Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Environmental constraints and pathologies that modulate equine placental genes and development.

Equine placental development is a long process with unique features. Implantation occurs around 40 days of gestation (dpo) with the presence of a transient invasive placenta from 25-35 to 100-120 dpo. The definitive, non-invasive placenta remains until term (330 days). This definitive placenta is diffuse and epitheliochorial, exchanging nutrients, gas and waste with the endometrium through microvilli, called microcotyledons. These are lined by an external layer of haemotrophic trophoblast. Moreover, histotrophic exchange remains active through the histotrophic trophoblast located along the areolae. Placental development is dependent on the maternal environment that can be affected by several factors (e.g. nutrition, metabolism, age, embryo technologies, pathologies) that may affect fetal development as well as long-term offspring health. The first section of the review focuses on normal placental development as well as definitive placental structure. Differences between the various regions of the placenta are also highlighted. The latter sections provide an overview of the effects of the maternal environment and reproductive pathologies, respectively, on trophoblast/placental gene expression and structure. So far, only pre-implantation and late gestation/term data are available, which demonstrate important placental plasticity in response to environmental variation, with genes involved in oxidative stress and tissue differentiation mostly involved in the pre-implantation period, whereas genes involved in feto-placental growth and nutrient transfers are mostly perturbed at term.

Lethal variants of equine pregnancy: is it the placenta or foetus leading the conceptus in the wrong direction?

Embryonic and foetal loss remain one of the greatest challenges in equine reproductive health with 5-10% of established day 15 pregnancies and a further 5-10% of day 70 pregnancies failing to produce a viable foal. The underlying reason for these losses is variable but ultimately most cases will be attributed to pathologies of the environment of the developing embryo and later foetus, or a defect intrinsic to the embryo itself that leads to lethality at any stage of gestation right up to birth. Historically, much research has focused on the maternal endometrium, endocrine and immune responses in pregnancy and pregnancy loss, as well as infectious agents such as pathogens, and until recently very little was known about the both small and large genetic variants associated with reduced foetal viability in the horse. In this review, we first introduce key aspects of equine placental and foetal development. We then discuss incidence, risk factors and causes of pregnancy loss, with the latter focusing on genetic variants described to date that can impact equine foetal viability.

Preliminary testing of psychometric properties of the 'student perceptions of veterinary interprofessional education and work scale' (SP-VIEWS).

Responsibility for the provision of veterinary care and services is increasingly shared between veterinary surgeons/veterinarians and registered veterinary nurses/veterinary technicians. Interprofessional education of these clinical professionals is not widespread but is growing. Understanding students' perceptions of veterinary interprofessional education and working is therefore important; however, no validated scale exists to assess this. This study aimed to create and test the psychometric properties of a 'Student perceptions of veterinary interprofessional education and work scale' (SP-VIEWS). A scale was built using scales previously validated in other contexts, plus statements informed by veterinary interprofessional research, and sent to veterinary and veterinary nursing students at six UK institutions. Exploratory Factor Analysis (EFA) on a randomly-selected half of the responses (n = 260) suggested a model with 16 items grouped within three factors: 'Benefits of learning with the other profession', 'Leadership and speaking up' and 'Teams and benefits of teamwork'. Confirmatory Factor Analysis (CFA) on the remaining 260 responses demonstrated appropriate fit based on conventional parameters, such as goodness of fit index. Overall internal consistency was good (Cronbach's alpha 0.82). CFA demonstrated that SP-VIEWS showed adequate, though not excellent, fit to the data. Future research should evaluate SP-VIEWS in other universities and countries.

Dynamic changes in gene expression and signalling during trophoblast development in the horse

Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, 30, 31 and 34 (n = 5), corresponding to the initiation of chorionic girdle trophoblast proliferation, differentiation and migration. Data were analysed using R packages limma and maSigPro, Ingenuity Pathway Analysis and DAVID and verified using qRT-PCR, promoter analysis, western blotting and migration assays. Microarray analysis showed gene expression (absolute log FC >2, FDR-adjusted P < 0.05) was rapidly and specifically induced in the chorionic girdle between days 27 and 34 (compared to day 27, day 30 = 116, day 31 = 317, day 34 = 781 genes). Pathway analysis identified 35 pathways modulated during chorionic girdle development (e.g. FGF, integrin, Rho GTPases, MAPK) including pathways that have limited description in mammalian trophoblast (e.g. IL-9, CD40 and CD28 signalling). Rho A and ERK/MAPK activity was confirmed as was a role for transcription factor ELF5 in regulation of the CGB promoter. The purity and accessibility of chorionic girdle trophoblast proved to be a powerful resource to identify candidate genes and pathways involved in early equine placental development.

