Analysis of testosterone pathway genes in dogs (78,XY; SRY-positive) with ambiguous external genitalia revealed a homozygous animal for 2-bp deletion causing premature stop codon in HSD17B3.

The genetic background of disorders of sex development (DSD) in dogs with a normal male sex chromosome set (78,XY) is poorly described. In this study, we present for the first time, an analysis of six genes of the testosterone pathway, encoding enzymes (CYP17A1, HSD3B2, HSD17B3, SRD5A2) and transcription factors (NR5A1, AR). The entire coding sequence and flanking regions of the introns, 5'-UTR and 3'-UTR were analyzed in five DSD dogs (78,XY, SRY-positive) with ambiguous external genitalia and in 15 control dogs. A homozygous deletion of 2 bp in exon 2 of HSD17B3 (hydroxysteroid 17-beta dehydrogenase 3) was found in a Dachshund dog with enlarged clitoris, vulva and abdominal gonads and decreased serum testosterone level. In silico analysis revealed that this deleterious variant causes truncation of the encoded polypeptide (from 306 to 65 amino acids) and deprivation of the active site of the encoded enzyme. Genotyping of 23 control Dachshund dogs showed a normal homozygous genotype. Thus, we assumed that the 2-bp deletion is the causative variant. Moreover, 24 SNPs (four in CYP17A1, three in HSD3B2, six in HSD17B3, five in SRD5A2, one in AR and five in NR5A1), two intronic indels (one in HSD3B2 and one in SRD5A2) and two microsatellite polymorphisms in exon 1 of AR were found. Six SNPs appeared to be novel. No association with DSD phenotype was observed. Identification of the first case of DSD in domestic animals caused by a deleterious variant of a gene involved in testosterone synthesis showed that these genes are important candidates in such studies.

The Viability of Serval (Leptailurus serval) and Pallas Cat (Felis manul) Oocytes after Cryopreservation Using the Rapid-I Method.

BACKGROUND: Vitrification by Rapid-I method could be essential for felid rescue programs to protect wild felid in the future. OBJECTIVE: This study was aimed at adapting the Rapid I method and evaluating the viability of serval and Pallas cat oocytes compared to oocytes of the domestic cat. MATERIALS AND METHODS: Oocytes after collection and in vitro maturation were vitrified using Cryotech medium (Cryotech, Japan) and a Rapid-I device (Vitrolife, Sweden). To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. RESULTS: Survival rate in the control group (domestic cat) was 75 %. In the experimental group, 70% (serval) and 60% (pallas cat) viable oocytes were found. CONCLUSION: The Rapid-I method can be applied successfully for the vitrification of wild felid oocytes.

Obtaining Wisent early blastocyst in vitro is a basic for protection and creation of biodiversity for this threatened species.

Wisent, or European bison (Bison bonasus), is listed as "vulnerable" on the IUCN Red List of Threatened Species and is therefore protected by international law. For the first time, a Wisent embryo has been obtained in vitro. This procedure creates a new opportunity to protect and increase Wisent reproductive potential and thereby opens new possibilities for the establishment of a controlled and broad reserve of the gene pool.

Identification and functional analysis of bull (Bos taurus) cauda epididymal fluid proteome.

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.

Analysis of bull (Bos taurus) seminal vesicle fluid proteome in relation to seminal plasma proteome.

