Use of biotech manipulation of oocytes enclosed in preantral follicles for in vitro evaluation of potential anti-zona pellucida to imunoesterilização bitches. / Utilização da biotécnica de manipulação de oócitos inclusos em folículos pré-antrais para a avaliação in vitro do potencial de anticorpos anti-zona pelúcida para a imunoesterilização de cadelas.

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

22/12/2008

RESUMO

The aim of this study was do evaluate the potential of anti-zona pellucida antibodies for immunosterilization in dogs, through analyzing the capability of anti-porcine zona pellucida (pZP) antibodies to cause atresia of preantral follicles (PF). For this purpose, the biotechnique of manipulation of oocytes enclosed in preantral follicles was employed to establish an in vitro experimental model, comprising preservation and in vitro culture methods for canine PF. In Phase I, the effects of storage temperature (4, 20 ou 38C), medium (saline solution or Minimum Essential Medium MEM) and time (2, 6, 12 ou 24 h) on follicular morphology and viability were evaluated. Classical Histology and transmission electron microscopy, and viability analysis using Trypan Blue and fluorescent labels (calcein-AM and ethidium homodimer-1) were used. Storage in MEM at 4C was the most efficient method, which enabled preservation of the percentages of viable follicles with intact ultrastructure similar to the fresh control for up to 12 h. In Phase II, the efficiency of the cryoprotectants dimethyl sulfoxide, ethylene glycol, glycerol and 1,2-propanediol for the cryopreservation of canine PF through slow freezing was assessed. It was observed that dimethyl sulfoxide (DMSO) at 1.5 M provided the preservation of the highest percentages of viable PF with intact ultrastructure after thawing. In phase III, the effects of anti-pZP serum on viability of canine PF cultured in vitro for 24 h, and also on canine sperm binding to homologous oocytes was evaluated. Fresh PF as well as PF cryopreserved in 1.5 M DMSO were used and results between then were similar. Supplementation of culture medium with 10% anti-pZP serum had no effect on follicles smaller than 100 μm as compared to controls (absence of serum or supplementation with pre-immune serum). However, antipZP serum led PF with diameter larger than 100 μm to degenerate, as detected using calcein-AM and ethidium homodimer-1. Anti-pZP serum also blocked sperm binding to canine ZP. In conclusion, it was demonstrated in vitro the potential of anti-pZP serum for immunosterilization (through induction of atresia to PF) and for immunocontraception (through sperm binding blocking) in dogs.

ASSUNTO(S)

cães folículos pré-antrais zona pelúcida imunocontracepção reproducao animal immunocontraception zona pellucida preantral follicles dogs

ACESSO AO ARTIGO

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