Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Available online at www.sciencedirect.com

Theriogenology 74 (2010) 1296 –1303 www.theriojournal.com

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos C. Silva-Fradea,b, R. Gameiroa, A. Martins Jrb, T.C. Cardosoa,* a

Laboratory of Animal Virology, UNESP – University of São Paulo State, College of Veterinary Medicine, São Paulo, 16050-680, Brazil b Laboratory of Animal Reproduction, UNESP – University of São Paulo State, College of Veterinary Medicine, São Paulo, 16050-680, Brazil Received 13 January 2010; received in revised form 31 May 2010; accepted 1 June 2010

Abstract Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 ⫾ 0.05, mean ⫾ SD), but there were differences (P ⬍ 0.05) between infected (0.18 ⫾ 0.05) and uninfected blastocysts (0.73 ⫾ 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 ⫾ 0.04; P ⬍ 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development. © 2010 Elsevier Inc. All rights reserved. Keywords: BoHV-5; Herpesvirus infection; Bovine embryos; Apoptosis

1. Introduction The developmental potential of embryos produced in vitro is affected by various factors, including the culture system, oocyte quality, the presence of serum, sperm quality, and pathogens [1]. Recently, bovine Herpesviruses type 5 (BoHV-5) was isolated from samples of frozen semen of an apparently healthy bull undergoing screening prior to export [2]. Moreover, we

* Corresponding author: Tel.: ⫹55 18 36363200. E-mail address: [email protected] (T.C. Cardoso). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.06.001

demonstrated the experimental infection of bovine oocytes, sperm, and embryos with BoHV-5 was possible, but did not interfere with either embryo quality or in vitro development [3]. Therefore, we inferred that BoHV-5 had unknown mechanisms to inhibit cell death and evade host surveillance. The notion that organisms use apoptosis as an antiviral defense has became widely accepted [4,5]. Infected cells undergo apoptosis and thus are eliminated, limiting viral propagation. It is known that Alphaherpesviruses sub-family, which includes BoHV-1 and BoHV-5, may not require as stringent suppression of

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

apoptosis, because they may evade apoptosis prior to its completion, due to their faster replication cycle [6]. Typical features of apoptotic cells include a series of cellular, morphological, and biochemical alterations, such as chromatin condensation, phosphatidylserine (PS) exposure, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation [7–10]. Annexin-V binding is known to result from the loss of phospholipid asymmetry and PS. In addition, PS exposure on the outer surface of the plasma membrane occurs in the early stages of apoptosis [10]. Another phenomenon related to apoptosis was host DNA condensation, which can be detected with a TUNEL assay [7–9]; fragments are labeled in situ at the 3’-OH end of DNA fragments, using the enzyme terminal deoxynucleotidyl transferase (TdT) in the presence of fluorescently labeled nucleotides (e.g., bromolated deoxyuridine triphosphate nucleotide (BrdU) [8]. The objective of the present study was to investigate some host-virus interactions, confirm virus replication, and some apoptotic events. For this purpose, bovine oocytes were experimentally exposed to BoHV-5. The presence of viral DNA in infected embryos was confirmed by in situ hybridization. The BoHV-5 viable particles were recovered from infected embryos by virus re-isolation in MDBK cells and the polymerase chain reaction (PCR) targeting the US9 gene, followed by sequencing to confirm virus infection. The TUNEL and annexin-V label were detected with an indirect immunofluorescence assay to measure apoptotic effects of virus infection. 2. Materials and methods 2.1. Oocytes collection, in vitro fertilization, and maturation Oocyte collection, in vitro maturation (IVM), sperm, and in vitro fertilization procedures were followed as previously described [3]. The study was conducted with presumptive zygotes produced from oocytes exposed, or not exposed to BoHV-5 (the latter underwent the same protocol, but without exposure to BoHV-5). Frozen-thawed sperm for IVF were derived from 0.5 mL straws of bovine (Bos indicus) semen collected from a single bull. Semen was centrifuged on a Percoll (Nutricell®, Campinas, SP, Brazil) gradient at 700 ⫻ g for 20 min. The resulting sperm pellet was washed in TALP medium (Tyrode medium added by bicarbonate buffer supplemented with 6 mg BSA [Sigma-Aldrich®, St. Louis, MO, USA] per milliliter) and centrifuged at 200 ⫻ g for 5 min. The pellet was diluted in IVF

