Microbiome analysis reveals the differences in gut fungal community between Dutch Warmblood and Mongolian horses.

Similar to gut bacterial community, gut fungal community are also an important part of the gut microbiota and play crucial roles in host immune regulation and metabolism. However, most studies have focused on the gut bacterial community, and research on the gut fungal community has been limited. Dutch Warmblood (DWH) and Mongolian horses (MGH) are important equine breeds, but little research has been done on their gut fungal community. Here, we assessed differences in gut fungal community between two horse species. Results showed that a total of 2159 OTUs were found in the Dutch Warmblood and Mongolian horses, of which 308 were common. Between-group analyzes of microbial diversity showed no differences in the alpha and beta diversity of gut fungal community between the two horse species. Microbiological taxonomic surveys showed that the dominant fungal phyla (Neocallimastigomycota and Ascomycota) and genera (unclassified_Neocallimastigaceae and Anaeromyces) were the same without being affected by species. Although the types of dominant fungal phyla did not change, the abundances of some fungal genera changed significantly. Results of Metastats analysis showed that there were a total of 206 fungal genera that were significantly different between the two horses, among which 78 genera showed an increase and 127 genera significantly decreased in Dutch Warmblood horses compared with Mongolian horses. In conclusion, this study investigated the composition and structure of the gut fungal community of Dutch Warmblood and Mongolian horses and found significant differences in gut fungal community between both breeds. Notably, this is the first exploration of the differences in the gut fungal community of both breeds, which may help to understand the distribution characteristics of the gut fungal community of different breeds of horses and reveal the differences in the traits of different horses.

Integrated proteomic and transcriptomic profiling identifies aberrant gene and protein expression in the sarcomere, mitochondrial complex I, and the extracellular matrix in Warmblood horses with myofibrillar myopathy.

BACKGROUND: Myofibrillar myopathy in humans causes protein aggregation, degeneration, and weakness of skeletal muscle. In horses, myofibrillar myopathy is a late-onset disease of unknown origin characterized by poor performance, atrophy, myofibrillar disarray, and desmin aggregation in skeletal muscle. This study evaluated molecular and ultrastructural signatures of myofibrillar myopathy in Warmblood horses through gluteal muscle tandem-mass-tag quantitative proteomics (5 affected, 4 control), mRNA-sequencing (8 affected, 8 control), amalgamated gene ontology analyses, and immunofluorescent and electron microscopy. RESULTS: We identified 93/1533 proteins and 47/27,690 genes that were significantly differentially expressed. The top significantly differentially expressed protein CSRP3 and three other differentially expressed proteins, including, PDLIM3, SYNPO2, and SYNPOL2, are integrally involved in Z-disc signaling, gene transcription and subsequently sarcomere integrity. Through immunofluorescent staining, both desmin aggregates and CSRP3 were localized to type 2A fibers. The highest differentially expressed gene CHAC1, whose protein product degrades glutathione, is associated with oxidative stress and apoptosis. Amalgamated transcriptomic and proteomic gene ontology analyses identified 3 enriched cellular locations; the sarcomere (Z-disc & I-band), mitochondrial complex I and the extracellular matrix which corresponded to ultrastructural Z-disc disruption and mitochondrial cristae alterations found with electron microscopy. CONCLUSIONS: A combined proteomic and transcriptomic analysis highlighted three enriched cellular locations that correspond with MFM ultrastructural pathology in Warmblood horses. Aberrant Z-disc mechano-signaling, impaired Z-disc stability, decreased mitochondrial complex I expression, and a pro-oxidative cellular environment are hypothesized to contribute to the development of myofibrillar myopathy in Warmblood horses. These molecular signatures may provide further insight into diagnostic biomarkers, treatments, and the underlying pathophysiology of MFM.

A comparative study of breed differences in the anatomical configuration of the equine vertebral column.

