Identification of loci associated with conception rate in primiparous Holstein cows.

BACKGROUND: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472). RESULTS: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production. CONCLUSIONS: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Validation of 46 loci associated with female fertility traits in cattle.

BACKGROUND: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7). RESULTS: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10- 8) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10- 8) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci. CONCLUSIONS: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves.

BACKGROUND: Bovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated. RESULTS: The four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined. CONCLUSIONS: Four analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

Identifying Regions of the Genome Associated with Conception Rate to the First Service in Holstein Heifers Bred by Artificial Insemination and as Embryo Transfer Recipients.

Heifer conception rate to the first service (HCR1) is defined as the number of heifers that become pregnant to the first breeding service compared to the heifers bred. This study aimed to identify loci associated and gene sets enriched for HCR1 for heifers that were bred by artificial insemination (AI, n = 2829) or were embryo transfer (ET, n = 2086) recipients, by completing a genome-wide association analysis and gene set enrichment analysis using SNP data (GSEA-SNP). Three unique loci, containing four positional candidate genes, were associated (p < 1 × 10-5) with HCR1 for ET recipients, while the GSEA-SNP identified four gene sets (NES ≥ 3) and sixty-two leading edge genes (LEGs) enriched for HCR1. While no loci were associated with HCR1 bred by AI, one gene set and twelve LEGs were enriched (NES ≥ 3) for HCR1 with the GSEA-SNP. This included one gene (PKD2) shared between HCR1 AI and ET services. Identifying loci associated or enriched for HCR1 provides an opportunity to use them as genomic selection tools to facilitate the selection of cattle with higher reproductive efficiency, and to better understand embryonic loss.

Identification by whole genome sequencing of genes associated with delayed postoperative hemorrhage in Scottish deerhounds.

BACKGROUND: Delayed postoperative hemorrhage (DEPOH) is an important health concern for Scottish deerhounds. HYPOTHESIS/OBJECTIVES: Identify genes associated with DEPOH in Scottish deerhounds. ANIMALS: Two hundred sixty-nine privately owned Scottish deerhounds. METHODS: Retrospective case-control study. DEPOH cases and controls were identified through an owner health survey. Genome-wide association analysis was performed using whole genome sequences from 8 cases and 17 controls. All cases and controls were genotyped for selected variants. RESULTS: Of 269 dogs, 10 met inclusion and exclusion criteria for DEPOH, while 62 controls had undergone similar surgical procedures without DEPOH. Genome-wide association analysis identified a single locus on chromosome 9 spanning 40 genes. One of these genes (SERPINF2 encoding alpha-2 antiplasmin) was directly linked to the pathophysiology of DEPOH. The entire cohort was genotyped for a missense SERPINF2 variant (c.605 C>T; p.A202V). Compared to dogs with the reference C/C genotype, the likelihood of DEPOH was significantly higher for dogs with the T/T genotype (odds ratio [OR] = 1235; 95% confidence interval [CI] = 23-6752; P = 0.0005) and with the C/T genotype (OR = 28; 95% CI = 1.4-542; P = 0.03). CONCLUSIONS AND CLINICAL IMPORTANCE: SERPINF2 is associated with DEPOH in Scottish deerhounds. Genetic testing might be able to identify dogs that are susceptible to DEPOH.

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa).

Pseudorabies virus (PRV)-the causative agent of Aujeszky's disease-was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk-based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA-SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

Identifying Loci Associated With Bovine Corona Virus Infection and Bovine Respiratory Disease in Dairy and Feedlot Cattle.

