Eveline M. Ibeagha-Awemu

A critical analysis of disease-associated DNA polymorphisms in the genes of cattle, goat, sheep, and pig

Genetic variations through their effects on gene expression and protein function underlie disease susceptibility in farm animal species. The variations are in the form of single nucleotide polymorphisms, deletions/insertions of nucleotides or whole genes, gene or whole chromosomal rearrangements, gene duplications, and copy number polymorphisms or variants. They exert varying degrees of effects on gene action, such as substitution of an amino acid for another, shift in reading frame and premature termination of translation, and complete deletion of entire exon(s) or gene(s) in diseased individuals. These factors influence gene function by affecting mRNA splicing pattern or by altering/eliminating protein function. Elucidating the genetic bases of diseases under the control of many genes is very challenging, and it is compounded by several factors, including host × pathogen × environment interactions. In this review, the genetic variations that underlie several diseases of livestock (under monogenic and polygenic control) are analyzed. Also, factors hampering research efforts toward identification of genetic influences on animal disease identification and control are highlighted. A better understanding of the factors analyzed could be better harnessed to effectively identify and control, genetically, livestock diseases. Finally, genetic control of animal diseases can reduce the costs associated with diseases, improve animal welfare, and provide healthy animal products to consumers, and should be given more attention.

Proteomics, Genomics, and Pathway Analyses of Escherichia coli and Staphylococcus aureus Infected Milk Whey Reveal Molecular Pathways and Networks Involved in Mastitis

Gram-negative and -positive bacteria elicit different response patterns by the host. The proteomic profiles of milk whey samples from cows naturally infected with Escherichia coli or Staphyloccocus aureus as compared to whey from healthy cows were determined by one-dimensional, liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics processing, and pathway analyses. Since mammary epithelial cells contribute to immune responses in mammary glands, the genes of selected proteins were measured in MAC-T cells by real time quantitative PCR (qPCR) after stimulation with heat inactivated E. coli strain P4 and S. aureus strain Smith CP bacteria. A total of 173 proteins were identified including 73 proteins differentially expressed among normal, E. coli, and S. aureus treatment groups. E. coli was more effective at significantly altering the concentration of the affected proteins. The mRNA of 23 proteins out of 24 measured by qPCR was significantly altered in MAC-T cells. Pathway analyses identified top canonical pathways significantly enriched in our samples, the most significant being the acute phase response signaling pathway. Also, top networks of genes with significant associations to identified proteins were identified. Our study has demonstrated a wider proteome profile of E. coli and S. aureus mastitic milk whey, identified more low abundant defense proteins than reported before, and has linked for the first time identified proteins to several network functions and Biocarta pathways.

Influence of stearoyl-coenzyme A desaturase 1 genotype and stage of lactation on fatty acid composition of Canadian Jersey cows

Bovine milk contains high proportions of saturated fatty acids (SFA) because of the extensive biohydrogenation of dietary fatty acids in the rumen. Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of C10 to C18 SFA into their monounsaturated (MUFA) counterparts in the mammary glands of ruminant animals; and 2 alleles (A and V) have previously been identified at the SCD1 locus. Genotypes at this locus were identified and fatty acid contents of milk were measured for 525 Canadian Jersey cows. Association analysis indicated that allele A is positively associated with higher C10 (C10I), C12 (C12I) and C14 (C14I) indices and, consequently, with greater contents of C10:1 and C12:1, but not C14:1, relative to allele V. Allele A was also positively associated with increased 305-d milk and protein yields. Allele A, however, had no influence on C16 (C16I), C18 (C18I), or conjugated linoleic acid indices (CLAI) compared with the V allele. Stage of lactation had an influence on desaturase indices and consequently on the MUFA contents of milk fat. The indices C10I, C12I, C14I, and CLAI increased from early to mid lactation as did their respective MUFA. Genetic selection for increased unsaturation of the hypercholesterolemic fatty acids in milk fat is feasible and may be accompanied by increased lactation milk and protein yields.

Stearoyl-CoA desaturase 1 genotype and stage of lactation influences milk fatty acid composition of Canadian Holstein cows.

