Ovarian and uterine changes during the oestrous cycle in female dogs.

CONTEXT: An accurate staging of sexual cycle is essential for the optimum timing of medical interventions. AIMS: Here, an updated insight into clinical, endocrinological and vagino-cytological parameters, and their correlation with histomorphology of ovarian and uterine tissue samples is presented. METHODS: Samples from 39 dogs were collected at various stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10), according to clinical observations. Final allocation of samples was done after histomorphological evaluation of all tissues. Peripheral oestradiol-17ß (E2) and progesterone (P4) concentrations were measured, P4 by both chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). KEY RESULTS: Differences were observed between determination of the stage of the oestrous cycle, either by clinical, endocrinological or histomorphological evaluation. Individuals considered to be in clinical and endocrinological oestrus, had entered the luteal phase according to histomorphology. P4 concentrations measured by two different assays differed, underlying the importance to understand that absolute P4 concentrations may deviate depending on the used assay. Comparison of E2 and P4 concentrations is suggested to be useful when defining the transition from early follicular phase to the time of ovulation. CONCLUSIONS AND IMPLICATIONS: Based on parallel histomorphological observations, combined with clinical and endocrinological findings on the same individuals, the present study emphasises that an accurate classification of the stage of the cycle in female dogs based solely on clinical and endocrinological assessments can be difficult. The histomorphological findings presented herein provide new insights into the transitional phases between the different stages of the oestrous cycle in the dog.

Rhodesian Ridgebacks have an increased risk to develop benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is an age-dependent primarily non-inflammatory enlargement of the accessory gland in the intact dog. The aim of the present study was to control a previously raised suspicion of a breed-related higher incidence of BPH in dogs of the Rhodesian Ridgeback breed. For this, 18 Labrador Retrievers/LR and 20 Rhodesian Ridgebacks/RR were assigned to the age groups 18-24 months (n = 12), 25-48 months (n = 13) and 49-72 months (n = 13). Prostate gland status was determined by rectal palpation, B-mode ultrasound, calculation of the prostate gland volume and semen analysis regarding haemospermia and was classified according to blood plasma concentrations of canine prostate-specific arginine esterase (CPSE) (normal ≤ 60 ng/ml, increased ≥ 61 ng/ml; Pinheiro et al., 2017). Concentrations of testosterone, 5α-dihydrotestosterone and estradiol were analysed in peripheral blood serum or plasma for detecting breed-specific conditions regarding the endocrine metabolism. Prostatic volume was significantly larger in RR irrespective of the CPSE status. In RR, BPH occurred more frequently and started at an earlier age compared with the LR. Breed-related specificities in steroid metabolism in the RR were indicated by correlations of 5α-dihydrotestosterone and estradiol with age and of testosterone with prostate gland volume. Although the incidence of sonographic signs of BPH and haemospermia did not fit with normal and increased CPSE concentrations, a breed-specific higher incidence of BPH in the RR breed could be clearly verified.

An investigation on the relevance of prolactin, insulin-like growth factor-1 and 25-hydroxyvitamin D3 (25-OHD3 ) in canine benign prostatic hyperplasia in a predisposed breed model.

Serum concentrations of prolactin (PRL), insulin-like growth factor-1 (IGF-1) and 25 hydroxyvitamin D3 (25-OHD3 ) were analysed to investigate their possible involvement in the pathogenesis of benign prostatic hyperplasia (BPH). For this, dogs of the Rhodesian Ridgeback (RR) breed were used because of a verified breed disposition for the development of BPH. Labrador Retrievers (LR) served as controls. The prostate gland status was characterised by the prostate gland volume, clinical signs of BPH (haemospermia and sonographic findings) and the plasma concentration of canine prostate-specific arginine esterase (CPSE). Breed specificity in the RR was expressed by a correlation of PRL with breed (p < 0.05). Similar relationships existed in the dogs with normal CPSE (CPSEn) with respect to the IGF-1 concentrations (LR: p < 0.05). The latter were negatively correlated with prostatic volume and age (both p < 0.05). Concentrations of 25-OHD3 were tendentially (p = 0.18) lower in the RR with increased CPSE (CPSEi) compared with the CPSEn LR and RR showing clinical signs of BPH. A negative correlation between serum 25-OHD3 and age (p < 0.05) existed in the CPSEi RR. Proof of 25-OHD3 in prostatic secretion proved to be a breed specific feature in the RR (p < 0.0001). For all RR dogs showing clinical signs of BPH, a close to significant (p = 0.06) positive correlation with prostate gland volume was found. The results of the present study reveal no clear hints towards the significance of PRL and IGF-1 in the pathogenesis of canine BPH. In the RR breed there were indications of a causal relationship with age-dependent changes in the vitamin D metabolism. The data suggest the possibility of preventing or treating canine BPH by administering vitamin D or substances involved in the intraprostatic vitamin D metabolism.

