Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
146 Poster Abstracts
F1 (B6D2) female mice were superovulated and mated with DBA
males. Early blastocysts were collected from pregnant females and
cultured in KSOM till the expanded blastocyst stage, that were loaded
into 0.25 ml ministraws with CZB-Hepes. Embryos were treated with
600 bar pressure for 30 min at 23 ºC in a computer controlled
pressurizing device (Cryo-Innovation Inc., Budapest, Hungary).
Embryos were collected immediately and 120 min after the treatment.
Then, mRNA was isolated and cDNA prepared for real-time PCR
analyses.
To assess the change in gene expression profiles, nine stress related
genes and two reference genes (H2afz, and Ppia) were selected.
Primers were designed, optimized and standards prepared for real
time PCR analyses. Based on the analyses, the genes Azin1, Gas5,
Gadd45g and Sod2 were up-regulated (mean±SD) 1.88±0.25,
1.55±0.36, 1.47±0.59 and 1.41±0.25 fold, respectively after
hydrostatic pressure treatment, compared to the levels of the same
genes in the untreated control. All studied genes, but Gadd45g, have
returned to the control expression levels after 120 minutes culture.
The up-regulation of Gadd45g (1.92±0.60 fold) shows that
hydrostatic pressure has a prolonged effect on the transcription of this
gene. H2afz and Ppia genes were not significantly affected (P>0,05)
by the treatment, and this supports our previous results on the stable
expression of these genes during mouse preimplantation development.
This study demonstrates, for the first time, that HHP activates certain
stress related genes in the mouse embryo. The effects don’t
compromise developmental competence of the blastocysts, moreover
it may contribute to increased survival rates at the subsequent
cryopreservation.
Supported by OMFB-00364/2007 and NKTH/KPI Kozma F.
TUDAS-1-2006-0005
P360
Sexing in vitro produced bovine embryos with semen
selected by percoll or swim-up
Wolf, C 1 , Brass, KE 2 *
1Equine Reproduction, Federal University of Rio Grande do Sul, Brazil; 2 Large
Animal Clinics, Federal University of Santa Maria, Brazil
Preimplantation genetic diagnosis (PGD) is becoming a current issue
in animal reproduction biotechnology due to economical reasons.
Predetermining the sex of offspring is one example of PGD. This
study aimed to determine the percentage of male and female bovine
embryos in vitro produced after oocyte fertilization with Percoll
density gradient centrifugation or with self-migration (swim-up)
selected semen. In experiment 1, sperm selection was performed by
90%-45% discontinuous Percoll density gradient centrifugation (T1)
and swim-up (T2). In experiment 2, along side the discontinuous
gradient, a 67.5% continuous density gradient, and centrifugation time
of 5 and 10 minutes were used. A total of 4 treatment groups was
defined (TI = continuous, 5 minutes, TII = discontinuous, 5 minutes,
TIII = continuous, 10 minutes and TIV = discontinuous, 10 minutes).
Polymerase chain reaction (PCR) was used to determine the sex of the
embryos. T1 (n=185) resulted in 48.65% (n=90) male embryos and
51.35% (n=95) female embryos and T2 (n=142) in 58.45% (n=83)
male and 41.55% (n=59) female embryos. In experiment 2, the
percentages of male and female embryos obtained in TI (n=93), TII
(n=70), TIII (n=82) and TIV (n=82) were 49.46% (n=46) and 50.54%
(n=47), 57.14% (n=40) and 42.86% (n=30), 36.59% (n=30) and
63.41% (n=52) and 48.78% (n=40) and 51.22% (n=42), respectively.
There was no difference on the percentage of males and females in all
treatment groups from experiments 1 and 2 when these were
individually compared to the expected percentage of 50% of each sex.
There was also no difference in male and female embryo percentage
between treatment groups in experiments 1 and 2.
P361
Granulocyte-macrophage colony-stimulating factor (GM-
CSF) enhances glucose uptake in bovine granulosa cells
Bücher, DD 1 *, Castro, MA 1 , Beltrán, F 1 , Correa, JE 2 , Concha, II 1
1Instituto de Bioquímica, Universidad Austral de Chile, Chile; 2 Instituto de
Reproducción Animal, Universidad Austral de Chile, Chile
The granulocyte-macrophage colony stimulating factor (GM-CSF) is
a pleiotropic cytokine capable of stimulating proliferation, maturation
and function of hematopoietic cells. Receptors for this cytokine are
composed of two subunits alpha and beta and are expressed on
myeloid progenitors and mature mononuclear phagocytes, monocytes,
eosinophils and neutrophils, as well as in other nonhematopietic cells.
We have previously demonstrated that bull spermatozoa express
functional GM-CSF receptors that signal for increased glucose and
vitamin C uptake and enhance several parameters of sperm motility in
the presence of glucose or fructose substrates. The ovarian follicular
development involves a complex exchange of endocrine signals
between the hypothalamic-pituitary-ovarian system and within each
ovarian follicle in a cell to cell interaction that precisely coordinates
the process in a paracrine or autocrine manner. It is possible to assume
that GM-CSF plays a fundamental role during folliculogenesis. The
aim of the present work was to analyze the expression of GM-CSF
receptors in bovine granulosa cells of follicles at different
developmental stages and study the effect of GM-CSF on glucose
uptake by these cells. Immunolocalization and immunoblotting
analysis demonstrated that both subunits of GM-CSF receptors are
expressed in granulosa cells at an early stage of development up to
preovulatory stage of folliculogenesis. Using functional analysis with
2-D-3H-deoxyglucose we demonstrated that this cytokine enhances
glucose uptake via facilitative hexose transporters (GLUTs) in
granulosa cells isolated from follicles up to 6 mm in diameter
cultivated under serum free conditions for 24 hours. The results
suggest that GM-CSF interacts with factors present in the ovarian
environment such as IGF-I and FSH, modulating the process of
folliculogenesis. (MECESUP; DID D-2006-24; Escuela de
Graduados, Facultad de Ciencias Veterinarias; FONDECYT
1060135).
P362
Effect of undernutrition and pregnancy on hepatic and
adipose tissue gene expression
Carriquiry, M 1 *, Sosa, C 2 , Abecia, JA 2 , Forcada, F 2 , Meikle, A 1
1Facultad de Agronomia-UDELAR, Uruguay; 2 Facultad de Veterinaria,
Zaragoza, Spain
Undernutrition decreases embryo survival and pregnancy rates in
ewes. The effect of plane of nutrition and physiological status (cyclic
or pregnant) on hepatic and adipose tissue mRNA expression of genes
involved in the somatotropic axis and leptin was investigated.
Twenty-four ewes were fed either 1 or 0.5 times their maintenance
requirements, estrus-synchronized, and mated with intact (n=12) or
vasectomized (n=12) rams, to establish four treatment groups in a 2 x
2 factorial combination of plane of nutrition and physiological status.
Ewes were slaughtered at day 14 of estrous cycle or pregnancy (Day
0=estrus) The abundance of mRNA for growth hormone receptor
(GHR), GHR1A, insulin-like growth factor-I (IGF-I), leptin (LEP),
and an endogenous control (ribosomal protein L19; RPL19) was
measured by quantitative real time RT-PCR using SYBR Green.
Abundance of mRNA of target genes was normalized to RPL19 and
expressed in relative amounts to an external control (delta-deltaCT -
method). Data were analyzed using PROC MIXED (SAS Institute)
and considered to differ when P