1297

medium (TALP medium supplemented with 3 mg/mL heparin and PHE solution: 2 mM penicillamine, 1 mM hypotaurine, and 250 mM epinephrine, Sigma-Aldrich) to a final concentration of 1 ⫻ 105 sperm/mL in drops of 100 ␮L. After 24 h of maturation, oocytes were transferred to drops containing IVF medium. For IVF, oocytes and sperm were co-incubated in the IVF medium for 20 h under the same conditions used for IVM. Afterwards, presumptive zygotes (PZ) were placed in in vitro culture medium (IVC) up to Day 7 (Day 0 ⫽ day of fertilization). After blastocyst production on Day 7 post-fertilization, only embryos graded as Code 1 (Excellent or Good) or Code 2 (Fair) following IETS guidelines [11] were used. Similarly, only oocytes and presumptive zygotes classified as good quality were used. All uninfected cells and reagents used in this study were assayed for bovine Herpesvirus types 1 and 5 (BoHV-1 and 5), bovine viral diarrhea (BVD), and others pathogens, e.g. Mycoplasma [12]. 2.2. Virus infection and embryo development Stocks of BoHV-5, isolated in 2007 from outbreaks in Araçatuba, SP, Brazil [12] were propagated in Madin-Darby bovine kidney (MDBK, ATCC CCL-2) cells, which were cultured in minimum essential medium (MEM, Sigma-Aldrich). The tissue culture infective dose per 50 ␮L (TCID50) of stock virus was determined by virus titration infection of confluent monolayers of MDBK cells [12] at multiplicity of infection (MOI) of 1. Aliquots of stock virus (100 ␮L) with 103.3 TCID50/50 ␮L were frozen at ⫺86 °C prior to use. Only COCs with several layers of compact cumulus cells and homogeneous cytoplasm were used, divided into drops of 15 to 20 oocytes each for experimental use. The culture consisted of oocytes maintained in 100 ␮L TCM-199 (GIBCO-BRL, Grand Island, NY, USA), supplemented with 10% FBS (Nutricell), 2.2 mg/mL sodium bicarbonate, 0.02 mg/mL sodium pyruvate, 0.05 mg/mL gentamicin sulfate, 0.5 ␮g/mL FSH (Pluset, Calier, Barcelona, Spain), and 50 ␮g/mL LH (Lutropin-V, Bioniche Inc., Belleville, ON, Canada) for 24 h at 39 °C in 5% CO2-air. Selected oocytes were washed in maturation medium (MM) and transferred to drops containing 100 ␮L of MM. Oocytes were experimentally infected by co-incubation with 10 ␮L BoHV-5 (102.3 TCID50 corresponding to 1 MOI) for 1 h at 39 °C in 5% CO2-air. The oocytes were subsequently washed three times and transferred to new, virus-free maturation drops for further in vitro development, as previously described.

1298

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

2.3. In situ hybridization

2.4. Virus recovery

The in situ hybridization assay, probe preparation and detection system were applied to detect the US9 gene among infected embryos, as described previously [13]. The probe (250 bp) was generated based on the sequence of US9 region (GenBank accession number AY064172). Procedures for DNA purification and PCR were previously described [12]. The PCR was performed using 12.5 ␮L of 2 ⫻ Platinum Taq Polymerase High Fidelity Master Mix (Invitrogen™, Carlsbad, CA, USA), and 10 pmoles of each primer (US9-For: 5=AGAGTCCACACAGCGTCGTCAA-3= and US9-Rev: 5=-CTACAGCGAGAGCGACAGCGAGA-3=). The reverse primer (US9-Rev was purchased biotinylated (Invitrogen). In addition, 2.5 ␮L of nuclease-free water and 8 ␮L of the DNA sample (positive reference strain AY064172) consisting of 3 ␮L of DMSO (SigmaAldrich) plus 5 ␮L of DNA were added to the master mix. In an automated thermocycler (Eppendorf, Hamburg, Germany) the reactions were incubated at 98 °C for 5 min; 34 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, and finally 72 °C for 5 min. The PCR products were visualised on a 1.5% (w/v) agarose gel after staining with ethidium bromide (0.5 ␮g/mL). After confirmation of the 250-bp product, another unstained gel was used to separate the PCR amplicon for purification using a GenElute Agarose Spin Column (Sigma-Aldrich).The purified PCR product was cloned into the TA-vector (pGEMTEasy, Promega, Madison, WI, USA), and ligation products were transformed into E. coli using heat shock. A confirmed positive colony was cultured, and plasmid DNA was prepared using a commerciale kit (Promega Mini-Prep). The final plasmid was confirmed using sequencing. The probe (2 ng/mL) was denatured in 98 ␮L of pre-hybridisation buffer (50% formamide, 5% bovine serum albumin, 1% N-lauroylsarcosine and 0.02% sodium dodecyl sulphate [Sigma-Aldrich]) at 98 °C for 8 min and immediately immersed in ice. The slides, containing fixed embryos were pre-heated, covered with 100 ␮L of heated probe, and incubated at 42 °C overnight under a plastic cover slip in a humidified chamber. The excess, unbound probe was removed by washing the slides three times in PBS for 10 min at room temperature, with two additional washes in PBS for 3 min each. After incubation in Biotin Blocking System (DakoCytomation, Carpinteria, CA, USA) for 30 min, slides were incubated using the ExtrAvidinFITC conjugate system (Sigma-Aldrich). All incubations were performed at room temperature for 30 min in the dark.

