Reagents and media
Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Invitrogen (Grand island, NY, USA) at the highest available purity. The basic dilutions and culture media were cell culture grade, which are routinely used in our laboratory.
Oocyte collection, in vitro fertilization and maturation
Oocyte collection, in vitro maturation (IVM), sperm and in vitro fertilization procedures were followed as previously described [11, 12]. The study was conducted with presumptive zygotes produced from oocytes exposed or unexposed (n = 30/per slide; in five repetitions) 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 x g for 20 min. The resulting sperm pellet was washed in TALP medium (Tyrode medium with added bicarbonate buffer and supplemented with 6 mg BSA per milliliter) and centrifuged at 200 X g for 5 min. The pellet was diluted in IVF medium (TALP medium supplemented with 3 mg/mL heparin and PHE solution: 2 mM penicillamine, 1 mM hypotaurine, and 250 mM epinephrine) to a final concentration of 1 x 105 sperm/mL in drops of 100 μL. After 24 h of maturation, the 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. Afterward, 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 the IETS guidelines  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 by the use of molecular search .
Virus infection and embryo development
Stocks of BoHV-5, isolated in 2007 from outbreaks in Araçatuba, SP, Brazil , were propagated in Madin-Darby bovine kidney (MDBK, ATCC CCL-2) cells, which were cultured in minimum essential medium (MEM) [25, 26]. The tissue culture infective dose per 50 μL (TCID50) of stock virus was determined by virus titration infection of confluent monolayers of MDBK cells [25, 26] at a multiplicity of infection (MOI) of 1. Aliquots of stock virus (100 μL) with 103.3 TCID50/50 μL 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 30 oocytes each for experimental use. The culture consisted of oocytes maintained in 100 μL TCM-199 (GIBCO-BRL, Grand Island, NY, USA), supplemented with 10% FBS, 2.2 mg/mL sodium bicarbonate, 0.02 mg/mL sodium pyruvate, 0.05 mg/mL gentamicin sulfate, 0.5 μg/mL FSH (Pluset, Calier, Barcelona, Spain), and 50 μg/mL LH (Lutropin-V, Bioniche Inc., Belleville, ON, Canada) for 24 h at 39°C in 5% CO2-air. Selected oocytes (n = 30) were washed in maturation medium consisted of 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 μg/mL FSH (Pluset®, Calier, Barcelona, Spain) and 50 μg/mL LH (Lutropin-V®, Bioniche Inc., Belleville, ON, Canada). Oocytes were transferred to drops containing 100 μL of MM in five repetitions in a total number of 150 oocytes. Oocytes were experimentally exposed by co-incubation with 10 μL 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 [11, 12].
BoHV-5 molecular detection
The viral DNA detection was applied to detect the US9 gene among exposed and unexposed derived-embryos, follicular liquid, oocytes and sperm, as described previously . A 250-bp amplicon was generated based on the sequence of US9 region (GenBank accession number AY064172). Procedures for DNA purification and PCR were previously described . The PCR was performed using 12.5 μL of 2× Platinum Taq Polymerase High-Fidelity Master Mix (Invitrogen™TM, 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 by Invitrogen. In addition, 2.5 μL of nuclease-free water and 8 μL of the DNA sample (positive reference strain AY064172) consisting of 3 μL of DMSO plus 5 μL 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 visualized on a 1.5% (w/v) agarose gel after staining with SYBR green at 0.5 μg/mL of concentration.
Embryos derived from oocytes directly exposed to BoHV-5 and observed during in vitro production were collected and freeze-thawed (1 h under −86°C and 1 h under room temperature) three times. Monolayer cultures of MDBK cells at 80% confluence were prepared, according to standard procedures, to be free of BoHV-1 and any other potential pathogens . Adsorption was allowed for 90 min at 38.5°C. Then, fresh medium was added, and for the next 7 dayays, cultures were examined for cytopathic effect (CPE). After an additional passage, the 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 plates previously seeded with MDBK cells. Serial dilutions, from 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 μL of supplemented MEM. The 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 .
MitoTracker green FM, jc-1 and Hoechst 33342 staining
To evaluate the number of embryos/slide, intensively labeled with MitoTracker Green FM, Jc-1 and Hoechst 33342 probes, 30 selected embryos, in five repetitions, from oocytes exposed or unexposed to BoHV-5 were washed in PBS and then fixed with 4% (w/v) formaldehyde. This procedure was similar for all dyes used to assay exposed and unexposed embryos. MitoTracker Green FM (Invitrogen, Eugene, OR, USA) was used to evaluate mitochondrial distribution and segregation patterns. The MitoTracker Green FM was diluted in DMSO at 10 nM per slide direct applied on fixed embryos (30 per slide, in a total of 150 analyzed) and incubated for 10 min at 38.5°C. The mitochondrial distribution in the cytoplasm of embryos appeared as increased areas of fluorescence intensity or aggregates detected by fluorescence. Mitochondrial activity was qualified based on Jc-1 (5,5´, 6,6´-tetrachloro-1,1´, 3,3´-tetraethyl-benzimidazoyl-carbocyanine iodide) staining. Jc-1 monomers were detected with a green filter. Jc-1 dimers that formed on mitochondrial membranes with high potential were detected via a red filter. Mitochondria distribution in the embryonic cytoplasm was evaluated by the intensity of the green/red fluorescence. To observe the embryo quality, 1 μg/mL per slide of Hoechst 33342 at 38.5°C for 30 min was used. The fixed embryos were washed again to remove excessive Hoechst 33342 and then mounted onto slides under coverslips to evaluate the nuclear configuration. To measure the fluorescence intensity (MitoTracker Green FM emission 500 nm, Jc-1 red filter 515 nm and green filter 488 nm, and Hoechst 33342, emission 488 nm, stained slides were observed under an AxioImager A.1 light and ultraviolet microscope connected to an AxioCam MRc camera (Carl Zeiss, Oberkochen, Germany), and micrographs were processed with AxioVision 4.8 software (Carl Zeiss).
