Robyn Elliene Ellerbrock

Leukospermia in dogs is characterized by increased white blood cells (WBC) in semen and is associated with reproductive tract inflammation, but diagnostic accuracy remains poorly defined. The objectives of this study were to assess the effect of varying WBC concentrations on sperm motility parameters and to analytically and clinically validate five diagnostic tests (hemacytometer, leukocyte esterase dipstick test, peroxidase stain, semen cytology, and CD45⁺ immunostaining). We hypothesized that increasing WBC concentrations would impair sperm motility and that all diagnostic methods would have diagnostic value to detect leukospermia. Analytical validation was performed in nine semen samples from healthy dogs by inducing leukospermia with increasing WBC concentrations and assessing sperm motility over time. Diagnostic metrics (sensitivity, specificity, positive and negative predictive values, likelihood ratios, and Youden index) were calculated. Clinical validation included 67 ejaculates, with diagnostic performance assessed using the same metrics, receiver operating characteristic (ROC) analysis, and univariable logistic regression with CD45⁺ immunostaining as the gold standard. Increasing WBC concentrations caused a dose-dependent reduction in sperm motility, with clinically relevant impairment observed at ≥2.5x10⁶ WBC/mL (p<0.05). Analytical validation demonstrated high sensitivity across diagnostic tests, with the peroxidase test performing best (sensitivity 96.3%, negative predictive value 94.1%). In the clinical phase, the hemacytometer and semen cytology showed poor diagnostic accuracy, whereas the peroxidase test demonstrated fair performance (AUC=0.72, 95% CI 0.54-0.91) with high sensitivity (83.3%), specificity (76.7%), and negative predictive value (97.9%). Overall, the peroxidase test demonstrated to have the most consistent analytical and clinical performance for diagnosing leukospermia in dogs.

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3β-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD), P450,17α hydroxylase, 17–20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-β, and 3βHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p > 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p < 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3β-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed.

It was the aim of this study to provide a more precise timeframe of development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localisation of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size by 23-fold from 45 to 60 days of gestation, paralleled by a greater increase in metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p<0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells was proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

Background Recent studies have shown that fluoroquinolones, specifically, enrofloxacin and its active metabolite (ciprofloxacin), cross the equine placenta without causing gross or histological lesions in the first and third trimester fetuses or resulting foal. However, it is possible that in utero exposure to fluoroquinolones may cause subtle lesions not detectable by standard means; thus, a more in‐depth assessment of potential toxicity is warranted. Objectives To utilise quantitative magnetic resonance imaging (qMRI), biomechanical testing, and chondrocyte gene expression to evaluate the limbs of foals exposed to enrofloxacin during the third trimester of pregnancy. Study design In vivo and control terminal experiment. Methods Healthy mares at 280d gestation were assigned into three groups: untreated (n=5), recommended therapeutic (7.5 mg/kg enrofloxacin, PO, SID, n=6), or supratherapeutic (15 mg/kg, PO, SID, n=6) doses for 14d. Mares carried and delivered to term and nursed their foals for ~30d. Two additional healthy foals born from untreated mares were treated postnatally with enrofloxacin (10 mg/kg PO, SID, for five days). By 30d, foal stifles, hocks, elbows, and shoulders were radiographed, foals were euthanised, and foal limbs were analysed by quantitative MRI, structural MRI, biomechanical testing and chondrocyte gene expression. Results Osteochondral lesions were detected with both radiography and structural MRI in foals from both enrofloxacin‐treated and untreated mares. Severe cartilage erosions, synovitis, and joint capsular thickening were identified in foals treated with enrofloxacin postnatally. Median cartilage T2 relaxation times differed between joints but did not differ between treatment groups. Main limitations A small sample size was assessed and there was no long‐term follow up. Conclusion While further research is needed to address long‐term foal outcomes, no differences were seen in advanced imaging, biomechanical testing, or gene expression by 30d of age, suggesting that enrofloxacin may be a safe and useful antibiotic for select bacterial infections in pregnant mares.