Haflinger Research Articles

The genetic diversity and structure of the Haflinger horse population in the Czech Republic

The genetic diversity and structure of the Haflinger horse population in the Czech Republic

DDB2-associated incidence of squamous cell carcinoma in Haflingers: risk minimization by genotyping

SCC (squamous cell carinomas) are among the most common eye neoplasms in horses. In recent studies Haflinger horses with a homozygous genotype for a missense variant in the DDB2 gene (damage specific DNA binding protein 2) had a significant increased risk of developing ocular SCC. The aims of this study were to determine the frequency of the SCC-associated risk allele in the DDB2 gene in Swiss and Austrian Haflinger populations and to validate the previously described phenotypic correlation. For this purpose, Haflingers presented at various horse clinics in Switzerland (n = 21, including 11 SCC cases), privately kept Haflingers (n = 52, including 1 SCC case), and Haflingers from a stud farm in the Austrian Tyrol (n = 53) were recruited. The individual DDB2 genotype of the animals was determined using a polymerase chain ceaction (PCR) test using hair follicle or whole blood samples. Of the 12 horses suffering from SCC, nine had ocular SCC and three had non-ocular SCC. Six of the nine Haflingers with ocular SCC and one of the three Haflingers with non-ocular SCC were homozygous for the DDB2 variant. Of the 113 clinically normal animals, 7/113 were homozygous (6 %) and 42/113 were heterozygous (37 %), which corresponds to an allele frequency of 24,8 % in the control cohort. The risk of ocular SCC occurring in Haflingers is significantly increased with the homozygous DDB2 genotype. However, not all animals with SCC carry this gene variant and not all DDB2 homozygous animals develop SCC, which can be explained by the multifactorial genesis of the disease. Due to the high frequency of the undesirable allele, we recommend taking the individual DDB2 genotype of breeding animals into account in order to avoid homozygous offspring with a greatly increased SCC risk by excluding high-risk matings.

Ocular and non-ocular squamous cell carcinomas in the Haflinger: Eight cases at the Institut Suisse de Médecine Equine (2015-2022)

This case series describes the clinical course of ocular and non-ocular squamous cell carinoma (SCC) in the Haflinger horse and is intended to raise awareness of the high recurrence rate and tendency to metastasize. Eight Haflingers with histologically confirmed SCC were included, five ocular and three non-ocular, who were presented at the Institut Suisse de Médecine Équine (ISME) Bern between July 2015 and January 2022. The ocular SCC cases were all presented because of an apparent mass, which in most cases was post-treatment recurrence. The occurrence of recurrences was observed between 3 weeks and 16 years after initial therapy. Four of five Haflingers with ocular SCC had an enucleation, three of which were clinically normal at the time of the completion of this study, one case was euthanized due to confirmed metastases and one due to lameness. The result of enucleations for therapy of ocular SCC was good if no metastases occurred. Of the three non-ocular SCC cases, only one case, a penile SCC, had an apparent mass. Therapy was initiated in this case, while the other two cases were euthanized shortly after diagnosis due to the poor prognosis of SCC in the appropriate locations (maxillary sinus, mandible). Metastases occurred three and two years after removal of the primary tumor in ocular SCC in the scapula, liver and lungs and in non-ocular SCC from the penis to the nostrils. Since a postmortem pathological examination was not carried out on all Haflingers, further metastases cannot be ruled out. Haflingers with SCC should be monitored by a veterinarian over the long term, as recurrences and/or metastases can still occur years later.

