Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
146 Poster Abstracts<br />
F1 (B6D2) female mice were superovulated and mated with DBA<br />
males. Early blastocysts were collected from pregnant females and<br />
cultured <strong>in</strong> KSOM till the expan<strong>de</strong>d blastocyst stage, that were loa<strong>de</strong>d<br />
<strong>in</strong>to 0.25 ml m<strong>in</strong>istraws with CZB-Hepes. Embryos were treated with<br />
600 bar pressure for 30 m<strong>in</strong> at 23 ºC <strong>in</strong> a computer controlled<br />
pressuriz<strong>in</strong>g <strong>de</strong>vice (Cryo-Innovation Inc., Budapest, Hungary).<br />
Embryos were collected immediately and 120 m<strong>in</strong> after the treatment.<br />
Then, mRNA was isolated and cDNA prepared for real-time PCR<br />
analyses.<br />
To assess the change <strong>in</strong> gene expression profiles, n<strong>in</strong>e stress related<br />
genes and two reference genes (H2afz, and Ppia) were selected.<br />
Primers were <strong>de</strong>signed, optimized and standards prepared for real<br />
time PCR analyses. Based on the analyses, the genes Az<strong>in</strong>1, Gas5,<br />
Gadd45g and Sod2 were up-regulated (mean±SD) 1.88±0.25,<br />
1.55±0.36, 1.47±0.59 and 1.41±0.25 fold, respectively after<br />
hydrostatic pressure treatment, compared to the levels of the same<br />
genes <strong>in</strong> the untreated control. All studied genes, but Gadd45g, have<br />
returned to the control expression levels after 120 m<strong>in</strong>utes culture.<br />
The up-regulation of Gadd45g (1.92±0.60 fold) shows that<br />
hydrostatic pressure has a prolonged effect on the transcription of this<br />
gene. H2afz and Ppia genes were not significantly affected (P>0,05)<br />
by the treatment, and this supports our previous results on the stable<br />
expression of these genes dur<strong>in</strong>g mouse preimplantation <strong>de</strong>velopment.<br />
This study <strong>de</strong>monstrates, for the first time, that HHP activates certa<strong>in</strong><br />
stress related genes <strong>in</strong> the mouse embryo. The effects don’t<br />
compromise <strong>de</strong>velopmental competence of the blastocysts, moreover<br />
it may contribute to <strong>in</strong>creased survival rates at the subsequent<br />
cryopreservation.<br />
Supported by OMFB-00364/2007 and NKTH/KPI Kozma F.<br />
TUDAS-1-2006-0005<br />
P360<br />
Sex<strong>in</strong>g <strong>in</strong> vitro produced bov<strong>in</strong>e embryos with semen<br />
selected by percoll or swim-up<br />
Wolf, C 1 , Brass, KE 2 *<br />
1Equ<strong>in</strong>e <strong>Reproduction</strong>, Fe<strong>de</strong>ral University of Rio Gran<strong>de</strong> do Sul, Brazil; 2 Large<br />
Animal Cl<strong>in</strong>ics, Fe<strong>de</strong>ral University of Santa Maria, Brazil<br />
Preimplantation genetic diagnosis (PGD) is becom<strong>in</strong>g a current issue<br />
<strong>in</strong> animal reproduction biotechnology due to economical reasons.<br />
Pre<strong>de</strong>term<strong>in</strong><strong>in</strong>g the sex of offspr<strong>in</strong>g is one example of PGD. This<br />
study aimed to <strong>de</strong>term<strong>in</strong>e the percentage of male and female bov<strong>in</strong>e<br />
embryos <strong>in</strong> vitro produced after oocyte fertilization with Percoll<br />
<strong>de</strong>nsity gradient centrifugation or with self-migration (swim-up)<br />
selected semen. In experiment 1, sperm selection was performed by<br />
