Expression of PTGS2, PGFS and PTGFR during downregulation and restart of spermatogenesis following GnRH agonist treatment in the dog.

Prostaglandins (PGs) and prostaglandin endoperoxide synthase (PTGS) are considered to be relevant for spermatogenesis and steroidogenesis. PTGS2, prostaglandin F synthase (PGFS) and PGF receptor (PTGFR) are investigated in the adult male dog using the model of the GnRH-agonist implant downregulated canine testis and its subsequent restart of spermatogenesis following abolition of treatment (3, 6, 9, 12 weeks after implant removal). On the mRNA level (ratio), PTGS2, PGFS and PTGFR expression did not differ between downregulation, different stages of recovery of spermatogenesis and untreated adult controls (CG). On the protein level, Sertoli and Leydig cells in all samples and some peritubular cells stained immunopositive for PTGS2. In the tubular compartment, the percentage of the PTGS2-immunopositive area (PIA) and the mean PTGS2-staining intensity (gray scale, GS) did not differ between groups but in the interstitial compartment, the PIA (p = 0.0494) and the GS (p < 0.0001) were significantly upregulated during early recrudescence (week 3/6). Comparing downregulation by two GnRH-agonist implants with juvenile controls (JG) and CG, the mRNA expression (ratio) did not differ. In the tubular compartment, the GS (p = 0.0321) was significantly higher at downregulation compared to CG and in the interstitial compartment, the PIA (p = 0.0073) and the GS (p = 0.0097) were significantly higher in JG compared to downregulation/CG. PTGS2, PGFS and PTGFR mRNA and PTGS2 protein are regularly expressed in the adult, juvenile and downregulated canine testis and downregulation and subsequent recrudescence affect PTGS2 protein expression mainly in Leydig cells. PTGS2 expression in the downregulated testis resembles the one in seasonal Syrian hamster but not juvenile canine testis.

Bacteriological findings in the canine uterus during Caesarean section performed due to dystocia and their correlation to puppy mortality at the time of parturition.

Canine intrauterine bacteriological flora during dystocia is unknown. Thus, frequency (bacterial growth (not) detected), quality (species and number of different bacterial isolates) and quantity (colony-forming units) of intrauterine bacteria in relation to in utero foetal death in 50 bitches undergoing emergency Caesarean section were investigated. Bacterial growth was quantified from single colonies, (+) (0.5), to strong growth, +++ (3) and was observed in 34 bitches (68%), with Staph. epidermidis (n = 12), Staph. intermedius-group (n = 7), ß-haemolytic streptococci (n = 6), Staph. aureus, α- and γ-haemolytic streptococci (n = 4 each) being most common and one to four bacteria per sample. Regarding the quantity, most often (n = 46) low growth was identified. In bitches with living pups only (group I), mean number of isolates was 0.78 ± 0.83 compared to 1.60 ± 1.10 (living + stillborn pups, group II) and 1.0 ± 1.15 (stillborn pups only, group III) and mean bacterial growth in groups I/II/III was + (1.0, quantity), + (1.4) and ++ (1.6). Taking just positive samples into consideration, mean number of bacterial isolates was significantly higher in group II compared to I (p = .0088). We concluded that the canine uterus cannot be considered free of bacteria during dystocia. Mean numbers of different bacterial isolates and quantity of bacterial growth are higher in bitches with in utero foetal death.

Blood calcium, glucose and haematology profiles of parturient bitches diagnosed with uterine inertia or obstructive dystocia.

