Preliminary study on effects of bovine frozen semen storage using a liquid nitrogen-independent method on the quality of post-thaw spermatozoa

• Rationale: a reliable supply of LN2 for storage of frozen semen is not guaranteed.
• Objective: the feasibility of using a mechanical freezer as an alternative to LN2 for storing frozen semen was evaluated.
• Conclusion: frozen bovine semen can be stored for a month in a −80 °C freezer without compromising post-thaw sperm quality.

Frozen semen of eight bulls was used to assess effects of storage temperature and length of storage time on frozen-thawed bovine sperm quality. In experiment 1, 25 straws of frozen semen of each bull were allocated to 3 groups. The control was still maintained in liquid nitrogen (LN2). The rest were abruptly moved from LN2 to −80 °C and −30 °C mechanical freezers, respectively. After thawing, it was found that the sperm motility, vitality and membrane integrity were comparable (P > 0.05) between the control and the −80 °C samples and were significantly inferior (P < 0.001) in the −30 °C samples, irrespective of storage time (1-day, 1-week and 1-month). In experiment 2, two out of the three parts (16–18 straws) of frozen semen of each bull were rapidly removed from LN2 and further kept in the freezer (−80 °C). One day before being thawed, half of the samples in the freezer were promptly put back to LN2. The results showed that the frozen-thawed sperm quality was not significantly affected (P > 0.05) both by storage temperature (−196 °C, −80 °C and −80 & −196 °C) and storage time [day-2, day-8 (1-week) and day-31 (1-month)]. At the same storage times, the quality measures at different temperatures were not significantly different from one another (P > 0.05). In conclusion, a −80 °C mechanical freezer was as effective as LN2 in preserving in vitro quality of frozen-thawed bovine spermatozoa throughout 1-month of storage. When required for use, frozen semen stored in the freezer could be thawed immediately or transferred to the LN2 tank for thawing elsewhere.