Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

Abstract

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.

Damage assessment of the equine sperm membranes by fluorimetric technique

“…Studies have been carried out to develop new techniques for seminal evaluation. Among these, great emphasis has been given to fluorescent probes by epifluorescence microscopy or flow cytometry system (Graham et al, 1990Andrade et al, 2007;Celeghini et al, 2008). Fluorescence is a sensible and specific indicator of the state of some molecules, so that it can be applied as a method to measure the metabolic changes in the living cells (Haugland, 2001;Santos et al, 2008).…”

Artificial Insemination in Pigs

“…Since the acrosome is important for the penetration of the oocyte, its integrity is considered vital for an optimal fertilising capacity. The acrosome integrity can be evaluated on the basis of its microscopic appearance, either by phase contrast evaluation of glutaraldehyde fixed spermatozoa or by various staining methods (de Andrade et al, 2007;Woelders, 1991). …”

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility

“…After, samples were stored into a refrigerated chamber at 17°C and evaluated at 0, 24, 48 and 72 hours. Spermatozoa ϐluorescence assessment was evaluated by the PI, AI, and MP evaluation using ϐluorescent probes and classiϐied according to describe by Andrade et al (2007).…”