191 IN VITRO DEVELOPMENT OF BLASTOCYSTS CONTINUES AFTER ARTIFICIAL INFECTION WITH BOVINE HERPESVIRUS TYPE 5

Abstract

Bovine herpesvirus-5 (BoHV-5), which is the second most important infectious brain disease affecting livestock in Latin America, has been detected in bull semen and aborted fetus; however, no reports are found regarding its presence in bovine embryos. Because it has 90% genomic similarity to BoHV-1, it is possible that BHV-5 can infect the genital system after viral reactivation, leading to reproductive disorders. This study was designed to investigate the effects of infection of bovine blastocysts (B) by BoHV-5. Hormones and fetal calf serum were tested by PCR and considered free of virus. Selected oocytes, obtained from ovaries at a local slaughterhouse, were washed in PBS with 10% fetal calf serum (Nutricell^®, Campinas, Brazil). The oocytes were transferred to 100-μL drops of maturation medium consisting of TCM 199 (Gibco^®, Grand Island, NY, USA), 0.5 μg mL^-1 of FSH (Pluset^®, Calier, Spain), and 50 μg mL^-1 of LH (Lutropin^®-V, Bioniche Inc., Belleville, Ontario, Canada) for 24 h at 39°C and 5% CO₂ in air. Afterward, frozen semen (500 μL) was thawed and placed on a Percoll gradient (45 and 90%) and centrifuged at 700 × g for 30 min. The resultant pellet was centrifuged (200 × g for 5 min) in TALP medium and the sperm pellet was then diluted in TALP plus phenylalanine (PHE) and heparin. After an 18-h fertilization period, presumptive zygotes were transferred to culture in CR2 medium up to Day 7 post-fertilization. The procedures were carried out under the same conditions previously reported for IVM. A total of 150 intact B were assigned into 2 experimental groups: I (free of virus) and II (with virus; 10² TCID₅₀/mL for a 1-h period). Then, B of both groups were washed and re-cultured for 72 h in drops of CR2 without virus. The percentage of embryos reaching the hatched blastocyst (HB) stage was observed and analyzed by the chi-square test. At this time, HB were fixed to investigate the presence of virus, degree of apoptosis, and oxidative stress. The virus detection was performed by using an in situ hybridization assay with a specific probe to the glycoprotein C gene of BoHV-5 labeled to biotin. The apoptosis was determined by the annexin V, 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), and propidium iodide (PI) markers, using immunofluorescence technique. The oxidative stress was realized by using monoclonal anti-AOP1 (antioxidant-like protein 1; Sigma^®, St. Louis, MO, USA) through immunoassay. More HB (P > 0.05) were found in group II (75.0%) than for group I (55.0%). In both groups, positive signs for the presence of the apoptosis and oxidative stress markers were observed. The mechanism of apoptosis was initiated independently of virus presence, as evidenced by positive signs also observed in group I. However, oxidative stress was intense in group II, suggesting an evident viral effect on the host cell without compromising embryonic development. These findings might indicate that BoHV-5 uses some mechanisms that keep the cell viable to allow its replication, as seen by the greater hatching rate of infected embryos (75%) compared with the control (55%).

FAPESP 07/57774-7 and BRASFRIGO (Birigui-SP).