Malgorzata A. Pozor

Treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis

The objective was to determine if long-term treatment with trimethoprim sulfamethoxazole (antimicrobial), pentoxifylline (anti-inflammatory/anti-cytokine) and altrenogest (synthetic progestin), would improve pregnancy outcome in mares with experimentally induced placentitis. Seventeen normal, pregnant pony mares were enrolled in the study at 280-295 d of pregnancy. Placentitis was induced in all mares by intra-cervical inoculation of Streptococcus equi subsp. zooepidemicus (10(7) CFU). Five mares served as infected, untreated control animals (Group UNTREAT). Twelve mares (Group TREAT) were infected and given trimethoprim sulfamethoxazole (30 mg/kg, PO, q 12h), pentoxifylline (8.5 mg/kg, PO, q 12h) and altrenogest (0.088 mg/kg, PO, q 24h) from the onset of clinical signs to delivery of a live foal or abortion. Blood samples were cultured from all foals at delivery and fetal stomach and thoracic contents were obtained for culture from dead fetuses. More mares in Group TREAT delivered viable foals (10/12; 83%; P < 0.05) than mares in Group UNTREAT (0/5; 0%). Ten of 12 foals (83%) in Group TREAT had negative blood cultures at birth. All foals in Group UNTREAT (5/5; 100%) had positive cultures from one or more samples (blood, stomach contents, and thoracic fluid). Bacteria were recovered from uterine culture samples in both groups. Streptococcus equi subsp. zooepidemicus was the predominant organism recovered from fetal/foal or mare culture samples. The authors inferred that administration of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest may improve the viability of foals from mares with experimentally induced placentitis.

Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1–0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.

Uterine artery blood flow remains unchanged in pregnant mares in response to short-term administration of pentoxifylline.

The objective of this study was to use Doppler ultrasound technology to determine whether pentoxifylline administration increased uterine blood flow in normal pregnant pony mares. Thirteen pregnant pony mares between 18 and 190 d of gestation (mean ± SEM, 101 ± 55) were utilized for the study during two trial periods. In each trial, pentoxifylline (17 mg/kg by mouth every 12h, diluted in syrup) was administered to half of the mares for 3 d, while the other mares were treated with syrup only. Doppler measurements were obtained from the right and left uterine arteries from each mare for 2 d prior to treatment and throughout the treatment period. The mean Resistivity Index (RI), Pulsatility Index (PI), Uterine Artery Diameter (D), and Total Arterial Blood Flow (TABF) from each day were compared over time and between groups. Administration of pentoxifylline did not alter uterine blood flow parameters compared with controls (values for all treatment days combined were RI: 0.517 ± 0.014 vs 0.543 ± 0.016; PI: 0.876 ± 0.048 vs 0.927 ± 0.057; D: 0.388 ± 0.018 vs 0.379 ± 0.023 cm; and TABF: 35.26 ± 7.38 vs 30.73 ± 5.29 mL/min). Uterine blood flow increased over the course of the 5 d study, irrespective of treatment, and was higher in mares of greater gestational age than in early gestational mares (RI: r(2) = 0.35; PI: r(2) = 0.37; D: r(2) = 0.66; and TABF: r(2) = 0.67 - P < 0.00001). We concluded that any immediate benefits of pentoxifylline administration in the pregnant mare were not mediated through enhanced uterine artery blood flow.

Effect of pentoxifylline treatment on testicular perfusion and semen quality in Miniature horse stallions.