The importance of the equine thoracolumbar vertebral column in orthopaedic disorders is well recognized and diagnostic imaging becomes more feasible, but little is known about variations in the anatomical configuration within breeds. In this descriptive post-mortem study, anatomical variations in three widely differing breeds: Warmblood horses, Shetland ponies and semi-feral Konik horses are described. The caudal cervical (C), thoracic (T), lumbar (L) and sacral (S) regions of the vertebral column of 30 Warmblood horses, 29 Shetland ponies and 18 Konik horses were examined using computed tomography and visualized by volume rendering. Homologous/morphologic variations in the caudal cervical area were frequently seen in Warmblood horses (43%), which was significantly more than in the other breeds (p < 0.001). The as standard described equine formula of 18 T, 6 L and 5 S vertebrae was seen in 78% of Konik horses, but only in 53% Warmblood horses and 38% Shetland ponies, which was significantly different (p < 0.05). Overall, Shetland ponies showed a higher tendency of thoracoization, lumbarization and more variations in the number of vertebrae and pairs of ribs. Ankylosed intertransverse joints (ITJs) between transverse processes of the lumbar vertebrae were most common between the second last and last lumbar vertebra and prevalence was significantly higher in Shetland ponies (61%), than in Warmblood horses (38%) and Konik horses (7%) (p < 0.0001). Cranial to the second last lumbar vertebra there were fewer ITJs ankylosed (14%) in Warmblood horses (p < 0.0095), and this decrease in number of ankylosed ITJs was different compared to the change in ankylosed ITJs in Shetland ponies (p < 0.005). ITJs occurred asymmetrically in 15% (12/77) of the cases. A limitation of the study was that clinical data of the horses were only incompletely available, precluding any conclusions about the potential clinical implications of anatomical variations. Knowledge of variation in osseous anatomy of the equine thoracolumbar vertebral column is important for the interpretation of diagnostic imaging. To assess the functional importance and clinical relevance of this variation, follow-up studies are necessary.

Comparison of pathological lesions in navicular bone (os sesamoideum distale) and analysis of remodelling capacity in warmblood and coldblood horses.

The problem of navicular bone lesions is better understood in warmblood horses. The aim of our research was to compare pathologic lesions of navicular bone of different types of horses divided into age groups. An extra goal was to compare radiographs with anatomical status of navicular bone dissected from the hoof capsule. The study group included 161 horses, out of which 100 individuals were warmbloods, and 61 - coldbloods. In total 280 navicular bones were analysed. The flexor surface, distal border fragments, enthesophytes and the process of obliteration of nutrient foramina were analysed. Some navicular bones were also subject to histology test. A greater tendency for the obliteration of nutrient foramina was observed in coldblood horses than in warmbloods. This fact can be explained as increased remodelling, covering the phenomenon of closure of synovial invaginations and canaliculi within the navicular bone. As a result, the synovial groove disappears and blood vessels cannot enter the distal border. New bone formation on the distal border is more frequently observed in coldblood horses. The development of enthesophytes both on the distal and proximal borders depends to a large extent on the age of horses of both types. Warmbloods, however, are more prone to have the lesions located on the flexor surface, leading as a consequence to deep erosions. They might be found even in very young horses in the form of grooves crossing the sagittal ridge. The frequency of lesions varies depending on the type of the horse. Post-mortem analysis of navicular bones helped interpret radiographs in a more precise manner.

Quantitative and qualitative aspects of standing-up behavior and the prevalence of osteochondrosis in Warmblood foals on different farms: could there be a link?

BACKGROUND: Osteochondrosis (OC) is a common, clinically important joint disorder in which endochondral ossification is focally disturbed. Reduced blood supply to growing cartilage is considered an important cause of the condition, which has both genetic and environmental origins. Housing conditions can influence cartilage injury through peak-pressure changes during limb sliding. Additionally, circulatory perturbation can cause the avascular necrosis of cartilage. In this study, we evaluated the type and frequency of limb sliding during standing up and the occurrence of OC in foals aged up to 12 months on different farms. METHODS: Standing-up behavior was observed in 50 weaned, group-housed, Dutch Warmblood foals aged 6-9 months at five farms using black-and-white surveillance cameras, and their standing-up behavior was scored using a predetermined ethogram. OC was scored using a categorical scale between 6 and 12 months of age in 50 foals in the weanling period, and in 48 from the weanling to yearling periods because two foals died in this time. RESULTS: At both 6 and 12 months of age, the total prevalence of OC differed between the farms: the lowest prevalence was observed on a farm with no sliding, and the highest prevalence was evident on a farm with a higher sliding frequency. The mean ratio of sliding versus normal standing-up behavior was 29% (range: 0-50%); i.e., foals experienced limb sliding during around 29% of standing-up maneuvres. The frequency of sliding instead of normal standing-up behavior differed significantly between the farms (range: 0-50%; P < 0.05), but significantly decreased when foals could better prepare themselves to stand, e.g., when there was an obvious provocation such as the announced approach of another foal (P < 0.05). CONCLUSIONS: Small but significant differences exist between farms in the sliding frequency and total OC incidence in Warmblood foals, but whether environmental factors are causally related to these differences requires further elucidation.

