Toward the most ideal case-control design with related and unrelated dogs in whole-exome sequencing studies.

With the recent development of whole-exome sequencing enrichment designs for the dog, a novel tool for disease-association studies became available. The aim of disease-association studies is to identify one or a very limited number of putative causal variants or genes from the large pool of genetic variation. To maximize the efficiency of these studies and to provide some directions of what to expect, we evaluated the effect on variant reduction for various combinations of cases and controls for both dominant and recessive types of inheritance assuming variable degrees of penetrance and detectance. In this study, variant data of 14 dogs (13 Labrador Retrievers and one Dogue de Bordeaux), obtained by whole-exome sequencing, were analyzed. In the filtering process, we found that unrelated dogs from the same breed share up to 70% of their variants, which is likely a consequence of the breeding history of the dog. For the designs tested with unrelated dogs, combining two cases and two controls gave the best result. These results were improved further by adding closely related dogs. Reduced penetrance and/or detectance has a drastic effect on the efficiency and is likely to have a profound effect on the sample size needed to elucidate the causal variant. Overall, we demonstrated that sequencing a small number of dogs results in a marked reduction of variants that are likely sufficient to pinpoint causal variants or genes.

Cecal drop reflects the chickens' cecal microbiome, fecal drop does not.

Microbiota in the gastro-intestinal tract are closely related to both the intestinal and overall health of the host. Experimental chickens have always been euthanized in order to identify and quantify the bacteria in cecal content. In this study, quantification and identification of the microbial populations in cecal drop, cecal content and fecal drop samples from chickens showed that cecal drop contains a bacterial community that is very similar (concerning bacterial diversity, richness and species composition) to cecal content, as opposed to the bacterial community found in fecal drop. Cecal drop analysis thus allows for longitudinal experiments on chickens' cecal bacteria. The varying results in the analysis of fecal samples question the method's reliability in reflecting the true cecal microbiota in chickens.

The ligaments of the canine hip joint revisited.

Numerous conventional anatomical textbooks describe the canine hip joint, but many contradictions, in particular regarding the ligament of the femoral head, are present. This paper presents a brief overview of the different literature descriptions. These are compared with own observations that have resulted in a revised description of the anatomy of the ligament of the femoral head in the dog. To this purpose, the hip joints of 41 dogs, euthanized for reasons not related to this study and devoid of lesions related to hip joint pathology, were examined. It was observed that the ligament of the femoral head is not a single structure that attaches only to the acetabular fossa, as generally accepted, but it also connects to the transverse acetabular ligament and is complemented by a strong accessory ligament that courses in caudal direction to attach in the elongation of the acetabular notch that extends on the cranioventral surface of the body of the ischium. The description of this accessory ligament in conventional anatomical handbooks is incomplete. This description of the accessory ligament of the femoral head could support the research unravelling the etiopathogenesis of hip instability.

Combined prevalence of inherited skeletal disorders in dog breeds in Belgium.

Canine hip dysplasia (CHD), canine elbow dysplasia (CED), and humeral head osteochondrosis (HHOC) are inherited traits with uneven incidence in dog breeds. Knowledge of the combined prevalence of these three disorders is necessary to estimate the effect of the currently applied breeding strategies, in order to improve the genetic health of the population. Official screening results of the Belgian National Committee for Inherited Skeletal Disorders (NCSID) revealed that an average of 31.8% (CHD, CED, or both; n = 1273 dogs) and 47.2% (CHD, CED, HHOC, or a combination of these three diseases; n = 250 dogs) of dogs are mildly to severely affected by at least one skeletal disorder. According to the current breeding recommendations in some dog breeds in Belgium, these animals should be restricted (mild signs) or excluded (moderate to severe signs) from breeding. The introduction of genetic parameters, such as estimated breeding values, might create a better approach to gradually reduce the incidence of these complex inherited joint disorders, without compromising genetic population health.

The effects of positioning, reason for screening and the referring veterinarian on prevalence estimates of canine hip dysplasia.

