Cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares.

CASE DESCRIPTION: 6 mares with pyometra secondary to transluminal cervical adhesions were examined. CLINICAL FINDINGS: Reasons for hospital admission included infertility (5 mares) and acute colic (1 mare). In the 6 mares, palpation per rectum of the reproductive tract revealed uterine distention, and transrectal ultrasonography confirmed the presence of echogenic fluid accumulation within the uterus. Cervical palpation during vaginal speculum examination indicated transluminal cervical adhesions. Three mares had severe distortion of the cervix as a result of diverticula and fibrosis. All 6 mares had a diagnosis of pyometra secondary to transluminal cervical adhesions. TREATMENT AND OUTCOME: Initially, the cervical adhesions were manually broken down to establish a patent cervical lumen to accommodate a uterine lavage catheter. A sample of the uterine content was obtained for bacteriologic culture and antimicrobial susceptibility testing, and the uterus was lavaged with 0.05% povidone-iodine solution to remove the mucopurulent exudate. Once the uterus was evacuated, cervical surgery was performed in standing mares following sedation and caudal epidural anesthesia. A full-thickness wedge-shaped defect was made in the dorsolateral aspect of the cervix that created a permanent opening to the uterus. Postoperative care included applying topical medication to the cervix to reduce the recurrence of adhesion formation. All 6 mares had patent cervices and resolution of pyometra following surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Cervical wedge resection enabled treatment of pyometra in mares with transluminal cervical adhesions, without the need for ovariohysterectomy.

Regulation of axonemal motility in demembranated equine sperm.

Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm.

Dexamethasone acutely down-regulates genes involved in steroidogenesis in stallion testes.

In rodents, livestock and primate species, a single dose of the synthetic glucocorticoid dexamethasone acutely lowers testosterone biosynthesis. To determine the mechanism of decreased testosterone biosynthesis, stallions were treated with 0.1mg/kg dexamethasone 12h prior to castration. Dexamethasone decreased serum concentrations of testosterone by 60% compared to saline-treated control stallions. Transcriptome analyses (microarrays, northern blots and quantitative PCR) of testes discovered that dexamethasone treatment decreased concentrations of glucocorticoid receptor alpha (NR3C1), alpha actinin 4 (ACTN4), luteinizing hormone receptor (LHCGR), squalene epoxidase (SQLE), 24-dehydrocholesterol reductase (DHCR24), glutathione S-transferase A3 (GSTA3) and aromatase (CYP19A1) mRNAs. Dexamethasone increased concentrations of NFkB inhibitor A (NFKBIA) mRNA in testes. SQLE, DHCR24 and GSTA3 mRNAs were predominantly expressed by Leydig cells. In man and livestock, the GSTA3 protein provides a major 3-ketosteroid isomerase activity: conversion of Δ(5)-androstenedione to Δ(4)-androstenedione, the immediate precursor of testosterone. Consistent with the decrease in GSTA3 mRNA, dexamethasone decreased the 3-ketosteroid isomerase activity in testicular extracts. In conclusion, dexamethasone acutely decreased the expression of genes involved in hormone signaling (NR3C1, ACTN4 and LHCGR), cholesterol synthesis (SQLE and DHCR24) and steroidogenesis (GSTA3 and CYP19A1) along with testosterone production. This is the first report of dexamethasone down-regulating expression of the GSTA3 gene and a very late step in testosterone biosynthesis. Elucidation of the molecular mechanisms involved may lead to new approaches to modulate androgen regulation of the physiology of humans and livestock in health and disease.

Total RNA isolation from stallion sperm and testis biopsies.

Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility.

Use of a modified Vinsot technique for partial phallectomy in 11 standing horses.

