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

Abstract

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 × 10 9 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 temperature.