Effects of therapeutic ultrasound and moderate heat on stallion testes.

Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of "proof of concept" studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for increasing intratesticular temperature in stallions. Six min of treatment using the Vetrison Clinic Portable TUS machine raised the intratesticular temperature by 8°C-12.5 °C. This protocol was applied to treat three scrotal testes in three Miniature horse stallions, three times, every other day. Contralateral testes served as controls. There were signs of slight tubular degeneration in treated testes two and three weeks after TUS treatment. The number of seminiferous tubules (STs) with exfoliated germ cells (GCs) was increased in one testis only, three weeks after treatment. The degree of apoptosis of GCs was higher in each treated testis in comparison to the contralateral control testis. Next, the ability of various heating devices to increase intratesticular temperatures to at least 43 °C in stallion testes was tested, using twenty testes obtained from castrations. ThermaCare® Lower Back & Hip Pain Therapy Heatwrap (TC heat wrap) reliably increased intratesticular temperatures and kept them continuously between 43 °C and 48 °C for seven to 8 h. In the follow-up in vivo study, the left testes of three Miniature horse stallions were treated with TUS, after which both testes of each stallion were treated with moderate heat provided by the TC heat wrap (three times, every other day, for 5 h each time). There were signs of moderate tubular degeneration in the samples from all treated testes obtained three weeks after treatments (Heat only or Heat/TUS): areas with hypospermatogenesis, spermatogenic arrest, vacuolized Sertoli cells, numerous STs with exfoliated GCs, increased degree of GCs apoptosis, and changes in three histomorphometric numeric attributes of STs. We concluded that TUS or TC wraps increase intratesticular temperature of the isolated stallion testes. Further, treatment with TUS or moderate heat may induce mild to moderate degenerative changes in stallion testes. However, to achieve more robust result - severe testicular degeneration, our treatment protocol has to be modified.

Evidence for anti-inflammatory effects of firocoxib administered to mares with experimentally induced placentitis.

PROBLEM: Minimal evidence exists supporting therapeutic selections for equine placentitis. The goal of this study was to characterize the anti-inflammatory effects of firocoxib when administered to mares with placentitis. METHODS: Mares (gestation D270-300) were assigned to: INFECT (n = 6; placentitis, no treatment), FIRO (n = 6; placentitis, firocoxib, 0.1 mg/kg, PO, daily), and NORM (n = 6; no infection/treatment). Allantoic fluid (8 hours, 24 hours, birth) and amniotic fluid (birth) were collected from mares after infection. Concentrations of IL-1ß, IL-6, TNF-α, IL-10, PGF2α , and PGE2 in fluids were measured by ELISA. mRNA expression of IL-1ß, IL-6, TNF-α, IL-8, IL-10, matrix metalloproteinases (MMPs) -1, 3, and 9 in fetal membranes/fetuses was quantified using real-time PCR. RESULTS: Allantoic TNF-α concentrations were lowest in FIRO at 8 hours and 24 hours post-infection; IL-6 concentrations were lower in FIRO than NORM at 8 hours, lower in FIRO than INFECT at 24 hours post-inoculation, and lower in NORM than FIRO or INFECT at birth. Marginal mean allantoic IL-ß and IL-10 concentrations were lower in FIRO and NORM than INFECT. Amniotic fluid cytokines were lowest in NORM with all measurements in that group being below the limit of detection. Allantoic PGF2α concentrations were lower in FIRO and INFECT than NORM at 8 hours post-inoculation, and lower in FIRO than INFECT or NORM at 24 hours post-inoculation. Allantoic PGE2 concentrations were lower in FIRO than INFECT. Amniotic PGF2α and PGE2 concentrations were lower in NORM than INFECT. In fetal membranes, group differences with respect to IL-1ß, IL-6, IL-8, and MMP1 were dependent on tissue type. CONCLUSIONS: Data suggest a suppressive effect of firocoxib administration on cytokine and prostaglandin production in mares with placentitis.