CASE DESCRIPTION: 6 geldings and 5 stallions were evaluated from January 2007 through April 2009 for the following conditions requiring phallectomy: chronic paraphimosis (n = 7), squamous cell carcinoma of the penis (3), and priapism (1). CLINICAL FINDINGS: None of the 7 horses with paraphimosis was able to retract the penis. Chronicity of the paraphimosis in 6 horses ranged from 2 weeks to 2 months and was unknown in the seventh horse. Horses with paraphimosis had been medically treated without success. The horse with priapism had developed the condition secondary to acepromazine administration 2 days prior to referral and was unsuccessfully treated once by intracavernosal administration of phenylephrine and irrigation of the cavernosal tissues prior to surgery. The 3 horses with squamous cell carcinoma of the penis had had the condition for 2 years and had been treated by repeated application of a cryogen or chemotherapeutic agent to the lesions. TREATMENT AND OUTCOME: All 11 horses underwent a partial phallectomy by means of a modified Vinsot technique. Modifications to the original technique included creation of a linear urethrostomy, alteration of the location and shape of the urethrostomy, application of a latex tourniquet, concurrent castration of stallions, and use of the procedure in standing horses. The procedure was technically easy to perform, well tolerated by the horses, and cosmetically acceptable to the owners, and had minimal postoperative complications. Long-term follow-up information was obtained from owners of 10 horses a median of 454 days after surgery; 2 owners reported mild urine scalding as the only adverse effect. CONCLUSIONS AND CLINICAL RELEVANCE: The modified Vinsot technique of partial phallectomy was effective and may be useful for horses that are unsuitable candidates for general anesthesia because of medical or owner financial constraints.

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH.

In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 ± 0.20 mg; mean slice thickness=515.00 ± 2.33 µm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32 °C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17ß (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.

Gene expression in the spermatogenically inactive "dark" and maturing "light" testicular tissues of the prepubertal colt.

In the testis of the 1.5-year-old horse, spermatogenesis initiates locally in grossly light, central areas that contrast with grossly dark, peripheral areas that are as yet inactive in spermatogenesis. Gene expression was compared between "light" and "dark" tissues of 1.5-year-old horse testes to identify mechanisms important to the initiation of spermatogenesis. Microarrays containing human cDNAs were used to assess expression levels of 9132 genes simultaneously in matched pairs of dark and light testis tissues from 3 prepubertal colts. In all 3 analyses, dysferlin (DYS), down-regulated in ovarian cancer 1 (DOC1), and Golgi apparatus protein 1 (GLG1) genes were preferentially expressed in dark tissues, while outer dense fiber of sperm tails (ODF2) and phosphodiesterase 3B (PDE3B) genes were more highly expressed in light testis tissue (>1.7 balanced difference value, Incyte GEM tools software). Expression levels of 88 additional genes appeared to be different between dark and light tissues in 2 of the 3 microarray analyses. The preferential expression of DYS, DOC1, ODF2, and PDE3B genes in dark or light testis tissues was confirmed on Northern blots and localized to cell types by in situ hybridization. Future studies to determine the role of genes regulated during the initiation of spermatogenesis may aid in elucidating molecular mechanisms during this critical time as well as in identifying new therapies for enhancing male fertility.

Effects of gas conditions, time of medium change, and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection.

This study was conducted to evaluate the effects of two different gas conditions (5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2), mixed gas), time of medium change (Day 3 or 4) and ratio of medium to embryo (2, 5 or 10 microl per presumptive zygote) on the development of horse oocytes fertilized by intracytoplasmic sperm injection and cultured in G1.2/2.2 medium. Oocytes from slaughterhouse-derived ovaries were matured in vitro for 24 h and fertilized by injection of frozen-thawed sperm using micromanipulation with a Piezo drill. Presumptive zygotes were randomly assigned to 5% CO(2) in air or mixed gas and fixed after 96 h of culture. Cleavage rates between two gas conditions were similar (67 and 63%), but the mean nucleus number of embryos in the mixed gas treatment was significantly (P<0.05) higher than that of embryos cultured in 5% CO(2) in air (15.2 versus 7.0, respectively). Further experiments were done with mixed gas incubation. Development of embryos was compared after change from G1.2 to G2.2 medium at Day 3 or 4. There was no significant difference in cleavage rate (56 and 65%, respectively) or development to the blastocyst stage after 7 days of culture (5% and 46%, respectively) between embryos changed on different days. To evaluate the effect of the ratio of medium to embryo, zygotes were cultured at a ratio of 2, 5 or 10 microl medium per zygote. There were no significant differences among ratio treatments in rates of cleavage or development to blastocyst.