Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
130 Poster Abstracts
integrity (PMI) (propidium iodide and carboxyfluorescein diacetate)
and sperm concentration. Samples were divided into three equal
aliquots, which were first diluted with TOG and, afterwards, with
TOG supplement with 10% of glycerol in proportions that permitted
final concentrations of 3, 5 or 7% of glycerol. Sperm samples from
the same ejaculate contained equal final volume (100 or 150 µL) and
sperm concentration (40 to 60 x 10 6 / mL). After load into 0.25 mL
straws, samples were placed in a programmed refrigerator at 5 o C for
60 minutes, then in liquid nitrogen vapour during 15 minutes and
immersed. Sperm samples were thawed at 46 o C for 12 seconds and
analyzed immediately (CASA and IMP), 30 (CASA) and 60 (CASA)
minutes post-thawing. Data were submitted to statistical analysis by
ANOVA and Tukey test, with p < 0.05 taken as significant. Among
all post-thaw moments, VAP (average path velocity), VSL (straight
line velocity), BCF (beat cross frequency) and STR (straightness)
were not different between 3, 5 and 7% of glycerol groups. Higher
values for TM (total motility), PM (progressive motility), R
(percentage of rapid spermatozoa) and PMI (plasma membrane
integrity) were obtained in all evaluated moments after thawing for
groups 5 and 7% of glycerol, with no statistical difference between
them. Values for ALH (amplitude of lateral head displacement)
increased after thawing only in group 3% of glycerol. Group 7% of
glycerol exhibited lower VCL (curvilinear velocity) values compared
to groups 3 and 5%. Since frozen-thawed sperm samples containing 5
or 7% of glycerol showed better results compared to 3% of glycerol
and considering that glycerol is known to be toxic for spermatozoa,
we can conclude that a concentration of 5% of glycerol is suitable for
freezing domestic cat spermatozoa.
P310
Preliminary studies on isolation and culture of the
epithelial, stromal and endothelial cells from the uterus of
domestic cat-technical report
Siemieniuch, M., Skarzynski, DJ.*
Institute of Animal Reproduction and Food Research, Olsztyn, Poland
The most of the previous experiments concerning feline female
reproductive regulations, were based on the hormones plasma levels,
morphological examination of ovaries and CL during laparoscopy and
behavioral changes in the animals. The local, immuno-endocrine
events within the feline ovary and/or uterus, and such interaction
between different cells of the endometrium have been largely ignored.
Thus, we decided to establish methodology for the isolation and
culture of different types of endometrial cells (epithelial, stromal and
endothelial cells) from uteri of domestic cat. The main goal of the
study is to establish the model for further examinations of local,
immuno-endocrine regulations in cat uterus and mechanisms of early
embryo development.
Uteri of queen were obtained by ovariohysterctomy. A polyvinyl
catheter was inserted into the uteri horn, and the end of the horn near
the corpus uteri was tied shut in order to retain an enzyme solution for
solubilizing the epithelial cells. 1-2 ml of enzyme solution (sterile
HBSS containing (dispase I, collagenase I, DN-ase IV and 0.1% BSA)
was then infused into the uterine lumen through the catheter.
Epithelial cells were isolated by incubation twice at 37,5˚C for 45 min
and 20 min with gentle shaking. The cell suspension obtained from
the first and second digestions was pooled and washed 3 times by
centrifugation in the gradient and finally resuspended in culture
medium (DMEM/Ham's F-12; supplemented with 10% calf serum).
After isolation of epithelial cells, the horns were longitudinally slit
and the surface was scratched. The rest of endometrial tissue were
digested in 10-20 ml of the above-described enzyme solution. After
20, 40 and 60 minutes stirring, dissociated cells were collected. For
isolation of endothelial cells, horns from intact uterus were cut and
minced into small pieces (1 mm 3 ). The mix cells were isolated as
described for stromal cells isolation. The pure population of
endothelial cells was isolated using Dynabeads® M-450 magnetic
beads coated by the lectin BS-1.
The cells of each cell type were separately seeded at a density of 1 x
10 5 viable cells/ml in 24-well plates. The culture medium was
changed every 2 days until confluency was reached. When the cells
were confluent the homogenity of cells was estimated using
immunofluorescent staining for specific markers of epithelial
(cytokeratin), stromal (vimentin) or endothelia cells (von Willebrand
VIII factor). The test revealed 95%, 90% and 95 % of purity of the
epithelial, stromal and endothelail cells in the culture, respectively.
P311
Retroflexion of the urinary bladder in a rottweiler bitch
during pregnancy
Sontas, BH*; Apaydin, SO; Toydemir, TSF; Kasikci, G; Ekici, H
Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine,
Istanbul University, Turkey
Clinical case A 2.5-year-old, pregnant rottweiler bitch, weighing 29
kg, was presented with a 24-hour’ history of a large mass of tissue
visible through the vulvar cleft and difficulty in parturition.
Accompanying complaints were loss of appetite and increased licking
of the mass. No previous trauma was reported, but the bitch had been
continuously barking through the night because of a snake in the
garden. The bitch had mated several times with a mixed-breed dog of
the same size two months before presentation and one year before the
bitch had whelped five live puppies without requiring any veterinary
assistance.
On clinical examination a large, firm, non-painful round ball-shaped
mass of tissue, approximately 10 cm in diameter, was identified
blocking the entrance of the vulva. The tissue was clean with no
haemorrhage or ulceration. Abdominal ultrasonography demonstrated
several fetuses without heart beats and the absence of the urinary
bladder in its anatomical position in the abdominal cavity.
Ultrasonography of the mass revealed anechoic appearance with no
fetal vesicles inside. Vaginal cytology showed the presence of
neutrophils and parabasal cells, typical of a bitch in the luteal phase.
Haematology, serum biochemical analysis and radiographic
evaluation of the mass or the caudal abdomen were not performed
because of the cost. Diagnosis of pregnancy, fetal death and
retroflexion of the urinary bladder confined to the vagina were made
according to the clinical and ultrasonographic findings. Removal of
the gravid uterus by ovariohysterectomy was performed and the
bladder was manually repositioned. The bitch recovered well and was
sent home after the surgical procedure. One week later, the bitch was
healthy with no complaints of dysuria, stranguria or urinary
incontinence. Two months after the surgery, the owner reported that
the bitch was clinically normal with no recurrence of the retroflexion.
To our knowledge, this is the first case of a vaginal mass occurring
after the retroflexion of the urinary bladder in a pregnant bitch.
Discussion A mass of tissue that is visible from the vulva is the most
common sign of vaginal hyperplasia, neoplasia or prolapse
(Manothaiudom and Johnston 1991). However, in the present case,
the mass was associated with the urinary bladder which was
retroflexed into the space between the vagina and pelvic wall because
of a perineal hernia. Perineal hernias occur most commonly in the
male when degenerative changes develop in the muscles of the pelvic
diaphragm as a result of hormonal influences or pelvic fractures
(Hayes et al. 1978, White and Herrtage 1986, Risselada et al. 2003,
Angeli et al. 2005).
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