Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey’s test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.     

Surgical Treatment of Priapism in a Stallion

Priapism occurred in a stallion after the administration of acetylpromazine. When conservative measures failed, the corpus cavernosum penis (CCP) was drained and irrigated, and a vascular shunt between the erectile bodies was created. Subsidence of erection resulted, but subsequently, the stallion was unable to retract or erect the penis. The stallion was castrated and the penis was surgically retracted into the sheath.     

Insemination with Stallion Semen Frozen in 0,5 ml Straws

Contents: An insemination trial using frozen semen is described. The freezing procedure was slightly modified from the Hannover method. The insemination dose consisted of 7 medium straws containing approx 1 10⁹ spermatozoa. A total of 28 mares of the Norwegian Trotter breed were inseminated during the 1991 season. During oestrus the mares were examined at 12 hour intervals, and the insemination was carried out after detection of ovulation. The pregnancy rate was 43% after the first insemination, increased to 68% after second and further to 75% after the third and last insemination. The foaling rate was 61.5%.     

Vestibuloplasty for Persistent Pneumovagina in Mares

Occasionally Caslick vulvoplasty does not resolve the condition of pneumovagina. This study aimed at evaluating the validity of surgical construction of artificial vestibular valve in standing mares. For 10 sedated mares with history of chronic pneumovagina, upside down U-form incision was made on the vestibular mucosa and sutured together to form a large valve after the cover. The surgical site was opened for another cover or foaling and sutured again. The condition of air aspiration into the vagina was monitored ultrasonographically after the surgery. In 9 of the 10 mares, the signs of pneumovagina improved after the surgery and 8 mares had normal foaling the next year. In addition, all mares which had the second surgery after the surgical site was incised for the second cover or foaling were conceived and had normal foaling in the following breeding season. The results revealed that construction of the vestibular valve can be a potential surgical option for the treatment of persistent pneumovagina.     

Hyperthermia and Delayed-Onset Myopathy after Recovery from Anesthesia in a Horse

A 13-year old Thoroughbred cross gelding (528 kg) underwent lameness investigation. Because of his temperament, general anesthesia was required to facilitate ultrasound of the left fore fetlock and intra-articular medication of three joints. Anesthesia was induced with ketamine/diazepam after acepromazine/detomidine premedication. Anesthesia was maintained for 40 minutes with a guaifenesin/detomidine/ketamine intravenous infusion. Recovery from anesthesia was initially uneventful, although of a moderate duration (70 minutes). Once standing, the horse proceeded to box walk in an agitated state and became recumbent on two occasions. The horse was manually restrained, at which time its rectal temperature was 41.8°C. Cooling measures were employed (fans, ice-water enemas, wet rugs, intravenous fluid therapy (IVFT), and topical application of surgical spirit) until rectal temperature reached 38.7°C. IVFT was continued for a further 16 hours. Four days after recovery from anesthesia, bilateral triceps, deltoideus, trapezius, and rhomboideus muscle swelling was observed. Blood creatinine kinase was elevated (24,898 IU/L). Treatment for postanesthetic myopathy was initiated (hot packing of the muscle groups, topical dimethylsulfoxide [DMSO] cream application, and oral phenylbutazone). Myoglobinuria was not observed at any time. Muscle swelling decreased over the following 3 days. The horse was discharged on day 11 and has since returned to work.     

Persistent Hematuria as a Result of Chronic Renal Hypertension Secondary to Nephritis in a Stallion

A 13-year-old Lusitano stallion was referred to our institution with a history of severe hematuria for 8.5 months. The origin of the hematuria was determined to be the left kidney. The diagnostic workup failed to identify obstructive, infectious, or neoplastic conditions. The history and ultrasonographic examination suggested a chronic condition. The stallion was subjected to left nephrectomy because of the persistent hematuria and anemia. A histopathological examination detected lesions, which were compatible with chronic nephritis and vascular renal hypertension. It is hypothesized that changes in the normal parenchymal architecture produced a vascular aberration that led to renal hypertension, with subsequent blood extravasation and hematuria. The horse recovered completely after nephrectomy.     

The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.     

