Spermatogonial stem cells (SSCs) are the only adult male germline stem cells capable of lifelong self-renewal and differentiation into spermatozoa. Scalable ex vivo survival is essential for endangered species germplasm banking, genetic resource conservation, and male infertility therapy. Here, chitosan (CO) or chitosan oligosaccharide (COS) was cross-linked into injectable, biodegradable 3D hydrogels loaded with the natural astaxanthin (AST). CCK-8 optimization identified 0.3% CO + 0.2% AST (CHAG) and 0.2% COS + 0.2% AST (COAG) as superior formulations. After 7 or 14 d of 3D culture, CHAG yielded significantly more colonies than controls (p < 0.01), with elevated EdU incorporation, alkaline phosphatase activity, and positive OCT4 and PLZF staining, confirming preserved stemness. Caspase-3 expression was markedly reduced, indicating the AST-mediated suppression of oxidative apoptosis. RNA-seq showed distinct transcriptome pathways (p < 0.01): CHAG up-regulated adhesion and ECM–receptor and cell cycle pathways, whereas COAG enriched immune-modulatory and signaling modules, enabling context-specific use. AST-loaded CO/COS hydrogels are inexpensive, cytocompatible, and scalable, doubling as a biomimetic niche that accelerates SSC proliferation while delaying senescence. The platform provides a robust, controllable 3D system for SSC expansion and establishes a pre-clinical basis for translating CO/COS/AST composites to reproductive stem cell biotechnology.

Testicular tissue cryopreservation is a key strategy for preserving fertility in adult males, yet optimal cryoprotective protocols have yet to be established. This study evaluated the effects of varying concentrations of chitosan oligosaccharide (COS, 0–25%) and chitosan (CS, 0–25%) on rabbit testicular tissues. Right testes from 35 male New Zealand rabbits were assigned to COS groups and left testes to CS groups. Each group was further divided into seven subgroups: fresh control, 0% control, and 5%, 10%, 15%, 20%, and 25% treatment groups. After one month of slow freezing, tissues were assessed by histomorphological scoring (cell swelling, cell loss, basement membrane integrity, and seminiferous tubule architecture) and seminiferous tubule diameter measurement. The 20% COS group achieved the highest histomorphological score, significantly exceeding the 0% control and comparable to the fresh control. Among CS treatments, the 15% group yielded the best score, also significantly higher than the 0% control. All frozen groups exhibited significantly reduced seminiferous tubule diameters compared with the fresh control, and these diameter changes did not correlate with the morphological scores. Concentrations exceeding the optimal level led to lose tubular structure and severe exfoliation of epithelial cells, suggesting that excessive osmotic stress may have contributed to the observed structural damage. COS demonstrated superior cryoprotection compared with CS under the present experimental conditions, which may be attributed to its lower molecular weight and improved diffusibility. These findings indicate that COS may represent a promising candidate for optimizing polysaccharide-based cryoprotectants in testicular tissue banking, although further functional validation is required.