Bitches with dystocia most often present with clinical signs of uterine inertia (UI). The aetiology of myometrial dysfunction in most of these cases is still not elucidated. We compared blood ionized calcium (iCa) and glucose concentrations in bitches diagnosed with primary UI (PUI, n = 14), secondary UI (SUI, n = 6) or obstructive dystocia (OD, n = 6), and we described their haematology profiles. Bitches diagnosed with UI had a patent birth canal and delivered no puppies yet (PUI) or only part of the whole litter (SUI). The OD group had no UI and showed strong abdominal contractions. Blood iCa did not differ between the PUI, SUI and OD groups and was not influenced by litter size. There was a significant positive relationship (R2  = .241, p = .013) between iCa concentrations and the dam's body weight. Glucose concentrations were also not significantly different between dystocia groups or influenced by body weight and litter size. Hypocalcaemia was detected in 11 bitches, and hypoglycaemia in two bitches. Pregnancy-associated anaemia was seen in about one-third of the bitches. Eight of 12 dogs had increased platelet counts, and ten had leukocytosis with mature neutrophilia. Although iCa did not differ between dystocia groups, low concentrations may have contributed to the development of UI in some of the small size bitches. Hypoglycaemia was uncommon, and therefore, we consider low glucose concentrations not to have played an important role in the pathogenesis of UI in our study population. Pregnancy-associated anaemia, thrombocytosis, leukocytosis and mature neutrophilia were common findings in otherwise healthy bitches diagnosed with different forms of dystocia.

Long-term effects of GnRH agonists on fertility and behaviour.

This review aimed to summarize the present knowledge about the effects of GnRH agonist slow-release implants (GnRH A-SRI) on fertility and behaviour in male and female dogs and cats with special focus on deslorelin. Following an initial stimulation of gonadotropin and testosterone secretion possibly associated with an improved semen quality, GnRH A-SRI induce long-term depression of fertility in male dogs and cats with, however, a large individual variation in onset and duration of efficacy especially in cats. The GnRH A-SRI furthermore interfere with testosterone-dependent/affected behaviour; a significant positive effect in reducing sexual behaviour and libido, hypersexuality, intermale dominance and excessive territorial urine marking has been described. Rates of improvement of the respective behaviour are comparable to those after surgical castration, making GnRH A-SRI a valuable option to predict castration-related effects on behaviour and to identify animals where surgical castration will not be beneficial. No effect has been seen in reducing aggression towards humans indicating the need for behavioural therapy to control this problem. Effects on spermatogenesis, steroidogenesis and behaviour have by now been shown to be fully reversible. Knowledge in females is more limited, and particularly, the initial induction of a possibly fertile oestrus and individual variation in duration of efficacy remain problems in bitches and queens treated for suppression of fertility. However, long-term suppression of oestrous cycle and fertility seems to be possible with induced effects shown to be reversible including restoration of normal fertility after the end of efficacy/GNRH A-SRI removal.

Effect of dietary vitamin E and selenium supplementation on semen quality in Cairn Terriers with normospermia.

Among others, selenium (Se) and vitamin E (VitE) have been provided to dogs to improve semen quality. However, scientific evidence documenting an effect in dogs is lacking. The aim of this study was to investigate the effect of supplementation of these antioxidants on various ejaculate parameters in a randomized, double-blinded trial using Cairn Terrier males exhibiting normal seminal quality parameters. Three dogs each were fed a standardized diet and supplemented with 0.1 mg Se, 100 mg VitE or 0.1 mg Se + 100 mg VitE/dog for 3 months. Ejaculate analyses (volume, progressive motility, vitality, morphology, concentration) were performed before inclusion (D0) and after 1, 2 and 3 months (+1, +2, +3). At the same time, glutathione peroxidase (GSH-PX) and VitE in seminal plasma (SP) and GSH-PX in blood samples were determined. Vitamin E levels in SP were below the detection limit (1.0 mg/L) in all samples. GSH-PX in blood (164.0-2794.4 IU/L) and SP (18.4-4326.0 IU/L) was highly variable. Supplementation only significantly affected the total percentage of sperm head abnormalities (p = .011). Time significantly affected the percentage of morphologically abnormal sperm (p = .025), sperm head abnormalities (p = .007), proximal droplets (p = .001) and GSH-PX in SP (p = .015). Additionally, a significant interaction between time and group was identified for the percentage of membrane-intact sperm (p = .048), head abnormalities (p = .018), acrosomal defects (p = .043) and proximal droplets (p = .002). Although some effects could be identified for selected parameters, we failed to identify a clear trend about how a 3 months VitE and/or Se supplementation affects semen parameters in normospermic Cairn Terriers.

Suppression of fertility in adult cats.

Cats are animals with highly efficient reproduction, clearly pointing to a need for suppression of fertility. Although surgical contraception is highly effective, it is not always the method of choice. This is predominantly because it is cost-intensive, time-consuming and irreversible, with the latter being of major importance for cat breeders. This article reviews the use of progestins, scleroting agents, immunocontraception, melatonin, GnRH antagonists and finally, GnRH agonists, in adult male and female cats in detail, according to the present state of the art. By now, various scientific and clinical options are available for the suppression of fertility in adult cats and the decision as to which should be chosen - independent of the legal registration of any state - depends on different facts: (i) feral or privately owned animal? (ii) temporary or permanent suppression of fertility wanted/needed? (iii) sex of the animal? New effective and available methods for hormonal contraception include melatonin implants for short-term post ponement of oestrus in adult queens and slow-release GnRH-agonist implants containing deslorelin (Suprelorin(®) ) for short- and long-term contraception in male and female companion and breeding cats.

The spatio-temporal distribution of aromatase cytochrome in ovary throughout the canine oestrous cycle.

CONTEXT: New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. AIMS: Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. METHODS: Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. KEY RESULTS: Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. CONCLUSIONS AND IMPLICATIONS: Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus.

Status of the down-regulated canine testis using two different GNRH agonist implants in comparison with the juvenile testis.

Testicular function in the dog was down-regulated using two different GNRH agonist implants, with adult and juvenile testes serving as controls. Treatment resulted in an increased percentage of the interstitial area and decreased area of Leydig cell nuclei. Expression of StAR and the steroidogenic enzymes cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1) and cytochrome P450 17α-hydroxylase-17,20-lyase (P450c17, CYP17A1) in Leydig cells was blocked at the mRNA and protein level, showing no differences between the two agonists. Staining for androgen receptor (AR) by immunohistochemistry was positive in Sertoli, Leydig and peritubular cells and some spermatogonia, with in situ hybridization confirming expression in Sertoli cells. At the mRNA level, expression of AR was not affected; however, translation was blocked (reduced percentage of AR-positive Sertoli cells), with the number of nuclei in basal position being decreased. In the juvenile testes, mRNA expression of StAR, CYP11A1 and CYP17A1 was higher compared with the other groups but distinctly lower for the AR. At the protein level, the expression was at the limit of detection for StAR; AR-positive Sertoli cells were not detected. Our observations show that the down-regulated testis is different from the juvenile one rather resembling the testicular status in seasonal breeders out of season.

Retrospective analysis of canine semen evaluations with special emphasis on the use of the hypoosmotic swelling (HOS) test and acrosomal evaluation using Spermac(®).

Routine semen evaluation includes volume, motility, vital staining for live-dead ratio and pathomorphology including Spermac(®) staining for evaluation of the acrosome. In recent years, depending on the species, also the hypoosmotic swelling (HOS) test has been applied routinely for evaluation of semen quality. In this respect, a significant correlation between the ability of spermatozoa to swell in HOS test and the fertilizing ability has been reported. Also for evaluation of dog semen, reference has been made to the HOS test; however, its correlation to conventional semen parameters so far is discussed controversially. In the present study, the results of 400 semen examinations from stud dogs presented at our clinic were evaluated for their correlations between conventional semen parameters (motility, live/dead ratio, pathomorphology), conventional semen parameters and age, Spermac(®) staining and HOS test, respectively. We found a significant correlation of age and sperm concentration (p < 0.01), total sperm count (p < 0.0001), percentage of progressively motile sperm (p < 0.01) and live spermatozoa (p = 0.012). Furthermore, several correlations between conventional semen parameters were identified. Percentage of sperm with normal acrosome identified by Spermac (®) staining correlated significantly with live spermatozoa (p < 0.0001) and percentage of progressively motile sperm (p < 0.01). A significant correlation was proven between curled tails in HOS test and age (p < 0.001), motility (p < 0.0001), live sperm (p < 0.0001), acrosomal status (p < 0.05), pathomorphology (p < 0.0001) and sperm concentration (p = 0.011). These results indicate that Spermac(®) staining and the HOS test are useful in improving canine semen analysis.