Estimation of the genetic parameters for temperament in Haflinger horses based on the results of a character test using a pedigree-based relationship matrix

Literature provides indications that the personality and temperament of a horse are sufficiently genetically determined. This background was the impetus to continue collecting phenotypic information on the temperament in the South Tyrolean Haflinger horses, not only to have marketing tools but also to be able to estimate genetic parameters for a future genetic evaluation. The latter should open the possibility to include temperament traits in the breeding goal. Therefore, the aim of the present study was to consider for the very first-time phenotypic information from character testing for estimating genetic parameters for temperament in Haflinger horses. For the present study, data from routine character testing of 210 South Tyrolean Haflinger mares between 2019 and 2021 were considered. A multivariate estimation of heritability (h2) and genetic correlations (r) was carried out for the criteria ‘Interest’, ‘Activity’ and ‘Excitement’. In addition, univariate and bivariate estimation runs were also carried out. The pedigree-based heritability for the behavioural trait Interest was lowest with 0.07 and a standard error of ±0.04. For the other two criteria, Activity and Excitement, h2 was 0.19 ± 0.05 and 0.21 ± 0.04, respectively. The genetic correlation between Interest and Activity and between Interest and Excitement was in both cases negative with −0.52 and −0.61, respectively while the genetic correlation between Excitement and Activity was positive (0.99). Results of the present study highlight the potential of considering temperament traits in breeding of Haflinger horses.

How frequently do and what time Thoroughbred and Haflinger breeding horses perform particular behaviors in paddock areas?

The purpose of this study was to evaluate the frequency and time of specific behaviors in Thoroughbred and Haflinger horses reared in outdoor paddock environments. Therefore, 2 Thoroughbred and 2 Haflinger horses were used in the study, and the data were collected by video recording (112 hours in total) between 09:00 and 16:00 (7 h/day) for 16 days. Some behaviors examined during this period; eating from the hay rack, drinking water, grazing on the pasture, walking, running, standing, being next to the other horse and other activities (Rolling, lie down, etc.). Considering the total number of behaviors performed in both horse breeds, it was observed that Thoroughbred horses (154.82 times - 7 h/day) made more daily movements than Haflingers (134.63 times - 7 h/day). When the behavior of the horses in the paddock is assessed based on breed, Thoroughbreds were seen to repeat eating from the hay rack, drinking water activities significantly (P < 0.05) more frequently than the other breed, as were Haflingers when grazing on the pasture. There was no statistically significant difference in breeds among hours (P > 0.05), but when the repetition or intensity results of the movements were examined according to the study hours, it was discovered that the hours with the largest total number of movements were between 11:00 and 15:00. As a result, it has been determined that the behavior activities of Thoroughbred horses is more in an outdoor paddock area during the 09:00-16:00 hours of the day compared to Haflinger horses.

Methodical investigation of mineral and trace element concentrations in equine feces with special consideration of the sampling location

Objective measurements of the mineral supply in horses are rarely performed. As a result, incorrect elements or an improper amount of elements are provided. The analysis of feces could represent a novel method to evaluate the nutritive supply. The prerequisite is a knowledge of methodological factors influencing the mineral concentration in the fecal samples. Within the scope of this investigation, the effects of different kinds of mineral supply and the influence of the sampling location on the concentration of minerals in equine feces samples were analyzed. Additionally, the methodical error of the measurement procedure was estimated. Six minerals and 22 trace elements in 30 fecal samples of 5 warmbloods, 3 haflinger horses and 2 ponies with differing mineral supplementation (none, calculated amount, mineral lick mass for free intake) were investigated. The samples derived from 3 locations (cranial rectum, ampulla recti and dung heaps) were dried and analyzed using ICP-OES and ICP-MS. Besides a descriptive data analysis, the effects of the fixed factors sampling location and mineral supplementation as well as repeatability and the relative error of the method were assessed. The element concentrations were not influenced by the sampling location, while they varied significantly depending on the type of mineral supplementation. The repeatability of analysis was high with an average measure of certainty of 0.949 (0.894-0.978) and an average median relative error of 0.18 (0.01-0.86). The study outlines a methodological framework for the determination of mineral and trace element concentrations in equine feces. The location of fecal sampling did not affect the results, therefore, preference may be given to noninvasive sample collection from a fecal pile. Furthermore, as the types of mineral supplementation result in significantly differing fecal element concentrations, varying concentrations in feces allow for conclusions concerning the amount of mineral intake of the individual horse.

