Double distilled water

Rhamnolipid nano-micelles were prepared following our previously published protocol [14 (link)]. Briefly, an aqueous solution of rhamnolipids at a concentration of 10 mg/mL was sonicated in phosphate-buffered saline (PBS, 10 mM, pH 7.4) using a probe sonicator (Hielscher Ultrasonics, Berlin, Germany) to form rhamnolipid nano-micelles. Ethyl alcohol 70% (v/v) was prepared by mixing 70 mL of absolute ethyl alcohol with 30 mL of double-distilled water (Merck KGaA, Darmstadt, Germany).

Within the framework of this investigation, an aqueous solution of formaldehyde weighing 37% was harnessed, where methanol constitutes 11% of its weight, acting as a preventive factor, and manufactured by KBR (India). The procurement of resorcinol (98%) was accomplished through Sigma-Aldrich (Germany). The catalytic agent employed for the aerogel synthesis was sodium hydrogen carbonate (NaHCO₃), procured from DUKSAN (South Korea). Minocycline, an integral component, was acquired from Hakim Pharmaceutical Co. (Iran). Acetone with a purity level of 99% from Dr. Mojallali’s Inc. (Iran), sulfuric acid with a concentration of 97–98% from Merck (Germany), oxygenated water with a purity of 30% from Merck (Germany), methanol from Merck (Germany), hydrochloric acid (HCl) at a concentration of 0.1 M from Merck (Germany), double distilled water, and natural graphite in flake form exhibiting an average dimension of 300 μm and a density of 2.3 g/cm³ with a purity of 98% from Superior Graphite Co. (China), were also used in this research.

The reagents used in this research are of high-purity analytical grade. Reagents include silver nitrate (Merck; 99% pure), de-ionized water (obtained from the Phytomedicine Research Lab, University College of Conventional Medicine), double distilled water (Department of Pharmacy, The Islamia University of Bahawalpur), and ethanol (Merck; 99.9% pure). Glassware like beakers, conical flasks, measuring cylinders, burette, and pipette used was made up of Pyrex brand. A mercury thermometer (360°C) was also used to measure temperature.
Jack bean urease bought from Sigma-Aldrich (Sigma Co., St. Louis, United States) was used for the enzyme assay. Thiourea, alkali (0.1% w/v NaOCl and 0.5% w/v NaOH), and reagents (0.005% w/v sodium nitroprusside and 1% w/v phenol) were used as reagents and chemicals.

The semen of three black-headed goat bucks (3–5 years old) was used in this study. The black-headed goat is a new breed based on Macheng Black Goat (Chinese local breed) and introduces Boer Goat lineage. It has a black head and a white body. Prior to this study, goat bucks were ejaculated two times per week during the breeding seasons. In this study, one ejaculate from each goat buck was collected using an artificial vagina in the presence of estrus goats. The semen volume of each goat buck collected was from 0.5~1.5 mL every time. The semen used was milky white and had no abnormal smell. The sperm concentration was >2 × 10⁹ sperm/mL, and the total motility was >0.8. The semen was pooled to minimize individual differences between goat bucks.
Tris-based solution composed of Tris 250 mmol/L, fructose 100 mmol/L, sodium citrate 75 mmol/L, penicillin 50,000 IU, streptomycin 50,000 IU, and double-distilled water 100 mL (chemicals all from Sigma, St. Louis, MO, USA) was used as the base extender. The mixed semen was diluted 10-fold with diluent and divided equally into five aliquots. FA was added to the base extender at concentrations of 25, 50, 100, and 200 μmol/L, while the control was the base extender without FA. All samples were stored in a constant temperature refrigerator at 17 °C (shaken and turned over every 12 h) and used for the experiment.

In July 2021, leaves were harvested from Olea europaea plants growing in the Ibbin area of Ajloun Governorate of Jordan from H Al-momani own farm. To confirm the leaves’ identity, they were compared against the verified sample held in the herbarium of the Faculty of Agriculture at the Jordan Uiversity of Sceince and Technology. Institutional, national, and international guidelines and legislation were observed during the leaf harvesting process.
To prepare the leaves for processing, they were washed in double-distilled water (Sigma-Aldrich), then left at room temperature to dry completely. Once dry, the leaves were placed into an electric grinder and ground to yield a coarse powder. A mixture was made using 5 g of powder and 50 mL of double-distilled water. To obtain the extract, the mixture was heated in a 70° C water bath 15 min. The mixture was then filtered and the recovered extract was stored at − 4° C until required.

Cetyltrimethylammonium bromide (CTAB ≥ 98%), ammonium hydroxide solution (NH₄OH), double distilled water (ddH₂O), tetraethyl orthosilicate (TEOS), calcium nitrate tetrahydrate (Ca(NO₃)₂ ∙ 4H₂O, 99%), copper chloride (CuCl₂, 99%), Pluronic P123 (EO₂₀PO₇₀EO₂₀, M_n ∼5800 Da), ibuprofen (>98% GC), Trizma^® base, primary standard and buffer, ≥99.9% (titration) were purchased from Sigma Aldrich, Milan, Italy and used as received. All solvents were purchased from Sigma Aldrich (Milan, Italy) in analytical grade.