Wojciech Niżański

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.

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 degrees 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 degrees 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.

Computer-Assisted Sperm Analysis in Dogs and Cats: An Update after 20 Years

In dogs and cats, computer-assisted sperm analysis (CASA) was originally described almost 20 years 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.

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.

Lipid peroxidation and antioxidant enzymes activity in avian semen

The present study compared the antioxidant system and lipid peroxidation in semen of two avian species: chicken and goose. The experiment was conducted on Greenleg Partridge roosters and White Koluda(®) ganders, each represented by 10 mature males. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma. In gander spermatozoa, the amount of MDA was 10 times greater (P<0.01) than in rooster spermatozoa. Each of the investigated antioxidant enzymes had greater (P<0.01) activity in goose than chicken sperm. Catalase activity was detected in seminal plasma and spermatozoa from both studied species for the first time. In seminal plasma, the activity of GPx was two times greater (P<0.01) in the White Koluda(®) than in chickens, whereas SOD activity was less (P<0.01) than in chickens. This is the first study describing the presence of CAT in avian semen and the occurrence of indicator of lipid peroxidation (LPO) in geese. Data from the present study clearly show the species-specific differences in the activity of antioxidant defense and LPO. The greater amount of lipid peroxidation and greater activity of antioxidant enzymes in goose semen might suggest that spermatozoa were under greater oxidative stress and the enzymes were not utilized for the protection of functionally and structurally impaired cells. In turn, in fresh chicken semen a lesser activity of antioxidant enzymes accompanied with a lesser lipid peroxidation amount and good semen quality could indicate that fowl spermatozoa were under oxidative stress, but the enzymes were employed to protect and maintain sperm quality.

Robertsonian Translocation in a Sex Reversal Dog (XX, SRY negative) May Indicate that the Causative Mutation for This Intersexuality Syndrome Resides on Canine Chromosome 23 (CFA23)

A Bernese mountain dog was subjected for clinical evaluation due to the presence of ambiguous external genitalia (enlarged clitoris). Anatomical and histological studies revealed the presence of one testicle, one ovotestis and a uterus. This dog was classified as a female-to-male sex reversal, with 2 normal X chromosomes and a lack of the Y chromosome-linked genes SRY and ZFY. It is the first case of this syndrome in this breed. Apparently a Robertsonian translocation, rob(5;23), was also identified in this dog and it is again the first case of this type of chromosome abnormality in this breed, as well as the first case of co-occurrence of the sex reversal syndrome along with a centric fusion in the dog. Since on the canine chromosome 23 (CFA23) 3 genes (FOXL2,PISRT1 and CTNNB1) involved in the sex determination process are present, further cytogenetic FISH studies were carried out with the use of BAC probes specific for this chromosome. It was found that a pericentromeric fragment of CFA23 was deleted as a result of the centric fusion. We hypothesize that a cis regulatory sequence for the sex determination genes on CFA23 (e.g. proximally located CTNNB1) is present in the deleted fragment. Thus, a causative mutation responsible for this sex reversal syndrome may reside on CFA23.

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.

Vitrification of domestic cat oocytes--effect on viability and integrity of subcellular structures.

Cryopreservation of female gametes is a crucial part of assisted reproduction techniques. The domestic cat is a model for both wild felids and human research. The aim of this study was to evaluate the effect of different vitrification protocols applied to feline oocytes at different maturational stages on the preservation of oocyte viability and integrity of subcellular structures. The vitrification solutions consisted of 20% ethylene glycol, 20% DMSO, 20% FCS, and 1.5 m Trehalose with and without 10% Ficoll PM-70. Markers for cell viability (PI/FDA), cytoskeleton organization (Anti-α-Tubulin-FITC antibody, Phalloidin-TRITC), as well as nuclear configuration (DAPI) were used for evaluation of vitrified-warmed oocytes. Our results show that 52% and 41% of live mature and immature oocytes, respectively and until 32% of microtubules, 28% of nuclear configuration and 36% of microfilaments in the normal pattern can be obtained with protocol described in this paper. According to our data, Ficoll PM-70 essentially improves the oocytes survival upon vitrification.

Possibilities of using the European bison (Bison bonasus) epididymal spermatozoa collected post-mortem for cryopreservation and artificial insemination: a pilot study

BackgroundEuropean bison is the largest mammal in Europe with the population of approximately 4000 individuals. However, there is no report of post-mortem spermatozoa collection and cryopreservation from this species and the aim of this study was to test if the epididymal spermatozoa collected post-mortem from European bison are suitable for cryopreservation and artificial insemination (AI).MethodsEpididymides were collected post-mortem from two European bison bulls at age of 8 (bull 1) and 11 year (bull 2). Epididymal sperm was harvested by making multiple incisions in caudae epididymidis, which were then rinsed with extender. The left epididymis of bull 1 was rinsed with BioXcell (IMV, France), whereas the right epididymis of bull 1 and the right and left epididymides of bull 2 were rinsed with the extender based on Tris, citric acid, glucose, egg yolk, glycerol, antibiotics and distilled water (extender II). The diluted semen was cooled to 5 degrees C, and frozen in liquid nitrogen vapour. Then, properties of the frozen/thawed semen were examined with the use of computer-assisted semen analysis system, and thirty cows and nine heifers of domestic cattle were artificially inseminated.ResultsMotility of fresh spermatozoa collected from the right epididymis of bull 1 was 70% (spermatozoa diluted with extender II), and from the left one was 60% (spermatozoa diluted with BioXcell), whereas motility of fresh spermatozoa collected from bull 2 was 90% (spermatozoa diluted with extender II). Spermatozoa motility just after thawing were 11 and 13% in bull 1, respectively for spermatozoa collected from the left and right epididymis and 48% in bull 2. As a result of AI of domestic cows and heifers with the frozen/thawed European bison spermatozoa, two pregnancies were obtained in heifers. One pregnancy finished with a premature labour after 253 days of pregnancy, and the second one after 264 days of pregnancy.ConclusionsThis is the first report showing pregnancy in the domestic cattle following AI with frozen-thawed European bison spermatozoa collected post-mortem. The protocol of spermatozoa collection, dilution, and cryopreservation presented in this paper may be useful for the creating genetic resource bank in the European bison.

Chromosome Instability in a Calf with Amelia of Thoracic Limbs

We report here on a case of a Holstein-Friesian male calf with the congenital total absence of thoracic limbs (amelia). cytogenetic study showed a high rate of chromosome instability, represented by chromosome or chromatid breaks and gaps in 46% of the analyzed metaphase spreads. Moreover, 12% of the spreads appeared to be polypolid. The number of micronuclei also was significantly higher when compared to control animals. This paper discusses the association between chromosome instability and limb malformation.