Feed intake patterns of sport ponies and warmblood horses following iso-energetic intake of pelleted fibre-rich mixed feed, muesli feed and semicrushed oat grains.

The aim of the study was to compare diverse feed intake patterns in sport ponies and warmblood-type horses after feeding iso-energetic amounts of three different concentrates: a pelleted fibre-rich mixed feed (PF), a muesli feed (MF) and semicrushed oat grains (OG). Four sport ponies and six warmblood-type horses received the concentrates and meadow hay according to maintenance (0.52 MJ metabolizable energy (ME) kg body weight (BW)0.75 /day). Both breeds were allocated at random to three groups and received once daily for 8 days either OG (1 g starch/kg BW/meal) or iso-energetic quantities of PF and MF according to a crossover design. On the 8th day, feed intake patterns (chewing frequency (CF) in number of chewings (NC), NC/s), feed intake time (FITDM , min/kg dry matter (DM); FIT(neutral detergent fibre)NDF , min/kg NDF) and chewing intensity (CI, NC/kg DM) were measured using modified halters. In warmbloods, PF tended to be ingested faster than MF and OG (10.3 ± 1.9, 13.0 ± 1.3, 14.4 ± 2.0 min/kg DM, respectively; p = 0.05). In ponies, a similar trend was observed (13.5 ± 2.5, 13.6 ± 2.0 and 15.4 ± 1.1 min/kg DM, respectively; p > 0.05). Ponies needed more time to ingest PF than warmbloods (p < 0.05). The CF of ponies was affected by the type of concentrate (PF and MF 1.43 ± 0.11 vs. OG 1.28 ± 0.093 NC/s; p < 0.05). The results indicate that concentrate intake of breeds follows a similar pattern and that breed differences were limited to PF only. In both breeds, PF induced chewing patterns different from those observed for OG and/or MF. However, an interpretation of these differences needs to take into consideration the diverse chemical compositions of the concentrates. Upcoming studies should address the throwback regarding PF and also examine the metabolic consequences of concentrate intake in ponies.

Immunoreactivity of lactic acid-treated mare's milk after simulated digestion.

The similarity of mare's milk to breast milk makes it an interesting substrate for the creation of dairy beverages. The aim of this study was to determine the immunoreactivity of the digested mare's milk products carried out by lactic acid fermentation with Lactobacillus casei LCY, Streptococcus thermophilus MK10 and Bifidobacterium animalis Bi30. Simulation of digestion with saliva, pepsin and pancreatin/bile salts was carried out. The immunoreactivity of the milk proteins was assessed by competitive ELISA. The separation of proteins was studied using a tricine SDS-PAGE method. It has been demonstrated that lactic acid fermentation significantly decreases the immunoreactivity of ß-lactoglobulin, ß-casein, κ-casein and bovine serum albumin. The level of reduction was connected to the type of bacterial strain. The simulated digestion processes caused the decline of immunoreactivity, and the decreases obtained in the experiment were as follows: lactoferrin: 95%, ß-lactoglobulin: 94%, ß-casein: 93%, α-lactalbumin: 82%, α-casein: 82%, bovine serum albumin: 76% and κ-casein: 37%. The results of the study indicated that microbial fermentation with tested strains is a valuable method for reducing the immunoreactivity of mare's milk proteins. However, further studies with other bacterial strains are needed to gain a higher level of elimination or total reduction of mare's milk immunoreactivity to possibly introduce fermented mare's milk into the diet of patients with immune-mediated digestive problems.

No effect of moderate or high concentrate allowance on growth parameters in weanling Warmblood foals fed late-cut haylage as forage.

