Fluorescent stain method for the simultaneous determination of mitochondrial potential and integrity of plasma and acrosomal membranes in boar sperm.

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology <or=10%, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen in liquid nitrogen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen : flash frozen semen; 100 : 0 (T100), 50 : 50 (T50), and 0 : 100 (T0). The samples were then submitted to a stain technique. To a 150-microl aliquot of diluted semen it was added 3 microl of PI (0.5 mg/ml), 2 microl of JC-1 (153 microm) and 50 microl of FITC-PSA (100 microg/ml). Samples were incubated at 38.5 degrees C for 8 min, in the dark. An 8-microl sample was put on a slide, coverslipped and immediately evaluated by epifluorescent microscopy. The association of fluorescent probes was divided into eight cell classes, according to plasma membrane integrity, intact acrosome and mitochondrial function. For plasma membrane integrity, detected by PI probe, the equation: (p < 0.0001) and R(2) = 0.97 was obtained. The intact acrosome, verified by the FITC-PSA probe, produced the equation: (p < 0.0001) and R(2) = 0.98. The mitochondrial potential, marked by JC-1, was estimated by the equation: (p < 0.001) and R(2) = 0.99. The group of spermatozoa with combined intact plasma membrane, intact acrosome and high mitochondrial potential (IPIAH), was estimated by the equation: (p < 0.0001) and R(2) = 0.97. The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa.