Feline Wharton's jelly-derived mesenchymal stem cells as a feeder layer for oocytes maturation and embryos culture in vitro.

Introduction: Due to their capacity to release growth factors and cytokines, co-culture using mesenchymal stem cells has been considered a good alternative to promoting the maturation of the oocytes and the embryo's development quality in vitro in different mammalian species. In this regard, we investigated the effect of feline Wharton's jelly MSCs as feeders layer in oocyte maturation-consequently, the development of resulting embryos in co-culture. Methods: Oocytes with dark cytoplasm and a few layers of cumulus cells were collected and subjected to in vitro maturation and embryo culture using commercial media with and without MSCs addition. The oocytes' nuclear maturation and the degree of cumulus expansion in different groups were assessed after 24 h; the development of the embryo was evaluated every 12 h until day eight. Results: Although MSCs increased the proportion of cumulus cells oocytes exhibiting cumulus expansion, there were no significant differences in the percentage of matured oocytes (metaphase II) among the groups (p > 0.05). However, the embryo development differs significantly, with a higher cleavage, morula, and blastocyst percentage in oocytes matured with MSC co-culture conditions than in commercial media alone (p < 0.05). Also, we observed higher morula and blastocyst rates in the embryos co-cultured with MSCs during the in vitro culture (p > 0.05). Conclusion: Based on our results, the co-culture with MSCs during the oocyte maturation resulted in better embryo development, as well as the MSCs addition during embryo culture returned an increased number of morula and blastocysts. Further research is needed to fully understand and optimize the use of MSCs in oocyte maturation and embryo development.

Mesenchymal Stem Cells: Generalities and Clinical Significance in Feline and Canine Medicine.

Mesenchymal stem cells (MSCs) are multipotent cells: they can proliferate like undifferentiated cells and have the ability to differentiate into different types of cells. A considerable amount of research focuses on the potential therapeutic benefits of MSCs, such as cell therapy or tissue regeneration, and MSCs are considered powerful tools in veterinary regenerative medicine. They are the leading type of adult stem cells in clinical trials owing to their immunosuppressive, immunomodulatory, and anti-inflammatory properties, as well as their low teratogenic risk compared with pluripotent stem cells. The present review details the current understanding of the fundamental biology of MSCs. We focus on MSCs' properties and their characteristics with the goal of providing an overview of therapeutic innovations based on MSCs in canines and felines.

Preliminary study on fluid bolus administration for prevention of spinal hypotension in dogs undergoing elective cesarean section.

Introduction: This study aimed to investigate the effect of fluid bolus administration during epidural anesthesia (coload) in female dogs scheduled for elective cesarean section (CS). Hypotension is one of the most common complications of epidural (EA) and spinal (SA) analgesia, and in the case of cesarean section, it may pose a significant risk for placental perfusion and subsequent fetal vitality and puppy survival. Methods: Pregnant bitches scheduled for elective CS underwent EA with (treatment group) or without (control group) intravenous fluid bolus administration. The following parameters were measured and compared between both groups: HR, RR, etCO2, SpO2, systolic, diastolic and mean arterial blood pressure were measured at three time points (T1: before surgery, T2: after the last puppy removal, and T3: end of surgery) in dams; vitality (Apgar score at 0, 5, and 20 min) and umbilical cord blood parameters (pH, pCO2, HCO3, base excess, lactate and glucose) in newborns. Results: The results indicated that crystalloid coloading increased maternal systolic, diastolic, and mean blood pressure (treatment vs. control group, 101.46 ± 9.18, 48.01 ± 13.47, and 67.07 ± 13.15 mmHg vs. 80.68 ± 7.29, 36.52 ± 8.75, and 180 52.30 ± 7.77, p < 0.05) with significantly fewer episodes of hypotension. Additionally, puppies in the treatment group received higher scores in the 5-min (7.91 ± 1.67 vs. 6.74 ± 2.20) and 20-min (9.38 ± 0.87 vs. 8.39 ± 2.50) assessments without the favorable effect on umbilical blood gas parameters. Discussion: Based on the obtained results, it can be stated that crystalloid coload offers an effective option in cases of hypotension during cesarean section, with clear benefits for both mothers and newborns.

