Search

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne's disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity., (Copyright © 2024 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Mastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry. Multi-omics approaches enable the comprehensive investigation of the complex interactions between multiple layers of information to provide a more holistic view of disease pathogenesis. Therefore, this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data (mRNA and lncRNA), small RNA sequencing data (miRNA) and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes., Results: Functional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis, provided further insights into subclinical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis. The abundant genomic and epigenomic signatures with significant alterations related to subclinical mastitis were observed, including 30,846, 2552, 1276 and 57 differential methylation haplotype blocks (dMHBs), differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs), respectively. Next, 5 factors presenting the principal variation of differential multi-omics signatures were identified. The important roles of Factor 1 (DEG, DEM and DEL) and Factor 2 (dMHB and DEM), in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed. Each of the omics within Factors 1 and 2 explained about 20% of the source of variation in subclinical mastitis. Also, networks of important functional gene sets with the involvement of multi-omics signatures were demonstrated, which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis. Furthermore, multi-omics integration enabled the association of the epigenomic regulatory factors (dMHBs, DELs and DEMs) of altered genes in important pathways, such as 'Staphylococcus aureus infection pathway' and 'natural killer cell mediated cytotoxicity pathway', etc., which provides further insights into mastitis regulatory mechanisms. Moreover, few multi-omics signatures (14 dMHBs, 25 DEGs, 18 DELs and 5 DEMs) were identified as candidate discriminant signatures with capacity of distinguishing subclinical mastitis cows from healthy cows., Conclusion: The integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis, which may ultimately lead to the development of more effective mastitis control and management strategies., (© 2024. His Majesty the King in Right of Canada.)

Background: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis., Results: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield)., Conclusions: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures., (© 2024. His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada.)

Peste des petits ruminants (PPR) cause severe economic losses to many countries of the world where the disease is endemic. It has been targeted for global eradication by 2030 following the successful eradication of rinderpest in 2011. The proposed eradication program would benefit from efficient and relatively reliable diagnostic tools for early PPR virus (PPRV) detection. A total of 33 eight to 12 months old West African Dwarf (WAD) goats were used. Nineteen goats infected by commingling with two PPR virus-positive animals formed the infected group (PPRV-infected goats) while 14 non-infected goats formed the control group (CTG). The suitability of hydroxyl naphthol blue (HNB) staining of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and haemagglutination (HA) assays was compared for their sensitivity to detect the PPRV in PPRV-infected goats and non-infected CTG. PPR disease severity in WAD goats at different days post infection (dpi) was evaluated by clinical scoring and haemagglutination titre (HAT). HNB staining RT-LAMP reaction and HA showed sensitivities of 100% and 73.68%, respectively, for PPRV detection. Expression of PPR clinical signs began from 3 dpi, attained peak at 5 dpi, thereafter showed irregular patterns till 24 dpi. Evaluation of HAT in PPRV-infected goats at 12 dpi ranged from 2 to 64 haemagglutination units (HAU), while CTG goats had 0 HAU. In conclusion, HA could be a good tool for rapid diagnosis of PPRV in a developing country setting. However, HNB staining RT-LAMP assay demonstrated high sensitivity for accurate diagnoses of PPRV and as an important diagnostic tool when precise phenotyping is desired., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection., (Crown Copyright © 2023. Published by Elsevier B.V. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the Open Government Licence (https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/).)

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5' untranslated region (5' UTR) of HOXB13 is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep., (© 2023 Li et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied., Results: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG., Conclusion: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance., (© 2022. The Author(s).)

Background: Long noncoding RNAs (lncRNAs) are now proven as essential regulatory elements, playing diverse roles in many biological processes including mammary gland development. However, little is known about their roles in the bovine lactation process., Results: To identify and characterize the roles of lncRNAs in bovine lactation, high throughput RNA sequencing data from Jersey (high milk yield producer), and Kashmiri cattle (low milk yield producer) were utilized. Transcriptome data from three Kashmiri and three Jersey cattle throughout their lactation stages were utilized for differential expression analysis. At each stage (early, mid and late) three samples were taken from each breed. A total of 45 differentially expressed lncRNAs were identified between the three stages of lactation. The differentially expressed lncRNAs were found co-expressed with genes involved in the milk synthesis processes such as GPAM, LPL, and ABCG2 indicating their potential regulatory effects on milk quality genes. KEGG pathways analysis of potential cis and trans target genes of differentially expressed lncRNAs indicated that 27 and 48 pathways were significantly enriched between the three stages of lactation in Kashmiri and Jersey respectively, including mTOR signaling, PI3K-Akt signaling, and RAP1 signaling pathways. These pathways are known to play key roles in lactation biology and mammary gland development., Conclusions: Expression profiles of lncRNAs across different lactation stages in Jersey and Kashmiri cattle provide a valuable resource for the study of the regulatory mechanisms involved in the lactation process as well as facilitate understanding of the role of lncRNAs in bovine lactation biology., (© 2022. The Author(s).)

Milk is an excellent source of nutrients for humans. Therefore, in order to enhance the quality and production of milk in cattle, it is interesting to examine the underlying mechanisms. A number of new investigations and research have found that, circRNA; a specific class of non-coding RNAs, is linked with the development of mammary gland and lactation. In the present study, genome wide identification and expression of the circRNAs in mammary epithelial cells of two distinct cattle breeds viz Jersey and Kashmiri at peak lactation was conducted. We reported 1554 and 1286 circRNA in Jersey and Kashmiri cattle, respectively, with 21 circRNAs being differentially expressed in the two breeds. The developmental genes of the established differentially expressed circRNAs were found to be largely enriched in antioxidant activity, progesterone, estradiol, lipid, growth hormone, and drug response. Certain pathways like MAPK, IP3K and immune response pathways were found significantly enriched in KEGG analysis. These results add to our understanding of the controlling mechanisms connected with the lactation process, as well as the function of circRNAs in bovine milk synthesis. Additionally, the comparative analysis of differentially expressed circRNAs showed significant conservation across different species., Competing Interests: Syed Mudasir Ahmad is an Academic Editor for PeerJ. Eveline M. Ibeagha-Awemu is employed by Agriculture and Agri-Food Canada., (©2022 Syed et al.)

