Effect of selection genotype on immune response to Brucella abortus RB51 in Holstein cattle.

Genetic selection for milk production traits in US Holsteins has affected numerous genes associated with reproduction and immunity. This study compares the transcriptomic response of peripheral blood mononuclear cells to an in vitro Brucella abortus strain RB51 (RB51) bacterial challenge between contemporary Holsteins and Holsteins that have not been selected for milk production traits since the mid-1960s. Total RNA was extracted from peripheral blood mononuclear cells from four contemporary and four unselected lactating, primiparous cows following 24-h incubation with or without stimulation with RB51 bacteria. RNA was sequenced and reads analyzed using tools from galaxy.scinet.usda.gov. A total of 412 differentially expressed genes (false discovery rate p < 0.05, log fold change > |1|) were identified. The upregulated genes (genes with higher expression in contemporary than unselected cattle) were enriched for 19 terms/pathways, including alanine, aspartate, and glutamate metabolism, indicating a cellular stress response. Downregulated genes (genes with higher expression in unselected than contemporary cows) were enriched for 37 terms/pathways, representing diverse immune responses, including natural killer cell-mediated immunity, interferon-γ production, negative regulation of interleukin-10 production, and cytokine receptor activity indicating a broad immune response with an emphasis on immune defense. These results provide evidence that differences exist between the two genotypes in response to in vitro bacterial challenge. This suggests that contemporary cows, genetically selected for milk production, may have reduced immune function, including limitations in response to intracellular bacteria.

The Association between Prepartum Rumination Time, Activity and Dry Matter Intake and Subclinical Hypocalcemia and Hypomagnesemia in the First 3 Days Postpartum in Holstein Dairy Cows.

Changes in prepartum behaviors such as total daily rumination (TDR), total daily activity (TDA) and dry matter intake (DMI) have the potential to be used as early indicators for cows at risk for subclinical hypocalcemia (SCH) or hypomagnesemia (HYM) after calving. Our objective was to investigate associations between average daily rate of change in total daily rumination (ΔTDR), total daily activity (ΔTDA) and dry matter intake (ΔDMI) from -3 days prepartum to calving with SCH and HYM at D0 or D3 relative to calving. Prepartum TDR, TDA and DMI were measured in 64 Holstein dairy cows. Blood samples were taken at D0 and D3 post-calving for the measurement of total plasma Ca and Mg concentration. Linear regression models were used to analyze the association between ΔTDR, ΔTDA and ΔDMI and SCH and HYM at D0 and D3 relative to calving. Potential confounding variables were offered to the models and backwards selection was used to determine which covariates to retain. No significant differences in prepartum ΔTDR, ΔTDA or ΔDMI were found between cows with or without SCH and HYM at D0 and D3. Our results suggest that the change in TDR, TDA and DMI in the last 3 days prepartum are not effective predictors for cows that will have SCH or HYM in the first 3 days postpartum.

Effect of Holstein genotype on ex-vivo interleukin-1ß response to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and heat-killed Gram-negative and Gram-positive bacteria.

Effects of Holstein genotype on interleukin-1ß response were assessed by ex-vivo stimulation of whole blood with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or sonicated, heat-killed Gram-negative or Gram-positive bacteria. Holstein genotypes were unselected Holsteins (UH, n = 14) not subjected to selection pressures since the mid-1960s and contemporary Holsteins (CH, n = 13). Milk yield of UH and CH cows differ by more than 4500 kg/lactation. Whole blood was mixed with 0.01 µg LPS, 10 µg LTA or 2.5 × 106 CFU of sonicated, heat-killed E. coli, K. pneumoniae, S. marcescens, S. aureus, S. dysgalactiae, or S. uberis per mL of blood and incubated (4 h, 37 °C). Plasma IL-1ß was quantified by ELISA and log10-transformed concentrations analyzed with a multivariate linear mixed effects model. Responses to bacteria were greater than responses to LPS or LTA. Responses to LPS, LTA and the Gram-negative stimulants were greater in UH than in CH cows while responses to the Gram-positive bacteria did not differ between Holstein genotypes. In both genotypes, strong correlations were detected among IL-1ß responses to the Gram-negative stimulants and to LTA. There were strong correlations among IL-1ß responses to the Gram-positive bacteria in CH cows but only between S. aureus and S. dysgalactiae in UH cows. The IL-1ß response to S. uberis was highly correlated with responses to all of the Gram-negative stimulants in CH cows but only with E. coli in the UH cows. The reduced immune response could make contemporary cows more susceptible to infection by Gram-negative bacteria. Results confirm selection practices since the mid-1960s have altered immune response in the Holstein, at least to Gram-negative bacteria, and validate the need for additional studies to further evaluate the impacts of these selection practices on immune function in contemporary Holsteins.

