In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes

Roseli Fernandes Gonçalves,Leonardo José Richtzenhain,Cássia Maria Barroso Orlandi,Magali D'Angelo,José Antonio Visintin,Rodrigo Martins Soares

doi:10.1590/1808-1657000272013

Abstract

The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.

Highlights

MATERIAL AND METHODSEmbryo cryopreservation techniques have improved over the last decade, providing a safe opportunity for embryo storage and increasing the commercial scale for embryo transfer (ET) and in vitro fertilization (IVF) process
Experiment 2 – Effect of co-culturing oocytes with uterine tube epithelial cells previously inoculated with bovine herpesvirus 1 and subsequently treated with trypsin
The Madin Darby Bovine Kidney (MDBK) cells that co-cultured with the control oocytes, free of virus, did not show evidence of cytopathic effect (CPE)

Summary

Introduction

Embryo cryopreservation techniques have improved over the last decade, providing a safe opportunity for embryo storage and increasing the commercial scale for embryo transfer (ET) and in vitro fertilization (IVF) process. This fact certainly brought more attention for the potential risks in terms of infectious diseases transmission, such as bovine rhinotracheitis. The disease agent is the bovine herpesvirus 1 (BoHV-1), which can be found in the ovaries, uterine tube, and follicular fluid It is apparently more seen in healthy animals, which increases the risk of transmission by the use of these techniques (D’angelo, 1998; Wrathall et al, 2006). Neither washing nor trypsin treatment seem to completely remove the virus from embryos (Edens et al, 2003; D’angelo et al, 2009)

Objectives

Results

Conclusion