insufficiently characterized, hindering the understanding of their impact on dairy production and health. This study aimed to systematically identify, classify and unveil the characteristics of SINEs within the bovine reference genome (ARS-UCD1.3). Multiple de novo identification tools, including RepeatModeler, SINE_scan, LongRepMarker, and SINE-finder, were employed for SINE identification and characterization. A total of 1.6 million copies of SINEs were identified, constituting approximately 10.8% of the genome. The SINEs were stratified into seven distinct families based on their structural attributes, denoted as BosSINEL, BosSINE1, BosSINE2, BosSINE3, BosSINE4, BosSINE5 and BosSINE6. BosSINE1 represents a novel family, whereas the remaining six families were previously documented in Repbase. Among these families, BosSINE3 emerges as the most prolific, boasting 254,586 complete copies (over 80% of the length of the consensus sequence), followed by BosSINEL (164,538 copies), BosSINE2 (162,207 copies) and BosSINE1 (150,799 copies). Conversely, the remaining families exhibit lower abundance, each harboring no more than 100,000 copies within the genome. Structural comparisons among SINE families elucidate distinctive features. Notably, BosSINEL lacks a tRNA-related region, including boxA and boxB, which is the hallmark element of the classic SINE structure according to SINEBase. Conversely, all other families exhibit tRNA-related regions. BosSINE1, the lengthiest at 328 bp (excluding Poly-A), possesses two tRNA-related regions, distinguishing it from other families. BosSINEL, BosSINE1, BosSINE2 and BosSINE3 exhibit long interspersed nuclear elements (LINE)-related regions, suggesting their retrotransposition by engaging with the enzymatic machinery encoded by LINEs. In contrast, BosSINE6, the shortest at 109 bp, features a GC-rich region resulting in a high GC content of 67%. Additionally, BosSINE4 contains an AT-rich region. Evolutionary analysis disclosed that BosSINEL is the youngest SINE family in the bovine genome, potentially still harboring active roles and contributing to the genetic diversity related to different dairy production and health performance. Conversely, BosSINE1 to BosSINE6 are comparatively aged in terms of evolution, likely fixed in the bovine genome with diminished or lost activities. In summary, this investigation unveils the comprehensive profile of SINE in the bovine genome, delineating both structural and evolutionary characteristics. Particularly noteworthy is the emergence of BosSINEL as the youngest SINE family, suggesting active roles in the bovine genome. Since lineage-specific TEs (such as Bov-A2 elements) have been reported to regulate interferon-inducible gene expression in bovine, our data warrants further investigations to reveal the potential of SINEs as markers for improving dairy production and health.

Abstract Unique genetic patterns in the genome of individuals who have experienced a selection process are called selection signatures. These signatures can identify genomic regions with phenotypic effects. In the case of dairy cattle, the Holstein breed was intensively selected to maximize productivity in temperate environments. In contrast, zebu cattle are better adapted to tropical and subtropical environments but with decreased productivity. The dairy Gir cattle is the most important dairy zebu in Brazil for its adaptation to the tropical environment. This study aimed to identify selection signatures between Holstein and Dairy Gir. Samples of 42 sires from the National Dairy Gir Breeding Program in Brazil underwent whole-genome sequencing (WGS), were aligned to the ARS_UCD1.2 bovine reference genome and processed according to the 1000 Bull genome project (1KBull) pipeline. WGS data from 307 Holstein animals from Canada and the United States from the 1KBull (Run 9) were used. Phasing was performed using Beagle, and selection signatures between populations were investigated using the extended haplotype homozygosity (XP-EHH) test with windows of 50 Kb containing ≥ 5 SNPs in the “rehh” R package. The top and bottom 0.5% extreme windows were considered potential selection signatures. Subsequently, a functional enrichment analysis (P-value &amp;lt; 0.05) of genes overlapping the selection signatures was performed. For the Holstein population, 153 selection signatures were detected, harboring 160 genes. Terms related to ovarian steroidogenesis, cardiovascular system, lipid absorption, sulfur, fatty acid, and lipid metabolism were enriched. Disruptions in sulfur metabolism were previously observed in cows with clinical mastitis in Holstein. Fatty acid and lipid metabolism are related to negative energy balance, which increases lipid mobilization, leading to loss of body condition and greater concentrations of non-esterified fatty acids in circulation. This may further affect fertility due to the excess accumulation of lipids in the oocytes and endometrium, mainly because the postpartum uterus is remodelling. The intensive selection for milk production in Holstein is negatively associated with reproduction. The potential link between disruptions in lipid metabolism with inflammation and reproductive health may ultimately lead to an increase in days open. For the Dairy Gir population, 198 selection signatures flanking 220 genes were detected. Unlike the Holstein population, the genes under selection pressure in the Gir population were predominantly enriched to cell differentiation and organization, amine and amino acid metabolism, and protein metabolism, digestion, and absorption. Exposure to heat stress may cause ruminal epithelium damage, affecting DNA replication and amino acid metabolism. Amino and fatty acids are important for directing energy production to promote damaged tissue repair. Thus, these regions are candidates for playing an important role in heat tolerance. In conclusion, different genetic regions under selection pressure between populations were identified, indicating breed-specific adaptations and mechanisms underlying milk production, metabolic disorders, and reproduction.