Johne's Disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), is an incurable disease of ruminants and other animal species and is characterized by an imbalance of gut immunity. The role of MAP infection on the epigenetic modeling of gut immunity during the progression of JD is still unknown. This study investigated the DNA methylation patterns in ileal (IL) and ileal lymph node (ILLN) tissues from cows diagnosed with persistent subclinical MAP infection over a one to 4 years period. DNA samples from IL and ILLN tissues from cows negative (MAPneg) ( n = 3) or positive for MAP infection (MAPinf) ( n = 4) were subjected to whole genome bisulfite sequencing. A total of 11,263 and 62,459 differentially methylated cytosines (DMCs), and 1259 and 8086 differentially methylated regions (DMRs) (FDR<0.1) were found between MAPinf and MAPneg IL and ILLN tissues, respectively. The DMRs were found on 394 genes (denoted DMR genes) in the IL and on 1305 genes in the ILLN. DMR genes with hypermethylated promoters/5'UTR [3 (IL) and 88 (ILLN)] or hypomethylated promoters/5'UTR [10 (IL) and 25 (ILLN)] and having multiple functions including response to stimulus/immune response ( BLK, BTC, CCL21, AVPR1A, CHRNG, GABRA4, TDGF1 ), cellular processes ( H2AC20, TEX101, GLA, NCKAP5L, RBM27, SLC18A1, H2AC20BARHL2, NLGN3, SUV39H1, GABRA4, PPA1, UBE2D2 ) and metabolic processes ( GSTO2, H2AC20, SUV39H1, PPA1, UBE2D2 ) are potential DNA methylation candidate genes of MAP infection. The ILLN DMR genes were enriched for more biological process (BP) gene ontology (GO) terms ( n = 374), most of which were related to cellular processes (27.6%), biological regulation (16.6%), metabolic processes (15.4%) and response to stimulus/immune response (8.2%) compared to 75 BP GO terms (related to cellular processes, metabolic processes and transport, and system development) enriched for IL DMR genes. ILLN DMR genes were enriched for more pathways ( n = 47) including 13 disease pathways compared with 36 enriched pathways, including 7 disease/immune pathways for IL DMR genes. In conclusion, the results show tissue specific responses to MAP infection with more epigenetic changes (DMCs and DMRs) in the ILLN than in the IL tissue, suggesting that the ILLN and immune processes were more responsive to regulation by methylation of DNA relative to IL tissue. Our data is the first to demonstrate a potential role for DNA methylation in the pathogenesis of MAP infection in dairy cattle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Suraj Bhattarai, Stephanie McKay, Xin Zhao and Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Eveline M. Ibeagha-Awemu, Nathalie Bissonnette, Mengqi Wang and Pier-Luc Dudemaine.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative infectious agent of Johne's disease (JD), an incurable granulomatous enteritis affecting domestic livestock and other ruminants around the world. Chronic MAP infections usually begin in calves with MAP uptake by Peyer's patches (PP) located in the jejunum (JE) and ileum (IL). Determining host responses at these intestinal sites can provide a more complete understanding of how MAP manipulates the local microenvironment to support its long-term survival. We selected naturally infected (MAPinf, n=4) and naive (MAPneg, n=3) cows and transcriptionally profiled the JE and IL regions of the small intestine and draining mesenteric lymph nodes (LN). Differentially expressed (DE) genes associated with MAP infection were identified in the IL (585), JE (218), jejunum lymph node (JELN) (205), and ileum lymph node (ILLN) (117). Three DE genes ( CD14 , LOC616364 and ENSBTAG00000027033 ) were common to all MAPinf versus MAPneg tissues. Functional enrichment analysis revealed immune/disease related biological processes gene ontology (GO) terms and pathways predominated in IL tissue, indicative of an activated immune response state. Enriched GO terms and pathways in JE revealed a distinct set of host responses from those detected in IL. Regional differences were also identified between the mesenteric LNs draining each intestinal site. More down-regulated genes (52%) and fewer immune/disease pathways (n=5) were found in the ILLN compared to a higher number of up-regulated DE genes (56%) and enriched immune/disease pathways (n=13) in the JELN. Immunohistochemical staining validated myeloid cell transcriptional changes with increased CD172-positive myeloid cells in IL and JE tissues and draining LNs of MAPinf versus MAPneg cows. Several genes, GO terms, and pathways related to metabolism were significantly DE in IL and JE, but to a lesser extent (comparatively fewer enriched metabolic GO terms and pathways) in JELN suggesting distinct regional metabolic changes in IL compared to JE and JELN in response to MAP infection. These unique tissue- and regional-specific differences provides novel insight into the dichotomy in host responses to MAP infection that occur throughout the small intestine and mesenteric LN of chronically MAP infected cows., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Antonio Facciuolo, Philip John Griebel and Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Eveline M. Ibeagha-Awemu, Nathalie Bissonnette, Duy N. Do, Pier-Luc Dudemaine and Mengqi Wang.)

Milk fat and protein contents are among key elements of milk quality, and they are attracting more attention in response to consumers' demand for high-quality dairy products. To investigate the potential regulatory roles of DNA methylation underlying milk component yield, whole genome bisulfite sequencing was employed to profile the global DNA methylation patterns of mammary gland tissues from 17 Canada Holstein cows with various milk fat and protein contents. A total of 706, 2420 and 1645 differentially methylated CpG sites (DMCs) were found between high vs. low milk fat (HMF vs. LMF), high vs. low milk protein (HMP vs. LMP), and high vs. low milk fat and protein (HMFP vs. LMFP) groups, respectively ( q value < 0.1). Twenty-seven, 56 and 67 genes harboring DMCs in gene regions (denoted DMC genes) were identified for HMF vs. LMF, HMP vs. LMP and HMFP vs. LMFP, respectively. DMC genes from HMP vs. LMP and HMFP vs. LMFP comparisons were significantly overrepresented in GO terms related to aerobic electron transport chain and/or mitochondrial ATP (adenosine triphosphate) synthesis coupled electron transport. A total of 83 (HMF vs. LMF), 708 (HMP vs. LMP) and 408 (HMFP vs. LMFP) DMCs were co-located with 87, 147 and 158 quantitative trait loci (QTL) for milk component and yield traits, respectively. In conclusion, the identified methylation changes are potentially involved in the regulation of milk fat and protein yields, as well as the variation in reported co-located QTLs.

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene expression post-transcriptionally by targeting either the 3' untranslated or coding regions of genes. They have been reported to play key roles in a wide range of biological processes. The recent remarkable developments of transcriptomics technologies, especially next-generation sequencing technologies and advanced bioinformatics tools, allow more in-depth exploration of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), including miRNAs. These technologies have offered great opportunities for a deeper exploration of miRNA involvement in farm animal diseases, as well as livestock productivity and welfare. In this review, we provide an overview of the current knowledge of miRNA roles in major farm animal diseases with a particular focus on diseases of economic importance. In addition, we discuss the steps and future perspectives of using miRNAs as biomarkers and molecular therapy for livestock disease management as well as the challenges and opportunities for understanding the regulatory mechanisms of miRNAs related to disease pathogenesis.

As the poultry industry recedes from the use of antibiotic growth promoters, the need to evaluate the efficacy of possible alternatives and the delivery method that maximizes their effectiveness arises. This study aimed at expounding knowledge on the effect of the delivery method of a probiotic product (Bacillus subtilis fermentation extract) on performance and gut parameters in broiler chickens. A total of 450 fertile eggs sourced from Cobb 500 broiler breeders were randomly allotted to 3 groups: in ovo probiotic (n = 66), in ovo saline (n = 66), and noninjection (n = 200) and incubated for 21 d. On day 18.5 of incubation, 200 μL of either probiotic (10 × 10 6  cfu) or saline was injected into the amnion. At hatch, chicks were reallotted to 6 new treatment groups: in ovo probiotic, in ovo saline, in-feed antibiotics, in-water probiotic, in-feed probiotics, and control (corn-wheat-soybean diet) in 6 replicate cages and raised for 28 d. Of all hatch parameters evaluated, only percentage pipped eggs was found significant (P < 0.05) with the noninjection group having higher percentage pipped eggs than the other groups. Treatments did not affect the incidence of necrotic enteritis on day 28 (P > 0.05). Irrespective of the delivery method, the probiotic treatments had no significant effect on growth performance. The ileum villus width of the in ovo probiotic treatment was 18% higher than the in ovo saline group (P = 0.05) but not statistically higher than other groups. The jejunum villus height was 23% higher (P = 0.000) in the in ovo probiotic group than in the control group. There was no effect of treatment on total cecal short-chain fatty acid concentration and cecal gut microbiota composition and diversity (P > 0.05), although few unique bacteria differential abundance were recorded per treatment. Conclusively, although probiotic treatments (irrespective of the delivery route) did not affect growth performance, in ovo delivery of the probiotic product enhanced intestinal morphology, without compromising hatch performance and gut homeostasis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The dynamic changes in the epigenome resulting from the intricate interactions of genetic and environmental factors play crucial roles in individual growth and development. Numerous studies in plants, rodents, and humans have provided evidence of the regulatory roles of epigenetic processes in health and disease. There is increasing pressure to increase livestock production in light of increasing food needs of an expanding human population and environment challenges, but there is limited related epigenetic data on livestock to complement genomic information and support advances in improvement breeding and health management. This review examines the recent discoveries on epigenetic processes due to DNA methylation, histone modification, and chromatin remodeling and their impacts on health and production traits in farm animals, including bovine, swine, sheep, goat, and poultry species. Most of the reports focused on epigenome profiling at the genome-wide or specific genic regions in response to developmental processes, environmental stressors, nutrition, and disease pathogens. The bulk of available data mainly characterized the epigenetic markers in tissues/organs or in relation to traits and detection of epigenetic regulatory mechanisms underlying livestock phenotype diversity. However, available data is inadequate to support gainful exploitation of epigenetic processes for improved animal health and productivity management. Increased research effort, which is vital to elucidate how epigenetic mechanisms affect the health and productivity of livestock, is currently limited due to several factors including lack of adequate analytical tools. In this review, we (1) summarize available evidence of the impacts of epigenetic processes on livestock production and health traits, (2) discuss the application of epigenetics data in livestock production, and (3) present gaps in livestock epigenetics research. Knowledge of the epigenetic factors influencing livestock health and productivity is vital for the management and improvement of livestock productivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Mengqi Wang and Eveline M. Ibeagha-Awemu.)