Feline umbilical cord mesenchymal stem cells: Isolation and in vitro characterization from distinct parts of the umbilical cord.

Mesenchymal stromal/stem cells (MSCs) are a particular population of cells that play an essential role in the regeneration potential of the body. As a source of MSCs, the umbilical cord (UC) has significant advantages, such as a no-risk procedure of tissue retrieval after birth and the easiness of MSCs isolation. In the presented study, the cells derived from the feline whole umbilical cord (WUC) and two separate parts of the UC tissue, including Wharton's jelly (WJ) and umbilical cord vessels (UCV), were investigated to check whether they exhibit MSCs characteristics. The cells were isolated and characterized based on their morphology, pluripotency, differentiation potential, and phenotype. In our study MSCs were successfully isolated and cultured from all UC parts; after one week of culture, the cells had a typical spindle shape consistent with MSCs morphology. Cells showed the ability to differentiate into chondrocytes, osteoblasts and adipocytes cells. Two markers typical of MSCs (CD44, CD90) and three pluripotency markers (Oct4, SOX2 and Nanog) were expressed in all cells cultures; but no expression of (CD34, MCH II) was evidenced by flow cytometry and RT-PCR. In addition, WJ-MSCs showed the highest ability of proliferation, more significant pluripotency gene expressions, and greater differentiation potential than the cells isolated from WUC and UCV. Finally, we conclude in this study that cat MSCs derived from all the parts are valuable cells that can be efficiently used in various fields of feline regenerative medicine, but cells from WJ can offer the best clinical utility.

Comparison of 2 anesthetic protocols and surgical timing during cesarean section on neonatal vitality and umbilical cord blood parameters.

BACKGROUND: The objective of this study was to evaluate the relationship between the mode of anesthesia, the time form the induction to the extraction of a puppy and the immediate postnatal vitality and umbilical cord blood gases parameters in cesarean section derived-puppies. Two different anesthetic protocols were used: inhalation using isoflurane (ISO) and combined-inhalation and epidural (EPI) with propofol being the induction agent. RESULTS: Significant differences were found in ISO group in pH values, pCO2 levels and Apgar scores between puppies at different extraction times (< 30 vs. ≥ 30 min). In ISO group puppies extracted later were more acidic (7.16 vs. 7.22), had higher levels of pCO2 (69 vs. 57 mmHg) and lower Apgar scores at birth (1.2 vs. 2.5). On the contrary, in EPI group no differences were observed between the delivery time, umbilical blood gas parameters and puppies' vitality. Furthermore, the dams from the EPI group required lower concentrations of isoflurane (MAC 1.11 ± 0.19 vs.1.37 ± 0.16, p < 0.001). CONCLUSIONS: Multiple pregnancies frequent in dogs lead to significant differences in extraction times between the first and the last puppy during cesarean section. Obtained results showed that the mode of anesthesia and the surgical time would influence the neonatal outcome during cesarean section in dogs. The higher concentration of isoflurane with the longer time of exposure had a negative effect on the initial newborn vitality as well as the umbilical cord blood gas parameters. Therefore, when performing CS in giant dog breeds or expecting many puppies in the litter, it is worth considering epidural component that allow for lower concentrations of inhalant agents, which may contribute to a better clinical condition of newborns.

Ultrastructural changes in feline oocytes during ovary storage for 24- and 48-hours.