Effect of Holstein genotype on ex-vivo cytokine response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during the periparturient period.

Effects of Holstein genotype on innate immune response were assessed with ex-vivo lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation of whole blood from unselected (UH, n = 10) and contemporary (CH, n = 11) Holsteins that differ in production by more than 4,500 kg/lactation. Blood was collected at -14, 7, 28, and 49 days in milk (DIM), mixed with a pathogen-associated molecular pattern (PAMP) molecule (0.01 or 1.0 µg LPS or 10 or 100 µg LTA per mL blood) and incubated (4 h, 37 °C). Plasma cytokines were quantified by ELISA, log10-transformed and analyzed by repeated measures with DIM as the repeated effect. Cytokine responses increased with PAMP dose and decreased as DIM increased. There was a genotype by LPS dose interaction for IL-1ß as response to the low dose was greater in UH but did not differ between genotypes for the high dose. The IL-1ß response was greater while the IL-6 response to LTA tended to be greater in UH than in CH cows. The more negative energy balance of CH cows did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of CH cows to LPS and to LTA.

Effect of Holstein genotype on immune response to an intramammary Escherichia coli challenge.

Selective breeding of US dairy cows since the mid-1960s has contributed to remarkable gains in milk yield per cow. This increased milk yield has been associated with an increase in health issues. Since 1964, the University of Minnesota has selectively bred a Holstein herd to maintain genetically static, unselected Holsteins (UH). Comparison of these UH cows with contemporary Holsteins (CH) has demonstrated that the UH cows not only produce less milk but also have fewer health concerns than their CH herdmates. The objective of this study was to determine the effects of Holstein genotype on innate immune response in an experimental intramammary Escherichia coli challenge model. Primiparous UH (n = 5) and CH (n = 7) cows received 430 cfu of E. coli strain P4 in 1 quarter. Blood and affected quarter milk samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 7, 9, and 11 d relative to E. coli infusion. Rectal temperatures were recorded at each milking through d 4 of the experiment. Milk bacterial counts, somatic cell count and BSA concentrations, complete blood cell counts, rectal temperature, and serum and milk whey cytokine (IL-1ß and IL-6) concentrations were used as metrics to determine infection severity. Longitudinal (repeated) data were analyzed using general linear models with PROC MIXED with day of study as the repeated effect. Whole blood transcriptomes were generated by RNA sequencing. Transcripts with a false discovery rate of P < 0.05 and a delta log2 expression value greater than 0.7 or less than -0.7 were used for functional enrichment analysis. Bacterial counts were consistently greater in milk from CH than UH cows from d 0.25 through d 2.5. Milk somatic cell count increased within 6 h (d 0.25) after E. coli administration in CH and UH cows but did not differ between genotypes after d 1. Rectal body temperature peaked at d 1 in CH and UH cows but was greater in CH cows. Milk BSA, IL-1ß, and IL-6 concentrations were greater in CH than UH cows after E. coli administration. Blood lymphocyte and neutrophil counts were decreased at 0.5 and 1 d in CH but not in UH cows. The number of differentially expressed transcripts at each of the postinfusion sampling times was consistently greater (4- to 90-fold) in CH than in UH cows. A key difference between the immune reaction of the 2 genotypes was that the immune response to E. coli was largely contained within the mammary gland of the UH cows but became more systemic in the CH cows. These data demonstrate that UH cows exerted more effective control of E. coli infused into the mammary gland and thus support the hypothesis that selection practices since the mid-1960s have resulted in CH cows with an immune system that is less effective in fighting intramammary infections. Identification of genetic factors associated with enhanced immune functions that differ between the UH and CH cows could contribute to efforts to reintroduce or enhance beneficial components that have been lost or reduced in the CH cows since the mid-1960s.

Prevalence, antibiotic resistance, virulence and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of U.S. dairy herds.

