Technique to a more accurate semen evaluation: Fluorochrome to differentiate the true acrosome reaction
1999
Sumonya Kanchanaphangkha | Sinchai Pianchop | Chongkon Chiansuwan (Chulalongkorn Univ., Bangkok (Thailand). Faculty of Veterinary Science)
Boar, bulls and buffalo bulls fresh and frozen semen are differentiated for viability/non viability and also for the true acrosome reaction using fluorochrome. The living and damaged or altered membrane spermatozoa are also measured. Normal, living spermatozoa are only noted when using 5 micro g/ml fluorochrome in the dry preparation. Evaluation 7 dats afterward (D7) reveals an increase in damaged spermatozoa (bull 41.3 percent buffalo bull 24.4 percent and boar 18.5 percent). Determination is irrelevant after 14 days (D14) using dry preparation in bull and buffalo bull semens (frozen semen). All sperms are fluorescence on D14. Damaged or altered membrane of the unfixed sperm allow the dye to penetrate into cell where it binds strongly to DNA. Boar semen (fresh) on day 14 has 16.5 percent more fluorescence sperms than D0. Fluorochrome concentrations (10 or 20 micro g/ml) using in wet preparation do not have a drastic effect on viability of spermatozoa. Number of viable sperms with normal acrosomal ridge (having potential to develop acrosome reaction) and all the nonviable sperms are about the same at D0, D7 and D14. Concentration of fluorochrome should be reduced to 5 micro g/ml in dry preparation and either 10 or 20 micro g/ml in wet preparation. Binding with the sperm DNA, fluorochrome concentrations used in our study can be examined incompletely through phase contrast microscope. To be albe to differentiate between true acrosome reaction and degenerative postmortem loss of acrosomal membrane, microscope equipped with UV-excitation has to be employed. In addition, we find that the dark field microscope gives the best result in differentiating acrosome status and reaction.
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