Two groups of Warmblood foals from the Bavarian federal stud participated in the study beginning from the age of approximately 6 months. The foals were offered a late 1st cut of haylage, oats and foal starter feed. For 2 months after weaning, group 'R' (15 foals) received an amount of oats to provide a total digestible energy supply meeting the recommendations of the German Society of Nutrition Physiology (GfE), whereas the other group 'A' (16 foals) was offered a higher amount of oats (surplus of approximately 1.3 kg/animal/day). Concentrates were fed individually twice daily; total daily haylage intake of all foals together was recorded. In both groups, individual concentrate intake, body weight (BW), body condition score (BCS) and several growth parameters were documented. Both groups showed an absolutely parallel development of the measured growth parameters and of BW and BCS. BW and BCS increased above the recommendations of GfE and Hois. The amount of concentrates offered was not ingested completely in both groups. The average metabolisable energy (ME) intake from concentrates amounted to 30.3 and 32.1 MJ ME/animal/day (group 'R') and 38.7 and 38.2 MJ ME/animal/day (group 'A') for the 7th and 8th month respectively. The mean haylage intake of all foals together equalled 26.2 MJ ME/animal/day. The parallel development of all documented growth parameters in both groups leads to the assumption that higher concentrate intake must have caused lower intake of haylage and vice versa, thus resulting in an overall comparable energy intake for each foal, independently of energy source. The calculated average daily energy intake for all foals together amounted to 60.5 and 61.4 MJ ME/animal for the 7th and 8th month. The mean crude protein intake in both groups together amounted to 640 and 647 g/animal/day for the 7th and 8th month.

Clinical and histopathological features in horses with neuroaxonal degeneration: 100 cases (2017-2021).

BACKGROUND: Adult horses with proprioceptive ataxia and behavior changes that have histologic lesions consistent with neurodegenerative disease have been increasingly recognized. HYPOTHESIS/OBJECTIVES: Describe the history, clinical findings and histopathologic features of horses presented to a referral institution with neuroaxonal degeneration. ANIMALS: One hundred horses with a necropsy diagnosis of neuroaxonal degeneration compatible with neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM). METHODS: Retrospective study of horses presented to the University of Pennsylvania, New Bolton Center, between 2017 and 2021 with a necropsy diagnosis of eNAD/EDM. RESULTS: Affected horses had a median age of 8 years (range, 1-22), and the majority were Warmbloods (72). Sixty-eight horses had behavioral changes, and all 100 had proprioceptive ataxia (median grade, 2/5). Fifty-seven horses had abnormal findings on cervical vertebral radiographs, and 14 had myelographic findings consistent with compressive myelopathy. No antemortem diagnostic test results were consistently associated with necropsy diagnosis of neurodegenerative disease. All 100 horses had degenerative lesions characteristic of eNAD in the brainstem gray matter, and 24 had concurrent degenerative features of EDM in the spinal cord white matter. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histopathologic findings in this large group of horses with neurodegenerative disease were most consistent with eNAD/EDM, but with a different signalment and clinical presentation from earlier descriptions. The increasing occurrence of neurodegenerative disease in horses and the safety risk posed emphasize the importance of focused research in affected horses.

Baselining physiological parameters in three muscles across three equine breeds. What can we learn from the horse?

Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species. Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism. Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters. Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds. Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.

Onchocerca sp. in an imported Zangersheide gelding causing suspensory ligament desmitis.

A 5-year-old imported Zangersheide gelding was evaluated for SC swellings over both forelimbs and lameness localized to the distal metacarpus. Ultrasound examination of the SC masses was compatible with verminous granulomas. Linear hyperechoic foci were present within the suspensory ligament branches of both forelimbs, suggestive of ligamentous parasitic infiltrates. A diagnosis of onchocerciasis was confirmed on biopsy of a SC mass. The gelding was treated with ivermectin and a tapering course of PO dexamethasone but was eventually euthanized. Necropsy confirmed the presence of SC eosinophilic granulomas and degenerative suspensory ligament desmitis, both with intralesional nematodes. Given the location and appearance of the nematode, a diagnosis of Onchocerca sp., most likely O. reticulata, was made. Onchocerciasis should be included as a differential diagnosis for multifocal suspensory ligament desmitis with these sonographic characteristics when paired with SC masses in imported European Warmbloods.