Embryos derived from directly exposed oocytes with BoHV-5 and observed during in vitro production were collected and frozen-thawed three times. Monolayer cultures of MDBK cells at 80% confluence were prepared according to standard procedures free of BoHV-1 and any other potential pathogens [3,12]. Adsorption was allowed for 90 min at 38.5 °C. Then, fresh medium was added and for the next 7 d, cultures were examined for cytopathic effects classified as syncytial formations (CPE). After a further passage, cultures with no evidence of CPE were considered negative. When CPE was observed, the respective cells were removed and submitted to virus titration and identification. Virus titration was conducted with infected embryo suspensions and 96-well plate previously seeded with MDBK cells. Serial dilutions, 10⫺2 to 10⫺8, of infected embryos suspensions were prepared and used for BoHV-5 titration onto a single well in triplicate. The plate was incubated for 1 h at 38.5 °C prior to adding 100 ␮L of supplemented MEM (Sigma-Aldrich). Plates were incubated for 7 d and examined every 24 h for evidence of CPE. Infectious virus was calculated according to the Spearmann-Kärber method, as described [12]. 2.5. Polymerase chain reaction (PCR) and nucleic sequencing Viral DNA for PCR was obtained from infected embryos derived from BoHV-5 infected oocytes and from MDBK cells infected with these embryos, as well as non-infected embryos, as described [13]. For this purpose, inoculated cells were harvested when the CPE reached ⬃80% of the MDBK monolayers. Total DNA was extracted from infected cells using viral RNA/ DNA PureLink extraction method (Invitrogen). The PCR was performed according to the manufacturer’s protocol for an in situ hybridization assay. After the 250-bp product was confirmed, an unstained gel was used to separate the PCR product to be purified using the GenElute agarose spin columns (Sigma-Aldrich) for nucleotide sequencing in a MEGABACE sequencer (GE Healthcare). Sequences were aligned using Lasergene DNA STAR (Version 7, Lasergene Corp., Madison, WI, USA). The degree of identity among sequences at the nucleotide and amino acids levels was determined using BIOEDIT v.7.0.5. The sequence was deposited in GenBank (accession number GU947818).

