D glucose

D-pinitol (purity: 95%, Lot No: 441252) and streptozotocin (STZ) were from Sigma Chemicals Co. (St. Louis, MO, USA). Paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), trypsin/EDTA solution, D-glucose, and 3-(4,5-dimethylthiazol)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NE, USA). Hoechst 33258 and PI were purchased from Beyotime (Shanghai, China). AGE-BSA was purchased from Abcam (Cambridge, UK). ELISA kits and necrosulfonamide (NSA) were purchased from R&D systems (Minneapolis, MN, USA). Annexin V/PI Kit was from Key Gen (Nanjing, China). The primers were synthesized by Sangon Biotechnology (Shanghai, China). The MFG-E8 antibody was purchased from MBL (Woburn, MA, USA) and R&D (Minneapolis, MN, USA), and antibodies RIPK1, MLKL, phospho-MLKL, and β-action were purchased from Abcam (Cambridge, UK). RIPK3 and HMGB1 antibodies were purchased from Proteintech (Wuhan, China). All other reagents are standard commercial high purity materials.

P407 and d-glucose were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). TRNzol Universal total RNA extraction reagent and RNase/DNase-free ddH₂O were purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing, China). TB Green Premix Ex Taq II was purchased from Takara Biomedical Technology Co., Ltd. and Trans DNA Marker II and DNase I were purchased from TransGen Biotech (Beijing, China). The Hematoxylin and Eosin (H&E) staining solution was purchased from Nanjing Jiancheng Bioengineering Institute. The Oil Red O staining solution was purchased from Servicebio Co., Ltd. (Wuhan, China).

1.0 mg/ml D-glucose (Solarbio, Beijing) stock solution was pipetted in deionized water at final concentrations of 25, 30, 35, 40, 45, 50, 55, and 60 μg/ml in 2 ml total volume and mixed with 2 ml of 5% phenol solution (v/v) and 10 ml of concentrated sulfuric acid (Shidande, Shanghai, P.R. China) separately. The mixture was placed in a water bath at 80°C and kept for 30 min. It was then cooled to room temperature, and the A values were measured at 486 nm using a spectrophotometer (Persee, Beijing). The standard curve of total sugar was obtained using the A value as the ordinate and the concentration as the abscissa.

7H9 liquid medium powder, isoniazid, deoxycholate and iodoacetamide (IAA) were purchased from Sigma (St Louis, MO). Middle Brook 7H10 Agar was purchased from Hopebio (Qingdao, China). Albumin, D-glucose, dithiothreitol (DTT), BCA protein assay kit were purchased from Solarbio (Beijing, China). Glycerol and CuSO₄ was from Xilong (Shantou, China). Sequencing grade trypsin was purchase from Promega (Fitchburg, WI). The Total RNA Isolation System and Reverse Transcription kits were purchased from TIANGEN (Beijing, China). SYBR mixture was purchased from Genstar (Beijing, China).

All chemicals and reagents were purchased from Sigma-Aldrich, China, unless specified otherwise.
The Modena solution was prepared in the laboratory which composed of 153 mM D-glucose, 26.7 mM trisodium citrate, 11.9 mM sodium hydrogen carbonate, 15.1 mM citric acid, 6.3 mM EDTA-2Na, 46.6 mM Tris, 1000 IU/mL penicillin G sodium salt (Solarbio, Beijing, China), 100 μg/mL polymyxin B, and 1 mg/mL streptomycin sesquisulfate (Solarbio, Beijing, China). Modena solution was used as the extender for liquid semen or thawing solution. The freezing extender (NSF) as described by Okazaki et al. [29 (link)] was used in this study with some modifications (mNSF). The first modification was done by adjusting the osmolarity to 400 mOsm/kg at a final concentration (mNSF1). The mNSF1 was further modified as mNSF2 by adding 1.5% (v/v, final concentrations: 0.75%) Orvus Es Paste (Miyazaki Chemical Sales Ltd., Tokyo, Japan) and 4% (v/v, final concentrations: 2%) glycerol.
In Experiment I, resveratrol was added to the mNSF1 at concentrations of 0, 25, 50, 75, 100, or 125 μM to determine the optimum concentration, which was used to the mNSF1, mNSF2, or Modena solution in the subsequent Experiments II and III.