Małgorzata Ochota

The effect of cysteine and superoxide dismutase on the quality of post-thawed chicken sperm.

The study was conducted to determine the influence of N-acetyl-l-cysteine (NAC) and superoxide dismutase (SOD) on chicken sperm motility, plasma membrane and acrosome integrity, mitochondrial activity, lipid peroxidation (LPO) and apoptotic changes after freezing-thawing process. Semen samples from fifteen Greenlegged Partridge roosters were pooled, diluted with EK extender without antioxidants (control), or supplemented with 5 mM NAC, or 200 U/mL SOD. Samples were subjected to cryopreservation. After thawing, sperm parameters evaluated by using CASA system and flow cytometry were assessed. The extender supplemented with NAC and SOD caused the increase (P < 0.01) in sperm motility and provided the higher percentage of rapid sperm (P < 0.01) compared to control. The addition of NAC increased the progressive motility of cells (P < 0.01). In NAC and SOD groups post-thaw plasma membrane integrity was higher (P < 0.05) and less spermatozoa showed apoptotic changes (P < 0.01, P < 0.05). Post-thaw percentage of sperm with high mitochondrial activity was the greatest (P < 0.05) with NAC addition. The SOD supplementation only reduced (P < 0.05) the percentage of sperm with LPO, following the cryopreservation. These results indicate that the addition of NAC (5 mM) and SOD (200 U/mL) is beneficial for the function of frozen-thawed chicken spermatozoa. The antioxidants prevented the reduction in motility, viability and mitochondrial membrane potential, as well as protected from apoptotic changes in sperm. Lipid peroxidation in sperm plasma membrane was decreased by SOD supplementation. Therefore, these antioxidants can be recommended as an additional component of chicken freezing extender.

Hypospadias in a Male (78,XY; SRY-Positive) Dog and Sex Reversal Female (78,XX; SRY-Negative) Dogs: Clinical, Histological and Genetic Studies

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5′- and 3′-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.

Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10(6)/mL, 5 × 10(6)/mL, and 1 × 10(6)/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10(6)/mL dilution rate, spermatozoa diluted to 1 × 10(6) sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.

A Lack of Association between Polymorphisms of Three Positional Candidate Genes (CLASP2, UBP1, and FBXL2) and Canine Disorder of Sexual Development (78,XX; SRY-Negative)

A disorder of sexual development (DSD) of dogs with a female karyotype, missing SRY gene, and presence of testicles or ovotestes is quite commonly diagnosed. It is suggested that this disorder is caused by an autosomal recessive mutation; however, other models of inheritance have not been definitely ruled out. In an earlier study it was hypothesized that the mutation may reside in a pericentromeric region of canine chromosome 23 (CFA23). Three positional candidate genes (CLASP2, UBP1, and FBXL2) were selected in silico in the search for polymorphisms in 7 testicular or ovotesticular XX DSD dogs, 8 XX DSD dogs of unknown cause (SRY-negative, with enlarged clitoris and unknown histology of gonads), and 29 normal female dogs as a control group. Among the 15 molecularly studied dogs with enlarged clitoris there were 3 new cases of testicular or ovotesticular XX DSD and 4 new cases of XX DSD with unknown cause (histology of the gonads unknown). Altogether, 11 (including 10 novel) polymorphisms in 5′- and 3′-flanking regions of the studied genes were found. The distribution analysis of these polymorphisms showed no association with the DSD phenotypes. Thus, it was concluded that the presence of the causative mutation for testicular or ovotesticular XX DSD in the pericentromeric region of CFA23 is unlikely.

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 drugs 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.

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.

Methods of Assessment of Cryopreserved Semen

Despite the significant progress, the post-thaw viability and fertility of the cryopreserved sperm are still reduced, as a consequence of accumulated cellular injuries that arise throughout the cryopreservation process. Many laboratory tests have already been carried out to verify these detrimental effects and their origin. Their is needed to well understand the whole process of cryopreservation and its influence on sperm function. As a consequence, it would lead to a subsequent improvement of sperm viability by means of reformulated protocols and approaches helping to minimize the detrimental effect of cryopreservation.

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.

Testicular disorder of sex development in four cats with a male karyotype (38,XY; SRY-positive)

The molecular background of disorders of sex development (DSD) in cats is poorly recognized. In this study we present cytogenetic, molecular and histological analyses of four cats subjected for the analysis due to ambiguous external genitalia. Three cases, with rudimentary penises and an abnormal position of the urethral orifice, represented different types of hypospadias. The fourth case had a normal penis, a blind vulva and spermatogenetically active testes. Histological studies showed structures typical of testes, but spermatogenic activity was observed in two cats only. All the cats had a normal male chromosome complement (38,XY) and the Y-chromosome linked genes (SRY and ZFY) were also detected. Fluorescent in situ hybridization (FISH), with the use of the feline BAC probe harboring the SRY gene, excluded the possibility of chromosome translocation of the Y chromosome fragment carrying the SRY gene onto another chromosome. Sequencing of four candidate genes (SRY--sex determining region Y; AR--androgen receptor; SRD5A2--steroid-5-alfa reductase 2 and MAMLD1--mastermind-like domain containing (1) revealed one SNP in the SRY gene, one common polymorphism in exon 1 of the AR gene (tandem repeat of a tri-nucleotide motif--CAG), six polymorphisms (5 SNPs and 1 indel) in the SRD5A2 gene and one SNP in the MAMLD1 gene. Molecular studies of the candidate genes showed no association with the identified polymorphisms, thus molecular background of the studied DSD phenotypes remains unknown.

Histopathological pattern of gonads in cases of sex abnormalities in dogs: An attempt of morphological evaluation involving potential for neoplasia

Disturbances in sex differentiation (DSD - disorder of sexual development) may result from disturbances in sex chromosomes or a disturbed development of gonads, or from genotypic disturbances. The objective of this article is to describe the histological structure of gonads in dogs showing sexual disturbances and a case of a cancer resembling gonadoblastoma in one of the animals. Among the 10 examined dogs with disturbances of sex development only a single case of a gonadoblastoma was observed. In animals with sex disturbances, similarly to humans, there exists a potential tendency for neoplastic lesions in dysgenetic gonads. As a rule, its frequency in population is confined due to the early procedure of castration of non-breeding dogs. In the present study dogs demonstrated phenotypical traits of bitches with developmental anomalies such as hyperplastic clitoris with vestigial os penis (baculum), or abnormalities in the location and structure of the vulva. The material for the study included canine gonads of various breeds, sampled from phenotypical bitches, aged 7 months to 4 years - patients of the Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, University of Environmental and Life Sciences in Wroclaw (Poland) in years 2006-2013. The organs were surgically removed from the abdomen and sent for histopathological examination for the purpose of determining their histological structure. The 10 examined cases of altered gonads included 6 bilateral cases of testes (60%), 2 cases of bilateral ovotestis (20%), one case of co-manifestation of testis and ovotestis (10%), and a single case of a testis and a neoplastically altered gonad (gonadoblastoma) (10%).