Bovine coronavirus (BCoV) is associated with respiratory and enteric infections in both dairy and beef cattle worldwide. It is also one of a complex of pathogens associated with bovine respiratory disease (BRD), which affects millions of cattle annually. The objectives of this study were to identify loci and heritability estimates associated with BCoV infection and BRD in dairy calves and feedlot cattle. Dairy calves from California (n = 1,938) and New Mexico (n = 647) and feedlot cattle from Colorado (n = 915) and Washington (n = 934) were tested for the presence of BCoV when classified as BRD cases or controls following the McGuirk scoring system. Two comparisons associated with BCoV were investigated: (1) cattle positive for BCoV (BCoV+) were compared to cattle negative for BCoV (BCoV-) and (2) cattle positive for BCoV and affected with BRD (BCoV+BRD+) were compared to cattle negative for BCoV and BRD (BCoV-BRD-). The Illumina BovineHD BeadChip was used for genotyping, and genome-wide association analyses (GWAA) were performed using EMMAX (efficient mixed-model association eXpedited). The GWAA for BCoV+ identified 51 loci (p < 1 × 10-5; 24 feedlot, 16 dairy, 11 combined) associated with infection with BCoV. Three loci were associated with BCoV+ across populations. Heritability estimates for BCoV+ were 0.01 for dairy, 0.11 for feedlot cattle, and 0.03 for the combined population. For BCoV+BRD+, 80 loci (p < 1 × 10-5; 26 feedlot, 25 dairy, 29 combined) were associated including 14 loci across populations. Heritability estimates for BCoV+BRD+ were 0.003 for dairy, 0.44 for feedlot cattle, and 0.07 for the combined population. Several positional candidate genes associated with BCoV and BRD in this study have been associated with other coronaviruses and respiratory infections in humans and mice. These results suggest that selection may reduce susceptibility to BCoV infection and BRD in cattle.

Functional Variants Surrounding Endothelin 2 Are Associated With Mycobacterium avium Subspecies paratuberculosis Infection.

Bovine paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), continues to impact the dairy industry through increased morbidity, mortality, and lost production. Although genome-wide association analyses (GWAAs) have identified loci associated with susceptibility to MAP, limited progress has been made in identifying mutations that cause disease susceptibility. A 235-kb region on Bos taurus chromosome 3 (BTA3), containing a 70-kb haplotype block surrounding endothelin 2 (EDN2), has previously been associated with the risk of MAP infection. EDN2 is highly expressed in the gut and is involved in intracellular calcium signaling and a wide array of biological processes. The objective of this study was to identify putative causal mutations for disease susceptibility in the region surrounding EDN2 in Holstein and Jersey cattle. Using sequence data from 10 Holstein and 10 Jersey cattle, common variants within the 70-kb region containing EDN2 were identified. A custom SNP genotyping array fine-mapped the region using 221 Holstein and 51 Jersey cattle and identified 17 putative causal variants (P < 0.01) located in the 5' region of EDN2 and a SNP in the 3' UTR (P = 0.00009) associated with MAP infection. MicroRNA interference assays, mRNA stability assays, and electrophoretic mobility shift assays were performed to determine if allelic changes at each SNP resulted in differences in EDN2 stability or expression. Two SNPs [rs109651404 (G/A) and rs110287192 (G/T)] located within the promoter region of EDN2 displayed differential binding affinity for transcription factors in binding sequences harboring the alternate SNP alleles. The luciferase reporter assay revealed that the transcriptional activity of the EDN2 promoter was increased (P < 0.05) with the A allele for rs109651404 and the G allele for rs110287192. These results suggest that the variants rs109651404 and rs110287192 are mutations that alter transcription and thus may alter susceptibility to MAP infection in Holstein and Jersey cattle.

Influenza C and D viral load in cattle correlates with bovine respiratory disease (BRD): Emerging role of orthomyxoviruses in the pathogenesis of BRD.

Bovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Orthomyxoviruses, influenza C virus (ICV) and influenza D virus (IDV) have recently been implicated to play a role in BRD. However, there are contradicting reports about the association of IDV and ICV to BRD. Using the largest cohort study (cattle, n = 599) to date we investigated the association of influenza viruses in cattle with BRD. Cattle were scored for respiratory symptoms and pooled nasal and pharyngeal swabs were tested for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, ICV and IDV by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, 2 logs higher IDV viral RNA in BRD-symptomatic cattle that are co-infected animals than those infected with IDV alone. Our results strongly suggest that ICV and IDV may be significant contributors to BRD.

