Combined approaches to identify genomic regions involved in phenotypic differentiation between low divergent breeds: Application in Sardinian sheep populations

Selective breeding has led to modifications in the genome of many livestock breeds. In this study, we identified the genomic regions that may explain some of the phenotypic differences between two closely related breeds from Sardinia. A total of 44 animals, 20 Sardinian Ancestral Black (SAB) and 24 Sardinian White (SW), were genotyped using the Illumina Ovine 50K array. A total of 68, 38 and 15 significant markers were identified using the case–control genome‐wide association study (GWAS), the Bayesian population differentiation analysis (F_ST) and the Rsb metric, respectively. Comparisons among the approaches revealed a total of 22 overlapping markers between GWAS and F_ST and one marker between GWAS and Rsb. Three markers detected by Rsb were also located near (<2 Mb) to highly significant regions identified by GWAS and F_ST analyses. Moreover, one candidate marker identified by GWAS and F_ST approaches was located in a run of homozygosity island that was shared by both breeds. We identified several genes involved in many phenotypic differences (such as stature and growth, reproduction, ear size, coat colour, behaviour) between the two analysed breeds. This study shows that combining several genome‐wide approaches could improve discovery of regions involved in the variability of breeding traits and responsible for the phenotypic diversity even between closely related breeds. Overall, the combination of such genome‐wide methods can be extended to other livestock breeds that share between them a similar genetic background, to understand the process that shapes the patterns of genetic variability between closely related populations.     

Diet-related risk factors for gastric dilatation-volvulus in dogs of high-risk breeds

A nested case-control study was conducted among 1634 dogs with complete diet information in a 5-year prospective study to determine diet-related risk factors for gastric dilatation-volvulus (GDV). Cases included 106 dogs that developed GDV; controls included 212 dogs without GDV that were frequency matched to cases by year of GDV onset. Proportionate energy consumed from major food types and from carbohydrates was determined. Dogs were categorized as consuming either a low volume or high volume of food based on the median number of cups of food fed per kg of body weight per meal. Dogs fed a larger volume of food per meal were at a significantly (P<0.05) increased risk of GDV, regardless of the number of meals fed daily. For both large- and giant-breed dogs, the risk of GDV was highest for dogs fed a larger volume of food once daily.     

Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability

It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P < 0.001), but there was no relationship with cooking temperature. Quality (A(260)/A(280), i.e., absorbance at 260 and 280 nm) was also affected by cooking (P < 0.001). For all 3 genes, large PCR amplicons (product size >800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (<200 bp) were present for all core temperatures. Cooking meat to high temperatures thus resulted in a reduced overall yield and probable fragmentation of DNA to sizes less than 800 bp. Although nuclear DNA is preferable to mitochondrial DNA for food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures.     

Targeted sequencing of candidate gene regions for myelofibrosis in dogs

BackgroundMyelofibrosis often lacks an identifiable cause in dogs. In humans, most primary myelofibrosis cases develop secondary to driver mutations in JAK2, CALR, or MPL.ObjectivesTo determine the prevalence of variants in JAK2, CALR, or MPL candidate regions in dogs with myelofibrosis and in healthy dogs.AnimalsTwenty‐six dogs with myelofibrosis that underwent bone marrow biopsy between 2010 and 2018 and 25 control dogs matched for age, sex, and breed.MethodsCross‐sectional study. Amplicon sequencing of JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10 was performed on formalin‐fixed, decalcified, paraffin‐embedded bone marrow (myelofibrosis) or peripheral blood (control) DNA. Somatic variants were categorized as likely‐benign or possibly‐pathogenic based on predicted impact on protein function. Within the myelofibrosis group, hematologic variables and survival were compared by variant status (none, likely‐benign only, and ≥1 possibly‐pathogenic). The effect of age on variant count was analyzed using linear regression.ResultsEighteen of 26 (69%) myelofibrosis cases had somatic variants, including 9 classified as possibly‐pathogenic. No somatic variants were detected in controls. Within the myelofibrosis group, hematologic variables and survival did not differ by variant status. The number of somatic variants per myelofibrosis case increased with age (estimate, 0.69; SE, 0.29; P = .03).Conclusions and Clinical ImportanceSomatic variants might initiate or perpetuate myelofibrosis in dogs. Our findings suggest the occurrence of clonal hematopoiesis in dogs, with increasing incidence with age, as observed in humans.     

Mapping genomic regions for reproductive traits in beef cattle: Inclusion of the X chromosome

Although the second largest chromosome of the genome, the X chromosome is usually excluded from genome-wide association studies (GWAS). Considering the presence and importance of genes on this chromosome that are involved in reproduction, the aim of this study was to evaluate the effect of its inclusion in GWAS on reproductive traits (scrotal circumference [SC], early pregnancy [P16] and age at first calving [AFC]) in a Nelore herd. Genotype data from 3,263 animals with the above-mentioned phenotypes were used. The results showed an increase in the variances explained by the autosomal markers for all traits when the X chromosome was not included. For SC, there was an increase of more than 10% for the windows on chromosomes 2 and 6. For P16, the effect was increased by almost 20% for windows on chromosome 5. The same pattern was found for AFC, with an increase of more than 10% for the most important windows. The results indicate that the noninclusion of the X chromosome can overestimate the effects of autosomes on SC, P16 and AFC not only because of the additive effect of the X chromosome itself but also because of its epistatic effect on autosomal genes.     

