Background: Cryopreserved semen and embryos are essential tools in livestock reproduction, enabling genetic improvement and herd management. Although these materials are theoretically stable in liquid nitrogen (LN2), viability often decreases over time, particularly in farm settings. Micro-ice crystals (MICs) are hypothesized to form under poor LN2 handling conditions, potentially compromising the survival of frozen genetic resources. However, the extent and impact of MIC accumulation have not been thoroughly quantified. Methods: This study evaluated MIC accumulation and its effects on the viability of cryopreserved bovine semen and embryos under different LN2 storage environments and conditions. MIC content was measured by filtering 10 L of LN2 through nonwoven fabric and weighing the retained crystals and debris. The viability of sperm and embryos were assessed using a computer-assisted semen analysis (CASA) and blastocoel re-expansion. Results: MIC content was 3.5 times higher in farm-stored LN2 than in laboratory LN2, with significantly more debris also detected. Progressive motility and velocity parameters (VCL, VAP, VSL) were similarly reduced. Blastocyst survival dropped significantly under farm conditions after six months (42.4%) compared to laboratory storage (84.4%, p < 0.05). These findings suggest a strong correlation between MIC accumulation and decreased post-thaw viability of cryopreserved materials. Conclusions: MICs formed in LN2 due to environmental exposure and poor handling can severely impair the viability of cryopreserved sperm and embryos. Regular filtration and improved LN2 management, especially in farm environments, are essential to reduce MIC-related damage. These practices may enhance the long-term usability and reliability of genetic resources in livestock breeding programs.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Animals, semen collection, and reagents
    Measurement of MICs in the LN2 containers
    233accordance with the regulations and permissions of theInstitutional Animal Care and Use Committee (IACUC) ofthe National Institute of Animal Science (NIAS) (ApprovalNo. NIAS 2023-0624). The chemicals of semen diluentsand media were purchased from Sigma-Aldrich (St. Louis,MO, USA).Measurement of MICs in the LN2 containersThe volume of LN2 in each cryopreservation containerwas measured using a 2 L plastic volumetric beaker, thenfiltered through two layers of non-woven fabric (35 ×35 cm) with a 0.3 μm pore size. As shown in Fig. 1, nonwovenpaper was fitted into a plastic funnel to filter MICsand particulates from LN2 during collection. The weightof the filter containing MICs was measured immediatelyusing a chemical balance (AB304-S, Mettler Toledo, USA).After overnight dehydration in a dry oven, the filter wasreweighed using the same balance.Cryopreservation and thawing of semen
    Production of in vitro-fertilized bovine embryos
    Blastocyst freezing and thawing
    Evaluation of cryopreserved sperm and blastocysts
    Statistical analysis
RESULTS
    Measurement of MICs in LN2 containers after sixMonths of storage in two-year-used tanks
    Motility kinetics of frozen semen preserved in LN2with MICs for two years
    Viability of blastocysts after six months of preservation
DISCUSSION
CONCLUSION
REFERENCES

• Bongki Kim(Department of Animal Resources Science, Kongju National University, Yesan 32439, Korea) Corresponding author
• Yu-Da Jeong(Hanwoo Research Center, National Institute of Animal Science, RDA, Pyeongchang 25340, Korea, Department of Animal Resources Science, Kongju National University, Yesan 32439, Korea)
• Ga-Yeong Lee(Animal Genetic Resource Research Center, National Institute of Animal Science, RDA, Hamyang 50000, Korea, Department of Animal Resources Science, Kongju National University, Yesan 32439, Korea)
• Sung Woo Kim(Dairy Science Division, National Institute of Animal Science, RDA, Cheonan 31000, Korea) Corresponding author