Identification of Loci and Pathways Associated with Heifer Conception Rate in U.S. Holsteins.

Heifer conception rate (HCR) is defined as the percentage of inseminated heifers that become pregnant at each service. The genome-wide association analyses in this study focused on identifying the loci associated with Holstein heifer (n = 2013) conception rate at first service (HCR1) and the number of times bred (TBRD) to achieve a pregnancy. There were 348 unique loci associated (p < 5 × 10-8) with HCR1 and 615 unique loci associated (p < 5 × 10-8) with TBRD. The two phenotypes shared 302 loci, and 56 loci were validated in independent cattle populations. There were 52 transcription factor binding sites (TFBS) and 552 positional candidate genes identified in the HCR1- and TBRD-associated loci. The positional candidate genes and the TFBS associated with HCR1 and TBRD were used in the ingenuity pathway analysis (IPA). In the IPA, 11 pathways, 207 master regulators and 11 upstream regulators were associated (p < 1.23 × 10-5) with HCR1 and TBRD. The validated loci associated with both HCR1 and TBRD make good candidates for genomic selection and further investigations to elucidate the mechanisms associated with subfertility and infertility.

Genomic Analysis of Spontaneous Abortion in Holstein Heifers and Primiparous Cows.

BACKGROUND: The objectives of this study were to identify loci, positional candidate genes, gene-sets, and pathways associated with spontaneous abortion (SA) in cattle and compare these results with previous human SA studies to determine if cattle are a good SA model for humans. Pregnancy was determined at gestation day 35 for Holstein heifers and cows. Genotypes from 43,984 SNPs of 499 pregnant heifers and 498 pregnant cows that calved at full term (FT) were compared to 62 heifers and 28 cows experiencing SA. A genome-wide association analysis, gene-set enrichment analysis-single nucleotide polymorphism, and ingenuity pathway analysis were used to identify regions, pathways, and master regulators associated with SA in heifers, cows, and a combined population. RESULTS: Twenty-three loci and 21 positional candidate genes were associated (p < 1 × 10-5) with SA and one of these (KIR3DS1) has been associated with SA in humans. Eight gene-sets (NES > 3.0) were enriched in SA and one was previously reported as enriched in human SA. Four master regulators (p < 0.01) were associated with SA within two populations. CONCLUSIONS: One locus associated with SA was validated and 39 positional candidate and leading-edge genes and 2 gene-sets were enriched in SA in cattle and in humans.

Loci and pathways associated with uterine capacity for pregnancy and fertility in beef cattle.

Infertility and subfertility negatively impact the economics and reproductive performance of cattle. Of note, significant pregnancy loss occurs in cattle during the first month of pregnancy, yet little is known about the genetic loci influencing pregnancy success and loss in cattle. To identify quantitative trait loci (QTL) with large effects associated with early pregnancy loss, Angus crossbred heifers were classified based on day 28 pregnancy outcomes to serial embryo transfer. A genome wide association analysis (GWAA) was conducted comparing 30 high fertility heifers with 100% success in establishing pregnancy to 55 subfertile heifers with 25% or less success. A gene set enrichment analysis SNP (GSEA-SNP) was performed to identify gene sets and leading edge genes influencing pregnancy loss. The GWAA identified 22 QTL (p < 1 x 10-5), and GSEA-SNP identified 9 gene sets (normalized enrichment score > 3.0) with 253 leading edge genes. Network analysis identified TNF (tumor necrosis factor), estrogen, and TP53 (tumor protein 53) as the top of 671 upstream regulators (p < 0.001), whereas the SOX2 (SRY [sex determining region Y]-box 2) and OCT4 (octamer-binding transcription factor 4) complex was the top master regulator out of 773 master regulators associated with fertility (p < 0.001). Identification of QTL and genes in pathways that improve early pregnancy success provides critical information for genomic selection to increase fertility in cattle. The identified genes and regulators also provide insight into the complex biological mechanisms underlying pregnancy establishment in cattle.