Nuevos métodos integrados de evaluación de la calidad seminal en vacuno

Abstract

[EN] he 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 conducte d with the ImageJ free software, and the statistical analysis of the data using the analysis of variance (ANOVA), followed by Tukey's test. In the s tudy 1 , the percentage motile sperm atozoa in the fluorescent subpopulations was determined . The fluorochromes allowed a rapi d determination of the plasma membrane and acrosome integ rity of sperm . The subpopulation with intact plasma membrane and acrosome displayed a hi gher percentage of mot ile sperm with the damaged membrane were motile. In the stud y 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 <0,05) in smears than it wet mounts treatment s. However, no significant differences in the sperm morphometry of the two treatments in the wet mounts were found. The subpopulation with acrosome normal, damaged membra ne presented significantly higher measures (P <0,05) compared with the subpopulation with acrosome normal, membrane intact , for both treatments in wet mounts. The percentage of t he head occupied by the acrosome in the smears wereas higher that those of the treatments in wet mounts. Finally, in the study 3, the effect of hypos - osmotic test (HOST), two types of solu tions based on citrate and TRIS , a nd the test water ( WT) on t he distribution of the sperm subpo pulations. The final osmolarity was adjusted to 150 mOsm/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 37°C. The response to HOST, the state and acrosome membrane integrit y were used to establish eight subpopulations . For both periods of incubation, the subpo pulation HOST negative, acrosome damaged, damaged membrane was significantly higher in the WT that in both HOST solutions evaluated (P <0.05). At 40 min, the subpop ulat ion, HOST positive, acrosome i ntact, intact membrane was lower in the WT t hat in the HOST when citrate - based solution was used . The new met hod of multiple fluorescent staining allowed, for the first time, to study in the same sperm atozoa the integrity of the plasma membrane and acrosome, functionality and motility, obtaining diffe rent the sperm subpopulations. The ability of these new methods of analysis to predict sperm fertility will be addressed in future studies.

[ES] Se trabajará sobre el desarrollo de nuevos métodos de evaluación de la calidad seminal en rumiantes, que estarán basados en una tinción múltiple fluorescente del espermatozoide, lo que permitirá evaluar simultáneamente diferentes parámetros con relevancia funcional y, potencialmente, mejorar la capacidad predictiva de la fertilidad del semen