Two-stage genome-wide association study for the identification of causal variants underlying hoof disorders in cattle.

Feet and legs disorders influence dairy cattle breeding by their effect on animal welfare, economic losses due to lower production and fertility, costs of treatment, and problems with herd management. In our study, we estimated heritabilities and performed a 2-step GWAS for 3 traits describing hoof health: hoof health status defined by a veterinarian (HSV), hoof health status defined by a claw trimmer (HSC), and the total number of hoof disorders (NHD), scored in 1,998 Fleckvieh and 979 Braunvieh cows. The individuals were genotyped with a high-density (HD) panel consisting of 76,934 SNP. For significant genomic regions, the SNP information was enhanced by SNP imputed from the whole-genome sequence of Fleckvieh and Braunvieh bulls from the 1000 Bulls Genome project. The heritabilities were estimated to be 0.035 for HSV, 0.249 for HSC, and 0.279 for NHD. Based on the first-stage GWAS with SNP from the HD panel, 7 significant genomic regions on 6 chromosomes were defined: (1) 120 SNP spanning 15,522 bp on BTA1, including the TOPBP1 gene; (2) 4,139 SNP spanning 1,426,046 bp on BTA7, including the RIOK2 and RGMB genes; (3) 167 SNP spanning 167,352 bp on BTA13, including the C13H20orf194 gene; (4) 2 regions on BTA14, one harboring 1,071 SNP spanning 380,024 bp, including RRM2B and NCALD, and the other comprising 632 SNP spanning 385,111 bp, including STK3; (5) 328 SNP on BTA15, spanning 235,567 bp between FAM168A and PLEKHB1; and (6) 1,549 SNP on BTA22, spanning 596,101 bp in the neighborhood of PTPRG. Then, we conducted a second-stage GWAS based on SNP from whole-genome sequences within the significant regions obtained in the first stage of the analysis. For HSV, the highest additive effect was estimated for 23 SNP located within a region on BTA15, close to FAM168A, corresponding to a predicted gene sequence. For HSC, the highest additive effect was attributed to 44 SNP located within a region of BTA22 corresponding to 4 predicted gene sequences, with rs135082893 within a sequence encoding a microRNA. Another potential causal mutation for HSC was rs134142607 on BTA13, within the exon of C13H20orf194. For NHD, 33 SNP with the highest estimated effect were located on BTA7 within a region of a predicted gene positioned between RIOK2 and RGMB. On BTA14, all significant SNP were located in introns of STK3, which is responsible for the "abnormal gait" phenotype in mice.

[Congenital flexural deformity in 93 calves - appearance, treatment techniques and results of pedigree analysis]. / Sehnenstelzfuß bei 93 Kälbern ­ Erscheinungsformen, Behandlungs­methoden und Ergebnis einer Pedigreeanalyse.

INTRODUCTION: Contracture of the flexor tendons (CFT) is very common in calves and it is usually diagnosed within the first few days after birth (congenital flexural deformity). However, CFT can appear even in older calves caused by chronic pain. The aetiology of CFT is still unknown. In this study, the distribution of sex, age, breed, the severity of flexural deformity, concurrent presence of other diseases, applied treatment methods for flexural deformity, and the outcome of calves with CFT, which were examined at the University Clinic for Ruminants in Vienna from 2001 to 2016, were evaluated retrospectively. 93 calves were admitted with CFT in the observation period. 70 (75.3%) calves were male and 78 (83.9%) of the affected animals were Simmental calves. The age of calves with CFT varied from one day to 41 days. Twenty-six calves suffered exclusively from CFT, and CFT was diagnosed as an additional finding in 67 calves. 91 animals (97.8%) showed CFT on the front limbs, 79 of them (84.9%) on both front limbs. The distribution of the severity scores was as follows: 69 calves (74.2%) had score 1, 17 calves (18.3%) had score 2, three calves (3.2%) had score 3. Three additional calves (3.2%) had a score 1 CFT on one front limb and a score 2 CFT on the other front limb, and one additional calf showed all three scores on both front limbs and one hind limb. 69 patients (74.2%) could be discharged with a significant improvement in CFT after treatment and 24 calves (25.8%) had to be euthanized due to other severe diseases. The results of the applied pedigree analysis do not show that a single gene mutation is the cause for the development of CFT, but rather a complex hereditary pattern has to be assumed. Depending on the severity of CFT and the presence of other concurrent diseases, an early and consistent therapy has to be carried out to achieve the highest possible success. Since animals with CFT are usually restricted in their movement, sufficient colostrum intake must be ensured within the first hours of life.

