Sodium bicarbonate

After the first BCB test, the BCB⁺ COCs were subjected to IVM. The COCs were cultured in Nunclon™Δ 4-well dishes (Thermo Fisher Scientific, Waltham, MA, USA) in 500 μL standard porcine IVM culture medium: TCM-199 (tissue culture medium) with Earle’s salts and l-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA), supplemented with 2.2 mg/mL sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan), 0.1 mg/mL sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL BSA (Bovine Serum Albumin) (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mg/mL cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10% (v/v) filtered porcine follicular fluid, and gonadotropin supplements at final concentrations of 2.5 IU/mL hCG (human Chorionic Gonadotropin) (Ayerst Laboratories, Inc., Philadelphia, PA, USA) and 2.5 IU/mL eCG (equine Chorionic Gonadotropin) (Intervet, Whitby, ON, Canada). Wells were covered with mineral oil overlay and cultured at 38 °C under 5% CO₂ in air for 22 h, and then for additional 22 h in medium without hormones. After cultivation, the second BCB staining test was performed, and BCB⁺ oocytes were used for further molecular analyses.

The IVM was conducted using oocytes that deemed positive in the first BCB test. Nunclon™Δ 4-well dishes (Thermo Fisher Scientific, Waltham, MA, USA) were used for the oocyte maturation in 500 μL standard porcine IVM culture medium: TCM-199 (tissue culture medium) containing Earle’s salts and l-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA), supplemented with 2.2 mg/mL sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan), 0.1 mg/mL sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL BSA (Bovine Serum Albumin) (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mg/mL cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10% (v/v) filtered porcine follicular fluid, and gonadotropin supplements of 2.5 IU/mL hCG (human Chorionic Gonadotropin) (Ayerst Laboratories, Inc., Philadelphia, PA, USA) and 2.5 IU/mL eCG (equine Chorionic Gonadotropin) (Intervet, Whitby, ON, Canada) final concentrations. For the first 22 h of maturation, the well plates were overlaid with mineral oil and placed in 38 °C, 5% CO₂. Then, the medium was changed for one without hormone supplementation, with the maturation allowed to proceed in the same conditions for another 22 h. Second BCB test was performed following the maturation, with BCB⁺ oocytes used for subsequent analyses.

Dibasic sodium phosphate, sodium dihydrogen phosphate, D,L-glyceraldehyde, human recombinant aldose reductase (HR-AR), AG, quercetin, critric acid monohydrate, natrium carbonicum, sodium azide, gelatin and sulphuric acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sodium bicarbonate, sodium chloride, potassium dihydrogen phosphate and ethanol were supplied by Nacalai Inc. (Kyoto, Japan). Tween 20, bovine serum albumin, glucose, O-phenylenediamine dihydrochloride, phosphate buffered saline, fetal bovine serum (FBS), steroyl myristoyl phosphatidylcholine glycine (SMPC Gly), 3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromidetetrazolium salt (MTT) were purchased from Sigma-Aldrich company, Ltd. (St. Louis, MO, U.S.A.). NADPH was provided by Oriental Yeast Co., Ltd. (Tokyo, Japan). Anti-AGE antibody and goat anti-mouse IgG, HRP conjugate-secondary antibody Millipore (Merck U.S.A) were purchased from Transgenic Inc. (Hyogo, Japan). Silica gel 60 F₂₅₄ TLC plates (Merck, U.S.A), silica gel 60 N (100–200 μm) and ODS were purchased from Kanto Chemical (Tokyo, Japan). DMSO was purchased from Wako Pure Chemical Industries, Ltd (Japan). Kaempferol-3-O-β-D-glucopyranoside was isolated from liquorice, and was identified by NMR and MS data [11 (link)].

SK-L cells [25 (link)] were propagated with Eagle’s minimum essential medium (EMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with sodium bicarbonate (Nacalai Tesque), 0.295% tryptose phosphate broth (Becton, Dickinson and Company, CA, USA), 10 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (Sigma-Aldrich, St. Louis, MO, USA), and 10% horse serum (HS; Thermo Fisher Scientific, Waltham, MA, USA). This cell line was kindly provided from National Institute of Animal Health (Tsukuba, Ibaraki, Japan).
A recombinant clone of CSFV vALD-A76, derived from a virulent strain, ALD-A76, was used in this study. The vALD-A76 stain was previously established in our laboratory [26 (link)].

COCs were cultured in 4-well Nunclon™ plates in 500 μl of standard IVM porcine culture medium TCM-199 (tissue culture medium) with Earle salts and l-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA), supplemented with 0.1 mg/ml sodium pyruvate (SigmaAldrich, St. Louis, MO, USA), 2.2 mg/ml sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan) 10 mg/ml BSA, (SigmaAldrich, St. Louis, MO, USA), 0.1 mg/ml cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10% filtered porcine follicular fluid (v/v) and gonadotropin supplements at final concentrations of 2.5 IU/ml of eCG (Intervet, Whitby, ON, Canada) and 2.5 IU/ml of hCG (Ayerst Laboratories, Inc., Philadelphia, PA, USA). All wells were flooded with a layer of mineral oil and maintained for 44 h at 38 °C in a 5% CO₂ atmosphere. After maturation, for the maturity assessment the BCB staining test was repeated, with only BCB+ oocytes used for further study.

The BCB⁺ COCs were matured in Nunclon™Δ 4-well dishes covered with mineral oil in 500 μl standard porcine IVM culture medium, TCM-199 (tissue culture medium) with Earle's salts, and L-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA) supplemented with 0.1 mg/ml sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 2.2 mg/ml sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan), 0.1 mg/ml cysteine Sigma-Aldrich (St. Louis, MO, USA), 10 mg/ml BSA (bovine serum albumin) (Sigma-Aldrich, St. Louis, MO, USA), 10% (v/v) filtered porcine follicular fluid and gonadotropin supplements at final concentrations of 2.5 IU/ml hCG (Ayerst Laboratories, Inc., Philadelphia, PA, USA), and 2.5 IU/ml eCG (Intervet, Whitby, ON, Canada). The COCs were cultured at 38°C under 5% CO₂ in humidified atmosphere for 22 h and then for additional 22 h in medium without hormones. Thereafter, the BCB staining test was performed once again and only BCB⁺ oocytes were taken for the following analysis.

The swine kidney-derived cell line (SK-L cells) [26 (link)] was cultured in Eagle’s minimum essential medium (EMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.295% tryptose phosphate broth (TPB) (Becton Dickinson, Franklin Lakes, NJ, USA), 10 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) (Sigma-Aldrich, St. Louis, MO, USA), sodium bicarbonate (Nacalai Tesque, Kyoto, Japan), and 10% horse serum (HS; Thermo Fisher Scientific, Waltham, MA, USA). SK-L cells were used for viral production, titration, and serological tests. The cell line was incubated at 37 °C in 5% CO₂.
A recombinant clone of CSFV live attenuated vaccine, vGPE⁻, was derived from pGPE⁻ [22 (link)], and a plasmid containing its full-length cDNA was used. A CSFV vALD-A76 was also derived from the full-length cDNA clone of a virulent strain, ALD-A76, which was developed in a previous study [27 (link)].