D aspartic acid

l‐Arginine (C₆H₁₄N₄O, ≥ 98%), l‐glutamic acid (C₅H₉NO₄, ≥ 99%), l‐proline, (C₅H₉NO₂, 99%), l‐asparagine (C₄H₈N₂O₃, 99%), l‐glutamine (C₅H₁₀N₂O₂, ≥ 98%), l‐aspartic acid (C₄H₇NO₄, ≥98%), and l‐threonine (C₄H₉NO₃, ≥ 98%) were bought from Sigma–Aldrich; l‐histidine (C₆H₉N₃O₂, 99%), and l‐serine (C₃H₇NO₃, 99%) were purchased from Alfa Aesar. l‐Alanine (C₃H₇NO₂, 99%), l‐cysteine (C₃H₇NO₂S, 99%), and glycine (C₂H₅NO₂, 99%) were purchased from Acros Organics. d‐aspartic acid (99%) was purchased from Sigma–Aldrich. Silk fibroin was purchased from ChemSrc, collagen (calf skin) was purchased from Merck, and gamma globulin (bovine blood) was purchased from Sigma–Aldrich. Deionized (DI) water from a Milli‐Q ultrapure system and acetone from Echo Chemical Co. were used throughout the experiments. By using high‐purity deionized water (18.2 MΩ cm⁻¹), all aqueous solutions were prepared. Without any further modification, all the chemicals were used as received. The single‐side polished highly doped p‐type (1 0 0) Silicon wafer was purchased from Twice Jin Limited, Taiwan. The thickness of the Si wafer is 675 ± 25 µm and the resistivity is 1–10 ohm‐cm.

d-Alanine, d-Arginine, d-Asparticacid, d-Asparagine, d-Glutamicacid, d-Histidine, d-Isoleucine, d-Leucine, d-Lysine, d-Methionine, d-Phenylalanine, d-Proline, d-Serine, d-Threonine, d-Tryptophan, d-Tyrosine, d-Valine, KBr (spectral grade), bovine serum albumin (BSA), glutathione and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). d-Cystine and d-Glutarnine were purchased from Aladdin Reagent Co, Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade. All aqueous solutions were prepared with Milli-Q water.

A 20 mM solution of sodium D-Aspartate was prepared by diluting a 1M sodium D-Asp solution, composed of 13.3 g of D-Aspartic acid (Sigma Aldrich, cat #219096) neutralized with NaOH in 100 mL distilled water, at a final pH of 6.6–7.5 in drinking water. Young male mice were treated at 7 or 5 weeks of age for 2 or 4 weeks, respectively, so that they were 9 weeks old at the end of the study. Adult 14- or 12-week-old males were treated for the same duration so that all were 16 weeks old at the end of the study. In both experiments, drinking water without D-Asp was administered to control mice.
During the experiments, all males were housed singly. For each of the two age groups, 9 or 16 weeks, 3 males were used for timepoint 0 (basal values) and 6 males (3 controls and 3 D-Asp-treated) were used for each timepoint (2 or 4 weeks). Therefore, a total of 30 males were used for sperm cryopreservation (Figure 1).