Expression of claudin-11 in canine prepubertal testes, and in canine adult testes showing normal spermatogenesis, impaired spermatogenesis, or testicular neoplasia.

The blood-testis barrier (BTB) consists of different cell-to-cell connections, including tight junction proteins like claudin-11 (CLDN11). For dogs, only limited data is published dealing with these proteins in general. Therefore, their physiological relevance, their postnatal expression, and their distribution pattern in pathological conditions, e.g. in altered spermatogenesis and testicular neoplasia were assessed. Canine testes from routine castrations, and those sent in for diagnostic purposes were investigated. Based on morphological evaluation, the dogs and testes were divided into groups: (1) dogs with normal spermatogenesis, (2) four months old prepubertal dogs, (3) intratubular seminoma, (4) diffuse seminoma, (5) Sertoli cell tumours (SCT), (6) Leydig cell tumours (LCT), and (7) dogs with impaired spermatogenesis (e.g. mixed atrophy). In order to examine possible alterations of the BTB components, immunohistochemistry (IHC) and immunofluorescence using a commercial antibody against CLDN11 was performed. Sertoli cell (SC) nuclei (SOX9) and peritubular myoid cells (smooth-muscle-actin, SMA) were also assessed using IHC. Additionally, semi-quantitative Western-blot (WB) and RT-PCR analyses of CLDN11 were conducted. In tubules with normal spermatogenesis, IHC of CLDN11 revealed a basolateral staining at BTB localisation. In prepubertal cords, CLDN11 was diffusely expressed along the cytoplasmic extensions of SCs supposing that the BTB was neither built up nor functional, yet. A shift from weakly expressed CLDN11 between/in residual SCs in intratubular seminoma to only small CLDN11 immunopositive stained spots in the cytoplasm of remaining SOX9-positive SCs in diffuse seminoma was detectable. Reduction or even loss of CLDN11 expression in diffuse seminoma was confirmed using RT-PCR and WB analyses, thus indicating that in seminoma, CLDN11 was downregulated at transcriptional level and completely lost its sealing function. Basal SCs in SCT still showed a CLDN11/SOX9 co-localisation, suggesting that luminal neoplastic SCs undergo de-differentiation during tumour progression. In LCT, no CLDN11 was detectable. Dogs with mixed atrophy showed an upregulation of CLDN11 in tubules with spermatogenic arrest on mRNA and protein level, leading to the conclusion that within these tubules regulatory mechanisms lost their equilibrium. For the first time, the spatial expression of CLDN11 in prepubertal canine testis, impaired spermatogenesis, intratubular seminoma and its absence in diffuse seminoma and LCT was shown. Since altered CLDN11 levels could be part of adaptive mechanisms to modify BTB integrity, further functional investigations to characterize the canine BTB need to be conducted.

Urospermia indicating ectopic ureters in breeding dogs - 3 cases.

An Entlebucher Mountain Dog (57 months old, case 1), a Labrador Retriever (24 months, case 2) and an Irish Soft-Coated Wheaten Terrier (31 months old, case 3) were presented for breeding soundness evaluation to the clinic. During semen collection in all 3 dogs, the pre-secretion and the sperm-rich fraction showed normal consistency and colour, whereas the prostatic secretion (3 rd ejaculate fraction) appeared strikingly yellow. In cases 1 and 2, a severely decreased sperm motility (asthenozoospermia) and an increased amount of abnormal spermatozoa (teratozoospermia), and in case 3, a moderately decreased total sperm count (oligozoospermia) were detected. Sonographical examination revealed abnormal findings regarding the uretero-vesical junction and ectopic ureters. Therefore it is concluded that urine admixture to the 3 rd ejaculate fraction may indicate the presence of ectopic ureters and may cause impairment of semen quality and fertility. The present cases raise questions regarding urospermia concerning: 1. its incidence in dogs in general and in connection with ectopic ureters and 2. its relevance as a cause of deficient ejaculate quality and subfertility or infertility.

Impaired spermatogenesis, tubular wall disruption, altered blood-testis barrier composition and intratubular lymphocytes in an infertile Beagle dog - a putative case of autoimmune orchitis.