Abstract Essential (E) amino acids (AA) may alter bovine mammary epithelial cell functions by impacting mRNA expression of genes through their upstream transcription factors. The objective of this research was to study the impact of EAA deficiencies on gene expression and to identify upstream transcriptional factors mediating their effects in bovine mammary epithelial cells. Epithelial cells were collected from the mammary glands of lactating dairy cows and cultured in combined media of DMEM/F12 and Medium 170, differentiated for 5 d, and subsequently treated in triplicate for 60 h with a control medium containing all EAA at normal plasma concentrations for cows (CTL), or a medium with 20 μM Lys (LY), 5 μM Met (LM), or 10 μM His (LH), which are each 25% of normal concentration. RNA was isolated from the cells and sequenced on an Illumina NovaSeq 6000 producing 150 base pair paired-end reads with a minimum sequencing depth of 150 million reads. Reads were cleaned with ‘fastp’ (Chen et al., 2018), aligned to the bovine reference genome (ARS-UCD1.2) using ‘STAR’ aligner (Dobin et al., 2013), and genes counted using ‘featureCounts’ (Liao et al., 2014). Differential expression of genes between treatment pairs was determined using the ‘DESeq2’ package (Love et al., 2014) in R. Genes with an absolute log2 fold change ≥ 1 and a false discovery rate of &amp;lt; 0.05 were considered differentially expressed. The number of differentially expressed genes relative to CTL was 780 for LY, 383 for LH, and 536 for LM. QIAGEN Ingenuity Pathway Analysis (IPA) identified four pathways consistently affected across LY, LM, and LH: kinetochore metaphase signaling, mitotic roles of polo-like kinase, cell cycle: G2/M DNA damage checkpoint regulation, and interferon signaling (Figure 1-3). Notably, the kinetochore metaphase signaling pathway was identified as the most significant across all treatments, suggesting a strong link to cell proliferation and apoptosis during EAA deficiency. The interferon signaling might be related to the Janus kinase 2/signal transducer and activator of transcription 5 (Jak2/STAT5) pathway which can promote the expression of genes encoding milk proteins such as β-casein and β-lactoglobulin. Furthermore, the IPA identified upstream transcription factors related to cell proliferation, apoptosis, autophagy, protein translation, endoplasmic reticulum biogenesis, and amino acid metabolism including NUPR1, FOXM1, MYC, CCND1, TP53, DDIT3, and ATF4 across all treatments. The findings suggest that AA deprivation regulates bovine mammary epithelial cell function by instigating amino acid response pathways and altering secretory cell number.

