Shetland Pony Mares Research Articles

LIPID PEROXIDATION AND THE TOTAL ANTIOXIDANT CAPACITY IN THE PLASMA OF SHETLAND PONY MARES AND STALLIONS INVOLVED IN RECREATIONAL HORSEBACK RIDING: ROLE OF PHOTOPERIOD AND EXERCISE

This study focuses on the photoperiod-induced variability in the levels of biomarkers of lipid peroxidation (2-thiobarbituric acid reactive substances, TBARS) and the total antioxidant capacity (TAC) in the blood of Shetland pony mares and stallions before and after exercise. We have analyzed the effect of photoperiods and exercise on the levels of TBARS and TAC in the blood of Shetland pony mares and stallions involved in recreational horseback riding in the central Pomeranian region (Pomeranian Voivodeship, northern part of Poland). Twenty-one healthy adult Shetland ponies (11 mares and 10 stallions) aged 6.5 ± 1.4 years old were used in this study. All horses participated in recreational horseback riding. Training started at 10:00 AM, lasted 1 hour, and consisted of a ride of cross country by walking (5 min), trotting (15 min), walking (10 min), trotting (10 min), walking (5 min), galloping (5 min), and walking (10 min). Blood was drawn from the jugular veins of the animals in the morning, 90 minutes after feeding, while the horses were in the stables (between 8:30 and 10 AM), and immediately after the exercise test (between 11 AM and 12 AM). Blood samples were taken once per season for one year: summer and winter. The level of lipid peroxidation was determined by quantifying the concentration of 2-thiobarbituric acid reacting substances (TBARS) for determining the malonic dialdehyde (MDA) concentration. The TAC level in samples was estimated by measuring the TBARS level after Tween 80 oxidation. In the stallions and mares before exercise (at rest) in the summer photoperiod, we obtained minimum TBARS values, while maximum levels were determined in the winter photoperiod. In our study, the TAC level illustrated the baseline levels of enzymatic and non-enzymatic antioxidant defense systems, which were different in the mares than in the stallions (statistically significant in the winter photoperiod) both before and after exercise (winter photoperiod). The results of the current study demonstrated that changes in the lipid peroxidation and the total antioxidant capacity in the blood of Shetland ponies are photoperiod-, sex-, and exercise-dependent. The maximum statistically significant values for the effect of the photoperiod-exercise combination defined as the F values were observed. The multivariate analysis of the influence of the three parameters: photoperiods, sex, and exercise on the lipid peroxidation and the total antioxidant capacity in the blood of the ponies indicated the leading role mainly of the photoperiod factor, and next the exercise and sex factors.

PHOTOPERIOD-DEPENDENT ALTERATIONS IN OXIDATIVELY MODIFIED PROTEINS IN THE PLASMA OF SHETLAND PONY MARES AND STALLIONS INVOLVED IN RECREATIONAL HORSEBACK RIDING

This study focuses on the photoperiod-induced variability in the levels of oxidatively modified proteins in the plasma of Shetland pony mares and stallions before and after exercise. We have analyzed the effect of photoperiods and exercise on the levels of aldehydic (AD) and ketonic (KD) derivatives of oxidatively modified proteins (OMP) in the blood of Shetland pony mares and stallions involved in recreational horseback riding in the central Pomeranian region (Pomeranian Voivodeship, northern part of Poland). Twenty-one healthy adult Shetland ponies (11 mares and 10 stallions) aged 6.5 ± 1.4 years old were used in this study. All horses participated in recreational horseback riding. Training started at 10:00 AM, lasted 1 hour, and consisted of a ride of cross country by walking (5 min), trotting (15 min), walking (10 min), trotting (10 min), walking (5 min), galloping (5 min), and walking (10 min). Blood was drawn from the jugular veins of the animals in the morning, 90 minutes after feeding, while the horses were in the stables (between 8:30 and 10 AM), and immediately after the exercise test (between 11 AM and 12 AM). Blood samples were taken once per season for one year: summer and winter. The level of oxidatively modified proteins (OMP) was evaluated by the content of protein carbonyl derivatives in the reaction with 2,4-dinitro-phenylhydrazine (DNFH). There was a statistically significant reduction in the levels of aldehydic derivatives of OMP in the plasma of ponies during the winter photoperiods only after exercise in both sexes. A decrease in the levels of ketonic derivatives of OMP in the summer photoperiod was observed. These changes were observed independently of the sex and only after exercise. Levels of aldehydic and ketonic derivatives of OMP varied depending on the photoperiod and exercise session in our studies. These changes were dependent on the baseline levels of the enzymatic and non-enzymatic antioxidant defense systems in the ponies, which differed between the mares and the stallions (statistically significant differences in the winter period) both before and after exercise (winter). Keywords: oxidatively modified proteins, plasma, exercise, seasonal alterations, photoperiods, Shetland ponies, mares and stallions.