Successful Treatment of Seminal Vesiculitis with Imipenem-Cilastatin in a Stallion

A 7-year-old thoroughbred stallion presented with severe colic, which was resolved surgically. On postsurgical palpation and rectal ultrasonography, the left seminal vesicle was enlarged, the left testicle and epididymis showed signs of inflammation with hydrocele. Antibiotics and anti-inflammatory treatment were administered. Fever and colic syndrome recurred, with severe inflammation of the left testis and epididymis, and unilateral orchiectomy was performed. Histopathological diagnosis confirmed chronic orchioepididymitis, and Klebsiella pneumoniae was identified and typed from all cultures. At breeding soundness examination 2 months after treatment with ceftiofur, K. pneumoniae was again isolated, and cytology revealed degenerated polymorphonuclear cells and red blood cells. Transurethral endoscopic examination was used to locate the infection in the left seminal vesicle, and ticarcillin-clavulanic was locally infused for 10 days. Nevertheless, the pathogen was once again isolated in the ejaculate, and the affected seminal vesicle was again treated locally for 5 consecutive days, using imipenem-cilastatin, a new antibiotic showing high sensitivity. Follow-up breeding soundness examination revealed an improvement in seminal characteristics and no pathogen isolation. Total pregnancy rate (31 of 36; 86%) and first cycle pregnancy rate (18 of 36; 50%) in the last breeding season reached levels similar to those prior to infection (65 of 82; 80% and 47 of 82; 57% respectively), suggesting that local treatment with imipenem-cilastatin in stallions with seminal vesicle infections is effective and, in this case, did not produce any adhesions or other undesirable effects, permitting the stallion to resume breeding with no apparent deleterious effect on fertility despite having only one testicle.     

Alternative Protocol Using Imipramine,Detomidine, and Oxytocin for Semen Collection in Stallion with Ejaculatory Dysfunction

A 7-year-old Quarter Horse stallion was admitted at the hospital with a history of ejaculatory failure for 12 months. The stallion revealed no physical or psychological abnormalities, as well as, normal libido and erection. In addition, there were no abnormalities in accessory sex glands or the aorta artery detected by transrectal ultrasonography. Based on clinical findings, the stallion was diagnosed with an idiopathic ejaculatory dysfunction; therefore, alternative attempts of semen collection were performed. Thermal compress on the basis of the stallion’s penis, semen collection on the ground, and imipramine hydrochloride treatment were performed unsuccessfully. However, a protocol consisted by the association of imipramine (3 mg/kg/v.o.), detomidine (0.02 mg/kg/i.v.), and oxytocin (20 U.I./i.v.) successfully produced ejaculation in this stallion. The semen obtained from ex copula ejaculation of the stallion was collected using a collector cup lined with a plastic bag, which was positioned over the prepuce of the stallion. Semen with good sperm quality (87% of total motility) was obtained using the proposed protocol. Semen was then processed for cryopreservation and post-thawed semen samples presented satisfactory sperm parameters. In conclusion, the association of imipramine, detomidine, and oxytocin can be considered for ex copula semen collection in stallions.     

Effect of general anesthesia on plasma cardiac troponin I concentrations in healthy horses

Objective

To evaluate the effect of general anesthesia on plasma cTnI concentrations in horses.

Animals, materials and methods

Thirty-two horses undergoing general anesthesia and either elective surgery or MRI without surgery were prospectively studied. Twenty-nine horses (22 surgical, 7 imaging) completed the study. Plasma cTnI concentrations were determined prior to anesthesia and at 6, 12 and 24 h following discontinuation of the inhalant anesthetic.

Results

All horses had cTnI values within the reference range at all time points. Six horses (21%) developed detectable cTnI 6 or 12 h following anesthesia. Risk factors for detectable cTnI include increasing age and dorsal recumbency. Horses with detectable cTnI had significantly lower mean and diastolic arterial blood pressures than those without detectable cTnI.

Conclusion

Uncomplicated general anesthesia with or without surgery does not result in cardiac troponin I elevations above the reference range in the first 24 h postoperatively.     

Successful Treatment of Penile Hematoma in a Colt

Traumatic injuries of the penis result from kick-related injuries, and injuries during coitus or masturbation are reported as common causes of penile hematoma in horses. In the present report, mating for the first time in a 2-year-old colt resulted in penile hematoma. The animal had difficulty in passing urine and had paraphimosis. Under general anesthesia a stab was given to drain the hematoma, and hydrotherapy with cold water along with antibiotics and analgesics was advised. Early surgical intervention and drainage of the hematoma helped in resolving the condition and resulted in normal functioning of the penis.     

Cardiovascular changes after administration of aerosolized salbutamol in horses: five cases

Prevention and treatment of intraoperative hypoxemia in horses is difficult and both efficacy and safety of therapeutic maneuvers have to be taken into account. Inhaled salbutamol has been suggested as treatment of hypoxia in horses during general anesthesia, due to safety and ease of the technique. The present report describes the occurrence of clinically relevant unwanted cardiovascular effects (i.e. tachycardia and blood pressure modifications) in 5 horses undergoing general anesthesia in dorsal recumbency after salbutamol inhalation. Balanced anesthesia based on inhalation of isoflurane in oxygen or oxygen and air and continuous rate infusion (CRI) of lidocaine, romifidine, or combination of lidocaine and guaifenesine and ketamine was provided. Supportive measures were necessary to restore normal cardiovascular function in all horses but no long-term adverse effects were noticed in any of the cases.     

