Genome-wide association study identifies genetic variants underlying footrot in Portuguese Merino sheep.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D. nodosus) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses. RESULTS: Phenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction (p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes. CONCLUSION: This is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

Impact of inbreeding and genetic parameter estimates for seminal traits in Lusitano horses.

The objectives of this study were to establish baseline information for seminal traits in Lusitano stallions, to assess the impact of inbreeding, interval between collections and age on semen quality during the breeding and non-breeding seasons, and to estimate the corresponding genetic parameters. A total of 2129 ejaculates by 146 Lusitano stallions used for artificial insemination, obtained from four equine reproduction centers distributed throughout Portugal, over a period of 14 years (2008-2021), were included in the study. The seminal traits analyzed, and the corresponding means and standard deviations, were gel-free volume (56.95 ± 28.76 mL), concentration (186.48 ± 104.68 × 106), motility (64.1 ± 16.9%), total number of spermatozoa (TNS) (9.271 ± 4.956 × 109) and total number of motile spermatozoa per ejaculate (TNMS) (5.897 ± 3.587 × 109). These results are in the normal range of values described for other breeds. In the stallions analyzed, the mean value for the inbreeding coefficient was 7.93 ± 5.29%, and for age it was 12.70 ± 6.83 years. A significant decline in sperm concentration, motility, TNS, and TNMS was observed as inbreeding increased. The season also influenced sperm concentration, motility, TNS and TNMS, with the highest values observed during the breeding season. When considering the impact of age on Lusitano seminal parameters, results showed a nonlinear relationship, with a positive effect until 18 years of age for volume, motility, TNS and TNMS and a negative effect after this age, with a slow decrease. However, age had a markedly negative effect on sperm concentration. The interval between semen collections only affected (P < 0.05) sperm motility, with a regression coefficient of +1.89 ± 2.17% per additional day. Genetic parameters were estimated with an Animal Model, and the estimated heritability (repeatability) was 0.27 (0.35) for volume, 0.02 (0.38) for sperm concentration, 0.24 (0.44) for motility, 0.29 (0.39) for TNS and 0.41 (0.41) for TNMS. These results suggest that it is possible to improve semen quality by selection and that the properties of semen produced by a stallion tend to remain consistent throughout its lifetime. Furthermore, the impact of inbreeding should be taken into consideration when selecting Lusitano stallions for fertility.

A metagenomics approach to characterize the footrot microbiome in Merino sheep.

In the Portuguese Alentejo region, Merino sheep breed is the most common breed, reared for the production of meat, dairy, and wool. Footrot is responsible for lameness, decreased animal welfare, and higher production losses, generating a negative economic impact. The disease is caused by Dichelobacter nodosus that interacts with the sheep foot microbiome, to date largely uncharacterized. In fact, Dichelobacter nodosus is not able to induce footrot by itself being required the presence of a second pathogen known as Fusobacterium necrophorum. To understand and characterize the footrot microbiome dynamics of different footrot lesion scores, a whole metagenome sequencing (WMGS) approach was used. Foot tissue samples were collected from 212 animals with different degrees of footrot lesion scores, ranging from 0 to 5. Distinct bacterial communities were associated with feet with different footrot scores identifying a total of 63 phyla and 504 families. As the severity of footrot infection increases the microorganisms' diversity decreases triggering a shift in the composition of the microbiome from a dominant gram-positive in mild stages to a dominant gram-negative in the severe stages. Several species previously associated with footrot and other polymicrobial diseases affecting the epidermis and provoking inflammatory responses such as Treponema spp., Staphylococcus spp., Streptococcus spp. and Campylobacter spp. were identified proliferating along with the lesions' severity. Although these bacteria are not able to initiate footrot, several evidences have been described supporting their association with the severity and incidence increase of footrot lesions caused by Dichelobacter nodosus and Fusobacterium necrophorum. Further investigation is required to establish the roles of particular taxa and identify which of them play a role in the disease process and which are opportunistic pathogens.

Osteochondrosis (Osteochondritis Dissecans) in Lusitano Horses: Prevalence and Characteristics.