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

2.6. Indirect immunofluorescence to assay annexin-V marker Embryos (n ⫽ 30 per treatment) were washed three times in PBS (pH ⫽ 7.4) and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 24 h at 4 °C. Samples were then rinsed with PBS and permeabilized with proteinase K (10 ␮g/mL, Invitrogen) for 15 min at room temperature. After pre-treatment with proteinase K (10 ␮g/mL) at 4 °C, slides were incubated overnight with primary antibodies (mouse anti-annexin-V) diluted in antibody diluent (DakoCytomation). The slides were then incubated for 24 h at 4 °C with secondary antibody (FITC-goat anti-mouse IgG; Zymed, South San Francisco, CA, USA). Omission of the primary antibody was used as a negative control. Subsequently all samples were counterstained with 6.25 mg/mL propidium iodide (PI, Sigma-Aldrich) for 15 min at room temperature before mounting the slides in the dark [13]. 2.7. TUNEL assay Staining for theTUNEL assay was performed following the manufacturer’s instructions (APO-BrdU TUNEL Assay Kit, Molecular Probes, Invitrogen). Briefly, bovine embryos were washed twice in wash buffer (ABO-BRDU Kit) and incubated at 4 °C for 12 h in prepared DNA-labeling solution (ABOBRDU Kit), containing TdT enzyme, BrdUTP, TdT reaction buffer, and distilled water. After washing twice in rinse buffer (ABO-BRDU Kit), embryos were incubated in the dark for 30 min at room temperature with antibody solution (fluorescein-labeled anti-BrdU monoclonal antibody and rinse buffer). The positive control consisted of bovine embryos pre-incubated with 10 ␮g/mL of DNAse (Invitrogen) for 1 h at 37 °C, whereas the negative control was incubated without the TdT enzyme [14]. 2.8. Semi quantification and data analysis The levels of annexin-V and the TUNEL assay labels were semi-quantified according to the intensity of the immunofluorescence reactions. Three standard filters were employed: DAPI filter (emission wavelength: 425 nm) was used to determine quality, propidium iodide (PI; emission wavelength, 538 nm), whereas fluorescein isothiocyanate (FITC) filter (emission wavelength, 512 nm) was used to detect TUNEL stained nuclei, annexin-V and in situ hybridization DNA. Bovine embryos were examined on two separate occasions by two observers without prior knowledge of classification. The intensity of labeling was

1299

graded semi-quantitatively as follows: (⫺) negative, (⫾) minimal, (⫹) mild, (⫹⫹) moderate, and (⫹⫹⫹) marked. Differences in the respective mean values were tested using ANOVA, with the main effects being morphological quality groups, followed by a multiple pairwise comparison using a Student’s t-test for independent samples and Bonferroni t-test. Differences of P ⬍ 0.05 were considered significant. The images were collected under an AxioImager A.1 light and ultraviolet microscope connected to AxioCam MRc (Carl Zeiss, Oberkochen, Germany), and micrographs were processed with Axiovision 4.7 software (Carl Zeiss). 3. Results 3.1. Embryos development and virus detection From 179 unexposed oocytes to virus infection and 185 submitted to BoHV-5 infection, 90.9 ⫾ 6.5 and 89.4 ⫾ 8.9 were cleaved, respectively. There were no significant differences relative to total number of unexposed oocytes when compared to those from exposed to BoHV-5. There was no significant effect of virus infection on embryonic development, including proportions of oocytes which developed into a morula (exposed oocytes 41.2 ⫾ 8.2; unexposed oocytes 41.1 ⫾ 7.7), blastocyst expanded blastocyst or hatching (exposed oocytes 29.3 ⫾ 8.2; unexposed 28.8 ⫾ 9.2). The quality of infected and uninfected embryos was considered equal, Code 1 (excellent or good) as shown (Figs. 1A and 2A). To confirm virus infection, BoHV-5 DNA was identified by in situ hybridization (Fig. 2C), PCR amplification among all infected embryos (Fig. 3C) and typical cytopathic effect in infected MDBK cells (Fig. 3B). Negative results represented by absence of CPE on MBBK cells, were found in unexposed oocytes and sperm used in the in vitro fertilization procedures (Fig. 3A). Moreover, the BoHV-5 titre used to expose bovine oocytes were 102.3 TCID50/50 ␮L; after in vitro fertilization and embryo development, the titre increased to 105.3 TCID50/50 ␮L, consistent with replication. Sequencing of US9 confirmed the presence of the BoHV-5 genome (GenBank accession number GU947818). 3.2. Expression of annexin-V and TUNEL stained There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 ⫾ 0.05), but there were differences (P ⬍ 0.05) between infected (0.18 ⫾ 0.05) and uninfected blastocysts (0.73 ⫾ 0.07). The intensity of TUNEL stained was classified as moderate related to

1300

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

Fig. 1. Day 7 bovine blastocysts in vitro cultured derived from control oocytes (not exposed to BoHV-5): (A) DAPI labeling (425 nm) under fluorescence showing good quality; (B) counterstained with PI labeling (538 nm); (C) in situ hybridization assay showing negative signals to US9 gene of BoHV-5 under fluorescence (512 nm). Scale bars 100 ␮m.

