Effect of the freezer and straw positioning on the post-thaw quality of bull spermatozoa: A comparative study

Cryopreservation is known to induce detrimental alterations in bull sperm structure and function. This study compared post-thaw sperm quality and the control and accuracy of cooling rates among three commercial semen freezers: Digitcool, Digitcool Alpha, and TurboFreezer M. Additionally, the effect of straw position within the freezing chamber (top, center, bottom) was evaluated. Twelve ejaculates from Holstein-Friesian bulls (n = 12) were diluted in an egg yolk-based TRIS extender, filled into 0.25 mL straws, and processed in a split-sample design using a uniform freezing protocol. Temperature profiles were recorded via thermocouples inserted into straws to confirm adherence to the programmed curves. Post-thaw sperm quality was assessed based on six parameters: total sperm motility after 30 and 120 min of incubation at 38°C (TRT30/120), morphologically intact spermatozoa (MIS), viable and acrosome-intact spermatozoa (VAI), spermatozoa with low membrane fluidity and low intracellular calcium (LMLC), and spermatozoa with high mitochondrial membrane potential (HMMP). Generalized linear mixed models revealed an effect of the freezing machine on all tested parameters (P < 0.001). The TurboFreezer M produced the highest overall sperm quality, particularly in the bottom position, with superior outcomes in TRT30 and VAI. Digitcool showed the best performance for MIS, while Digitcool Alpha excelled in LMLC. Straw positions (top, center, bottom) did not significantly influence sperm quality in any respect. In conclusion, although the choice of freezing machine can significantly affect in vitro sperm quality, all devices demonstrated the ability to produce high-quality cryopreserved semen when used with standardized protocols.