Photoperiod-dependent changes in oxidative stress markers in the blood of Shetland pony mares and stallions involved in recreational horseback riding

ABSTRACT The objective of the current study was to determine the photoperiod-induced variations and the impact of exercise on oxidative stress biomarkers [2-thiobarbituric acid reactive substances (TBARS), aldehydic (AD) and ketonic (KD) derivatives of oxidatively modified proteins (OMP), total antioxidant capacity (TAC), and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)] and biomarkers of metabolic alterations [glucose, urea, and uric acid and the activity of lactate dehydrogenase (LDH)] in the blood of Shetland pony mares and stallions involved in recreational horseback riding. Twenty-one healthy adult Shetland ponies (11 mares and 10 stallions) aged 6.5 ± 1.4 years old from the central Pomeranian region in Poland were used in this study. Blood samples were taken once per season for one year: spring (3 April), summer (5 July), autumn (1 October) and winter (25 January). A MANOVA analysis revealed that the photoperiod factor had a leading role in alterations of these biomarkers, while the exercise and sex of the ponies exerted a lower impact. The lipid peroxidation biomarkers, for example, the plasma TBARS level, indicated the maximum adjusted coefficient of determination R2 ad = 0.77. Before exercise (at rest), the plasma of the stallions and mares exhibited minimum values of TBARS levels in the spring and summer photoperiods and maximum levels in autumn and winter. A statistically significant reduction in the levels of both aldehydic and ketonic derivatives of OMP in the blood of ponies was observed during the autumn and winter periods; additionally, the level of ketonic derivatives of OMP declined after exercise in spring. TAC was statistically significant in the spring and winter photoperiods both before and after exercise. SOD activity did not have a pronounced photoperiod-induced pattern but was dependent on the sex and exercise. CAT activity varied and was statistically significant only in the plasma of the mares after exercise in the spring, summer, and winter photoperiods. The minimum GPx activity in the blood of the mares before exercise (at rest) was observed in autumn, while the maximum was noted in winter and summer. Photoperiod- and exercise-induced alterations in markers of oxidative stress and antioxidant defences may contribute to the adaptation of animals to exercise, depending on sex. The seasonal variations in the antioxidant defences demonstrated in our study, as well as substrates of energy metabolism in the blood of mares and stallions, depending on exercise capacity, could be an important aspect in the ability of endogenous adaptive mechanisms of animals to react in advance to environmental changes associated with seasons.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes

Simple SummaryIn pregnant individuals, maternal overnutrition is associated with disturbances in the expression of specific genes and nutrient transporters in the early embryo, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of several genes, nutrient transporters and DNA methyltransferases (DNMTs; play an important role in regulating gene expression) were determined in extra-embryonic membranes after recovery on day 28 of gestation. The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after embryo removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven embryos were assessed. Maternal overfeeding resulted in elevated expression of several genes, DNMTs and nutrient transporters following embryo transfer from an HE to a control mare. The expression of two amino acid transporters was also elevated in the endometrium after embryo transfer from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in day-seven embryos, or glucose uptake by membranes of day-28 embryos. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes (IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN), DNMTs (DNMT1 and DNMT3B), glucose (SLC2A1), fructose (SLC2A5) and amino acid (SLC7A2) transporters following ET from an HE to a control mare. Expression of amino acid transporters (SLC1A5 and SLC7A1) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Effect of a long-term high-energy diet on cardiovascular parameters in Shetland pony mares.

