Reproduction

Question 1

Relationship between equine herpesvirus-1 viremia and abortion or equine herpesvirus myeloencephalopathy in domesticated horses: A systematic review

Answer 1

34 studiesthat met the inclusion criteria, of which30evaluated viremia and neurologic outcomes and8evaluated viremia and abortion. Viremiawas generally detectable before the onset of either EHM or abortion, butno conclusive evidencecould be drawn about the correlation between the occurrence of these outcomes and themagnitude or durationof viremia. The incidence rates of EHM and abortion varied among the studies, depending on thevirus strains,dose,analytical methods, andsample sizesused. The paper concluded that moreconsistent and reliableexperimental models and data collection methods are needed to better understand the role of viremia in EHV-1 pathogenesis and to test future therapeutic and preventive measures. Duration of viraemia may be more important than level of viraemia for development of EHM

Question 2

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism

Answer 2

Embryos exposed to 10 mmol/L glucose during early culture (10-17 group) showed higher expression of glycolysis-related genes (e.g., PFKP and LDHA) and a higher BAX/BCL2 ratio, indicative of increased glycolytic activity and apoptosis.
5mmol/l is best in the first days of culture.
Equine embryos are adapted to high glucose environments vs other species

Question 3

Prevalence of the RAPGEF5 c.2624C>A and PLOD1 c.2032G>A variants associated with equine familial isolated hypoparathyroidism and fragile foal syndrome in the US Thoroughbred population (1988–2019)

Answer 3

-EFIH (RAPGEF5): Low and stable allele frequency of 0.8% across the study period (1988–2019). Combined carrier frequency of approximately 1.5% (658 foals out of 20,000 born annually).
-FFS (PLOD1): Low allele frequency increased slightly from 0.4% (1988–2000) to 0.9% (2001–2019), though not statistically significant. Carrier frequency increased from 0.8% to 1.8%. (not significant)
-No homozygous individuals for either variant were detected, likely due to the fatal nature of these recessive conditions.
-No significant regional differences in allele frequency or carrier prevalence were observed
-The variants have existed for at least 30 years, with no evidence of selection for or against these alleles in the Thoroughbred population.

Question 4

Molecular cytogenetic screening of sex chromosome abnormalities in young horse populations

Answer 4

The study utilized Fluorescence In Situ Hybridization (FISH) for screening:
• FISH enhances detection sensitivity, even in cases with low-incidence abnormal metaphases.
• Results revealed a higher prevalence of chromosomal abnormalities (3.8% overall, compared to ~2% in previous studies).

Key Chromosomal Abnormalities Identified
• X Monosomy (63,X): Represents 57.9% of all diagnosed abnormalities in the study.
• X Trisomy (65,XXX): Identified in both simple mosaics (e.g., 64,XX/65,XXX) and complex mosaics (e.g., 63,X/64,XX/65,XXX).21.05% of chromosomal abnormality cases.One case of male sex chromosome aneuploidy (64,XY/65,XXY)
• SRY-Negative XY (64,XY SRY-Negative): Rare but significant finding in one mare: External phenotype appeared normal, but internal reproductive organs were underdeveloped (presence of testicular tissue).
• Novel Reciprocal Translocation [64,X,t(1p;Xp)(1q;Xq)]: First documented reciprocal translocation between the X chromosome and chromosome 1 in horses. Found in a Hucul mare with a normal external and internal phenotype but expected reproductive challenges (e.g., REEL).

Question 5

Serum cortisol and thyroid hormone concentrations and survival in foals born from mares with experimentally induced ascending placentitis

Answer 5

• Sick foals from mares with placentitis had reduced T3, FT3, and FT4 levels at birth and during the first week, consistent with Nonthyroidal Illness Syndrome (NTIS).
• Premature foals showed lower cortisol and thyroid hormone levels, suggesting immature fetal development impacts thyroid function.
• T3:cortisol and T4:cortisol ratios were lower in nonsurviving sick foals, suggesting these ratios are better prognostic markers than hormone levels alone.

Mechanisms Behind Hormone Reduction
• Lower thyroid hormones may result from inflammation (e.g., cytokines like IL-1β, IL-6, TNF-α): Cytokines suppress TSH release, inhibit T4 to T3 conversion, and reduce hormone protein binding.
• Cortisol supports thyroid gland maturation near birth; premature foals lack sufficient cortisol, affecting thyroid function.

• Unlike some studies, this research showed no direct link between thyroid hormone levels and survival but emphasized the importance of dynamic hormonal changes.

Question 6

Plasma metabolomic profiling of healthy pregnant mares and mares with experimentally induced placentitis

Answer 6

Early and late metabolomic changes were observed:
• 4 hours post-inoculation: Elevated metabolites (e.g., pyruvate, glucose, lactate, histidine, phenylalanine) linked to energy and nitrogen metabolism.
• Day 4 post-inoculation: Reduced metabolites involved in energy (pyruvate, citrate) and nitrogen metabolism (histidine, phenylalanine, tyrosine) coinciding with clinical signs of placentitis.

• Early increases in metabolites may reflect immediate systemic inflammatory or metabolic response to infection.
• Later reductions likely result from increased metabolic demand or microbial utilization during placentitis progression

Question 7

The association between the endometrial biopsy grade and selected epidemiological and reproductive variables in a population of sub-fertile mares

Answer 7

• Significant association between increasing mare age and higher biopsy grade.
• No significant associations found between biopsy grade and: Breed (Thoroughbred vs. non-Thoroughbred). Barren status. Presence of uterine fluid retention. Acute endometritis or positive bacterial cultures. History of dystocia.
Discrepancies with prior studies may reflect the small sample size (41) and retrospective design.

• Endometrial biopsies are strongly indicated in older mares due to higher risks of fibrosis and poor reproductive outcomes.

Question 8

The therapeutic effect of stem cell-derived exosomes in the treatment of chronic endometritis as assessed by histopathological, Doppler and hormonal expression in Arabian mares

Answer 8

• Exosomes from mesenchymal stem cells (MSCs) exhibit therapeutic potential through paracrine signaling, involving hormones, chemokines, and exosomal microvesicles.
Histological improvements post-treatment included:
• Increased epithelial thickness.
• Re-epithelialization.
• Enhanced blood vessel formation, crucial for tissue repair and angiogenesis.
•Post-treatment biopsies demonstrated significant regression of fibrosis and restoration of normal glandular structures.

Doppler assessments post-treatment revealed:
• Elevated blood flow rate and peak systolic velocity, indicative of improved vascular function.
• Enhanced uterine artery perfusion, supporting recovery from chronic endometritis.
• These vascular changes are attributed to vasoactive factors associated with embryo implantation.

