title: 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 degrees 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 degrees 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 x 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.
authors.affiliation: Division of Animal Resources Development, Animal Sciences Group, Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands. henri.woelders@wur.nl
authors.forename: H
authors.initials: H
authors.lastname: Woelders
authors.forename: A
authors.initials: A
authors.lastname: Matthijs
authors.forename: C A
authors.initials: CA
authors.lastname: Zuidberg
authors.forename: A E N
authors.initials: AE
authors.lastname: Chaveiro
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes; D007053:Ice; D008297:Male; D008853:Microscopy / Q000662:veterinary*; D008954:Models, Biological; D009995:Osmosis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013076:Sperm Count; D013094:Spermatozoa / Q000502:physiology; D013552:Swine; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D007053:Ice