The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PTX-treated stallions between Treatment 1 and Post-treatment periods. Arterial diameter, as well as Total Arterial Blood Flow (TABF), decreased in controls between Baseline and Treatment 1 (P < 0.05). A similar decrease in arterial diameter was delayed in Group TREATED, but reached significance during Post-treatment (P < 0.05), whereas TABF did not change in this group. Furthermore, TABFR had a transient tendency to increase during Treatment 1 (P < 0.1), whereas it steadily decreased in controls and reached significance in the Post-treatment period (P < 0.05). Both RI and PI were negatively correlated with end diastolic velocity (EDV) in both groups (P < 0.0001). There were positive correlations between RI and peak systolic velocity (PSV) in treated stallions during Treatment 1 (RI: r = 0.53, P = 0.021; PI: r = 0.59, P = 0.007). Also, there were negative correlations between Time Averaged Maximum Velocity (TAMAX) and Doppler indexes in treated stallions during Treatment 2 period (RI: r = -0.49, P = 0.006; PI: r = -0.47, P = 0.008), and during Post-treatment periods (RI: r = -0.40, P = 0.049; PI: r = -042, P = 0.039). Transient hydrocele occurred in all treated stallions (a potential complication of high-dose PTX). Semen end points were not significantly affected by PTX treatment. In conclusion, PTX delayed the seasonal decrease of testicular perfusion in stallions. Sperm quality and quantity were not significantly affected; perhaps they would have been enhanced by prolonged treatment.

Placental abnormalities in equine pregnancies generated by SCNT from one donor horse

Placental changes associated with SCNT have been described in several species, but little information is available in this area in the horse. We evaluated the ultrasonographic, gross, and histopathological characteristics of placentas from three successful and five unsuccessful equine SCNT pregnancies, established using cells from a single donor horse. Starting at approximately 6-month gestation, the pregnancies were monitored periodically using transrectal (TR) and transabdominal (TA) ultrasonography (US) to examine the placentas, fetal fluids, and fetuses. Of the five mares that aborted, one mare did so suddenly without any abnormal signs detected by US and four had enlarged umbilical vessels visible on TA-US before abortion. Placental edema (TR-US) and intravascular thrombi in the umbilical cords were seen (TA-US) in two of these four mares; one mare aborted shortly after acute placental separation was identified on TA-US. In three mares that delivered live foals, TA-US showed engorged allantoic vessels and enlarged umbilical vessels. Two of these mares had placental thickening visible on TR-US, interpreted as a sign of placentitis, that subsided after aggressive medical treatment. Seven of the eight placentas were submitted for gross and histopathological examinations after delivery. All placentas had some degree of edema, abnormally engorged allantoic vessels, and enlarged umbilical vessels. Placentitis, large allantoic vesicles, cystic pouches in the fetal part of the cord, and hemorrhages and thrombi in the umbilical vessels were detected only in placentas from mares that aborted. Equine pregnancies resulting from SCNT may be associated with placental pathologies that can be detected using ultrasonography. However, interpreting their severity is difficult. Although placental abnormalities have been observed in SCNT pregnancies in other species, to the best of our knowledge, placentitis has not been previously reported and may be an important complication of equine SCNT pregnancies, leading to pregnancy loss.

Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa.

REASONS FOR PERFORMING STUDY The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. OBJECTIVES To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal labelling with a fluorescein-conjugated lectin - a method that has been validated for acrosome status evaluation in stallions. STUDY DESIGN In vivo experimental design. METHODS Semen from 6 mature Miniature horse stallions of known fertility was collected on 5 separate occasions. To increase the number of reacted acrosomes, portions of each ejaculate were incubated with the calcium ionophore, A23187. Ejaculates were divided and semen samples were processed according to recommendations for fluorescein-conjugated peanut lectin, Pisum sativum agglutin, Dip Quick, and Spermac staining methods. Slides were evaluated independently by 2 separate investigators. Spermatozoa were classified as having intact, reacting, reacted or defective acrosomes. RESULTS All parameters obtained by both investigators, using all 3 staining methods were highly correlated (P<0.001). There was no statistical difference (P>0.05) between investigators or staining method for the percentages of intact or reacted acrosomes. However, there was a significant difference between investigators and staining methods for determining reacting acrosome percentages (P<0.05). CONCLUSIONS Dip Quick and Spermac stains are useful for determining intact vs. reacted acrosomes for stallion spermatozoa.

