Colorazione multipla fluorescente e sistema "CASA" degli spermatozoi di suino : valutazione della loro vitalità in diluitori a breve e lunga conservazione

The aim of this study was to test a simple and consistent method for the simultaneous evaluation of the integrity of the plasma, the acrosome and the mitochondrial sheath membranes of boar spermatozoa extended in six short-term (A-F) and three long-term (G-I) commercial extenders. Sperm samples collected from three boars of proven fertility were evaluated examining motility by Computer Assisted Sperm Analyzer (CASA), viability using Propidium Iodide (PI) staining, acrosome intactness by fluorescein isothiocyanate- conjugated peanut agglutin (FITCPNA) staining and mithocondrial activity by 5,5’, 6,6’-tetrachloro-1,1’, 3,3’ tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) staining. The results obtained from the fluorescent multiple staining, were compared with sperm motility assessed by CASA and with sperm morphology. Sperm evaluations along with bacteriological contamination (CFU) were performed during a 6-12 days period, depending on the labelled preservation period of each extender. In our study it was found that the frequency of viable spermatozoa with nonreacted acrosome and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2>0.9) regardless of the extender used. In all extender the frequency of motile spermatozoa significantly decreased with the preservation period (p<0.05). The rate of progressively motile spermatozoa was very low in all the assayed extenders (14,95% ±0,89 since day 0) but this motility could have been influenced by the diluition effect. The fluorescent multiple staining test is a potent indicator of sperm motility because it analyses the mitochondrial integrity independently from observable alteration of motility and our results indicated that the low progressive motility was only apparent since the mitochondrial sheath of most spermatozoa was intact. CFU number remained stable during the study period for all extenders and there was no significant correlation between CFU, percentage of motility and dead spermatozoa. In conclusion, the integration of the data obtainable from the fluorescent multiple staining test and the CASA analysis are required for a correct evaluation of boar sperm viability during preservation.