Thermographic Image of the Hoof Print in Leisure and Cross-Country Warmblood Horses: A Pilot Study.

BACKGROUND: The field of veterinary medicine lacks information on equine thermal hoof printing, and few data on the same subject are available in dogs. In human medicine, thermography is used to detect heat emitted by the foot when it comes in contact with a flat surface to detect the abnormalities of the foot balance. The hypothesis states that the thermal pattern of the hoof print in Warmblood horses is detectable and it does not vary among the four limbs in leisure and cross-country Warmblood horses in terms of mean temperature of the hoof print surface. A pilot study was conducted to investigate the accuracy of thermography in temperature detection of the hoof print and to investigate the occurrence of possible differences in the mean value of six selected areas and whether there are any differences in the mean temperature of the hoof print between leisure and cross-country Warmblood horses. METHODS: The study included sixty non-lame Warmblood horses with all limbs taken into consideration (n = 240). The selection criteria for the horses were: no alterations in posture and no muscle group asymmetry during visual examination, no lateral or medial deviation of the carpus or hock, no reaction to the flexion tests, negative reactions to the hoof tester, no lameness during walking, trotting or lunging, no anti-inflammatory medication in the last three weeks prior to examination and rectal temperature between 37 °C and 38 °C. The hoof print of each hoof was measured with the horse in the standing position, all four limbs on the ground, using a FLIR E50 thermal camera. Six areas of temperature from the hoof print were taken into consideration, and for each of them, the mean value was identified using FLIR Tools software for photo interpretation. The One-Way ANOVA test was used to test the differences between the mean temperatures obtained for each selected area from all limbs and to compare the hoof print temperature values between the leisure horses and cross-country horses. Data were statistically processed using SAS Studio. RESULTS: Thermography can detect the temperature emitted by the hoof but the thermal patterns of the hoof print show no difference for all four studied limbs. No significant statistical differences were noticed between the mean temperatures identified for each studied area. Also, there were no statistical differences between the mean temperature of the selected areas from the forelimbs and hindlimbs from the horses used for leisure and those used for cross-country. Based on this aspect, the mean temperature of one selected area can be determined in any of the four limbs, without visible variations. CONCLUSIONS: Thermography can detect the hoof print on a flat surface and the mean temperature for each studied area can be proposed as a reference temperature value. There were no differences in the mean temperature of the hoofprint between leisure and cross-country Warmblood Horses. Further investigations are required to clarify whether there are any differences in the thermal pattern of hoof prints from other breeds or from horses with musculoskeletal conditions.

Genomic loci associated with performance limiting equine overriding spinous processes (kissing spines).

Commonly known as "Kissing Spines" (KS), the pathological mechanisms underlying impingement and overriding of spinous processes (ORSPs) in horses are poorly understood. Thoroughbreds, Warmbloods, and stock-type breeds, including Paint Horses and Quarter Horses are at increased risk for developing clinical signs of KS. A total of 155 stock-type and Warmblood horses presented at collaborating veterinary clinics and hospitals were examined using a strict clinical and radiographical phenotyping scheme to grade each horse from 0 for unaffected controls to 4 for severe KS. Following genotyping with the Illumina Equine SNP70 array (Illumina, Inc.) a Genome Wide Association Study (GWAS) using 61,229 filtered individual Single Nucleotide Polymorphisms (SNPs) was performed to the KS grade phenotype. Two significantly associated SNPs (BIEC2-668062 and BIEC2-668013) on chromosome 25 defined a ~1.4 Gb candidate region containing approximately 17 coding genes (EquCab3) and 195 ENSEMBL annotated variants. Investigation of the best associated SNP (BIEC2-668062) on chr25 demonstrates a significant correlation with an increase in one KS grade, on average, per A allele in this population. A significant effect of breed group, age, height or sex was not observed in this population. These preliminary results demonstrate the potential for KS diagnosis and preventative measures for WB/ST individuals supported by increased genetic risk for more severe KS grade. We propose further research including other affected breeds and evaluating causative variants, as well as the effect of BIEC2-668062 in these populations.