BackgroundChanges in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown.ObjectivesDetermine the order in which EMS‐associated changes in cardiovascular parameters arise.AnimalsTwenty Shetland pony mares.MethodsHigh‐energy (HE) diet mares were fed 200% of net energy requirements for 1 (n = 3) or 2 (n = 7) consecutive diet‐years, with 17 weeks of hay‐only between years. Noninvasive BP measurements and echocardiograms were performed during both years. Resting 24‐hour ECGs and measurements of autonomic tone (splenic volume and packed cell volume [PCV]) were performed at the end of diet‐year 1. Results were compared to control mares receiving a maintenance diet for 1 (n = 7) or 2 (n = 3) consecutive years.ResultsIn year 1, HE mares had significantly higher values than control mares for mean relative left ventricular wall thickness (P = .001). After 2 diet‐years, mean systolic (P = .003), diastolic (P < .001) and mean arterial BP (P = .001), heart rate (HR; P < .001), and mean left ventricular wall thickness (P = .001) also were significantly increased in HE compared to control mares. No pathological arrhythmias or differences in splenic volume or PCV were detected.Conclusions and Clinical ImportanceIngesting a HE diet first induced minor changes in BP, and progressed to left‐sided cardiac hypertrophy in Shetland pony mares. These findings are of interest given the increasing incidence of obesity in horses.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Simple SummaryObesity has been associated with altered reproductive activity in mares and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

EquineReview

Introduction: this edition of the Equine Review looks at atypical myopathy, facial nerve paralysis, Fasciola hepatica and the effect of long-term overfeeding of a high-energy diet in Shetland pony mares.

Effect of long-term overfeeding of a high-energy diet on glucose tolerance in Shetland pony mares.

BackgroundOverfeeding is associated with obesity and insulin dysregulation (ID), which are both risk factors for equine metabolic syndrome. How chronic overfeeding affects development of these factors is poorly understood.ObjectivesTo examine the influence of long‐term high‐energy diet provision on body condition and ID.AnimalsEleven Shetland pony mares.MethodsIn a 3‐phase study, the high‐energy group (n = 7) was fed 200% of net energy (NE) requirements (hay; concentrate: 36% sugar and starch, 13% fat) for 24 weeks, followed by 17 weeks hay‐only feeding before resuming the high‐energy diet (n = 4) for an additional 29 weeks. Mares were weighed weekly. Oral glucose tolerance tests were performed 3 to 4 times per dietary period. Results were compared with those of a control group (phase 1, n = 4; phases 2 and 3, n = 6) that received 100% NE requirements, using a general linear mixed model with post hoc Bonferroni testing.ResultsThe mean body weight of the high‐energy group increased by 27% per high‐energy feeding period. During both feeding periods, area under the curve (AUC) for plasma glucose concentration decreased (P < .01), whereas AUC for plasma insulin concentration increased. Mean basal plasma glucose concentration and peak plasma insulin concentrations were higher (P < .05) in the high‐energy group than in the control group.Conclusion and Clinical ImportanceFeeding a high‐energy diet to healthy nonobese Shetland pony mares led to more efficient glucose metabolism within 5 weeks, followed by significant hyperinsulinemia and obesity. Hyperinsulinemic status was reversed during 17 weeks of hay‐only feeding, regardless of body condition, but returned rapidly after restarting the high‐energy diet.

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.

Long-term adaptation capacity of ponies: effect of season and feed restriction on blood and physiological parameters

