RESUMO

FREEZABILITY of BULL SEMEN CORRELATED WITH SEMINAL PLASMA PROTEINS

Trabalho aceito para apresentação como poster no Congresso Internacional de Reprodução Animal, ICAR 2000, em Estocolmo - Suécia, em Julho de 2000.

The present work had as objective to study the protein profile of bull seminal plasma (electrophoretical and total protein concentration), taken from semen–donor bulls, zebu and taurine bulls, searching to semen freezability. Were used 29 bulls semen donors at the Artificial Insemination Centre, from diferent breeders and from the subspecies Bos taurus indicus (zebu bull) and Bos taurus taurus (taurine bull). The animals were divided in 04 groups, high (>80%), good (79-50%), medium (49-30%) and low (<29%) semen freezability. The freezability profile was based on the retrospective description of production during 5 years. The semen was collected using the method of artificial vagina, and immediately, an aliquot of 1,0 ml of the semen was separated and centrifugation at 1500 g for 20 minutes at 4oC the seminal plasma (SP) was obtained. The SP was dialysed in cellulose membrane, in TRIS-Glycine buffer pH 7,4 for 48 hours on 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 to 10,0mg/mL, with dilution in Tris-HCl buffer 62mM pH 6.8 increased of 20% of glycerol and 4% of SDS and 0,05% B-mercaptoethanol. After that, the samples have been boiled for 5 minutes. For the determination of the eletrophoretics profile, SDS-PAGE technique described by Laemmli (1970)was used, with 13% gel. The gel stain was achieved with the use of Coomassie Brilliant Blue 250-R. The results of dosage and densitometry of the seminal plasma proteins had been submitted to statistical analysis for the Entirely Randomized Design and the averages to the Tukey Test. Significant difference was found in the total protein concentration among the group with semen freezability, The high semen freezability group had presented an amount around 19% higher than low freezability group. The zebu bulls had presented an amount around 20% more protein in the seminal plasma than taurine bulls in the in the high semen freezability group. The low freezability animals group, did not present differences between the subspecies. In the electrophoretic protein profile of seminal plasma, the maximum of 33 bands has been found. The bands with 45, 24 and 10.8KDa presented significant differences in densitometry values between the high/good and medium/low semen freezability groups, found a higher concentration in the high/good groups. The band 14.4KDa showed superior concentration value in the medium/low freezability groups. Beyond of that, it’s require further studies, in order to identify these proteins present in the seminal plasma, and compare with the others proteins, already mentioned in the literature as fertility markers, therefore they seem to participate in metabolic mechanisms that assist the semen cryopreservation.

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