Cause of death in dogs according to breed: a necropsy survey of five breeds

The final necropsy diagnoses, obtained between 1985 and 1999, were compared among 1,206 golden retrievers, boxers, German shepherd dogs, Labrador retrievers, and rottweilers. A significantly larger proportion of boxers and golden retrievers died of neoplastic disease than the other three breeds. The ages at death were not significantly different among golden retrievers, German shepherd dogs, and Labrador retrievers. However, rottweilers and boxers died at a younger age from both neoplastic and nonneoplastic diseases. Although boxers were already known to be at a higher risk for neoplasia, a similar index of suspicion for neoplasia should now be used for golden retrievers.     

Comparison of two radiographic techniques for evaluation of hip joint laxity in 10 breeds of dogs

OBJECTIVE: To evaluate hip joint laxity in 10 breeds of dogs via 2 radiographic techniques. ANIMALS: 500 clinically normal dogs. DESIGN: Prospective study. PROCEDURE: Radiographs obtained via routine hip joint evaluations performed in sedated dogs of 10 popular breeds were randomly selected from a database. Measurements of distraction index (DI) and hip-extended index (HEI) on 1 hip joint radiograph randomly chosen from each dog were made. RESULTS: Mean age of dogs was 20.7 months. Mean HEI was 0.17 (range, 0.0 to 0.72) and mean DI was 0.44 (range, 0.07 to 0.96). Borzois had uniformly tight hip joints as judged by use of both methods and were considered the gold standard by which hip joint laxity was judged (all Borzois had DI < or = 0.32). Overall, DI was significantly greater than HEI. Within each breed, mean DI was always greater than mean HEI. Significant differences were detected among breeds for HEI; however, compared with DI, the magnitude of differences among breeds was less. CONCLUSIONS AND CLINICAL RELEVANCE: Distraction radiography detected the greatest range and magnitude of passive hip laxity in the 10 breeds of dogs. The difference in values between breeds known to have high prevalence of canine hip dysplasia and those in Borzois was greater for DI than for HEI. Breeds must be evaluated individually because of inherent differences in hip joint laxity.     

Improving understanding of early behavioral indicators of lumbosacral disease in working dogs using 3D visualization of skeletal movements during working tasks: Feasibility study

An improved understanding of the early behavioral indicators of lumbosacral disease in working dogs may allow earlier interventions and help reduce premature retirement because of disability. However, recognition of early behavioral indicators can be challenging in stoic, high-drive working dogs because they often mask clinical signs. The purpose of this feasibility study was to develop a technique for visualizing canine skeletal movements during working tasks and to describe veterinary clinical specialist opinions on the utility of the visualization technique. Three detection-trained police dogs with a recent history of working task deficits and suspected lumbosacral disease were recruited for the study. Conventional and motion capture video recordings were acquired as dogs performed walking and search high working tasks. Whole-body computed tomography (CT) scans were acquired using clinical multislice CT scanners. Image data from motion capture recordings and whole-body CT scans were analyzed and merged. Three-dimensional (3D) computer animation video clips of skeletal movements were created for each dog and each task, using multiple viewing angle perspectives. Interactive meetings with veterinary clinical specialist reviewers were used to refine point placements for the final renderings. Veterinary clinical specialists reviewed final 3D animation movie clips and recorded their opinions on the utility for the visualization technique. Veterinary clinical specialists reported that the computer animations helped them recognize behavioral characteristics that they had not initially noticed in physical examinations. Potential applications for this visualization technique include creating educational training aids for veterinary students, owners, and handlers; assisting veterinarians in planning rehabilitative treatments; and assisting researchers in developing computer models for biomechanical analyses. Future controlled prospective studies are needed in a large number of normal and affected working dogs to improve accuracy of the visualization technique and test the effect of the technique on observer performance.     

山羊痘病毒温度敏感株基因组DNA复制非必需区的筛选

提取山羊痘病毒温度敏感株基因组DNA，用HindⅢ酶切，随机克隆到pUC18质粒中，限制性内切酶片段电泳分析和序列测定结果表明，克隆到4个不同大小的HindⅢ片段pUA、pUB、pUC和pUD。随后，将它们和羊痘病毒Pellor（AY077835）株进行了同源性比较，发现核苷酸同源性为90。0％～94．5％，氨基酸同源性为83．0％～91．4％。选取克隆或亚克隆后的单一酶切位点，插入P11P7.5-LacZ报告基因，构建了pUAl、pUBl、pUCl和pUDl共4个重组载体质粒。通过脂质体分别转染山羊痘病毒感染的BHK-21细胞，在X-gal存在条件下经蓝白斑筛选重组病毒，获得1株重组病毒，证明筛选出了1个复制非必需区片段。     

Plasma leptin concentration in dogs: effects of body condition score, age, gender and breeds

Leptin is a cytokine produced by adipocytes, and plays a key role in the regulation of energy balance. In the present study, we measured plasma leptin concentrations of 166 normal and obese dogs visiting veterinary practices, and clarified the influence of age, gender and breed on plasma leptin levels in dogs. Leptin levels were higher in the dogs with higher body condition scores. There was no noticeable influence of age, gender and breed, but those in optimal puppies and obese Miniature Dachshund tended to be lower than those in corresponding groups. We conclude that plasma leptin is a reliable marker of adiposity in dogs regardless of age, gender and breed variations, and thereby useful as a blood biochemistry test for health examinations and treatment of obesity.     

