Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after sedation with xylazine

Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after administration of xylazine was examined. In experiment 1, the mean (+/- SD) spermatozoal concentration in urine collected by cystocentesis before ejaculation was 0.322 +/- 0.645 X 10(6)/ml. After ejaculation, motile spermatozoa were present in the urine collected by cystocentesis from 12 of 15 dogs, and the concentration of spermatozoa in the urine (5.139 +/- 7.014 X 10(6)/ml) was higher (P less than 0.025) than the concentration in the urine collected before ejaculation. The percentage of the total number of spermatozoa that were displaced during ejaculation and flowed into the urinary bladder (retrograde flow) ranged from 0 to 99.75% (24.67 +/- 33.98%). In experiments 2 and 3, administration of xylazine to sexually rested dogs induced retrograde flow of spermatozoa into the urinary bladder. In experiment 2, all dogs had spermatozoa in urine collected after xylazine administration, with motile spermatozoa present in the urine from 9 of 10 dogs. In experiment 3, urine collected from dogs before administration of xylazine was azoospermic or contained few, nonmotile spermatozoa (0.063 +/- 0.135 X 10(6)/ml), whereas urine collected after administration of xylazine had more (P less than 0.025) and motile spermatozoa (3.717 +/- 4.273 X 10(6)/ml). In experiment 4, administration of xylazine to dogs after ejaculation did not increase the concentration of spermatozoa in the urine. Results indicate that spermatozoa flow into the urinary bladder of dogs during ejaculation or after administration of xylazine to sexually rested dogs.     

Effect of administrating oxytocin or prostaglandin F2alpha on characteristics of the canine ejaculate

To test the hypothesis that male dogs treated with smooth muscle contracting drugs have an increase in the total number of spermatozoa in the ejaculate but no change in all other ejaculate characteristics, such as progressive motility of spermatozoa or percentage morphologically normal spermatozoa, dogs were treated with oxytocin or prostaglandin F2alpha (PGF2alpha) and compared to saline treatments. Semen was collected from each of the 3 dogs once every 3 to 4 d for a total of 6 collections per dog. Ten minutes before each collection, 1 of 3 injections (oxytocin 10 IU [0.5 mL], IM; PGF2alpha 2.5 mg [0.5 mL], IM; or saline 0.5 mL, IM) was administered. Compared to the saline controls, neither treatment had any significant effect on any measured variable when collected in this manner with an estrus bitch present. Therefore, the use of these drugs does not appear to be a viable treatment to increase the number of spermatozoa.     

Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.     

Intratubal insemination with fresh semen in dogs

The number of spermatozoa required to obtain conception by intratubal insemination in dogs was examined. Three groups consisting of 5, 8 and 8 dogs received 0.5 x 10 (6), 2.0 x 10(6) and 4.0 x 10(6) spermatozoa, respectively, into each uterine tube. No conception occurred in the 5 animals inseminated with 0.5 x 10(6) spermatozoa, but conception occurred in 6/8 (75.0%) and 3/8 (37.5%) dogs inseminated with 2.0 x 10(6) and 4.0 x 10 (6) spermatozoa, respectively. Among the pregnant animals, three aborted (33.3%) and the mean number of newborns was small, 2.5 +/- 0.5 (SE). One acardiacus anceps was observed with normal fetus in one animal with a Caesarean delivery.     

Yohimbine prevents the retrograde flow of spermatozoa into the urinary bladder of dogs induced by xylazine