Convergent evolution of pregnancy-specific glycoproteins in human and horse.

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions.

Prevalence of Microbial Isolates Cultured from Endometrial Swab Samples Collected from United Kingdom Thoroughbred Mares from 2014 to 2020.

Determining whether endometrial microbial isolates are pathogens, contaminants, or even part of the "normal" microbiome is extremely complex, particularly given the absence of "gold standard" tests for endometritis. Population-level benchmarking and temporal monitoring can provide novel insights and a wider context to improve understanding. This study aimed to (i) estimate the prevalence of endometrial isolates from swabs of Thoroughbred broodmares in Newmarket, UK between 2014 and 2020; and (ii) evaluate the effects of year, mare age, and cytology findings on isolate prevalence. Generalised linear mixed models with a logit link, both null models and models using year of sampling, mare age, or cytology findings as predictors, were fitted to estimate isolate prevalence. Over the 7-year period, data were available from 18,996 endometrial-swab samples from 6050 mares on 290 premises. The overall isolate prevalence was 35.5% (95% confidence interval (CI) 33.0-37.9), and this varied significantly between years. The most prevalent isolates were ß-hemolytic Streptococcus (17.9; 95% CI: 17-19) and E. coli (10.3%; 95% CI: 9.0-11.6). Isolate prevalence increased with mare age except for E. coli isolates, and with increasing category of cytology findings except for α-hemolytic Streptococcus isolates. The results provide novel estimates of isolate prevalence and highlight knowledge gaps around potential complexities in the interpretation of findings.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses.

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15-1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15-1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

Does inbreeding contribute to pregnancy loss in Thoroughbred horses?

BACKGROUND: Excessive inbreeding increases the probability of uncovering homozygous recessive genotypes and has been associated with an increased risk of retained placenta and lower semen quality. No genomic analysis has investigated the association between inbreeding levels and pregnancy loss. OBJECTIVES: To compare genetic inbreeding coefficients (F) of naturally occurring Thoroughbred Early Pregnancy Loss (EPLs), Mid and Late term Pregnancy Loss (MLPL) and Controls. The F value was hypothesised to be higher in cases of pregnancy loss (EPLs and MLPLs) than Controls. STUDY DESIGN: Observational case-control study. METHODS: Allantochorion and fetal DNA from EPL (n = 37, gestation age 14-65 days), MLPL (n = 94, gestational age 70 days-24 h post parturition) and Controls (n = 58) were genotyped on the Axiom Equine 670K SNP Genotyping Array. Inbreeding coefficients using Runs of Homozygosity (FROH) were calculated using PLINK software. ROHs were split into size categories to investigate the recency of inbreeding. RESULTS: MLPLs had significantly higher median number of ROH (188 interquartile range [IQR], 180.8-197.3), length of ROH (3.10, IQR 2.93-3.33), and total number of ROH (590.8, IQR 537.3-632.3), and FROH (0.26, IQR 0.24-0.28) when compared with the Controls and the EPLs (p < 0.05). There was no significant difference in any of the inbreeding indices between the EPLs and Controls. The MLPLs had a significantly higher proportion of long (>10 Mb) ROH (2.5%, IQR 1.6-3.6) than the Controls (1.7%, IQR 0.6-2.5), p = 0.001. No unique ROHs were found in the EPL or MLPL populations. MAIN LIMITATIONS: SNP-array data does not allow analysis of every base in the sequence. CONCLUSIONS: This first study of the effect of genomic inbreeding levels on pregnancy loss showed that inbreeding is a contributor to MLPL, but not EPL in the UK Thoroughbred population. Mating choices remain critical, because inbreeding may predispose to MLPL by increasing the risk of homozygosity for specific lethal allele(s).

The equine umbilical cord in clinically healthy pregnancies.