BACKGROUND: Colonization of dairy cows by Staphylococcus aureus (S. aureus), especially those which are multi-drug resistant and toxin producing, is a concern for animal health and well-being as well as public health. The objective of this study was to investigate the prevalence, antibiotic resistance, gene content and virulence determinants of S. aureus in bulk tank milk samples (BTM) from U.S. dairy herds. RESULTS: BTM samples were collected, once in winter and once in summer, from 189 U.S. dairy herds. Of 365 BTM samples cultured, the sample and herd prevalence of S. aureus in BTM was 46.6% (170 of 365 samples) and 62.4% (118 of 189 herds), respectively. Among a subset of 138 S. aureus isolates that were stored for further analysis, 124 were genome sequenced after being confirmed as S. aureus using phenotypic tests. The most commonly identified antimicrobial resistance-associated gene was norA (99.2%) and mecA gene responsible for methicillin resistance (MRSA) was identified in one isolate (0.8%). The most frequently detected putative virulence genes were aur (100%), hlgB (100%), hlgA, hlgC, hlb (99.2%), lukE (95.9%) and lukD (94.3%). In the 53 staphylococcal enterotoxin positive isolates, sen (37.9%), sem (35.5%), sei (35.5%) and seg (33.1%) were the most frequently detected enterotoxin genes. Among the 14 sequence types (ST) and 18 spa types identified, the most common was ST2187 (20.9%) and t529 (28.2%), respectively. The most predominant clone was CC97 (47.6%) followed by CC unknown (36.3%). The single MRSA isolate belonged to ST72-CC8, spa type t126 and was negative for the tst gene but harbored all the other virulence genes investigated. CONCLUSION: Our findings indicated a high prevalence of S. aureus in BTM of U.S. dairy herds, with isolates showing little evidence of resistance to antibiotics commonly used to treat mastitis. However, isolates often carried genes for the various enterotoxins. This study identified predominant genetic clones. Despite lower prevalence, the presence of MRSA and multi-drug resistant strains in BTM poses a significant risk to animal and public health if their number were to increase in dairy environment. Therefore, it is necessary to continuously monitor the use of antibiotics in dairy cows.

Forming 4-Methylcatechol as the Dominant Bioavailable Metabolite of Intraruminal Rutin Inhibits p-Cresol Production in Dairy Cows.

Rutin, a natural flavonol glycoside, elicits its diverse health-promoting effects from the bioactivities of quercetin, its aglycone. While widely distributed in the vegetables and fruits of human diet, rutin is either absent or inadequate in common animal feed ingredients. Rutin has been supplemented to dairy cows for performance enhancement, but its metabolic fate in vivo has not been determined. In this study, plasma, urine, and rumen fluid samples were collected before and after the intraruminal dosing of 100 mg/kg rutin to 4 Holsteins, and then characterized by both targeted and untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis. In plasma and urine, 4-methylcatechol sulfate was identified as the most abundant metabolite of rutin, instead of quercetin and its flavonol metabolites, and its concentration was inversely correlated with the concentration of p-cresol sulfate. In rumen fluid, the formation of 3,4-dihydroxyphenylacetic acid (DHPAA) and 4-methylcatechol after rapid degradation of rutin and quercetin concurred with the decrease of p-cresol and the increase of its precursor, 4-hydroxyphenylacetic acid. Overall, the formation of 4-methylcatechol, a bioactive microbial metabolite, as the dominant bioavailable metabolite of rutin and quercetin, could contribute to their beneficial bioactivities in dairy cows, while the decrease of p-cresol, a microbial metabolite with negative biological and sensory properties, from the competitive inhibition between microbial metabolism of rutin and tyrosine, has the potential to reduce environmental impact of dairy operations and improve the health of dairy cattle.

Effects of anionic supplement source in prepartum negative dietary cation-anion difference diets on serum calcium, feed intake, and lactational performance of multiparous dairy cows.

