*4.3. NMR Analysis*

Samples were prepared according to a modified Bligh and Dyer extraction method [75,76]. Lyophilized gonads (~100 mg) were added to 400 μL methanol, 400 μL deionized filtered water, and 400 μL chloroform. The solution was mixed and placed on ice for 10 min before centrifugation at 10000 rpm for 20 min at 4 ◦C. The polar and lipophilic phases were separated and dried by a SpeedVac concentrator (SC 100, Savant, Ramsey MN, USA). The lipid extracts were dissolved in 700 μL of CD3OD/CDCl3 (1:2 mix) containing 0.03% *v*/*v* tetramethylsilane (TMS, δ = 0.00) as internal standard. The aqueous extracts were dissolved in 160 μL 0.2 M Na2HPO4/NaH2PO4 buffer (pH 7.4) and 540 μL D2O containing 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP δ = 0.00) as internal standard). Both extracts were transferred into 5 mm NMR tubes for NMR analyses.

### NMR Spectroscopy and Data Processing

NMR spectra were obtained using a Bruker Avance III 600 Ascend NMR spectrometer (Bruker, Ettliger, Germany) operating at 606.13 MHz for 1H observation with a z axis gradient coil and automatic tuning-matching (ATM). Experiments were performed at 300 K in automation mode after loading individual samples on a Bruker Automatic Sample Changer, interfaced with the software IconNMR (Bruker). For aqueous extracts, a one-dimensional zgcppr Bruker standard pulse sequence was applied to suppress the residual water signal. A number of 32 repetitions of scans (with 4 dummy scans) were collected into 64 k data points with relaxation delay set to 5 s, a spectral width of δ 20.0276 (12,019.230 Hz), 90◦ pulse of 11.11 μs. For lipid extracts, a one-dimensional zg Bruker standard pulse sequence was acquired, with repetitions of 64 scans (with 4 dummy scans), a spectral width of δ 20.0276 (12,019.23 Hz) and 90◦ pulse of 10 μs. For both aqueous and lipid extracts, the FIDs were multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz before Fourier transformation, phasing and baseline correction. Metabolites identified by the 1H NMR spectra were assigned on the basis of analysis of 2D NMR spectral analysis (2D 1H J *res*, COSY, HSQC, and HMBC) and by comparison with literature data [23,28,40]. Whenever possible, also quantitative analysis of free metabolites was determined by integrating selected unbiased NMR signals, using TSP for chemical shift calibration and quantification [25]. In particular, signals corresponding to valine (δ 1.05), alanine (δ 1.48), acetate (δ 1.92), succinate (δ 2.41), β-alanine, (δ 2.56), hypotaurine (δ 2.65), betaine (δ 3.90), taurine (δ 3.41), glycine (δ 3.57), homarine (δ 8.04), formate (δ 8.46), and trigonelline (δ 9.13) were integrated. All chemical reagents for analysis were of analytical grade. D2O, CDCl3, CD3OD (99.8 atom%D), TSP, 3-(trimethylsilyl)-propionic-2,2,3,3,d4 acid, and tetramethylsilane, TMS (0.03 *v*/*v*%) were purchased from Armar Chemicals (Döttingen, Switzerland).
