Correction: Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

[This corrects the article DOI: 10.1371/journal.pone.0218963.].

Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

Detection of bovine herpesvirus 1 in genital organs of naturally infected cows.

Bovine herpesvirus 1 (BoHV-1) is a causative agent of respiratory diseases in cattle, and infection with BoHV-1 can cause reproductive failure. There are few studies regarding infections in natural conditions in the reproductive organs of bovine animals. In this context, this study investigated the presence of BoHV-1 in the uterus, oviducts, and ovarian tissues of naturally infected cows. The three genital structures were evaluated for the presence or absence of BoHV-1 by immunofluorescence assay using confocal scanning laser microscopy. Blood and genital organ samples of 75 cows unvaccinated against BoHV-1 were used. Fragments of uterus, oviduct, and ovarian tissue were processed and analyzed by confocal scanning laser microscopy. Neutralization by antibodies was observed in 54.7% (41/75) of the serum samples tested. BoHV-1 were detected in the uterus of all the seropositive cows. The oviducts contained BoHV-1 in 73.2% of the samples and the ovaries contained BoHV-1 in 58.5% of the samples from seropositive animals. The presence of the virus was not observed in any of the genital organs of seronegative animals. There was no correlation between the antibody titer and the detection of BoHV-1 in positive tissue in the different genital organs or with the number of infected structures per animal. The detection of BoHV-1 in 100% of the uterus samples from seropositive cows suggests that this organ may be a source of infection for the fetus, resulting in abortion. Further studies on the mechanism by which BoHV-1 infects the fetus via the uterine route should be performed.

Use of two new formulations as bovine embryo manipulation solution.

This study aimed to evaluate the effect of two Embryo Manipulation Solutions (EMS and EMS supplemented) in maintenance of the viability of embryos, initially using structures derived from mice (first phase). Next, the efficiency of these solutions in routines of bovine embryo transfer was evaluated (second stage). Mice embryos were used in the stages of early blastocyst, and compact morula grades I and II. These embryos were initially randomly distributed and maintained for four hours in three solutions: Modified phosphate buffered saline (PBS; Control); EMS (treatment 1), and EMS supplemented (treatment 2). Subsequently, they were cultured in TCM 199 medium and evaluated in terms of total number of cells, morphometric characteristics, ultra structural aspects, detection of cell apoptosis, and quantification of Hsp70.3 gene expression. In the second phase, these same solutions were tested in the transfer of quality I and II bovine embryos (excellent and good). These embryos were transferred fresh to 58 recipients. The results showed that the total number of cells in embryos expanded blastocyst (ExB), the number of apoptotic cells, the cell, nuclear, nucleolar diameter and the nucleus/nucleolus ratio was similar among the treatments. The pregnancy rate shown on second phase was also similar. However, the EMS supplemented expressed more Hsp70.3 than EMS. The expression of Hsp70.3 was also greater for embryos in EMS than that of EMS supplemented. The McII embryos, EMS and EMS supplemented samples also expressed more Hsp70.3 compared to control embryos. In conclusion, the tested solutions can be used in routine embryo transfer techniques, replacing modified PBS solution as an effective media in maintaining embryo viability.