INTRODUCTION: La contracture des tendons fléchisseurs (CTF) est très fréquente chez les veaux et elle est généralement diagnostiquée dans les premiers jours suivant la naissance (malformation congénitale en flexion). Cependant, la CTF peut apparaître même chez les veaux plus âgés en raison d'une douleur chronique. L'étiologie de la CTF est encore inconnue. Dans cette étude, la répartition du sexe, de l'âge, de la race, la sévérité de la déformation en flexion, la présence concomitante d'autres maladies, les méthodes de traitement appliquées pour la déformation en flexion et les résultats chez les veaux atteints de CTF, examinés à la Clinique universitaire des ruminants de Vienne de 2001 à 2016, ont été évalués rétrospectivement. 93 veaux ont été admis avec une CTF au cours de la période d'observation. 70 veaux (75,3%) étaient des mâles et 78 (83,9%) des animaux atteints étaient des veaux Simmental. L'âge des veaux atteints de CTF variait d'un jour à 41 jours. Vingt-six veaux ont souffert exclusivement de CTF et une CTF a été diagnostiquée comme une découverte supplémentaire chez 67 veaux. 91 animaux (97,8%) présentaient une CTF sur les membres antérieurs, dont 79 (84,9%) sur les deux membres antérieurs. La répartition des scores de gravité était la suivante: 69 veaux (74,2%) ont obtenu un score de 1, 17 veaux (18,3%) ont obtenu un score de 2, trois veaux (3,2%) ont obtenu un score de 3. Trois autres veaux (3,2%) ont obtenu un score 1 CTF sur un membre antérieur et un score de 2 CTF sur l'autre membre antérieur et un veau présentait les trois scores sur les deux membres antérieurs et un membre postérieur. 69 patients (74,2%) ont pu quitter l'hôpital avec une amélioration significative de la CTF après le traitement et 24 veaux (25,8%) ont dû être euthanasiés en raison d'autres maladies graves. Les résultats de l'analyse du pedigree ne montrent pas qu'une seule mutation du gène soit la cause du développement de la CTF, mais plutôt qu'un schéma héréditaire complexe doit être supposé. En fonction de la gravité de la CTF et de la présence d'autres maladies concomitantes, un traitement précoce et cohérent doit être mis en œuvre pour obtenir le meilleur succès possible. Comme les mouvements des animaux atteints de CTF sont généralement limités, un apport suffisant de colostrum doit être garanti dès les premières heures de la vie.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

Short communication: Genotyping of cows to speed up availability of genomic estimated breeding values for direct health traits in Austrian Fleckvieh (Simmental) cattle--genetic and economic aspects.

The aim of this study was to quantify the impact of genotyping cows with reliable phenotypes for direct health traits on annual monetary genetic gain (AMGG) and discounted profit. The calculations were based on a deterministic approach using ZPLAN software (University of Hohenheim, Stuttgart, Germany). It was assumed that increases in reliability of the total merit index (TMI) of 5, 15, and 25 percentage points were achieved through genotyping 5,000, 25,000, and 50,000 cows, respectively. Costs for phenotyping, genotyping, and genomic estimated breeding values vary between €150 and €20 per cow. The gain in genotyping cows for traits with medium to high heritability is more than for direct health traits with low heritability. The AMGG is increased by 1.5% if the reliability of TMI is 5 percentage points higher (i.e., 5,000 cows genotyped) and 6.53% higher AMGG can be expected when the reliability of TMI is increased by 25 percentage points (i.e., 50,000 cows genotyped). The discounted profit depends not only on the costs of genotyping but also on the population size. This study indicates that genotyping cows with reliable phenotypes is feasible to speed up the availability of genomic estimated breeding values for direct health traits. But, because of the huge amount of valid phenotypes and genotypes needed to establish an efficient genomic evaluation, it is likely that financial constraints will be the main limiting factor for implementation into breeding program such as Fleckvieh Austria.

Short-term intermittent hypoxia reduces the severity of acute mountain sickness.

Intermittent hypoxia (IH) is a promising approach to induce acclimatization and hence lower the risk of developing acute mountain sickness (AMS). We hypothesized that a short-term IH protocol in normobaric hypoxia (7 × 1 h to 4500 m) effectively increases the hypoxic ventilatory response (HVR) and reduces the incidence and severity of AMS. Therefore, 26 men (25.5 ± 4.4 years), assigned in a double-blinded fashion to the hypoxia group (HG) or placebo group (PG), spent 8 h at 5300 m before (PRE) and 2 days after cessation of the IH protocol (POST). Measurements included the evaluation of the Lake Louise Score (LLS) and the HVR. The severity of AMS decreased from PRE to POST in the HG (from 6.0 ± 2.7 at PRE to 4.1 ± 2.1 at POST), whereas the LLS in the PG stayed high (from 5.7 ± 2.9 to 5.5 ± 2.8, respectively). The HVR in the HG increased from 0.73 ± 0.4 L/min/% at PRE to 1.10 ± 0.5 L/min/% at POST and did not increase in the PG. The reduction of the LLS was inversely related to the changes in the HVR (r = -0.434), but the AMS incidence was not different between the HG and the PG at POST. In conclusion, short-term IH reduced the severity of AMS development during a subsequent 8-h exposure to normobaric hypoxia.

