I am working in Canine frozen sperm-oocyte penetration assay for last 2 months. Still, I could not get better result for aceto-orcein staining method.. Can anyone recommend the staining protocol for Canine oocyte penetration test?
Dear Palanisamy,
you study the sperm zona pellucida penetration or the sperm oocyte cytoplasm penetration?
Pavel Travnik
Prof. Pavel,
Thanks for your reply. Actually, I want to identify the "spermatozoa-zona pellucida penetration" and "nuclear maturation status of oocyte".. I also tried to Hoechst 33342 staining with oocyte but I couldn't get the result.. if you give protocol for the sperm oocyte cytoplasm penetration, it will also more helpful for me..
Dear Palanisamy,
these topics need different methods and there is no reason to combine them – the spermatozoa penetrate the zona pellucida of metaphase II oocytes, where no nucleus is present. For the zona penetration test the aceto-orcein method is not suitable, because the acetic acid solves the zona pellucida and the localization of spermatozoa is not possible. For this purpose I suppose simple phase contrast or dark field observation of non-fixed sample (in culture medium or saline, under coverslip). For the nuclear status of the oocyte observation the aceto-orcein method is very suitable.
Best regards
Pavel Travnik
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