Genomic selection methodologies and genome-wide association studies use powerful statistical procedures that correlate large amounts of high-density SNP genotypes and phenotypic data. Actual 305-d milk (MY), fat (FY), and protein (PY) yield data on 695 cows and 76,355 genotyping-by-sequencing-generated SNP marker genotypes from Canadian Holstein dairy cows were used to characterize linkage disequilibrium (LD) structure of Canadian Holstein cows. Also, the comparison of pedigree-based BLUP, genomic BLUP (GBLUP), and Bayesian (BayesB) statistical methods in the genomic selection methodologies and the comparison of Bayesian ridge regression and BayesB statistical methods in the genome-wide association studies were carried out for MY, FY, and PY. Results from LD analysis revealed that as marker distance decreases, LD increases through chromosomes. However, unexpected high peaks in LD were observed between marker pairs with larger marker distances on all chromosomes. The GBLUP and BayesB models resulted in similar heritability estimates through 10-fold cross-validation for MY and PY; however, the GBLUP model resulted in higher heritability estimates than BayesB model for FY. The predictive ability of GBLUP model was significantly lower than that of BayesB for MY, FY, and PY. Association analyses indicated that 28 high-effect markers and markers on Bos taurus autosome 14 located within 6 genes (DOP1B, TONSL, CPSF1, ADCK5, PARP10, and GRINA) associated significantly with FY., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus intramammary infection is one of the most common causes of chronic mastitis in dairy cows, whose development may be associated with epigenetic changes in the expression of important host defense genes. This study aimed to construct a genome-wide DNA methylation profile of the mammary gland of Chinese Holstein cows ( n = 3) following experimentally induced S. aureus mastitis, and to explore the potential gene regulatory mechanisms affected by DNA methylation during S. aureus mastitis. DNA was extracted from S. aureus -positive ( n = 3) and S. aureu s-negative ( n = 3) mammary gland quarters and subjected to methylation-dependent restriction-site associated DNA sequencing (Methyl-RAD Seq). Results showed that C m CGG/C m CWGG DNA methylation sites were unevenly distributed and concentrated on chromosomes 5, 11, and 19, and within intergenic regions and intron regions of genes. Compared with healthy control quarters, 9,181 significantly differentially methylated (DM) C m CGG sites and 1,790 DM C m CWGG sites were found in the S. aureus -positive quarters ( P < 0.05, |log2FC| > 1). Furthermore, 363 C m CGG differently methylated genes (DMGs) and 301 C m CWGG DMGs (adjusted P < 0.05, |log2FC| > 1) were identified. Gene ontology and KEGG enrichment analysis indicated that C m CGG DMGs are involved in immune response pathways, while the C m CWGG DMGs were mainly enriched in gene ontology terms related to metabolism. The mRNAs of 526 differentially methylated C m CGG genes and 124 differentially methylated C m CWGG genes were also significantly differentially expressed (RNA-Seq data) in the same samples, herein denoted differentially methylated and expressed genes (DMEGs) ( P < 0.05). Functional enrichment analysis of DMEGs revealed roles related to biological processes, especially the regulation of immune response to diseases. C m CGG DMEGs like IL6R , TNF , BTK , IL1R 2, and TNFSF8 enriched in several immune-related GO terms and pathways indicated their important roles in host immune response and their potential as candidate genes for S. aureus mastitis. These results suggest potential regulatory roles for DNA methylation in bovine mammary gland processes during S. aureus mastitis and serves as a reference for future epigenetic regulation and mechanistic studies., (Copyright © 2020 Wang, Liang, Ibeagha-Awemu, Li, Zhang, Chen, Sun, Karrow, Yang and Mao.)

Innate responses provide the first line of defense against viral infections, including the influenza virus at mucosal surfaces. Communication and interaction between different host cells at the early stage of viral infections determine the quality and magnitude of immune responses against the invading virus. The release of membrane-encapsulated extracellular vesicles (EVs), from host cells, is defined as a refined system of cell-to-cell communication. EVs contain a diverse array of biomolecules, including microRNAs (miRNAs). We hypothesized that the activation of the tracheal cells with different stimuli impacts the cellular and EV miRNA profiles. Chicken tracheal rings were stimulated with polyI:C and LPS from Escherichia coli 026:B6 or infected with low pathogenic avian influenza virus H4N6. Subsequently, miRNAs were isolated from chicken tracheal cells or from EVs released from chicken tracheal cells. Differentially expressed (DE) miRNAs were identified in treated groups when compared to the control group. Our results demonstrated that there were 67 up-regulated miRNAs, 157 down-regulated miRNAs across all cellular and EV samples. In the next step, several genes or pathways targeted by DE miRNAs were predicted. Overall, this study presented a global miRNA expression profile in chicken tracheas in response to avian influenza viruses (AIV) and toll-like receptor (TLR) ligands. The results presented predicted the possible roles of some DE miRNAs in the induction of antiviral responses. The DE candidate miRNAs, including miR-146a, miR-146b, miR-205a, miR-205b and miR-449, can be investigated further for functional validation studies and to be used as novel prophylactic and therapeutic targets in tailoring or enhancing antiviral responses against AIV.

High blood cholesterol concentration, mainly caused by high dietary cholesterol, is a potential risk factor for human health. Dairy products are important sources of human dietary cholesterol intake. Therefore, monitoring bovine milk cholesterol concentration is important for human health benefit. Genetic selection for improvement of cow milk cholesterol content requires understanding of the genetics of milk cholesterol. For this purpose, we performed analyses of additive and dominance effects of 126 potentially functional SNPs within 43 candidate genes with milk cholesterol content [expressed as mg of cholesterol in 100 g of fat (CHL&#95;fat) or in 100 mg of milk (CHL&#95;milk)]. The additive and dominance effects of SNPs rs380643365 in AGPAT1 (P = 0.04) and rs134357240 in SOAT1 (P = 0.035) genes associated significantly with CHL&#95;fat. Moreover, five (rs109326954 and rs523413537 in DGAT1, rs109376747 in LDLR, rs42781651 in FAM198B and rs109967779 in ACAT2) and four (rs137347384 in RBM19, rs109376747 in LDLR, rs42016945 in PPARG and rs110862179 in SCAP) SNPs were significantly associated with CHL&#95;milk (P < 0.05) based on additive and dominance effect analyses respectively. Rs109326954 and rs523413537 in DGAT1 explained a considerable portion of the phenotypic variance of CHL&#95;milk (7.54 and 6.84% respectively), and might be useful in selection programs for reduced milk cholesterol content. Several significantly associated SNPs were in genes (such as ACAT2 and LDLR) involved in cholesterol metabolism in the liver or cholesterol transport, suggesting multiple mechanisms regulating milk cholesterol content. Nine and seven SNPs identified by additive or dominance effect analyses associated significantly with milk yield and fat yield respectively. Further analyses are required to better understand the consequences of these variants and their potential use in genomic selection of the studied traits., (© 2020 Stichting International Foundation for Animal Genetics.)

Background: Exploration of the bioactive components of bovine milk has gained global interest due to their potential applications in human nutrition and health promotion. Despite advances in proteomics profiling, limited studies have been carried out to fully characterize the bovine milk proteome. This study explored the milk proteome of Jersey and Kashmiri cattle at day 90 of lactation using high-resolution mass spectrometry based quantitative proteomics nano-scale LC-MS/Q-TOF technique. Data are available via ProteomeXchange with identifier PXD017412., Results: Proteins from whey were fractionated by precipitation into high and low abundant proteins. A total of 81 high-abundant and 99 low-abundant proteins were significantly differentially expressed between Kashmiri and Jersey cattle, clearly differentiating the two breeds at the proteome level. Among the top differentiating proteins, the Kashmiri cattle milk proteome was characterised by increased concentrations of immune-related proteins (apelin, acid glycoprotein, CD14 antigen), neonatal developmental protein (probetacellulin), xenobiotic metabolising enzyme (flavin monooxygenase 3 (FMO3), GLYCAM1 and HSP90AA1 (chaperone) while the Jersey milk proteome presented higher concentrations of enzyme modulators (SERPINA1, RAC1, serine peptidase inhibitor) and hydrolases (LTF, LPL, CYM, PNLIPRP2). Pathway analysis in Kashmiri cattle revealed enrichment of key pathways involved in the regulation of mammary gland development like Wnt signalling pathway, EGF receptor signalling pathway and FGF signalling pathway while a pathway (T-cell activation pathway) associated with immune system regulation was significantly enriched in Jersey cattle. Most importantly, the high-abundant FMO3 enzyme with an observed 17-fold higher expression in Kashmiri cattle milk seems to be a characteristic feature of the breed. The presence of this (FMO3) bioactive peptide/enzyme in Kashmiri cattle could be economically advantageous for milk products from Kashmiri cattle., Conclusion: In conclusion, this is the first study to provide insights not only into the milk proteome differences between Kashmiri and Jersey cattle but also provides potential directions for application of specific milk proteins from Kashmiri cattle in special milk preparations like infant formula.

Mycobacterium avium spp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), also known as paratuberculosis, in ruminants. The mechanisms of JD pathogenesis are not fully understood, but it is known that MAP subverts the host immune system by using macrophages as its primary reservoir. MAP infection in macrophages is often studied in healthy cows or experimentally infected calves, but reports on macrophages from naturally infected cows are lacking. In our study, primary monocyte-derived macrophages (MDMs) from cows diagnosed as positive (+) or negative (-) for JD were challenged in vitro with live MAP. Analysis using next-generation RNA sequencing revealed that macrophages from JD(+) cows did not present a definite pattern of response to MAP infection. Interestingly, a considerable number of genes, up to 1436, were differentially expressed in JD(-) macrophages. The signatures of the infection time course of 1, 4, 8, and 24 h revealed differential expression of ARG2, COL1A1, CCL2, CSF3, IL1A, IL6, IL10, PTGS2, PTX3, SOCS3, TNF , and TNFAIP6 among other genes, with major effects on host signaling pathways. While several immune pathways were affected by MAP, other pathways related to hepatic fibrosis/hepatic stellate cell activation, lipid homeostasis, such as LXR/RXR (liver X receptor/retinoid X receptor) activation pathways, and autoimmune diseases (rheumatoid arthritis or atherosclerosis) also responded to the presence of live MAP. Comparison of the profiles of the unchallenged MDMs from JD(+) vs. JD(-) cows showed that 868 genes were differentially expressed, suggesting that these genes were already affected before monocytes differentiated into macrophages. The downregulated genes predominantly modified the general cell metabolism by downregulating amino acid synthesis and affecting cholesterol biosynthesis and other energy production pathways while introducing a pro-fibrotic pattern associated with foam cells. The upregulated genes indicated that lipid homeostasis was already supporting fat storage in uninfected JD(+) MDMs. For JD(+) MDMs, differential gene expression expounds long-term mechanisms established during disease progression of paratuberculosis. Therefore, MAP could further promote disease persistence by influencing long-term macrophage behavior by using both tolerance and fat-storage states. This report contributes to a better understanding of MAP's controls over the immune cell response and mechanisms of MAP survival., (Copyright © 2020 Ariel, Gendron, Dudemaine, Gévry, Ibeagha-Awemu and Bissonnette.)

Background: MicroRNAs (miRNAs) in milk-derived exosomes may reflect pathophysiological changes caused by mastitis. This study profiled miRNAs in exosomes from both normal milk and mastitic milk infected by Staphylococcus aureus (S. aureus). The potential targets for differentially expressed (DE) miRNAs were predicted and the target genes for bta-miR-378 and bta-miR-185 were also validated., Results: Total RNA from milk exosomes was collected from healthy cows (n = 3, the control group) and S. aureus infected cows (n = 6, the SA group). Two hundred ninety miRNAs (221 known and 69 novel ones) were identified. Among them, 22 known and 15 novel miRNAs were differentially expressed. Target genes of DE miRNAs were significantly enriched in intracellular protein transport, endoplasmic reticulum and identical protein binding. The expression of two miRNAs (bta-miR-378 and bta-miR-185) with high read counts and log 2 fold changes (> 3.5) was significantly higher in mastitic milk infected with S. aureus. One target gene (VAT1L) of bta-miR-378 and five target genes (DYRK1B, MLLT3, HP1BP3, NPR2 and PGM1) of bta-miR-185 were validated., Conclusion: DE miRNAs in exosomes from normal and S. aureus infected milk were identified. The predicted targets for two DE miRNAs (bta-miR-378 and bta-miR-185) were further validated. The linkage between the validated target genes and diseases suggested that we should pay particular attention to exosome miRNAs from mastitic milk in terms of milk safety.

Lactation persistency (LP), defined as the ability of a cow to maintain milk production at a high level after milk peak, is an important phenotype for the dairy industry. In this study, we used a targeted genotyping approach to scan for potentially functional single nucleotide polymorphisms (SNPs) within 57 potential candidate genes derived from our previous genome wide association study on LP and from the literature. A total of 175,490 SNPs were annotated within 10-kb flanking regions of the selected candidate genes. After applying several filtering steps, a total of 105 SNPs were retained for genotyping using target genotyping arrays. SNP association analyses were performed in 1,231 Holstein cows with 69 polymorphic SNPs using the univariate liner mixed model with polygenic effects using DMU package. Six SNPs including rs43770847, rs208794152, and rs208332214 in ADRM1; rs209443540 in C5orf34; rs378943586 in DDX11; and rs385640152 in GHR were suggestively significantly associated with LP based on additive effects and associations with 4 of them (rs43770847, rs208794152, rs208332214, and rs209443540) were based on dominance effects at P < 0.05. However, none of the associations remained significant at false discovery rate adjusted P (FDR) < 0.05. The additive variances explained by each suggestively significantly associated SNP ranged from 0.15% (rs43770847 in ADRM1) to 5.69% (rs209443540 in C5orf34), suggesting that these SNPs might be used in genetic selection for enhanced LP. The percentage of phenotypic variance explained by dominance effect ranged from 0.24% to 1.35% which suggests that genetic selection for enhanced LP might be more efficient by inclusion of dominance effects. Overall, this study identified several potentially functional variants that might be useful for selection programs for higher LP. Finally, a combination of identification of potentially functional variants followed by targeted genotyping and association analysis is a cost-effective approach for increasing the power of genetic association studies., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2019.)

The objective of this study was to characterize GnRHR gene in Sokoto (n = 70) and Kalahari Red (n = 70) goats. Three SNPs, (g.-29T > G, g.48 G > A and g.209 T > G), were detected in Sokoto Red (SR) and one (g.48 G > A) in Kalahari Red (KR) goats. All the mutations occurred within the 5'UTR and Exon one of the gene and the g.209 T > G was non-synonymous and, therefore, resulted in an amino acid change from methionine to arginine at Position 70 of the GnRHR polypeptide. The homozygous mutant genotypes at the three SNP loci were not detected in both breeds but minor allele frequencies were ≥ 0.1 for the three SNP loci in SR goats. Frequency of the T allele, however, was 0.93 at the only SNP locus detected in KR goats. There was a strong linkage disequilibrium (LD; r 2 >0.98) among the detected mutations in SR goats resulting in two haplotypes (T-G-T and G-A-G) with a frequency of 86% and 13%, respectively. There was no significant association between genotypes at the polymorphic loci and litter size (P > 0.05) in the two breeds. The non-synonymous mutation (g.209T>G) appears to have changed the nucleotide binding region and area spanning exposed/buried regions on the predicted secondary structure of the two variants of the receptor. This change led to variation in the tertiary structure between the two variants of the receptor and can influence the function of the transmembrane receptor. Comparison of the GnRH receptor domains for goats, sheep, cattle and swine confirmed that the seven transmembrane domains of the receptor are conserved in all the farm animals considered., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

This study aimed to explore the roles of microRNAs (miRNAs) in calf rumen development during early life. Rumen tissues were collected from 16 calves (8 at pre-weaning and 8 at post-weaning) for miRNA-sequencing, differential expression (DE), miRNA weighted gene co-expression network (WGCNA) and miRNA-mRNA co-expression analyses. 295 miRNAs were identified. Bta-miR-143, miR-26a, miR-145 and miR-27b were the most abundantly expressed. 122 miRNAs were significantly DE between the pre- and post-weaning periods and the most up- and down-regulated miRNAs were bta-miR-29b and bta-miR-493, respectively. Enrichment analyses of the target genes of DE miRNAs revealed important roles for miRNA in rumen developmental processes, immune system development, protein digestion and processes related to the extracellular matrix. WGCNA indicated that bta-miR-145 and bta-miR-199a-3p are important hub miRNAs in the regulation of these processes. Therefore, bta-miR-143, miR-29b, miR-145, miR-493, miR-26a and miR-199 family members might be key regulators of calf rumen development during early life., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

