Pony Mares Research Articles

Application of the Alivecor KardiaMobile smartphone modulated ECG device for use in horses.

Electrocardiography is the method used to monitor the electrical impulses in the heart. These diagnostics are increasingly making use of smartphone-based technologies. The objective of this research was to determine whether the Alivecor KardiaMobile (ECGAKM) smartphone-modulated electrocardiographic device, a novel ECG device, can be used to obtain reliable electrocardiogram (ECG) readings in horses. The device was initially tested in 36 Nooitgedacht pony mares to determine the best site of application, method of skin preparation, and ECGAKM device orientation for reliable ECG tracings. Once the most reliable site for ECG acquisition was determined, the device was then applied, in this manner, to 31 Nooitgedacht pony mares and compared with a standard telemetric ECG system (ECGTV). The ECGAKM device was best applied in the fourth intercostal space on the left hemithorax in a vertical orientation and with the skin dampened with 70% ethanol. Mean values determined for RR and QT intervals between the ECGAKMS and ECGTV were not significantly different, however, mean values for the duration of the QRS complexes were significantly different for the two devices. There is acceptable agreement between the ECGTV and ECGAKM devices with regards to the measurement of the PQ; RR and QT intervals but not the QRS duration. The automatically calculated heart rate is not an accurate measure of true heart rate. The Alivecor KardiaMobile (ECGAKM) device can be considered as a simplified screening ECG device in situations where the more standardised system is either unavailable or impractical, but has some limitations.

Effect of breed and embryo sexing on the efficiency of a commercial OPU-ICSI program in mares

Breeders of Polo pony mares favour females over males as their sport performance is shown to be superior. This sex preference in breeding Polo horses is a challenge for stud veterinarians as sex sorted semen in horses is not available commercially due to technical difficulties. An alternative is the sexing of embryos. Ovum pick-up (OPU) and ICSI have become popular in show jumping (SJ) horses over the last decade, with the overall efficiency of the process improving steadily, with reported embryo per OPU session of > 1.5 embryos. The in vitro production of embryos not only allows a higher yield of blastocysts compared with in vivo derived embryos, but also their cryopreservation with similar pregnancy results similar to those achieved with fresh embryos. In addition, the possibility of sexing embryos before freezing would be an attractive option to the Polo breeding industry. Although there are several reports on the efficiency of an OPU-ICSI commercial program in SJ, information in Polo mares OPU-ICSI programs is scarce. The objective of this study was to describe the OPU-ICSI outcomes of a commercial program of Polo mares (n=77 cycles) compared to show jumping mares (n=73 cycles), including the likelihood of pregnancy after transferring sexed cryopreserved embryos. A general linear model of variance was created to test the effect of breed (Polo vs. SJ)on antral follicles available for aspiration, recovered oocytes, and number of embryos per OPU-ICSI session. Furthermore, the effect of embryo sexing prior to cryopreservation on pregnancy rate was determined by chi-square test. All OPUs were performed in the UK and oocytes were shipped to an external laboratory in Europe. Pre-implantation gender diagnosis was performed by extended embryo culture and sexing of extruded cells in 53 embryos of Polo mares. Polo mares had a lower (P<0.001) number of punctured follicles and recovered oocytes (14.1±0.6 and 8.2±0.5) compared to SJ mares (19.2±0.7 and 11.0±0.5), respectively. The mean number of embryos per OPU-ICSI session was similar (P>0.1) for SJ and Polo mares (1.38 and 1.41, respectively). Embryos derived from SJ mares (15/22, 68%) were more likely (P<0.05) to develop into a pregnancy than embryos from Polo mares (22/51, 43%). The ongoing pregnancy rate of sexed embryos (6/26, 23.1%) was lower (P=0.016) than that of unsexed embryos (14/25, 56%) from Polo mares, which explains the overall lower pregnancy outcome of Polo compared to SJ mares (unsexed embryos). In conclusion, Polo mares had a lower antral follicle pool than SJ mares, but their oocytes showed similar developmental potential to those of SJ mares. Embryo sexing decreased the chances of having a viable pregnancy after an OPU-ICSI session.

Molecular mechanisms underlying equine endometrial cup regression: a transcriptomic analysis

The gonadotropin-secreting equine endometrial cups (EC) have a major role in pregnancy maintenance, but an intriguingly short lifespan. First seen in the maternal endometrium at days (d) 38-40 post-ovulation, the EC undergo degeneration via poorly understood mechanisms starting around d 60-70, with complete dehiscence at approximately 120-150 d of gestation. Pregnancy losses during the EC physiological lifespan and EC pathological persistence after abortion or uncomplicated parturition usually lead to irregular estrous cyclicity and subfertility, with only anecdotal and inconsistent therapeutic options currently available. A holistic understanding of the cellular signaling leading to EC regression may provide insight for development of effective therapies for retained or persistent EC. We therefore hypothesized that comparing the transcriptome of EC during development and regression could elucidate key regulators and molecular pathways leading to EC trophoblast cell demise. Samples of EC were dissected from the adjacent endometrium of pregnant pony mares during early development (DEC, 42-47 d post-ovulation, n = 5), and regression (REC, 96 – 120 d, n = 5). Next generation RNA sequencing was performed with Illumina NovaSeq6000 and reads mapped to EquCab3.0 (STAR-2.5.2b). Differentially expressed genes (DEGs) were identified with Cuffdiff-2.2.1 (FDR < 0.05). A total of 2,504 DEG were identified, being 1,648 upregulated and 856 downregulated in REC when compared to DEC. Pathway Analysis of DEGs was also performed (IPA, Qiagen). Interestingly, the main pathways affected in REC included oxidative phosphorylation (P = 1.08e-38);mitochondrial dysfunction (P = 2e-25); estrogen receptor signaling (P = 2.2e-10); Sirtuin signaling pathway (P = 9.64e-16), which has central regulatory roles in cell senescence and apoptosis; and Granzyme A Signaling (3.29e-15), associated with Cytotoxic T Lymphocyte and Natural Killer-mediated apoptosis via caspase-independent pathways. Upstream regulator analysis highlighted the roles of mitochondrial protein ubiquinol-cytochrome c reductase complex-3; rapamycin-insensitive companion of mTOR, an essential molecule in cellular homeostasis; Torin-1, a potent mTOR inhibitor; dead box protein 5, which may act as a co-activator of estrogen receptor signaling; and transducing-like enhancer protein-3, a member of a family of proteins that regulate cell fate through the Notch signaling pathway. Importantly, the main upstream regulators and downstream molecules are also involved in cancer cell biology (P = 1.32e-07 – 1.71e-122), including the downstream effectors of neoplastic and epithelial cell death secreted phosphoprotein 1 (Spp1), and secreted protein acidic and cysteine rich (Sparc). Along with aromatase, Spp1 and Sparc were upregulated in REC in comparison to DEC. In summary, we have highlighted key molecular pathways and regulators of EC regression, which merit further molecular and functional investigations. Since multiple identified molecules have also been targeted in human oncological treatments, further investigation of their effectiveness in hastening EC demise is warranted.

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.