Although the prevalence of canine hip dysplasia (HD) has been the subject of a number of published studies, estimates vary widely. This study evaluated several possible causes for these differences. Sixty Belgian, Dutch and German veterinarians were asked to submit all hip radiographs obtained for screening purposes (irrespective of HD status) over a 2-year period, resulting in a database of 583 dogs. Each set of radiographs was accompanied by information on the reason for screening (breeding soundness examination, clinical complaint, assistance dogs, or other reasons), and dog breed, date of birth and age. Dog positioning exerted an effect at multiple levels. The agreement among different observers regarding correct or incorrect positioning was limited and incorrect positioning itself reduced the inter-observer agreement for radiographic hip conformation. Dysplastic dogs were more commonly positioned incorrectly than non-dysplastic dogs. The clinical complaint population had a high prevalence of dysplastic dogs (>70%) compared with the breeding population (11%) and the assistance dogs (6%). There was a significantly lower prevalence of HD among cases referred by veterinarians who frequently submitted hip-extended radiographs for evaluation (P = 0.002) compared to those who refer less frequently. However, this was likely to be selection bias, as radiographs that were from dogs suspected to be dysplastic were not submitted by frequent senders. The prevalence of dysplastic dogs varied widely between breeds (16.7-71.4%). Dogs diagnosed with dysplasia were significantly older than dogs considered healthy (P = 0.001) and dogs classified as borderline dysplastic (P = 0.035). Inter-observer agreement for hip conformation was moderately low, resulting in >7% variation in prevalence estimates for dysplasia.

Prevalence of hip dysplasia, elbow dysplasia and humeral head osteochondrosis in dog breeds in Belgium.

The official screening results of the Belgian National Committee for Inherited Skeletal Disorders, an affiliate of the Belgian Kennel Club, have been used to estimate the prevalence of hip dysplasia, elbow dysplasia and humeral head osteochondrosis in the dog breeds in Belgium, and these have been compared with reported prevalence data from other countries. In some breeds, the prevalence of hip and elbow dysplasia is very high, both in Belgium and in other countries. Comparisons of the prevalence of hip dysplasia are not always feasible because different systems are used to evaluate the quality of the hips and because there is no strict consensus on what should be considered a diseased hip joint.

Interobserver agreement in the diagnosis of canine hip dysplasia using the standard ventrodorsal hip-extended radiographic method.

OBJECTIVES: To determine the agreement between observers and to investigate the effect of observer experience in diagnosing canine hip dysplasia and providing final scoring of hips using the standard ventrodorsal hip-extended radiographic method. The agreement of the final scoring, with a presumed correct assessment based on the Norberg angle, is also investigated. METHODS: Thirty observers were requested to read 50 ventrodorsal hip-extended radiographs of 25 dogs according to Federation Cynologique International criteria. Groups of experienced (nine members) and inexperienced (21 members) observers were used. RESULTS: For providing the distinction between dysplastic versus non-dysplastic dogs, the average interobserver agreement was 72 per cent and was significantly higher (P<0.0001) than the score that could be expected by chance without any agreement between observers. For providing the final score (A, B, C, D or E), an average interobserver agreement of 43.6 per cent was found. In the experienced group, an agreement score of 76 per cent was found for the distinction between AB versus non-AB and an agreement score of 81 per cent was found for the distinction between C versus non-C. The agreement score was significantly higher (P<0.0001) for the experienced group than for the inexperienced group in all cases. Agreement between the presumed correct assessment based on the Norberg angle and the observer's evaluation was low (P=0.35), irrespective of whether the observers were experienced (71.8 per cent correct assessments) or inexperienced (69 per cent correct assessments). CLINICAL SIGNIFICANCE: Although interobserver agreement is low, observer experience increases agreement.

Comparison of subchondral lesion size between clinical and non-clinical medial trochlear ridge talar osteochondritis dissecans in dogs.

In this retrospective study of nine dogs exhibiting bilateral medial trochlear ridge talar osteochondritis dissecans (MTRT-OCD) and unilateral hind limb lameness, we compared subchondral lesion size in limbs with visible lameness, with contralateral lesions that were not associated with any visible lameness. All MTRT-OCD lesions were imaged by radiography and computed tomography (CT). The dimensions of subchondral bone lesions were measured (length, width and depth) using CT software. Similar to a method used in humans, the estimated volume (length x width x depth) and cross sectional area (length x width) were calculated and compared. We found that MTRT-OCD lesions causing visible lameness were significantly larger, and were associated with more joint thickening and degenerative joint disease than contralateral lesions that were not associated with any apparent lameness. As in the disease of shoulder osteochondritis dissecans, there is probably a correlation between the size of MTRT-OCD lesions and the symptoms of lameness.

Estimation of phenotypic and genetic parameters for weight gain and weight at fixed ages in the double-muscled Belgian Blue Beef breed using field records.