Impairment of blood-testis barrier integrity can be observed during inflammation, infection, trauma and experimental autoimmune orchitis, which is inducible in rodents. In the present study, an initially fertile two-year-old Beagle dog was presented with a decline in total sperm number resulting in azoospermia within five months, verified by twice-monthly semen analyses. The dog was clinically healthy with bilateral small testes and showed normal thyroid function. Bacterial cultures of semen were negative and serum biochemical analyses showed no abnormal findings. To determine causes of azoospermia, the dog was castrated. Histological examinations of hematoxylin-eosin stained testicular sections revealed impaired spermatogenesis, seminiferous tubules with spermatogenic arrest or Sertoli-cell-only syndrome as well as focal interstitial and even intratubular lymphocytic infiltrations. Germ cell sloughing, apoptosis and giant cells were also observed in some tubules. Subsequent immunostainings of smooth-muscle-actin, claudin3, claudin11 and connexin43 demonstrated, for the first time, a mechanical and functional disruption of the tubular wall and alterations of blood-testis barrier proteins in these tubules. Presence of claudin3 and claudin11 in canine testis was confirmed using RT-PCR and sequencing and/ or Western-blot analyses. All findings suggested a possible spontaneous autoimmune orchitis to be the underlying cause for the observed azoospermia.

Zoonotic intestinal helminths interact with the canine immune system by modulating T cell responses and preventing dendritic cell maturation.

Parasite co-evolution alongside the mammalian immune system gave rise to several modulatory strategies by which they prevent exaggerated pathology and facilitate a longer worm survival. As little is known about the immunoregulatory potential of the zoonotic canine parasites Ancylostoma caninum and Toxocara canis in the natural host, the present study aimed to investigate whether their larval excretory-secretory (ES) products can modulate the canine immune system. We demonstrated TcES to increase the frequency of CD4+ Foxp3high T cells, while both AcES and TcES were associated with elevated Helios expression in Foxp3high lymphocytes. ES products were further capable of inducing IL-10 production by lymphocytes, which was mainly attributed to CD8+ T cells. ES treatment of PBMCs prior to mitogen stimulation inhibited polyclonal proliferation of CD4+ and CD8+ T cells. Moreover, monocyte-derived ES-pulsed dendritic cells reduced upregulation of MHC-II and CD80 in response to lipopolysaccharide. The data showed that regulation of the canine immune system by A. caninum and T. canis larvae comprises the modification of antigen-specific and polyclonal T cell responses and dendritic cell maturation.

[Evaluation of the blood progesterone concentration in the bitch measured by chemiluminescence immunoassay at the day of ovulation]. / Evaluation der mittels Chemilumineszenztest gemessenen Blut-Progesteronkonzentration bei der Hündin am Tag der Ovulation.

OBJECTIVE: Modifications of human test systems used in veterinary laboratory practice could lead to reference-range adaptions for their veterinary use. In 2012 the manufacturer of a widely used chemiluminescence immunoassay modified the test for progesterone measurement leading to a reference range adaption for the breeding-time detection in the bitch. MATERIAL AND METHODS: The aim of the present study was to evaluate the mean progesterone concentration by using the modified chemiluminescence immunoassay at the time of ovulation in the bitch and to compare this with previously used reference ranges. Moreover, internal and external quality controls were performed and progesterone concentrations measured in different laboratories with different methods were compared at a national and international level. RESULTS: In the present study, it could be demonstrated that the concentration of progesterone of 0-6 ng/ml measured by the modified test was clearly lower than that measured by the previously delivered test. National and international quality control assurance showed a good agreement of progesterone measurements between different laboratories and with the modified test. In seven bitches, the mean progesterone concentration on the day of ultrasonographically detected ovulation was 3.4 ± 0.9 ng/ml (2.0-4.5 ng/ml). CONCLUSION: This analysis indicates the need to change the widely accepted reference value for ovulation from 5-8 ng/ml to ~ 3.5 ng/ml for the currently used method. Particularly in veterinary endocrinology, the routine evaluation of reference values should be standard for good laboratory practice. However, the respective reference range is laboratory specific. CLINICAL RELEVANCE: Reference ranges of the progesterone concentration indicating the day of ovulation should be provided by the respective laboratory.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs.

BACKGROUND: Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS: We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION: This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Specific order in the appearance of protein tyrosine phosphorylation patterns is functionally coordinated with dog sperm hyperactivation and capacitation.

