Oral and Poster Abstracts

spermatozoon, average value). The same tendency was registered
both in the case of thermo resistance test during a period of three
hours of incubation at 37 °C and in the case of acrosomal integrity
distinguished through the FITC-PNA/PI-staining and examined at
flow cytometry. Acrosomal integrity was better (p0.05 ). Altogether, from 50 cases of pregnancy, 28 cases (56%)
were in the right horn and 22 cases (44%) were in the left horn. It is
concluded that insemination of cattle with left ovarian ovulation may
increase the rate of female fetuses at pregnancy and parturition.
However, this effect has not been observed in ewes.
Key words: sex ratio, sexing, left and right uterine horn pregnancy
879 Detection of Leptospira spp. in Semen and Vaginal Fluids of
Goats and Sheep by Polymerase Chain Reaction
W. Lilenbaum 1 , R. Varges 1 , F. Brandao 1 , A. Cortez 2 , S. Souza 2 , P.
Brandao 2 , L. Richtzenhain 2 , S. Vasconcellos 2
1
Universidade Federal Fluminense, Microbiology, Niterói - RJ -
Brasil, Brazil
1
Universidade de Sao Paulo, Preventive Medicine and Public Health,
Sao Paulo, Brazil
The purpose of the present study was to evaluate the use of PCR for
the detection of Leptospira spp. in semen and vaginal fluids of goats
and sheep. Thirteen goat herds and seven sheep flocks in the state of
Rio de Janeiro, Brazil were screened for leptospirosis, using
serologic approaches. In this first step, approximately 20% of the
adult animals in each herd/flock were randomly selected; overall,
248 caprine and 292 ovine serum samples were tested by a
microscopic agglutination test - MAT. From those, three herds and
three flocks with great proportion of seroreactive animals (>30% in
each herd/flock) were identified, and 19 goats (16 females and three
bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive
(specific anti-Leptospira titres >400, based on MAT), were selected
for more detailed studies. From those animals, samples of vaginal
fluids or semen were collected for bacteriological and molecular
assays. Diluted semen and vaginal fluid samples were centrifuged
(3000 x g for 10 min) and the supernatant used for both molecular
and bacteriological assays. Blood samples were also centrifuged
(1000 x g for 10 min) and examined for Leptospira antibodies by
MAT. Bacterial DNA was extracted by a phenol and guanidine thiocyanate
method. The PCR assay for the detection of Leptospira spp.
is genus-specific and based on protocol that employs the primers
Lep1 (5’-GGCGGCGCGTCTTAAACATG-3’) and Lep2 (3’-
TTAGAACGAAGTTACCCCCCTT-5’). For both species of
animals, the most common reactions were to serovars Hardjo,
Shermani, and Grippotyphosa. Although leptospires were detected
by darkfield microscopy in three vaginal fluid samples (from two
goats and one ewe), pure isolates were not obtained by
bacteriological culture of semen or vaginal fluids. However, seven
vaginal fluid samples (four goats and three ewes) and six semen
samples (all from rams) were positive on PCR. Based on these
findings, in addition to analogous findings in cattle, we inferred that
there is potential for venereal transmission of leptospirosis in small
ruminants.
Key words: leptospirosis, PCR, reproduction, sheep, goats
880 Use of 3D-Cell Culture of Bovine MEC in Study of the Role of
Hormones and Growth Factors in the Formation of
Differentiated Acinar Structures
M. Gajewska, M. Kozlowski, P. Jasinski, K. Hajduk, T. Motyl
Warsaw University of Life Sciences, Faculty of Veterinary Medicine,
Department of Physiological Sciences, Warsaw, Poland
Mammary gland epithelium is comprised of individual acinar units,
which are notable for hollow lumen, surrounded by polarized
epithelial cells. The development and maintenance of this polarized
structure is critical for the form and function of epithelial
cells.Mammary epithelial cells supported on a laminin-rich
extracellular matrix (ECM) form three-dimensional (3D) acinar
structures that mature to form polarized and functional monolayers
surrounding a lumen and having the ability to produce milk proteins.
We have used the 3D laminin-rich ECM cell cultures to study the
regulation of bovine mammary epithelial cells (MEC) differentiation
in vitro, on the model of BME-UV1 cell line. The role of hormones
(prolactin - PRL, growth hormone GH, and progesterone) and
growth factors (EGF, IGF-I and TGF-1) was examined in the context
of MEC polarization and functional differentiation.
Immunofluorescence, confocal microscopy and Western-blotting
techniques were used in order to determine the expression of chosen
polarization markers: E-cadherin and ZO-1, and a marker of
functional differentiation: milk protein L-casein.
Western-blot analysis showed an increased expression of L-casein in
bovine MECs cultured in ECM environment in comparison to a
classical monolayer cell culture. The 3-D cell culture conditions also
exhibited an increased expression of E-cadherin and ZO-1 proteins.
Confocal images of immunocytochemical staining revealed that
proper polarization of cells forming 3D-acinar structures was
obtained when hormones: PRL, or GH or progesterone were added to
the medium. However administration of growth factors: IGF, EGF or
TGF-1 caused a disturbed pattern of E-cadherin and ZO-1 staining,
which may suggest a failure in proper polarization of acinar
structures cultured on ECM.
The results of our study show that all used hormones exhibit a positive
role in the formation of fully polarized 3D-acinar structures, with a
hollow lumen and the ability to produce L-casein. The addition of EGF
or IGF-I to the cell culture led to a partial failure in cell differentiation,
causing a prolonged cell proliferation and not full lumen clearance,
while TGF-1 treated cells showed morphology of small disorganized
spheres. To summarize, our study has shown that the process of acini
formation by bovine MEC requires the involvement of studied
hormones, especially PRL and GH.
Key words: 3D-MEC culture, growth factors, prolactin, GH,
differentiation
881 Regulation of Autophagy in Bovine Mammary Epithelial Cells
A. Sobolewska 1 , M. Gajewska 1 , J. Zarzynska 2 , B. Gajkowska 3 ,
T. Motyl 1
Reproduction and Biotechnology 197