Hormonal alterations:
• Decrease in oestradiol levels post-treatment, aligning with recovery from inflammatory conditions.
• Increased progesterone levels, supporting endometrial receptivity and embryo implantation.

Exosomal treatment accelerated tissue regeneration via:
• Angiogenesis: Delivery of nutrients, oxygen, and stem cells to injured tissues.
• Enhanced cellular proliferation in damaged areas, facilitated by growth factors like VEGF and IGF.
• Re-epithelialization and restoration of histological features, critical for normal uterine function.

• Supports pregnancy establishment through improved uterine conditions.

Question 9

Image analysis quantification of sperm acrosome associated 3 protein expression in domesticated and free-roaming equine ovaries

Answer 9

Granulosa Cells:
• Higher SPACA3 immunoexpression was observed in granulosa cells across all follicular stages (primordial, primary, secondary, tertiary) in domesticated horses compared to free-roaming mares.
• Differences in SPACA3 expression between domesticated and free-roaming mares might reflect environmental and physiological variations, such as immune status or energy availability.
Theca Cells:
• SPACA3 expression in theca cells was higher in tertiary follicles for domesticated mares but did not differ significantly in secondary follicles.
• These differences likely arise from distinct functional roles and developmental processes between granulosa and theca cells.

Potential Mechanisms
• Granulosa cells, which are actively involved in follicular development and regulated by factors like follicle-stimulating hormone (FSH) and estrogen, showed higher SPACA3 levels, supporting their role in fertility.
• The study’s findings align with granulosa cells’ known plasticity and their capacity to differentiate into other cell types, which might be influenced by SPACA3 expression.

• Free-roaming mares exhibited lower SPACA3 expression, potentially due to limited resources affecting homeostasis and immune function.
• SPACA3’s consistent and significant expression in domesticated mares suggests it is a promising target for developing immunosterilants aimed at controlling free-roaming horse populations.
• Immunosterilants targeting SPACA3 could provide a humane, non-surgical method to manage overpopulation without disrupting herd dynamics.

Question 10

Evidence for Origin of Lavender Foal Syndrome among Egyptian Arabian horses in Egypt

Answer 10

lavendar foal syndrome, egyptian arabians, PCR

Question 11

Transcriptomic analysis of the chorioallantois in equine premature placental separation

Answer 11

• The transcriptomic analysis revealed that ECM remodeling, cell adhesion, and associated pathways are central to PPS pathogenesis.
• ECM components like collagen and proteoglycans are significantly upregulated in PPS cases, indicating altered placental integrity and attachment dynamics.

• 1204 DEGs: Collagen components (e.g., COL1A2, COL5A1, COL6A1) and proteoglycans (e.g., VCAN, DCN, BGN) were prominently upregulated.
• Gene Ontology (GO) Analysis: Terms like cell adhesion, ECM organization, and collagen fibril assembly were enriched.
• Pathways such as ECM-receptor interaction, integrin signaling, and focal adhesion play critical roles in maintaining CA integrity.
Predicted Upstream Regulators:
• Identified regulators (e.g., HIF1A, TGFB2, JUNB) highlight the role of hypoxia, inflammation, and ECM stabilization. Hypoxia-inducible factor (HIF1A) may initiate ECM production and cell adhesion changes in response to vascular compromise. Transforming growth factor beta (TGFB2, TGFB3) promotes collagen synthesis and inhibits ECM degradation.

Histological and Immunohistochemical Observations
• Placental histology in PPS revealed edema and thickened allantoic layers, correlating with increased ECM deposition.
Immunohistochemistry showed higher expression of:
• HIF1A: Localized to the nuclei in vascular and connective tissues, reflecting hypoxia-driven transcriptional activity.
• Versican (VCAN) and Collagen (COL1A2): Indicative of ECM remodeling and structural reinforcement in PPS placentas.

Mechanistic Insights
• ECM upregulation and the suppression of ECM degradation (via protease inhibitors like TIMP1) may explain the failure of placental rupture during delivery.
• Hypoxia appears to be a key initiating factor, activating pathways involving growth factors and inflammation mediators.
• The failure of matrix metalloproteinases (MMPs) to degrade ECM adequately suggests disrupted matrix turnover in PPS cases.

Question 12

Peritoneal fluid analysis in equine post-partum emergencies admitted to a referral hospital: A retrospective study of 110 cases

Answer 12

Post-Partum Hemorrhage (PPH):
• Increased peritoneal packed cell volume (PCV).
• Reduced nucleated cell count (WBCC).
• Absence of degenerate neutrophils in fluid cytology.
• PPH mares often presented older and showed clinical signs of colic.
• Peripheral blood analysis revealed reduced red blood cell count (RBCC), hemoglobin (Hb), and protein levels due to hemorrhage.
• Survival rate was favorable (82%) with medical management.

Gastrointestinal Trauma (GIT):
• Elevated WBCC and presence of degenerate neutrophils in peritoneal fluid.
• Plant material or bacteria present in fluid cytology (though not consistently observed in this study).
• Severe cases like ruptured viscera had rapid progression to systemic inflammatory response syndrome (SIRS), requiring immediate intervention.
• Survival to discharge was poor (15%), reflecting the severity of these cases.

Urogenital Trauma (UGT):
• Peritoneal fluid showed higher WBCC compared to PPH, likely indicative of septic peritonitis.
• Presence of concurrent hemorrhage in some cases, emphasizing the importance of cytological evaluation even when fluid appears hemorrhagic.
• Survival to discharge was moderate (69%), with surgical intervention often required.

Question 13

Diagnostic characteristics of refractometry cut-off points for estimating immunoglobulin G concentration in mare colostrum

Answer 13

•Both optical (OR) and digital refractometers (DR) were validated as effective tools for assessing colostrum quality by estimating immunoglobulin G (IgG) concentrations.
Optimal Cut-off Points: align closely with previous studies and indicate poor colostrum quality (<60 g/L IgG).
• DR: ≤23.75%
• OR: ≤23.90%

Digital Refractometer (DR): Sensitivity: 93.3% Specificity: 87.9% Positive Predictive Value (PPV): 63.6% Negative Predictive Value (NPV): 98.3%
Optical Refractometer (OR): Sensitivity: 93.3% Specificity: 81.8% PPV: 53.8% NPV: 98.2%
• Both refractometers demonstrated high accuracy, particularly in ruling out poor-quality colostrum due to high NPV values.
• Strong correlation observed between IgG concentrations measured by RID and refractometers for both Dr and OR
• High correlation between DR and OR readings ( =0.992), confirming interchangeability for colostrum assessment.
• Moderate correlation between serum and colostrum IgG concentrations (=0.542), supporting colostrum evaluation as an indirect predictor of neonatal immunity.