Hot topic: Effect of breeding strategies using genomic information on fitness and health.

A complex deterministic approach was used to model the breeding goal and breeding structure for the Austrian Fleckvieh (dual-purpose Simmental) breed. The reference breeding goal corresponded to the current total merit index (TMI-R), where dairy traits have a relative weight of 37.9% and fitness traits of 43.7% (beef traits 16.5%; milkability 2%). The breeding program was characterized by 280,000 cows under performance recording, 3,200 bull dams, 100 test bulls with a test capacity of 25%, and 15 proven bulls and 8 bull sires per year. The annual monetary genetic gain (AMGG) was generated mainly by increases in milk fat and milk protein yield (80.6%) and only to a small extent by fitness traits (6.6%). The inclusion of direct health traits (early reproductive disorders, cystic ovaries, and mastitis) with their economic weights increased the relative AMGG for fitness traits from 6.6 to 11.2%. The presently slightly negative AMGG for fertility index and udder health changed in a positive direction. Increasing the weight on the direct health traits by 50% resulted in a further shift toward fitness and health. The effect of strategies using genomic information in a total merit index (TMI) with varying weights on fitness and health traits was also analyzed. The conventional progeny-testing scheme was defined as the reference breeding program. A breeding program was considered to be genomically enhanced (GS50) when 50% of inseminations of herdbook cows and of bull dams were from young bulls with a genomic TMI, and a second program (GS100) did not rely on progeny-tested bulls at all. For GS50, a clear shift of the relative gain in AMGG toward fitness and health traits was observed for all 3 TMI scenarios, as a result of larger progeny groups and a shorter generation interval. For GS100, where no gene flow from progeny-tested bulls was assumed, the genetic gain per generation was lower for the fertility and udder health index but higher per year. The results based on natural genetic gain per year showed that no positive genetic response for fertility and udder health index were achieved for TMI-R (without the inclusion of direct health traits) in GS50 and GS100. The direction of the genetic trend was determined by the weights given to fertility and udder health indices within the TMI. When appropriate weights generated a clear positive trend, GS50 and GS100 reinforced this trend.

Recording of direct health traits in Austria--experience report with emphasis on aspects of availability for breeding purposes.

A project to establish an Austria-wide health-monitoring system for cattle was launched in 2006. Veterinary diagnostic data subject to documentation by law [Law on the Control of Veterinary Medicinal Products (Tierarzneimittelkontrollgesetz)] are standardized, validated, and recorded in a central database. This Austria-wide project is a collaboration among agricultural and veterinary organizations as well as universities, and is also supported by the Austrian government. In addition to providing information for herd management and preventive measures, further objectives of the project include estimating breeding values for health traits and monitoring the overall health status of Austria's cattle. To ensure a high level of participation from farmers and veterinarians, data security issues are extremely important. Valid data are the prerequisite for the efficient use of health records. The challenge hereby is to distinguish between farms with low frequencies of diseases and incomplete documentation and recording. Measures were undertaken to establish a routine monitoring system for direct health traits. A routine genetic evaluation for direct health traits as part of the joint breeding value estimation program between Germany and Austria was introduced for Fleckvieh in December 2010, based on diagnostic data from 5,428 farms with 147,764 Fleckvieh cows. In 2010 to 2011, the reporting of direct health traits as a compulsory part of performance recording and the breeding program was introduced as well. The overall challenge is the availability of sufficient valid direct health data for reliable breeding values. Practical experience gained in Austria in setting up a health registration system, focusing mainly on the availability of direct health data for breeding purposes with its successes and difficulties, is described.

Characterization of the porcine AMPK alpha 2 catalytic subunitgene (PRKAA2): genomic structure, polymorphism detection and association study.

AMP-activated protein kinase (AMPK), known as a key regulator of cellular energy homeostasis, plays an important role in regulation of glucose and lipid metabolism, and protein synthesis in mammals. The characterization of porcine PRKAA2 encoding the alpha 2 catalytic subunit of AMPK is reported in this study. PRKAA2 was assigned to porcine chromosome 6q by analysis of radiation hybrids (IMpRH panel), and its genomic structure was determined by BAC sequencing. PRKAA2 spans more than 62 kb and consists of nine exons and eight introns. A total of 25 polymorphisms were identified by re-sequencing approximately 7 kb, including all the exons, exon-intron boundaries and 5' and 3' gene flanking regions using twelve founder animals of a Mangalitsa x Piétrain intercross. Neither of two single nucleotide polymorphisms (SNPs) found in the coding region caused an amino acid substitution. Two SNPs (NM_214266.1: c.236+142A>G and NM_214266.1: c.630C>T) in PRKAA2 were genotyped in the Mangalitsa x Piétrain F(2) cross (n = 589) and two commercial populations [Piétrain (n = 1173) and German Landrace (n = 536)] and evaluated for association with traits of interest (muscle development and fat deposition). Single SNP and haplotype analyses revealed weak associations between the PRKAA2 genotypes and loin muscle area in the investigated populations.