Anti-Müllerian hormone as a biomarker for acute testicular degeneration caused by toxic insults to stallion testes

Recently, anti-Müllerian hormone (AMH) was validated as a reliable marker of testicular damage caused by various chemotherapy drugs in humans and in mice. In horses, the reference values of AMH concentrations in normal stallions, during different seasons of a year, have been recently reported. However, this hormone was not evaluated in subfertile or infertile stallions with testicular damage. Therefore, the objective of this study was to investigate the effects of experimentally induced testicular degeneration on the concentration of AMH in stallions. Severe but transient testicular degeneration was induced in six Miniature horse stallions, in two, separate experiments (three stallions in each experiment), by the administration of a single dose of the contraceptive compound RTI-4587-073(l). Six different stallions served as controls (three stallions in each experiment). Treated and control stallions were switched between the experiments. Concentrations of AMH were determined in 78 samples of blood plasma collected during the first experiment and in 24 samples collected during the second experiment. Furthermore, the expression of AMH in 30 samples of testicular parenchyma, collected from these stallions during the second experiment, was also evaluated, using immunohistochemistry (IHC) and objectively analyzed using computerized methods. During the first experiment, the concentrations of AMH in blood increased significantly in treated stallions (P < 0.05), reaching a 62-151% change from the baseline by day 10 after treatment, before gradually decreasing to the pretreatment levels. There was no change in blood AMH concentration in control stallions. Only a trend to increase AMH concentration was observed in treated stallions during the second experiment (P = 0.055). The labeling for immunoreactive AMH in the Sertoli cells gradually increased after treatment, which was confirmed by the significantly increased IHC optic density score value (P < 0.05) and significantly decreased percentage contribution of negative pixels at fourth week after treatment (P < 0.05). We concluded that AMH is a promising candidate as a biomarker of testicular damage in stallions caused by toxic insults that lead to testicular degeneration.

Disposition of firocoxib in late pregnant and early postpartum mares

Pregnancy induces several physiologic changes that might impact the bioavailability, distribution, metabolism, and excretion of drugs. The objective of this study was to determine the effects of pregnancy on the disposition of oral firocoxib in mares. Seven pony mares received oral firocoxib paste at a dose of 0.1 mg/kg during late pregnancy and again 12 to 33 days postpartum. Firocoxib concentrations were measured in plasma by HPLC with ultraviolet detection. Maximum plasma concentrations were significantly lower in pregnant (50.0 ± 21.8 ng/mL) than in postpartum (73.7 ± 25.6 ng/mL) mares. Plasma concentrations 24 h after administration, time to maximum plasma concentrations, and area under the plasma concentration versus time curve were not significantly different between late pregnancy and the postpartum period in mares.

Anatomical variations in epididymal–testicular fusion in stallions and their possible clinical significance

REASONS FOR PERFORMING STUDY Fusion anomalies of the epididymis with the testis may be clinically relevant in horses. However, anatomical variations in epididymal-testicular fusion have not been classified, and their clinical significance is unknown. OBJECTIVES To describe anatomical variations and clinical significance of epididymal-testicular fusion in stallions. STUDY DESIGN Anatomical study of testes from castrations, and description of 2 clinical cases with atypical epididymal-testicular fusion. METHODS A total of 104 testes were obtained from equine castrations. Eight patterns of epididymal-testicular fusion were identified. Two clinical cases with epididymal dislocation were also described. RESULTS Close attachment of the entire epididymis to the testis was the most common pattern of fusion (40%). Ninety-five per cent of cryptorchid testes and 34% of scrotal testes in the studied sample had elongated proper ligaments of the testes. Dislocation of the epididymal tail was observed in 2 stallions that had atypically long proper ligaments inserted on the dorsal aspect of the testes. CONCLUSIONS Patterns of epididymal-testicular fusion can vary in stallions. Elongated proper ligaments of the testes occur mostly in cryptorchid testes but are also found in stallions with scrotal testes. Epididymal dislocation may develop in stallions with long proper ligaments that are inserted dorsally on the testes.