Incidence of subclinical hypocalcemia in early postpartum dairy cows continues to be an animal welfare concern and an economic burden for producers. Feeding prepartum negative dietary cation-anion difference (DCAD) diets produces metabolic acidosis, which supports mobilization of bone calcium and reduces the incidence of hypocalcemia. Achieving a sufficient degree of metabolic acidosis without reducing dry matter intake (DMI) can be difficult. This study compared the ability of MegAnion (MA; Origination O2D Inc., Maplewood, MN), a new DCAD supplement designed to be more palatable than typical anionic salt sources, and another palatable commercial DCAD product, SoyChlor (SC; Landus Cooperative, Ralston, IA), to reduce urine pH (a surrogate for metabolic acidosis) without reducing prepartum DMI. A secondary objective was to assess the effect of these anionic supplements on postpartum serum calcium concentrations and DMI. Prepartum multiparous Holstein (HO) and crossbred (XX) cows were blocked by breed and expected calving date and randomly assigned within breed to total mixed rations (TMR) with MA or SC and DCAD values of -215 mEq/kg of DM. Cows (n = 56; 15 MA-HO, 12 SC-HO, 15 MA-XX, 14 SC-XX) consumed the treatment TMR for at least 19 d and completed the 28 d in milk (DIM) phase of the study. Urine and blood samples were collected weekly and at 1, 2, and 3 DIM. Data were analyzed as a randomized block design by repeated measures with week or DIM as the repeated effect. Prepartum urine pH decreased from 8.15 ± 0.27 before treatment to 6.12 ± 0.14 during treatment, was not affected by anionic supplement, and increased immediately after calving when all cows consumed the same early-lactation TMR. Prepartum serum calcium concentrations were not affected (2.34 vs. 2.33 ± 0.02 mmol/L) by treatment, whereas nonesterified fatty acids were lower (86 vs. 120 ± 10 mmol/L) and insulin was greater (215 vs. 174 ± 10 pmol/L) in cows fed MA than in cows fed SC. These differences are supported by the numerically greater prepartum DMI (1.2 kg/d) and energy balance (1.8 Mcal/d) of cows fed MA. However, pre- and postpartum DMI and other production variables, including body weight, body condition score, milk yield, and energy balance, were not affected by treatment. This lack of difference indicates that MA provides another effective source of anionic salts for diets designed to reduce urine pH and induce metabolic acidosis in prepartum dairy cows.

Single molecule sequencing of nucleic acids in real time (SMRT) to characterize transcriptomes and mRNA isoforms / Secuenciación de moléculas individuales de ácidos nucleicos en tiempo real (SMRT) para caracterizar transcriptomas e isoformas de ARNm

Abstract Our efforts are oriented to assess bovine Y-chromosome gene expression patterns. One set of genes that are of interest are the so-called X-degenerate Y-chromosome genes that are located in the male-specific region of the Y-chromosome (MSY). This region contains 95% of the DNA of the Y chromosome. These genes are single copy and have an X-chromosome homolog. Both, the Y-encoded and X-encoded homologs have ubiquitous expression profiles. However, some genes, like SRY that regulates male sex determination, have functions that are more specific. Identifying DNA sequence differences between these homologs will allow evaluation of their spatial and temporal expression patterns. Identification of the Y-encoded mRNAs and their isoforms will allow our understanding of tissue specific expression of isoforms in male tissues. The latter will facilitate our evaluation of gene function in male sex differentiation and fertility. Hence, we hypothesized that each of these X-degenerate gene homologs generate isoforms and that differential expression patterns exist between sexes and across tissues. To investigate the latter we used a new generation sequencing (NGS) technology that generates long sequencing reads with a range between 1000 to 10,000 base pairs in length. Single molecule real time (SMRT) isoform sequencing (IsoSeq) of several tissues (liver, lung, adipose, muscle, hypothalamus and testis) was carried out. Transcript sequences were used for bioinformatics analysis and isoform characterization. Given the focus of this manuscript the SMRT technology we are only presenting results obtained with the analysis of the bUTY and bUTX genes.

