The use of a 4.7 mg deslorelin slow release implant in male dogs in the field.

OBJECTIVE: Slow-release GnRH agonist implants (SRI) are used for reversible medical downregulation of testicular function in male dogs as an alternative to surgery. The 4.7 mg deslorelin SRI should reduce testosterone after 6-8 weeks and induce castration-like effects for 6 months (mon). However, some individual variation is described in the field in regard to onset and duration of effect. For this reason, we aimed to study the effects of the 4.7 mg deslorelin SRI in a larger cohort. MATERIAL AND METHODS: In total 50 intact, healthy male dogs (12-48 months, mon; 9-40 kg) were treated with a 4.7 mg deslorelin SRI into the umbilical area (TG, n=45) or served as untreated controls (CG, n=5). CG dogs were surgically castrated after measurement of testicular dimensions and blood sampling for testosterone. In TG, SRIs remained for 5 mon in place and subsequently 3-7 male dogs were surgically castrated at removal (week, W 0) or 1, 2, 3, 4, 5, 6, 7, 8 or 10 weeks later. Examination parameters were testicular dimensions (before treatment, at 4, 8, 12 W, 5 mon, weekly until castration), testosterone (before treatment, at 8 W, 5 mon, castration) and testicular histology (castration). RESULTS: Whereas examination parameters did not differ between CG and TG before treatment, testicular volume and testosterone was significantly reduced at all time points during treatment. In all but 3 (8 W) and 2 male dogs (5 mon) testosterone was basal during treatment before removal, whereas the parameters were significantly reduced compared to pre-treatment in the respective dogs. After implant removal, testosterone and testicular volumes increased. However, different to earlier studies, the "restart" was more variable with individual basal testosterone until W7, but also physiological testosterone concentrations in W2. Similarly, histological testicular findings at castration were quite variable: besides an arrest on spermatogonia and spermatocytes, elongated spermatids with normal spermatogenesis were found in individual dogs. CONCLUSION: Our study confirms the efficacy of the deslorelin SRI, but also individual variation especially regarding reversibility of effects on endocrine and germinative testicular function. CLINICAL RELEVANCE: Deslorelin SRIs offer a suitable alternative to surgical castration with individual variation to be considered when used in clinical practice.

Detection of spermatogonial stem cells in testicular tissue of dogs with chronic asymptomatic orchitis.

Chronic asymptomatic idiopathic orchitis (CAO) is an important but neglected cause of acquired infertility due to non-obstructive azoospermia (NOA) in male dogs. The similarity of the pathophysiology in infertile dogs and men supports the dog's suitability as a possible animal model for studying human diseases causing disruption of spermatogenesis and evaluating the role of spermatogonial stem cells (SSCs) as a new therapeutic approach to restore or recover fertility in cases of CAO. To investigate the survival of resilient stem cells, the expression of the protein gene product (PGP9.5), deleted in azoospermia like (DAZL), foxo transcription factor 1 (FOXO1) and tyrosine-kinase receptor (C-Kit) were evaluated in healthy and CAO-affected canine testes. Our data confirmed the presence of all investigated germ cell markers at mRNA and protein levels. In addition, we postulate a specific expression pattern of FOXO1 and C-Kit in undifferentiated and differentiating spermatogonia, respectively, whereas DAZL and PGP9.5 expressions were confirmed in the entire spermatogonial population. Furthermore, this is the first study revealing a significant reduction of PGP9.5, DAZL, and FOXO1 in CAO at protein and/or gene expression level indicating a severe disruption of spermatogenesis. This means that chronic asymptomatic inflammatory changes in CAO testis are accompanied by a significant loss of SSCs. Notwithstanding, our data confirm the survival of putative stem cells with the potential of self-renewal and differentiation and lay the groundwork for further research into stem cell-based therapeutic options to reinitialize spermatogenesis in canine CAO-affected patients.