Variation in neighbouring genes of the dopaminergic and serotonergic systems affects feather pecking behaviour of laying hens.

Feather pecking is a behavioural disorder of laying hens and has serious animal welfare and economic implications. One of the several aetiological hypotheses proposes that the disorder results from redirected exploratory behaviour. Variation in the gene encoding the dopamine D4 receptor (DRD4) has been shown to be associated with exploratory behaviour in several species, including in a passerine bird species. We therefore considered DRD4 as a candidate gene for feather pecking. We have annotated DRD4 in the chicken genome and have re-sequenced it in 140 animals belonging to: experimental layer lines divergently selected for high and low propensity to feather pecking; the unselected founder population; and two commercial lines with low and high propensity to feather pecking. We have identified two sub-haplotypes of DRD4 that are highly significantly associated with feather pecking behaviour in the experimental (P = 7.30 x 10(-7)) as well as in the commercial lines (P = 2.78 x 10(-6)). Linkage disequilibrium (LD) extends into a neighbouring gene encoding deformed epidermal autoregulatory factor 1 (DEAF1). The product of DEAF1 regulates the transcription of the gene encoding the serotonin (5-hydroxytryptamine) 1A receptor. Thus, DEAF1 represents another candidate gene for feather pecking. Re-sequencing of five animals homozygous for the 'low-pecking' sub-haplotype and of six animals homozygous for the 'high-pecking' sub-haplotype delineated an LD block of 14 833 bases spanning the two genes. None of the variants in the LD block is obviously functional. However, the haplotype information will be useful to select against the propensity to feather pecking in chicken and to elucidate the functional implications of the variants.

Multiple-marker mapping for selective DNA pooling within large families.

Selective DNA pooling is a very powerful method for quantitative trait loci (QTL) mapping. It considerably reduces genotyping costs while maintaining high statistical power. Applied to a daughter design, milk samples of offspring with extreme phenotypic values for a trait of interest are assigned to high and low groups, respectively, and within each group the pooled DNA is used for densitometric estimation of allele frequencies in the 2 groups. A single-marker test for linkage between marker and QTL considers marker allele frequency differences between high and low groups. Single-marker across-sire test statistics are strongly affected by the number of sires that are heterozygous for a given marker and the QTL status (homozygous or heterozygous) of these sires, which decreases the accuracy of QTL mapping. Here we propose a simple method to deal with this problem by taking information from multiple linked markers into account. In particular, given the single-marker test statistics, a multiple-marker method was developed to predict test statistics for markers for which a sire was homozygous (or at any other location on the chromosome). Power and map resolution of the proposed method were assessed by simulation, and we show that for the same data set, multiple-marker mapping performed better than the commonly used single-marker analyses.

Characterization of a 320-kb region containing the HEXA gene on bovine chromosome 10 and analysis of its association with BSE susceptibility.

Bovine spongiform encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible prion diseases. Recently, variants in the promoter region of the prion protein (PRNP) gene have been shown to have a considerable effect on the susceptibility to BSE. However, a previous genome scan revealed other putative BSE-susceptibility loci. Here, we analysed such a region on BTA10, which contains the functional candidate gene HEXA. Three hundred and twenty kilobases that, besides HEXA, also contain ARIH1, BRUNOL6 and PARP6 were characterized and screened for polymorphisms. Genotyping of 38 SNPs in Holstein-Friesian animals from the UK (350 diseased and 270 controls) revealed two intronic SNPs that were associated with BSE incidence, with experiment-wise P-values of 3.5 x 10(-3) and 7.7 x 10(-3) respectively. Both SNPs were in strong linkage disequilibrium and the rare alleles had a protective effect. These alleles were contained in a haplotype dubbed 'UK-protective' that was significantly overrepresented in the controls with a permuted P-value of 2 x 10(-3). An association study in German Holstein animals (73 diseased and 627 controls) revealed an opposite effect of the 'UK-protective' haplotype in this population, i.e. it was overrepresented in the diseased animals, although not significant after correction for multiple testing. These findings indicate a causal variant for BSE susceptibility on BTA10 in linkage disequilibrium with the markers studied. Candidate gene analyses of the surrounding region and additional association studies will help to clarify the origin of the protective effects and to identify causal variants for BSE susceptibility on BTA10.

Genetic parameters for semen production traits in Austrian dual-purpose Simmental bulls.

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.