Resumen Nuestros esfuerzos están orientados a evaluar patrones de expresión génica del cromosoma Y bovino. Los genes de interés son los denominados genes X-degenerados que se encuentran en la región específica masculina del cromosoma Y (MSY). Esta región contiene el 95% del ADN del cromosoma Y. Estos genes son de copia única y tienen un homólogo en el cromosoma X. Ambos homólogos tienen perfiles amplios de expresión. Sin embargo, algunos genes, como el SRY que regula la determinación del sexo masculino, tienen funciones más específicas. La identificación de las diferencias de secuencia de ADN entre estos homólogos permitirá evaluar sus patrones de expresión espacial y temporal. La identificación de los ARNm codificados en el cromosoma Y y de sus isoformas permitirán analizar la expresión específica de sus isoformas en tejidos masculinos. Esto último facilitará nuestra evaluación de función génica en la diferenciación sexual masculina y la fertilidad. Por lo tanto, planteamos la hipótesis de que cada uno de estos genes homólogos degenerados del X genera isoformas y que existen patrones de expresión diferencial entre sexos y tejidos. Para investigar esto último, utilizamos una tecnología de secuenciación de nueva generación (NGS) que genera lecturas de secuenciación largas con un rango de longitud de 1000 a 10,000 pares de bases. Se secuenciaron los transcriptomas en varios tejidos (hígado, pulmón, adiposo, muscular, hipotálamo y testículo). Se utilizaron las secuencias generadas para el análisis bioinformático y la caracterización de isoformas. Siendo el foco de este manuscrito la tecnología SMRT, solo presentamos los resultados obtenidos con el análisis de los genes bUTY y bUTX.

Effect of bovine genotype on innate immune response of heifers to repeated lipopolysaccharide (LPS) administration.

This pilot study provides a preliminary assessment of the impact of genotype on acute innate immune pro-inflammatory, metabolic and endocrine responses to repeated lipopolysaccharide (LPS) administered to growing heifers. Heifers (n = 4/genotype) were from unselected (stable milk yield since 1964, UH) or contemporary (CH) Holstein cows that differed in milk yield (6200 vs 11,100 kg milk/305 d) or from contemporary Black Angus (CA) cows bred to contemporary Red Angus bulls. Heifers were challenged with iv administration of 0.5 µg LPS/kg body weight on day 1 (Challenge 1) and d 5 (Challenge 2) of study to assess endotoxin tolerance. Plasma was collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to each LPS administration. Rectal body temperature (BT) was measured before each blood sampling and at 5 and 7 h. Data were analyzed by repeated measures with sampling time as the repeated effect. Each genotype had at least one pro-inflammatory response that indicated it might have a more robust response than the other genotypes. The CH heifers had a greater TNF-α response, UH heifers had greater IL-6 and XO responses and CA heifers had greater BT and SAA response to LPS than the other genotypes. There was a genotype by time by interaction as cortisol peaked earlier in CH and UH than in CA heifers. Glucose response was less in CA and insulin response was greater in CH heifers. Endotoxin tolerance to LPS was evident as pro-inflammatory, cortisol, glucose and insulin responses were less during Challenge 2 than during Challenge 1. Differences among genotypes during Challenge 1 were eliminated during Challenge 2 except for the greater SAA response in CA heifers and indicate the potential for differential impacts of genotype on the development of endotoxin tolerance. Specific reasons for these effects of genotype are not clear from these data but the results support the hypothesis for differential innate immune signaling among these bovine genotypes.

Genome changes due to artificial selection in U.S. Holstein cattle.

BACKGROUND: The availability of a unique unselected Holstein line since 1964 provided a direct comparison between selected and unselected Holstein genomes whereas large Holstein samples provided unprecedented statistical power for identifying high-confidence SNP effects. Utilizing these unique resources, we aimed to identify genome changes affected by selection since 1964. RESULTS: Direct comparison of genome-wide SNP markers between a Holstein line unselected since 1964 and contemporary Holsteins showed that the 40 years of artificial selection since 1964 resulted in genome landscape changes. Among the regions affected by selection, the regions containing 198 genes with fertility functions had a larger negative correlation than that of all SNPs between the SNP effects on milk yield and daughter pregnancy rate. These results supported the hypothesis that hitchhiking of genetic selection for milk production by negative effects of fertility genes contributed to the unintended declines in fertility since 1964. The genome regions subjected to selection also contained 67 immunity genes, the bovine MHC region of Chr23 with significantly decreased heterozygosity in contemporary Holsteins, and large gene clusters including T-cell receptor and immunoglobulin genes. CONCLUSIONS: This study for the first time provided direct evidence that genetic selection for milk production affected fertility and immunity genes and that the hitchhiking of genetic selection for milk production by negative fertility effects contributed to the fertility declines since 1964, and identified a large number of candidate fertility and immunity genes affected by selection. The results provided novel understanding about genome changes due to artificial selection and their impact on fertility and immunity genes and could facilitate developing genetic methods to reverse the declines in fertility and immunity in Holstein cattle.