In the double-muscled Belgian Blue beef (DM-BBB) breed, selection focuses on muscular conformation and not on weight gain and higher weight. There are very few studies on growth in the DM-BBB using field records. Therefore, farms have no available useful figures on weight at fixed ages and weight gain for the DM-BBB. This study describes and evaluates live weights of DM-BBB animals. All the data were gathered on farms in Belgium. It was found that a male DM-BBB weighs an average of 51 kg at birth, 98 kg at 3 months, 242 kg at 7 months, 430 kg at 13 months and 627 kg at 20 months. Between the age of 7 and 20 months, weight gain is more than 1200 g a day. Females weigh 47 kg at birth, 96 kg at 3 months, 189 kg at 7 months and 332 kg at 13 months. For males, estimates of heritability for weights at 7, 13 and 20 months were between 0.21 and 0.36. The heritability for weight gain between 13 and 20 months was 0.13. This demonstrates that it is possible to select for higher weights and for increased growth between 13 and 20 months. Animals having high weights at a young age (7 and 13 months) tend to have also high weight at slaughtering age (20 months; r(g) between 0.81 and 0.98), but no additional growth between 13 and 20 months (r(g) between -0.09 and 0.00). High weight at 20 months is partially due to growth between 13 and 20 months (r(g) = 0.49).

Regional binding index of the radiolabeled selective 5-HT2A antagonist 123I-5-I-R91150 in the normal canine brain imaged with single photon emission computed tomography.

The pattern of the specific 5-HT2A (5-hydroxytryptamine 2A receptor) antagonist 123I-5-I-R91150 was measured in 10 healthy dogs without neurologic and behavior abnormalities. Eight cortical regions (left and right fronto-, temporo-, parieto-, and occipitocortical area), one global subcortical region (including the thalamic system) were compared with a reference region lacking receptors; that is, the cerebellum. The 123I labeled radioligand was injected intravenously 100-200 minutes before acquisition. Both transmission and emission data were obtained with a triple head gamma camera equipped with high-resolution fanbeam collimators. The emission data were corrected for scatter and attenuation. To delineate different cerebral regions more accurately, the regions of interest (ROI) defined in a former study on brain perfusion measured with 99mTc-ethyl cysteinate dimer (ECD) in the same dogs were used. The co-registration of the 99mTc-ECD and the 123I-5-I-R91150, obtained from each dog, was realized with the help of corresponding transmission maps. By normalizing each regional cerebral activity to the activity observed in the cerebellum, the regional radioactivity (binding index) could be relatively quantified. Highest brain uptake was noted in the frontocortical brain areas (right: 1.85, left: 1.89), followed by the temporocortical region (right: 1.58, left: 1.56). Least uptake was noted in the more caudal and middle brain regions [occipito- (right: 1.46, left: 1.41), parietocortical (right: 1.30, left: 1.26), and striatal region (1.19)]. No gender nor age influence was noted in this series. The 123I labeled serotonin-2A receptor ligand seems to have similar cortical binding in the normal canine brain, as shown in humans and other animal species. A frontocortical to occipitocortical (rostrocaudal) binding index gradient was identified within the dog, which has not been seen in imaging studies from humans and other animal species. The significance of these results will need further investigation. This normative data can be used to compare regional brain uptake of the 123I-radioligand to dogs with behavioral disorders related to the serotonergic system, in future studies.

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

Bovine melanocortin receptor 4: cDNA sequence, polymorphisms and mapping.

A cDNA encoding the bovine melanocortin receptor 4 (MC4R) was cloned and sequenced. Comparing human, pig and rat homologues showed a 87, 85 and 89% identity on the DNA level, respectively, and over 90% on the protein level. The bovine MC4R gene was mapped to BTU 24 by radiation hybrid mapping. Two nucleotide changes were identified by single stranded conformation polymorphism (SSCP) and sequencing. The substitutions proved to be a T to C and G (allele B) to A (allele A) resulting, respectively, in a conservative valine to alanine substitution (Val 145 Ala) and an alanine to threonine (Ala 172 Thr). Using PCR-RFLP, 13 different cattle breeds were screened for the presence of the Ala 172 Thr substitution. With the exception of one Red Pied animal, allele A could only be detected in Red Holstein animals.

Evaluation of the genetic variability of 23 bovine microsatellite markers in four Belgian cattle breeds.

The polymorphism of 23 microsatellites in the four main cattle breeds in Belgium (Holstein Friesian, Belgian Blue, Belgian Red Pied and East Flemish) was analysed. Heterozygosity, polymorphism information content, the effective number of alleles, exclusion probability and the probability of genotypic identity for two random individuals were calculated for all microsatellites and all breeds. The Belgian Blue breed is generally a little less polymorphic in comparison with the other three breeds. Estimates of the genetic distances between these breeds confirmed the widely accepted proposition that the Belgian Blue is the most genetically distinct of these breeds. The three other breeds are likely to become one population, given current breeding strategies. Exclusion probabilities in parentage control cases are > 0.9999 in all four breeds when all 23 microsatellites are used and > 0.98 with only the two most polymorphic multiplexes.