Use in combination with serum IgG measurement

Question 14

Abnormal mare behaviour is rarely associated with changes in hormonal markers of granulosa cell tumours: A retrospective study

Answer 14

2914 cases that were submitted due to behaviour issues
• 2506 (86%) did not have any of the measured hormones reach GCT-like concentrations.
• 408 cases had either one (63%), two (25.5%), or three (11.5%) hormones with concentrations consistent with those from confirmed GCT cases.
• Testosterone had the lowest percent of GCT-like values among the cases (7.7%), compared with AMH (9.4%), inhibins (9.6%) and inhibin B (8.7%).

Stallion-like Behavior:
• Most strongly associated with elevated ovarian hormones, including testosterone, AMH, inhibins, and inhibin B.
• This behavior was reported in up to 82% of cases with GCT-like hormonal profiles, consistent with previous literature identifying it as a hallmark of GCT.
Other Behaviors:
• Aggression, oestrous behavior, and generalized abnormal behaviors were not strongly correlated with elevated hormone levels.
• Hormonal imbalances were present in less than 10% of cases submitted for abnormal behaviors.

Hormonal Patterns:
• Testosterone and AMH showed the highest correlation with reported stallion-like behaviors.
• Hormonal variability and the presence of marginally increased levels were less predictive for behaviors outside of stallion-like presentation.

Clinical Implications
• Hormonal assays remain critical for diagnosing GCT but are not reliable standalone indicators for behavioral abnormalities.
• Incorporating transrectal palpation or ultrasound findings increases diagnostic confidence.
Management:
• Cases presenting with stallion-like behaviors and abnormal hormonal profiles should undergo further diagnostic evaluations for GCTs.
• For mares with aggressive or oestrous behaviors but normal hormone levels, alternative non-ovarian pathologies should be explored.

Question 15

Recumbency decreases mare and foal survival following in-hospital dystocia management

Answer 15

Impact of Recumbency on Survival
Mare Survival:
• Recumbency at hospital admission significantly decreased mare survival rates compared to ambulatory mares.
• Survival for recumbent mares: 58.5% vs. ambulatory mares: 91.8%. OR 6.9
• Common causes of death for recumbent mares included: Neuromuscular trauma. Fractures. Hemorrhage. Reproductive or gastrointestinal damage.
• Recumbency often linked to prolonged dystocia, leading to exhaustion, trauma, and complications.
Foal Survival:
• Foals from recumbent mares had significantly lower survival rates (4.9%) compared to foals from ambulatory mares (38.5%). OR 22.7
• Higher survival rates in foals delivered through controlled vaginal delivery (CVD) than Caesarean section (CS), attributed to shorter preparation times with CVD.

Dystocia Duration and Outcomes
Prolonged stage II parturition negatively affected both mare and foal outcomes.
• Median dystocia duration: Survivors: 65 minutes. Non-survivors: 120 minutes.
• Recumbency correlated with significantly longer dystocia durations.

• Post-dystocia fertility in Thoroughbred mares was not significantly impacted by ambulation status at admission: Ambulatory mares: 71% fertility. Recumbent mares: 65% fertility.
• Mares categorised as old (>15 years) at the time of hospital admission were 71% less likely to survive than young (<8 years) mares (OR 0.29)
• Mares which underwent CVD for the dystocia resolution procedure were more likely to survive than mares that underwent CS (OR 3.57)
• CVD was more likely to result in the delivery of a live foal than CS (OR 1.54)

Question 16

Reproductive performance of a cohort of Standardbred mares under a commercial breeding system

Answer 16

Key reproductive metrics:
• Foaling Rate (FR): 73.1%, aligning with prior reports for Standardbreds.
• First-Cycle Pregnancy Rate (FCPR): 61.4%, higher than Thoroughbred mares under natural mating systems (e.g., 53.6% in New Zealand).
• End of Season Pregnancy Rate (SPR): 84.7%, comparable or higher than previous studies in Standardbreds and Thoroughbreds.
• Metrics provide a benchmark for reproductive performance under artificial insemination (AI) systems.

Factors Affecting Reproductive Success
• Mare Age: Older mares (≥14 years) had significantly lower FCPR and SPR compared to younger mares. Likely due to: Degenerative reproductive changes. Decreased oocyte viability. Increased susceptibility to endometritis.
• Mare Location: Mares located on-farm had significantly higher FCPR and SPR compared to off-farm mares. On-farm management advantages include: Access to experienced stud staff. Reduced stress from transport or unfamiliar environments. Off-farm mares often lack intensive reproductive care, possibly affecting outcomes.
• Semen Type: Fresh-extended semen yielded the highest FCPR and SPR. Chilled-transported semen showed reduced FCPR but comparable SPR. Thawed–frozen semen resulted in the lowest pregnancy rates for both metrics, consistent with prior studies highlighting reduced fertility with frozen semen.

Pregnancy Losses
• Early embryonic loss (14–42 days post-insemination): 5.9%, comparable to Thoroughbreds.
• Pregnancy loss after 42 days to term: 7.8%, higher than some reports for Thoroughbreds.
• Possible reasons for higher late-term losses: Inconsistent reporting in stud book records. Less robust data collection for off-farm pregnancies.

Gestation Length
• Median gestation length: 350 days, similar to prior Standardbred studies (349 days) but slightly longer than Thoroughbreds (344 days).
• Variability in gestation length affects rebreeding timing and foaling outcomes.
Time to Conception
• Median time to conception: 82 days from the start of the breeding season.
• Shorter conception intervals were observed in:
-Non-foaling mares.
-Younger mares (<14 years).
-Mares inseminated with fresh semen.
-On-farm mares.

Comparative Insights
• Artificial breeding systems, particularly AI with fresh or chilled semen, appear more efficient than natural mating for first-cycle pregnancy outcomes.

Question 17

Ultrasound-guided fetal thorax compression to reduce post-fixation twins in the mare

Answer 17

High Success in Dizygotic Twins:
• Successfully reduced 90% (9/10) of dizygotic twin pregnancies to singleton pregnancies.
• 70% (7/10) resulted in healthy singleton foals at term.
• Success appeared unaffected by fixation type (unilateral or bilateral) or post-procedure administration of altrenogest. 8/10 dizygotic twins were fixed unilaterally while the remaining two were fixed bilaterally. Notably, all mares with unilaterally fixed dizygotic twins were discharged with one live and one dead fetus. Two of the unilaterally fixed pregnancies were lost after discharge from the clinic. The twin reduction technique was successful in one of two mares with bilaterally fixed dizygotic twin pregnancies. This mare subsequently delivered a healthy singleton foal of normal size.
• Success rates exceeded those of other methods like:
-Transvaginal ultrasound-guided reduction (33%-49% success).
-Cranio-cervical dislocation (63% success).