Genetic and genomic characterization followed by single-step genomic evaluation of withers height in German Warmblood horses.

Reliability of genomic predictions is influenced by the size and genetic composition of the reference population. For German Warmblood horses, compilation of a reference population has been enabled through the cooperation of five German breeding associations. In this study, preliminary data from this joint reference population were used to genetically and genomically characterize withers height and to apply single-step methodology for estimating genomic breeding values for withers height. Using data on 2113 mares and their genomic information considering about 62,000 single nucleotide polymorphisms (SNPs), analysis of the genomic relationship revealed substructures reflecting breed origin and different breeding goals of the contributing breeding associations. A genome-wide association study confirmed a known quantitative trait locus (QTL) for withers height on equine chromosome (ECA) 3 close to LCORL and identified a further significant peak on ECA 1. Using a single-step approach with a combined relationship matrix, the estimated heritability for withers height was 0.31 (SE = 0.08) and the corresponding genomic breeding values ranged from - 2.94 to 2.96 cm. A mean reliability of 0.38 was realized for these breeding values. The analyses of withers height showed that compiling a reference population across breeds is a suitable strategy for German Warmblood horses. The single-step method is an appealing approach for practical genomic prediction in horses, because not many genotypes are available yet and animals without genotypes can by this way directly contribute to the estimation system.

First reported case of fragile foal syndrome type 1 in the Thoroughbred caused by PLOD1 c.2032G>A.

BACKGROUND: Warmblood Fragile Foal Syndrome Type 1 (WFFS) is an autosomal recessive disorder reported previously only in warmbloods and thought to be caused by a variant in the gene procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1, c.2032G>A, p.Gly678Arg). Given the presentation of this Thoroughbred case, we hypothesised that a similar genetic mechanism caused this phenotype. OBJECTIVES: To describe the pathological and genetic findings on a foal presenting to a veterinary practice in the UK with skin lesions similar to other Ehlers-Danlos Syndromes, including those documented for warmbloods with WFFS. STUDY DESIGN: A single case report describing a genetic investigation. METHODS: A Thoroughbred foal presenting as dystocia was euthanised for multiple skin lesions and developmental abnormalities. DNA extracted from the foal was tested for the PLOD1 variant (c.2032G>A, p.Gly678Arg) using the commercially available assay. To confirm causality and further interrogate potential novel causes of Ehlers-Danlos Syndrome, 1799 functional candidate genes, including PLOD1, were analysed using whole genome sequencing data generated from DNA extracted from the foal's muscle. These data were compared to 34 control samples from at least 11 other breeds. Variants were prioritised for further evaluation based on predicted impact on protein function. RESULTS: Post-mortem evaluation concluded that this foal suffered from a condition of collagen dysplasia. The foal was homozygous for the c.2032G>A PLOD1 variant. Only two other missense variants identified from whole genome sequencing data were also computationally predicted to be deleterious to protein function, (NPHP3 c.1253T>C, p.Leu418Pro, EPDR1 c.154G>C, p.Glu52Gln). Neither of these genes have been linked to similar phenotypes, or Ehlers-Danlos Syndrome in humans or other species and thus further investigation of these variants as the cause of EDS was not warranted. MAIN LIMITATIONS: This study is a single case report in the Thoroughbred with no additional cases from this breed yet identified to replicate this finding. CONCLUSIONS: Given the clinical presentation similar to WFFS, homozygosity for the PLOD1 variant, and absence of another more plausible causal variant from the WGS experiment, we conclude that PLOD1 c.2032G>A is the likely cause of this foal's condition. This is the first documented evidence of fragile foal syndrome caused by the PLOD1 variant in a breed outside of warmbloods, the Thoroughbred. We therefore recommend a change in the name of this disorder to fragile foal syndrome type 1 (FFS) and utilisation of genetic testing in Thoroughbreds to avoid producing affected foals.

Magnetic Resonance Imaging Findings of the Proximal Metacarpal Region in Warmblood Horses: 36 Lame and 26 Control Limbs (2015-2021).