Use of Xylocaine to facilitate embryo transfer to Welsh pony recipient

Embryo transfer is a commonly used procedure in equine breeding worldwide. Embryos are trans-cervically transferred into the recipient uterus on Day 5 or 6 after ovulation and expected pregnancy rates range from 65 to 85% and 50 to 75% at days 14 and 45, respectively. Poorer results in pony mares may be attributed to the difficult passage of the cervix and the subsequent contamination of the cervix and/or release of inflammatory products. Paracervical block, i.e. local anesthesia of the cervical area is routinely used for gynecological procedures, such as intrauterine device placement, in women (Mody et al. Obstet Gynecol. 2018; 132(3): 575–582) but has not been reported for similar procedures in the equine. The present study aimed to evaluate the benefits of the application of an anesthetic gel on the cervix before transfer to facilitate the introduction of the transfer gun. Twenty-one grade 1 embryos (one morula and 20 blastocysts), 166-1300µm in diameter (mean±SEM=422±52µm), were collected from Welsh B pony mares at day 7 after ovulation. After collection, embryos were evaluated and conventionally transferred transcervically to recipient pony mares, under 8 years ofage, at day 5, 6, or 7 after ovulation (1 embryo per recipient). In the xylocaine group (XYL), 11 embryos were transferred 10 min after the application of 3ml of xylocaine gel (Xylocaine® gel 2%, AstraZeneca) at the base of the recipient's cervix. Ten other transfers were routinely performed without xylocaine as a control group (CTRL). Pregnancies were monitored by ultrasonography at day 14 and until an embryonic heartbeat was detected at day 21 days. In XYL, 8 (73%) and 6 (55%) recipients were pregnant on day 14 and 21, respectively. In CTRL, 6 (60%) and 4 (40%) recipients were pregnant on day 14 and 21, respectively. While, slightly better in XYL, both at day 14 and day 21, pregnancy rates were not statistically different between groups (P=0.66 and P=0.67 respectively). No significant difference was also obtained between the day of transfer for both groups (P>0.05). Transfers in XYL were subjectively found to be smoother by the operators. It can be concluded that the application of xylocaine on the recipient's cervix does not interfere with early gestation development and may ease cervical catheterization in pony mares. Further work with larger numbers is required to evaluate its potential benefits on transfer outcomes.

Ultrasonographic examination of equine fetal growth parameters throughout gestation in pony for Equine-Assisted Therapy.

Equine-Assisted Therapy (EAT) is gaining popularity. Ultrasound examination is used to decrease the abortion rate in horses. In this study, to monitor fetal well-being throughout the gestation for EAT, we measured fetal heart rate (FHR), fetal eye orbit (FEO), fetal gonad length (FGL), fetal kidney length (FKL), and the combined thickness of the uterus and placenta (CTUP) by ultrasonography in pony mares. Additionally, we measured the plasma progesterone (P4) and estradiol (E2) concentrations in pregnant horses using enzyme immunoassay. The FGL peaked at week 32 and then decreased to term, and a strong correlation (r=0.72, P<0.001) between the FGL and E2 concentration was observed. A strong correlation with gestational age was detected among the FEO (r=0.96, P<0.001), FKL (r=0.85, P<0.001), and CTUP (r=0.96, P<0.001). The P4 concentration peaked at week 10, decreased to low levels (below 5 ng/ml), and peaked before parturition. In conclusion, this study provides information on fetal growth throughout gestation in pony mares for EAT. In addition, it revealed the relationship between ultrasonographic profile and plasma hormone concentrations during gestation.

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.

A Study on Some Serum Biological Constituents during Different Days of Normal Oestrous Cycle of Manipuri Pony Mares

Background: The aim of this study was to estimate the serum biochemical (glucose, cholesterol, total protein, calcium, phosphorus and magnesium) and hormonal constituents (estrogen and progesterone) in female Manipuri pony mares during the different days of oestrous cycle. Methods: A total of 50 numbers of blood samples were collected from 10 numbers of apparently healthy, non-pregnant on the different days of oestrous cycle i.e. day 0, 4, 8, 14 and 18 by using commercial diagnostic kits. Result: The mean serum levels of glucose, cholesterol and phosphorus were found to be non-significant on the different days of oestrous cycle whereas the mean serum level of total protein and calcium was found significantly different (P less than 0.01). The serum magnesium level differed significantly (P less than 0.05) amongst the different days of oestrous cycle. Both estrogen and progesterone were also found to be significant (P less than 0.01) between the different days of oestrous cycle. The results showed significant influence on serum biochemical and hormonal assay during different days of oestrous cycle in female Manipuri pony.

A Study on Oestrus and Parturition Behaviour of Manipuri Pony Mares

Background: The aim of this research was to study the behavioural signs exhibited during the different phases of oestrus and parturition in Manipuri pony mares. Methods: A total of ten each for oestrus and pregnant female Manipuri Ponies were selected for the behavioural signs study and a total data of 30 numbers of set questionnaires were analysed for studying the oestrus and foaling during various seasons and months of Manipuri Pony mares. Result: The behavioural oestrus signs observed in female Manipuri pony were raised tail, vulva swelling, vaginal mucous secretion, stallion seeking, inappetance and male acceptance. Pre-partum sign includes swelling of vulva, waxing, udder development, teats engorgement with milk discharges. Restlessness, isolation from human and herd mates, abdominal straining were shown during first stage of parturition. Abdominal straining with expulsion of fetus was displayed in the second stage followed placenta expulsion at the third stage of parturition. The occurrences of timing of birth were lesser between 6 pm and midnight (20.00%). The highest occurrence of oestrus was recorded during spring season (46.67%, 56.67%) respectively and in months it occurred highest during March to June (16.67%).

Clinical Outcome of Transcervical Infusion of a Combination of Procaine Penicillin and Gentamicin in Late-term Pregnant Mares

Transcervical intrauterine infusion of antibiotics may more effectively treat pathogens associated with fetal and neonatal disease in pregnant mares than standard systemic routes. The objective of this study was to assess the safety of transcervical antibiotic infusion by characterizing the gestational outcome in nine healthy pregnant pony mares following a single transcervical infusion of 2.4 million IU of procaine penicillin and 200 mg of gentamicin in a 10 mL volume during late gestation. Assessment of fetal-placental health was performed through serial measurement of the combined thickness of the uterus and placenta (CTUP) and fetal heart rate and mares and foals were closely monitored in the periparturient period. Fetal heart rate and CTUP remained unchanged after infusion, with no evidence of fluid accumulation or significant increase at the time-points 24, 48, and 72 hours. All mares foaled without complication 12–58 days after antibiotic infusion at a mean gestational age of 322.7 ± 12.7 days. Two out of nine foals displayed signs of mild neonatal maladjustment syndrome that responded to minimal supportive care and all foals survived to weaning without further complications.

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.

Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology.

Simple SummaryNotwithstanding extensive research into fertility problems in mares, pregnancy rates have remained low mainly because of endometrial inflammation (endometritis). In the field of equine research, endometrial explants have been used to carry out in vitro studies of the mare’s endometrium. However, there has been no wide-ranging assessment of relative stability of this model over time. The aim of this study was to perform an in-depth transcriptomic assessment of endometrial explants over a culture period of 72 h and assess if they are representative of the whole mare. Explants at 24 h demonstrated significant changes when compared to biopsies at 0 h as expected. Even though gene expression changes were seen between 24 and 48 h of culture, prior to this window changes were dominated by the effects of explanting and culture and subsequently, transcription was generally compromised. Our results, therefore have defined the optimal period when explants can be used to study equine endometritis and how the endometrium is modulated during inflammation. It highlights the use of abattoir derived samples to understand the physiology and pathophysiology of the equine endometrium, negating the need to collect repeated uterine biopsies from living mares.Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2α as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h.

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.

Análise de componentes principais de medidas morfométricas de equinos da raça Pônei Brasileiro

O Pônei Brasileiro é uma raça recente que iniciou o registro dos animais a partir do ano de 1970, e vem sendo selecionada continuamente principalmente na sua morfologia para aperfeiçoamento e padronização racial. O objetivo do estudo foi avaliar através da análise componentes principais medidas morfométricas de garanhões e éguas pôneis da raça Pônei Brasileiro para identificar as que mais contribuem para a explicação da variância total em cada sexo. Foram tomadas 14 medidas lineares de um total de 281 animais adultos e foram realizadas a análise descritiva, análise de variância, correlações e análise de componentes principais. As medidas morfométricas dos animais apresentaram grande variação nos dois sexos. As correlações entre as variáveis foram positivas e, em geral, de alta magnitude. Para os garanhões foram necessários apenas dois componentes e para as fêmeas foram necessários apenas três componentes para explicar pelo menos 80% da variação total. Após metodologia para descarte de variáveis foram identificadas as variáveis largura da cabeça, comprimento do pescoço e altura do tórax como as medidas que explicam a maior parte da variação dos animais da raça Pônei Brasileiro e devem ser utilizadas para seleção e controle racial.