This study was carried out to determine whether yohimbine antagonizes the retrograde flow of spermatozoa into the urinary bladder of dogs caused by xylazine. Adult dogs were assigned to one of four groups of six dogs each and treated as follows: saline control, xylazine (2.2 mg/kg, i.m.), yohimbine (0.2 mg/kg, im.), yohimbine/xylazine (yohimbine, 0.2 mg/kg, i.m., followed 10 min later by xylazine. 2.2 mg/kg, i.m.). Pre- and post-treatment urine were collected by cystocentesis from all dogs. The mean (± SD) adjusted total number of spermatozoa in the post-treatment urine of xylazine-treated dogs (141.02 ± 136.75 × 10⁶) was 15 times higher ( P < 0.05) than the number in the post-treatment urine of control dogs (9.16 ± 20.26 × 10⁶), 1763 times higher ( P < 0.05) than the number in the urine of yohimbine-treated dogs (0.08 ± 0.20 × 10⁶), and 56 times higher ( P < 0.05) than the total number in the post-treatment urine of yohimbine/xylazine-treated dogs (2.54 ± 4.54 × 10⁶). These results confirm that xylazine induces a significant ( P = 0.007) displacement of spermatozoa into the urinary bladder of dogs and demonstrate that pre-treatment with yohimbine prevents this effect.     

Ultrasonographic diagnosis of non-palpable Sertoli cell tumours in infertile dogs

Three previously fertile dogs were examined with a history of recent infertility. Semen evaluation revealed marked spermatozoal abnormalities, including lesions of the mid-piece region, poor spermatozoal motility and a low total spermatozoal output. The mean number of live normal spermatozoa was 21·1 ± 17·4 × 10⁶ spermatozoa. Examination of the testes with diagnostic B-mode ultrasound demonstrated solitary mass lesions which in each case were not palpable. Plasma oestrogen concentrations were elevated (mean, 43·0 ± 8·s 5 pg/ml), and histologically the masses were shown to be Sertoli cell tumours. Several months after unilateral orchidectomy there was an increase in the percentage of normal spermatozoa and an increase in the spermatozoal motility and total spermatozoal output. The mean number of live normal spermatozoa was 149·8 ± 22·9 × 10⁶ spermatozoa and all the dogs subsequently returned to fertility. Ultrasonographic examination of the testes should be considered part of the routine investigation of the male reproductive tract.     

Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds

Objectives : Studies in many species indicate that variation of spermatozoan head morphology is a sensitive biomarker for abnormal chromatin structure and resultant clinical fertility. This preliminary study evaluated spermatozoan head morphometry in different dog breeds and assessed whether morphometric parameters could reflect spermatozoan DNA fragmentation in dogs. Methods : Spermatozoan morphometry and DNA quality (measured by TUNEL flow cytometry) were assessed in semen from 11 dogs of three Italian breeds (Cirneco dell’Etna, Piccolo Levriero Italiano and Segugio Maremmano). Results : Morphometric data showed that Segugio dogs had significantly larger (33·67%) spermatozoa and that Piccolo Levrieros had a higher incidence of long (46·75%) and elliptical spermatozoan heads (11·5%) when compared with the samples from other breeds. Moreover, the predominance of elliptical spermatozoa in one dog (23%) was significantly related to the percentage of spermatozoa with fragmented DNA (12·6%), whereas in another dog, where no more than 1% of spermatozoa was elliptical, only 0·36% of spermatozoa had damaged DNA. It is noteworthy that the breeding record of the former dog in the previous 12 months showed poor fertility and fecundity. Clinical Significance : These data suggest that spermatozoan head morphometry could be breed related and that there is a significant correlation between DNA fragmentation and elliptical spermatozoa in individual animals. This finding, albeit limited in our study to a single case, is possibly related to clinical infertility.     

Effect of Body Weight, Age and Breeding History on Canine Sperm Quality Parameters Measured by the Hamilton-Thorne Analyser

During the last decade, several computer assisted sperm analysis (CASA) systems have been validated for canine sperm quality assessment. Regarding the impressive possibilities of these systems, further research is required to determine which CASA measurements are of clinical importance in canine andrology. In the present study, the sperm quality parameters obtained by the Hamilton-Thorne Semen Analyser (Ceros 12.1; HTR) were correlated with the body weight and the age of the dogs. Moreover, the sperm quality parameters of dogs with a different breeding history were compared. The sperm-rich fraction was collected from 111 dogs of 50 different breeds, which were presented at our department. Immediately after collection, the concentration, the total sperm output (TSO) and 13 different sperm motility and velocity characteristics were measured by the HTR. The percentage of live spermatozoa and the spermatozoal morphology were examined on eosin/nigrosin stained smears. Based on their breeding history, the dogs were divided in three groups: 'fertile' (n = 60), 'subfertile' (n = 17) or 'not used for breeding' (n = 34). Significant (p < 0.05) correlations were established between the body weight of the dogs and the TSO (r = 0.245) and velocity curvilinear (VCL; r = -0.220), respectively. The age was negatively correlated with the percentage of normal spermatozoa (r = -0.203; p < 0.05). The correlations with all the other evaluated sperm parameters were low and not significant. Significant differences between the 'fertile' and the 'subfertile' group were found for all of the evaluated sperm quality parameters (except for BCF, LIN, STR and MEDIUM). In conclusion, dogs tend to produce ejaculates with a lower percentage of normal spermatozoa with increasing age and dogs with higher body weights produce ejaculates with a higher TSO and a lower VCL. Significantly poorer sperm characteristics were found for dogs with lower in vivo fertility results.     

Influence of theophylline supplementation on oocyte penetration of canine epididymal spermatozoa

The influence of supplementation of theophylline to the medium regarding the penetration of canine epididymal spermatozoa into immature oocytes was examined. In the control medium, sperm penetration into the oocytes was observed in 8 of 13 dogs (61.5%), and the mean penetration rate was 22.0%. The penetration rate of individual dogs ranged from 0 to 64.9%. Supplementation of 0.1, 1.0 and 2.5 mM theophylline to the medium did not significantly affect sperm penetration. Sperm penetration was induced by supplementation of 2.5 mM theophylline in two dogs that showed no sperm penetration in the control group. Penetration of the epididymal spermatozoa into the oocytes was shown to vary among individual dogs in cases of the absence and presence of theophylline.     

Disappearance of spermatoza from the ejaculates of vasectomized dogs.

Ejaculates of bilaterally vasectomized dogs contained spermatozoa as long as 21 days after vasectomy, indicating that spermatozoa in the canine ejaculate originated from both the epididymides and the vasa deferentia, not from the epididymides alone.     

Hyperactivation and acrosome reaction in vitro in spermatozoa ejaculated by cryptorchid dogs after orchiopexy.

Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38 degrees C under 5% CO2 in air in canine capacitation medium at a concentration of 30 x 10(6) sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean +/- SD; 47 +/- 12%) and ND (80 +/- 9%) after three hours of incubation (p less than 0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58 +/- 5%) and ND (61 +/- 16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26 +/- 4%) was significantly lower than ND (46 +/- 5%) after eight hours of incubation (p less than 0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.     

Antisperm responses in male dogs with chronic Brucella canis infections

Male Beagles infected with Brucella canis for greater than or equal to 3 months developed serum antibodies that agglutinated normal canine spermatozoa. Titers were highest in dogs that had been infected for 4 to 6 months. Lower spermagglutinin titers were detected in sera collected 10 months after inoculation. Antibodies were also observed in seminal plasma of chronically infected dogs. Seminal plasma from infected, but not from clinically normal dogs, caused head-to-head agglutination of normal sperm. In contrast to macroagglutination of sperm by serum antibodies, agglutination by seminal plasma antibodies was detected only by microscopic examination. Seminal plasma agglutinins were not inactivated by heat (56 C, 1 hour) or by reduction with 2-mercaptoethanol. When seminal plasma and sperm were mixed with 2 hemolytic units of guinea pig complement, spermatozoa were not inactivated. Spermagglutinin activity was present in the first 2 spectral absorption peaks of Sephadex G-200 fractionated seminal plasma. Fractions that had the highest spermagglutinin titers contained mostly immunoglobulin A. Seminal plasma from infected dogs also contained cytophilic factors for normal splenic macrophages that caused sperm adherence to macrophages. Dogs with a bacteremia lasting greater than 4 months had cutaneous delayed-type hypersensitivity reactions when tested with soluble canine testicular extracts. Reactions did not occur in normal dogs. Dogs with testicular atrophy had the most severe skin test responses. Seemingly, isoimmune responses to sperm antigens are involved in infertility caused by B canis infection of male dogs.     

The lack of effect of parvovirus vaccination on the seminal characteristics of dogs

The onset of ejaculation and development of normal seminal characteristics in six young dogs vaccinated and seroconverting against canine parvovirus did not differ from the accepted range, and by 45 weeks of age the ejaculates were considered to be normal. At one year of age three of the dogs were given a large antigenic stimulus by vaccination once a week for four weeks; this produced no change in the output or characteristics of spermatozoa.     

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs

Background: During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis(lipids were extracted, transmethylated and analyzed by chromatography).Results: Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.Conclusions: These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.     

Some observations on the dilution, cooling and freezing of canine semen

Canine semen was obtained by digital manipulation from two donor dogs and twelve stud dogs. The sperm-rich fraction was diluted with either of two different diluents and the survival of spermatozoa before and after freezing was determined. It appeared that there was no difference in the post-thaw survival rate in either of the diluents used.     

Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa

OBJECTIVE: To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. SAMPLE POPULATION: Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. PROCEDURE: Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. RESULTS: Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. CONCLUSIONS AND CLINICAL RELEVANCE: Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.     

Scrotal width as an index of testicular size in dogs and its relationship to body size

Measurements of scrotal width were strongly correlated with testicular weight in 71 dogs (r = 0–919) and thus provided a convenient index of testicular size and potential sperm production. However, to be useful in trying to predict male fertility this measurement had to be adjusted for bodyweight. A graph is presented which allows estimation of ‘average’ scrotal width and its 95 per cent confidence limits for potentially fertile dogs of bodyweight between 5 and 50 kg. With one exception, dogs with scrotal widths below the 95 per cent confidence limits had very few or no spermatozoa in smears from the cauda epididymidis.     

Pentoxifylline induces capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa

To evaluate effects of different concentrations of pentoxifylline, as phosphodiesterase inhibitor, on quality of motility, capacitation and acrosome reaction, Ejaculated spermatozoa were collected from crossbred dogs. The sperm were incubated at concentrations of 0.1, 1, 10 and 100 mM pentoxifylline for 2 h. Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Capacitation and acrosome reaction were also evaluated by chlortetracycline fluorescence staining. SMI as quality index of sperm was significantly increased in concentrations of 10 and 100 mM pentoxifylline during 1 and 2 h compared to control. The number of capacitated or acrosome reacted spermatozoa significantly (P < 0.05) were higher than controls at high concentrations of pentoxifylline (10 and 100 mM) during 1 and 2 h. In conclusion, high concentration of pentoxifylline is able to induce capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa.     

Effects of the Inclusion of Equex STM into Tris-Based Extender on the Motility of Dog Spermatozoa Incubated at 5°C

The aim of this study was to estimate the effects of Equex STM on sperm motion characteristics in chilled dog semen extended in Tris-based diluent. Thirty-two ejaculates were collected from 12 proven German shepherd stud dogs. The sperm-rich fractions were diluted in Tris-based extender with 1% (v/v) Equex STM (sample A) and in Tris-based extender with no addition of detergent (sample B). The extended semen was incubated for 240 h at 5°C and the motility parameters were evaluated by CASA system at 24-h intervals. Addition of Equex STM to Tris-based extender led to an initial activation of motion activity of spermatozoa, followed by a rapid decrease, shortening the lifespan of spermatozoa incubated at 5°C. Computer-assisted sperm analysis clearly showed that Equex STM-induced changes of sperm motion characteristics resemble the hyperactivation (HA) of spermatozoa associated with the capacitation process.     

The effect of season of the year on the characteristics and composition of dog semen

Semen (collected by digital manipulation) and peripheral blood samples were obtained twice weekly from five fully grown Beagle dogs for a twelve month period from 1st August to 31st July. Sperm concentration, sperm output and glycerylphosphorylcholine concentrations of the second fraction showed evidence of a seasonal variation with highest values recorded in the months of March, April, May and June. The libido of the dogs, volume of ejaculate, percentage dead and abnormal spermatozoa, sperm motility and plasma testosterone concentration showed no evidence of seasonal change.