Evaluation of Temporal Development of the Acrosome Reaction In Vitro in Spanish Black Berrendo Breed Bulls by Epifluorescence Methods.

摘要

Black Berrendo cattle breed belongs to the Andalusian heritage bovine livestock and itis a endangered one, protected by the EU programs of cattle conservation. There are onlyseminal frozen doses from 10 bulls of this breed. It is important to know their ability to becapacitated in vitro for later use in IVF as a technique for recovery of their populations.The aim of this study was to evaluate the evolution of the acrosome reaction in vitrothrough the time in a defined culture medium by epifluorescence microscopy methods inseminal frozen samples of Spanish Black Berrendo bulls.Samples (0.25 ml.) of frozen semen from 5 Berrendo Black breed bulls were thawedand incubated with Sp-TALP culture medium enriched with 100 ?gr/ml of sodium heparin ascapacitating agent. Aliquots of 100 ?l were taken at 0, 60 and 120 minutes. These were stainedwith Propidium Iodide and FITC-PNA following a modified protocol in our lab and extendedon slides. Slides were mounted and observed in a fluorescence microscope with excitationfilter BP 450-490 nm and a emission filter of BP 515-560nm. Acrosome reaction waspositive in those sperm cells in which a green color in the acrosomal region and a faint redcoloration in the rest of the head were found and negative one in those in which only the faintred coloration appeared. The capacitating percentage was evaluated on individual bulls indifferent times of capacitating analyzing 200 cells three times. The results showed somesignificant differences at 0.05% for different times. After 60 minutes of incubation thecapacitating percentage is the adequate to use for IVF. The values obtained in this Andalusianbovine breed were consistent with previous studies in other breeds. In the study 50% of frozensperm samples of this breed were suitable to use for IVF. This technique proves to be quickand effective way to evaluate bovine semen capacitated in vitro.