Failure in Monozygotic Twins:
• Uniformly unsuccessful (0/6) in monozygotic twin pregnancies, with both fetuses dying post-procedure in all cases.
• Hypothesized that shared placental vascularization compromises the viability of the co-twin after one fetus dies.

Challenges and Limitations
• Multiple Attempts: 80% of cases required repeated procedures (1-12 sessions) due to challenges in isolating and anchoring the fetus, necessitating prolonged hospitalization.
• Operator experience is crucial for success, particularly in cases requiring sustained manual pressure.
• Narrow Gestational Window: Most effective between 55-80 days of gestation. Challenges arise earlier (vesicle too turgid) or later (fetus harder to reach as the uterus descends).

Question 18

Using mycobacterium cell wall fraction to decrease equine chorionic gonadotropin after abortion

Answer 18

• MCWF treatment (2x 7 days apart beginning 5 days post abortion) significantly reduced circulating eCG concentrations, reaching below critical thresholds (10 IU/mL) associated with hormonal suppression of cyclicity.
• Reduction was achieved within 28 days post-treatment, faster than the natural degradation of endometrial cups (100–150 days of gestation). In contrast, control mares showed slower eCG decline.
• 80% of MCWF-treated mares developed pre-ovulatory follicles within 24 days of treatment initiation, compared to 33% in controls.
• Treated mares were ovulation-ready within one month, allowing for potential re-breeding during the same season.
• Peripheral IFN-γ and IL-1β levels increased significantly in treated mares, supporting the hypothesis that MCWF stimulates a pro-inflammatory immune response critical for endometrial cup degradation.

Mechanistic Insights
• Immunological Role: Endometrial cup degradation in natural conditions involves an influx of immune cells like lymphocytes, macrophages, and neutrophils.
• MCWF likely enhances this process by stimulating cytokine production (e.g., IFN-γ), promoting immune cell activation and facilitating trophoblast destruction.
• The observed cytokine delay in serum may indicate localized immune activity preceding systemic changes.

• Fertility post-treatment was not assessed in this study, but successful return to oestrus suggests promising potential for subsequent breeding success.

Question 19

Successful vitrification of equine embryos >300 microns without puncture or aspiration

Answer 19

• Demonstrates that equine embryos up to 480 μm in diameter can be successfully vitrified without puncture or aspiration.
• This approach simplifies the vitrification process and broadens its applicability by removing the need for invasive micromanipulation.

Pregnancy rates for vitrified embryos:
• 81.8% for embryos ≤300 μm.
• 80% for embryos between 300–480 μm.
• 0% for embryos >500 μm.
• The success threshold for survival is ≤480 μm, with the largest embryo resulting in a viable pregnancy measuring 480 μm.

• Day 7 embryo collection is recommended for achieving high recovery rates of embryos ≤480 μm, which are suitable for this method.

Question 20

Does inbreeding contribute to pregnancy loss in Thoroughbred horses?

Answer 20

Mid and Late Pregnancy Loss (MLPL):
• MLPL cases exhibited significantly higher inbreeding metrics (FROH, total ROH, and long ROH proportions) compared to early pregnancy loss (EPL) cases and controls.
• Longer ROHs (>10 Mb) were more prevalent in MLPL cases, indicating recent inbreeding as a likely factor. (higher proportion of long (>10 Mb) ROH (2.5%) than the Controls (1.7%)= recent inbreeding. no difference in short ROH)

Early Pregnancy Loss (EPL):
• No significant differences in inbreeding metrics were observed between EPL cases and controls.
• Suggests that EPL may not be as strongly linked to inbreeding as MLPL.

• No specific ROHs were uniquely associated with either EPL or MLPL, suggesting that multiple genetic factors or other mechanisms contribute to pregnancy loss.

Mechanistic Insights
• Inbreeding increases the likelihood of homozygosity for deleterious recessive alleles, potentially leading to pregnancy loss during mid-to-late gestation when fetal development places higher demands on maternal and fetal physiology.
• Examples of known homozygous mutations linked to equine conditions include congenital hepatic fibrosis and fragile foal syndrome, underscoring the importance of genetic surveillance.

Question 21

The equine umbilical cord in clinically healthy pregnancies

Answer 21

Median
• total= 53.5 cm
• amniotic = 29.5 cm
• allantoic = 25.0 cm

Umbilical Cord Length (UCL):
• No evidence of inadvertent selection for increased UCL based on sires or breeding practices.
Amniotic UCL Growth:
• Exhibits an exponential increase until ~200 days of gestation, followed by a plateau.
• This growth period aligns with fetal hindlimbs entering the uterine horn and decreased fetal positional changes, emphasizing the role of fetal movement in UCL development.
Allantoic UCL:
• Fixed earlier in gestation, showing no significant correlation with gestational age after ~100 days.

Fetal Measurements:
• Fetal crown-rump length (CRL) and weight showed predictable linear relationship with age: predicted CRL = -17.60 + 0.38 x gestational age, p < 0.001. Gestational age accounted for 99.0% of the variability in the CRL.
• sigmoidal relationships with gestational age
• significant positive correlation with total (rho = 0.49, p = 0.01) and amniotic UCL (rho = 0.50, p = 0.01), but not allantoic UCL (p = 0.8)

Correlations and Associations
• UCL and Maternal/Parental Factors: No correlation was found between UCL and maternal age, parity, or fetal sex.
-These findings contradict earlier research suggesting longer UCL in multiparous and older mares or male fetuses.
-Potential sire effects, hypothesized in prior studies, could not be explored due to insufficient data on repeat pregnancies by the same stallion.
• Allantoic vs. Amniotic UCL:
- No correlation between these two UCL components, suggesting independent programming mechanisms or localized influences such as fetal activity.

Question 22

Inflammatory markers for differentiation of endometritis in the mare

Answer 22

• Cytokines and chemokines in low-volume lavage (LVL) fluid distinguished healthy mares from those with acute endometritis and chronic endometrial fibrosis (CEF).
Significant markers include:
• Acute Endometritis: Elevated IFN-γ.
• CEF: Marked increases in IL-17, IL-10, IL-1β, TNF-α, CCL2, CCL3, CCL5, and CCL11.
• CCL2 ≥ 550 pg/mL predicted endometritis (acute or chronic) with 100% accuracy. But addition of CD14 expression improved the probability of identifying a reproductively healthy mare.