The aims of the present study were to characterize a slow capacitation system that records initial changes in the sperm membrane state, and, using a canine model, to order the specific protein tyrosine phosphorylation signaling in the sequence of capacitational events and to associate them with hyperactivated motility. Dog sperm washed through Percoll were incubated in complete bicarbonate Tyrode medium for 6 hours in 5% CO(2). Capacitation was evaluated using chlortetracycline staining. Tyrosine phosphorylation patterns were assessed by immunocytochemistry. Parallel to this, a computer-assisted motility analysis was performed. Significant changes in the percentage of capacitated and acrosome-reacted cells were first observed after 90 minutes, increasing in a linear manner during further incubation (P <.05). Changes in the percentage of capacitated cells were accompanied by motility changes. During incubation, a strictly sequential phosphorylation of sperm tail (midpiece, principal piece, and end piece) and head proteins was observed. According to an analysis of kinetics, phosphorylation of head proteins occurred after the tail became completely phosphorylated. Changes in head phosphorylation progressed at the same rates as capacitation and acrosome reaction. Sperm motility, curvilinear velocity, average path velocity, straight line velocity, and lateral head displacement were correlated positively or negatively with phosphorylation of midpiece or end piece proteins, respectively. The bicarbonate-stimulated increases in cyclic adenosine monophosphate levels and changes in protein phosphatase activity may be involved in the signaling system that controls membrane changes and motility in dog sperm. Phosphorylation kinetics of sperm proteins are potentially useful for diagnostic purposes to characterize the response of individual males to fertilizing conditions.

Canine distemper virus infection leads to an inhibitory phenotype of monocyte-derived dendritic cells in vitro with reduced expression of co-stimulatory molecules and increased interleukin-10 transcription.

Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper.

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation.

BACKGROUND: Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation. METHODS: Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing's were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution. RESULTS: The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct. CONCLUSIONS: Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.

A noncoding melanophilin gene (MLPH) SNP at the splice donor of exon 1 represents a candidate causal mutation for coat color dilution in dogs.

Coat color dilution in several breeds of dog is characterized by a specific pigmentation phenotype and sometimes accompanied by hair loss and recurrent skin inflammation, the so-called color dilution alopecia or black hair follicular dysplasia. Coat color dilution (d) is inherited as a Mendelian autosomal recessive trait. In a previous study, MLPH polymorphisms showed perfect cosegregation with the dilute phenotype within breeds. However, different dilute haplotypes were found in different breeds, and no single polymorphism was identified in the coding sequence that was likely to be causative for the dilute phenotype. We resequenced the 5'-region of the canine MLPH gene and identified a strong candidate single nucleotide polymorphism within the nontranslated exon 1, which showed perfect association to the dilute phenotype in 65 dilute dogs from 7 different breeds. The A/G polymorphism is located at the last nucleotide of exon 1 and the mutant A-allele is predicted to reduce splicing efficiency 8-fold. An MLPH mRNA expression study using quantitative reverse transcriptase-polymerase chain reaction confirmed that dd animals had only about approximately 25% of the MLPH transcript compared with DD animals. These results provide preliminary evidence that the reported regulatory MLPH mutation might represent a causal mutation for coat color dilution in dogs.

Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen.

Due to the similarity of plasma membrane changes induced by capacitation and cryopreservation, the parameters describing sperm response to capacitating conditions can be used for evaluating the cryopreservation response in many animal systems. In dog sperm, the response of the total sperm population to ionophore treatment has been shown to be an indication of the freezability of semen samples. Another sperm functional characteristic decisive for cryopreservability is cell volume regulation, due to the generation of essential osmotic gradients across the plasma membrane during the freeze-thaw cycles. In the present study, cryopreservation-induced changes in the membrane functional integrity were examined by monitoring the osmotically induced response of cell volume and the response to an ionophore in live cell populations. Cell volume measurements were performed on Percoll-washed suspensions of freshly diluted and frozen-thawed dog spermatozoa. The proportion of live acrosome-reacted cells was evaluated by flow cytometry after incubation under capacitating conditions in the presence of the calcium ionophore, A23187. During freezing-thawing, significant membrane changes occurred related to the disturbance of volume control ability and the loss of a proportion of live acrosome-reacted cells (P < 0.05). There were significant differences between individuals with respect to the degree of functional and structural membrane changes after thawing. Significant correlations were found between acrosomal integrity and functional membrane integrity. When assessed in freshly diluted semen, these parameters correlated with those of frozen-thawed semen samples, pointing to the similarities between mechanisms of cryopreservation-related changes and those mechanisms that mediate changes in membrane permeabilities and in cell volume regulation. The detection of changes in the sperm plasma membrane by monitoring the sperm cell volume represents a simple, rapid and sensitive method to estimate sperm quality after the cryopreservation procedure. The individual variability in response to osmotic stress or to calcium ionophore treatment appears to reflect the subtle differences in the sperm membrane functionality which are crucial for the prediction of cryopreservability.