Biomarkers for identifying the early phases of osteoarthritis secondary to medial patellar luxation in dogs

The levels of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in synovial fluid (SF) and serum in cases of canine osteoarthritis (OA) were measured. OA was induced by a surgically-created medial patellar luxation in the left stifle of 24 dogs. SF and blood samples were collected at 1.5- and 3-month intervals, respectively. Every 3 months, one dog was euthanatized to collect tissue samples from both stifles. TRAP levels in SF and serum were measured using a spectrophotometer, and TRAP-positive cells in joint tissues were identified by enzyme histochemistry. MMP-2 and TIMP-2 in SF and serum were detected by Western blotting and ELISA, respectively. TRAP in SF from the stifles and serum was significantly increased (p < 0.05) after 3 months. TIMP-2 in SF and serum was significantly decreased (p < 0.05), whereas MMP-2 in SF was significantly increased (p < 0.05) during the progression of OA. Histochemistry revealed an increased number of TRAP-positive cells in tissues from OA-affected joints. Assays measuring TRAP, MMP-2, and TIMP-2 in SF and serum, and methods that detect increased numbers of TRAP-positive cells in the joint tissues can play an important role in identifying the early phases of degenerative changes in canine joint components.     

Comparison of the hanging-drop technique and running-drip method for identifying the epidural space in dogs

Objective

To compare the running-drip and hanging-drop techniques for locating the epidural space in dogs.

Identification of genomic regions that exhibit sexual dimorphism for size and muscularity in cattle

Sexual dimorphism, the phenomenon whereby males and females of the same species are distinctive in some aspect of appearance or size, has previously been documented in cattle for traits such as growth rate and carcass merit using a quantitative genetics approach. No previous study in cattle has attempted to document sexual dimorphism at a genome level; therefore, the objective of the present study was to determine whether genomic regions associated with size and muscularity in cattle exhibited signs of sexual dimorphism. Analyses were undertaken on 10 linear-type traits that describe the muscular and skeletal characteristics of both males and females of five beef cattle breeds: 1,444 Angus (AA), 6,433 Charolais (CH), 1,129 Hereford, 8,745 Limousin (LM), and 1,698 Simmental. Genome-wide association analyses were undertaken using imputed whole-genome sequence data for each sex separately by breed. For each single-nucleotide polymorphism (SNP) that was segregating in both sexes, the difference between the allele substitution effect sizes for each sex, in each breed separately, was calculated. Suggestively (P ≤ 1 × 10⁻⁵) sexually dimorphic SNPs that were segregating in both males and females were detected for all traits in all breeds, although the location of these SNPs differed by both trait and breed. Significantly (P ≤ 1 × 10⁻⁸) dimorphic SNPs were detected in just three traits in the AA, seven traits in the CH, and three traits in the LM. The vast majority of all segregating autosomal SNPs (86% in AA to 94% in LM) had the same minor allele in both males and females. Differences (P ≤ 0.05) in allele frequencies between the sexes were observed for between 36% (LM) and 66% (AA) of the total autosomal SNPs that were segregating in both sexes. Dimorphic SNPs were located within a number of genes related to muscularity and/or size including the NAB1, COL5A2, and IWS1 genes on BTA2 that are located close to, and thought to be co-inherited with, the MSTN gene. Overall, sexual dimorphism exists in cattle at the genome level, but it is not consistent by either trait or breed.     

Development of a genomic tool for breed assignment by comparison of different classification models: Application to three local cattle breeds

Assignment of individual cattle to a specific breed can often not rely on pedigree information. This is especially the case for local breeds for which the development of genomic assignment tools is required to allow individuals of unknown origin to be included to their herd books. A breed assignment model can be based on two specific stages: (a) the selection of breed-informative markers and (b) the assignment of individuals to a breed with a classification method. However, the performance of combination of methods used in these two stages has been rarely studied until now. In this study, the combination of 16 different SNP panels with four classification methods was developed on 562 reference genotypes from 12 cattle breeds. Based on their performances, best models were validated on three local breeds of interest. In cross-validation, 14 models had a global cross-validation accuracy higher than 90%, with a maximum of 98.22%. In validation, best models used 7,153 or 2,005 SNPs, based on a partial least squares-discriminant analysis (PLS-DA) and assigned individuals to breeds based on nearest shrunken centroids. The average validation sensitivity of the first two best models for the three local breeds of interest were 98.33% and 97.5%. Moreover, results reported in this study suggest that further studies should consider the PLS-DA method when selecting breed-informative SNPs.