•Gene-level differences in biopsy samples (only IL-17 mRNA) were less pronounced compared to protein concentrations, highlighting post-translational regulation or variability in mRNA stability.

Chronic Inflammation:
• Persistent cytokine elevation (e.g., TNF-α, CCL2) aligns with chronic endometrial fibrosis and its association with infertility.
• Similar patterns of IL-17, TNF-α, and IL-1β are observed in other species with chronic reproductive disorders, suggesting conserved mechanisms.
Acute vs. Chronic Endometritis:
• IFN-γ elevation in acute cases emphasizes its role in immune activation, while chronic cases showed a broader inflammatory cytokine profile indicative of ongoing tissue remodeling and fibrosis.

Question 23

Conductivity of mammary gland secretions is a sensitive and specific predictor of parturition in mares

Answer 23

• Conductivity of mammary gland secretions (MGS) was the most sensitive and specific single marker for predicting parturition within 24 hours.
• Coupling conductivity with pH significantly increased the odds ratio for accurate prediction (from 25.4 to 62.3).

Physiological Basis:
• Decline in conductivity reflects changes in electrolyte concentrations (e.g., reductions in sodium and increases in potassium and calcium) during the final stages of gestation.
• Conductivity showed a strong correlation with pH (r = 0.88), supporting their combined use for enhanced predictive power.

Impending parturition was associated with a reduction in conductivity and pH (p < 0.05),
• decrease conductivity of ≤4.8 mS/cm,
• decrease pH ≤6.4 units,
• Increased Brix index ≥23.6%

Sensitivity, specificity, PPV, and NPV for parturition in ≤24 h for:
• conductivity (82%, 91%, 77%, and 92%)
• pH (79%, 84%, 81%, and 71%),
• Brix (72%, 79%, 66%, and 83%) .
• combining conductivity and pH= (80%, 95%, 90%, and 88%)

Question 24

Effects of repeated embryo flushing without PGF2α administration on luteal function, percentage of unwanted pregnancies, and subsequent fertility in mares

Answer 24

Luteal Function:
• Embryo flushing (EF) caused a subtle but significant reduction in progesterone concentrations compared to non-flushed cycles.
• The degree of progesterone reduction was greatest in cycles flushed on Day 9 post-ovulation, potentially due to increased sensitivity of the corpus luteum (CL) to luteolytic stimuli at later stages.
• Full luteolysis was rare (9.3%), mostly associated with uterine inflammation or bacterial contamination during EF.
Unwanted Pregnancies:
• The risk of unwanted pregnancy in negative EF cycles was 16.7%, and 18.5% when fewer embryos were recovered than the number of CLs.
• These outcomes were concentrated in a single mare, suggesting individual variation or procedural factors like incomplete flushing or suboptimal uterine massage.
Subsequent Fertility:
• Repeated EF without administering PGF2α did not negatively impact fertility over seven cycles.

Physiological Insights
Cervical Manipulation and Uterine Inflammation:
• EF-induced progesterone reductions could stem from oxytocin or low-level prostaglandin release during cervical dilation.
• Sub-luteolytic prostaglandin doses might contribute to partial luteolysis without affecting dioestrus.
Day of EF:
• Flushing later in dioestrus (Day 9) increased the likelihood of progesterone reduction, possibly due to CL age and responsiveness to inflammatory signals.

Question 25

Endometrial expression of antimicrobial peptides as markers of subclinical endometritis in mares

Answer 25

• The study identified increased expression of two AMPs, DEFB4B (beta-defensin 4B) and LYZ (lysozyme), in mares with subclinical endometritis (EM) compared to reproductively normal mares (NM).
• SLPI (secretory leukocyte peptidase inhibitor) showed no significant difference in expression between groups, suggesting a more limited role in detecting subclinical endometritis.
• LYZ expression correlated moderately and positively with both mare age and endometrial biopsy grade (Kenney and Doig classification), supporting its use as a marker of endometrial ageing or pathology.

Mechanistic Insights
DEFB4B:
• Part of the beta-defensin family, which contributes to antimicrobial defense by permeabilizing bacterial and fungal cell membranes.
• Also acts as a regulatory molecule, attracting leukocytes to infection sites, supporting its increased expression in subclinical inflammation.
LYZ:
• An enzyme with bactericidal activity, found in uterine secretions and other body fluids.
SLPI:
• A protease inhibitor involved in protecting epithelial tissues and regulating inflammatory responses.

Question 26

Selecting potential biomarkers of plasma proteins in mares with acute endometritis

Answer 26

Identification of Plasma Biomarkers:

Six proteins were identified as potential biomarkers for diagnosing equine endometritis:
• Fetuin B (FETUB): Best predictive value with an AUC of 0.94. Downregulation linked to impaired mucosal barriers and susceptibility to bacterial infections.
• Von Willebrand Factor (vWF): Significant elevation indicative of inflammatory and vascular changes. Acts as a positive APP. Promotes vascular inflammation, cell adhesion, and immune cell recruitment,
• Protein C (PROC): Downregulation associated with coagulation overactivity and prolonged inflammation. Potentially reflects systemic impacts of chronic endometrial inflammation on coagulation pathways.
• Insulin-like Growth Factor Binding Protein 3 (IGFBP-3): Lower levels associated with disrupted metabolic and cellular processes. Plays a role in cellular survival, proliferation, and differentiation. Reduction may indicate metabolic dysregulation and chronic inflammatory effects.
• Interleukin 1 Receptor Accessory Protein (IL1RAP): Decreased levels, indicative of immune dysfunction and chronic inflammation. Central to IL-1-mediated inflammatory signaling. dysregulation.
• Type II Keratin (KRT2): Elevated, reflecting endometrial damage and inflammation. Reflects cellular damage and structural changes in the endometrium, aligning with severe inflammation and tissue remodeling.

Combined Biomarker Effectiveness:
• Using all six proteins together yielded an AUC of 1.0, demonstrating perfect discrimination between healthy and diseased mares.

The identified proteins reflect key processes in endometritis:
• Immune regulation.
• Coagulation and vascular health.
• Cellular damage and repair.

Diagnostic Potential:
• These biomarkers provide a non-invasive, blood-based diagnostic tool for detecting equine endometritis.
• Particularly beneficial for identifying subclinical cases that lack overt symptoms.