BACKGROUND: Excessive umbilical cord length (UCL) is associated with equine pregnancy loss. However, a lack of UCL reference values makes it difficult to define excessive UCL. Further, factors associated with differences in UCL are poorly understood. OBJECTIVES: To (i) report the total, allantoic and amniotic UCL in healthy term pregnancies in Thoroughbreds, (ii) describe the relationship between gestational age and UCL, fetal weight and crown rump length (CRL) using clinically normal pregnancies (CNPs) from mares dying during gestation, and (iii) identify associations between UCL and maternal age and parity, paternal age, and fetal sex. STUDY DESIGN: Cross-sectional. METHODS: Data including UCLs, fetal weight, CRL and maternal age, parity, paternal age and fetal sex were taken from CNPs from Thoroughbred mares dying during gestation (n = 32), and placentas from HTPs (n = 34) in England. Correlations were assessed using Spearman's rank with significant correlations estimated by locally weighted scatter plot smoothing (LOWESS). Regression plots were fitted to highly correlated variables to further assess and quantify relationships. Differences in UCL between categorical variables were assessed using Kruskall Wallis and Mann-Whitney U tests. RESULTS: The median total, amniotic and allantoic HTP UCLs were 53.5 cm (interquartile range [IQR] 16), 29.5 cm (IQR 7) and 25.0 cm (IQR 8) respectively. Gestational age and amniotic UCL were moderately correlated (rho = 0.53, p = 0.04), with LOWESS estimating an exponential increase followed by plateauing at around Day 200. Nonlinear associations were observed between fetal weight and gestational age and CRL (adjusted r2 = 0.98 and 0.95 respectively). A linear association was observed between gestational age and CRL: predicted CRL = -17.60 + 0.38 × gestational age, p < 0.001. MAIN LIMITATIONS: Limited availability of CNPs from mares dying during gestation. Estimated relationships can only approximate growth. CONCLUSIONS: This study provides important UCL and fetal size reference values, which may aid in assessing abnormalities. For the first time, associations between UCL and gestational age have been described.

Gestation Length is Associated With Early-Life Limb Deformities in Thoroughbred Foals.

Flexural and angular limb deformities (LD) are an important cause of early-life morbidity and mortality/euthanasia in Thoroughbred foals. The majority are congenital in origin but, to date, their precise aetiology is poorly understood. We hypothesized that maternal- and pregnancy-level factors, particularly those with potential to influence in-utero growth and development, could play an important role. The aim of this study was therefore to investigate associations between such factors and early-life LD in Thoroughbred foals. A birth cohort was established on seven farms across the United Kingdom and Ireland and details of veterinary interventions for LD in foals in the first six months of life prospectively recorded. Details of dams' signalment, breeding history and reproductive and veterinary history in the breeding season(s) of interest were retrieved retrospectively from stud farm and veterinary records. Associations between mare- and pregnancy-level factors and LD in offspring were assessed using multivariable logistic regression. Records were available for 275 pregnancies in 235 mares, over two breeding seasons. Pregnancies resulted in the birth of 272 live foals, 21% of which (n = 57/272, 95% CI, 16-26) required veterinary intervention for LD in the first six months of life. Odds of LD decreased by 4% per day increase in gestation length between 314 and 381 days (OR 0.96, 95% CI, 0.93-0.99, P = .01). Longer gestation length appears to reduce the odds of early-life LD, including within the normal range of gestation length for Thoroughbred foals. Further work is required to elucidate biological mechanisms behind this association.

Antimicrobial resistance of endometrial bacterial isolates collected from UK Thoroughbred mares between 2014 and 2020.

BACKGROUND: Antimicrobial resistance (AMR) is important in equine reproduction, as antimicrobials have historically been widely used in the management of breeding mares. However, evidence of the characteristics of AMR in uterine isolates is limited in the UK. The objective of this retrospective study was therefore to describe temporal changes in AMR patterns of bacteria isolated from the endometrium of Thoroughbred broodmares in south-east England between 2014 and 2020. METHOD: Endometrial swabs were processed for microbiology and antimicrobial susceptibility testing (AST). For frequently isolated bacteria, changes in AMR patterns over time were assessed using a logistic regression model. RESULTS: From 18,996 endometrial swabs, 30.5% were positive for microbial culture. AST was performed on 2091 isolates, representing 1924 swabs collected from 1370 mares located at 132 premises. Beta-haemolytic Streptococcus (BHS, 52.5%) and Escherichia coli (25.8%) were most frequently isolated. In BHS, resistance to enrofloxacin (p = 0.02), nitrofurazone (p < 0.001) and oxytetracycline (p < 0.01) increased significantly between 2014 and 2020, while resistance to trimethoprim-sulfamethoxazole (p < 0.001) decreased. In E. coli, resistance to nitrofurazone increased (p = 0.04) and resistance to gentamycin (p = 0.02) and trimethoprim-sulfamethoxazole (p < 0.001) decreased. LIMITATIONS: Variations in the specimen collection protocols might have affected the frequency of isolates detected. CONCLUSION: Between 2014 and 2020, AMR changed in this bacterial population. However, there was no significant increase in resistance to penicillin (99.6% BHS susceptible), gentamycin (81.7% E. coli susceptible) or ceftiofur.