Abstract Most embryo losses occur in the first trimester of pregnancy in cows, resulting in negative economic impact for dairy production systems. The current scientific paradigm implies the conceptus evades rejection by the maternal immune system. However, the cellular and molecular mechanisms behind the maternal immune response to the growing embryo have not been fully characterized. Hence, this study examined gene expression profiles of peripheral white blood cells (PWBC) from pregnant cows at 21 days after an embryo was transferred, (Bos taurus taurus), and cows that were treated equally but lost the embryo. We investigated the effect of pregnancy status on differential gene expression in PBMCs. We obtained and compared the total transcripts of PWBC from heifers that became pregnant at day 21 (N = 5) or failed to become pregnant after the embryo transfer (N = 5t). The total RNA transcripts, where an average of 96 % of the total reads were successfully mapped to the bovine reference genome. Each sample generates between 32 to 43 million reads. Sequencing data can be accessed by GEO with the accession number GSE210665. There was no need to remove samples in this study. Based on the length and abundance of the reads that cover the bovine genomic, we conclude that deep sequencing is suitable for the experiment outcome. A total of 13.167 genes were tested for differential expression by controlling the false discovery rate (FDR). A positive fold change denotes an overexpression and a negative fold change value a downregulation gene between treatments. After statistical analysis, 682 of these genes present a P-value smaller than 0.01, a total of 302 genes were upregulated, and 382 downregulated. The top 7 expression fold change top upregulated (logFC &amp;gt; 2) genes were COL1A2, LOC100337213, H2AC18, HTRA1, MMP14, CD5L, and LOC100297044. The top 4 downregulated (logFC&amp;lt; − 2) were ADAMDEC1, MYO1A, RPL39, and Histone H4. These data show differentially expressed genes associated with pregnancy status were both upregulated and downregulated as a response to early pregnancy, or a failure to become pregnant after embryo transfer. Our results show that these are key maternal genes for promoting tolerance allowing developing of the embryo. In summary, this study presented the genes related to positive regulation of inflammatory chemokine activity and immune defense response in the maternal PWBC.

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼104 median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼1010 cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

Abstract Heifer pregnancy (HPG) is lowly heritable, yet economically relevant in beef cattle production systems. Hence, identifying single nucleotide polymorphisms (SNP) associated with phenotype could increase the amount of information used in the genetic evaluation of the trait. Using RNA-sequence data from lactating Holstein cows, a set of SNP (n = 69) were discovered among embryo mortality conceptuses (Conceptus-SNP) associated with maternal recognition of pregnancy on day 16 after artificial insemination (AI). To further study these SNP, our objective was to conduct analyses to identify markers associated with HPG in Red Angus. Phenotypic records and genotypes were provided by the Red Angus Association of America (RAAA). For phenotypes, a total of 59,387 cattle records, from the June 2022 RAAA genetic evaluation, were used in the analysis. For genotype data, a 47,860 SNP panel (excluding SNP on the X-chromosome) were formed by combining the historical RAAA genotype data (96,162 animals with genotypes) and genotypes from whole genome sequence of 30 prominent Red Angus AI sires. Markers were mapped to the ARS-UCD1.2 bovine reference genome and then imputed to 47,860 SNP panel using findhap software. A subset of SNP (n = 2,169 SNP; Conceptus-SNP and the multi-breed selected reduced panel [MSRP]) were extracted from the imputed genotypes file. Using PLINK software, genotype quality control procedure was performed so that animals with call rate &amp;lt; 0.9 were removed. A marker was removed if: it had minor allele frequency &amp;lt; 0.01, not in Hardy–Weinberg equilibrium (P &amp;lt; 0.00001), or in high linkage disequilibrium (r2 &amp;gt; 0.95). The final genotype file contained 96,667 animals with 2,137 SNP. A Bayes Cπ single-step super hybrid model (π = 0.99), using the BOLT software package, was used to estimate marker’s posterior probability of inclusion (PPI), the marker significance criterion in predicting HPG. A marker’s PPI was calculated as the average probability from four different Gibbs sampling chains of 75,000 iterations that included 5,000 iterations as burn-in. Markers rs42576394 (18:60583784) and rs42942055 (14:34249373) had PPI of 25.5 and 7.1%, respectively; suggesting association with HPG. None of the Conceptus-SNP had a detectable association with HPG; however, these Conceptus-SNP may be much more informative for genotype to phenotype association analyses for the trait of stayability. Such a trait would be measured with lactating mature beef cows and potentially more similar to the lactating Holstein cows from which the markers were discovered.

