Optimization of Sodium Alginate Concentration and Evaluation of Individual Versus Group In Vitro Culture of Porcine Preantral Follicles in a Serum-Free Medium

The increasing biomedical and conservation interest in porcine species has driven the development of advanced in vitro follicle culture systems designed to preserve genetic diversity and accurately model key stages of folliculogenesis. This study assessed a three-dimensional (3D) alginate-based system for the in vitro culture of porcine preantral follicles, aiming to overcome the structural limitations of conventional two-dimensional (2D) methods. A total of six experimental groups were established, consisting of group-cultured (four follicles/well) or individually cultured (one follicle/well) follicles maintained either without alginate (0%) or encapsulated in 0.5% or 1% alginate for 14 days in media supplemented with FSH, EGF, and IGF-I, with LH added from day 9. Follicular development was assessed by morphometric evaluation, image-based and histological analyses, and quantification of steroid hormones in media collected every 48 h. Group-cultured follicles encapsulated in 0.5% alginate most effectively maintained their 3D architecture, reached the largest diameters, and progressed more uniformly compared with other groups. In contrast, follicles cultured without alginate rapidly lost structural integrity, showed granulosa cell migration, and decreased in size, whereas those encapsulated in 1% alginate exhibited restricted growth. Estradiol and testosterone concentrations increased over time in the 0.5% alginate group, were lowest without alginate, and intermediate in 1% alginate. Individually cultured follicles exhibited reduced growth and lower total hormone production compared with group-cultured follicles; however, when normalized per-follicle, steroid secretion, particularly in the 0.5% alginate group, was enhanced, indicating increased steroidogenic efficiency on a per-follicle basis. These findings indicate that 0.5% alginate provides an optimal balance between structural support and physiological steroidogenesis during preantral follicle culture. This 3D system improves the biological relevance of porcine follicle culture and may support future applications in reproductive biology, conservation, and genetic resource preservation.