The African continent is home to diverse populations of livestock breeds adapted to harsh environmental conditions with more than 70% under traditional systems of management. Animal productivity is less than optimal in most cases and is faced with numerous challenges including limited access to adequate nutrition and disease management, poor institutional capacities and lack of adequate government policies and funding to develop the livestock sector. Africa is home to about 1.3 billion people and with increasing demand for animal proteins by an ever growing human population, the current state of livestock productivity creates a significant yield gap for animal products. Although a greater section of the population, especially those living in rural areas depend largely on livestock for their livelihoods; the potential of the sector remains underutilized and therefore unable to contribute significantly to economic development and social wellbeing of the people. With current advances in livestock management practices, breeding technologies and health management, and with inclusion of all stakeholders, African livestock populations can be sustainably developed to close the animal protein gap that exists in the continent. In particular, advances in gene technologies, and application of genomic breeding in many Western countries has resulted in tremendous gains in traits like milk production with the potential that, implementation of genomic selection and other improved practices (nutrition, healthcare, etc.) can lead to rapid improvement in traits of economic importance in African livestock populations. The African livestock populations in the context of this review are limited to cattle, goat, pig, poultry, and sheep, which are mainly exploited for meat, milk, and eggs. This review examines the current state of livestock productivity in Africa, the main challenges faced by the sector, the role of various stakeholders and discusses in-depth strategies that can enable the application of genomic technologies for rapid improvement of livestock traits of economic importance.

Jersey and Kashmiri cattle are important dairy breeds that contribute significantly to the total milk production of the Indian northern state of Jammu and Kashmir. The Kashmiri cattle germplasm has been extensively diluted through crossbreeding with Jersey cattle with the goal of enhancing its milk production ability. However, crossbred animals are prone to diseases resulting to unsustainable milk production. This study aimed to provide a comprehensive transcriptome profile of mammary gland epithelial cells at different stages of lactation and to find key differences in genes and pathways regulating milk traits between Jersey and Kashmiri cattle. Mammary epithelial cells (MEC) isolated from milk obtained from six lactating cows (three Jersey and three Kashmiri cattle) on day 15 (D15), D90 and D250 in milk, representing early, mid and late lactation, respectively were used. RNA isolated from MEC was subjected to next-generation RNA sequencing and bioinformatics processing. Casein and whey protein genes were found to be highly expressed throughout the lactation stages in both breeds. Largest differences in differentially expressed genes (DEG) were between D15 vs D90 (1,805 genes) in Kashmiri cattle and, D15 vs D250 (3,392 genes) in Jersey cattle. A total of 1,103, 1,356 and 1,397 genes were differentially expressed between Kashmiri and Jersey cattle on D15, D90 and D250, respectively. Antioxidant genes like RPLPO and RPS28 were highly expressed in Kashmiri cattle. Differentially expressed genes in both Kashmiri and Jersey were enriched for multicellular organismal process, receptor activity, catalytic activity, signal transducer activity, macromolecular complex and developmental process gene ontology terms. Whereas, biological regulation, endopeptidase activity and response to stimulus were enriched in Kashmiri cattle and, reproduction and immune system process were enriched in Jersey cattle. Most of the pathways responsible for regulation of milk production like JAK-STAT, p38 MAPK pathway, PI3 kinase pathway were enriched by DEG in Jersey cattle only. Although Kashmiri has poor milk production efficiency, the present study suggests possible physicochemical and antioxidant properties of Kashmiri cattle milk that needs to be further explored., Competing Interests: The authors have declared that no competing interests exist.

The stearoyl-CoA desaturase 1 (SCD1) A293V and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphisms have been associated with significant variation in bovine milk fatty acid composition and unsaturation indices in western cattle breeds. This study aimed to estimate the milk fatty acid variability in indigenous Borgou and White Fulani cattle breeds of Benin, and the effects of the SCD1 A293V and DGAT1 K232A polymorphisms on milk and fatty acid composition and unsaturation indices. Thus, 85 Borgou and 96 White Fulani cows were genotyped for the SCD1 A293V and DGAT1 K232A polymorphisms and their milk and fatty acid composition and unsaturation indices were determined. Borgou presented milk with higher linoleic acid (P < 0.001), oleic acid (P < 0.05), C18 index (P < 0.001), total unsaturation index (P < 0.05), and lower total saturated fatty acid (SFA) compared to White Fulani. The SCD1 VV genotype was associated with higher protein and lactose contents in White Fulani (P < 0.05). In Borgou, the SCD1 AV genotype was associated with higher C14 and total unsaturation indices (P < 0.01), while the SCD1 V allele was associated with decrease in C14 index (P < 0.05). In White Fulani, the SCD1 VV genotype was associated with lower C18:1 cis-9 content (P < 0.05) while the DGAT1 K allele was associated with increased total SFA (P < 0.05), and decreased C18 index (P < 0.05), total unsaturation index (P < 0.01) and total monounsaturated fatty acid (P < 0.01). The SCD1 A293V and DGAT1 K232A may serve as genetic markers to improve milk fatty acid traits in Borgou and White Fulani breeds.

This study aimed to characterize the long non-coding RNA (lncRNA) expression in the bovine mammary gland and to infer their functions in dietary response to 5% linseed oil (LSO) or 5% safflower oil (SFO). Twelve cows (six per treatment) in mid lactation were fed a control diet for 28 days followed by a treatment period (control diet supplemented with 5% LSO or 5% SFO) of 28 days. Mammary gland biopsies were collected from each animal on day-14 (D-14, control period), D+7 (early treatment period) and D+28 (late treatment period) and were subjected to RNA-Sequencing and subsequent bioinformatics analyses. Functional enrichment of lncRNA was performed via potential cis regulated target genes located within 50 kb flanking regions of lncRNAs and having expression correlation of >0.7 with mRNAs. A total of 4955 lncRNAs (325 known and 4630 novel) were identified which potentially cis targeted 59 and 494 genes in LSO and SFO treatments, respectively. Enrichments of cis target genes of lncRNAs indicated potential roles of lncRNAs in immune function, nucleic acid metabolism and cell membrane organization processes as well as involvement in Notch, cAMP and TGF-β signaling pathways. Thirty-two and 21 lncRNAs were differentially expressed (DE) in LSO and SFO treatments, respectively. Six genes ( KCNF1 , STARD13 , BCL6 , NXPE2 , HHIPL2 and MMD ) were identified as potential cis target genes of six DE lncRNAs. In conclusion, this study has identified lncRNAs with potential roles in mammary gland functions and potential candidate genes and pathways via which lncRNAs might function in response to LSO and SFA.

This study investigated the effect of supplementing the diet of calves with two direct fed microbials (DFMs) (Saccharomyces cerevisiae boulardii CNCM I-1079 (SCB) and Lactobacillus acidophilus BT1386 (LA)), and an antibiotic growth promoter (ATB). Thirty-two dairy calves were fed a control diet (CTL) supplemented with SCB or LA or ATB for 96 days. On day 33 (pre-weaning, n = 16) and day 96 (post-weaning, n = 16), digesta from the rumen, ileum, and colon, and mucosa from the ileum and colon were collected. The bacterial diversity and composition of the gastrointestinal tract (GIT) of pre- and post-weaned calves were characterized by sequencing the V3-V4 region of the bacterial 16S rRNA gene. The DFMs had significant impact on bacteria community structure with most changes associated with treatment occurring in the pre-weaning period and mostly in the ileum but less impact on bacteria diversity. Both SCB and LA significantly reduced the potential pathogenic bacteria genera, Streptococcus and Tyzzerella&#95;4 (FDR ≤ 8.49E-06) and increased the beneficial bacteria, Fibrobacter (FDR ≤ 5.55E-04) compared to control. Other potential beneficial bacteria, including Rumminococcaceae UCG 005, Roseburia and Olsenella, were only increased (FDR ≤ 1.30E-02) by SCB treatment compared to control. Furthermore, the pathogenic bacterium, Peptoclostridium, was reduced (FDR = 1.58E-02) by SCB only while LA reduced (FDR = 1.74E-05) Ruminococcus&#95;2. Functional prediction analysis suggested that both DFMs impacted (p < 0.05) pathways such as cell cycle, bile secretion, proteasome, cAMP signaling pathway, thyroid hormone synthesis pathway and dopaminergic synapse pathway. Compared to the DFMs, ATB had similar impact on bacterial diversity in all GIT sites but greater impact on the bacterial composition of the ileum. Overall, this study provides an insight on the bacteria genera impacted by DFMs and the potential mechanisms by which DFMs affect the GIT microbiota and may therefore facilitate development of DFMs as alternatives to ATB use in dairy calf management.