The high genetic heterogeneity and environmental effects of subfertility in livestock species make the elucidation of the genetic mechanisms associated with reproductive efficiency a difficult task. Network and co-expression network meta-analyses were applied alongside genetic variant calling and long non-coding RNA (lncRNA) characterization to identify functionally relevant target genes and regulatory subnetworks associated with fertility in dairy cattle. In total, 505 lncRNAs (441 previously annotated in the bovine reference genome ARS-UCD 1.2 and 64 novel lncRNAs) were identified. Seven differentially expressed genes between high-fertile (HF) and sub-fertile (SF) Holstein cows were identified in the network meta-analysis (CA5A, ENSBTAG00000051149, ENSBTAG00000003272, DEFB7, DIO2, TRPV3, and COL4A4). Additionally, seven functional candidate differentially co-expressed (DcoExp) modules with a differential regulatory pattern (|z-score|>2) were identified between HF and SF cows. The functional candidate genes and DcoExp modules identified were associated with fertility relevant processes such as the regulation of embryonic implantation and proliferation, interaction and molecule transfer between the fetus and the cow, and the immune system. These results help to better understand the genetic mechanisms associated with reproductive efficiency in dairy cattle through the identification of potential biomarkers and genetic variants associated with differentially expressed regulatory gene and lncRNAs regulatory element networks.

Several ocular pathologies in cattle, such as ocular squamous cell carcinoma and infectious keratoconjunctivitis, have been associated with low pigmentation of the eyelids. The main objective of this study was to analyze the transcriptome of eyelid skin in Hereford cattle using strand-specific RNA sequencing technology to characterize and identify long noncoding RNAs (lncRNAs). We compared the expression of lncRNAs between pigmented and unpigmented eyelids and analyzed the interaction of lncRNAs and putative target genes to reveal the genetic basis underlying eyelid pigmentation in cattle. We predicted 4,937 putative lncRNAs mapped to the bovine reference genome, enriching the catalog of lncRNAs in Bos taurus. We found 27 differentially expressed lncRNAs between pigmented and unpigmented eyelids, suggesting their involvement in eyelid pigmentation. In addition, we revealed potential links between some significant differentially expressed lncRNAs and target mRNAs involved in the immune response and pigmentation. Overall, this study expands the catalog of lncRNAs in cattle and contributes to a better understanding of the biology of eyelid pigmentation.

Creation of the bovine reference assembly paved the way to develop the high-throughput genotyping arrays of the single nucleotide polymorphisms (SNPs) based on the available map coordinates that facilitated major advances in gene mapping and selection programs. The assembly flaws, however, may cause false results in the downstream gene mapping studies. The most recent bovine reference genome (ARS-UCD1.2) is built on the long-read sequences that provides improved quality and continuity. By applying population genetic metrics in this study, we aimed to evaluate the map coordinates to which SNP markers were assigned. We employed a three-step approach by combining the recombination and linkage disequilibrium analyses to test if the markers fit into the assigned physical map coordinates. We applied the method to the bovine 50k array in a large pedigree of Holstein cattle and revealed a panel of 65 candidate markers, most of which were re-located either on a different chromosome or re-mapped as far as several million base pairs away on the same chromosome. This list of candidates accounts for 0.1% of the SNPs in the widely used 50k genotyping array and we foresee a reasonably larger set of markers being misplaced in the BovineHD 700K BeadChip. We suggest pre-removal of the candidate misplaced markers to reduce false signals in association mapping studies.