Endoscopic Laser Ablation of a Midline Cyst of the Colliculus Seminalis as a Treatment for Anejaculation in a Stallion.

An 18-year-old Appaloosa stallion presented with a history of ejaculatory dysfunction, which had recently progressed to an inability to ejaculate (anejaculation). Transrectal ultrasound evaluation revealed the presence of a prominent midline cyst of the colliculus seminalis, which was compressing the most terminal parts of the deferent ducts. Both ducts were enlarged and filled with hyperechoic content. The stallion was diagnosed with a complete occlusion of the deferent ducts because of the compression from the midline cyst of the colliculus seminalis. To date, there are no established treatments for this condition in stallions, although several procedures have been successfully used in men affected by similar problems. Therefore, we proposed performing one of these procedures-a laser ablation of the cyst to collapse it and to open the ejaculatory path. The stallion was placed under general anesthesia, and an endoscopic ultrasound-guided laser ablation of the cyst was performed. No immediate or long-term complications were observed. After the expulsion of accumulated material, the stallion produced normal semen and successfully impregnated mares during the next breeding season.

Fetal Membrane Removal in the Mare: Proactive Versus Reactive Approaches.

Retained fetal membranes are the most common postpartum condition in mares. Although the incidence of retained fetal membranes is low, the consequences for the health of the mare can be severe (metritis, endotoxemia, laminitis, death). Oxytocin administration is often the first line of therapy for management of retained fetal membranes. Removal of fetal membranes using umbilical vessel infusion and manual membrane removal are effective tools for reducing risks associated with abnormally heavy membranes, retained membranes, or for mares that are geographically limited for veterinary care.

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.

Relationship between echotextural and histomorphometric characteristics of stallion testes.

The goal of this study was to investigate correlations between objective measures of testicular echotexture and histomorphometric attributes related to the histological composition of stallion testes. Fifty-four scrotal testes were obtained from three groups of stallions during routine castrations: colts <1 yr old (n = 18), young stallions 1-5 yrs old (n = 27), mature stallions > 5 yrs old (n = 9). In addition, two scrotal testes with degeneration, 16 retained inguinal and 10 retained abdominal testes were surgically obtained. Cross-sectional and longitudinal ultrasonograms were obtained for each testis. Mean numerical pixel values (NPVs) as well as pixel standard deviations (PSDs) were determined for each image (ImageJ-1.5 software). Histomorphometric attributes of the seminiferous tubules (STs) were derived (three tissue samples per each testis) using image analysis software [relative STs area: RSTA = ST area/total cross-sectional area (TA) x 100%; relative STs lumen: RSTL = ST lumen area/TA x 100%; individual ST area; ISTA; individual ST lumen: ISTL; seminiferous epithelium height: SHE]. Degree of fibrosis was graded semi-quantitatively (0-3) in samples from 17 testes. All measures of testicular echotexture as well as all histomorphometric attributes of STs had highest values when obtained from the scrotal testes of young and mature stallions (P < 0.05). The NPVs and PSDs from the ultrasonographic images of the scrotal testes were significantly correlated with all histomorphometric attributes of STs (P < 0.001). However, there was no correlation between the majority of these measures and attributes if each group of the scrotal testes was analyzed separately. The NPVs from the ultrasonographic images of the retained inguinal testes were correlated with RSTA, RSTL, ISTA, and ISTL, while the NPVs from the retained abdominal testes were not correlated with any of the histomorphometric attributes of the STs. Testes with high degree of fibrosis had variable values of pixel intensity and pixel heterogeneity. Based on the results of this study, we concluded that the pixel intensity and pixel heterogeneity of stallion testes increase during the first year of life and remain stable in young and mature stallions. These changes occur in parallel to the development of the seminiferous tubules. Testicular echogenicity in stallions does not seem to reflect degree of testicular fibrosis. Retained abdominal testes have lower and less heterogeneous echogenicity than scrotal testes from stallions that are more than one year old. While pixel analysis cannot replace biopsy in assessing testicular histomorphology in young and mature stallions, testicular echogenicity is a good indicator of peri-pubertal growth and expansion of the seminiferous tubules.

