RT Journal Article T1 Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum-free medium containing FSH, EGF and IGF-I T2 Determinación de la concentración apropiada de sodioalginato utilizado para el cultivo in vitro de folículos preantrales de gato en unsuero-medio libre que contiene FSH, EGF e IGF-I A1 Fontanillas Pérez, Juan Carlos A1 González Gil, Alfredo A1 García-Cuenca Ariati, Isabel A1 Picazo González, María Dolores A1 Fuertes Recuero, Manuel AB Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum-free medium containing FSH, EGF and IGF-I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G-0%), 0.5% (G-0.5%) or 1% (G-1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF-I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48 h and samples were stored at −20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24 h. G-0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 μm; p < .05) than G-0.5% and G-1% follicles (157.89 ± 8.47 μm and 95.23 ± 1.67 μm, respectively) which maintained three-dimensional organization, being larger in G-0.5% than in G-1% (p < .05). G-0.5% follicles attained the multi-layer preantral follicle stage on day 7 of culture, whereas G-1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p < .05) in G-0% than in G-1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G-0.5% were comprised between those of G-0% and G-1% (p > .05). In conclusion, two-layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF-I can develop up to the multi-layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three-dimensional organization, and experienced regression with compromised steroidogenesis, respectively. PB Wiley SN 0936-6768 YR 2023 FD 2023 LK https://hdl.handle.net/20.500.14352/98168 UL https://hdl.handle.net/20.500.14352/98168 LA eng NO Fuertes‐Recuero, M., et al. «Determination of the Appropriate Concentration of Sodium Alginate Used for in Vitro Culture of Cat Preantral Follicles in a Serum‐free Medium Containing FSH , EGF and IGF‐I». Reproduction in Domestic Animals, vol. 58, n.o 5, mayo de 2023, pp. 670-78. DOI.org (Crossref), https://doi.org/10.1111/rda.14336. NO Author contributionsM. Fuertes-Recuero: Preantral follicle isolation and culture, hormone analyses, image analyses and writing the manuscript. A. González-Gil: Design of experiments, preantral follicle isolation and culture, image analyses, writing and critical review of manuscript. J.C. Fontanillas Pérez: Hormone analyses and critical review of manuscript. I. García-Cuenca Ariati: Hormone analyses and critical review of manuscript. R.A. Picazo: Design of experiments, preantral follicle isolation and culture, image analyses, writing, critical review of manuscript and funding search. NO Universidad Complutense de Madrid DS Docta Complutense RD 18 jul 2024