90%-45% discont<strong>in</strong>uous Percoll <strong>de</strong>nsity gradient centrifugation (T1)<br />
and swim-up (T2). In experiment 2, along si<strong>de</strong> the discont<strong>in</strong>uous<br />
gradient, a 67.5% cont<strong>in</strong>uous <strong>de</strong>nsity gradient, and centrifugation time<br />
of 5 and 10 m<strong>in</strong>utes were used. A total of 4 treatment groups was<br />
<strong>de</strong>f<strong>in</strong>ed (TI = cont<strong>in</strong>uous, 5 m<strong>in</strong>utes, TII = discont<strong>in</strong>uous, 5 m<strong>in</strong>utes,<br />
TIII = cont<strong>in</strong>uous, 10 m<strong>in</strong>utes and TIV = discont<strong>in</strong>uous, 10 m<strong>in</strong>utes).<br />
Polymerase cha<strong>in</strong> reaction (PCR) was used to <strong>de</strong>term<strong>in</strong>e the sex of the<br />
embryos. T1 (n=185) resulted <strong>in</strong> 48.65% (n=90) male embryos and<br />
51.35% (n=95) female embryos and T2 (n=142) <strong>in</strong> 58.45% (n=83)<br />
male and 41.55% (n=59) female embryos. In experiment 2, the<br />
percentages of male and female embryos obta<strong>in</strong>ed <strong>in</strong> TI (n=93), TII<br />
(n=70), TIII (n=82) and TIV (n=82) were 49.46% (n=46) and 50.54%<br />
(n=47), 57.14% (n=40) and 42.86% (n=30), 36.59% (n=30) and<br />
63.41% (n=52) and 48.78% (n=40) and 51.22% (n=42), respectively.<br />
There was no difference on the percentage of males and females <strong>in</strong> all<br />
treatment groups from experiments 1 and 2 when these were<br />
<strong>in</strong>dividually compared to the expected percentage of 50% of each sex.<br />
There was also no difference <strong>in</strong> male and female embryo percentage<br />
between treatment groups <strong>in</strong> experiments 1 and 2.<br />
P361<br />
Granulocyte-macrophage colony-stimulat<strong>in</strong>g factor (GM-<br />
CSF) enhances glucose uptake <strong>in</strong> bov<strong>in</strong>e granulosa cells<br />
Bücher, DD 1 *, Castro, MA 1 , Beltrán, F 1 , Correa, JE 2 , Concha, II 1<br />
1Instituto <strong>de</strong> Bioquímica, Universidad Austral <strong>de</strong> Chile, Chile; 2 Instituto <strong>de</strong><br />
Reproducción Animal, Universidad Austral <strong>de</strong> Chile, Chile<br />
The granulocyte-macrophage colony stimulat<strong>in</strong>g factor (GM-CSF) is<br />
a pleiotropic cytok<strong>in</strong>e capable of stimulat<strong>in</strong>g proliferation, maturation<br />
and function of hematopoietic cells. Receptors for this cytok<strong>in</strong>e are<br />
composed of two subunits alpha and beta and are expressed on<br />
myeloid progenitors and mature mononuclear phagocytes, monocytes,<br />
eos<strong>in</strong>ophils and neutrophils, as well as <strong>in</strong> other nonhematopietic cells.<br />
We have previously <strong>de</strong>monstrated that bull spermatozoa express<br />
functional GM-CSF receptors that signal for <strong>in</strong>creased glucose and<br />
vitam<strong>in</strong> C uptake and enhance several parameters of sperm motility <strong>in</strong><br />
the presence of glucose or fructose substrates. The ovarian follicular<br />
<strong>de</strong>velopment <strong>in</strong>volves a complex exchange of endocr<strong>in</strong>e signals<br />
between the hypothalamic-pituitary-ovarian system and with<strong>in</strong> each<br />
ovarian follicle <strong>in</strong> a cell to cell <strong>in</strong>teraction that precisely coord<strong>in</strong>ates<br />
the process <strong>in</strong> a paracr<strong>in</strong>e or autocr<strong>in</strong>e manner. It is possible to assume<br />
that GM-CSF plays a fundamental role dur<strong>in</strong>g folliculogenesis. The<br />
aim of the present work was to analyze the expression of GM-CSF<br />
receptors <strong>in</strong> bov<strong>in</strong>e granulosa cells of follicles at different<br />
<strong>de</strong>velopmental stages and study the effect of GM-CSF on glucose<br />
uptake by these cells. Immunolocalization and immunoblott<strong>in</strong>g<br />
analysis <strong>de</strong>monstrated that both subunits of GM-CSF receptors are<br />
expressed <strong>in</strong> granulosa cells at an early stage of <strong>de</strong>velopment up to<br />
preovulatory stage of folliculogenesis. Us<strong>in</strong>g functional analysis with<br />
2-D-3H-<strong>de</strong>oxyglucose we <strong>de</strong>monstrated that this cytok<strong>in</strong>e enhances<br />
glucose uptake via facilitative hexose transporters (GLUTs) <strong>in</strong><br />
granulosa cells isolated from follicles up to 6 mm <strong>in</strong> diameter<br />
cultivated un<strong>de</strong>r serum free conditions for 24 hours. The results<br />
suggest that GM-CSF <strong>in</strong>teracts with factors present <strong>in</strong> the ovarian<br />
environment such as IGF-I and FSH, modulat<strong>in</strong>g the process of<br />
folliculogenesis. (MECESUP; DID D-2006-24; Escuela <strong>de</strong><br />
Graduados, <strong>Facultad</strong> <strong>de</strong> <strong>Ciencias</strong> Veter<strong>in</strong>arias; FONDECYT<br />
1060135).<br />
P362<br />
Effect of un<strong>de</strong>rnutrition and pregnancy on hepatic and<br />
adipose tissue gene expression<br />
Carriquiry, M 1 *, Sosa, C 2 , Abecia, JA 2 , Forcada, F 2 , Meikle, A 1<br />
1<strong>Facultad</strong> <strong>de</strong> Agronomia-UDELAR, Uruguay; 2 <strong>Facultad</strong> <strong>de</strong> Veter<strong>in</strong>aria,<br />
Zaragoza, Spa<strong>in</strong><br />
Un<strong>de</strong>rnutrition <strong>de</strong>creases embryo survival and pregnancy rates <strong>in</strong><br />
ewes. The effect of plane of nutrition and physiological status (cyclic<br />
or pregnant) on hepatic and adipose tissue mRNA expression of genes<br />
<strong>in</strong>volved <strong>in</strong> the somatotropic axis and lept<strong>in</strong> was <strong>in</strong>vestigated.<br />
Twenty-four ewes were fed either 1 or 0.5 times their ma<strong>in</strong>tenance<br />
requirements, estrus-synchronized, and mated with <strong>in</strong>tact (n=12) or<br />
vasectomized (n=12) rams, to establish four treatment groups <strong>in</strong> a 2 x<br />
2 factorial comb<strong>in</strong>ation of plane of nutrition and physiological status.<br />
Ewes were slaughtered at day 14 of estrous cycle or pregnancy (Day<br />
0=estrus) The abundance of mRNA for growth hormone receptor<br />
(GHR), GHR1A, <strong>in</strong>sul<strong>in</strong>-like growth factor-I (IGF-I), lept<strong>in</strong> (LEP),<br />
and an endogenous control (ribosomal prote<strong>in</strong> L19; RPL19) was<br />
measured by quantitative real time RT-PCR us<strong>in</strong>g SYBR Green.<br />
Abundance of mRNA of target genes was normalized to RPL19 and<br />
expressed <strong>in</strong> relative amounts to an external control (<strong>de</strong>lta-<strong>de</strong>ltaCT -<br />
method). Data were analyzed us<strong>in</strong>g PROC MIXED (SAS Institute)<br />
and consi<strong>de</strong>red to differ when P