Incidence of disease, injury and death in Thoroughbred foals and yearlings on stud farms in the UK and Ireland.

BACKGROUND: Up-to-date estimates of early-life morbidity and mortality in Thoroughbreds are lacking. METHODS: A birth cohort was established on Thoroughbred stud farms across the UK and Ireland. All veterinary interventions for disease or injury between birth and 18 months of age or leaving the study were recorded. Multilevel Poisson regression models with farm and foal as random effects were fitted to estimate incidence rates. RESULTS: Data were available for 3328 foal-months at risk for 275 foals on seven farms. The overall rates of disease and injury requiring veterinary intervention and mortality were 11.9 cases/100 foal-months at risk (95% confidence interval [CI] 8.6-16.2) and 0.2 cases/100 foal-months at risk (95% CI 0.1-0.4), respectively. Almost half (n = 133/273, 49%, 95% CI 43-55) of the live-born cohort required veterinary intervention for musculoskeletal disease or injury, equating to 5.8 cases/100 foal-months at risk (95% CI 4.1-8.2), predominantly reported as developmental orthopaedic disease (DOD). LIMITATIONS: Convenience sampling of participants may affect the generalisability of the findings. CONCLUSIONS: Rates of musculoskeletal disease and injury, in particular DOD, on Thoroughbred stud farms were high. Further work to identify modifiable risk factors and further understanding of the economic impact of these conditions and long-term consequences for musculoskeletal health and performance is required.

Mixed-Effects Modelling of the Risk Factors Associated with Multiple Pregnancies in Thoroughbred Mares.

Multiple pregnancies (MPs) are commonly diagnosed during breeding management of mares. Whilst some studies have reported on factors associated with the risk of MPs, few have utilised multivariable data analysis to control for confounding variables. A prospective cohort study of Thoroughbred broodmares was conducted with information collected on 27 factors. Mixed-effects logistic regression was used to determine risk factors for MPs. Mare, stallion, stud, and veterinarian were evaluated as random effects. The prevalence of MPs in 1754 mares and 2245 pregnancies was 16.06% (95% confidence interval [CI] = 14.54, 17.58). Multiple ovulations (OR = 15.57, 95% CI = 11.88, 20.53) and treatment with cloprostenol (OR = 1.35, 95% CI = 1.015, 1.80) were associated with increased odds of MPs following multivariable analysis. Mares that foaled at the start of the breeding season (OR = 0.66, 95% CI = 0.47, 0.94), conceived at the second or more oestrus cycles (OR = 0.60, 95% CI= 0.43, 0.84), or identified with a uterine cyst (OR = 0.63, 95% CI = 0.40, 0.97) were at reduced odds of conceiving MPs. Mare, stallion, stud, and veterinarian were not associated with MPs. These findings provide possible explanations as to why the prevalence of MPs but not MOs have increased over the last decade.

Multivariable analysis to determine risk factors associated with abortion in mares.

Risk factors associated with equine reproductive efficiency have been identified along with those associated specifically with early pregnancy loss (EPL). In contrast, no studies have reported risk factors associated with abortion (loss between day 70 and 300 post-cover). Given the causes of abortion differ to those of EPL, likely too will the risk factors. A retrospective cohort study was carried out to identify risk factors associated with abortion in UK and Irish based Thoroughbreds, collecting data on 20 exposure variables over a five-year period. A generalized linear mixed model was utilized to evaluate the associations between exposure variables and abortion, with clustering of observations accounted for at the mare and farm level. Variables with a likelihood ratio test (LRT) p value <0.2 were entered into the model in a forward stepwise approach. Pregnancy outcome was available on 4,439 pregnancies from 2,510 mares. Having had two or more prior abortions (odds ratio (OR) 7.91, 95% confidence interval (CI) 2.86, 21.88), conceiving on the second or subsequent covered estrous cycle (OR 1.84, 95% CI 1.22, 2.78) and conceiving multiple conceptuses (OR 1.68, 95% CI 1.02, 2.76) were associated with an increased risk of abortion compared to null parous, first estrous cycle covers and singleton conceptions respectively. Increasing paternal age (OR 0.95, 95% CI 0.90, 0.99) was associated with a decreasing risk of abortion. Mare and farm variance were not significant in the final model, LRT p=0.43. These findings provide evidence-based data to inform Thoroughbred breeding management practices to help mitigate abortion risk.

