RESUMO
The aim of the present study was to investigate the distribution of BHV1 (Bovine Herpesvirus 1) in the semen from a naturally infected bull and more particularly, to determine if there were viral particles associated with sperm that could interfere with in-vivo or in-vitro fertilization. Frozen semen from a single ejaculate that had been previously identified as being contaminated with BHV1 was used. The contents of 12 ministraws were mixed. Two aliquotes served as controls, the first was held at 0 degrees C in ice water while the second was held at 4 degrees C in the centrifuge during the procedure. The remaining semen was separated into seminal plasma and sperm cell fractions by centrifugation. The seminal plasma was kept at 0 degrees C until viral titration. The pellet was treated by 10 consecutive washes in PBS and by a trypsin treatment after the fifth wash. Subsequently, the last pellet was macerated to break the cell membranes. Aliquotes for viral titration were taken from all washing fluids, from pellets after the fourth, sixth and tenth washes, and from the last crushed pellet. These aliquotes were kept in ice water until titration. The virus was titrated on secondary cell cultures of fetal calf kidney. The titers were 4.3, 4.2 and 4.4 log TCID50/ml, respectively, in the 2 controls and the seminal plasma. Titers declined from 3.8 log to 0 from the first to the tenth wash. The titer was 2.2 log in the resuspended pellet of sperm cell fraction prior to trypsin treatment. No virus was detected in the sixth pellet, the tenth one or from the crushed cells. It was concluded that a significant proportion of the BHV1 particles was associated with the sperm cells. Ten washes and a trypsin treatment could remove the adsorbed virus. No viral particles were detected within the sperm cells.
