**Background:** Portunus trituberculatus is a commercially important marine crab species in East Asia, but its germplasm resources are threatened by environmental degradation and disease. Sperm cryopreservation offers a solution for long-term genetic resource conservation, yet no standardized protocol existed for this species. This study aimed to optimize each step of the cryopreservation process—sperm collection, cryoprotectant selection, equilibration, cooling, and thawing—to maximize post-thaw sperm viability.
**Methods:** Sperm were collected from spermathecae of post-mating female crabs using three methods: 0.2% trypsin digestion (37°C, 5 min), mesh-rubbing (250 mesh screen), and mechanical grinding (glass homogenizer). Sperm viability was assessed using 2% eosin B staining under light microscopy (×1000), classifying sperm into four categories. Five cryoprotectants (glycerol, DMSO, methanol, ethylene glycol, propylene glycol) were tested at six concentrations (5–30% v/v) in sterile Ca²⁺-free artificial seawater (1:1 dilution). Equilibration times at 4°C were tested from 0 to 75 minutes, with refined gradients from 5 to 25 minutes. Cooling was performed using a two-step method with straws placed at heights of 1.5–11.5 cm above liquid nitrogen for 0–20 minutes. Thawing temperatures ranged from 22°C to 67°C in a water bath. Long-term storage stability was assessed over 30 days. Gene expression (ALF, DIC, caspase 1, acrosin) was quantified by qPCR (GAPDH as reference), and enzyme activities (total SOD, CAT, GSH, ACP, AKP) were measured using commercial kits. Statistical analyses used one-way ANOVA with Tukey's post hoc tests and t-tests (significance at p < 0.05).
**Key Results:** The mesh-rubbing method yielded the highest proportion of normal sperm (>60%) with a survival rate of 64.3%, compared to trypsin digestion (8.2% survival, >90% acrosome-reacted) and mechanical grinding (10.7% survival, severe damage). Among cryoprotectants, 20% glycerol produced the highest post-thaw survival rate at 41.79%, significantly better than all other concentrations and types (p < 0.05). The worst performer was 5% propylene glycol (6.35% survival). The optimal equilibration time at 4°C was 15 minutes (40.98% survival); shorter or longer times reduced viability, with direct liquid nitrogen input yielding only 7.65% survival. The best cooling procedure was 15 minutes at 3.5 cm above the liquid nitrogen surface (44.79% survival), not significantly different from 12.5 minutes at the same height (43.22%, p > 0.05). The optimal thawing temperature was 42°C (49.44% survival), followed by 37°C (45.56%). Thawing at 67°C was worst (23.89%, p < 0.05). Over 30 days of liquid nitrogen storage, sperm survival remained stable at approximately 50% with no significant differences (p > 0.05). Gene expression analysis showed significant decreases in ALF, DIC, caspase 1, and acrosin mRNA in frozen sperm compared to fresh (p < 0.05). Enzyme activities were significantly reduced: total SOD from 31.81 ± 2.82 to 13.13 ± 1.89 U/mL, CAT from 25.51 ± 0.81 to 6.60 ± 0.23 U/mL, GSH from 80.21 ± 3.09 to 27.10 ± 1.63 U/mL, ACP from 41.32 ± 2.07 to 8.41 ± 0.66 U/mL, and AKP from 0.41 ± 0.20 U/mL (fresh value not clearly reported for AKP) (all p < 0.05).
**Clinical Implications:** This study establishes the first optimized cryopreservation protocol for P. trituberculatus sperm, achieving a stable ~50% post-thaw survival rate—double the 25% reported in prior work. The protocol (mesh-rubbing collection, 20% glycerol in Ca²⁺-free artificial seawater, 15 min equilibration at 4°C, 15 min cooling at 3.5 cm above liquid nitrogen, 42°C water bath thawing) provides a practical foundation for germplasm banking of this species. The significant reductions in gene expression (ALF, DIC, caspase 1, acrosin) and antioxidant enzyme activities (SOD, CAT, GSH) indicate that cryopreservation induces oxidative stress and molecular damage, which may reduce fertilization capacity. Future work should explore combined cryoprotectants and antioxidant supplementation to further improve outcomes. The protocol has direct applications for selective breeding, artificial reproduction, and conservation of genetic diversity in P. trituberculatus and may inform cryopreservation strategies for other crustacean species.