*4.2. Biochemical Analysis*

The ovary organic matter content of *R. pulmo* was evaluated using approximately 10 mg (±0.01 mg) of dry tissue reduced to ash for 4 h at 500 ◦C in a muffle furnace (BICASA B.E. 34). This content was expressed as the percentage of organic matter of total tissue dry weight (DW)). The weight of organic matter (OM) was determined as the difference between the gonad DW and the ash weight [69]. Female gonads biochemical composition was performed in order to detect contents in protein, carbohydrate, and total lipid (*n* = 15). Ovary tissue was frozen in liquid nitrogen, temporarily stored at −20 ◦C, and briefly transferred one hour before lyophylisation to −80 ◦C to facilitate freeze-drying (48 h) for the biochemical and NMR analyses.

Carbohydrate, protein, and lipid quantification was performed by colorimetric determination at 480 nm, 750 nm, and 520 nm, respectively. In order to calculate the carbohydrate content in the ovarian tissue, approximately 10 mg (±0.1 mg) of each lyophilized sample was homogenized in 3 mL of double distilled water [70] with glucose as a standard. The content of proteins was estimated by employing approximately 10 mg (±0.1 mg) of each lyophilized tissue sample homogenized in 2 mL of 1N NaOH [71] with albumin as a standard. Finally, total lipids were determined by homogenizing approximately 10 mg (±0.1 mg) of each lyophilized tissue sample in 3 mL of chloroform–methanol (2:1) with cholesterol as a standard [72]. Quantities were expressed as μg mg<sup>−</sup><sup>1</sup> of OM.

Cholesterol content was evaluated by homogenizing approximately 150 mg (±0.1 mg) of each lyophilized sample in 4 mL of distilled water and was calculated by the colorimetric enzymatic method using the commercial kit (10028 Cholesterol, SGM, Rome, Italy) based on Jacobs et al. [73] with known amounts of cholesterol standard. Finally, triglycerides were estimated by homogenizing approximately 150 mg (±0.1 mg) of each lyophilized tissue sample in 4 mL of distilled water and were measured by the colorimetric enzymatic method using the commercial kit (10160 Triglycerides, SGM, Rome, Italy) based on Bucolo and David [74].
