RESUMO

SPERM MEMBRANE AND SEMINAL PLASMA PROTEIN PROFILES RELATED TO BULL SEMEN FREEZABILITY

Enviado para publicação para Journal of Andrology, em Abril de 2000

The present work aimed to study the protein profiles (electrophoretical and total protein concentration) of bull seminal plasma (SP) and sperm membrane (SM) and their relation to sperm freezability. Semen samples were taken from 29 semen–donor bulls (zebu and taurine) at the Lagoa da Serra Artificial Insemination Centre. The animals were divided in 04 groups: high (H = >80%), good (G = 79-50%), regular (R = 49-30%) and low (L = <29%) semen freezability. The freezability profile was based on the previous 5-year production history. Semen was collected using the artificial vagina method, and immediately, an aliquot of 1.0 ml was separated and centrifuged at 1500 g for 20 minutes at 4oC to obtain SP, which was dialysed in a cellulose membrane, in 10mM TRIS-Glycine buffer pH 7.4 for 48 hours at 4oC under constant and slow agitation. Before and after dialysis, an amount of each sample was separated for total protein assay (Bradford, 1976). From these amounts, a volume was taken for the standardization of the samples at 10.0mg/mL, by dilution in 62mM Tris-HCl buffer pH 6.8 supplemented with 20% glycerol, 4% SDS and 0.05% β-mercaptoethanol. After that, the samples were boiled for 5 minutes. Electrophoretic profiles (EP) were determined by SDS-PAGE (Laemmli, 1970). The results for total protein (TP) content and densitometry of seminal plasma proteins were submitted to statistical analysis using an Entirely Randomized Design and the averages were compared by Tukey Test. Significant difference for TP concentration was found between the H and L groups. The H group presented an amount about 19% higher than the L group. Within the H group, zebu bulls presented about 20% more protein in the SP than taurine bulls. The L group, did not present differences between subspecies. For the EP of SP, a maximum of 33 bands was found. Seminal plasma bands of 45, 24 and 10.8KDa presented significant differences in densitometry values between the H/G and R/L groups, showing a higher concentration within the H/G groups. The 14.4KDa SP band was found in higher concentration within the R/L groups. The EP of SM proteins showed significant difference for the 95, 58, 45 and 36KDa bands with higher concentrations in the L group. Thus, sperm cryopreservation is a process that depends directly on the behaviour of biological membranes and indirectly on the environment the seminal plasma. Therefore, we suggest that the 45, 24, 14.4 and 10.8KDa seminal plasma proteins may be used as biochemical markers of different semen freezability patterns in bulls and that they may interact with the sperm membrane and act during the cryopreservation process, being either harmful (14.4KDa) or beneficial (45, 24 and 10.8KDa).

palavras-chaves: semen cryopreservation, biochemical markers, fertility, bovine