Question 27

Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos

Answer 27

• Transfer of frozen-thawed in vitro-produced (IVP) embryos resulted in a 1.6% incidence of monozygotic multiple pregnancies (4/254).
• No multiple pregnancies were observed after transfer of in vivo-derived embryos (0/413), consistent with historical data suggesting natural monozygotic twinning is rare in horses.
• All multiple pregnancies in this study (twins or triplets) resulted in pregnancy loss:
• Single embryonic vesicles detected at early ultrasonographic examinations do not exclude monozygotic multiples.
• Follow-up scans after the appearance of the embryo proper are essential to identify multiples.

Question 28

Application of droplet digital PCR in diagnosing of X monosomy in mares

Answer 28

• Prevalence: X monosomy is the most frequent chromosomal abnormality linked to disorders of sex development in mares.
• Affects approximately 3% of infertile mares in cytogenetic studies.
• Leads to infertility, irregular or absent estrous cycles, and underdeveloped gonads.
• Unlike humans (e.g., Turner Syndrome), where X monosomy often results in mosaic karyotypes, horses frequently exhibit nonmosaic monosomy (63,X).

Utility of Droplet Digital PCR (ddPCR)
• Cost-effective: ddPCR costs approximately 10% of traditional karyotyping.
• Practicality: Requires minimal sample handling and is compatible with biological samples like saliva and hair follicles, unlike cytogenetics, which needs fresh blood samples delivered within 24 hours.
• Speed: ddPCR provides faster results, facilitating timely diagnosis.

Applications:
• Effective in detecting nonmosaic X monosomy, as demonstrated in an infertile mare.
• Reliable for population screening, as shown in 70 mares where two cases of X monosomy were detected.
Limitations:
• ddPCR cannot unequivocally detect mosaicism (e.g., 63,X/64,XX or 63,X/65,XXX), necessitating cytogenetic follow-up for ambiguous cases.
• Less effective for diagnosing complex chromosomal anomalies involving multiple cell lines.

Question 29

hCG is more effective than the GnRH agonist buserelin for inducing the first ovulation of the breeding season in mares

Answer 29

When given when a DF >35mm present following sulipride administration from the time 2 follicles 25mm.
hCG was significantly more effective than GnRHa in inducing the first ovulation of the breeding season:
• hCG: 90.1% success rate.
• GnRHa: 38.0% success rate.
• This difference is attributed to hCG’s direct LH-like action, bypassing the requirement for endogenous LH release, which is limited during the transitional phase in mares.

Ovulation induction was more successful in the late transitional phase compared to the early transitional phase:
• Late transition (Phase 2): 80.0% ovulation rate.
• Early transition (Phase 1): 53.1% ovulation rate.
• The improved response during the late phase reflects the higher pituitary LH content and greater follicular steroidogenic activity as mares approach natural ovulation.

Mares showing both uterine edema (grades 2 or 3) and teaser stallion acceptance for at least three consecutive days were more likely to ovulate:
• Full heat: 78.0% ovulation rate.
• Incomplete heat: 42.9% ovulation rate.
• Estrous behavior and uterine edema are indicative of sufficient estrogen levels and follicular readiness for ovulation.

Mechanistic Insights
• hCG Mechanism: Directly mimics LH, binding to luteinizing hormone receptors on the follicle and inducing ovulation without relying on pituitary LH stores.
• GnRHa Mechanism: Requires functional pituitary LH release, which is limited in the early transition due to low hypothalamic GnRH stimulation and basal pituitary LH stores.
• GnRHa effectiveness improves in cyclic mares or mares treated later in the transitional phase when LH availability increases.

Practical Implications
• hCG is the agent of choice for inducing ovulation in transitional mares, especially during early phases when pituitary LH reserves are low.
• Behavioral observations and ultrasound findings are critical in identifying suitable candidates for ovulation induction, minimizing failures.

Question 30

Fetal morphological features and abnormalities associated with equine early pregnancy loss

Answer 30

the majority of pregnancy failure occurs between the initial diagnosis and day 65 of gestation and is commonly referred to as ‘early pregnancy loss’ (EPL)

EPL embryos/fetuses often exhibited:
• A mismatch between embryonic/fetal size (CRL) and gestational age.
• Neural tissue abnormalities, including:
-Failed neural tube closure (specific but rare).
-Disruption in the prosencephalon, mesencephalon, and rhombencephalon.
-Presence of haemosiderin, indicating pre-mortem hemorrhage.
• Subcutaneous hemorrhage (“red patching”) localized in the neck, shoulder, and thoracic regions.

Intrauterine Growth Retardation (IUGR):
• Found in 3/8 embryos/fetuses with CRL measurements.
• Affected specimens had significantly smaller sizes relative to developmental age, suggesting impaired growth.
• Gestational and developmental ages were inconsistent in some EPL samples, likely due to uterine retention.
CNS Abnormalities:
• CNS anomalies were common in EPL cases, contrasting with normal pregnancies:
• Observed macroscopically in 10/13 EPL embryos (compared to 2/11 controls).
• Histological changes included extracellular edema, cellular disruption, and hemorrhage.

Mechanistic Insights
• Impaired endometrial function and histotroph production might contribute to IUGR and EPL, aligning with prior studies linking endometrial pathology to pregnancy loss.
• The identified case of failed neural tube closure represents a specific abnormality, likely linked to developmental disruptions or genetic factors.

Question 31

Incidence and causes of pregnancy loss after Day 70 of gestation in Thoroughbreds

Answer 31

• Overall, 7.3% of Day 70 pregnancies failed to produce a live foal at 24 hours post-parturition.
• Loss incidence between Day 70–300 was 4.0%, between Day 301–315 was 0.3%, and during stillbirth or perinatal death was 1.4%.

Umbilical Cord-Related Pathologies:
• The most frequently diagnosed cause, accounting for 44.8% of examined cases and 1.5% of all Day 70 pregnancies.
• Includes umbilical cord torsion (UCT), excessive length, and entanglement, consistent with prior studies in the UK.
Infectious Placentitis:
• A minor contributor in this cohort, with an incidence risk of 0.3%, notably lower than international reports, where placentitis is more common.
Other Diagnosed Causes:
• Noninfectious placental disease: 0.4% incidence.
• Fetal-origin pathologies: 0.2%, including cases like carpal contracture and scoliosis.
• Equine herpesvirus (EHV): Rare, with yearly variations and an overall 0.3% incidence risk.
• Unexplained Losses: 11.2% of cases lacked a primary diagnosis, often due to incomplete submissions, autolysis, or insufficient pathological evidence.