Effect of the Power Ranking of Mares on the Stereotypies and the Response Behaviors Against Restraining of the Desire to Eat in the Offspring of Haflinger Horse

Effect of the Power Ranking of Mares on the Stereotypies and the Response Behaviors Against Restraining of the Desire to Eat in the Offspring of Haflinger Horse

Comparison of gluteus medius muscle activity in Haflinger and Noriker horses with polysaccharide storage myopathy.

Type 1 polysaccharide storage myopathy caused by genetic mutation in the glycogen synthase 1 gene is present in many breeds including the Noriker and Haflinger horses. In humans, EMG has already been used to document changes in the muscle activity patterns of patients affected by human glycogen storage disorders. Therefore, the aim of the present study was to describe gluteus muscle activity with surface electromyography (sEMG) in Haflinger and Noriker horses with known GYS1 mutation status during walk and trot. Thirty‐two horses (11 Haflinger and 21 Noriker horses) with homozygous non‐affected (GG), heterozygous affected (GA) and homozygous affected (AA) status of GYS1 mutation without overt clinical signs of any myopathy were selected for the current study. Using surface electromyography gluteus medius muscle activity at walk and at trot was measured, and muscle activity was described in relation to the maximum observed value at the same sensor and the same gait. In order to further describe the signals in detail comprising both frequencies and amplitudes, the crossings through the baseline and the 25, 50 and 75 percentile lines were determined. The result of the relative muscle activity did not show a consistent difference between affected and non‐affected horses. Genetically affected (GA and AA) horses showed significantly less density of muscle activity for both gaits and horse breeds except for the crossings per second at the baseline and 75 percentile at walk in the Haflinger horses and 75 percentile at trot in the Noriker horses. The medians of all calculated density values were significantly lower in the GA Haflingers compared to the GG Haflingers (p = 0.012) and also in the AA Norikers compared to the GG Norikers (p = 0.011). Results indicate that the GYS1 mutation reduces the number of functional muscle fibres detected by sEMG measurements even in the absence of overt clinical signs.

Serum Amyloid A (SAA) Concentration after Vaccination in Horses and Mules

Serum amyloid A (SAA) is a sensitive acute-phase response (APR) marker in equids. Prominent APRs with elevations of SAA concentrations ([SAA]) have been reported after vaccination. The authors hypothesized that vaccination with an inactivated EHV-1/-4 vaccine would cause increase in [SAA] and antibody responses and that higher [SAA] would be positively correlated with the antibody titer in both equids. Twelve Haflinger horses and 12 mules were included in this longitudinal prospective study. All horses and mules were vaccinated with a commercially available EHV-1/-4 vaccine. Blood was sampled before and after vaccination to measure [SAA] and virus-neutralizing response (VN-T). In horses and mules, significantly higher [SAA] were measured on days 1, 3, and 5 after EHV-1/-4 vaccination; [SAA] on day 1 after vaccination were only measured in animals that developed fever, where mean [SAA] were significantly higher in horses than in mules (horses: 1,365.75 ± 87.64 mg/L, mules: 615.5 ± 153.444 mg/L) (P > .05). Four horses and 2 mules developed fever after vaccination, lasting for ≤24 hours. Increased antibody responses (VN-T) on days 7 and 14 after vaccination were observed in all animals, whereas mules showed higher overall antibody responses. Nevertheless, [SAA] did not correlate with the intensity of the antibody responses (VN-T) stimulated by the vaccine (P < .05). EHV-1/-4 vaccination caused a prominent APR, higher in horses than in mules, but [SAA] did not correlate with antibody responses. Measuring [SAA] after vaccination could help identify severe APRs that may require longer resting intervals before training or competition.