The existing knowledge on the bull seminal vesicle proteome, a major seminal plasma constituent, and its relationship with seminal plasma is limited. This knowledge is prerequisite for a better understanding of seminal plasma variability, which is linked to semen quality. The objective of this study was to characterize the proteomes of seminal vesicle fluid and seminal plasma and to compare them to better understand the origin of seminal plasma proteins. We collected ejaculates and seminal vesicle fluid postmortem from 6 mature Holstein Friesian bulls. We performed the analysis and identification of proteins using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. We identified 105 proteins in bull seminal vesicle fluid and 88 proteins in seminal plasma. For both seminal vesicles and seminal plasma proteins described in our study, top biological functions were cellular movement, cell death and survival, and cellular growth and proliferation. Additionally, seminal vesicle fluid proteins were involved in protein degradation and synthesis. Seminal plasma proteins were also involved in cellular assembly and organization and cell-to-cell signaling and interactions. Proteins of both fluids were involved in the following canonical pathways: glycolysis, gluconeogenesis, liver X receptor/farnesoid X receptor, and farnesoid X receptor/retinoid X receptor activation. Additionally, seminal vesicle fluid proteins appeared to be involved in oxidative stress response mediated by nuclear factor E2-related factor 2. Our results described the bull seminal vesicle fluid proteome for the first time and allowed for significant expansion of the current knowledge on the bull seminal plasma proteome. Moreover, analysis indicated that both bull seminal vesicle fluid and seminal plasma proteomes contained interconnected protein groups related to protective functions, glycolysis, and the morphology and physiology of the spermatozoa. These proteins and their interactions could be targeted in future research.

Survival rate after vitrification of various stages of cat embryos and blastocyst with and without artificially collapsed blastocoel cavity.

Embryo vitrification is a modern technique for cryopreservation in assisted reproductive programs. From all the embryos, blastocysts are the most challenging during cryostorage due to their size, multicellular structure and the presence of blastocoelic fluid. The aim of this study was to evaluate the suitability for vitrification of various developmental stages of feline embryos and the influence of the artificial shrinkage (AS) of expanded blastocyst on post-vitrification survival rates. The AS procedure is the manual puncture of the trophectoderm allowing for the reduction of blastocoelic fluid prior to vitrification and thus preventing the ice crystal formation. The vitrified embryos were divided into groups of 2-cell, 4- to 8-cell, >8-cell, morulae, compacted and expanded blastocyst, based on morphological assessment and vitrified in groups of 1-3 embryos per Cryotop. The post-warming survival was similar regardless the embryo developmental stage prior vitrification; however, development to blastocysts was only noted in 4- to 8-cell and >8-cell vitrified embryos (13% and 27%, respectively). Following warming, the significantly more viable blastocysts were noted in vitrified compacted versus expanded blastocyst and in expanded blastocyst subjected to AS procedure versus expanded blastocyst without AS (total survival: 58.3% vs. 33.3% and 64.3% vs. 38.5%, respectively; re-expansion rate within 2 hr post-warming: 41.7 vs. 6.7% and 50% vs. 7.7%, respectively). One-fifth of vitrified expanded blastocyst showed morphological damage immediately after warming procedure, whereas no visible damage was noted in compacted blastocyst and artificially collapsed expanded ones. The obtained results suggest that the most suitable for vitrification are feline embryos containing four to 16 blastomeres and compacted blastocyst. In addition, the reduction of blastocoel cavity in expanding blastocyst by artificial collapse improved the blastocyst vitrification outcome.

Mitochondrial characteristics in oocytes of the domestic cat (Felis catus) after in vitro maturation and vitrification.

CONTENTS: The objective of this study was to evaluate mitochondria in immature and in vitro-matured domestic cat oocytes and to assess for the first time the effect of vitrification on mitochondrial traits. Mitochondrial distribution and aggregation were assessed using confocal microscopy after staining with the fluorescent dye-MitoTracker® Red CMXRos. Only cells at the germinal vesicle and the metaphase II stages of nuclear development, representing immature and mature oocytes, respectively, were included in our study. Our study shows that 80% of immature and 100% of mature oocytes exhibit a peripheral pattern of mitochondria distribution, indicating that, in contrast to the situation in other species, the mitochondria of cat oocytes are not dispersed throughout the cell after in vitro maturation but instead maintain a strong affinity for the oocyte periphery near the membrane. However, a loss of aggregation was observed during in vitro maturation-78% of immature oocytes showed homogeneous granulation versus only 18% of mature oocytes (p < .001). The increased intensity of MitoTracker® Red CMXRos staining after in vitro maturation (p < .05) may be tentatively attributed to an increase in mitochondrial activity but could likewise reflect a concomitant appearance of sulphhydryl groups in cytoplasm (known to be targeted by the dye). Mitochondrial distribution did not change upon vitrification; however, dye intensity decreased (p < .05) and mitochondrial aggregation was intensified in both immature and mature vitrified cat oocytes.

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.

Sequence analysis of three canine adipokine genes revealed an association between TNF polymorphisms and obesity in Labrador dogs.

Obesity is an emerging health problem in purebred dogs. Due to their crucial role in energy homeostasis control, genes encoding adipokines are considered candidate genes, and their variants may be associated with predisposition to obesity. Searching for polymorphism was carried out in three adipokine genes (TNF, RETN and IL6). The study was performed on 260 dogs, including lean (n = 109), overweight (n = 88) and obese (n = 63) dogs. The largest cohort was represented by Labrador Retrievers (n = 136). Altogether, 24 novel polymorphisms were identified: 12 in TNF (including one missense SNP), eight in RETN (including one missense SNP) and four in IL6. Distributions of five common SNPs (two in TNF, two in RETN and one in IL6) were further analyzed with regard to body condition score. Two SNPs in the non-coding parts of TNF (c.-40A>C and c.233+14G>A) were associated with obesity in Labrador dogs. The obtained results showed that the studied adipokine genes are highly polymorphic and two polymorphisms in the TNF gene may be considered as markers predisposing Labrador dogs to obesity.

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.

Evaluation of spermatozoal function-useful tools or just science.

Conventional microscopic semen analysis does not provide precise information on the fertilizing potential of a male. The traditional basis for semen evaluation is that male fertility is dependent on production of a "proper" concentration/number of motile, morphologically normal spermatozoa in excess to achieve conception. Many independent studies, especially in human medicine, have demonstrated that the absolute number of spermatozoa does not accurately determine fertility, but their functional competence. Many functional tests of spermatozoa are developed over the last decades. Computer-assisted sperm analysis (CASA) and flow cytometry have become the gold standard for semen assessment in specialized andrology laboratories. Other functional assays, such as gamete interaction tests, provide additional information regarding the real fertilizing potential of sperm cells. From this point of view, such tests are valuable diagnostic tools in fertility disorders and may be helpful to make a decision which method of treatment to use: pharmacological therapy, intrauterine insemination, introduction of classic IVF, ICSI or exclusion from a breeding programme. The most useful gamete interaction tests include induced acrosome reaction, zona pellucida binding assay, oocyte penetration assay and hyaluronan binding assay. In recent years, andrology has entered into a new era of sophisticated OMICS methods. Genomics, epigenomics, transcriptomics and proteomics brought high hopes for rapid progress in clinical diagnostics.

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.

Computed Tomography of the Prostate Gland in Healthy Intact Dogs and Dogs with Benign Prostatic Hyperplasia.

To date, there is only scarce data on the evaluation of the prostate gland in dogs using computed tomography (CT). The aims of our study were to describe CT features of BPH in dogs and to determine the size of the prostate gland in healthy male dogs and dogs with benign prostatic hyperplasia (BPH) through CT. Additionally, we aimed to compare and establish the most useful parameters for CT measurements of the prostate in patients with BPH. The study population consisted of 20 healthy intact male dogs and 20 male intact dogs with confirmed BPH. Pre- and post-contrast CT studies were evaluated. The most common CT features in dogs with recognized BPH were symmetrical prostatomegaly and heterogeneity of the prostatic parenchyma. The mean prostatic density (D) was 56HU (±4.39) in pre-contrast CT images and 84HU (±8) in post-contrast images in dogs with BPH. The mean prostatic length (L) was 43.87 mm (±11), the mean width (W) amounted to 48.95 mm (±8.76) and the mean height (H) reached 44.9 mm (±9.48) in clinically affected patients. The mean ratios were: rL - 2,12 (±0.5); rW - 2.39 (±0.53) and rH - 2.16 (±0.39) in the BPH group. The prostate should be considered to be enlarged when rL exceeds 3.05; rW exceeds 3.38 and rH exceeds 2.94. Our findings indicated that CT is a useful tool in diagnosing prostate disorders, including BPH. The heterogeneity, density and ratios of prostatic length, width and height can be useful parameters in the diagnosis of BPH.

X monosomy in a virilized female cat.

An infertile Siamese female cat was subjected for clinical, histological, cytogenetic and molecular studies due to ambiguous external genitalia (vulva, vagina, rudimentary penis and scrotum-like structure) and masculine behaviour. An elevated oestrogen activity and a detectable level of testosterone were found. The cat underwent laparotomy. The gonads and the uterus were removed and subjected for histological studies, which showed ovaries with corpora lutea and a some primordial follicles. Chromosome studies of lymphocyte and fibroblast cultures, with the use of Giemsa staining, G-banding and whole X chromosome painting by fluorescence in situ hybridization, revealed pure X monosomy. Molecular analysis showed the absence of the SRY gene. Our study revealed for the first time that X monosomy in cats may be associated with virilization, in spite of the lack of the SRY gene.

Pharmacological treatment for common prostatic conditions in dogs - benign prostatic hyperplasia and prostatitis: an update.

The two most frequent prostatic diseases in dogs are benign prostatic hyperplasia (BPH) and prostatitis. Prostatitis requires prolonged antibiotic treatment. In acute prostatitis, the blood-prostate barrier is broken, thus facilitating the penetration of antibiotics, whereas in chronic prostatitis, the barrier prevents the penetration of many drugs into the gland. The selection of antibiotic agents is based on the sensitivity test and the drug's ability to penetrate into the gland. Many protocols for the treatment of BPH are available. In non-breeding dogs, surgical and optionally pharmacological castration by means of GnRH agonists may be performed. In breeding dogs, drugs retaining fertility are used. Recently, androgen receptor antagonistic treatment with osaterone acetate has been applied. Other drugs used for BPH treatment include progestagens, oestrogens, antioestrogens and 5α-reductase inhibitors. Some of these compounds may provoke severe side effects. The efficiency of GnRH antagonists used for the treatment of prostatic diseases, such as neoplasia and BPH, in humans has been recently investigated in dogs. This androgen deprivation therapy (ADT) is devoid of an initial exacerbation of androgen-dependent symptoms, which is typical for GnRH agonistic treatment. In many cases, BPH and prostatitis must be treated simultaneously as these conditions may develop in combination.

Diagnosis of common prostatic conditions in dogs: an update.

Prostatic diseases account for 3-10% of intact male dogs presented to veterinary surgeons. Conditions vary according to severity and frequency ranging from the most common, such as prostatic hyperplasia and cysts to the rarer conditions such as prostatic abcesses and neoplasia. Different causes of prostatic disease can often not be distinguished by evaluation of clinical signs, as these are not very distinctive and may be similar for many prostatic conditions. Understanding which additional diagnostic tools to use for each of the possible conditions is essential in making a correct diagnosis leading to the proper treatment. This article will discuss the different etiologies, age groups of dogs and the decision-making process which will help the practitioner to choose the right investigative tools, treatments and prognosis when dealing with prostatic disease.

A case of leucocyte chimerism (78,XX/78,XY) in a dog with a disorder of sexual development.

A 1-year-old Shih Tzu dog was presented for examination because of abnormal external genitalia. A residual penis with a prepuce was located in a position typical of a male. The dog had no palpable testicles or scrotum. The ultrasound examination revealed the presence of the prostate, but the gonads remained undetectable. Cytogenetic analysis performed on chromosome preparations obtained from lymphocyte culture showed two cell lines - 78,XX and 78,XY. Molecular analysis of 14 polymorphic microsatellite markers allowed us to distinguish leucocyte chimerism from whole body chimerism. The presence of 3 or 4 alleles was confirmed in DNA isolated from blood, while in DNA isolated from hair follicles only 1 or 2 alleles were detected. The case was classified as leucocyte 78,XX/78,XY chimerism. Our study showed that XX/XY leucocyte chimerism might be associated with disorder of sexual development in dogs. Furthermore, it is emphasized that the use of cytogenetic study, in combination with analysis of polymorphic markers in DNA isolated from different somatic cells, facilitates distinguishing between leucocyte and whole body chimerism.

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).

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.