Question 32

The clinical features and short-term treatment outcomes of scirrhous cord: A retrospective study of 32 cases

Answer 32

• Common clinical signs: unilateral scrotal swelling, thickened cord, draining tract, pyrexia, and sometimes lameness.
• Ultrasound is a valuable diagnostic tool for identifying cord abnormalities, such as enlargement and abscesses.
• Microbial Involvement: Contrary to previous beliefs, Staphylococcus species were rarely isolated; other bacteria like Streptococcus spp. and gram-negative organisms were more common.
-Suggests antimicrobial therapy should be guided by culture and sensitivity testing.
• Surgical excision of infected tissue was effective, with all 32 horses surviving to discharge.
• Variability in surgical and antimicrobial protocols reflects the retrospective nature of the study but highlights the need for individualized treatment based on culture results.

Clinical and Procedural Insights
• SC may develop more frequently in older horses, possibly due to increased tissue size and vascularity, though data is limited.
• Right-sided scirrhous cords were more commonly affected, an observation requiring further study.

Question 33

Perioperative pain management protocols of veterinarians in the United States for horses undergoing routine orchiectomy (castration)

Answer 33

• Over 70% of surveyed veterinarians in the US routinely administered NSAIDs at the time of castration and recommended post-operative use for 1–7 days (median: 3 days).
• Veterinarians who believed horses experienced higher pain levels post-castration were more likely to recommend NSAID use.
• More recent graduates (post-2000) and those working in multi-veterinarian practices were significantly more likely to recommend NSAID analgesia.

• Pain ratings for castration varied widely among veterinarians (median scores ranged from 4–6), reflecting a lack of consensus.
• Studies consistently show behavioral evidence of pain for over 8 hours post-castration, suggesting that relying solely on short-term anesthetics (e.g., xylazine, butorphanol) is insufficient.
• NSAID usage rates in this study (~73%) are higher than reported in studies from the UK (~37% in 2005) and Australia (~43% in 2018), possibly reflecting changing practices or geographic differences.
• BEVA guidelines (2020) recommend NSAID use before and for at least 3 days post-castration, but no similar guidelines exist in the US, highlighting a gap in standardization.
• A minority of veterinarians (6.8%) provided no analgesia beyond anesthesia at surgery, with some acknowledging significant post-operative pain yet offering limited management.
• Evidence-based pain management guidelines for equine castration are needed in the US to address welfare concerns and standardize care.

Question 34

Early castration in foals: Consequences on physical and behavioural development

Answer 34

• Both groups underwent castration using an open technique with primary closure under general anesthesia.
• Early castration (EC) at 3 days of age was associated with smoother procedures and fewer perceived difficulties compared to traditional castration (TC) at 18 months, according to the surgeons.
• No significant perioperative complications
• The EC group demonstrated quick recovery, with no foals exhibiting behavioral issues or rejection by their dam.
• No significant differences in temperament traits during testing at 1 and 3 years of age.
• Morphometric data, including weight and body dimensions, were similar across groups from birth to 40 months, except for a minor interaction in hip width.

Benefits and Limitations
• Early castration reduced the risk of complications associated with later surgeries, such as adhesions and eventration, and was technically easier due to smaller tissue size.
• The study used a small, homogenous population (22 Welsh ponies).
• Long-term impacts on parameters like bone development were not assessed, necessitating further studies.

Question 35

Characterisation of the testicular transcriptome in stallions with age-related testicular degeneration

Answer 35

Preliminary study- pretty flawed

• Upregulation of inflammatory pathways and tissue remodeling genes highlights the potential role of chronic inflammation in age-related testicular degeneration.

• Chronic, low-grade inflammation (e.g., cytokine-mediated pathways) was identified as a significant factor.
• Inflammation-related genes (e.g., NFKB, CSF1) linked to apoptosis, fibrosis, and tissue remodeling
• Genes promoting cell survival and inhibiting apoptosis were upregulated, indicating a compensatory mechanism against cell loss.
• Aberrant expression of stem cell niche factors like BMP4 and CSF1 suggests a disrupted balance in stem cell self-renewal and differentiation.

Histology: Abnormal spermatogenesis, characterised by loss of the normal architecture and vacuolisation of the seminiferous epithelium, absence of late-stage germ cells or Sertoli cells only within the seminiferous tubules, loss of the normal associations between germ
cell stages, and shedding of immature spermatogenic cells into the seminiferous tubule lumen, was present in at least 50% of the observed tubules from several different sections of the testes

• Principal component analysis (PCA) showed separation between and clustering within the three fertile testis samples and three of the four testicular degeneration samples components accounting for 71.9% of the variance among the data. The
fourth degenerate testis sample (Degen_Testis_83) was distinct from both the normal and degenerate clusters. This sample was obtained from the oldest horse in the data set (the 30-year-old Appaloosa) and represented end stage disease.

• Upregulated: inflammation and chemokinesis, tissue and collagen remodelling, cellular differentiation and mitogenesis and germ cell maturation
• Downregulated: encoded proteins that regulate cellular growth, development, self-renewal and/or differentiation. Of these, one gene (SHISA6) is reported to stimulate spermatogonial stem cell (SSC) differentiation, two are involved in cell–cell adhesion, two regulate apoptosis/cell death and two are involved in metabolism

• The most significantly altered canonical pathway in age-related testicular degeneration relates to tissue fibrosis, extracellular matrix deposition and collagen formation. This pathway is predicted to be highly upregulated in the diseased tissue compared with normal adult tissue .
• Other highly upregulated pathways included interferon signalling and complement signalling pathways (both P < 1E 4), acute phase response signalling (P < 0.001) and cytokine production (P < 0.05).
• Pathways involved in apoptosis, cell death and cell survival were also significantly differentially regulated in normal versus diseased tissue

Question 36

Vaccination against GnRH as a prelude to surgical castration of horses

Answer 36

Reduction in Testicular Dimensions:
• The administration of the anti-GnRH vaccine Improvac® led to a 50% reduction in testicular volume by Day 57 and a further reduction to ~33% of baseline in colts castrated on Day 100.
• Reduction in testicular size positively correlated with decreased post-surgical complications, including scrotal and preputial swelling.
Testosterone Suppression:
• Serum testosterone concentrations (STC) decreased to undetectable levels by Day 28 following vaccination.
• STC remained suppressed until castration in both vaccinated groups (Day 57 and Day 100), demonstrating effective endocrine downregulation.
Post-Surgical Complications:
• Vaccinated colts experienced significantly less scrotal swelling compared to controls.
• Pre-treatment with GnRH vaccination likely mitigates inflammatory responses associated with larger testicular volumes and adnexa.
Safety and Immune Response:
• Improvac® was well-tolerated with no adverse reactions at the injection site or systemic side effects.
• Antibody responses were robust, confirming effective immunogenicity.