Domesticated horses are increasingly kept under semi-natural housing conditions, whereas their adaptation capacity is not fully investigated. In all, 10 Shetland pony mares were held under semi-extensive conditions for 1 year. In winter animals were allocated into two feeding groups (60% and 100% of maintenance energy requirement, respectively). Triiodothyronine, thyroxine, non-esterified fatty acids (NEFA), total bilirubin, total protein, triglyceride, glucose, insulin and hair length were measured at monthly intervals, whereas BW, body condition score, cresty neck score and resting heart rate were recorded every 2 weeks. From summer to winter all Ponies showed a reduction in resting heart rate (P<0.001) and triiodothyronine (P<0.001) but an increase in NEFA (P<0.001), thyroxine (P<0.001) and triglyceride (P<0.001) concentrations. Feed restriction led to a reduced resting heart rate (P=0.009), increased NEFA (P<0.001) and total bilirubin (P=0.008) concentrations. Thyroid hormones did not differ between feeding groups (P>0.05). Refeeding of restrictively fed ponies resulted in a rapid increase in resting heart rate and BW and a return of blood parameters to reference values. Adequately supplied animals adapted without difficulty to varying environmental conditions, whereas feed restriction in ponies during winter resulted in reduced resting heart rates suggesting a reduced basal metabolic rate. The energy restriction was compensated by mobilizing body fat reserves which led to changes in blood parameters. Refeeding in feed restricted animals revealed a remarkably quick recovery of physiological and blood parameters to reference values. We therefore suggest that year round-outdoor housing can be a suitable housing system for robust horse breeds provided that an adequate food supply is available.

Thyroid hormones correlate with field metabolic rate in ponies, Equus ferus caballus.

During winter, free-living herbivores are often exposed to reduced energy supply at the same time that energy needs for thermoregulation increase. Several wild herbivores as well as robust horse breeds reduce their metabolism during times of low ambient temperature and food shortage. Thyroid hormones (THs) affect metabolic intensity and a positive effect of THs on basal metabolic rate (BMR) has been demonstrated in mammals and birds. As BMR and field metabolic rate (FMR) are often assumed to be intrinsically linked, THs may represent a reliable indicator for FMR. To test this hypothesis, 10 Shetland pony mares were kept under semi-extensive central European conditions. During the winter season, one group was fed 60% and one group 100% of their maintenance energy requirements. We measured FMR, locomotor activity, resting heart rate and TH levels in summer and winter. FMR, locomotor activity, resting heart rate and total T3 concentrations decreased substantially in winter compared with summer, whereas total T4 increased. Food restriction led to a reduced FMR and resting heart rate, while THs and locomotor activity were not affected. Across both seasons, FMR, resting heart rate and locomotor activity were positively correlated with total T3 but negatively and more weakly correlated with total T4.

Temporal stability of social structure and behavioural synchronization in Shetland pony mares (Equus caballus) kept on pasture

The temporal stability of synchronization was studied for two consecutive years in a herd of 10 Shetland pony mares kept on pasture. Direct field observations lasted 1.5–2.5 h per session between 8:00 am and 8:00 pm, adding up to an observation time of 74 h in the first and 134 h in the second year. Behaviour, nearest neighbour identity and distance were recorded individually every 15 min by point sampling. Rank order remained very stable between years (τb = 0.91; P < 0.01). All mares showed preferences for the proximity of primarily one specific group member. K-coefficients of synchronization, i.e. non-random synchronization, were higher in the second year of observation (2010: κ = 0.22; 2011: κ = 0.54; t = 2.69; P = 0.007). Among the different parameters evaluated, the Kappa coefficient proved to be most reliable for assessment of synchronization at the group level, because it is not inherently affected by time budgets. A high degree of behavioural synchronization might be the basis of stable group cohesion. Preferences between individuals should be considered for re-groupings in managed horse groups.

Saving energy during hard times: energetic adaptations of Shetland pony mares.

Recent results suggest that wild Northern herbivores reduce their metabolism during times of low ambient temperature and food shortage in order to reduce their energetic needs. It is, however, not known whether domesticated animals are also able to reduce their energy expenditure. We exposed 10 Shetland pony mares to different environmental conditions (summer and winter) and to two food quantities (60% and 100% of maintenance energy requirement) during low winter temperatures to examine energetic and behavioural responses. In summer, ponies showed a considerably higher field metabolic rate (FMR; 63.4±15.0 MJ day(-1)) compared with food-restricted and control animals in winter (24.6±7.8 and 15.0±1.1 MJ day(-1), respectively). During summer, locomotor activity, resting heart rate and total water turnover were considerably elevated (P<0.001) compared with winter. Animals on a restricted diet (N=5) compensated for the decreased energy supply by reducing their FMR by 26% compared with control animals (N=5). Furthermore, resting heart rate, body mass and body condition score were lower (29.2±2.7 beats min(-1), 140±22 kg and 3.0±1.0 points, respectively) than in control animals (36.8±41 beats min(-1), 165±31 kg, 4.4±0.7 points; P<0.05). While the observed behaviour did not change, nocturnal hypothermia was elevated. We conclude that ponies acclimatize to different climatic conditions by changing their metabolic rate, behaviour and some physiological parameters. When exposed to energy challenges, ponies, like wild herbivores, exhibited hypometabolism and nocturnal hypothermia.