Single nucleotide polymorphisms in the coding region of the bovine stearoyl-CoA desaturase 1 gene have been predicted to result in p.293A (alanine at amino acid 293) and p.293V (valine at amino acid 293) alleles at the stearoyl-CoA desaturase1 locus. The objectives of this study were to evaluate the extent to which genotypes at the stearoyl-CoA desaturase 1 locus and stage of lactation influence milk fatty acid composition in Canadian Holstein cows. Cows with the p.293AA genotype had higher C10 index, C12 index and C14 index and higher concentrations of C10:1 (10 carbon fatty acid with one double bond), C12:1 (12 carbon fatty acid with one double bond) and myristoleic acid (C14:1) compared with the p.293AV or p.293VV cows. Cows had higher C18 index and total index, and lower C10 index, C12 index, C14 index and CLA index during early lactation compared with the subsequent lactation stages. Early lactation was also characterized by higher concentrations of oleic acid (C18:1 cis-9), vaccenic acid (C18:1 trans-11), linoleic acid (C18:2), monounsaturated fatty acids and total polyunsaturated fatty acids, and lower concentrations of capric acid (C10:0), C10:1, lauric acid (C12:0), C12:1, myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0) and total saturated fatty acids compared with the subsequent lactation stages. Neither the stearoyl-CoA desaturase 1 genotype nor the stage of lactation had an influence on conjugated linoleic acid concentrations in milk.

Epigenetic marks: regulators of livestock phenotypes and conceivable sources of missing variation in livestock improvement programs

Improvement in animal productivity has been achieved over the years through careful breeding and selection programs. Today, variations in the genome are gaining increasing importance in livestock improvement strategies. Genomic information alone, however, explains only a part of the phenotypic variance in traits. It is likely that a portion of the unaccounted variance is embedded in the epigenome. The epigenome encompasses epigenetic marks such as DNA methylation, histone tail modifications, chromatin remodeling, and other molecules that can transmit epigenetic information such as non-coding RNA species. Epigenetic factors respond to external or internal environmental cues such as nutrition, pathogens, and climate, and have the ability to change gene expression leading to emergence of specific phenotypes. Accumulating evidence shows that epigenetic marks influence gene expression and phenotypic outcome in livestock species. This review examines available evidence of the influence of epigenetic marks on livestock (cattle, sheep, goat, and pig) traits and discusses the potential for consideration of epigenetic markers in livestock improvement programs. However, epigenetic research activities on farm animal species are currently limited partly due to lack of recognition, funding and a global network of researchers. Therefore, considerable less attention has been given to epigenetic research in livestock species in comparison to extensive work in humans and model organisms. Elucidating therefore the epigenetic determinants of animal diseases and complex traits may represent one of the principal challenges to use epigenetic markers for further improvement of animal productivity.

Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells.

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.

Single nucleotide polymorphisms in the open reading frame of the stearoyl-CoA desaturase gene and resulting genetic variants in Canadian Holstein and Jersey cows

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) from their saturated counterparts in the mammary gland and adipose tissue of ruminant animals. We hypothesize that single nucleotide polymorphisms (SNPs) in the SCD gene account for some of the differences in SCD activity, and consequently for some of the variations in CLA and MUFA content of milk fat between Holsteins and Jersey cows and within these two breeds. We analyzed the open reading frame of the SCD gene of 44 Holsteins and 48 Jerseys for SNPs by sequencing. Three SNPs: 702A → G, 762T → C and 878C → T were identified in both breeds and a further SNP, 435G → A, was unique to Holsteins. The SNPs characterized four different genetic variants in Holsteins: A (G435A702T762C878), A1 (A435A702T762C878), B (G435G702C762T878) and B1 (A435G702C762T878), with only variants A and B in Jerseys. SNP 878C → T resulted in a non-synonymous codon change while the rest resulted in synonymous codon changes giving rise to two protein variants, A having alanine and B having valine. Allele A was the most prevalent in the two breeds. These differences may, therefore, contribute to existing variations in CLA and fat content between and within Canadian Holstein and Jersey cows.

MicroRNA roles in signalling during lactation: an insight from differential expression, time course and pathway analyses of deep sequence data

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.

High density genome wide genotyping-by-sequencing and association identifies common and low frequency SNPs, and novel candidate genes influencing cow milk traits

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.

Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows

BackgroundFatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk.ResultsThirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3′UTR SNP (FADS2-23, rs109772589), and another 3′UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6.ConclusionOur study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3’UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as potential genetic markers in breeding programs to increase milk FAs that are of benefit to human health.