Anatomical Stages of Penile Erection in the Agouti (Dasyprocta leporina) Induced by Electro-Ejaculation

The reproductive system of the male agouti is not well documented. This study describes the specific anatomical features of the free part of the penis occurring during penile erection in the agouti. Electro-ejaculation was used to induce erection in three male agoutis that had previously produced offspring. Results proved that there were four stages in the erection process. Stage 1 involved protrusion of the penis from the preputial orifice. The lateral penile cartilages were then spread (stage 2). During stage 3, there was the blooming of the head of the glans penis (penile flower) and eversion of the intromittent sac. The protrusion of the keratinaceous styles and ejaculation occurred during stage 4. This information could assist in semen collection for use in reproductive techniques for the agouti such as artificial insemination.     

Comparison of the Effects of Four Freezing Methods on Motility Characteristics,Morphology, and Viability of Postthaw Stallion Epididymal Sperm

Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freeze-thaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period.     

Motility of Fresh and Frozen-Thawed Stallion Sperm from Three Segments of the Epididymal Cauda and the Effect of Post-Thaw Seminal Plasma Addition on Motility

Preservation of epididymal spermatozoa is an advantageous method to preserve genetic material of endangered species or valuable breeding animals after sudden death and injuries. Despite lower pregnancy rates, fertilization with epididymal sperm has been proven successful. Variable sperm quality after cryopreservation among individual stallions and the usually terminal chance to preserve epididymal sperm are opportunities for the development of a freezing procedure suitable for the majority of stallions. To evaluate the effect of the preservation procedure, we analyzed the sperm motion characteristics after every step of processing. In addition, we investigated the influence of seminal plasma on motility of frozen-thawed semen. We compared three segments of the cauda epididymidis and harvested spermatozoa by retrograde flushing (most caudal part) and mincing (more cranial segments) to augment the number of spermatozoa. During processing, there were differences in sperm motion characteristics depending on the segment of the cauda epididymidis. Distinct increases in motility due to different extenders and the effect of seminal plasma suggest that motion characteristics of raw and frozen-thawed spermatozoa are strongly influenced by microenvironment and must be interpreted with caution.     

Reduced birth weight,cleft palate and preputial abnormalities in a cloned dog

The aim of the present study was to report a novel developmental abnormality in a cloned dog. A fibroblast cell line was established from an 8-year-old male German shepherd dog. In vivo matured oocytes were retrieved from a large breed dog, and the nucleus was removed from each oocyte. A donor cell was injected into an enucleated oocyte, and the oocyte-cell couplet was fused electrically. After chemical activation, the resulting embryos were transferred into a naturally estrus-synchronized recipient dog, and two cloned pups were delivered by Cesarean section 60 days later. One cloned pup (Clone 1) was healthy, but the other (Clone 2) had a birth weight of only 320 g and cleft palate, failure of preputial closure at the ventral distal part, and persistent penile frenulum. Clone 2 was raised by stomach feeding until Day 40 after birth, where palatoplasty was performed. The abnormalities in external genitalia in Clone 2 resulted in persistent penile extrusion that was surgically corrected. This complex developmental abnormality has not been reported in dogs previously.     

Electrochemotherapy for the Treatment of Multiple Melanomas in a Horse

A 22-year-old Camargue mare was referred for showing signs of discomfort during chewing, building up of food in the mouth, and foul odor from the mouth. On physical examination, the mare presented bilateral pigmented labial masses affecting the upper and lower lips, more in the form of plaques at the mucocutaneous junction as well as multiple perivulvar and perianal lumps. The skin lesions were biopsied for histopathologic examination and the clinical suspect of melanoma was confirmed. The owner elected the lesions to be treated with electrochemotherapy using the drug cisplatin. After two sessions, the nodules were shrunk by 50% and the horse’s ability to feed normally was restored: at that time, the owner had the treatment discontinued because of financial issues. The horse is still in partial remission after 1 year from the end of the treatment. Electrochemotherapy can be successfully used to palliate large melanomas in horses.     

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.     

Case of Bilateral Seminoma in a Trotter Stallion

This report describes a bilateral seminoma in a stallion. After slaughter, histological examination revealed that the tumour consisted predominantly of polyhedral tumour cells with large nuclei, obvious nucleoli and a small border of cytoplasm. The mitotic index was low and Ki67 staining revealed 4% nuclear staining. To our knowledge, this paper is the first using Ki67 staining as a method to evaluate the mitotic rate in a testicular seminoma in the stallion.