Comparison of slow and rapid freezing in freeze-dry ram spermatozoa

Semen lyophilization is an interesting technique that might be a cheap alternative to a long-term storage in liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 demonstrating, for the first time, the birth of healthy mouse pups from epididymal freeze-dried (mouse) spermatozoa. Authors follow lyophilisation technique, commonly used in pharmaceutical/food industry, namely deep-freezing, that requires direct immersion of the semen sample into liquid nitrogen before vacuum drying. In this work, we focused on the freezing phase in order to improve and make the technique more reliable. We have compared two different protocols: i) Rapid-freezing, where the semen is plunged directly into liquid nitrogen (LN group); ii) Slow-freezing, where the sample is frozen with a freezing rate of 1°C/min until -50°C degrees (SL group). Then, both frozen samples were lyophilized. Subsequently, after (in a range between 1 – 3 months) dry spermatozoa from LN and SL-groups were used for Intracytoplasmic Sperm Injection (ICSI) and the embryo development was evaluated at 24h (2-Cells stage) and 7 days (expanded blastocyst) post-ICSI. Moreover, a part of the semen, immediately after freezing phase, has been dedicated for the evaluation of acrosome integrity through the Pisum Sativum Agglutinin (PSA) staining. LN-group semen showed the acrosome completely melted, while the SL-group showed a better integrity of the acrosome which was comparable to that of the normal frozen (vital) spermatozoa. ICSI outcomes point out at 24h post-ICSI the SL-group showed a higher number of cleaved embryos than LN-group [42/100 (42%) versus 19/75 (25.3%), P=0.0253, SL and LN respectively]. At 7 days after fertilization the blastocyst rate in SL-group was higher [7/100 (7%)] than in LN-group [2/75 (2.7%), P=0.0238]. Our data shows that lyophilisation can be conveniently achieved in ram spermatozoa with liquid nitrogen – free way, thus simplifying the procedure. This data supports the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen.