In vitro fertilizing potential of urethral and epididymal spermatozoa collected from domestic cats (Felis catus)

The aim of this study was to provide a comparative analysis of in vitro fertilizing potential of frozen-thawed urethral and epididymal feline spermatozoa. Both types of semen were collected from 7 cats and cryopreserved in liquid nitrogen. To perform in vitro fertilization, both urethral and epididymal samples from the same individual were thawed and spermatozoa were co-incubated with in vitro matured cat oocytes. Obtained embryos were cultured in vitro for 7 days in a commercial medium. Cleavage rate, morula rate and blastocyst rate were calculated. Experiment was run in 10 replicates. The examined parameters showed no significant differences between urethral and epididymal spermatozoa (p>0.05). Cleavage rate and embryo’s development were highly variable between replicates, even for the different sperm samples collected from one individual. There was no significant correlation between fertilizing capacity of two types of spermatozoa collected from the same male. In this study we confirmed that cryopreserved urethral spermatozoa have equally good fertilizing potential as epididymal ones, and both can be successfully used for in vitro fertilization in cats with the use of commercial medium

Morphological analysis of testicles in cats with disorders of sex development

Disorders of sex development (DSD) are rare in cats. They can be caused by chromosomal aberrations, gene mutations or other undefined factors. The aim of the present study was to compare the histological structure and immunohistochemical reactivity of testes in cats with DSD and in healthy cats. The research material consisted of the gonads of four cats – phenotypic males with an incorrect structure of the reproductive system. The control group consisted of the testes of four healthy cats – routinely castrated phenotypical males. The material was fixed with formalin and embedded in paraffin; the sections were stained with hematoxylin and eosin. The immunohistochemical investigation were performed using monoclonal and polyclonal antibodies directed against desmin, vimentin, actin of smooth muscles, S100 protein and MCM3 protein. The results obtained allow concluding that the testes of cats with DSD differed in certain respects, mainly in the number of blood vessels, from the normal testes. Moreover, the results of immunohistochemical examination indicate that in the testes of cats with DSD the number of supporting cells is lower, the amount of interstitial cells is comparable and spermatogenesis is correct es compared to those determined in the control gonads. The number of blood vessels in cats with DSD is reduced by about 30%. It confirms the recommendations for castration of these animals in order to eliminate the potential inheritance of sex development disorders

Methyl paraben as a sex pheromone in canine urine – is the question still open?

The literature concerning the issue of canine sex pheromones includes reports presenting completely conflicting opinions about the chemical composition of the canine urine in the context of semiochemical communication. At present, the predominant report cited by many different authors is the article published in Science in 1979 by Goodwin at al., presenting methyl p-hydroxybenzoate (methyl paraben) as the main canine sex pheromone. While it has been proved that pure methyl paraben lacks semiochemical activity as do commercially available products containing this substance (Eau D’Estrus, Synbiotics, USA), in view of the conflicting published reports the aim of this study was to revaluate using modern techniques the presence of methyl p-hydroxybenzoate in canine urine during different phases of the ovarian cycle. Ten female dogs of different breeds were used. Urine samples from bitches collected during various stages of the ovarian cycle were examined with using the SPME and GC/MS methods. Methyl paraben was not detected in any of the samples. In conclusion, because of the lack of methyl-p-hydroxybenzoate in the samples examined, the present study confirmed negative opinions on the possibility of this substance playing a crucial role in semiochemical communication during reproduction in dogs (Canis familiaris)

Computer-assisted sperm analysis in dogs and cats: an update after 20 years

Contents In dogs and cats, computer-assisted sperm analysis (CASA) was originally described almost 20years ago. Subsequently, numerous CASA systems were validated and used for various applications in dogs and to a lesser extent in cats. CASA systems offer an accurate, rapid, objective and simultaneous assessment of different semen parameters allowing the visualization of subtle changes in sperm characteristics, which cannot be identified by conventional semen analysis. The main problems of these computerized measuring devices are the relatively high investment costs and the need for standardization and validation before any practical use is possible. In comparison with automated motility and concentration assessment, automated morphometry and morphology assessment is more complex and time-consuming. Once validated, CASA systems can be routinely used in veterinary centres for assessment of fertility and for the improvement of sperm diluters, cooling and cryopreservation procedures in dogs and cats. Furthermore, information obtained by CASA systems could also be important when monitoring for example the effect of environmental stress on spermatozoa and for toxicity studies. In cats, CASA is less documented, and most studies describe the characteristics of epididymal sperm, which is frequently used for in vitro fertilization in cats. Implementation of the CASA technique in cat reproduction could be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids

The influence of antibiotic treatment of bitches in oestrus on their attractiveness to males during mating

The aim of this study was to evaluate the influence of the antibiotic treatment, including the mode of drugs administration, on bitches’ attractiveness to the stud dogs during mating. Moreover, we tried to estimate the possibility of aversive effect of the drug vehicle on the male behavior. In experiment I, four bitches in oestrus without antibiotic treatment (group A), four bitches treated with intravaginal antibiotic (group B) and four bitches treated with intramuscular antibiotic (group C) were presented to four stud dogs. In experiment II, bitches in oestrus (n=5) were presented to the males (n=2) before and after the application to the females’ vulva the antibiotic carrier – Miglyol 840 (Sasol, Germany). In both experiments the presence of the typical sexual behavior of the males (sniffing, licking the vulva and anal region, mating attempts) was evaluated. In experiment III the reaction of the males to the samples containing oestrual discharge from the bitches untreated and treated with antibiotics was evaluated. In the last part of study the aversion reaction to the samples containing antibiotic and the antibiotic carrier was evaluated. The results of experiments showed that females treated with the antibiotics were less attractive to males than untreated females, regardless of the method of administration. We did not observe adverse effect of the antibiotic carrier but samples from the bitches treated with antibiotics were significantly less attractive to the males. We concluded that the reason for reduced attractiveness of the bitches in oestrus after antibiotic treatment was the changes in semiochemical signal emitted by treated females as a consequence of elimination of the vaginal bacterial flora, which seems to be involved in creation of the typical, recognizable by the stud dogs, oestrual signal but also by the possible covering effect of used drugs

Supplementary Material for: A Rare Case of Testicular Disorder of Sex Development in a Dog (78,XX; <b><i>SRY</i></b>-Negative) with Male External Genitalia and Detection of Copy Number Variation in the Region Upstream of the <b><i>SOX9</i></b> Gene

Testicular or ovotesticular disorder of sex development (DSD) in genetic females (78,XX; <i>SRY</i>-negative) has been reported quite frequently in numerous dog breeds and is usually diagnosed due to the presence of female external genitalia with an enlarged clitoris. The molecular background of this disorder, diagnosed also in human and other mammals, is not fully understood. However, it has recently been proposed that a copy number variation (CNV) in the region upstream of the <i>SOX9</i> gene is associated with it. We described a rare case of this disorder in a French Bulldog with abdominal testes and male external genitalia (a slightly malformed penis). FISH studies showed a female karyotype, lack of a translocation involving the Y chromosome, and a distinct size variation in the CNV region (CNVR) upstream of the <i>SOX9</i> gene, located on chromosome 9 (CFA9). A large FISH variant on a single CFA9 and a lack of the variant on its homologue was observed. Surprisingly, in the mother of this DSD dog, 2 normal-sized variants were identified which means that the CNV in the DSD dog was de novo. Our observations are in agreement with earlier suggestions that a high number of copies at the CNVR upstream of <i>SOX9</i> may be associated with this type of DSD