Nuevos métodos integrados de evaluación de la calidad seminal en vacuno y su relación con la fertilidad

The general aim of the present study was to apply a method of fluorescent multiple analysis developed by the group TECNOGAM in the study of the motility and morphometry of the spermatic subpopulations and improving hypo-osmotic test. Three studies were conducted using frozen semen of Friesian bulls as animal model. In three studies the image analysis was conducted with the Image J free software, and the statistical analysis of the data using the analysis of variance (ANOVA), followed by Tukey's test. In the study 1, the percentage of motile spermatozoa in the fluorescent subpopulations was determined. The fluorochromes allowed a rapid determination of the plasma membrane and acrosome integrity of sperm. The subpopulation with intact plasma membrane and acrosome displayed a higher percentage of motile sperm with the damaged membrane were motile. In the study 2, the morphometric parameters of the head, nucleus and acrosome of the sperm subpopulations and of 3 treatments: liquid samples immobilized with formaldehyde (1) and with shock for heat 65°C - Trumorph® (2), dried, fixed smears dyed with IP/PSA (3) were compared.The area of the head, nucleus and acrosome were lower (P less than 0,05) in smears than it wet mounts treatments. However, no significant differences in the sperm morphometry of the two treatments in the wet mounts were found. The subpopulation with acrosome normal, damaged membrane presented significantly higher measures (P less than 0,05) compared with the subpopulation with acrosome normal, membrane intact, for both treatments in wet mounts. The percentage of the head occupied by the acrosome in the smears whereas higher than those of the treatments in wet mounts. Finally, in the study 3, the effect of hypos-osmotic test (HOST), two types of solutions based on citrate and TRIS, and the test water (WT) on the distribution of the sperm subpopulations. The final osmolarity was adjusted to 150 mOsm per kg in all the cases. The pH was adjusted to 7.0 based only on the TRIS solution. The subpopulations were determined at 5 and 40 min from incubation to 37degrees C. The response to HOST, the state and acrosome membrane integrity were used to establish eight subpopulations. For both periods of incubation, the subpopulation HOST negative, acrosome damaged, damaged membrane was significantly higher in the WT that in both HOST solutions evaluated (P less than 0.05). At 40 min, the subpopulation, HOST positive, acrosome intact, intact membrane was lower in the WT that in the HOST when citrate-based solution was used. The new method of multiple fluorescent staining allowed, for the first time, to study in the same spermatozoa the integrity of the plasma membrane and acrosome, functionality and motility, obtaining different the sperm subpopulations. The ability of these new methods of analysis to predict sperm fertility will be addressed in future studies