QuanTP: A Software Resource for Quantitative Proteo-Transcriptomic Comparative Data Analysis and Informatics.

Next-generation sequencing technologies, coupled to advances in mass-spectrometry-based proteomics, have facilitated system-wide quantitative profiling of expressed mRNA transcripts and proteins. Proteo-transcriptomic analysis compares the relative abundance levels of transcripts and their corresponding proteins, illuminating discordant gene product responses to perturbations. These results reveal potential post-transcriptional regulation, providing researchers with important new insights into underlying biological and pathological disease mechanisms. To carry out proteo-transcriptomic analysis, researchers require software that statistically determines transcript-protein abundance correlation levels and provides results visualization and interpretation functionality, ideally within a flexible, user-friendly platform. As a solution, we have developed the QuanTP software within the Galaxy platform. The software offers a suite of tools and functionalities critical for proteo-transcriptomics, including statistical algorithms for assessing the correlation between single transcript-protein pairs as well as across two cohorts, outlier identification and clustering, along with a diverse set of results visualizations. It is compatible with analyses of results from single experiment data or from a two-cohort comparison of aggregated replicate experiments. The tool is available in the Galaxy Tool Shed through a cloud-based instance and a Docker container. In all, QuanTP provides an accessible and effective software resource, which should enable new multiomic discoveries from quantitative proteo-transcriptomic data sets.

Symposium review: Microbial endocrinology-Why the integration of microbes, epithelial cells, and neurochemical signals in the digestive tract matters to ruminant health.

The union of microbiology and neurobiology, which has been termed microbial endocrinology, is defined as the study of the ability of microorganisms to produce and respond to neurochemicals that originate either within the microorganisms themselves or within the host they inhabit. It serves as the basis for an evolutionarily derived method of communication between a host and its microbiota. Mechanisms elucidated by microbial endocrinology give new insight into the ways the microbiota can affect host stress, metabolic efficiency, resistance to disease, and other factors that may prove relevant to the dairy industry.

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

BACKGROUND: We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis. RESULTS: The Holstein breed displayed a more robust response to LPS than the Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2, and 8 h. Several immune-associated genes were expressed at greater levels (FDR < 0.05) in Holstein cultures including TLR4 at all time points and a number of pro-inflammatory genes such as IL8, CCL20, CCL5, and TNF following LPS exposure. Despite extensive breed differences in the transcriptome, MIRA-Seq unveiled relatively similar patterns of genome-wide DNA methylation between breeds, with an overall hypomethylation of gene promoters. However, by examining the genome in 3Kb windows, 49 regions of differential methylation were discovered between Holstein and Angus fibroblasts, and two of these regions fell within the promoter region (-2500 to +500 bp of the transcription start site) of the genes NTRK2 and ADAMTS5. CONCLUSIONS: Fibroblasts isolated from Holstein cattle display a more robust response to LPS in comparison to cultures from Angus cattle. Different selection strategies and management practices exist between these two breeds that likely give rise to genetic and epigenetic factors contributing to the different immune response phenotypes.

Effect of artificial selection on runs of homozygosity in u.s. Holstein cattle.

The intensive selection programs for milk made possible by mass artificial insemination increased the similarity among the genomes of North American (NA) Holsteins tremendously since the 1960s. This migration of elite alleles has caused certain regions of the genome to have runs of homozygosity (ROH) occasionally spanning millions of continuous base pairs at a specific locus. In this study, genome signatures of artificial selection in NA Holsteins born between 1953 and 2008 were identified by comparing changes in ROH between three distinct groups under different selective pressure for milk production. The ROH regions were also used to estimate the inbreeding coefficients. The comparisons of genomic autozygosity between groups selected or unselected since 1964 for milk production revealed significant differences with respect to overall ROH frequency and distribution. These results indicate selection has increased overall autozygosity across the genome, whereas the autozygosity in an unselected line has not changed significantly across most of the chromosomes. In addition, ROH distribution was more variable across the genomes of selected animals in comparison to a more even ROH distribution for unselected animals. Further analysis of genome-wide autozygosity changes and the association between traits and haplotypes identified more than 40 genomic regions under selection on several chromosomes (Chr) including Chr 2, 7, 16 and 20. Many of these selection signatures corresponded to quantitative trait loci for milk, fat, and protein yield previously found in contemporary Holsteins.