BackgroundReference-guided read alignment and variant genotyping are prone to reference allele bias, particularly for samples that are greatly divergent from the reference genome. A Hereford-based assembly is the widely accepted bovine reference genome. Haplotype-resolved genomes that exceed the current bovine reference genome in quality and continuity have been assembled for different breeds of cattle. Using whole genome sequencing data of 161 Brown Swiss cattle, we compared the accuracy of read mapping and sequence variant genotyping as well as downstream genomic analyses between the bovine reference genome (ARS-UCD1.2) and a highly continuous Angus-based assembly (UOA_Angus_1).ResultsRead mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. We discovered 22,744,517 and 22,559,675 high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. The concordance between sequence- and array-called genotypes was high and the number of variants deviating from Hardy-Weinberg proportions was low at segregating sites for both assemblies. More artefactual INDELs were genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. Using the 161 sequenced Brown Swiss cattle as a reference panel, we imputed sequence variant genotypes into a mapping cohort of 30,499 cattle that had microarray-derived genotypes using a two-step imputation approach. The accuracy of imputation (Beagle R2) was very high (0.87) for both assemblies. Genome-wide association studies between imputed sequence variant genotypes and six dairy traits as well as stature produced almost identical results from both assemblies.ConclusionsThe ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. Although differences in read mapping and genotyping accuracy between both assemblies are negligible, the choice of the reference genome has a large impact on detecting signatures of selection that already reached fixation using the composite likelihood ratio test. We developed a workflow that can be adapted and reused to compare the impact of reference genomes on genome analyses in various breeds, populations and species.

Bovine Respiratory Syncytial Virus (BRSV) is a primary viral cause of Bovine Respiratory Disease (BRD) in young calves, which is responsible for substantial morbidity and mortality. Infection with BRSV induces global gene expression changes in respiratory tissues. If these changes are observed in tissues which are more accessible in live animals, such as whole blood, they may be used as biomarkers for diagnosis of the disease. Therefore, the objective of the current study was to elucidate the whole blood transcriptomic response of dairy calves to an experimental challenge with BRSV. Calves (Holstein–Friesian) were either administered BRSV inoculate (103.5 TCID50/ml × 15 ml) (n = 12) or sterile phosphate buffered saline (n = 6). Clinical signs were scored daily and whole blood was collected in Tempus RNA tubes immediately prior to euthanasia, at day 7 post-challenge. RNA was extracted from blood and sequenced (150 bp paired-end). The sequence reads were aligned to the bovine reference genome (UMD3.1) and EdgeR was subsequently employed for differential gene expression analysis. Multidimensional scaling showed that samples from BRSV challenged and control calves segregated based on whole blood gene expression changes, despite the BRSV challenged calves only displaying mild clinical symptoms of the disease. There were 281 differentially expressed (DE) genes (p < 0.05, FDR < 0.1, fold change > 2) between the BRSV challenged and control calves. The top enriched KEGG pathways and gene ontology terms were associated with viral infection and included “Influenza A”, “defense response to virus”, “regulation of viral life cycle” and “innate immune response”. Highly DE genes involved in these pathways may be beneficial for the diagnosis of subclinical BRD from blood samples.

In the current study, allele specific expression analysis was performed in two subspecies cows (Bos taurus and Bos indicus) at SNP and gene levels. RNA-Seq data of 21,078,477 and 20940063 paired end reads from pooling of whole blood samples (Leukocyte) from 40 US Holstein (Bos Taurus) and 45 Cholistani cows (Bos indicus) obtained from SRA database in NCBI. Quality control and trimming of row RNA-Seq data were processed by FASTQC and Trimmomatic softwares. The transcriptome was assembled by TopHat2 software in two cow's population by aligning and mapping the RNA-Seq reads on bovine reference genome. The SNPs were discovered by Samtools software and ASE analysis was performed by Chi-square test. Results showed that 50183 and 137954 SNPs were discovered on the assembled transcriptome of Holstein and Cholistani cow samples, respectively, and 15308 SNPs were common in both breeds. 10158 SNPs from 50183 (20%) in Holstein and 31523 SNPs from 137954 (23%) in Cholistani cows were identified as ASE-SNPs. Reference allele and alternative allele count in Holstein and Cholistani cows were 3041 and 7155, respectively. Among 131 discovered SNPs in 41 genes with different expression in Holstein and Cholistani cows, 31 ASE-SNPs (5 in Holstein; 26 in Cholistani cows) were discovered.

