Quantitative and morphological study of preantral follicles from prepubertal gilts
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Data
2012
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Resumo
Background: The development of animal reproductive biotechniques is intended of raising reproduction effi ciency. The
mammal ovary contains thousands of follicles, of which approximately 99.9% are eliminated by means of the atresia,
apoptosis and cellular necrosis process. In other to reduce this follicular loss, the methods for isolation and characterization
of preantral follicles have been studied as a premise to culture systems of these structures. The purpose of this study was
quantify and evaluate the quality of preantral ovarian follicles from prepubertal gilts after mechanical isolation procedure.
Furthermore, it aims to analyze the preantral follicular histological morphology.
Materials, Methods & Results: Ovaries (n = 20) from prepubertal gilts were divided in two halves and used each one
for isolation and histological processes. The tissue chopper previously regulated for the performance of seriated cuts at
200 μm intervals was used for mechanical isolation. The marker bisbenzimidine Hoechst 33342 was added to the follicular
pool for evidence the presence of granulosa cells around oocyte and the viability of the isolated PAF was evaluated by using
propidium iodide. For the histological evaluation, the ovarian halves were fi xed, dehydrated and diaphanized, and after
enclosed in paraffi n, each of them was divided into 5 blocks and sectioned in series 6 μm thick. At each 1000 μm interval,
2 slides were removed per block, totaling 200 slides that were dyed with periodic acid Schiff (PAS) and hematoxylin. The
total PAF calculated in situ for each follicular stage (primordial, primary and secondary) was estimated according with
the preantral follicles number evaluated on histological sections. The number of isolated preantral follicles per ovary was
(599,160 ± 74,089), presented positive correlation (P < 0.05) between ovarian weight. After isolation, 76.4% of preantral
follicles were viable (P < 0.05), while 64.3% had granulosa cells around the oocyte (P < 0.05). The average number of
PAF estimated in histological cuts was 131,938 ± 14,615 per ovary, oscillating between 67,599 and 291,898, out of which
117,968 (89.41%) primordial follicles, 3,448 (2.61%) primary follicles, and 10,522 (7.98%) secondary follicles. The verifi
ed follicles diameter was primordial (33.1 μm), primary (47.6 μm) and secondary (79.1 μm).
Discussion: The PAF quantifi cation estimated in situ per ovary was different from results obtained on mechanical isolation,
showing that used techniques do not complement. However, both procedures highlighted the specie reproductive
potencial. The greater percentage of oocytes was included in primordial follicles (89.4%; P < 0.05). Follicular diameter
and the number of granulosa cells increased (P < 0.05) according to the development stage. These data are important to
defi ne which interval of cut can be adopted before utilizing the mechanical isolation procedure with tissue chopper, avoiding
sections damages to the follicular structure, which contributes to the smaller viability of isolated PAF. In summary, the
results reported that it is possible to obtain a great number of viable PAF from of prepubertal gilts ovaries after mechanical
isolation, providing material to supply the demand for studies relating to folliculogenesis and in vitro culture techniques.
Descrição
Palavras-chave
Primordial follicles, Oocyte, Porcine, Viability
Citação
ALVES; Benner Geraldo; ALVES, Kele Amaral; ARAÚJO, Valdevane Rocha; BELETTI, Marcelo Emílio; GAMBARINI, Maria Lúcia; JACOMINI, José Octávio. Quantitative and morphological study of Preantral follicles from prepubertal gilts. Acta Scientiae Veterinariae, Porto Alegre, v. 40, n. 4, p. 1079, 2012.