Osteochondrosis (osteochondritis dissecans), OC(D), is an important joint disorder that has been studied through the years in different breeds but, to our knowledge, there are no reports of its prevalence in Lusitano horses. The aim of this study was to assess the prevalence of OC(D) in Lusitanos, define the characteristics of the disorder in this breed and compare with Spanish Purebred horses. This is a retrospective study that analysed the radiographs from the metacarpometatarsophalangeal, tarsocrural and femoropatellar joints that were taken in 302 Lusitano horses (1-12 years; obtained from 2007 until 2019). The classification of the radiographs for OC(D) was performed using a 0-4 scale and scored by three veterinarians. Radiographical evidence of OC(D) was diagnosed in 53.3% of the horses examined. Most of the findings were lesions of score 1 or 2 corresponding to flattening, (36.8%). The distal intermediate ridge of the tibia in the tarsocrural joint was the most affected predilection site (31.0%, n=187/604 joints assessed). Overall, the tarsocrural joints were more likely to be affected, followed by metatarso-metacarpophalangeal joints and finally the femoropatellar joints. The prevalence of horses with scores 3 or 4 (fragments) in any of the joints was 16.6%. A positive correlation was found between the scores of contralateral joints. We can conclude that the prevalence of OC(D) in Lusitano horses is similar to the prevalence in Spanish Purebred, but there is a lower percentage of fragmentation. The present phenotypic description can provide valuable information for further quantitative and molecular genetic studies.

Prevalence of Radiographic Signs of Osteoarthritis in Lusitano Purebred Horses.

Radiographic prevalence and correlation of radiographic findings has not been performed in Lusitano Purebred horses. The aim of this study was to (1) evaluate the prevalence of primary osteoarthritis radiographic findings in Lusitano Purebred horses; (2) to assess correlations between radiographic findings in different joints of the same limb and different limbs; and (3) elucidate the effect of age in the radiographic findings. A radiographic protocol of the stifle, tarsi, fetlocks and distal limbs was done in 98 Lusitanos and the classification of the radiographs was performed using a 0-4 scale developed and applied blindly by three veterinarians. The distal interphalangeal, proximal interphalangeal, metacarpophalangeal, metatarsophalangeal, tarsometatarsal, distal intertarsal, proximal intertarsal/, tibiotarsal and femorotibial-patellar joints were evaluated. Most joints presented no abnormal findings or minor abnormal radiographic findings (82.86% grade ≤1). The most affected joint was tarsometatarsal and more severe lesions were found in tarsometatarsal and distal intertarsal. Femorotibial-patellar radiographic changes were rare (2.13%). A strong/moderate correlation was found between contralateral joints with exception hindlimb fetlocks. A moderate correlation was found between fore and hindlimbs for distal limb joints. When analyzing ipsilateral as well as diagonal distal limbs, a strong/moderate correlation was also found. The total score progressed in 0.2 score points per each year of age, revealing that age can be a statistically significant predictor for radiographic changes. Overall, Lusitano horses presented a low prevalence of severe radiographic sings of primary osteoarthritis. Findings in contralateral joints tend to be correlated.

Genetic and Environmental Factors Influencing Gestation Length in Lusitano Horses.

Breeding and parturition records collected over a period of 35 years in the Alter Real stud of Lusitano horses were used to calculate gestation length (GL). The 1027 gestations by 209 mares mated to 60 stallions had a mean GL of 338.1 ± 9.26 days. The mixed model analysis of variance indicated that the sex of the foal and inbreeding of the dam and foal had no significant effect on GL (P > .05). On the other hand, GL increased linearly with mare age, with an estimated regression coefficient of 0.155 ± 0.069 days/year (P < .05). Year and conception month affected GL (P < .05), with longer gestations observed when breeding occurred between January and April, followed by a decline of about 5 and 10 days in GL of mares bred in May and in June-July, respectively. Important differences were observed between stallions (P < .05), with most stallions resulting in a distribution of GL in an interval of ±5 days. The inclusion of the mare as a random effect in the mixed model resulted in an estimated repeatability of GL of 0.427, indicating that mares tend to be regular in having long or short gestations across their lifetime. Variance components estimated in an Animal Model resulted in heritability estimates of 0.39 for maternal genetic effects and 0.19 for direct genetic effects, with no association between the two components. Overall, the mare seems to have the major genetic influence on GL in Lusitano horses, but environmental factors, such as month of conception and also the age of the mare, have a noticeable effect.