Objectives: This study aims to evaluate the distribution and severity of bone and soft tissue lesions in the proximal metacarpal region of warmblood horses in lame and control groups. Correlation between lesions and ability to return to work was evaluated in the lame group. Methods: This restrospective analysis evaluated 62 horses with MRI examination of the proximal metacarpal region between Sept 2015 and Feb 2021. There were 36 lame limbs and 26 control limbs. The control group included seven contralateral limbs. Results: Proximal suspensory ligament (PSL) size was not different between the lame and control groups. Hyperintensity seen on T1W/T2*W GRE images within the dorsal collagenous part of the PSL and hyperintense Short-TI Inversion Recovery (STIR) signal within the dorsal collagenous part of the PSL or within the McIII were only present within the lame group. Palmar cortical McIII resorption and dorsal margin irregularity of the PSL and McIII sclerosis were more severe within the lame limbs, but mild gradations were also seen in control limbs. Intermediate gradings for a subset of lesions were commonly seen in the non-lame contralateral to lame limbs. Return to work in the lame group is not statistically different for any measured observation(s), and 19/33 of the lame horses returned to work at similar or higher levels. Conclusion and clinical importance: Fifty-eight percent in this group of warmblood horses returned to work within a variable time frame. The majority (81%) of lame limbs showed bone and soft tissue abnormalities, but no enlargement of the PSL was noted in lame horses, and no correlation was seen between the severity or type of lesions and the ability to return to work. The presence of STIR hyperintensity within the proximal McIII or dorsal collagenous part of the PSL and hyperintensity within the dorsal collagenous part of the PSL on T1W GRE and T2*W GRE images, as well as significant palmar cortical McIII resorption are considered clinically relevant lesions. Contralateral limbs may not truly represent the normal condition, showing nonclinical variations and adaptive remodeling.

Apparent Breed Predilection for Equid Herpesvirus-1-Associated Myeloencephalopathy (EHM) in a Multiple-Breed Herd.

Equid herpesvirus type 1 (EHV-1) causes several outbreaks of abortion and/or equid herpesvirus-associated myeloencephalopathy (EHM) worldwide each year. EHM is of great concern, as permanent neurological gait anomalies can leave a horse unfit for future use. The study assesses the risk factors associated with the occurrence of EHM. During an unmitigated outbreak, 141 adult horses/ponies of several distinct breeds were evaluated-using multiple Bayesian logistic regression calculating the odds ratios for breed, age, and sex. In total, 33 of the 141 horses showed signs of EHM. Fjord horses and warmblood horses were overrepresented among those developing EHM. The pony breeds, Welsh and Shetland ponies, were underrepresented. In addition, age and sex were not associated with the risk for EHM. The main limitation was that it was a retrospective analysis with some flaws of documentation. It can be concluded that breed was a significant risk factor for developing EHM during this outbreak.

Warmblood fragile foal syndrome causative single nucleotide polymorphism frequency in horses in Ireland.

BACKGROUND: Warmblood Fragile Foal Syndrome (WFFS) is an autosomal recessive disorder caused by a mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene. Homozygosity for the mutation results in defective collagen synthesis which clinically manifests as the birth of non viable or still born foals with abnormally fragile skin. While the mutation has been identified in non Warmblood breeds including the Thoroughbred, to date all homozygous clinically affected cases reported in the scientific literature are Warmblood foals. The objective of this study was to investigate the carrier frequency of the mutation in the Thoroughbred and sport horse populations in Ireland. METHODS: A test was developed at the UCD School of Veterinary Medicine using real-time PCR to amplify the PLOD1 gene c.2032G > A variant. A subset of the samples was also submitted to an external laboratory with a licensed commercial WFFS genetic test. RESULTS: Warmblood Fragile Foal Syndrome genotyping was performed on hair samples from 469 horses representing 6 different breeds. Six of 303 (1.98%) sport horses tested and three of 109 (2.75%) Thoroughbreds tested were heterozygous for the WFFS polymorphism (N/WFFS). The WFFS polymorphism was not identified in the Standardbred, Cob, Connemara, or other pony breeds. CONCLUSIONS: The study identified a low frequency of the WFFS causative mutation in sport horses and Thoroughbreds in Ireland, highlighting the importance of WFFS genetic testing in order to identify phenotypically normal heterozygous carriers and to prevent the birth of nonviable foals.

A retrospective study on semen quality parameters from four different Dutch horse breeds with different levels of inbreeding.

A high degree of inbreeding has been reported to negatively impact semen quality in Friesian horses and Shetland ponies. Both breeds are characterized by a closed studbook, small population size, and high incidence of inbreeding. The Dutch Warmblood studbook (KWPN: Koninklijk Warmblood Paardenstamboek Nederland) is a much larger studbook with two distinct populations: the KWPN-Riding horses, managed as an 'open' studbook, and the KWPN-Harness horses, representing a much smaller subpopulation within the KWPN breed and managed as an 'almost closed' studbook. It was recently reported that the degree of inbreeding in KWPN-Harness horses has increased in recent decades due to the small gene pool; however, the degree of inbreeding is still lower than that of Friesian horses and Shetland ponies. We hypothesized that a high or rising degree of inbreeding might negatively impact semen quality. In the present study, we retrospectively compared semen quality parameters of stallions from four different breeds or types (Friesian Horses, Shetland Ponies, KWPN-Riding horses, and KWPN-Harness horses), each reported with different degrees of inbreeding. Semen concentration, and percentages of motile, morphologically normal and live spermatozoa, and the total number of morphologically normal, progressive motile spermatozoa per ejaculate (TNM) were analyzed for 2832 semen evaluations performed over a 15-year period. KWPN-Harness horses had a significantly lower sperm concentration, % motile spermatozoa and % live spermatozoa than KWPN-Riding horses but the % motile and % morphologically normal spermatozoa and TNM in both KWPN-Harness and KWPN-Riding horses were significantly higher than in Friesian horses and Shetland ponies. These results suggest a lower semen quality in KWPN-Harness than KWPN-Riding horses, potentially as a result of a higher coefficient of inbreeding. The negative trend observed in the KWPN-Harness horses may be a warning sign, and breeders or stud books should monitor the degree of inbreeding carefully to avoid a further reduction in semen quality, to the levels observed in Friesian horses and Shetland ponies.

Subclinical Ultrasonographic Abnormalities of the Suspensory Ligament Branches Are Common in Elite Showjumping Warmblood Horses.

Background: There is limited information concerning the ultrasonographic appearance of suspensory ligament branches (SLB) in sports horses. Publications exist on clinical injuries that lead to loss of training days and retirement, but not on the appearance of SLBs in high level showjumping Warmbloods. Objectives: To demonstrate the prevalence of subclinical SLB abnormalities in regularly competing high-level showjumpers; to grade each branch 0-3; compare forelimb vs. hindlimb and medial vs. lateral SLBs; subjectively assess periligamentous fibrosis; measure cross sectional area (CSA) and to gather competition follow-up data. Study Design: Prospective cross-sectional study using ultrasonograms. Methods: Sixty elite showjumping Warmbloods without recent history or clinical signs of SLB desmopathy were examined ultrasonographically. Eight static images of each SLB were acquired in transverse and longitudinal sections, anonymised and stored. Images were then assessed and graded by two experienced blinded clinicians based on a published ordinal scale (grade 0-3). A branch score was assigned based on the most severe grade of any image slice in each branch series. SLB cross-sectional area measurement was obtained from each SLB using the transverse image proximal to insertion. Results: The frequency of grade 2 (moderate) ultrasonographic abnormalities was high. Combined data showed a prevalence of 58% (554/960) grade 2 SLBs. Interobserver agreement was good (kappa = 0.65). Periligamentous fibrosis was over represented in hindlimbs (64%). Combined observer data showed there was no statistical difference in branch scores based on limb or laterality. Follow-up over 12 months revealed only two horses were excluded from competition due to SLB injury. Main Limitations: Sample size was small. Images were obtained in static mode, limbs were not clipped of hair, lameness evaluation was under FEI veterinary inspection and not performed by the authors and follow-up evaluation was from FEI competition records and communication only, and was limited to 1 year. Conclusions: Regularly competing elite showjumping Warmbloods have a high prevalence of subclinical SLB ultrasongraphic abnormalities, which may not contribute to causing lameness, poor-performance or be viewed entirely negatively at prepurchase examination. Judicious interpretation of moderate severity SLB abnormalities is advised due the low incidence of clinical injury demonstrated during the 1 year follow-up.