Received July 23, 2024
Accepted January 25, 2025

Objective: To develop and evaluate the effectiveness of a 3D-printed prototype to hold semen straws during the freezing process under safe and reproducible conditions. Methods: A prototype capable of holding ten straws in liquid nitrogen vapor (LN2) was 3D printed. A second support that is commonly used was assembled from pieces of expanded polyethylene (EPS), respecting the identical distance between the straws and the LN2 surface. Temperatures were registered with a thermocouple placed inside a straw. Semen samples were frozen in the presence of cryoprotectant using the prototype (N=20) and the EPS support (N=20) in two independent series of measurements. Sperm parameters (motility, vitality, and DNA fragmentation) were measured for fresh and frozen-thawed samples. Results: The temperature cooling curves measured on the prototype were remarkably reproducible. The prototype material withstood over 300 freezing cycles without damage. The mean motility and vitality of fresh (64.2%, 72.0%) and frozen-thawed (25.7%, 38.8%) samples were significantly different (p<0.001) using either support. Recovery rates of motility, vitality, and sperm DNA fragmentation in frozen-thawed sperm samples were equal regardless of straw position on the prototype or support type used. Conclusion: The developed device allows a homogeneous, quantifiable, reproducible cooling of the straws in liquid nitrogen vapor. The recovery rates are comparable to those reported in the literature for both tested supports. The designed 3-D printed prototype favors the safe handling of the straws, an explicit way of describing freezing conditions, and a better intra-operator and inter-laboratory reproducibility of the cryopreservation process.