This study aimed to investigate the potential regulatory roles of miRNAs in calf ileum developmental transition from the pre- to the post-weaning period. For this purpose, ileum tissues were collected from eight calves at the pre-weaning period and another eight calves at the post-weaning period and miRNA expression characterized by miRNA sequencing, followed by functional analyses. A total of 388 miRNAs, including 81 novel miRNAs, were identified. A total of 220 miRNAs were differentially expressed (DE) between the two periods. The potential functions of DE miRNAs in ileum development were supported by significant enrichment of their target genes in gene ontology terms related to metabolic processes and transcription factor activities or pathways related to metabolism (peroxisomes), vitamin digestion and absorption, lipid and protein metabolism, as well as intracellular signaling. Integration of DE miRNAs and DE mRNAs revealed several DE miRNA-mRNA pairs with crucial roles in ileum development (bta-miR-374a- FBXO18, bta-miR-374a- GTPBP3 , bta-miR-374a- GNB2 ) and immune function (bta-miR-15b- IKBKB ). This is the first integrated miRNA-mRNA analysis exploring the potential roles of miRNAs in calf ileum growth and development during early life.

This study aimed to identify single nucleotide polymorphisms (SNPs) associated with milk cholesterol (CHL) content via a genome wide association study (GWAS). Milk CHL content was determined by gas chromatography and expressed as mg of CHL in 100 g of fat (CHL&#95;fat) or in 100 mg of milk (CHL&#95;milk). GWAS was performed with 1,183 cows and 40,196 SNPs using a univariate linear mixed model. Two and 20 SNPs were significantly associated with CHL&#95;fat and CHL&#95;milk, respectively. The important regions for CHL&#95;fat and CHL&#95;milk were at 41.9 Mb on chromosome (BTA) 17 and 1.6-3.2 Mb on BTA 14, respectively. DGAT1, PTPN1, INSIG1, HEXIM1, SDS, and HTR5A genes, also known to be associated with human plasma CHL phenotypes, were identified as potential candidate genes for bovine milk CHL. Additional new potential candidate genes for milk CHL were RXFP1, FAM198B, TMEM144, CXXC4, MAML2 and CDH13. Enrichment analyses suggested that identified candidate genes participated in cell-cell signaling processes and are key members in tight junction, focal adhesion, Notch signaling and glycerolipid metabolism pathways. Furthermore, identified transcription factors such as PPARD, LXR, and NOTCH1 might be important in the regulation of bovine milk CHL content. The expression of several positional candidate genes (such as DGAT1, INSIG1 and FAM198B) and their correlation with milk CHL content were further confirmed with RNA sequence data from mammary gland tissues. This is the first GWAS on bovine milk CHL. The identified markers and candidate genes need further validation in a larger cohort for use in the selection of cows with desired milk CHL content.

MicroRNAs (miRNA) regulate mRNA networks to coordinate cellular functions. In this study, we constructed gene co-expression networks to detect miRNA modules (clusters of miRNAs with similar expression patterns) and miRNA⁻mRNA pairs associated with blood (triacylglyceride and nonesterified fatty acids) and milk (milk yield, fat, protein, and lactose) components and milk fatty acid traits following dietary supplementation of cows' diets with 5% linseed oil (LSO) ( n = 6 cows) or 5% safflower oil (SFO) ( n = 6 cows) for 28 days. Using miRNA transcriptome data from mammary tissues of cows for co-expression network analysis, we identified three consensus modules: blue, brown, and turquoise, composed of 70, 34, and 86 miRNA members, respectively. The hub miRNAs (miRNAs with the most connections with other miRNAs) were miR-30d, miR-484 and miR-16b for blue, brown, and turquoise modules, respectively. Cell cycle arrest, and p53 signaling and transforming growth factor⁻beta (TGF-β) signaling pathways were the common gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched for target genes of the three modules. Protein percent ( p = 0.03) correlated with the turquoise module in LSO treatment while protein yield ( p = 0.003) and milk yield ( p = 7 × 10 -04 ) correlated with the turquoise model, protein and milk yields and lactose percent ( p < 0.05) correlated with the blue module and fat percent ( p = 0.04) correlated with the brown module in SFO treatment. Several fatty acids correlated ( p < 0.05) with the blue (CLA:9,11) and brown (C4:0, C12:0, C22:0, C18:1n9c and CLA:10,12) modules in LSO treatment and with the turquoise (C14:0, C18:3n3 and CLA:9,11), blue (C14:0 and C23:0) and brown (C6:0, C16:0, C22:0, C22:6n3 and CLA:10,12) modules in SFO treatment. Correlation of miRNA and mRNA data from the same animals identified the following miRNA⁻mRNA pairs: miR-183/ RHBDD2 ( p = 0.003), miR-484/ EIF1AD ( p = 0.011) and miR-130a/ SBSPON ( p = 0.004) with lowest p -values for the blue, brown, and turquoise modules, respectively. Milk yield, protein yield, and protein percentage correlated ( p < 0.05) with 28, 31 and 5 miRNA⁻mRNA pairs, respectively. Our results suggest that, the blue, brown, and turquoise modules miRNAs, hub miRNAs, miRNA⁻mRNA networks, cell cycle arrest GO term, p53 signaling and TGF-β signaling pathways have considerable influence on milk and blood phenotypes following dietary supplementation of dairy cows' diets with 5% LSO or 5% SFO.

A better understanding of the factors that regulate growth and immune response of the gastrointestinal tract (GIT) of calves will promote informed management practices in calf rearing. This study aimed to explore genomics (messenger RNA (mRNA)) and epigenomics (long non-coding RNA (lncRNA)) mechanisms regulating the development of the rumen and ileum in calves. Thirty-two calves (≈5-days-old) were reared for 96 days following standard procedures. Sixteen calves were humanely euthanized on experiment day 33 (D33) (pre-weaning) and another 16 on D96 (post-weaning) for collection of ileum and rumen tissues. RNA from tissues was subjected to next generation sequencing and 3310 and 4217 mRNAs were differentially expressed (DE) between D33 and D96 in ileum and rumen tissues, respectively. Gene ontology and pathways enrichment of DE genes confirmed their roles in developmental processes, immunity and lipid metabolism. A total of 1568 (63 known and 1505 novel) and 4243 (88 known and 4155 novel) lncRNAs were detected in ileum and rumen tissues, respectively. Cis target gene analysis identified BMPR1A , an important gene for a GIT disease (juvenile polyposis syndrome) in humans, as a candidate cis target gene for lncRNAs in both tissues. LncRNA cis target gene enrichment suggested that lncRNAs might regulate growth and development in both tissues as well as posttranscriptional gene silencing by RNA or microRNA processing in rumen, or disease resistance mechanisms in ileum. This study provides a catalog of bovine lncRNAs and set a baseline for exploring their functions in calf GIT development., Competing Interests: The authors declare no conflict of interest.