Seasonal changes of total body water and water intake in Shetland ponies measured by an isotope dilution technique1

Water is an essential nutrient necessary to support life, and adequate water supply is crucial for animal survival and productivity. The present study was designed to determine seasonal changes in the water metabolism of horses under outdoor conditions. Total body water (TBW) and total water intake (TWI) of 10 adult Shetland pony mares were estimated at monthly intervals for 14 mo by using the deuterium dilution technique. During the last 4 mo, 5 ponies were fed restrictively to simulate natural feed shortage in winter, and 5 ponies served as controls. The TBW (kg) was closely related to body mass [TBW (kg) = -2.86 + 0.67 × body mass (kg); P < 0.001; n = 105] explaining 86% of the variation. In contrast to TBW (kg), TBW (%) remained relatively stable across all measurements (57.8 to 71.2%). The TWI showed an increase in summer and a decrease in winter [TWI (mL·kg(-1)·d(-1)) = 15.07 + 23.69 × month - 1.45 × month(2) (R(2) = 0.64, P < 0.01)]. However, TWI measured at ambient temperatures (Ta) < 0°C did not follow the same trend as TWI at Ta > 0°C. Therefore, removing TWI values measured at Ta < 0°C from the analysis resulted in high correlations with locomotor activity (r = 0.87), Ta (r = 0.86), and resting heart rate (r = 0.88). The multiple regression among TWI, Ta, and heart rate explained 84% of the variation in TWI [TWI (mL·kg(-1)·d(-1)) = -13.38 + 1.77 × heart rate (beats/min) + 2.11 × Ta (°C); P < 0.001]. Feed restriction had no effect on TWI and TBW. The TBW content was unaffected by season and physical activity. The established regression equation for TBW and body mass can be used to predict TBW from body mass in ponies under field conditions. The comparison of TWI with published data on drinking water intake revealed that ponies had 1.7 to 5.1 times greater total water intakes when other sources of water such as feed and metabolic water were included. The TWI was highly influenced by environmental conditions and metabolic rate. Contrary to expectation, water supply during the cold seasons might be more critical than under summer conditions when water content of grass is high to allow for the compensation of limited availability of drinking water.

Genome-wide association study of insect bite hypersensitivity in two horse populations in the Netherlands

BackgroundInsect bite hypersensitivity is a common allergic disease in horse populations worldwide. Insect bite hypersensitivity is affected by both environmental and genetic factors. However, little is known about genes contributing to the genetic variance associated with insect bite hypersensitivity. Therefore, the aim of our study was to identify and quantify genomic associations with insect bite hypersensitivity in Shetland pony mares and Icelandic horses in the Netherlands.MethodsData on 200 Shetland pony mares and 146 Icelandic horses were collected according to a matched case–control design. Cases and controls were matched on various factors (e.g. region, sire) to minimize effects of population stratification. Breed-specific genome-wide association studies were performed using 70 k single nucleotide polymorphisms genotypes. Bayesian variable selection method Bayes-C with a threshold model implemented in GenSel software was applied. A 1 Mb non-overlapping window approach that accumulated contributions of adjacent single nucleotide polymorphisms was used to identify associated genomic regions.ResultsThe percentage of variance explained by all single nucleotide polymorphisms was 13% in Shetland pony mares and 28% in Icelandic horses. The 20 non-overlapping windows explaining the largest percentages of genetic variance were found on nine chromosomes in Shetland pony mares and on 14 chromosomes in Icelandic horses. Overlap in identified associated genomic regions between breeds would suggest interesting candidate regions to follow-up on. Such regions common to both breeds (within 15 Mb) were found on chromosomes 3, 7, 11, 20 and 23. Positional candidate genes within 2 Mb from the associated windows were identified on chromosome 20 in both breeds. Candidate genes are within the equine lymphocyte antigen class II region, which evokes an immune response by recognizing many foreign molecules.ConclusionsThe genome-wide association study identified several genomic regions associated with insect bite hypersensitivity in Shetland pony mares and Icelandic horses. On chromosome 20, associated genomic regions in both breeds were within 2 Mb from the equine lymphocyte antigen class II region. Increased knowledge on insect bite hypersensitivity associated genes will contribute to our understanding of its biology, enabling more efficient selection, therapy and prevention to decrease insect bite hypersensitivity prevalence.