uninfected embryos (Fig. 4D) and minimal in infected embryos (Fig. 4C). In addition, similar results were observed for annexin-V label, whereas uninfected embryos had more intense label which was different (0.79 ⫾ 0.04; P ⬍ 0.05) between them and infected embryos (Fig. 4A and B). 4. Discussion Bovine Herpesvirus type 1 has been associated with reproductive failure, including abortion and temporary infertility due to postular vulvovaginitis and balanoposthitis. Moreover, BoHV-1 could also cause infertility by directly affecting fertilization [15]. Although BoHV-1 and 5 are considered closely related, the first case of a

reproductive disorder caused by BoHV-5 was only reported recently [2]; natural infection was determined to be transmitted by frozen semen containing BoHV-5 [2]. In the present study, experimental infection of bovine gametes and embryos with BoHV-5 seemed to not interfere with in vitro production of embryos. These results were consistent with our previous results, when no effects of BoHV-5 exposure were detected on development of in vitro produced bovine embryos [3]. These achievements were in contrast to the hypothesis that BoHV-1 could directly affect the fertilization process, supported by the observation of a strong decrease in embryonic development rate when bovine IVF was performed in the presence of BoHV-1 [15]. Therefore, the potential for BoHV-5 to induce in cattle reproduc-

Fig. 2. Day 7 bovine blastocysts in vitro cultured derived from oocytes exposed to BoHV-5: (A) DAPI labeling under fluorescence (425 nm) showing good quality; (B) counterstained with PI labeling (538 nm); (C) in situ hybridization assay showing positive signals to US9 gene of BoHV-5 under fluorescence (FITC-512 nm). Scale bars 100 ␮m.

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

1301

Fig. 3. Characteristic of MDBK monolayers and electrophoresis agarose gel of US9 PCR amplified products: (A) Uninfected cells; (B) Cytopathic effect observed after two consecutive passages on MDBK cells; (C) MW-molecular weight 1 Kb plus; lanes 1 and 2 represent PCR results from unexposed oocytes and bovine semen; lane 3 positive PCR amplification of 7 d embryos derived from exposed oocytes (250 bp); lane 4 negative PCR amplification of embryos derived from unexposed oocytes.

tive pathology must be not underestimated, and there should be a review regarding appropriate sanitary measures required for international marketing of biological products. The successful replication of a virus within a cell requires a remarkable cascade of interactions between virus and host. As part of their arsenal, many viruses have the ability to modulate the apoptotic pathways of the host, through manipulation of a variety of key apoptotic proteins [5]. In this study, infected embryos had minimal TUNEL labeling, indicating that BoHV-5 replication did not interfere with host DNA integrity. A landmark of cellular self-destruction in by apoptosis is activation of nucleases that eventually degrade the host nuclear DNA into fragments of approximately 200-bp in length, caused by either physiological embryo development and/or by virus infection [14]. In addition to host DNA fragmentation, apoptosis is an active physiological process involving chromatin condensation, reduction of cell volume, and formation of membrane vesicles called apoptotic bodies; this results in the fragmentation and elimination of unnecessary, damaged and/or dangerous individual cells [6], Due to the extreme complexity of the apoptotic process, a single method cannot be used to reliably determine the levels of apoptosis in cells. It is possible to detect early and later apoptosis through the annexin-V assay, which follows PS exposure among the BoHV-5 infected embryos. In the

present study, annexin-V was minimally present in infected embryos, but was more intense in uninfected embryos. However, bovine embryos derived from unexposed oocytes to BoHV-5 revealed mild to moderate intensity of the annexin-V label. This was a very early phenomenon during apoptosis, preceding nuclear condensation and breakdown of the intracellular cytoskeleton and nuclear matrix constituents of bovine embryos [16]. In addition, blastomere apoptosis was low in in vitro produced equine, porcine and bovine blastocysts. Despite the higher level of apoptosis in in vitro produced bovine embryos than their in vivo counterparts, the present study revealed a minor level of embryo apoptosis when oocytes were exposed to BoHV-5 [16]. Persistent infection is a hallmark of the Herpesviridae family [17]. It is noteworthy that BoHV-5 can be reactivated from this persistent status and excreted without clinical signs, consistent with the low incidence of apoptotic embryos derived from oocytes exposed to virus infection in the present study. Moreover, cattle are considered natural host of BoHV-5 and latently infected animals constitute natural reservoirs of virus that could potentially contaminate susceptible animals after reactivation [17]. In conclusion, biological products derived from latently infected sources represent a potential source of infection for in vitro procedures. In summary, the present study confirmed that BoHV-5 infected bovine oocytes. Furthermore, we concluded that experimental infection may not interfere with via-

