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.

Bedding is an important source of teat end exposure to environmental mastitis pathogens. To better control environmental mastitis, we need an improved understanding of the relationships among bedding selection and management, bedding bacteria counts (BBC), and udder health (UH). The objectives of this cross-sectional observational study were (1) to describe BBC, bedding characteristics, udder hygiene scores, bulk tank milk (BTM) quality, and UH in US dairy herds using 1 of 4 bedding materials; (2) describe the relationship between BBC and herd measures of UH; and (3) identify benchmarks for monitoring bedding hygiene. Local dairy veterinarians and university researchers enrolled and sampled 168 herds from 17 states. Herds were on a Dairy Herd Improvement Association (DHIA) testing program and used 1 of 4 bedding types for lactating cows: new sand, reclaimed sand, manure solids (MNS), or organic non-manure materials. Each herd was sampled twice (winter and summer) in 2016. Samples and data collected included unused and used bedding, BTM samples, udder hygiene scores, DHIA test data, and descriptions of facilities and herd management practices. Bedding was cultured to determine the total bacteria count and counts of Bacillus spp., coliforms, Klebsiella spp., non-coliform gram-negative organisms, streptococci or streptococci-like organisms (SSLO), and Staphylococcus spp. Bedding dry matter, organic matter, and pH were also measured. Bulk tank milk samples were cultured to determine counts of coliforms, NAS, SSLO, Staphylococcus aureus, and Mycoplasma spp. Udder health measures included DHIA test-day average linear score (LS); the proportion of cows with an intramammary infection (IMI), where infection was defined as LS ≥4.0; the proportion of cows with a new IMI, where new IMI was defined as LS changing from <4.0 to ≥4.0 in the last 2 tests; the proportion of cows with a chronic infection, where chronic was defined as LS ≥4.0 on the last 2 tests; and the cumulative incidence of clinical mastitis in the 30-d period preceding sample collection. Although much variation existed within and among bedding types, mixed linear regression showed the use of MNS bedding to be generally associated with higher BBC, dirtier udders, increased coliform and SSLO counts in BTM, and poorer UH measures compared with organic non-manure materials, reclaimed sand, or new sand bedding materials. While controlling for important farm traits and management practices, mixed linear regression showed that increased counts of coliforms, Klebsiella spp., SSLO, and Staphylococcus spp. in both unused and used bedding were associated with poorer values for 1 or more herd-level measures of UH. Achievable benchmarks identified for counts of coliforms (unused: ≤500 cfu/cm3; used: ≤10,000 cfu/cm3), Klebsiella spp. (0 cfu/cm3 for unused and used), Staphylococcus spp. (0 cfu/cm3 for unused and used), and SSLO (unused: 0 cfu/cm3; used: ≤500,000 cfu/cm3) can be used to monitor bedding hygiene in most bedding materials, with minor variations suggested for SSLO in unused MNS (≤1,000 cfu/cm3).

Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4alpha (HNF4alpha), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or any of the treatments. Abundance of PPARalpha mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARalpha mRNA abundance from 24 to 136 DIM because the amount of PPARalpha mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-21 mRNA increased markedly in early lactation but, like HNF4alpha mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-21 via uncoupling the somatotropin-IGF-axis in early lactation.

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. 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. 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.

Objectives of this study were to (1) describe the intramammary infection (IMI) prevalence and pathogen profiles in quarters of cows approaching dry-off in US dairy herds, (2) compare IMI prevalence in quarters of cows exposed to different bedding material types, and (3) identify associations between bedding bacteria count and IMI in cows approaching dry-off. Eighty herds using 1 of 4 common bedding materials (manure solids, organic non-manure, new sand, and recycled sand) were recruited in a multi-site cross-sectional study. Each herd was visited twice for sampling. At each visit, aseptic quarter-milk samples were collected from 20 cows approaching dry-off (>180 d pregnant). Samples of unused and used bedding were also collected. Aerobic culture was used to determine the IMI status of 10,448 quarters and to enumerate counts (log10 cfu/mL) of all bacteria, Staphylococcus spp., Streptococcus spp. and Streptococcus-like organisms (SSLO), coliforms, Klebsiella spp., noncoliform gram-negatives, Bacillus spp., and Prototheca spp. in unused (n = 148) and used (n = 150) bedding. The association between bedding bacteria count and IMI was determined using multivariable logistic regression with mixed effects. Quarter-level prevalence of IMI was 21.1%, which was primarily caused by non-aureus Staphylococcus spp. (11.4%) and SSLO (5.6%). Only modest differences in IMI prevalence were observed between the 4 common bedding material types. Counts of all bacteria in unused bedding was positively associated with odds of IMI caused by any pathogen [ALL-IMI; odds ratio (OR) = 1.08]. A positive association was also observed for counts of SSLO in unused bedding and SSLO-IMI (OR = 1.09). These patterns of association were generally consistent across the 4 common bedding materials. In contrast, the association between counts of all bacteria in used bedding and ALL-IMI varied by bedding type, with positive associations observed in quarters exposed to manure solids (OR = 2.29) and organic non-manure (OR = 1.51) and a negative association in quarters exposed to new sand (OR = 0.47). Findings from this study suggest that quarter-level IMI prevalence in late-lactation cows is low in US dairy herds. Furthermore, bedding material type may not be an important risk factor for IMI in late lactation. Higher levels of bacteria in bedding may increase IMI prevalence at dry-off in general, but this relationship is likely to vary according to bedding material type.

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.