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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A073 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION
VIABILITY OF CANINE PREANTRAL AL FOLLICLES CULTURED
IN VITRO AFTER CONSER
ONSERVATION IN AN OOCYTE
TRANSPOR
ANSPORTER
Rodrigo José Sousa Gonçalves 1 , Vanessa Raquel Pinto Barros 2 , Anderson Pinto Almeida 3 , Ana Beatriz Graça Duarte 4 , Valdevane Rocha Araújo 5 , Cleidson
Mano
anoel Gomes Silv
ilva 6 , Gio
iovanna Quin
uintino Rodr
drigues
7 , Ger
erlane Modest
desto Da Silv
ilva 8 , José Ric
icar
ardo De Figueir
igueiredo
edo 9 & Mar
aria Helena
Tavar
ares Matos
10
1,2,10
UNIVERSIDADE FEDERAL DO VALE DO SÃO FRANCISCO, PETROLINA, PE, BRAZIL. 3,4,5,6,7,8,9 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.
Maintenance of follicular quality after collection and transportation of the ovaries to the laboratories of reproductive techniques
is a limiting factor for the success of in vitro culture of preantral follicles (PAF) in the canine species. In this way, the aim of this study was
to assess the viability of canine PAF cultured in vitro after storage for 12 or 24 h in an oocyte transporter ® - Compact, Wta-Brazil (TO). After
collection of canine ovaries,180 secondary preantral follicles with a diameter higher than 200 mm were mechanically isolated, and destined to
conservation into the oocyte transporter at 39°C in α-Minimum Essential Medium supplemented with BSA (3 mg/mL), glutamine (2 mM),
hypoxanthine (2 mM), ITS (insulin 10 µg/mL, transferrin 5.5 µg/mL, and selenium 5 ng/mL), ascorbic acid (50 ag/ml) and recombinant FSH
(100 ng/ml). The PAF were further destined to in vitro culture using the same conservation medium. Six treatments were used: PAF assessed
by fluorescence microscopy (FM) after isolation (T1), PAF cultured after isolation (T2 - cultured control), PAF destined to (FM) after 12
(T3) or 24 h (T4) of conservation, PAF destined for the culture after 12 (T5) or 24 h (T6) of conservation. PAF were individually cultured in
100 mL drops of medium, at 39°C and 5% CO2 for 7 days. Every 2 days, 60 ìl of the culture medium was changed by a fresh medium. The
follicular viability was analyzed using the fluorescent markers calcein-AM (4µM) and ethidium homodimer-1 (2µM) for viable and nonviable
PAF, respectively. At the end of the isolation, preservation in the TO and the culture for 7 days, PAF of all the treatments (100%) analyzed
by FM were stained in green by calcein-AM demonstrating that all them are viable the that explains the absence of statistical analysis of this
experiment. In conclusion, canine preantral follicle preserved for 12 and 24 h in an oocyte transporter maintain the viability in vitro after
culture for 7 days.
Keywords: canine, preantral follicle, preservation.
A074 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION
ASCORBIC ACID IMPROVES
THE SURVIV
VIVAL AND IN VITRO GROWTH OF ISOLATED CAPRINE PREANTRAL AL FOLLICLES
Gerlane Modesto da Silva 1 , Carlos Henrique Lobo 2 , Valdevane Rocha Araújo 1 , Ana Beatriz Graça Duarte 1 , Arlindo Alencar Moura 2 & José Ricardo de
Figueiredo 1
1
UECE, FORTALEZA, CE, BRAZIL. 2 UFC, FORTALEZA, CE, BRAZIL.
Ascorbic acid (AA) may act to regulate the matrix metalloproteinases and their inhibitors associated with the turnover of collagen
on the basement membrane during follicle growth (Murray et al., 2001; Journals of Reproduction and Fertility 121, 89-96). The present study
aims to investigate the influence of AA on the survival, growth, antral formation, extruded oocytes and mRNA expression of the matrix
metalloproteinases-9 (MMP9) and their tissue inhibitor-2 (TIMP2) on caprine preantral follicles (FOPA). Isolated FOPA were individually
cultured without (MEM+) or with AA at 50 µg/mL (AA50) or 100 µg/mL (AA100) during 18 days. To culture at least 32 follicles were used
per treatment. Three pools of 10 follicles from non cultured (fresh control), MEM+ alone, AA50 and AA100 were collected to quantified the
genes MMP9 and TIMP2 by real-time polymerase chain reaction (qPCR). The percentage of survival, antrum formation, extrusion rate and
maturation were compared by x2 test (StatView for Windows). The diameter and growth rates were compared by Kruskal-Wallis test. For qPCR
differences between the control and treatments was assessed with the T test of SAS 9.0. At the end of culture, AA50 significantly increased the
percentage of follicular survival (59.38% vs. MEM+: 29.03%; AA100: 44.83%; P < 0.05) and antral formation (65.63% vs. MEM+: 34.78%;
AA100: 52.00%; P < 0.05) compared with MEM+ alone. After 6 days of culture, AA50 (319.14±89.70) increased follicular diameter (mm),
compared with the other treatments (MEM+: 229.59±78.13; AA100: 269.80±68.50; P < 0.05). At day 12, both concentrations of AA (AA50:
452.38±136.81; AA100: 451.98±165.45) increased follicular diameter when compared with control medium (242.11±131.69; P < 0.05).
Moreover, mean increase in follicular diameter daily (µm/day) was higher in the presence of both concentrations of AA (AA50: 18.15±10.57
and AA100: 20.94±10.43) than in MEM+ alone (11.38±9.46; P < 0.05). Lower percentages of extruded oocytes were observed in medium
containing AA50 (18.75%) compared with control medium (56.52%); P < 0.05. With respect to the maturation, all fully grown oocytes showed
an intact germinal vesicle. The qPCR assays showed that AA50 show mRNA expression for MMP9 was higher 2.4 times when compared to
MEM+ alone (P < 0.05). In addition, mRNA expression for TIMP2 gene was higher 2.08 times in AA100 than in MEM+ (P < 0.05). In
conclusion, AA at 50 µg/ml increase the caprine FOPA viability and development after in vitro culture and influences the enzyme MMP9
involved with basement membrane remodeling.
Keywords: mmp9, timp2, ascorbic acid.
N
s373