Effect of sample stratification on dairy GWAS results.

BACKGROUND: Artificial insemination and genetic selection are major factors contributing to population stratification in dairy cattle. In this study, we analyzed the effect of sample stratification and the effect of stratification correction on results of a dairy genome-wide association study (GWAS). Three methods for stratification correction were used: the efficient mixed-model association expedited (EMMAX) method accounting for correlation among all individuals, a generalized least squares (GLS) method based on half-sib intraclass correlation, and a principal component analysis (PCA) approach. RESULTS: Historical pedigree data revealed that the 1,654 contemporary cows in the GWAS were all related when traced through approximately 10-15 generations of ancestors. Genome and phenotype stratifications had a striking overlap with the half-sib structure. A large elite half-sib family of cows contributed to the detection of favorable alleles that had low frequencies in the general population and high frequencies in the elite cows and contributed to the detection of X chromosome effects. All three methods for stratification correction reduced the number of significant effects. EMMAX method had the most severe reduction in the number of significant effects, and the PCA method using 20 principal components and GLS had similar significance levels. Removal of the elite cows from the analysis without using stratification correction removed many effects that were also removed by the three methods for stratification correction, indicating that stratification correction could have removed some true effects due to the elite cows. SNP effects with good consensus between different methods and effect size distributions from USDA's Holstein genomic evaluation included the DGAT1-NIBP region of BTA14 for production traits, a SNP 45kb upstream from PIGY on BTA6 and two SNPs in NIBP on BTA14 for protein percentage. However, most of these consensus effects had similar frequencies in the elite and average cows. CONCLUSIONS: Genetic selection and extensive use of artificial insemination contributed to overlapped genome, pedigree and phenotype stratifications. The presence of an elite cluster of cows was related to the detection of rare favorable alleles that had high frequencies in the elite cluster and low frequencies in the remaining cows. Methods for stratification correction could have removed some true effects associated with genetic selection.

Genome-wide association analysis of thirty one production, health, reproduction and body conformation traits in contemporary U.S. Holstein cows.

BACKGROUND: Genome-wide association analysis is a powerful tool for annotating phenotypic effects on the genome and knowledge of genes and chromosomal regions associated with dairy phenotypes is useful for genome and gene-based selection. Here, we report results of a genome-wide analysis of predicted transmitting ability (PTA) of 31 production, health, reproduction and body conformation traits in contemporary Holstein cows. RESULTS: Genome-wide association analysis identified a number of candidate genes and chromosome regions associated with 31 dairy traits in contemporary U.S. Holstein cows. Highly significant genes and chromosome regions include: BTA13's GNAS region for milk, fat and protein yields; BTA7's INSR region and BTAX's LOC520057 and GRIA3 for daughter pregnancy rate, somatic cell score and productive life; BTA2's LRP1B for somatic cell score; BTA14's DGAT1-NIBP region for fat percentage; BTA1's FKBP2 for protein yields and percentage, BTA26's MGMT and BTA6's PDGFRA for protein percentage; BTA18's 53.9-58.7 Mb region for service-sire and daughter calving ease and service-sire stillbirth; BTA18's PGLYRP1-IGFL1 region for a large number of traits; BTA18's LOC787057 for service-sire stillbirth and daughter calving ease; BTA15's CD82, BTA23's DST and the MOCS1-LRFN2 region for daughter stillbirth; and BTAX's LOC520057 and GRIA3 for daughter pregnancy rate. For body conformation traits, BTA11, BTAX, BTA10, BTA5, and BTA26 had the largest concentrations of SNP effects, and PHKA2 of BTAX and REN of BTA16 had the most significant effects for body size traits. For body shape traits, BTAX, BTA19 and BTA3 were most significant. Udder traits were affected by BTA16, BTA22, BTAX, BTA2, BTA10, BTA11, BTA20, BTA22 and BTA25, teat traits were affected by BTA6, BTA7, BTA9, BTA16, BTA11, BTA26 and BTA17, and feet/legs traits were affected by BTA11, BTA13, BTA18, BTA20, and BTA26. CONCLUSIONS: Genome-wide association analysis identified a number of genes and chromosome regions associated with 31 production, health, reproduction and body conformation traits in contemporary Holstein cows. The results provide useful information for annotating phenotypic effects on the dairy genome and for building consensus of dairy QTL effects.