Question 37

Preservation of viable Taylorella equigenitalis in different commercially available transport systems

Answer 37

• Among 11 tested transport systems, System K demonstrated superior preservation of Taylorella equigenitalis viability after 10 days of storage at refrigerated temperatures:
• System K retained 95% viability, outperforming the reference Amies charcoal system (System F), which retained 77% viability.

Temperature Effects:
• Storage at ambient temperatures (20°C) significantly reduced bacterial viability compared to refrigerated conditions (5°C):
• Refrigerated conditions preserved higher viability across all systems

PCR Results:
• While bacterial viability declined over time in most systems, quantitative PCR detection of T. equigenitalis DNA showed consistent recovery, indicating that DNA integrity is less affected by storage conditions.
• PCR remains effective for identifying T. equigenitalis DNA in all tested systems, offering flexibility in transport medium choice when culture is not the primary diagnostic method.

• System K’s composition (amphotericin B, vancomycin, colistin) inhibits bacterial and fungal overgrowth without compromising T. equigenitalis viability, making it a promising option for extended transport. Its performance suggests potential for clinical use, especially in scenarios requiring longer transport times.

Question 38

A simplified fixed-time insemination protocol using frozen-thawed stallion spermatozoa stored at 17°C for up to 24h before insemination

Answer 38

• The stallion significantly influenced sperm motility, but there was no effect of media on motility parameters.
• Storage time significantly affected sperm motility after centrifugation but not after microfluidic sorting.
• Storage time had no effect on the overall embryo recovery rate (52%, n = 42).

Microfluidic sperm selection improved motility parameters (total, progressive, and rapid motility) compared to unprocessed samples.
• Sperm motility remained above 55% for 24 hours post-thaw when processed through the microfluidic device and stored in SpermSafe™, a protein-free medium.
• This approach reduced the frequency of veterinary monitoring for ovulation, requiring only one ultrasound examination prior to insemination.
• Low-dose deep uterine insemination using the selected sperm population resulted in satisfactory fertilization outcomes.

Advantages of the Protocol
• Reduced Management Requirements:
-Fewer ultrasound scans (once daily instead of every 6 hours) lower the cost and labor associated with frozen–thawed semen insemination.
-Fixed-time insemination eliminates the need for precise timing of insemination relative to ovulation, simplifying logistics.
• Improved Logistics
• Minimized Post-Breeding Inflammation:
-The study observed a low incidence of intrauterine fluid accumulation, which typically requires treatment in conventional protocols.

Question 39

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions

Answer 39

• Sperm processed by SpermFilter® and resuspended in egg yolk-based (EY) or skimmed milk-based with pentoxifylline (SM-P) extenders demonstrated superior post-cooling motility, membrane integrity (PMI), and mitochondrial potential (HMMP) compared to unprocessed samples.
• EY extender yielded the best results for both motility and HMMP, outperforming SM-P and unprocessed semen across “bad-cooler” stallions.

• SpermFilter® caused less mechanical damage to sperm than centrifugation, leading to higher PMI and better overall sperm quality.
• Centrifugation was associated with reduced membrane integrity and increased susceptibility to damage.

Effect of Supernatant Removal:
• Removing metabolic waste and aged extender from cooled semen reduced oxidative stress and rejuvenated sperm by replenishing nutrients, thus improving longevity and functionality.
Role of Extenders:
• EY extender: Chemically complex, enhances mitochondrial activity and motility through stabilization of membrane potential.
• SM-P extender: Pentoxifylline acts as a methylxanthine phosphodiesterase inhibitor, stimulating motility via increased cyclic adenosine monophosphate (cAMP) concentrations.

Question 40

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare

Answer 40

• Performing TUA earlier in gestation (Days 25–34) resulted in significantly higher foaling rates compared to later gestational stages.
• Foaling rates were;
-64.7% for Days 25–29,
-47.4% for Days 30–34,
- 39.7% for Days 35–39.
-Beyond Day 40, foaling rates dropped below 30%, emphasizing the importance of early intervention.
• Gestational age had a greater effect on foaling rates in unilaterally fixed twins compared to bilaterally fixed twins.
• Bilaterally fixed twins showed slightly higher chances of achieving singleton pregnancies after TUA, though foaling rates were similar across both groups.
• Single puncture procedures resulted in higher pregnancy rates compared to multiple punctures.
• Aspiration of large fluid volumes negatively affected foaling outcomes, highlighting the need to minimize fluid removal.
• Puncturing the embryo or fetal structures during TUA was associated with a lower foaling rate (21.1% compared to 40.9% when punctures were avoided).

Medication and Operator Experience:
• Increased doses of butorphanol during sedation improved procedural outcomes by minimizing movement and visceral pain.
• Use of butylscopolamine (Buscopan®) reduced pregnancy rates, likely due to excessive uterine relaxation.
• Operator experience positively influenced initial pregnancy rates (5–7 days post-TUA) but not final foaling rates.

Practical Recommendations
• The best outcomes were achieved when TUA was performed between Days 28–32, balancing the risk of natural reduction in unilateral twins and procedural success rates.
• Limit punctures and fluid aspiration volumes during the procedure.
• Avoid direct puncture of embryos or fetuses to improve singleton pregnancy outcomes.
• Use moderate doses of detomidine and butorphanol for sedation while avoiding butylscopolamine to ensure uterine contractility and minimize procedural risks.
•Prompt treatment before Day 40 is advised to maximize success rates. After Day 40, alternative twin reduction methods (e.g., thoracic compression) may be required.

Question 41

In vitro synergy of eugenol on the antifungal effects of voriconazole against Candida tropicalis and Candida krusei strains isolated from the genital tract of mares

Answer 41

In vitro study only

• The combination of eugenol and voriconazole demonstrated significant synergistic effects against genital Candida isolates:
• 83.3% of C. tropicalis isolates and 77.7% of C. krusei isolates exhibited synergistic interactions.
• Synergy was measured using the fractional inhibitory concentration index (FICI),
• Eugenol substantially reduced the minimum inhibitory concentration (MIC) of voriconazole:
• These reductions suggest enhanced effectiveness of voriconazole when combined with eugenol, even against resistant strains.

• Eugenol exhibited fungistatic and fungicidal properties independently
• Its mechanism of action likely involves disruption of fungal cell membrane integrity and inhibition of ATPase activity.
• Eugenol maintained synergistic effects even in voriconazole-resistant strains, highlighting its potential to overcome drug resistance.