1302

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303

Fig. 4. A representative picture showing total numbers of cells examined under fluorescence (FITC, 512 nm) for annexin-V and TUNEL assays: (A) Day 7 bovine blastocysts derived from exposed oocytes to BoHV-5 infection showing negative (⫺) to minimal (⫾) intensity of signals to annexin-V and (C) for TUNEL assay; (B) Day 7 blastocysts derived from unexposed oocytes to BoHV-5 infection showing mild (⫹⫹) to moderate (⫹⫹⫹) intensity of signals to annexin-V and (D) for TUNEL assay. Scale bars 100 ␮m.

bility, in order to facilitate viral replication. However, multiple mechanisms are involved in inhibition of apoptotic responses in embryos and most of them remain unclear. Acknowledgements This work was supported by FAPESP (Grants, 2007/ 57774-7; 2008/50382-2). The authors are grateful to Brasfrigo for ovary donation. Funding for TC Cardoso (310485/2009-6) was provided by CNPq (Brazilian Council for Research). References [1] Warthall AE, Simmons HA, Van Soom A. Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilization with virus-infected semen. Theriogenology 2006;65:247–74. [2] Kirkland PD, Poynting AJ, Gu X, Davis RJ. Infertility and venereal disease in cattle inseminated with semen containing bovine Herpesvirus type 5. Vet Rec 2009;165:111–3. [3] Silva-Frade C, Martins Júnior A, Borsanelli AC, Cardoso TC. Effects of bovine Herpesvirus type 5 on development of in vitro produced bovine embryos. Theriogenology 2010;73: 324 –31.

[4] Hay S, Kannourakis G. A time to kill: viral manipulation of the cell death program. J Gen Virol 2002;83:1547– 64. [5] Thomson BJ. Viruses and apoptosis. Int J Exp Pathol 2001;82: 65–76. [6] Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 1993;14:133–51. [7] Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501. [8] Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993;207: 202–5. [9] Oberhammer FA, Hochegger K, Froschl G, Tiefenbacher R, Pavelka M. Chromatin condensation during apoptosis is accompanied by degradation of lamin A ⫹ B, without enhanced activation of cdc2 kinase. J Cell Biol 1994;126: 827–37. [10] Martin SJ, Reutelingsperger CP, Mcgahon AJ, Rader JA, Van Schie RC, Laface DM, Green DR. Early redistribution of plasma membrane phosphatidylserine is general features of apoptosis regard less of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995;182: 1545–56. [11] Robertson A, Nelson RE. Certification and identification of the embryo. In: Stringfellow DA, Seidel SM (eds.), Manual of the

C. Silva-Frade et al. / Theriogenology 74 (2010) 1296 –1303 International Embryo Transfer Society, IETS, Savoy, IL 1998;3:103–16. [12] Ferrari HF, Luvizotto MCR, Rahal P, Cardoso TC. Detection of bovine Hespesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue based diagnostic test of bovine encephalitis. J Virol Meth 2007; 143:335– 40. [13] Cardoso TC, Gomes DE, Ferrari HF, Silva-Frade C, Rosa ACG, Andrade AL, Luvizotto MCR. A novel in situ polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues. J Virol Meth 2010;163:509 –12. [14] Antunes G, Chaveiro A, Santos P, Marques A, Jin HS, FM. Silva Influence of apoptosis in bovine embryo’s develop-

1303

ment. Reprod Dom Anim 2010;45:26 –32. [Epub 2008 Nov 28]. [15] Tanghe S, Vanroose G, Van Soom A, Duchateau L, Ysebaert MT, Kerkhofs P, Thiry E, van Drunen Little-van den Hurk S, Van Oostveldt P, Nauwynck H. Inhibition of bovine spermzona binding by bovine herpesvirus-1. Reprod Res 2005;130: 251–9. [16] Pomar FJ, Teerds KJ, Kidson A, Colenbrander B, Tharasanit T, Aguilar B, Roelen BA. Differences in the incidence between in vivo and in vitro produced blastocysts of farm animal species: a comparative study. Theriogenology 2005; 63:2254 – 68. [17] Paroli M, Schiafella E, Di Rosa F, Barnaba V. Persisting viruses and autoimmunity. J Neuroimmunol 2000;107:201– 4.