Risk factors for insect bite hypersensitivity in Friesian horses and Shetland ponies in The Netherlands

Insect bite hypersensitivity (IBH) is an equine skin allergy caused by bites of Culicoides spp. and impacts on the welfare of affected horses. The aim of this study was to identify and quantify risk factors for IBH. Data from 3453 Friesian horse mares and 7074 Shetland pony mares scored for IBH by inspectors during obligatory foal inspections were analysed using breed-specific multivariable logistic regression models. The combined effect of month and year of scoring, Province and inspector were significantly associated with IBH in both breeds. In Shetland pony mares, withers height and coat colour were also significantly associated with IBH, while body condition had a nearly significant effect. The outcomes from this study on risk factors might contribute to the development of more efficient measures to reduce the prevalence of IBH.

Genome‐wide association study of insect bite hypersensitivity in Dutch Shetland pony mares

Insect bite hypersensitivity (IBH) is the most common allergic disease present in horses worldwide. It has been shown that IBH is under genetic control, but the knowledge of associated genes is limited. We conducted a genome-wide association study to identify and quantify genomic regions contributing to IBH in the Dutch Shetland pony population. A total of 97 cases and 91 controls were selected and matched on withers height, coat colour and pedigree to minimise the population stratification. A blood sample was collected from participating Shetland pony mares, their IBH phenotype was scored and the owner filled in a questionnaire. A total of 40 021 single-nucleotide polymorphisms (SNPs) were fitted in a univariable logistic model fitting an additive effect. Analysis revealed no effects of population stratification. Significant associations with IBH were detected for 24 SNPs on 12 chromosomes [-log(10) (P-value) > 2.5]. Odds ratios of allele substitution effects of the unfavourable allele were between 1.94 and 5.95. The most significant SNP was found on chromosome 27, with an odds ratio of 2.31 and with an allele frequency of the unfavourable allele of 0.72 in cases and 0.53 in controls. Genome-wide association studies on additional horse populations are desired to validate the identified associations, to identify the genes involved in IBH and to develop genomic tools to decrease IBH prevalence.

Distribution of ventilation in pregnant Shetland ponies measured by Electrical Impedance Tomography

The regional distribution of ventilation in conscious standing pregnant Shetland pony mares was investigated by Electrical Impedance Tomography (EIT). Six ponies were repeatedly examined a minimum of four weeks prior to (antepartum, AP) until three weeks after parturition (postpartum, PP). From the cross-sectional ventilation image the ventral to dorsal (V/D), left to right (L/R) ventilation distribution ratio and the relative ventilation in four horizontal regions of interest (ROI) placed symmetrically in the chest was analyzed. Antepartum V/D was 0.74 ± 0.09 on day -28 ± 3 (AP28) and decreased to 0.68 ± 0.10 on day -3 ± 2 (AP3). Postpartum V/D increased significantly (p<0.05) to 0.96 ± 0.08 on day 7 ± 2 (PP7). The L/R ventilation distribution remains unaffected. Ventilation in the most ventral ROI was significantly lower on days AP28 and AP3 compared to PP7. These results suggest that in Shetland ponies late pregnancy compromises the ventilation in ventral (dependent) lung regions. We demonstrated the feasibility of a repeated EIT measurement in standing conscious ponies.

