Dotriacontane

Eicosane, docosane, dotriacontane, tritetracontane, heptacosane, and tetracosane were purchased from Sigma, Missouri, USA. Trypan blue solution was purchased from Glibco (Waltham, MA, USA). Cell cytotoxicity assay kit was purchased from Dojindo Molecular Technologies, INC (CK04: Cell Counting Kit-8, Tokyo, Japan). Antibodies used in the immunoblotting study includes Anti-RSV-G (Abcam, #ab94966, Cambridge, UK), β-actin (Santa Cruz, SC 4777, Dallas, TX, USA), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell signaling technology, 7074P2, Danvers, MA, USA), HRP-conjugated anti-mouse IgG (Gene Tex, GTX213111-01, Taichung, Taiwan).

The concentration of cholesterol and FA profile were determined in fat extracted from the yolk, according to the method proposed by Folch et al. [44 (link)]. Cholesterol was separated from fat after saponification with potassium hyroxide KOH and extraction with ethyl ether, by the modified method of the International Dairy Federation [45 ]. The samples were analyzed on a PU4600 chromatograph (Unicam, Cambridge, UK) with a flame ionization detector (FID), under the following conditions: glass column length—1 m, inner diameter—4 mm, film thickness—0.25 m, temperature: detector—300 °C, injector—290 °C, column—260 °C, carrier gas—argon, flow rate—50 cm³/min, and internal standard—dotriacontane (Sigma, St. Louis, MO, USA). The extracted fat was esterified with a chloroform, methanol, and sulfuric acid mixture, as described by Peisker [46 (link)]. The resulting FA methyl esters (FAMEs) were analyzed on a 7890A gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) with a FID and a Supelcowax 10 capillary column (column length—30 m, internal diameter—0.32 mm, film thickness—0.25 m, carrier gas—helium, and temperature: detector—250 °C, injector—230 °C, and column—195 °C). FA peaks were identified by comparing their relative retention times with those of individual FAME reference standards (Supelco) diluted in hexane (1:1, 1:2, 1:3, and 1:4 v/v).

Wax profiling was carried out as described previously [38 (link)]. Briefly, fresh or frozen samples (100 mg) were immersed in chloroform for 30 s to extract the wax, and 20–50 µL (10 mg/50 mL) of n-Tetracosane (Sigma) was added as an internal standard. The extracted solution was dried with nitrogen and derived with 20 μL pyridine and 20 μL N,O-bis(trimethylsilyl) fluoroacetamide (BSTFA) (40 min at 70 °C). The derivate samples were then analyzed using gas chromatography–mass spectrometry (GC-MS).
For cutin analysis, samples were immersed in chloroform and methanol (1:1, v/v) in a glass vial for 2 weeks to remove phospholipids. Samples were then air-dried and reacted with methanol/HCl at 80 °C for 2 h. Saturated NaCl was used to end the reaction. Cutin was extracted with hexane, the extracted solution was dried with nitrogen, and the samples were derived with 20 μL pyridine and 20 μL BSTFA (40 min at 70 °C). Then, 20–50 μL (10 mg/50 mL) of dotriacontane (Sigma) was added to each sample as an internal standard. The derivate samples were then analyzed using GC-MS.