Arsenazo 3

Control and delor-treated Leydig cells were lysed in cold RIPA buffer (Thermo Scientific) and then sonicated for 60 s on ice and centrifuged at 10,000 g for 15 min. Ca2+ was estimated in the supernatants using Arsenazo III (Sigma–Aldrich) according to the modified method of Michaylo and Ilkova (1971 (link)). The intensity of the purple complex formed with the reagent was read at 600 nm in a spectrophotometer (Labtech LT-4000MS; Labtech International, Uckfield, UK) with Manta PC analysis software. The proteins were estimated by modified Lowry’s method (Lowry et al. 1951 (link)). Concentrations of Ca2+level in samples of Leydig cells after treatment with d103 and d106 in various doses and combinations were compared with the control, which was arbitrarily set at 1. The Ca2+ levels were calculated as μg/ml.

Six disk-shaped samples (1 mm thickness and 7 mm diameter) from each material (n = 6) were prepared using silicone molds, and individually light-cured for 40 s. The samples were stored in 2 mL distilled water, and storage solutions were exchanged after 28 and 45 days with equal volume replacement. The calcium release was determined by mixing the storage solutions with Arsenazo III in 20 mM HEPES at pH 7.4 (Sigma Aldrich, St. Louis, USA). The analysis of calcium release through this solution was performed using a UV-Visible spectrophotometer (Powerwave 340; Biotek, St. Paul, USA) with 656 nm wavelength for 3 s adopting the Arsenazo III colorimetric method [21 (link)]. Aliquots of 5 μL of the samples (diluted 1 : 10 and partially neutralized) were added to 50 μL of deionized water before UV-Vis analysis. For calibration, standards containing 40 to 200 μg Ca/mL solutions (Sigma Aldrich) were used.

All the chemicals used were analytic grade. Sucrose (S0389), CaCl₂ (C5080), succinic acid (S7501), L‐glutamic acid (G12519), malic acid (M0625), EGTA (E0396), ADP (01905), safranine‐O (S2255), and Arsenazo III (A92775) were from Sigma Chem Co (MO, USA). Bovine serum albumin was from GoldBio (E217100, MO, USA). H₃PO₄, KCl and MgCl₂ were from J.T. Baker Chemical Co. (NJ., USA), Ruthenium red was from Merck KGaA (R2751, Darmstadt, Germany). Cyclosporin A was from Sandimmum (Sandoz, Basilea, Swiss). Calcium Green‐5N was from Thermo Fisher Scientific (C3737 MA, USA).

Control or treated sperm suspensions (20 μL; final concentration 2 × 10⁶ cells/mL) were centrifuged at 150 g for 15 min and lysed in RIPA buffer at 4 °C for 30 min, followed by sonication for 60 s on ice. The lysate was centrifuged at 10,000 g for 15 min. The concentration of Ca²⁺ was estimated in the supernatants using Arsenazo III (Sigma–Aldrich, Saint Quentin Fallavier, France) according to the modified method by [31 (link)]. The intensity of the purple complex formed with the reagent was read at 600 nm in a spectrophotometer (Labtech LT-4000MS; Labtech International Ltd., Uckfield, UK) with Manta PC analysis software. The protein concentration was estimated in the pellet by modified Lowry’s method [32 (link)]. The Ca²⁺ levels were calculated as μg/mL. The experiment was performed at 2 times (5 and 20 min) after different treatments (chemerin 50 ng/mL, chemerin 150 ng/mL, chemerin 500 ng/mL, chicken CMKLR1 Ab (10 mg/mL) and chicken CMKLR1 Ab (10 mg/mL) + chemerin 500 ng/mL). It was repeated 6 times (about 2 times per week) using different batches of adult chicken semen.

A Pierce BCA protein assay kit (Thermo Fisher) was used following the manufacturerʼs instructions. Calcium quantification was performed on HCl-extracted samples using Arsenazo III reagent^{27 (link)}. Briefly, samples were completely dissolved by boiling with reflux in 1 M HCl, then neutralized to pH 7.0 by addition of 1 M NaOH. Neutralized samples were assayed by addition of one volume of 100 μM Arsenazo III (Sigma) and spectrophotometric reading at 595 nm. GAG was assayed on matrix extracted from 152 cm² monolayers using a commercially available kit (Blyscan, Biocolor Life Science Assays, County Antrim, UK).