2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A253 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
IN VITRO MATUR
TURATION TION OF IMMATURE OOCYTES
VITRIFIED IN DIFFERENT CONDITIONS
Fabiana For
orell
ell, Ner
erissa Albino
lbino, Jamir Machado Junior
unior, Fabiano Car
armina
minatti Zago & Alc
lceu Mezzalir
zzalira
CAV/UDESC, LAGES, SC, BRAZIL.
Vitrification of immature oocytes is an important tool for use in both the IVF and cloning, though the efficiency of this technique
is still low, limiting its application in commercial scale. For application in nuclear transfer, besides of survival of oocytes, vitrification of a large
number of structures is required. The vitrified oocytes undergo various morphological and molecular changes during cooling and warming,
which may potentially require another maturation conditions than those for non-cryopreserved oocytes. Thus, this study aimed to compare
survival rates after in vitro maturation and embryo development of vitrified oocytes and matured under different conditions. Bovine oocytes were
obtained from bovine ovaries and vitrified in groups of 15 COCs, loaded in a 5 µL vitrification solution microdrop in beveled-cut straws (Forell
et al. Rep. Fert. Dev, 2009, 21 (1) 115). After warming all recovered COCs were submitted to IVM for 24 h at 38.8°C with 5% CO2 in air.
Groups of 40 COCs allocated in 400 uL of medium, were subjected to three treatments: 1) ESS M199 +10% +0.5 ug/mL FSH +5 ug/mL LH
(standard laboratory system) 2) M199 +10 % ESS + FSH + LH +10 ng/mL EGF, or 3) M199 +10% FCS +10 ng/mL EGF. After IVM, cumulus
cells were mechanically removed and the surviving oocytes were parthenogenetically activated (5 min in ionomycin, and 4 h in 6DMAP) and
in vitro cultured in SOFaaci with atmosphere of 5% CO2 +5% O2 in N2, for 7 days. Oocytes non-vitrified (control) were also subjected to the
same treatments. The results were analyzed by chi-square test with significance level of 5%. The survival rate after the denudation did not differ
between experimental groups, 44% (266/315), 49% (157/321) and 52% (165/320) respectively for groups 1, 2 and 3. There was no difference
in cleavage rates of groups 1 - 36% (48/133), 2 - 45% (59/132) and 3 - 39% (65/165). The blastocyst rates were 3.8%, 3.8% and 9.7%
respectively for groups 1 (n = 133), 2 (n = 132) and 3 (n = 165) and the rates of group 3 was higher (P < 0.05) than others. In fresh control groups
was not statistically significant difference between the experimental groups in the cleavage rate (83%, 84% and 76%) or blastocyst rate (43%,
42% and 48%) for groups 1 (n = 121 ), 2 (n = 125) and 3 (n = 121) respectively. Thus, among the tested systems, the maturation medium
composed of FCS and EGF improves blastocyst rates for vitrified immature oocytes. [Funded by PNPD/CAPES].
Keywords: vitrification, in vitro maturation, EGF.
A254 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
DNA METHYLATION TION PAT TERN OF THE IGF2 AND XIST GENES IN OOCYTES OBTAINED OF THE PREANTRAL
AL
FOLLICLES FROM NELLORE COWS (
(BOS
TAUR
URUS
US INDICUS)
Luís Fernando Soares Gomes 1 , Isabela Rebouças Bessa 2 , Fernanda Castro Rodrigues 3 , Pablo José Silva Rua 4 , Otávio Bravim 5 ,
Juliana Mayumi Azevedo 6 , Margot Alves Nunes Dode 7 & Maurício Machaim Franco 8
1,3,4
FACULDADE DE MEDICINA VETERINÁRIA, UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL. 2,6 FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA,
UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 5 UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 7,8 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF,
BRAZIL.
This study focus on evaluating the methylation pattern in a differentially methylated region (DMR) of the last exon of IGF2 gene
in oocytes of 65-90 µm, and in a region of exon 1 of the XIST gene in oocytes =20 µm, and 65-90 µm obtained of the preantral follicles from
nellore cows. To do this work, ovaries from abattoir were used and the preantral follicles were isolated with aid of tissue chopper. The isolation
of oocytes enclosed in preantral follicles was performed by treatment with collagenase type II (Sigma, St. Louis, USA). Next the oocytes were
photographed to measure their diameters. After, the oocytes were separated based on their size for molecular analysis. Genomic DNA was
extracted from 65 oocytes =20 µm and 62 oocytes of 65-90 µm and it was treated with sodium bisulfate using the EZ DNA methylation kit ®
(Zymo Research) and amplified by PCR nested to the genes IGF2 and XIST. PCR products were recovered from agarose gel and purified by
GeneClean III kit (MP Biomedicals, LLC). The purified products were cloned using pGEMT-Easy vector kit (Promega) and transformed into
Escherichia coli (XL-1 Blue). The clones were sequenced by the dideoxy method using an ABI 3130xl sequencer. Only clones sequences with
=90% of conversion efficiency were used. There were sequenced and analyzed 28 clones for IGF2 65-90 µm group, 19 clones for XIST =20
µm group and 27 clones for XIST 65-90 µm group. Methylation pattern was calculated with BiQ Analyzer program (Bock et al., 2005) and
statistical analysis was performed by the Mann-Whitney test using the Prophet program, version 5.0 (BBN Systems and Technologies,
1996). For IGF2, the oocytes of late secondary follicles of 65-90 µm presented 31,09 ± 31,01% of methylation. For XIST, the oocytes of
primordial follicles =20 µm and the oocytes of late secondary follicles of 65-90 µm presented 15,17 ± 32,82% and 4,6 ± 3,46% (P = 0,0901)
of methylation respectively. It is concluded that the genomic region of the XIST gene studied undergoes epigenetic reprogramming during
gametogenesis.
Keywords: bovine, methylation, oocyte.
N
s463