Gonadotropin-releasing hormone receptor (GnRHR) gene is considered a candidate gene for litter size due to its critical role in regulating the activities of hypothalamo-pituitary-gonadal axis which synthesizes and releases gonadotropins. This study was designed to identify mutations within the caprine GnRHR gene and investigate their association with litter size at various parities. Polymorphisms scanning and genotyping of GnRHR gene in West African Dwarf (WAD) goats (n = 226) revealed three single nucleotide polymorphisms (SNPs), one mutation (g.-29T > G) was detected within 5'UTR region while two others (g.48G > A and g.209T > G) were identified in exon 1. Mutation at g.209T > G locus resulted in amino acid change from Methionine to Arginine at position 70 on the polypeptide residue. Based on heterozygosity and polymorphism information content (PIC), WAD goat population diversity at the SNP loci was moderate. Strong linkage disequilibrium (LD) (r 2  > 0.98) existed among the detected mutations resulting in three observed haplotypes, two (T-G-T and G-A-G) had cumulative frequency of > 97%. The mutation within 5'UTR region of GnRHR gene (g.-29T > G) is novel, being reported in goats for the first time. Association analysis revealed a significant (p < 0.05) association between allele G at g.-29T > G with higher mean litter size for homozygous (GG) mutant does compared with heterozygotes (GT) or homozygotes (TT), while the relationship between SNPs at the two loci detected in exon 1 and litter size was not significant.

Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (| r | > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (| r | > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to milk and mammary gland traits included cell differentiation, G-protein coupled receptor activity, and intracellular signaling transduction. Overall, this study uncovered regulatory networks in which miRNAs interacted with each other to regulate lactation traits., Competing Interests: The authors declare no conflict of interest.

Background: Mammary glands of dairy cattle produce milk for the newborn offspring and for human consumption. Long intergenic noncoding RNAs (lincRNAs) play various functions in eukaryotic cells. However, types and roles of lincRNAs in bovine mammary glands are still poorly understood., Results: Using computational methods, 886 unknown intergenic transcripts (UITs) were identified from five RNA-seq datasets from bovine mammary glands. Their non-coding potentials were predicted by using the combination of four software programs (CPAT, CNCI, CPC and hmmscan), with 184 lincRNAs identified. By comparison to the NONCODE2016 database and a domestic-animal long noncoding RNA database (ALDB), 112 novel lincRNAs were revealed in bovine mammary glands. Many lincRNAs were found to be located in quantitative trait loci (QTL). In particular, 36 lincRNAs were found in 172 milk related QTLs, whereas one lincRNA was within clinical mastitis QTL region. In addition, targeted genes for 10 lincRNAs with the highest fragments per kilobase of transcript per million fragments mapped (FPKM) were predicted by LncTar for forecasting potential biological functions of these lincRNAs. Further analyses indicate involvement of lincRNAs in several biological functions and different pathways., Conclusion: Our study has provided a panoramic view of lincRNAs in bovine mammary glands and suggested their involvement in many biological functions including susceptibility to clinical mastitis as well as milk quality and production. This integrative annotation of mammary gland lincRNAs broadens and deepens our understanding of bovine mammary gland biology.

Recently we showed that 5% linseed oil (LSO) and 5% safflower oil (SFO) supplementation of cow's diets reduced milk fat yield by 30·38 and 32·42% respectively, accompanied by differential expression of genes and regulation by microRNAs (miRNA). This research communication addresses the hypothesis that epigenetic regulation could be involved in the observed milk fat reduction. Thus, this study investigated the gene expression pattern of major epigenetic modifying enzymes in response to dietary supplementation with LSO or SFO. Twenty-six Canadian Holstein cows in mid lactation were randomly assigned to two groups (13/group) and fed a control diet for 28 d (day -28 to -1) (control period- CP) followed by a treatment period (TP) (control diet supplemented with 5% LSO (LSO treatment) or 5% SFO (SFO treatment) of 28 d (day +1 to +28). After treatment, cows in the two groups were returned to the control diet for another 28 d (day +29 to +56) (post treatment period-PTP). Milk samples were collected on day -1 (CP), +7, +28 (TP) and +56 (PTP) for RNA isolation and measurement of the expression of thirteen epigenetic modifying genes including two DNA methytrasferases (DNMT1, DNMT3A), four histone acetylases (HAT1, KAT2A, KAT5 and CREBBP), five histone deacetylases (HDAC1, HDAC2, HDAC3, SIRT1 and SIRT2) and two histone methytransferases (EHMT2 and PRMT1) by qPCR. Linseed oil supplementation significantly repressed the expression of EHMT2, HDAC2 and HDAC3 on day +7 (P < 0·05) and KAT2A and SIRT2 on day +28 (P < 0·05) as compared with the control period (day -1) while SFO had no effect. When LSO was withdrawn, the expression of some of the genes increased slightly but did not reach control (day -1) levels at the end of the PTP. Our study demonstrated a significant role of LSO in the epigenetic regulation of fatty acid synthesis as compared to SFO. The effect of LSO may be related to its higher degree of unsaturation and might represent a different regulatory mechanism which needs further investigation.

Background: Genotyping-by-sequencing (GBS) has emerged as a powerful and cost-effective approach for discovering and genotyping single-nucleotide polymorphisms. The GBS technique was largely used in crop species where its low sequence coverage is not a drawback for calling genotypes because inbred lines are almost homozygous. In contrast, only a few studies used the GBS technique in animal populations (with sizeable heterozygosity rates) and many of those that have been published did not consider the quality of the genotypes produced by the bioinformatic pipelines. To improve the sequence coverage of the fragments, an alternative GBS preparation protocol that includes selective primers during the PCR amplification step has been recently proposed. In this study, we compared this modified protocol with the conventional two-enzyme GBS protocol. We also described various procedures to maximize the selection of high quality genotypes and to increase the accuracy of imputation., Results: The in silico digestions of the bovine genome showed that the combination of PstI and MspI is more suitable for sequencing bovine GBS libraries than the use of single digestions with PstI or ApeKI. The sequencing output of the GBS libraries generated a total of 123,666 variants with the selective-primer approach and 272,103 variants with the conventional approach. Validating our data with genotypes obtained from mass spectrometry and Illumina's bovine SNP50 array, we found that the genotypes produced by the conventional GBS method were concordant with those produced by these alternative genotyping methods, whereas the selective-primer method failed to call heterozygotes with confidence. Our results indicate that high accuracy in genotype calling (>97%) can be obtained using low read-depth thresholds (3 to 5 reads) provided that markers are simultaneously filtered for genotype quality scores. We also show that factors such as the minimum call rate and the minor allele frequency positively influence the accuracy of imputation of missing GBS data. The highest accuracies (around 85%) of imputed GBS markers were obtained with the FIMPUTE program when GBS and SNP50 array genotypes were combined (80,190 to 100,297 markers) before imputation., Conclusions: We discovered that the conventional two-enzyme GBS protocol could produce a large number of high-quality genotypes provided that appropriate filtration criteria were used. In contrast, the selective-primer approach resulted in a substantial proportion of miscalled genotypes and should be avoided for livestock genotyping studies. Overall, our study demonstrates that carefully adjusting the different filtering parameters applied to the GBS data is critical to maximize the selection of high quality genotypes and to increase the accuracy of imputation of missing data. The strategies and results presented here provide a framework to maximize the output of the GBS technique in animal populations and qualified the PstI/MspI GBS assay as a low-cost high-density genotyping platform. The conclusions reported here regarding read-depth and genotype quality filtering could benefit many GBS applications, notably genome-wide association studies, where there is a need to increase the density of markers genotyped across the target population while preserving the quality of genotypes.

The study examined microRNA (miRNA) expression and regulatory patterns during an entire bovine lactation cycle. Total RNA from milk fat samples collected at the lactogenesis (LAC, day1 [D1] and D7), galactopoiesis (GAL, D30, D70, D130, D170 and D230) and involution (INV, D290 and when milk production dropped to 5 kg/day) stages from 9 cows was used for miRNA sequencing. A total of 475 known and 238 novel miRNAs were identified. Fifteen abundantly expressed miRNAs across lactation stages play regulatory roles in basic metabolic, cellular and immunological functions. About 344, 366 and 209 miRNAs were significantly differentially expressed (DE) between GAL and LAC, INV and GAL, and INV and LAC stages, respectively. MiR-29b/miR-363 and miR-874/miR-6254 are important mediators for transition signals from LAC to GAL and from GAL to INV, respectively. Moreover, 58 miRNAs were dynamically DE in all lactation stages and 19 miRNAs were significantly time-dependently DE throughout lactation. Relevant signalling pathways for transition between lactation stages are involved in apoptosis (PTEN and SAPK/JNK), intracellular signalling (protein kinase A, TGF-β and ERK5), cell cycle regulation (STAT3), cytokines, hormones and growth factors (prolactin, growth hormone and glucocorticoid receptor). Overall, our data suggest diverse, temporal and physiological signal-dependent regulatory and mediator functions for miRNAs during lactation.