Despite established microscopic markers of feline oocyte quality, little is known about their ultrastructural traits. To the best of our knowledge, there is no published report analysing the effect of 24 and 48 h ovarian storage time on the domestic cat oocytes characteristics at the ultrastructural level. Oocytes (n = 30) were classified using the light microscopy as good or bad quality and then proceeded for TEM observations. The location, shape, size and distribution of each organelle was noted in each examined oocyte. In in good quality oocytes the cytoplasmic organelles were generally easier to identify, and furthermore its distribution pattern was more obvious to spot than in bad quality ones. Whereas bad quality oocytes were typically characterised by the lower visibility of the cellular structures and cytoplasmic architecture was less apparent and often arranged without a predictable pattern. In good quality oocytes obtained from fresh ovaries cytoplasmic vacuoles (CVs) occupied a significantly larger area (0,72 vs. 0.18 CVs/µm2, respectively) than in bad quality ones, whereas in bad quality and stored oocytes more cytoplasm was occupied by lipid droplets (LDs) than in fresh good oocytes (0,22 ± 0,09 vs. 0,09 ± 0,05 respectively). It can be concluded that ultrastructure changes in feline oocytes during 24 and 48 h ovarian storage cannot be assessed in light microscopy. The ultrastructure of oocytes was seriously disturbed after 48 h of ovary storage, despite being classified as good quality. However, further investigations utilizing more cells are necessary to confirm reported traits of ultrastructure changes in stored and non-stored oocytes of good and bad quality.

A Comparative Study of the Effects of Osaterone Acetate and Deslorelin Acetate on Sperm Kinematics and Morpho-Functional Parameters in Dogs.

Benign prostatic hyperplasia (BPH) is one of the most common problems in older male dogs that often has a huge impact on their health and welfare. This article presents a comparison between osaterone acetate (Ypozane®; Virbac®)(OA) and deslorelin acetate (Suprelorin®; Virbac®)(DA), medications that are the main therapeutic alternative to castration in dogs with BPH. Forty dogs were divided into four groups: I-negative control (five dogs without BPH); II-positive control (10 individuals diagnosed with BPH); III-15 dogs treated with DA, and IV-10 individuals treated with OA. Semen fractions were collected on days 0 (day of treatment), 7, 14, and 21, and weeks 8, 12, 16, and 20. Macroscopic, microscopic and CASA analyses were performed. Both DA and OA significantly affected the properties of the canine ejaculate. The DA lead to the lack of libido and had lesser effects to the sperm function before it caused azoospermia, whereas OA had no effect on libido and only temporary reduction in seminal plasma volume was observed, which resulted in temporary deterioration in the percentage of motile and progressive spermatozoa.

Dead or Alive? A Review of Perinatal Factors That Determine Canine Neonatal Viability.

The perinatal period has a critical impact on viability of the newborns. The variety of factors that can potentially affect the health of a litter during pregnancy, birth, and the first weeks of life requires proper attention from both the breeder and the veterinarian. The health status of puppies can be influenced by various maternal factors, including breed characteristics, anatomy, quality of nutrition, delivery assistance, neonatal care, and environmental or infectious agents encountered during pregnancy. Regular examinations and pregnancy monitoring are key tools for early detection of signals that can indicate disorders even before clinical signs occur. Early detection significantly increases the chances of puppies' survival and proper development. The purpose of the review was to summarize and discuss the complex interactions between all elements that, throughout pregnancy and the first days of life, have a tangible impact on the subsequent fate of the offspring. Many of these components continue to pose challenges in veterinary neonatology; thus, publications presenting the current state of knowledge in this field are in demand.

Is an epidural component during general anaesthesia for caesarean section beneficial for neonatal puppies' health and vitality?

Regional centro-axial block is a recommended technique for uncomplicated caesarean section in human medicine. Since the application of regional anaesthesia as the only technique in veterinary medicine is impractical, the objective of the study was to assess and compare the epidural component of caesarean section (CS) on maternal and fetal outcomes. Bitches (n = 36) undergoing elective CS were enrolled in this study. Females were randomly assigned into two groups: Gr I (Isoflurane, n = 20) and Gr IE (Isoflurane plus Epidural, n = 16). Anaesthesia was induced with propofol, and maintained with isoflurane in oxygen. In the IE group, epidural anaesthesia was also performed using lidocaine. The maternal intraoperative parameters were compared at three time points: T1 - just before the skin incision, T2 - after the last puppy removal, and T3 - at the end of surgery. At least 100 mcl of mixed umbilical cord blood was collected for gas analysis. The modified Apgar scoring system (AS) was used to objectively score newborn health and vitality immediately after birth (0 min), 5 and 20 min after birth. Systolic, diastolic, and mean blood pressure were lower in the IE group at T1, T2, and T3 compared to I group (p < 0.05). In the combined anaesthesia group blood pressure remained stable but low at all time points. Throughout surgery, the IE group required a lower concentration of isoflurane (p < 0.05). The median values of the umbilical blood gas results were found to be similar (p > 0.05) in both investigated groups. The initial results of the Apgar score were comparable in the I and IE groups (p > 0.05). However, subsequent AS measurements revealed significant differences between both groups. Puppies from the IE group received better AS scores at 5 and 20 min compared to the I group (median AS 8 and 9 vs. 5 and 8, respectively). The results obtained demonstrated that epidural anaesthesia administration reduced the requirement for isoflurane in dams undergoing caesarean section and despite episodes of maternal hypotension did not affect the results of neonatal umbilical blood gas. Furthermore, newborns from the epidural anaesthesia group improved more quickly postnatally, developing a satisfactory condition in a shorter time.

Umbilical Cord Blood Gas Parameters and Apgar Scoring in Assessment of New-Born Dogs Delivered by Cesarean Section.

The article presents the results of the clinical evaluation (Apgar scores, AS) and umbilical cord blood gas analysis (UCBGA) obtained from clamped umbilical cords of newborn pups delivered by the elective Caesarean section. The study was planned as a controlled clinical study, the newborns were allocated into one of the groups, I-critical neonates (severe distress, AS ≤ 3), II-weak neonates (moderate distress, AS 4-6) and III-healthy neonates (no distress, AS ≥ 7). The following parameters were evaluated: pH (pH units), carbon dioxide partial pressure (pCO2; mmHg), oxygen partial pressure (pO2; mmHg), actual bicarbonate (cHCO3-; mmol/L), total carbon dioxide (cTCO2; mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b); mmol/L), oxygen saturation (csO2; %), lactate (Lac; mg/dL), hematocrit (Hct; %PCV), hemoglobin (cHgb; g/dL), glucose (Glu; mg/dL), ions (Na, K, Ca, Cl). The majority of puppies had low AS at birth (AS 4-6 in 38.1% and AS ≤ 3 in 57.1% of the neonates), but most of them (85.7%) improved by the 20th min. reaching AS of 7 and more. Moreover, puppies with lower AS (≤ 3) were at higher risk of death within the first 24h (20.8% did not survive). The positive correlation was found between Apgar score measured at 0 min and pH (r = 0.46, p = 0.01), and between Apgar score (at 0 min) and base excess in whole blood measured [BE(b)] r = 0.36, p = 0.03). Whereas, a negative correlation was detected between Apgar score at 0 and 5th min and glycemia (r = -0.42, p = 0.05, r = -0.34, p = 0.02 respectively. Overall, the puppies with higher glucose levels had lower Apgar scores and were at higher risk of death. Furthermore, in our study, the newborn puppies had mild acidemia with elevated pCO2 levels and the HCO3 at the lower range of normal limits, suggesting the mixed component in the acidemic state. Adaptation to extra-uterine life is crucial and any practical improvement in neonatal diagnostics and care would be beneficial for newborn puppy survival.

B-Mode and Doppler Ultrasonographic Findings of Prostate Gland and Testes in Dogs Receiving Deslorelin Acetate or Osaterone Acetate.

This article presents B-mode and color Doppler imaging of the prostate and testes in dogs suffering from benign prostate hyperplasia (BPH), and receiving deslorelin acetate (SuprelorinTM) or osaterone acetate (YpozaneTM). The study was planned as a controlled clinical trial, dogs were divided into negative control (healthy dogs, n = 10), positive control (dogs with BPH, n = 10), and study groups, III (n = 15), receiving deslorelin acetate (DA), and IV (n = 10), receiving osaterone acetate (OA). The B-mode appearance of the prostate parenchyma improved in all investigated dogs from the DA group, and in 60% of OA dogs. Prostate volume was reduced more quickly with OA (from D14), but lasting for a shorter time (on average up to week 20), compared to DA that reduced the prostate volume more slowly (>8 weeks), but the reduction remained longer (>24 weeks). The systolic peak velocity (SPV) and mean velocity (Vmean) were higher in all dogs diagnosed with BPH, compared to Control Group I. The indices did not change in both Control Groups I and II, whereas in study Groups III and IV they decreased throughout the study period compared to day 0 and Control Group II. In Group III the highest reduction was noted from day 21 to week 8, whereas in Group IV the lowest Vmean was recorded before day 21. Testicular parenchyma and volume changed significantly in Group III receiving DA, and the velocity of blood flow in the testicular artery correlated positively with testicular volume only in this group (III). The present study proved the usefulness of B-mode and color Doppler US imaging techniques for diagnosis and progress assessment of dogs suffering from BPH. The blood flow kinetics (mainly SPV) demonstrated a time association between the blood flow changes registered in the prostatic artery, and the subsequent volumetric and sonographic improvement of the prostate parenchyma. The reduction in flow indices was noted prior to the reduction in prostate volume, suggesting that the sonographic recovery of the prostate tissue, occurs secondarily to the regression of the prostate vascular system. Both investigated medications (osaterone acetate and deslorelin acetate) led to a significant sonographic improvement. Deslorelin acetate reduced prostate volume more slowly, but its effect lasted longer than for osaterone acetate.

Comparison of Clinical Effectiveness of Deslorelin Acetate and Osaterone Acetate in Dogs with Benign Prostatic Hyperplasia.

This article presents the results of a randomized clinical trial, designed to compare the efficacy and therapeutic profiles of YpozaneTM (osaterone acetate-OA) or SuprelorinTM (deslorelin acetate-DA) in male dogs with clinical signs of benign prostate hyperplasia (BPH). Forty-five intact male dogs were used in the study. The Group I (negative control) included 10 healthy dogs, the Group II (positive control) included 10 dogs with confirmed BPH and no treatment, whereas Group III and IV consisted of dogs with BPH and treated either with DA (15 dogs) or OA (10 dogs). The clinical response, testosterone and estradiol levels, hematology, biochemistry, and adverse effects incidence were evaluated. Both OA and DA proved to be effective for BPH treatment in dogs, as they allowed for the clinical remission in all treated dogs. The complete alleviation of BPH symptoms was noticed sooner with the use of OA (in 80% of dogs from day 7) compared to DA (in 40% of dogs within the first 21 days). The recurrence of clinical signs related to BPH was observed from week 24 in dogs treated with OA, whereas no relapse was noticed in dogs treated with DA at the end of the 36 weeks of the observation period. In 5 dogs (33%) treated with DA, a flare-up effect (increase in the clinical signs associated with BPH) was noticed on day 7. Despite individual differences in the clinical action, both medications were effective and safe options for the treatment of symptoms related to BPH in dogs.

Dystocia after Unwanted Mating as One of the Risk Factors in Non-Spayed Bitches-A Retrospective Study.

This article presents a retrospective study on dystocia cases in bitches that were unintentionally mated and carried an unwanted pregnancy in the last 39 years. The evaluated medical records include 76 cases of difficult labour, which is 8.3% of 914 dystocia cases recorded during the period. Of these bitches, 38.2% (29/76) were 8 years, and 18.4% (14/76) were younger than 12 months. In 67/76 cases (88.2%), conservative (pharmacological and manual) obstetrical assistance proved to be unsuccessful, and caesarian section (CS) had to be performed, in contrast to the remaining recorded cases of dystocia (in which the pregnancy was intended and expected) when CS was performed significantly less often, in 71.5% (599/838) of cases. In unplanned pregnancies, 46.6% (110/236) of delivered pups were dead compared to only 26.4% (864/3273) dead pups in planned pregnancies. p value < 0.05 was considered significant. Despite the widespread availability of the spaying procedure nowadays and its safety, unplanned and unwanted pregnancies in dogs are still a concern in clinical practice. However, throughout the years investigated here, we observed an apparent decrease in the occurrence of dystocia after unintended mating, with much less recorded cases from year 2004 (71 vs. 5). Most probably, this is due to the increasing popularity of surgical castration in both females and males, and rising societal awareness of its importance, giving hope that some improvement in the welfare of dogs has already been achieved.

Time of early cleavage affects the developmental potential of feline preimplantation embryos in vitro.

The study compared developmental competence of embryos, based on the timing of the first cleavage and morula formation, with their subsequent ability to reach the blastocyst stage and the resulting blastocyst morphology and quality. Cleaved embryos were separated at 18 hours post insemination (hpi), 24 hpi, and 30 hpi and then cultured in droplets to follow the individual embryo development. The significantly higher percentage of the blastocyst formation was noted in the group I of embryos cleaving within less than 18 hpi (12.7%) compared with the group II cleaving within 18 to 24 hpi (10.7%), None of the late-cleaving embryos (group III >24-30 hpi) reached blastocyst stage in our experiment. Interestingly, the hatching ability was similar regardless the time of the first cleavage (I: <18 hpi; 6.4% and II: 18-24 hpi; 5.4%). The ability to hatch was correlated with the time of morula formation; only embryos that reached morula on Day 4 or Day 5 were able to develop into hatching blastocyst (8.4% and 3.3%, respectively). The differential cell staining revealed significantly more blastomeres in blastocysts obtained from embryos cleaving within 18 to 24 hpi than the blastocysts obtained from embryos cleaving less than 18 hpi (188.6 ± 21.9 vs. 129.3 ± 16). Embryos cleaving within 18 to 24 hpi also demonstrated the higher number and percentage of embryoblast cells (97.2 ± 12.6, 51.6 ± 2.9 vs. 46.6 ± 9, 36.3 ± 6.6). The presented results confirmed the association among the onset of the first cleavage, time reaching morula, and subsequent blastocyst formation and quality.

Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine.

Characteristics of urethral and epididymal semen collected from domestic cats--A retrospective study of 214 cases.

This study was designed to describe and compare basic semen characteristics and sperm motility parameters obtained via computer-assisted sperm analysis (CASA) in feline semen collected from the urethra and epididymis, on the basis of large, unselected population of domestic cats. The semen collected from 214 males was subjected for routine semen assessment and CASA evaluation. Semen collected by urethral catheterization (CT) and by epididymal slicing (EP) has comparable characteristics according to total sperm count (47.7 ± 42.1 and 52.9 ± 45.0), subjective motility (71.1 ± 17.0 and 69.3 ± 13.9), viability (74.9 ± 13.4 and 76.7 ± 10.6), and morphology (52.6 ± 19.0 and 47.2 ± 17.4). The study of a large feline population confirmed a high incidence of teratospermy in cats, which negatively affects sperm motility parameters assessed by CASA. A lack of a correlation between CT and EP semen for total sperm count and viability, as well as occasional gross differences between the morphology of CT and EP semen of the same cat suggests that many factors may affect sperm cells, and the fertility and/or infertility of patients should not be assessed after examining only one sample. Additionally, technical problems with assessment of EP samples (understated results) suggest that CT semen is more appropriate for an analysis by CASA than EP.

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%).

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.

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.