Introduction: Sperm cryopreservation is a critical biotechnology for genetic resource conservation and artificial insemination in domestic animals. However, conventional glycerol-based methods cause severe damage to the plasma membrane, mitochondria, and sperm DNA due to intracellular ice crystal formation, osmotic stress, and increased reactive oxygen species (ROS), ultimately reducing post-thaw viability and fertility. The studies included in this meta-analysis cover multiple economically important species, including cattle, sheep, goats, and particularly poultry (roosters), all of which hold significant value for both economic productivity and biodiversity conservation in Iran. This systematic review and meta-analysis synthesizes evidence from 11 comparative studies to evaluate the efficacy of optimized cryopreservation protocols (e.g., DMA pellets, dry thawing, nano-enhanced extenders, and programmable freezing) versus standard glycerol-based methods on sperm viability and motility.

Aims: The primary objective is to quantify the pooled effect size of optimized cryopreservation methods specifically on post-thaw viability and progressive motility compared to standard glycerol‑based protocols. The secondary objectives are to identify sources of heterogeneity, to assess publication bias, to provide predictions for future studies, and to offer practical, actionable recommendations for implementing these technologies in Iran, particularly for poultry gene banking and the preservation of native breeds.

Materials and Methods: Following PRISMA 2020 guidelines, we systematically searched PubMed, Scopus, Web of Science, Google Scholar, SID, MagIran, and reference lists up to November 2025. Search keywords included: sperm cryopreservation, motility, DMA, nanoparticles, and poultry.

Inclusion criteria were: comparative studies in domestic animals, reporting mean ± SD for post-thaw viability/motility, with a sample size of at least 6 (n ≥ 6). Quality assessment of the included studies was performed using a risk-of-bias tool. Data were analyzed using CMA v4 with a random-effects model. Effect size was calculated as the standardized mean difference (SMD). Heterogeneity was assessed using the statistic and Q-test. Publication bias was evaluated via Funnel Plot and Egger's test.

Results: Eleven studies (comprising 276 sperm samples) were included in the final meta-analysis. Optimized methods significantly improved viability (SMD = 4.514, 95% CI: 3.253–5.776, Z = 7.014, p < 0.001; I² = 86.2%) and motility (SMD = 3.161, 95% CI: 2.378–3.943, Z = 7.919, p < 0.001; I² = 75.7%). The Q-test also revealed significant heterogeneity (p < 0.001), which is entirely expected given the diversity of animal species and cryoprotectant types employed across the primary studies. However, Egger's test indicated no significant publication bias for either outcome (p > 0.05).

Discussion: The pooled analysis showed very large effect sizes favoring optimized methods for both viability and motility. The high heterogeneity was expected and partly explained by species differences and the type of cryoprotectant used. From a biological perspective, DMA offers distinct advantages over glycerol due to its lower molecular weight and faster membrane permeability, which reduces equilibration time and minimizes cytotoxicity. Programmable freezing, by precisely controlling the cooling curve, prevents thermal shock and preserves the structural integrity of the spermatozoa.

Moreover, the incorporation of nanoparticles (e.g., selenium or titanium dioxide) into extenders effectively scavenges ROS, thereby inhibiting lipid peroxidation and optimizing sperm energy metabolism. The dry-thawing method further reduces cold-shock injury by minimizing the time spermatozoa spend in the critical temperature zone (−15 to 0°C), thus preventing recrystallization damage. The findings strongly support the adoption of DMA pellets, dry thawing, and nano‑enhanced extenders in breeding programs and gene banks, especially in Iran.

Conclusion: Optimized protocols substantially outperform standard methods. Given Iran's diverse climatic conditions and the urgent need to protect native poultry breeds from environmental and epidemiological threats, implementing these advanced protocols in national genetic resource centers and the country's gene bank is essential. Furthermore, standardizing rapid dry-thawing protocols across artificial insemination centers nationwide would be a highly effective step toward increasing practical fertility rates, reducing sperm wastage in commercial flocks, and ultimately supporting national food security goals. Therefore, we recommend the widespread adoption of these optimized methods to enhance fertility outcomes and ensure robust genetic preservation in domestic animals.