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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A211 CLONING, TRANSGENESIS AND STEM CELLS
EFFECT OF TRICHOSTAIN A ON PRODUCTION OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER
AND SUBSEQUENT GESTATIONS
TIONS
Luiz Sérgio Almeida Camar
amargo
go, Car
arolina Cap
apobiango R. Quin
uintão
tão, Anna Car
arolina Denic
enicol,
Lilian Tam
amy Iguma, Bruno Camp
ampos Car
aravalho
alho, Luiz Gusta
ustavo Bruno
Siqueira & João Henrique Moreira Viana
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL.
Histone deacetylation is associated to a compact and less permissive chromatin, which may impair an efficient nuclear reprogramming
of somatic cell nucleus. The inhibition of histone deacetylation can be achieved by histone deacetylases inhibitors, as Trichostatin A. This study
evaluated the effect of zygotes treatment with trichostatin A on production and quality of nuclear-transferred bovine embryos. Oocytes were
matured in vitro, denuded and exposed to Hoechst 33342 (Sigma, St Louis, USA) and cytochalasin (Sigma) before enucleation. Zygotes were
reconstructed with somatic cells from adult Gyr cow, fused with double electric pulse of 2.4 kV/cm for 30 µsec and activated with ionomycin
(Sigma) for 5 min. After ionomycin activation the zygotes were randomly separated in two groups: Trichostatina (TRICHO, n = 132) - zygotes
were cultured for 4h in 6-DMAP followed by 7h in CR2aa medium plus with 2.5% fetal calf serum, both supplemented with 50nM Trichostatin
A (Sigma); Control (CONT, n = 116) - zygotes were cultured in the same conditions described above but without Thichostatin A supplementation.
Parthenogenetic embryos (PART group, n = 287) were used as control for activation and embryo culture. Embryos from all groups were cultured
in CR2aa supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5%O2 and 90%N2. Embryos at 168h postactivation
(hpa) from TRICHO and CONT groups were transferred to synchronized recipients. Analyses of cleavage at 72hpa, blastocyst at
168hpa, total cell number and apoptotic cell index were performed by ANOVA and means compared by SNK test. Data are shown as
mean±SEM. Gestation and birth rates were analyzed by Fischer’s Exact Test. No difference on cleavage (87.7±4.1%, 83.3±4.0% and 79.3±4.1%)
and blastocysts (38.4±4.7%, 34.2%±6.0% and 30.5±3.7) rates was found among TRICHO, CONT and PART groups, respectively. Embryos
from TRICHO group presented lower (P < 0.05) index of apoptotic cells (0.065±0.009; n = 17) than embryos from CONT group (0.129±0.021;
n = 13), but similar (P > 0.05) to those from PART group (0.083±0.011; n = 19). No difference (P > 0.05) on total cell number was found among
groups. Although it is not statistically significant (P > 0.05), pregnancy rate at 60 days with embryos from TRICHO group (64.2%; 9/14) was
numerically higher than those ones from CONT group (45.4%; 5/11). One pregnancy from TRICHO group went to term (7.1%; 1/14) but the
calf died in the second day after birth. No offspring was obtained with embryos from CONT group. In conclusion, exposure of clone zygotes
to 50 nM of trichostatin A decreases apoptosis in embryos, which may favor pregnancy rate. [Financial support: Embrapa Project 01.07.01.002,
CNPq 403019/2008-7 and Fapemig].
Keywords: cloning, histone deacetylases inhibitor, apoptosis.
A212 CLONING, TRANSGENESIS AND STEM CELLS
EFFICIENCY OF NUCLEAR TRANSFER IN EQUINE: DIRECT INJECTION OF THE DONOR CELL NUCLEUS INTO THE
RECIPIENT CYTOPL
OPLAST
ASTS
Claudia Barbosa Fernandes 1 , Camila Gabriela Pereira Gonçalves 1 , Lilian Rigatto Martins 2 & Fernanda da Cruz Landim Alvarenga 3
1
USP FMVZ, SAO PAULO, SP, BRAZIL. 2 UFMT, MATO GROSSO, MT, BRAZIL. 3 UNESP FMVZ, BOTUCATU, SP, BRAZIL.
The aim of this study was to evaluate the efficiency of NT with donor cell nucleus direct injection in equine recipient cytoplasts.
There were used 150 equine compact and expanded oocytes in vitro matured (IVM) for 30h or kept for 24h under the roscovitine action before
the period of IVM. After 30 h of IVM equine oocytes were denuded and incubated during 30 min in H-MEM medium, with FBS, Hoechst
33342 and cytochalasin B. The oocytes enucleation and the recipient cytoplasts reconstitution with equine fibroblasts were performed between
32 and 34 h after the beginning of IVM. The metaphaseI (MI) and metaphaseII (MII) oocytes enucleated and reconstituted successfully were
incubated for one hour until the artificial activation, using Roscovitine + Ionomycin (I+R) and Ionomycin + 6 DMAP (I+6DMAP). After the
activation period, the oocytes were cultured in DMEM/F12 with FBS, myoinositol and gentamicin at 39ºC in 5% CO2, 5% O2 and 90% N2
during 3 days. The assessment of the activation and nuclear decondensation formation rates were performed with Hoechst 33342 in inverted
microscopy. There was used the Analysis of Deviance to select the best logistic regression model to explain the percentage of equine oocytes with
nuclear decondensed formation after NT. The equine oocytes classified as compact that were exposed or not to roscovitine, in MI and MII at the
time of enucleation, had 0%, 20% and 37.5%, 25% of nuclear decondensation rate after activation with I+R and 0% 30% and 22.2%, 50% after
I+6DMAP, respectively. The equine oocytes classified as expanded that were exposed or not to roscovitine in MI and MII at the time of
enucleation, had 14.2%, 30% and 0%, 14.2% of nuclear decondensation rate after activation with I+R and 12.5 %, 20% and 20%, 33.3% after
I+6DMAP respectively. We can observe that the only significant effect in chromatin decondensation rates was the stage of nuclear maturation,
regardless of the oocyte classification, the artificial activation protocol and the exposure or not to roscovitine, the percentage of oocytes with the
decondensed nucleus formation on the third day of culture was significantly higher for MII (P = 0.033) compared to MI. There was no
significant interaction among any other factors, however, for the equine the technique when the donor cell nucleus was directly injected into the
recipient cytoplast resulted in unsatisfactory cloned embryos production. [Acknowledgement: FAPESP].
Keywords: cloning, artificial activation, nuclear maturation.
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