Indirect immunofluorescence to assay SOD1, AOP-1, Hsp70.1 and BoHV-5 antigens
Exposed and unexposed embryos (n = 30/slide; in five repetitions) were washed three times in PBS and fixed in 4% formaldehyde for 24 h at 4°C. The samples were then rinsed with PBS and permeabilized with proteinase K (10 μg/mL, Invitrogen) for 15 min at room temperature. After pre-treatment with proteinase K (10 μg/mL) at 4°C, the slides were incubated overnight with primary antibodies against mitochondrial superoxide dismutase, anti-oxidative protein 1 and stress response heat shock protein 7 (mouse anti-SOD1; anti-AOP-1 and anti-Hsp70.1, respectively) diluted 1:50 in antibody diluent (PBS plus 0.1% of Tween 20). The viral antigens were detected by reacting exposed and unexposed embryos to monoclonal anti-BoHV-5 diluted in PBS plus 0.1% Tween 80 at 1:5 . 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 1 mg/mL of DAPI (4`-6-diamino-2-phenylindole; Sigma-Aldrich®) for 15 min at room temperature before mounting the slides in the dark .
Determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1)
The abundance of transcripts for genes related to mitochondrial activity antioxidant protection, manganese-superoxide dismutase (SOD1) sense 5´-CCCATGAAGCCTTTCTAATCCTG-3´ and antisense 5´-TTCAGAGGCGCTACTATTTCCTTC-3´ primers (accession no. L22092.1) and antioxidant protein like 1 (AOP-1) sense 5´-CCTAGGTTATTTAGCGCGT-3´ and antisense 5´-TTTCCGCTAGCGCTTATT-3´primers were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR), and these primers generated amplicons of 297 and 310 bp, respectively. The stress response gene transcription was performed by amplification of heat shock protein 70.1 (HSP 70.1 accession no. U09861) sense 5´-AAGGTGCTGGAGTAGGCT-3´ and antisense 5´-ACTTGGAAGTAAACAGAAGC-3´primers producing an amplicon of 312 bp. Total RNA was isolated from 7 pools of 10 exposed and unexposed embryos using the PureLink® viral RNA/DNA extraction kit, according to the manufacturer’s protocol (Invitrogen). The total RNA was eluted in 20 μL of ultra-pure water and treated with 0.5 IU DNAse. The reverse transcriptase reaction (RT) was immediately performed using 0.5 μg oligo (dT) primers (Invitrogen). The reaction mix consisted of 200 μM of each dNTP, 1 x RT buffer, 2 μL DTT 0.1 M, 40 IU RNase inhibitor and 200 IU SuperScript II (Invitrogen). The RT reaction was performed at 42°C for 52 min, with a final incubation at 70°C for 15 min. Polymerase chain reaction was conducted, as previously described in the Materials and Methods section. The PCR products were visualized on a 1.5% (w/v) agarose gel after staining with SYBR green (Invitrogen) at 0.5 μg/mL of concentration.
Semi quantitation and data analysis
The levels of SOD1, AOP-1, Hsp70.1 and viral antigens were semi-quantitated according to the intensity of the immunofluorescence reactions. Two standard filters were employed: a DAPI filter (emission wavelength: 425 nm) was used to determine quality and a fluorescein isothiocyanate (FITC) filter (emission wavelength, 512 nm) was used to detect SOD1, AOP-1, Hsp70.1 and BoHV-5 antigens. Bovine embryos were examined on two separate occasions by two observers without prior knowledge of the classification. The intensity of labeling was graded and only embryos considered marked lebeled were included in a semi-quantitative analysis. Differences in the respective mean values (n = 30 in five repetitions) were tested using ANOVA, with the primary effects as morphological quality groups, followed by a multiple pair-wise comparison using Student’s t-test for independent samples and the 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 an AxioCam MRc (Carl Zeiss, Oberkochen, Germany), and the micrographs were processed using the Axiovision 4.7 software (Carl Zeiss). The results are expressed as the mean +/− S.E.M. p values < 0.05 were considered significant.