Abstract Pregnancy failure in dairy cattle appears to be attributable to losses during embryogenesis, mainly within the first month. Models have estimated that pregnancies lost after day 30 of fertilization cost producers US $550/cow. The reasons for these losses are not well understood, and the transcriptome profile of the reproductive tract during the establishment of pregnancy may point to mechanisms. Thirty-eight cows in lactations 1 to 3 were artificially inseminated, and endometrial biopsies were collected on day 15 after artificial insemination to investigate the effect of parity on the preimplantation pregnancy outcome. Pregnancy status was determined by the presence of interferon-tau in the uterine flushing, revealing 19 pregnant (P) and 19 non-pregnant (NP) cows. Nineteen biopsy samples [9 cows in lactation 1 (6 P and 3 NP), 4 in lactation 2 (2 P and 2 NP), and 6 in lactation 3 (3P and 3NP)] with an average RNA integrity number of 7 were selected for RNA sequencing with an Illumina HiSeq analyzer. Sequence reads were assembled to the ARS_USD1.2.99 bovine reference genome using the CLC genomics workbench software. On average, the samples generated ~56 million reads and 94.99% were mapped to the reference genome. Differential gene expression analysis between P and NP cows identified 187, 60, and 136 differentially expressed genes (DEG) in lactation 1, 2 and 3, respectively (P &amp;lt; 0.01, FDR &amp;lt; 0.05, FC &amp;gt; ±2). Metabolic pathway enrichment analysis identified several DEG upregulated in lactations 2 and 3 associated with IL-17 signaling pathway, although expression of IL-17 itself was not different. This pathway is part of the protective response against extracellular bacteria and likely participates in pregnancy maintenance through local regulation of immune function. Further functional genomic analyses will be performed to determine additional metabolic pathways, key regulator genes, and functional SNPs associated with the establishment of pregnancy.

Abstract Reduced fertility is one of the main causes of economic losses in dairy farms. The financial losses are estimated in US$938 per stillbirth case in Holstein herds. The identification of genomic regions associated with stillbirth could help to develop better management and breeding strategies aiming to reduce the frequency of undesirable gestation outcomes. The same cow may show different outcomes in different gestations. Therefore, evaluation of a single time-point may provide a biased indication of the genetic causes of stillbirth. A weighted single-step genomic best linear unbiased prediction (WssGBLUP) was performed using the BLUPF90 software. A total of 14,145 cows (with three or more delivery records) were genotyped for 42,909 SNP markers mapped against the ARS-UCD1.2 bovine reference genome. Three rounds of WssGBLUP were performed using 50,541 stillbirth records (4.8±1.3 records per animal). In total, 5,796 stillbirth cases were observed out of the 50,541 records (~11%). The statistical model included: calf size, calf sex, and dam age class (&amp;lt; 36, 36–50, 50–63, 63–75, ³75 months) as fixed effects; and herd-year-season, cow additive genetic merit, cow permanent environment, and service sire as random effects. Nine windows explaining more than one percent of the total genetic variance were identified, where the top 3 windows explained 7.86% (BTA13:37558814-38550118), 4.92% (BTA18:59897080-60892357) and 2.17% (BTA12:80568320-81508005). The top candidate window, on BTA13, harbors the OVOL2 gene, which codifies a zinc-finger protein crucial for embryo development during the angiogenesis, heart formation, hematopoietic, endothelial, neural tube and placental development. Additionally, OVOL2 is crucial during the spermatogenesis and was already identified as down-regulated between ectopic and eutopic endometrial tissues in woman with endometriosis. These results pinpoint new genomic candidate regions for stillbirth in Holstein, helping to better understand the genetic mechanisms associated with fertility problems.