Nanoencapsulated Quercetin in Sperm Cryopreservation: A Systematic Review of Molecular Mechanisms and Delivery System Effectiveness

This project constitutes a systematic review conducted to synthesize and critically evaluate current evidence regarding the role of nanoencapsulated quercetin in improving sperm quality following cryopreservation. Cryopreservation-induced oxidative stress remains a major limitation in reproductive biotechnology, typically reducing post-thaw sperm quality by 40–60% due to excessive reactive oxygen species (ROS) generation, lipid peroxidation, mitochondrial dysfunction, and DNA fragmentation. Although quercetin is a potent flavonoid antioxidant with well-documented ROS-scavenging capacity, its practical application in semen extenders is constrained by poor aqueous solubility, rapid metabolism, and limited bioavailability. Nanoencapsulation technologies have emerged as a promising strategy to overcome these pharmacokinetic limitations and enhance intracellular delivery. The primary objective of this review is threefold: (1) to systematically map the molecular mechanisms through which quercetin exerts protective effects on spermatozoa during cryopreservation; (2) to evaluate the physicochemical and biological advantages conferred by nanoencapsulation systems; and (3) to comparatively assess different nanoparticle delivery platforms using a structured multi-criteria evaluation framework that integrates antioxidant effectiveness, preparation feasibility, cost efficiency, safety profile, and material accessibility. The review follows PRISMA 2020 guidelines. A structured literature search was conducted in Scopus and Web of Science databases for studies published between 2015 and 2025. Eligibility criteria were defined using a modified PICO framework, including mammalian spermatozoa as the population, nanoencapsulated quercetin as the intervention, free quercetin or untreated control as comparison, and post-thaw sperm quality parameters (motility, viability, membrane integrity, mitochondrial function, and DNA integrity) as outcomes. Methodological quality was assessed using the Mixed Methods Appraisal Tool (MMAT) 2018. Due to heterogeneity in experimental protocols, narrative synthesis was employed rather than meta-analysis. This review introduces two conceptual innovations. First, it proposes a four-layer hierarchical mechanistic framework linking primary antioxidant pathways (free radical scavenging, enzyme upregulation, mitochondrial stabilization, membrane protection, and inflammatory modulation) to measurable reproductive outcomes. Second, it develops a standardized multi-criteria scoring system enabling structured comparison of six nano-delivery systems, including nanostructured lipid carriers (NLC), liposomes, metal oxide nanoparticles, chitosan-based systems, whey protein-based systems, and free quercetin. Expected outcomes of this research include: • Clarification of dose-dependent mechanistic pathways (optimal range 10–50 µM); • Quantitative synthesis of improvements in post-thaw sperm parameters; • Identification of delivery systems balancing biological effectiveness and practical feasibility; • Evidence-based recommendations for laboratory optimization and potential field application in livestock reproductive programs. Overall, this registration documents the methodological framework, analytical approach, and theoretical synthesis strategy employed in the review to enhance transparency, reproducibility, and scientific rigor in veterinary reproductive biotechnology research.