Estrous cycle characteristics, luteal function, secretion of oxytocin (OT) and plasma concentrations of 15-keto-13,14-dihydro-PGF2α (PGF2α-metabolite) after administration of low doses of prostaglandin F2α (PGF2α) in pony mares

In the present study, the kinetics of the prostaglandin F2α (PGF2α)-metabolite 15-keto-13,14-dihydro-PGF2α after a single intramuscular application of various doses of the natural PGF2α dinoprost at Day 7 of the cycle in the mare were investigated. Effects of low doses on estrous cycle length and life span of corpus luteum were examined, because release of PGF2α is still under discussion to have detrimental influence on success rates of transcervical transfer of equine embryos. Eight Shetland pony mares were each randomly assigned to each of four treatments: (a) 0.8 mg/100 kg (group T1), (b) 0.4 mg/100 kg (group T2), (c) 0.2 mg/100 kg BM dinoprost i.m. (group T3), and (d) 1 ml physiological saline i.m. (group CO). Treatments were administered as single doses on Day 7 of the estrous cycle. Administration of dinoprost caused dose-dependent rises of plasma concentrations of PGF2α-metabolite, although values of individual mares showed great variation within groups. Prostaglandin treatments resulted in a distinct decrease of plasma progesterone concentrations to values between 1.6 and 7.9 ng/ml within 24 h. Treatment groups had significantly lower progesterone area under the curve (AUC: T1 942.8±175.9, T2 1050±181.2 and T3 1117.9±179.8 ng/ml/h) when compared with controls (CO 1601.9±227.6; t-test, P<0.05). There was a small, but significant negative correlation between AUC of progesterone and of PGF2α-metabolite ( R=−0.4; P<0.05). Administration of PGF2α caused secretion of oxytocin in three (T1, T2) and two (T3) mares out of eight ranging from 19.3 to 63.1 pg/ml. The AUC of oxytocin was positively correlated with AUC of PGF2α-metabolite ( R=0.4, P<0.05) and negatively correlated with AUC of progesterone ( R=−0.4, P<0.05). Administration of dinoprost yielded significantly shorter intervals from treatment to estrus and ovulation (values in parentheses), respectively, when compared with controls: T1 3.9±0.7 days (12.1±0.7 days), T2 4.5±0.6 (12.3±0.6), T3 4.9±0.5 (12.3±0.6), and CO 8.9±0.6 days (16.5±0.8 days) ( t-test, P<0.01) (Fig. 2). Different doses of PGF2α caused similar effects. Data suggest that progesterone concentrations at applications influence efficacy of treatments more than doses administered, as demonstrated by their high correlation with estrous cycle patterns. It is important to note that differences we achieved are gradual and that all mares responded to treatment by luteolysis and premature estrus, regardless of doses applied.

Male, female and management risk factors for non-return to service in Dutch mares

The “effect” of stallion, mare and management-related factors on the odds of pregnancy per cycle in the horse were identified and quantified from the breeding records of Dutch Warmblood ( n=4491), Friesian ( n=1467) and Shetland-pony mares ( n=3267) mated either naturally or by artificial insemination to one of the 88 stallions between 1992 and 1996. A mare was considered to be pregnant when she did not return to oestrous within 28 days of the last insemination. For Dutch Warmblood horses, the percentage of mares that did not return for service within 28 days (NR28) varied between studfarms and ranged from 61 to 82%. The NR28 for mares inseminated with fresh semen ranged from 67 to 74% and for mares inseminated with frozen/thawed semen this percentage was 59. Mares served at a second cycle had lower odds not to return than mares served at the third or subsequent cycle (OR=0.84). For Friesian horses, the NR28 for young mares was higher than that for older mares. Mares served before 1 May in any year had lower odds of non-return than mares served after 1 July (OR=0.69). The NR28 of mares inseminated once per cycle was 6% lower than that of mares inseminated three times or more per cycle. For Shetland ponies, the NR28 also varied between studfarms and ranged from 62 to 78%. Stallions ≤3 years old had lower odds of non-return compared to older stallion (≥11) (OR=0.57). Mares served before 1 July had lower odds of non-return. Other significant factors for this breed were age of the mare, cycle number and insemination frequency. Stallion factors accounted for 5.9, 2.0 and 14.7% of the variation in the NR28 for Dutch Warmblood, Friesian horses and the Shetland ponies, respectively.