Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at ¿5 and ¿15 °C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a `normal apical ridge¿ (NAR) were 72.5 ± 3.8 and 75.8 ± 2.0 versus 46.3 ± 4.8 and 36.0 ± 3.7 (means ± S.E.M., n = 4). In ejaculates of 15 unselected AI boars, after seeding at ¿5 °C, the post-thaw % live and % NAR were 66.3 ± 10.4 and 74.8 ± 7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 × 109 cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8 ± 1.9% versus 40.7 ± 1.7% (means ± S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3 ± 3.0% versus 59.3 ± 2.9% (means ± S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperatur