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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A003 STUDENT COMPETITION
EFFECT OF ETHANOL EXTR
TRACT OF AZADIRACHT
CHTA INDICA IN THE SYNCHRONIZA
ONIZATION OF THE CELL CYCLE CLE OF BOVINE
FIBROBLASTS
Natana Chaves Rabelo 1 , Carolina Capobiango R. Quintão 2 , Ana Paula Moreira 3 , Savana Giacomini Brito 4 , Michele Munk Pereira 3 , Juliane Dornellas Nunes 2 ,
Ana Luisa Sousa Azevedo
edo 2 , Nadia Barb
arbosa Rezende ende Rap
aposo
2,3 , Lilian Tam
amy Iguma 2 , João Henr
enrique Mor
oreir
eira Viana
2 & Luiz Sérgio Almeida Camar
amargo
2
1
CENTRO DE ENSINO SUPERIOR DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL. 2 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 3 UNIVERSIDADE FEDERAL DE JUIZ DE
FORA, JUIZ DE FORA, MG, BRAZIL. 4 UNIVERSIDADE PRESIDENTE ANTONIO CARLOS, JUIZ DE FORA, MG, BRAZIL.
The success of somatic cell nuclear transfer (SCNT) depends initially on the cell cycle synchrony between the donor nucleus and
the recipient cytoplasm. Many agents have been tested for cell cycle synchronization, but with limited success rate. Components of the
Azadirachta indica A. Juss plant (popularly known as Neem) may be an alternative for cell cycle synchronization of donor cell for SCNT. The
aim of the present study was to evaluate the effect of different concentrations of ethanol extract of Neem on synchronization at G0/G1 phases of
the cell cycle of bovine fibroblasts. The extract was obtained by dynamic maceration and rotary evaporation. Bovine fibroblast cells collected
from a Gyr cow were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity
in air. After cells reaching about 70% confluence, the extract was at the following concentrations: 0 µg/mL, 50 µg /mL, 100 µg /mL, 200 µg /mL
and 300 µg /mL, with exposure times of 12 and 24 h. Simultaneously, a control group with serum starvation (cells cultured in DMEM plus 0.5%
FCS for three days) was prepared. To examine the cell cycle, we performed flow cytometry analysis (FacsCallibur, Becton Dickinson, San Jose,
CA, USA), and DNA histograms were analyzed with the WinMDI software to determine the percentage of cells at G0/G1, S and G2 phases.
Three repetitions were performed in triplicate for each concentration. Results were analyzed by analysis of variance and means compared by
Student Newman Keuls. Concentrations of 100 µg/mL (88.6 ± 0.3%) e 200 µg/mL (88.4±0.4%), both at 24 h of exposure, resulted in more (P
< 0.05) cells arrested in G0/G1 phase than the other concentrations, but they were similar to the serum starvation group (89.6 ± 0.3%). In
conclusion, ethanol extract from Azadirachta indica A. Juss is able to synchronize cell cycle of bovine fibroblast, arresting in G0/G1 phase.
Further studies are needed to evaluate the reversibility of such arrest and the cell viability for SCNT. [Financial Support. CNPq, FAPEMIG and
Innovation Network Project on Animal Reproduction (01.07.01.002)].
Keywords: cell cycle, flow cytometry, nuclear transfer.
A004 STUDENT COMPETITION
INFLUENCE OF HIGH OR LOW INTAKE OF DRY MAT TER/ENERGY ON IN VITRO PRODUCTION OF BOVINE
EMBRYOS
Alexandre Barbieri Prata 1 , Ricardo Silva Surjus 1 , Marta Borsato 1 , Mariana Curci Martins da Silveira 1 , Maria Clara Costa Mattos 1,2 , Gerson Barreto Mourão 3 ,
Flávio Augusto Portela Santos 1 , Andrea Cristina Basso 1,2 , José Henrique Fortes Pontes 1 & Roberto Sartori 1
1
ESALQ/USP, PIRACICABA, SP, BRAZIL. 2 IN VITRO BRAZIL, MOGI MIRIM, SP, BRAZIL. 3 ESALQ/USP, PIRACICABA, SP, BRAZIL.
The aim of this study was to evaluate the influence of high or low dry matter (DM) intake and/or energy on oocyte quality and
embryo production in vitro. Nonlactating Nellore cows (n = 32, 4 to 10 years old) weighing 489.5 ± 11.3 kg and with a BCS of 3.25 (scale from
1 to 5) were used. The cows were confined without access to pasture, being two animals per stall. Mineral salt was provided in the diet and water
was offered ad libitum. After 15 days on the adaptation diet, groups of cows were blocked by initial body weight (BW) and randomly divided
in four experimental groups. The maintenance group (M) received a diet to provide 1.2% of DM per kg of BW. The restriction group (0.7M)
received the equivalent of 70% of the Group M diet (0.84% of DM per kg of BW). The high intake group (1.5M) received the equivalent of 150%
of the M group (1.8% of DM per kg of BW). The energy group (E) received a diet with the DM similar to the M group, however, with an energy
level equivalent to the 1.5M group. The cows were offered all diets in a crossover design study. There were four sessions of ovum pick up
(OPU), 42 days apart. Recovered oocytes were classified and taken to the In vitro Brazil laboratory, where all procedures for in vitro embryo
production were performed. Data were analyzed by PROC GLIMMIX of SAS and the results are presented as least squares means ± SE, always
following the order of treatments M, 0.7M, 1.5M and E. More total (20.2 ± 2.0b; 23.0 ± 2.3a; 21.5 ± 2.2ab and 20.1 ± 2.0b; P = 0.02) and viable
(14.4 ± 1.6b; 17.0 ± 1.9a; 15.7 ± 1.7ab and 14.1 ± 1.6b; P = 0.006) oocytes were recovered per cow per OPU session in Group 0.7M than in
Groups M and E. However, there was no difference in the number of atretic and degenerate oocytes per treatment (5.7 ± 0.6; 5.7 ± 0.6; 5.6 ± 0.6
and 5.7 ± 0.6; P = 0.99). Although Group 0.7M had a higher number of cleaved oocytes than Group M (10.7 ± 1.4b; 13.4 ± 1.7a; 12.6 ± 1.6ab
and 11.7 ± 1.5ab; P = 0.04), the number of blastocysts was similar among treatments (5.4 ± 0.8; 6.9 ± 0.9; 5.9 ± 0.8 and 6.6 ± 0.9; P = 0.15).
There was also no difference among groups regarding the percentage of viable oocytes (72.8, 75.4, 72.9 and 71.7%; P = 0.41) or blastocysts
(31.9; 30.6; 31.1 and 34.0%; P = 0.67) per total oocytes, or percentage of blastocysts per cleaved oocytes (52.7; 53.2; 46.5 and 56.4%; P = 0.14).
Although there was an apparent advantage of food restriction on the number of total, viable, and cleaved oocytes compared to other groups, such
feature was not translated into blastocyst production. Likewise, contrary to our initial hypothesis, high DM or energy intake for a period of 42
days did not seem to compromise the in vitro embryo production from Nellore cows. [Acknowledgment. Fapesp and CNPq].
Keywords: nutrition, in vitro production, embryo.
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