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.

Indenopyride derivative RTI-4587-073(l): a candidate for male contraception in stallions.

The objective of this study was to determine whether an indenopyridine derivative RTI-4587-073(l) was a good candidate for male contraception in horses. We hypothesized that a single administration of RTI-4587-073(l) causes significant suppression of testicular function in stallions without affecting sexual behavior. Three Miniature horse stallions received a single dose of 12.5 mg/kg RTI-4587-073(l) orally (group "treated"), whereas three other Miniature horse stallions received placebo only (group "control"). Semen was collected and evaluated from all stallions twice a week for three baseline weeks and 13 post-treatment weeks. Sexual behavior was video-recorded and analyzed. Testicular dimensions were measured using ultrasonography, and blood samples were drawn for endocrine evaluation once before treatment and once a week during the post-treatment period. Single administration of RTI-4587-073(l) caused severe oligoasthenozoospermia (low sperm number and low motility), shedding large numbers of immature germ cells in semen, and increased FSH concentrations in treated stallions. These effects were fully reversible within ∼71 days. However, libido and copulatory behavior remained unchanged throughout the entire experiment. We concluded that RTI-4587-073(l) was a promising candidate for male contraceptive in domestic stallions. Further research should be performed to test this compound for fertility control in wildlife and humans.

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.

Diagnosis and treatment of equine placentitis.

Equine placentitis is a complex disease that has devastating consequences for horse owners. Placentitis is a significant cause of foal loss annually. Prompt diagnosis and treatment of the disease are paramount for survival of the affected neonate. This article discusses current information on pathogenesis of the disease as well as diagnostic and therapeutic options.

Management of hydrops amnion in a mare resulting in birth of a live foal.

CASE DESCRIPTION: A 19-year-old Thoroughbred mare was evaluated at 265 days of gestation with a markedly distended abdomen and edema of the ventral portion of the abdomen. CLINICAL FINDINGS: The uterus was distended over the pelvic rim, making transrectal palpation of the fetus impossible. Transabdominal ultrasonography revealed excessive amounts of fetal fluid. Results of analysis of fluid obtained via amnio- and allantocentesis confirmed that the amniotic cavity was large. TREATMENT AND OUTCOME: The mare was monitored for signs of weakness of the prepubic tendon and abdominal wall. The fetus and placenta were monitored for signs of stress and pending abortion. Flunixin meglumine and altrenogest were administered to the mare. Parturition was attended and occurred at 321 days' gestation. Postpartum complications in the mare included hypovolemic shock and cardiac arrhythmias. Both conditions were treated, and the mare recovered. The foal was considered small, had bilateral angular limb deformities, and was unable to nurse. The foal was given plasma for failure of passive transfer of immunity. Ten months later, the foal underwent procedures to correct limb deformities. CONCLUSIONS AND CLINICAL RELEVANCE: Hydrops conditions are rare in horses, with hydrops allantois occurring more frequently than hydrops amnion; reportedly result in fetal or neonatal death; and may result in death of or injury to the mare. Close monitoring of maternal and fetal health in combination with supportive treatment of the mare can result in the safe progression of a hydrops pregnancy and the birth of a live foal.

Treatment strategies for mares with placentitis.

Equine placentitis, and resultant preterm labor, are important sources of fetal and neonatal loss. The primary cause of equine placentitis is infection of the placenta with Streptococcus equi subspecies zooepidemicus, which ascends through the caudal reproductive tract. Current treatment protocols for mares affected with placentitis are empirical. This paper reviews treatment approaches for resolving placentitis and preterm labor in both equine and non-equine species. Specific therapies reviewed include antimicrobial, anti-inflammatory, tocolytic, and progestin agents.