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.

Transient suppression of ovulatory ovarian function in pony mares after treatment with slow-release deslorelin implants

Behavior during the estrous cycle of mares can affect their performance and therefore inhibition of cyclical ovarian activity is indicated. We hypothesized that implants containing the GnRH analog deslorelin downregulate GnRH receptors and inhibit ovulation in mares. The estrous cycles of Shetland mares were synchronized with 2 injections of a PGF2α analog. One day after the second injection (day 0), mares received 9.4 (group D1, n = 6) and 4.7 mg deslorelin (D2, n = 5) as slow-release implants or 1.25 mg short-acting deslorelin as a control (C, n = 5). Ultrasonography of the reproductive tract and ovaries and observation of estrous behavior and collection of blood samples for analysis of progesterone and LH concentrations were performed every second day until day 10 and thereafter at 5-d intervals. Stimulation tests with the GnRH-agonist buserelin were performed on days 10 and 45. Until day 50, there were less spontaneous ovulations in group D1 (P < 0.01) and estrous behavior was reduced in groups D1 and D2 compared with group C (P < 0.05). The time until first ovulation (D1 62.0 ± 8.6, D2 44.2 ± 14.1, C 22.2 ± 3.1 d, P < 0.05) and the number of days with estrous behavior (P < 0.05) differed among groups. On day 10 after treatment, a GnRH stimulation test revealed interactions between group and time (P < 0.001) in plasma LH concentration that were no longer detectable on day 45 after treatment. In conclusion, long-acting deslorelin implants result in a transient downregulation of pituitary GnRH receptors that is associated with inhibition of ovulation and estrous behavior in Shetland mares.

Effect of age and the individual on the gastrointestinal bacteriome of ponies fed a high-starch diet.

Bacteria residing in the gastrointestinal tract of mammals are crucial for the digestion of dietary nutrients. Bacterial community composition is modified by age and diet in other species. Although horses are adapted to consuming fibre-based diets, high-energy, often high-starch containing feeds are increasingly used. The current study assessed the impact of age on the faecal bacteriome of ponies transitioning from a hay-based diet to a high-starch diet. Over two years, 23 Welsh Section A pony mares were evaluated (Controls, 5–15 years, n = 6/year, 12 in total; Aged, ≥19 years, n = 6 Year 1; n = 5 Year 2, 11 in total). Across the same 30-week (May to November) period in each year, animals were randomly assigned to a 5-week period of study and were individually fed the same hay to maintenance (2% body mass as daily dry matter intake) for 4-weeks. During the final week, 2g starch per kg body mass (micronized steam-flaked barley) was incorporated into the diet (3-day transition and 5 days at maximum). Faecal samples were collected for 11 days (final 3 days hay and 8 days hay + barley feeding). Bacterial communities were determined using Ion Torrent Sequencing of amplified V1–V2 hypervariable regions of 16S rRNA. Age had a minimal effect on the bacteriome response to diet. The dietary transition increased Candidatus Saccharibacteria and Firmicutes phyla abundance and reduced Fibrobactres abundance. At the genera level, Streptococcus abundance was increased but not consistently across individual animals. Bacterial diversity was reduced during dietary transition in Streptococcus ‘responders’. Faecal pH and VFA concentrations were modified by diet but considerable inter-individual variation was present. The current study describes compositional changes in the faecal bacteriome associated with the transition from a fibre-based to a high-starch diet in ponies and emphasises the individual nature of dietary responses, which may reflect functional differences in the bacterial populations present in the hindgut.

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.