Lactation persistency (LP), defined as the rate of declining milk yield after milk peak, is an economically important trait for dairy cattle. Improving LP is considered a good alternative method for increasing overall milk production because it does not cause the negative energy balance and other health issues that cows experience during peak milk production. However, little is known about the biology of LP. A genome-wide association study (GWAS) and pathway enrichment were used to explore the genetic mechanisms underlying LP. The GWAS was performed using a univariate regression mixed linear model on LP data of 3,796 cows and 44,100 single nucleotide polymorphisms (SNP). Eight and 47 SNP were significantly and suggestively associated with LP, respectively. The 2 most important quantitative trait loci regions for LP were (1) a region from 106 to 108 Mb on Bos taurus autosome (BTA) 5, where the most significant SNP (ARS-BFGL-NGS-2399) was located and also formed a linkage disequilibrium block with 3 other SNP; and (2) a region from 29.3 to 31.3 Mb on BTA 20, which contained 3 significant SNP. Based on physical positions, MAN1C1, MAP3K5, HCN1, TSPAN9, MRPS30, TEX14, and CCL28 are potential candidate genes for LP because the significant SNP were located in their intronic regions. Enrichment analyses of a list of 536 genes in 0.5-Mb flanking regions of significant and suggestive SNP indicates that synthesis of milk components, regulation of cell apoptosis processes and insulin, and prolactin signaling pathways are important for LP. Upstream regulators relevant for LP positional candidate genes were prolactin (PRL), peroxisome proliferator-activated receptor gamma (PPARG), and Erb-B2 receptor tyrosine kinase 2 (ERBB2). Several networks related to cellular development, proliferation and death were significantly enriched for LP positional candidate genes. In conclusion, this study detected several SNP, genes, and interesting regions for fine mapping and validation of candidate genes and SNP for potential use in selection for improved LP. This study also provided further insights on the biology of LP which will help to prioritize selected candidate genes for functional validation and application., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

High-throughput sequencing technologies have increased the ability to detect sequence variations for complex trait improvement. A high throughput genome wide genotyping-by-sequencing (GBS) method was used to generate 515,787 single nucleotide polymorphisms (SNPs), from which 76,355 SNPs with call rates >85% and minor allele frequency ≥1.5% were used in genome wide association study (GWAS) of 44 milk traits in 1,246 Canadian Holstein cows. GWAS was accomplished with a mixed linear model procedure implementing the additive and dominant models. A strong signal within the centromeric region of bovine chromosome 14 was associated with test day fat percentage. Several SNPs were associated with eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, CLA:9c11t and gamma linolenic acid. Most of the significant SNPs for 44 traits studied are novel and located in intergenic regions or introns of genes. Novel potential candidate genes for milk traits or mammary gland functions include ERCC6, TONSL, NPAS2, ACER3, ITGB4, GGT6, ACOX3, MECR, ADAM12, ACHE, LRRC14, FUK, NPRL3, EVL, SLCO3A1, PSMA4, FTO, ADCK5, PP1R16A and TEP1. Our study further demonstrates the utility of the GBS approach for identifying population-specific SNPs for use in improvement of complex dairy traits.

Abundant miRNAs have been identified in milk and mammary gland tissues of different species. Typically, RNA in milk can be extracted from different fractions including fat, whey and cells and the mRNA transcriptome of milk could serve as an indicator of the transcriptome of mammary gland tissue. However, it has not been adequately validated if the miRNA transcriptome of any milk fraction could be representative of that of mammary gland tissue. The objectives of this study were to (1) characterize the miRNA expression spectra from three milk fractions- fat, whey and cells; (2) compare miRNome profiles of milk fractions (fat, whey and cells) with mammary gland tissue miRNome, and (3) determine which milk fraction miRNome profile could be a better representative of the miRNome profile of mammary gland tissue. Milk from four healthy Canadian Holstein cows in mid lactation was collected and fractionated. Total RNA extracted from each fraction was used for library preparation followed by small RNA sequencing. In addition, miRNA transcripts of mammary gland tissues from twelve Holstein cows in our previous study were used to compare our data. We identified 210, 200 and 249 known miRNAs from milk fat, whey and cells, respectively, with 188 universally expressed in the three fractions. In addition, 33, 31 and 36 novel miRNAs from milk fat, whey and cells were identified, with 28 common in the three fractions. Among 20 most highly expressed miRNAs in each fraction, 14 were expressed in common and 11 were further shared with mammary gland tissue. The three milk fractions demonstrated a clear separation from each other using a hierarchical cluster analysis with milk fat and whey being most closely related. The miRNome correlation between milk fat and mammary gland tissue (rmean = 0.866) was significantly higher than the other two pairs (p < 0.01), whey/mammary gland tissue (rmean = 0.755) and milk cell/mammary gland tissue (rmean = 0.75), suggesting that milk fat could be an alternative non-invasive source of RNA in assessing miRNA activities in bovine mammary gland. Predicted target genes (1802) of 14 highly expressed miRNAs in milk fractions were enriched in fundamental cellular functions, infection, organ and tissue development. Furthermore, some miRNAs were highly enriched (FDR <0.05) in milk whey (3), cells (11) and mammary gland tissue (14) suggesting specific regulatory functions in the various fractions. In conclusion, we have obtained a comprehensive miRNA profile of the different milk fractions using high throughput sequencing. Our comparative analysis showed that miRNAs from milk fat accurately portrayed the miRNome of mammary gland tissue. Functional annotation of the top expressed miRNAs in milk confirmed their critical regulatory roles in mammary gland functions and potentially to milk recipients.

Background: Nutritional strategies can decrease saturated fatty acids (SFAs) and increase health beneficial fatty acids (FAs) in bovine milk. The pathways/genes involved in these processes are not properly defined. Next-generation RNA-sequencing was used to investigate the bovine mammary gland transcriptome following supplemental feeding with 5% linseed oil (LSO) or 5% safflower oil (SFO). Holstein cows in mid-lactation were fed a control diet for 28 days (control period) followed by supplementation with 5% LSO (12 cows) or 5% SFO (12 cows) for 28 days (treatment period). Milk and mammary gland biopsies were sampled on days-14 (control period), +7 and +28 (treatment period). Milk was used to measure fat(FP)/protein(PP) percentages and individual FAs while RNA was subjected to sequencing., Results: Milk FP was decreased by 30.38% (LSO) or 32.42% (SFO) while PP was unaffected (LSO) or increased (SFO). Several beneficial FAs were increased by LSO (C18:1n11t, CLA:10t12c, CLA:9c11t, C20:3n3, C20:5n3, C22:5n3) and SFO (C18:1n11t, CLA:10t12c, C20:1c11, C20:2, C20:3n3) while several SFAs (C4:0, C6:0, C8:0, C14:0, C16:0, C17:0, C24:0) were decreased by both treatments (P < 0.05). 1006 (460 up- and 546 down-regulated) and 199 (127 up- and 72 down-regulated) genes were significantly differentially regulated (DE) by LSO and SFO, respectively. Top regulated genes (≥ 2 fold change) by both treatments (FBP2, UCP2, TIEG2, ANGPTL4, ALDH1L2) are potential candidate genes for milk fat traits. Involvement of SCP2, PDK4, NQO1, F2RL1, DBI, CPT1A, CNTFR, CALB1, ACADVL, SPTLC3, PIK3CG, PIGZ, ADORA2B, TRIB3, HPGD, IGFBP2 and TXN in FA/lipid metabolism in dairy cows is being reported for the first time. Functional analysis indicated similar and different top enriched functions for DE genes. DE genes were predicted to significantly decrease synthesis of FA/lipid by both treatments and FA metabolism by LSO. Top canonical pathways associated with DE genes of both treatments might be involved in lipid/cholesterol metabolism., Conclusion: This study shows that rich α-linolenic acid LSO has a greater impact on mammary gland transcriptome by affecting more genes, pathways and processes as compared to SFO, rich in linoleic acid. Our study suggest that decrease in milk SFAs was due to down-regulation of genes in the FA/lipid synthesis and lipid metabolism pathways while increase in PUFAs was due to increased availability of ruminal biohydrogenation metabolites that were up taken and incorporated into milk or used as substrate for the synthesis of PUFAs.