Oral and Poster Abstracts

to the extender: A-gentamicin (250 µg/mL), tylosin (50 µg/mL),
lincomycin (150 µg/mL), spectinomycin (300 µg/mL); or Bpenicillin
(500 UI), streptomycin (500 UI), lincomycin (150 µg/mL),
spectinomycin (300 µg/mL). Some authors have shown that
Leptospira spp. remain viable in semen after thawing, in spite of
antibiotic treatment. Amicacine sulphate has been shown to be
efficient against ubiquitous microbiota control when added to the
semen extender, being easier to prepare and cheaper than treatments
A and B. The present trial aimed at studying the efficacy of three
antibiotic treatments A, B e C (amicacine sulphate -1,500 µg/mL)
added to the semen extender for inactivation of Leptospira spp., in
experimentally contaminated semen. Samples were collected from 8
bulls (3-7 years old), all negative to leptospirosis, using an artificial
vagina. Three hundred µL of semen were added to the extender
treated with A, B or C antibiotic protocols and experimentally
contaminated with 3.0 µL cultures of avirulent Leptospira spp
serovars Hardjo (strain Hardjoprajitno) and Wolffi (strain 3705), and
virulent Grippothyphosa (strain Moskava V), with 10 6 leptospires
/mL. Samples were incubated at 30°C for 30 min and frozen in liquid
nitrogen for 48h. Samples were thawed at room temperature,
incubated at 30°C for 30 min and cultured in EMJH 30oC for 10
days. PCR was performed using proteinase K extraction and Lig 1
and Lig 2 primers. Controls without antibiotic were 100% positive in
PCR, but only 2/8 and 5/8 cultures for avirulent and virulent strains
were positive, respectively. From the total of samples, 60/96
(62.5%) were positive in PCR and 17/97 (17.7%) in culture. A, B or
C treatments significantly reduced contamination when compared to
the control; treatment A produced a greater reduction than B, but a
decrease similar to C. Treatment C was more effective against
Hardoprajitino and Wolffi than against Grippothyphosa. These
positive results in PCR or bacteriologic examination confirmed the
risk of leptospirosis transmission by semen when antibiotic
treatment is the unique sanitary measure available.
This paper was supported by FAPESP: Fundaçao de Amparo a
Pesquisa do Estado de Sao Paulo, Brazil
Key words: bovine semen, antibiotic extender, Leptospira spp
899 Looking for a Needle in a Haystack - Identification of Bovine
Mammary Gland Stem Cells
M. Kozlowski 1 , M. Gajewska 1 , J. Bierla 1 , B. Gajkowska 2 , T. Motyl 1
1
Warsaw University of Life Sciences, Faculty of Veterinary Medicine,
Department of Physiological Sciences, Warsaw, Poland
2
Polish Academy of Science, Medical Research Center, Warsaw,
Poland
At birth, bovine mammary parenchyma consists of a rudimentary
duct network. Full mammary epithelium development occurs during
pregnancy and lactation. The cycle of pregnancy-associated
mammary gland proliferation, differentiation, and involution occurs
many times during the lifespan of diary cattle. The development and
subsequent redevelopment of the mammary gland with each
successive lactation; involution cycle provides a strong indication
that a self-renewing population exists within the mammary
parenchyma. The aim of our study is identification of bovine
mammary gland progenitor cells using morphological, biochemical
and molecular markers. Mammary glands were obtained from 2
years old Holstein heifers. Morphological characteristic of cell in
mammary gland tissue was performed using electron microscopy
according to Chepko and Smith designation. Isolation of mammary
gland epithelial cells was carried out by collagenase/hyaluronidase
digestion assay. Isolated cells were stained with Hoechst and
analyzed by FACS to define side population (SP). In the present
morphological studies we identified small light cells (SLC) similar
to those previously described in mouse mammary gland. Organelles
of these cells are small and show no structural evidence of
specialized function. This may be a combined population of stem
and primary progenitor cells. Hoechst 3342 efflux is one of the
features of haematopoetic stem cells. Cells with this ability are
referred to as the side population (SP). We used this assay to look for
the side population in bovine mammary gland. We managed to
identify a small fraction of cells (0,48%) that exhibited a SP
phenotype. The formation of the side population was blocked by 20
µM verapamil, which was consistent with previous studies showing
a requirement for AMB transporter family function in the SP
phenotype. The results of our study confirmed the existence of a
small population of stem and progenitor cells in the bovine
mammary gland. Our group is planning to continue the work on
further characteristic of these cells in order to create a transcriptomic
profile of the side population found in bovine mammary gland.
Key words: mammary gland, stem cells, side population
900 Comparative Pharmacokinetic Study of Progesterone in Cows
Following Single Administration of Two Intravaginal Devices
S. Floc’H 1 , A. Lagarde 2
1 CEVA santé Animale, Livestock Business Unit, Libourne, France
2 CEVA santé Animale, Project Department, Libourne, France
The objective of this study was to compare the kinetic profiles of
progesterone following single administration of two intravaginal
devices according to a cross over design.
Six Prim Holstein non lactating ovariectomised cows were included.
The study has been carried out according to a single dose,
randomised, two periods in a cross-over design and a wash-out
period of 7 days was allowed between each treatment period.The test
items [A = PRID ® from CEVA S.A.; France and B = CIDR ® Eazi
Breed from INTER AG; New Zealand] has been administered at D0
of each period as a single intravaginal insertion which is the
recommended route for use in bovine females. The devices stayed in
place for 7 days. Blood specimens were collected at the following
sampling times for each treatment period: just before the treatment,
1h, 3h, 6h, 9h, 25h, 33h, 49h, 57h, 73h, 81h, 97h, 105h, 121h, 129h,
145h, 153h, 169h (just before removal device), 170h, 171h post
administration. Plasma progesterone concentrations were measured
by RIA at INRA Nouzilly (France) and described by Saumande J. in
Steroid Biocem. Molec. Biol. 1991.The main pharmacokinetic
parameters of plasma progesterone for treatment A and treatment B
were: Cmax of 4.8 ± 0.7 ng/ml and 3.73 ± 0.6 ng/ml respectively,
and AUCtot of 666.580 ng.h/ml and 576.3 ng.h/ml respectively. No
statistically significant difference was noticed for AUCtot
(p=0.33737) while a statistically significant difference was noticed
for Cmax (p=0.00599) on behalf of treatment A. A large release of
progesterone at the beginning of the treatment leads most of the time
to dominant follicle atresy by a sudden and massive inhibition of
FSH (Follicle Stimulating Hormone) and LH which are necessary for
follicle growth and maturation. Therefore the start of a new follicular
wave can occur allowing the presence of a mature dominant follicle
at the time of device removal. This prevents persistent dominant
follicles and therefore allows good fertility at induced oestrus. In the
present study, the kinetic profiles of progesterone were compared
following single intravaginal administration of test item A (PRID ® )
and test item B (CIDR ® ). Items were administered for a treatment
period of 7 days according to a single dose. It was concluded that a
statistically significant difference (p = 0.006) was observed on Cmax
in favour of PRIDv compared to CIDR ®
Key words: progesterone, intravaginal device, pharmacokinetic
901 A Comparison of the Sexual Behaviour of Holstein-Friesian
Cows between Housed and Pasture Environments
M. Palmer 1 , G. Olmos 2 , L. Boyle 2 , J. Mee 2
1
University of Edinburgh, Royal (Dick) School of Veterinary Studies,
Edinburgh, United Kingdom
1
Teagasc, Moorepark Dairy Production Department, Fermoy, Co.
Cork, Ireland
This experiment compared the sexual ethogram of 46 spring-calved
cows in two management environments [cubicle housing with a
total mixed ration (HOUSED) and rotational pasture with
concentrate supplementation (GRASS)]. Cows were blocked and
randomly allocated to treatment and were monitored by one
observer from ten days postcalving for nine weeks on the same
farm. The occurrence of nine behaviours (ano-genital sniffing given
or received, chin resting given or received, head-to-head butting,
mounting attempts, evasion of mounting attempts, mounting and
standing to be mounted) was recorded during the thrice daily (6.00,
12.00 and 19.00h) 20 minute visual observation sessions. Milk
sampling for progesterone analysis (EIA) was carried out on
Mondays, Wednesdays and Fridays to determine the dates of true
standing oestrus. Data in the 24h before and after standing oestrus,
(seven peri-oestral observation periods), were analysed by proc
FREQUENCY, GENMOD, NPAR1WAY, TTEST and
UNIVARIATE, as appropriate, in SAS. In both treatment groups
Reproduction and Biotechnology 203