Protein-rich fibrous eggshell membrane derived ZnO 3D-nanohybrid for cryoprotectant, reproductive scavengers against Zn deficiency and hypokalemic effects in rats

Zinc deficiency is associated with impaired spermatogenesis, reduced sperm quality, and male infertility, highlighting the need for multifunctional biomaterials capable of supporting sperm preservation and reproductive health. Herein, we report, for the first time, the synthesis of protein-rich eggshell membrane (ESM)-supported ZnO nanocomposites with distinct ZnO nanostructures uniformly anchored onto a three-dimensional fibrous ESM scaffold. The hypothesis of this study was that ESM-supported ZnO nanocomposites could provide controlled Zn²⁺ release, antimicrobial protection, and enhanced sperm preservation while simultaneously improving reproductive and metabolic health in vivo. To evaluate this hypothesis, two experimental phases were conducted. First, the antimicrobial activity and sperm cryoprotective performance of ESM-ZnO nanocomposites were investigated under heat-shock and freeze–thaw conditions. Antibacterial and antifungal activities were assessed alongside sperm viability, reactive oxygen species (ROS) generation, DNA integrity, motility, and morphological characteristics. Subsequently, the biological efficacy of the optimized formulation was validated in male Sprague-Dawley rats through reproductive, biochemical, and histological analyses. The results demonstrated that the thermally synthesized ESM-ZnO-T nanocomposite exhibited superior physicochemical characteristics, improved colloidal stability, sustained Zn²⁺ release behavior, and enhanced antimicrobial efficacy. ESM-ZnO-T effectively preserved sperm viability, morphology, motility, and DNA integrity, achieving an area under the ROC curve (AUC) of 0.84 following cryopreservation. In vivo investigations further revealed improved reproductive hormone profiles, spermatogenesis, sperm morphology, and testicular histology compared with control groups. Moreover, ESM-ZnO-T contributed to the regulation of hypokalemia and hyperoxaluria-associated biochemical alterations while supporting normal liver and kidney function. Overall, the developed ESM-ZnO-T nanocomposite represents a promising multifunctional biomaterial platform for antimicrobial protection, sperm cryopreservation, and reproductive health management, providing a potential strategy for addressing zinc deficiency-associated male infertility.