Restart of steroidogenesis in dogs during recrudescence of testicular function following downregulation with a GnRH-agonist implant.

To date, no details are available concerning the restart of steroidogenesis following the downregulation of testicular endocrine and germinative function by gonadotrophin-releasing hormone (GnRH)-agonist implants. This restart was assessed by determining the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase,17,20-lyase (P450c17). The re-establishment of steroidogenesis was initiated by the removal of the GnRH-agonist implant (18.5 mg azagly nafarelin, Gonazon) at 5 months after treatment. Testes were removed at 3-week intervals (weeks 0-24) and four groups were formed according to the stage of spermatogenesis as revealed by the most developed germ cells observed (developmental group [DG] spermatocytes to DG elongated spermatids). Five dogs served as untreated controls. Positive immunostaining for StAR, P450scc and P450c17 was restricted to Leydig cells. Western blot indicated the specifity of the respective antibodies with hints of a expression of canine-specific P450scc and P450c17 proteins. A significant effect of group was observed for a percentage of the immunopositive area (PIA) as an indicator of active Leydig cells for StAR (P<0.05), P450scc (P<0.001) and P450c17 (P<0.001), with PIA being lowest for the DG spermatocytes. With regard to the strength of the immunopositive signal, a significant effect of group was found for P450scc (P<0.01) and P450c17 (P<0.05), with the lowest intensity being observed in DG spermatocytes. At the mRNA level, the upregulation from DG spermatocytes to DG round spermatids was clearly evident but was only significant for P450scc (P<0.05). Thus, downregulation affects the whole cascade of steroidogenesis, whereas withdrawal of inhibition results in a rapid restart, in part indicating a rebound phenomenon.

Development of semen quality following reversible downregulation of testicular function in male dogs with a GnRH agonist implant.

Slow-release GnRH agonist implants have shown to be an effective and reversible alternative to surgical castration. Testicular function is downregulated with an arrest of spermatogenesis on the level of spermatogonia/primary spermatocytes but is fully restored after abolition of downregulation. Aim of this study was to assess the quality of ejaculates after active abolishment of downregulation by implant removal and to follow recrudescence of spermatogenesis. Five dogs - which served as their own controls - were treated with a slow-release implant containing the GnRH agonist azagly-nafarelin. Implants were removed during full downregulation (testosterone <0.1 ng/ml), and attempts to collect ejaculates started from week 4 onwards to week 29. First ejaculates could be obtained between weeks 8 and 12 with the first fully elongated spermatozoa observed in week 10. Volume, %motility and total sperm count increased and %pathomorphology decreased during the course of the study with all ejaculates being in the normal range by week 29. Our data indicate that onset of recrudescence of spermatogenesis coincides with the first testosterone increase after active abolishment of downregulation. Semen quality was fully regained with a significant improvement of %pathomorphology (p < 0.05) and a tendency of improved %motility. However, these observations on an improved semen quality need further validation and no final conclusions can be drawn yet.

The use of a slow release GnRH-agonist implant in female ferrets in season for oestrus suppression.