Date of birth and purchase price as foals or yearlings are associated with Thoroughbred flat race performance in the United Kingdom and Ireland.

Background: Thoroughbred breeders aim to have foals born early in the season, but scientific evidence on the advantages for race performance is scarce and contradictory. Methods: The association between date of birth and purchase price as foal/yearling, with race performance by the end of the second and third years of life of Thoroughbreds racing in flat races in the United Kingdom (UK) and Ireland (IRE) was assessed using negative binomial and zero-inflated negative binomial models on the entire 2014-2015 UK/IRE foal crops (n = 28,282). Results: In total, 6666 and 9456 horses raced in UK/IRE flat racing by the end of their second and third years of life. Prize money and prize money per start decreased with each additional day beyond 1 January that the foal was born. Purchase price as foal and yearling was negatively associated with the number of races run, while it was positively associated with prize money and prize money per start by the end of the third year of life. Conclusions: Foals born early in the season had higher earnings by the end of their second and third years of life than foals born later. Differences were more marked among males than females. The most expensive horses sold as foals or yearlings ran fewer races but earned more prize money and prize money per start than less expensive horses. Results from this population-based analyses may inform strategies and management practices aiming to maximise horses' racing performance potential and increase financial returns.

Bayesian accuracy estimates and fit for purpose thresholds of cytology and culture of endometrial swab samples for detecting endometritis in mares.

The overall aim of this work was to identify the potential impact of misclassification errors associated with routine screening and diagnostic testing for endometritis in mares. Using Bayesian latent class models (BLCM), specific objectives were to: 1) estimate the diagnostic accuracy of cytology and culture of endometrial swab samples to detect endometritis in mares; 2) assess the impact of different cytology thresholds on test accuracy and misclassification costs; and 3) assess the sensitivity (Se) and specificity (Sp) of a diagnostic strategy including both tests interpreted in series and parallel. Diagnostic and pre-breeding endometrial swab samples collected from 3448 mares based at breeding premises located in the South East of England between 2014 and 2020 were retrospectively analysed. Culture results were classified as positive according to three different case definitions: (A) > 90% of the growth colonies were a monoculture; (B) pathogenic or pathogenic and non-pathogenic bacteria were identified; and (C) any growth was observed. Endometrial smears were graded based on the percent of polymorphonuclear cells (PMN) per high power field (HPF). A hierarchical BLCM was fitted using the cross-tabulated results of the three culture case definitions with a cytology threshold fixed at > 0.5% PMN. Fit for purpose cytology thresholds were proposed using a misclassification cost analysis in the context of good antimicrobial stewardship and for varying endometritis prevalence estimates. Median [95% Bayesian credible intervals (BCI)] cytology Se estimates were 6.5% (2.2-11.6), 6.4% (2.2-10.8) and 6.3% (2.2-10.8) for scenario A, B and C, respectively. Median (95% BCI) cytology Sp estimates were 88.8% (83.1-94.8), 88.9% (83.9-93.8) and 88.8% (84.0-93.8) for scenarios A, B and C, respectively. Median (95% BCI) culture Se estimates were 37.5% (29.9-46.0), 42.3% (33.8-51.1) and 46.4% (35.7-55.9) for scenarios A, B and C, respectively. Median (95% BCI) culture Sp estimates were 92.8% (84.3-99.0), 91.5% (82.5-98.0) and 90.8% (80.1-97.4) for scenarios A, B and C, respectively. Regardless of the culture case definition, Se and Sp of cytology (> 0.5% PMN) was lower than previously reported for swab samples in studies using histology as the reference standard test. The misclassification cost term decreased as the cytology threshold increased for all scenarios and all prevalence contexts, suggesting that, regardless of the endometritis prevalence in the population, increasing the cytology threshold would reduce the misclassification costs associated with false positive mares contributing to good antimicrobial stewardship.

Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares-A Preliminary Study.

Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student t tests. The mean percentage of endometrial CD4+FOXP3+ Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4+FOXP3+CD25+ Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus (p = 0.0947). The mean proportion of CD3-NKp46+ lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4+FOXP3+CD25+ Tregs and CD3-NKp46+ NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.

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

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

Functions of ectopically transplanted invasive horse trophoblast.

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, ii) whether equine chorionic gonadotropin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and iii) whether eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% confidence interval 55-94) and secreted sufficient eCG for the hormone to be measurable in the recipients' circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to 3 months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares.