Equine Veterinary Journal

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

Abstract Background Abnormal or undesired mare behaviours are often assumed to be associated with ovarian abnormalities. Objectives We aimed to determine the incidence of abnormal behaviours and their association with concentrations of one or more ovarian hormones associated with a granulosa cell tumour (GCT). Study design Retrospective descriptive. Methods A total of 2914 hormonal profile samples submitted with the words behave, behaviour, or behaving in the submission history were analysed. The association between reported abnormal behaviours and concentrations of testosterone, anti‐Müllerian hormone (AMH), inhibins and inhibin‐B were assessed. Statistical analysis was performed using a Chi‐squared test of association. Results Of the 2914 cases that were submitted due to behaviour issues, 2506 (86%) did not have any of the measured hormones reach GCT‐like concentrations. The remaining 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 behaviour was significantly associated with increased concentrations of all four hormones. In contrast, aggression, oestrous and other abnormal behaviours were significantly less likely to be associated with increased concentrations of the hormones. Main limitations Retrospective study, using sample submission history. Conclusion Overall, the abnormal behaviours among mares, except the stallion‐like behaviour, were not associated with increased ovarian hormones. These results highlight the common misassumption about the involvement of the ovaries in ‘abnormal behaviours’ or ‘undesirable behaviours’ of mares.

Active immunisation against GnRH as treatment for unilateral granulosa theca cell tumour in mares

Abstract Background Stallion‐like or aggressive behaviour in mares affected by unilateral granulosa theca cell tumour (GTCT) is well‐known, but use of a GnRH‐vaccine as an alternative to surgical removal of the neoplastic ovary has not been investigated. Objectives To determine the effect of immunisation against GnRH on ovarian size, testosterone concentration, Anti‐Müllerian hormone (AMH) concentration, and owner‐reported behaviour in four mares affected by unilateral GTCT. Study design Retrospective case report. Methods A presumptive diagnosis of GTCT was made in four mares based on clinical signs, behavioural changes, transrectal palpation, and ultrasonography. All mares were vaccinated twice with the GnRH‐vaccine Improvac® on day 0 and on day 13‐33. Further booster vaccinations were administered if aggressive behaviour recurred between days 15 and 498. Before and parallel to the vaccinations, serum levels of oestradiol, progesterone (P4), testosterone, and AMH were evaluated and transrectal ultrasonography was performed. Results In all horses, analysis of serum levels of oestradiol, progesterone, testosterone, and AMH confirmed the clinical diagnosis of GTCT. Serum levels of testosterone dropped to baseline levels following the first two of three vaccination in all mares. In addition, AMH serum values decreased shortly after the second vaccination in three of four mares, and in one of the four mares returned to baseline levels. No further GTCT linked behaviour was reported by the owners and the affected ovaries diminished in size in all four cases. Main limitations This report is a case series with a limited number of animals, no controls and no standardised immunisation protocol. Conclusions Repeated vaccinations with the GnRH‐vaccine Improvac® mitigated owner‐reported behavioural abnormalities and stopped tumour growth in four mares affected by unilateral GTCT over the entire observation period which extends to 7 years in one mare.

Equine granulosa cell tumours among other ovarian conditions: Diagnostic challenges

Abstract Background Granulosa cell tumours (GCT) are the most common ovarian tumours in mares. While the classical presentation may not represent diagnostic challenges, diagnosis is not easy in the early stages. Objectives Illustrate the variability in the presentation and serum biomarkers associated with ovarian abnormalities in the mare. Study design Retrospective case series . Methods Nonclassical cases of GCTs and other ovarian conditions were identified and behaviour, GCT endocrine results, palpation and ultrasonographic findings are described and the diagnostic value of each is discussed. Results Mares in this case series with GCTs had been presenting clinical signs ranging from no behavioural changes to behaviours including aggression, stallion‐like and inability to work under saddle. Hormonal profiles of endocrinologically functional GCTs can be erratic and unpredictable. The clinical form and ultrasonographic appearance may also vary with time from an initially enlarged/anovulatory follicular structure that later develops a multicystic ‘honeycomb’ appearance. Mares with GCTs can also present with persistent anovulatory follicles or apparent luteal tissue that are unresponsive to treatment. If both ovaries are of relatively normal size and symmetry, but hormonal biomarkers are markedly increased (AMH >10 ng/mL, inhibin B and/or testosterone >100 pg/mL; 0.37 nmol/L), it is likely that a functional GCT is present. Still, it can be a challenge to decide which ovary to remove. Post‐surgical endocrine testing can be helpful, especially if histopathology is not performed or a GCT is not found. Main limitations Cases limited to 14. Conclusions Granulosa cell tumours present with a wide variety of clinical signs that do not fit what is commonly described as ‘classic’. Only if AMH, testosterone and inhibin B concentrations are markedly increased, and there is an abnormally enlarged ovary, the diagnosis of a GCT is more confident. In the presence of normal size ovaries, normal hormonal biomarkers and abnormal behaviour, it is more likely that the ovaries are not involved.

Epithelial–mesenchymal transition in an EcPV2 ‐positive vulvar squamous cell carcinoma of a mare

Abstract Background Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. Objective To describe a case of naturally occurring EcPV2‐induced VSCC, by investigating tumour ability in undergoing the epithelial‐to‐mesenchymal transition (EMT). Study design Case report. Methods A 13‐year‐old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real‐time qPCR, real‐time reverse transcriptase (RT)‐qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT‐related and innate immunity‐related genes was investigated through RT‐qPCR. Results Real‐time qPCR, RT‐qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT‐related transcription factor HIF1α. With RT‐qPCR, significantly increased gene expression of EBI3 (45.0 ± 1.62, p  < 0.01), CDH2 (2445.3 ± 0.39, p  < 0.001), CXCL8 (288.7 ± 0.40, p  < 0.001) and decreased gene expression of CDH1 (0.3 ± 0.57, p  < 0.05), IL12A (0.04 ± 1.06, p  < 0.01) and IL17 (0.2 ± 0.64, p  < 0.05) were detected. Main limitations Lack of ability to generalise and danger of over‐interpretation. Conclusion The results obtained were suggestive of an EMT event occurring within the neoplastic lesion.