The jill is a long-day breeder with a constant oestrus without mating. Persistent oestrogen production results in clinical signs of hyperoestrogenism including pancytopenia and death if untreated. As spaying is thought to be related to the development of hyperadrenocorticism, a non-invasive, safe and effective long-term treatment is needed for oestrus suppression in jills. Seven jills in oestrus were treated with a 4.7mg deslorelin implant. Blood samples for estradiol-17ß (E2) and progesterone (P4) determination were obtained before as well as 4 and 8 weeks after treatment; data are given as geometric mean (deviation factor, DF). Mean E2 was 280.2 pmol/L (1.7) before, 36.4 pmol/L (1.4) 4 and 21.6 pmol/L (1.1) 8 weeks after treatment (p < 0.0001). P4 before treatment was 1.4 nmol/L (2.6), 57.8 nmol/L (1.9) on week 4 and 3.8 nmol/L (2.6) on week 8 (p < 0.0001) indicating ovulation had occurred after implant insertion. Oestrous signs within the observation period of up to 32 months remained suppressed.

Immune cell characterization in spontaneous autoimmune orchitis in dogs.

With a prevalence of up to 35% in dogs with reproductive problems, azoospermia is one of the most important reasons for male infertility. Non-obstructive azoospermia, without clinical symptoms, but histopathological damage of the testicular tissue and immune cell infiltration is referred to as spontaneous autoimmune orchitis (AIO) in the literature. Published cases in dogs describe immune cell infiltration; however, there is no consent about the involved immune cell types. We aimed to characterize immune cells in testicular biopsies of dogs with AIO (n = 9) and to compare them to those in testicular specimens from healthy control dogs with normospermic ejaculates (CG; n = 5). Immunohistochemistry was performed using specific antibodies against CD3, PAX5, MAC387, IgG and IgM to proof the presence of T lymphocytes, B lymphocytes, macrophages and early and late plasma cells, respectively. Presence of immune cells in healthy testicular tissue was low and restricted to T lymphocytes and macrophages in the interstitium with the latter also being found within the blood vessels. Different to this, AIO samples revealed presence of all investigated immune cells, underlining lymphoplasmacytic nature of chronic asymptomatic immune-mediated orchitis. Canine spontaneous AIO is characterised by a significantly increased number of immune cells, namely ≥33 immune cells/mm2 (sensitivity/specificity: 100% based on our data). The pathogenesis of canine AIO is hypothesized to be as follows: 1. Macrophages initiate AIO via T lymphocyte activation. 2. T lymphocytes lead to a "delayed type immunological response" and development of AIO. 3. Invaded B lymphocytes later differentiating to plasma cells are responsible for the second humoral immunological response and cause progression of AIO. Different to the situation in CG, T lymphocytes and plasma cells were identified within the seminiferous tubules indicating that disruption of spermatogenesis in AIO might be related to invading immune cells. Testicular biopsies provide an essential tool in the diagnosis of spontaneous AIO.

[Squeezing of the spermatic cord using a Pean clamp is not suitable for elimination of testicular function in the rabbit]. / Die Quetschung des Samenstrangs eignet sich beim Kaninchen nicht zur Ausschaltung der Hodenfunktion.

OBJECTIVE: The aim of this study was to evaluate the effect of three different methods for squeezing of the spermatic cord as bloodless castration in rabbits according to the "Burdizzo"-method used for ruminants. MATERIALS AND METHODS: In 18 anaesthetized male rabbits (group 1-3) spermatic cords were squeezed twice using three different methods according to Burdizzo: using a Pean haemostat for 3 minutes (group 1, n=10) or 10 minutes (group 2, n=4) or by use of a Doyen intestine clamp for 3 minutes (group 3, n=4). Seven males serving as controls underwent surgical castration under general anaesthesia. Animals were daily examined until day 7, and on days 14 and 90. Blood samples for testosterone measurement were taken on days 1 and 90. On day 90, all animals of groups 1-3 were surgically neutered and testis examined histologically in comparison with group 4. RESULTS: A swelling of the squeezing site was obvious in all animals of group 1-3 which was - like pain reaction - most obvious in group 1. Temporary changes in testicular consistency, as well as inflammation signs were observed in testes, epididymides and spermatic cords. During the course of the study, testes size increased (p<0.0001) independently of the group. Testosterone was within the physiological range in group 1-3 and differed significantly from group 4 (p=0.0002). Histology revealed normal spermatogenesis and fully elongated sperm in testes and epididymides. CONCLUSION: None of the three bloodless methods used led to testicular shrinking, basal testosterone concentration and disturbed spermatogenesis which would be a suitable marker for testicular atrophy indicating castration. CLINICAL RELEVANCE: Although bloodless castration seems to be an interesting alternative to surgery in rabbits, none of the methods used was successful in inducing castration effects.

Induction of abortion with aglepristone in cats on day 45 and 46 after mating.

The aim of this study was to test for the efficacy and safety of the use of aglepristone for pregnancy termination on day 45 in cats. Six healthy cats were treated with 10 mg/kg aglepristone sc on day 45 and 46 after mating; six other cats served as untreated controls. The effect of treatment was monitored by general examination, vaginal cytology, ultrasonography and blood sampling for haematology and progesterone determination. Besides, interoestrus interval and next pregnancy including litter size were recorded. The efficacy of treatment was approximately 67% (4/6) with abortion occurring 4-7 days after the first injection and a sanguineous discharge and erythrocytes in vaginal smears for at least 6 days afterwards. The two treated cats that did not abort gave birth to two kittens on day 67 and had a stillbirth of a single kitten on day 71, respectively. As expected enlargement of the mammary glands and lactation were observed in all treated cats. No other treatment-induced side effects were observed. Progesterone levels at abortion were high (30-140 nmol/l), but were decreased on day 55. Aglepristone treatment did not affect fertility in following cycles. Finally, it can be concluded that late-term pregnancy termination with aglepristone is possible but due to a success rate of 67% an ultrasonographical examination 7 days after treatment is an inherent necessity to control the effect of treatment.

[Luteal insufficicency in the bitch - symptoms, diagnosis, consequences and therapy. A review of the literature]. / Luteale Insuffizienz bei der Hündin - Symptome, Diagnose, Folgen und Therapie. Eine Übersicht der Literatur.

Insufficient progesterone synthesis, so called hypoluteoidism or luteal insufficiency, is one of the possible reasons for infertility in the bitch. Confirming this diagnosis may be difficult if the dynamic changes of progesterone during the reproductive cycle are not taken into account. The bitch ovulates at progesterone concentrations of about 5-10ng/ml (15.7-31.4 nmol/L). The concentrations increase to >25ng/mL (78.5 nmol/L) within 3-4 weeks and then subsequently decrease after a plateau of 7-14 days. In the pregnant bitch, progesterone rapidly drops to <2ng/ml (6.3 nmol/L) approximately 24-48 hours before parturition induced by PGF2α secretion. Luteal insufficiency, characterized as an early decrease of progesterone secretion, is most commonly observed between days 20 and 35 of pregnancy. Progesterone concentrations of approximately 2ng/ml (6.3nmol/L) are thought to be necessary for maintaining pregnancy. Lower concentrations result in resorption and abortion, respectively. In bitches suspected to have luteal insufficiency, weekly progesterone determinations using quantitative tests should be performed from 5-7 days after mating or at least from the date of early pregnancy diagnosis. The frequency has to be increased in the case of progesterone concentrations below 10ng/ml (31.4 nmol/L). Progesterone administration is indicated in the case of viable foetuses and progesterone concentrations <5 ng/ml (15.7 nmol/L) before day 58/60 of pregnancy or after the detection of a rapid progesterone decline of about 10-15ng/ml (31.4-47.1 nmol/L) between days 20 and 35 with viable foetuses in the sonographic examination. Either natural or synthetic progestins can be used. However, synthetic progestins have a greater risk potential for side effects (masculinisation of female puppies and cryptorchidism in male puppies), especially when administered between days 20 and 35 of pregnancy. Administration of natural progesterone should be stopped 2-3 days before expected parturition otherwise it would result in a prolonged duration of pregnancy with dystocia and stillbirth.

Uterine expression of smooth muscle alpha- and gamma-actin and smooth muscle myosin in bitches diagnosed with uterine inertia and obstructive dystocia.

Primary uterine inertia (PUI) is the most common type of dystocia in dogs. We hypothesized that PUI develops because of lower than normal expression of the basic contractile elements in the uterus, i.e., smooth muscle (SM) α- and γ-actin and SM-myosin, and that the expression of these proteins is influenced by the number of fetuses present in utero. Full-thickness inter-placental uterine biopsies were collected during Cesarean sections from dogs with PUI (n = 11), and from bitches with obstructive dystocia (OD) still presenting strong labor contractions (designated as the control group, n = 7). Relative gene expression was determined by semi-quantitative real-time (TaqMan) PCR, and protein localization by immunohistochemistry. Gene expression between PUI and OD bitches, and between PUI bitches carrying small, large, or average number of fetuses according to their breed, were compared. Uterine SM-γ-actin and SM-myosin mRNA levels were significantly higher in PUI than in OD dogs, while SM-α-actin did not differ. PUI bitches carrying large litters had lower uterine SM-γ-actin gene expression than those with small litters (P = 0.008). Immunostaining for SM-actin isoforms and SM-myosin was present in the myometrium, and localization pattern and staining intensity appeared similar in the PUI and OD groups. All proteins stained in blood vessels, and SM-γ-actin was also present in endometrial luminal and glandular epithelium. In conclusion, higher uterine SM-γ-actin and SM-myosin gene expression in PUI bitches, compared with OD dogs, might be an indication of abnormal progression with labor. Whether this is the cause of PUI due to an intrinsic error of the myometrium not becoming committed to labor, or the consequence of inadequate endocrine or mechanical stimuli, is not clear. Litter size was previously shown to be one of the risk factors for the development of uterine inertia in dogs, and our findings suggest possible differing uterine pathophysiology of PUI with respect to litter size.

Reversible downregulation of endocrine and germinative testicular function (hormonal castration) in the dog with the GnRH-agonist azagly-nafarelin as a removable implant "Gonazon"; a preclinical trial.

Downregulation of anterior pituitary GnRH-receptors by application of a slow release GnRH-implant offers an effective and reversible alternative to surgical castration of the male dog. Aim of the present study was to test the efficacy and the underlying mechanisms of a new non-biodegradable controlled-release device implant (Gonazon((R)), Intervet, containing 18.5mg of the GnRH-agonist Azagly-Nafarelin). Eight male beagle dogs were implanted s.c. at the para-umbilical region. In four dogs implant removal was after 180 days (group 1), in the other four dogs after 365 days (group 2). Eleven weeks after implantation availability of LH was reduced (p<0.0001) by 70%. After an initial increase lasting for about 4 days, testosterone (T) and estradiol (E2) concentrations decreased (p<0.0001) to basal levels within 17.5+/-8.4 days. Size of testes was decreased by about 82% after 17 weeks, size of prostate by about 46% after 5 weeks (p<0.0001). Five to 7 weeks after implantation all dogs were aspermic. Testosterone and estradiol concentrations, together with testicular and prostatic size remained suppressed in all dogs in group 1 and one dog of group 2 until implant removal. The other three dogs of group 2 escaped from down-regulation between 223 and 324 days. Effects on the availability of LH, T, E2 and on testicular and prostatic size were fully reversible after implant removal or escape from down-regulation. In six dogs semen quality was back to pre-treatment values after about 29 weeks, however, one dog developed oligozoospermia while another one stayed azoospermic, probably due to an obstruction within the epididymal duct.

Recrudescence of spermatogenesis in the dog following downregulation using a slow release GnRH agonist implant.

The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH-agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross-sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig-cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [X(g) (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre-treatment values following implant removal.