Water bucket colour preferences in horses

There is a general agreement that horses have dichromatic colour vision with similar capabilities to those of human beings with red-green colour deficiencies. However, whether colour perception has an impact on equine preference for drinking water and how pronounced the colour stimulus might be for horses is still unknown. This study aimed to investigate the preference of horses for different coloured buckets (red, light green, yellow, green, light blue, turquoise) with different tones, in terms of lightness and darkness, measured by the amount of water consumed from each of them in an outdoor arena. Three Thoroughbred and 3 Haflinger horses were used in the study, and data were collected between 09:00 - 16:00 h. The results showed that the average water consumption during this period can reach up to 25.74 L (at an average environmental temperature of 27.35 °C), and significantly more water had been consumed from the turquoise-coloured bucket than from the other colours. It was also observed that the light toned colours (turquoise, light blue) were preferred to dark ones (green, yellow, red). It is suggested to use water buckets that are turquoise and light blue. Further studies are needed to better understand the relationship between water consumption and colour vision.

Skeletal metastasis from a squamous cell carcinoma of the nictitating membrane in a Haflinger horse

SummaryA 12‐year‐old Haflinger gelding with a history of a persistent cough was referred for evaluation of a severe lameness of the left forelimb. An excision of the right nictitans had been performed 2 years prior to presentation, and a squamous cell carcinoma (SCC) with embolic neoplastic cells in several blood vessels had been confirmed by histopathology. The origin of the lameness could not be localised with regional analgesia; therefore, a nuclear scintigraphic examination was performed. This revealed an area of marked increased radiopharmaceutical uptake at the level of the caudodorsal border of the left scapula. Further examination, including ultrasound‐guided biopsy of the suspect region, confirmed the presence of SCC invading the scapula. Due to poor a prognosis, the horse was subjected to euthanasia. Prior to euthanasia, the gelding was tested homozygous for the missense variant in the damage‐specific DNA‐binding protein 2 (DDB2) gene, which is reported as a risk factor for the development of nictitans SCC in Haflinger horses. Post‐mortem evaluation revealed multiple SCC metastasis, affecting the scapula, the liver and the lungs. To the authors' knowledge, this is the first reported case of bone metastasis following a primary periocular SCC in a horse.

P.73Correlation of gluteus medius muscle activities at walk and trot with myopathy changes on biopsies of the exact same locations in horses without clinical signs of type 1 polysaccharide storage myopathy

In the native Austrian breed of Haflinger horses, abnormal polysaccharide accumulations and signs of myopathy in skeletal muscle are commonly caused by type 1 polysaccharide storage myopathy (T1PSSM), based on the genetic mutation of the glycogen synthase 1 (GYS1) gene. In the absence of clinical signs, mild histological myopathy changes in muscle fibres on biopsy as well as changes in the gluteus muscle activity pattern could be documented in Haflinger horses. Surface electromyography (sEMG) of the gluteus medius muscle during walk and trot as well as superficial muscle biopsies in the exact same location taken immediately after sEMG measurements are available of 13 Haflinger horses with homozygous non-affected status (GG, n=5) and heterozygous affected status (GA, n=6) of the GYS1 gene. Thus the aim of the present study is to investigate the correlation between sEMG activity parameters and histology scores for myopathy, to elucidate which histology changes are potentially useful to quantify the functional effect. Parameters used for sEMG signal analysis comprising frequency and amplitude information were the crossings per second (C/s) of the signal through the baseline and the 25, 50, and 75 percentile lines of the sEMG signal at the same location during the same gait. Of the biopsies of the same horses at the same location taken after the sEMG measurement myopathy changes were scored for anguloid atrophy, necrosis or macrophages, central nuclei, and fiber size variation, and numbers of fibers with characteristic cPAS inclusions were expressed as percentage of total fibers counted. There was a significant negative correlation between C/s parameters and myopathy scores over all horses; however, this was not significant within the GA horses alone for any of the myopathy characteristics documented. This may indicate that the functional relevance of the histology parameters in these clinically not affected horses is more of a qualitative than of a quantitative nature.

P.68Breed specifics of muscle function changes in the Austrian Haflinger and Austrian Noriker horses with polysaccharide storage myopathy (PSSM)

P.68Breed specifics of muscle function changes in the Austrian Haflinger and Austrian Noriker horses with polysaccharide storage myopathy (PSSM)

Comparison of a mouth aspirator and a mechanical vacuum aspirator for the collection of Culicoides midges (Diptera: Ceratopogonidae) in equids

AbstractIn a controlled field trial, a mechanical vacuum and a mouth aspirator were compared with respect to Culicoides midge catching results. The two collection methods were equally applied to two Haflinger horses. Once every hour between 2 hr before sunset and sunset, midges were aspirated for 3 min directly off from the coat of each horse at the same time. In total, 16 replicate measurement days were made. To get insight into the number of (blood‐fed) midges collected on different body parts, four replicate measurement days were done in which the hourly 3‐min samples for each collecting method were separately collected and counted per body part. Mean number of Culicoides midges collected on the legs and on the belly of the horse was two times and significantly higher by the mechanical vacuum aspirator compared to the mouth aspirator. Although the mean number of Culicoides midges from the other part of the host sampled (head/neck/manes/back/flanks) was comparable between the two methods, the mean number of blood‐fed Culicoides midges was four times and significantly higher collected by the mouth aspirator compared to the mechanical vacuum aspirator. In conclusion, it can be recommended to use a mechanical vacuum aspirator instead of a mouth aspirator for the legs and belly area, where a systematic, stroke‐by‐stroke‐vacuum procedure is recommended. For the rest of the body, it is strongly recommended to use a targeted procedure by visual guidance of the presence of midges. For now, based on this experiment, we can only recommend using the mouth aspirator for this targeted approach. However, it might be worth assessing the use of the mechanical vacuum aspirator in a targeted way for future studies.

Genetic risk for squamous cell carcinoma of the nictitating membrane parallels that of the limbus in Haflinger horses.

Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye, with a higher incidence documented in Haflinger horses. Recently, a missense variant in the gene damage specific DNA binding protein 2 (DDB2, p.Thr338Met) on ECA12 was identified as a risk factor for the development of limbal SCC in Haflinger horses. SCC also occurs on the nictitating membrane; therefore, investigating the role of this missense variant in nictitating membrane SCC is warranted. In this study, a common ancestor was identified among Haflinger horses affected with limbal SCC or with nictitating membrane SCC, thus supporting a recessive risk factor for the development of cancer at both ocular locations. Analysis of genotype data from Haflinger horses with and without nictitating membrane SCC revealed that the same region on ECA12 associated with limbal SCC was also associated with nictitating membrane SCC (P<2.04×10-5 ). Fine mapping of this locus using 25 cases and 49 controls supported the hypothesis that DDB2:c.1013C>T, p.Thr338Met, is a risk factor for nictitating membrane SCC, as 88% of our cases were homozygous for this variant and no other polymorphism was more strongly associated (P=4.13×10-14 ). These data indicate that the genetic risk is the same for the development of both limbal and nictitating membrane SCC in Haflinger horses and validates utilization of genetic testing of the DDB2 variant for both clinical management and the guidance of mating decisions.

Population Networks Associated with Runs of Homozygosity Reveal New Insights into the Breeding History of the Haflinger Horse.

Within the scope of current genetic diversity analyses, population structure and homozygosity measures are independently analyzed and interpreted. To enhance analytical power, we combined the visualization of recently described high-resolution population networks with runs of homozygosity (ROH). In this study, we demonstrate that this approach enabled us to reveal important aspects of the breeding history of the Haflinger horse. We collected high-density genotype information of 531 horses originating from 7 populations which were involved in the formation of the Haflinger, namely 32 Italian Haflingers, 78 Austrian Haflingers, 190 Noriker, 23 Bosnian Mountain Horses, 20 Gidran, 33 Shagya Arabians, and 155 Purebred Arabians. Model-based cluster analysis identified substructures within Purebred Arabian, Haflinger, and Noriker that reflected distinct genealogy (Purebred Arabian), geographic origin (Haflinger), and coat color patterns (Noriker). Analysis of ROH revealed that the 2 Arabian populations (Purebred and Shagya Arabians), Gidran and the Bosnian Mountain Horse had the highest genome proportion covered by ROH segments (306-397 Mb). The Noriker and the Austrian Haflinger showed the lowest ROH coverage (228, 282 Mb). Our combined visualization approach made it feasible to clearly identify outbred (admixture) and inbred (ROH segments) horses. Genomic inbreeding coefficients (FROH) ranged from 10.1% (Noriker) to 17.7% (Purebred Arabian). Finally it could be demonstrated, that the Austrian Haflinger sample has a lack of longer ROH segments and a deviating ROH spectrum, which is associated with past bottleneck events and the recent mating strategy favoring out-crosses within the breed.

Hoof Quality of Anglo-Arabian and Haflinger Horses.

IntroductionFoot quality is essential to the horse’s movement. The barefoot approach favours the animal’s welfare. Environment and selection determine hoof characteristics.Material and MethodsHoof characteristics of eight Anglo-Arabian (AA) and nine Haflinger (HA) horses were studied. After a preliminary visual analysis of feet, nail samples were collected after trimming for physico-chemical analysis. The parameters were submitted to analysis of variance. A principal component analysis and a Pearson correlation were used to compare mineral contents.ResultsThe hooves of both breeds were healthy and solid. The hooves of HA horses were longer than those of AA horses (14.90 ±0.30 cm vs 13.10 ±0.60 cm), while the AA hoof was harder than the HA hoof both in the wall (74.55 ±2.95 H vs 60.18 ±2.67 H) and sole (67.00 ±5.87 H vs 43.0 ±4.76 H). In comparison with the sole, the AA hoof wall also had a lower moisture percentage (12.56 ±0.67% vs 20.64 ±0.76%), while crude protein and ash contents were similar in both regions. The AA hoof showed a higher Se content, while the HA hoof had a higher level of macroelements. The negative correlations of K with Cu, Fe, Ni, Pb, and Zn in the AA hoof may indicate osmoregulation activity.ConclusionThe hoof morphology of AA and HA horses met the literature parameters for mesomorphic horses. Both breeds had healthy and well-conformed hooves, useful for sport and recreation activities.

P6048 Investigating a single nucleotide polymorphism in DDB2 as a risk factor for squamous cell carcinomas of the nictitans in the Haflinger and Belgian horse breeds

P6048 Investigating a single nucleotide polymorphism in DDB2 as a risk factor for squamous cell carcinomas of the nictitans in the Haflinger and Belgian horse breeds

Individuality of centre of body movement at walk and trot within the Haflinger breed

Kinematic measurements of fourteen Haflinger horses without lameness, walking and trotting on a treadmill were taken to document the location of the centre of the body (CB), defined as the centre between markers on the head, on the withers, on the sacral bone and on the lateral wall of all four hooves in relation to the sacral bone marker. During walk and trot, there are three dimensional CB position (x: forward-backward, y: side-to-side, and z: up and down). For each horse minimum of eight motion cycles were considered in walk as well as in trot. For all three axes, mean CB location, its standard deviation and its 95% confidence interval (Cl) were calculated. For statistical analysis, Shapiro-Wilk test and Spearman's correlation test were carried out. Mean body mass was 463 +/- 42 kg, CI (439, 487); mean height at the withers was 131 +/- 5 cm, CI (128, 134); mean height at the sacrum was 128 +/- 2 cm, Cl (127, 130). Mean CBx was in front of the sacrum (walk 74 +/- 2 cm, Cl (72, 75); trot 73 +/- 2 cm, CI (72, 74); walk vs trot p=0.008). Mean CBz was below the sacrum (-71 +/- 2 cm, CI (-73, -70) in walk; -69 +/- 2 cm, CI (-70, -68) in trot; walk vs trot p=0.001). Positive correlations were found between MeanCBx and trunk length in walk and trot, which could highlight the biomechanical importance of the trunk as it plays a crucial role in deceleration and acceleration. The analysis of the body location centre may be used to identify differences between horses of the same breed, and thus support evaluation of the quality of the horse during locomotion.

Genetic diversity in Czech Haflinger horses

Genetic diversity in Czech Haflinger horses