The In Vitro Contractile Response of Canine Pregnant Myometrium to Oxytocin and Denaverine Hydrochloride.

In pregnant bitches, the response to oxytocin and denaverine hydrochloride in dystocia management is usually poor. To better understand the effect of both drugs on myometrial contractility, the circular and longitudinal muscle layers were examined in an organ bath. For each layer, three myometrial strips were stimulated twice, each with one of three oxytocin concentrations. The effect of denaverine hydrochloride was studied once in direct combination with oxytocin and alone with subsequent oxytocin administration. Contractions were recorded and evaluated for average amplitude, mean force, area under the curve (AUC), and frequency. Effects of different treatments were analyzed and compared within and between layers. In the circular layer, oxytocin significantly increased amplitude and mean force compared to untreated controls regardless of stimulation cycles or concentrations. In both layers, high oxytocin concentrations caused tonic contractions, while the lowest concentration created regular rhythmic contractions. Longitudinal layer tissue responded to oxytocin with a significantly decreased contractility when stimulated twice, presumably a sign of desensitization. Denaverine hydrochloride neither affected oxytocin induced contractions nor showed a priming effect to subsequent oxytocin. Thus, no benefit of denaverine hydrochloride on myometrial contractility was found in the organ bath. Our results suggest a better efficiency of low-dose oxytocin in canine dystocia management.

Insights into Canine Infertility: Apoptosis in Chronic Asymptomatic Orchitis.

Chronic asymptomatic orchitis (CAO) is a common cause of acquired non-obstructive azoospermia in dogs. To understand the impact and mode of action of apoptosis, we investigated TUNEL, Bax, Bcl-2, Fas/Fas ligand, and caspase 3/8/9 in testicular biopsies of CAO-affected dogs and compared the results to undisturbed spermatogenesis in healthy males (CG). TUNEL+ cells were significantly increased in CAO, correlating with the disturbance of spermatogenesis. Bcl-2, Bax (p < 0.01 each), caspase 9 (p < 0.05), Fas, caspase 8 (p < 0.01 each), and caspase 3 (p < 0.05) were significantly increased at the mRNA level, whereas FasL expression was downregulated. Cleaved caspase 3 staining was sporadic in CAO but not in CG. Sertoli cells, some peritubular (CAO/CG) and interstitial immune cells (CAO) stained Bcl-2+, with significantly more immunopositive cells in both compartments in CAO compared to CG. Bcl-2 and CD20 co-expressing B lymphocytes were encountered interstitially and in CAO occasionally also found intratubally, underlining their contribution to the maintenance of CAO. Our results support the crucial role of the intrinsic and extrinsic apoptotic pathways in the pathophysiology of canine CAO. Autoprotective Bcl-2 expression in Sertoli cells and B lymphocytes seems to be functional, however, thereby also maintaining and promoting the disease by immune cell activation.

Involvement of Oxytocin and Progesterone Receptor Expression in the Etiology of Canine Uterine Inertia.

An altered oxytocin and progesterone receptor (OXTR and PGR, respectively) expression was postulated in canine uterine inertia (UI), which is the lack of functional myometrial contractions. OXTR and PGR expressions were compared in uterine tissue obtained during C-section due to primary UI (PUI; n = 12) and obstructive dystocia (OD, n = 8). In PUI, the influence of litter size was studied (small/normal/large litter: PUI-S/N/L: n = 5/4/3). Staining intensity in immunohistochemistry was scored for the longitudinal and circular myometrial layer and summarized per dog (IP-Myoscore). Mean P4 did not differ significantly between PUI (n = 9) and OD (n = 7). OXTR and PGR expressions (ratios) were significantly higher in PUI (OXTR: p = 0.0019; PGR: p = 0.0339), also for OXTR in PUI-N versus OD (p = 0.0034). A trend for a higher PGR IP-Myoscore was identified (PUI-N vs. OD, p = 0.0626) as well as an influence of litter size (lowest PGR-Myoscore in PUI-L, p = 0.0391). In conclusion, PUI was not related to higher P4, but potentially increased PGR availability compared to OD. It remains to be clarified whether OXTR is upregulated in PUI due to a counterregulatory mechanism to overcome myometrial quiescence or downregulated in OD due to physiological slow OXTR desensitization associated with an advanced duration of labor. Identified OXTR differences between myometrial layers indicate the need for further research.

What Happens in Male Dogs after Treatment with a 4.7 mg Deslorelin Implant? II. Recovery of Testicular Function after Implant Removal.

Although deslorelin slow-release implants are widely used in the clinic, detailed published information about the recovery of testosterone concentrations (T), semen quality, and testicular and prostatic volume (TV, PV) after treatment is still missing. This article aims to characterize changes during restart after a five-months treatment and subsequent implant removal. Seven male Beagle dogs were treated with deslorelin (treatment group, TG), and three saline-treated dogs served as controls (CG). Deslorelin implants were removed after five months (D ex), followed by detailed andrological examinations for TV, PV, semen collection, and blood sampling for T-analysis with/without GnRH/hCG stimulation tests. TV, PV, and T increased rapidly after D ex in TG, not differing from CG from D91 (TV), D49 (PV), and D14 (T). The first sperm-containing ejaculates were collected between D49 and 70, whereas the samples were normospermic between D84 and 133. A T increase (>0.1 ng/mL) subsequent to the GnRH/hCG stimulation test was observed from D28/29 onwards, respectively. Histological assessment of testicular tissue at the end of the observational period (D149 after implant removal) revealed normal spermatogenesis. Our data confirm that the restart of endocrine and germinative testicular function is highly variable, but nevertheless, all of the effects induced were reversible.

What Happens in Male Dogs after Treatment with a 4.7 mg Deslorelin Implant? I. Flare up and Downregulation.

Although registered since 2007, knowledge about changes in testosterone concentrations (T), testicular and prostatic volumes (TV, PV) and semen quality, as well as the time point of infertility following treatment with a 4.7 mg deslorelin (DES) slow-release implant, is limited. Therefore, seven sexually mature male dogs were treated with DES (TG); three male dogs treated with saline served as controls (CG). The study assessed local tolerance, TV, PV, semen parameters and T subsequent to GnRH/hCG stimulation in regular intervals. Local tolerance was good. In TG, T was increased right after treatment, but decreased four hours afterwards. Subsequently, TV, PV, semen quality and T decreased over time in TG, but not CG. T was basal (≤0.1 ng/mL) from D28 onwards. Response to GnRH/hCG stimulation was variable, with two TG dogs having increased T post-stimulation on all study days independent of pre-treatment concentrations. A(zoo)spermia in TG was observed from D35-D77 in all seven dogs. Whereas treatment was still effective in six TG dogs five months after implant insertion, it was fully reversed in one dog in terms of T and spermatozoa on the last examination. These results indicate high variation in individual dogs, necessary to consider when advising dog owners.

Chronic Immune-Mediated Orchitis Is the Major Cause of Acquired Non-obstructive Azoospermia in Dogs.

Azoospermia, the lack of spermatozoa in the ejaculate, is the most common finding in infertile but otherwise healthy male dogs and represents an increasing reproductive health issue in men, too. The diagnosis can be further classified as non-obstructive azoospermia and obstructive azoospermia due to an obstruction of the deferent ducts. Although non-obstructive azoospermia comprises more than half of azoospermic cases in men and is a common cause of infertility in the male dog, knowledge of the underlying etiology and pathophysiology is still strongly limited, and much uncertainty exists about the true incidence and possible treatment options. Therefore, this study aims to investigate and characterize infertile canine patients in detail by combining results of andrological examinations (clinical parameters, semen analysis, bacterial examination of semen, and Brucella canis serology), endocrine analysis (luteinizing hormone, testosterone, estradiol-17ß, and thyroid function), analysis of the alkaline phosphatase in seminal plasma, and histological assessment of testicular biopsies of 10 azoospermic dogs. Our results not only verify non-obstructive etiology for 9/10 cases of canine azoospermia but also further identified significant histopathological changes of the testicular tissue with severely disrupted spermatogenesis, including fibrotic remodeling, vacuolization, Sertoli-cell-only syndrome, tubular shadows, and an increase of the interstitial and vascular area. In addition, three dogs showed local and six dogs generalized immune-cell infiltration, indicating chronic immune-mediated orchitis. Only in one case (no. 1) that no immune cells were found, and obstructive azoospermia was suspected due to low alkaline phosphatase activity. Furthermore, the detection of anti-thyroideal antibodies in two dogs indicates an autoimmune thyroid disease and a correlation between the occurrence of thyroidal disorders and azoospermia. Our results confirm previous findings and contribute additional evidence suggesting that chronic immune-mediated orchitis is the major cause of infertility in dogs. Further studies should focus on uncovering underlying inflammatory processes behind spermatogenic failure in these cases and identify possible treatment options to (re-)initialize spermatogenesis.

Testicular effects of a postnatal GnRH antagonist in domestic cats.

The aim of this study was to describe the histological effects of two high postnatal doses of the potent third-generation GnRH antagonist, acyline in the domestic cat testicle. Secondly, the physical, endocrine, and steroidogenic findings of this pharmaceutical protocol are also reported. Twelve postnatal littermate male kittens were administered acyline in a dose of 2.2 mg/100 g SC weekly for 2 weeks (ACY; n = 6), or placebo (PL; n = 6). All the animals were followed up until puberty when they were castrated. Serial faecal samples were collected until the age of 10 weeks for testosterone (T) measurement. The kittens achieved puberty without either age (236.5 ± 19.7 vs. 221.7 ± 23.7 days) or body weight (3.05 ± 0.15 vs. 2.78 ± 0.28 kg, P > 0.05) differences between ACY and PL, respectively. Acyline suppressed faecal T concentrations for 3 weeks (P < 0.01). From the fourth week on, both groups had low concentrations up to the end of the follow-up period (P > 0.05). Histological assessment of the testes showed that ACY cats presented a reduced height of the epithelium (P < 0.01) due to the diminished number of germinal cells accompanied by an enlarged luminal area (P < 0.01) with cellular debris (P < 0.01). The immunostaining of P450c17 also appeared partially diminished in ACY testes.

Investigations on the potential role of prostaglandin E2 in canine uterine inertia.

Prostaglandin (PG) E2 plays a crucial role in the endocrine network of canine parturition and we hypothesized that PGE2, 15-hydroxyprostaglandin dehydrogenase (HPGD) and PG-transporter (PGT) might be involved in the development of primary uterine inertia (PUI). We investigated PTGE synthase (PTGES), PTGE receptors 2/4 (PTGER2/4), HPGD and PGT expression on the mRNA- and protein-level in interplacental (IP) and uteroplacental (UP) tissues of bitches presented with dystocia undergoing emergency caesarean section. Groups were formed retrospectively based on strict criteria: PUI (n = 12; small/normal/large litter - PUI-S/N/L: n = 5/4/3), and obstructive dystocia (OD, n = 8). Respective mRNA expressions (ratio) between PUI and OD in IP and UP, between PUI dogs with different litter sizes, between PUI-N and OD in IP, and overall between IP and UP were compared. PTGES, PTGER2, PTGER4, HPGD and PGT mRNA expressions did not differ significantly between PUI and OD in IP or UP. PUI-N PTGES mRNA expression was higher than PUI-S/L (P = 0.0203/P = 0.0186) and OD (P = 0.0314). Higher PTGES (P = 0.0112) and a tendency for higher PTGER2 (P = 0.059) mRNA-expressions were detected in UP versus IP. Other than hypothesized, we did not find a difference in PGE2 production and signaling between PUI and OD, indicating that altered uterine PTGES, PTGER2, PTGER4, HPGD and PGT expression was likely not causative for PUI. However, higher PTGES expression in PUI-N compared to OD might point to a possible role of PGE2 during the course of parturition. Higher PTGES expression in PUI-N compared to PUI-S/L indicates an influence of litter size, the underlying cause and biological relevance of which remain to be clarified.

Do uterine PTGS2, PGFS, and PTGFR expression play a role in canine uterine inertia?

The aetiology of primary uterine inertia (PUI), which is the most common cause of canine dystocia, is still not elucidated. Prostaglandins (PGs) play a crucial role in parturition. We hypothesized that the expression of prostaglandin endoperoxidase synthase 2 (PTGS2), PGF2α synthase (PGFS), and corresponding receptor (PTGFR) is altered in PUI. We investigated PTGS2, PGFS, and PTGFR mRNA expression, and PTGS2 and PGFS protein expression in interplacental (IP) and uteroplacental sites (UP) in bitches with PUI, obstructive dystocia (OD), and prepartum (PC). PTGS2, PGFS, and PTGFR mRNA expression did not differ significantly between PUI and OD (IP/UP). PTGFR ratio in UP was higher in PC than in OD (p = 0.014). PTGS2 immunopositivity was noted in foetal trophoblasts, luminal and superficial glandular epithelial cells, smooth muscle cells of both myometrial layers, and weakly and sporadically in deep uterine glands. PGFS was localized in luminal epithelial cells and in the epithelium of superficial uterine glands. PTGS2 and PGFS staining was similar between PUI and OD, while PGFS protein expression differed between OD and PC (p = 0.0215). For PTGS2, the longitudinal myometrial layer of IP stained significantly stronger than the circular layer, independent of groups. These results do not support a role for PTGS2, PGFS, and PTGFR in PUI. Reduced PGFS expression in IP during parturition compared with PC and the overall lack of placental PGFS expression confirm that PGFS is not the main source of prepartal PGF2alpha increase. The difference in PTGS2 expression between IP myometrial layers warrants further investigation into its physiological relevance.

Expression of prostaglandin (PG) D synthase lipocalin and hematopoietic type and PG D receptor during restart of spermatogenesis following downregulation using a slow release GnRH agonist implant in the dog.

Prostaglandin D and the associated prostaglandin D synthases (PGDS) and receptor (DP) are considered to be involved in spermatogenesis. However, the interplay of the PGDS-DP system in male reproduction is far from being understood. The expression of PGDS lipocalin (L) and hematopoietic (H) type and DP was studied in the GnRH agonist-downregulated canine testis (week, w 0) and during recrudescence of spermatogenesis after implant removal (w 3, 6, 9, 12). H-PGDS, L-PGDS and DP were present in the adult (CG), juvenile (JG) and downregulated canine testis at the mRNA level. PGDS immunohistochemistry revealed positive staining in the cytoplasm of Leydig cells (LCs) of all samples i.e., no difference between groups. mRNA expression (ratio) of L-, H-PGDS and DP did not differ between groups w 0-12 and CG. In contrast, significant differences were found for L-PGDS (p = 0.0388), H-PGDS (p < 0.001) and DP (p < 0.001) for the groups at downregulation (w0, suprelorin group, SG, profact group, PRG) compared with the control groups (JG, CG). L-PGDS expression was lowest in JG, whereas H-PGDS was significantly lower in CG compared with JG and at downregulation (p < 0.001 to p < 0.01). The highest ratio for H-PGDS and DP was observed in the dogs treated with buserelin acetate (PRG). Our data show that the PGDS-DP system is expressed in juvenile and adult canine testes and that downregulation of the testicular endocrine and germinative function significantly affects H-PGDS, L-PGDS and DP mRNA expression indicating a role in the regulation of spermatogenesis.