J. Vet. Med. A zyxwvut 41,3747 (1994) zyxwvuts 0 1994 Paul Parey ScientificPublishers, Berlin and Hamburg ISSN 0931 zyxwvutsrq - 184X zyxwvutsrq From the Department zyxwvut of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden zyx Membrane Damage during Dilution, Cooling and Freezing- Thawing of Boar Spermatozoa Packaged in Plastic Bags K. ORTMAN and H. RODRIGUEZ-MARTINEZ" Address of author": Department of Obstetrics & Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, P.O. Box 7039, Uppsala, Sweden. With 2figures and 2 tables (Received forpublication October 2, 1993) Summary The objective of the present investigation was to determine the degree of membrane damage in boar spermatozoa during the course of a cryopreservation procedure, including thawing. Ejacu- lates from four fertile Swedish Yorkshire boars were frozen under controlled conditions in Teflon@- plastic bags (2.5 ml) using 3 % glycerol as cryoprotectant. Membrane integrity was monitored using supravital flourescent dyes and confirmed by scanning electron microscopy. The results show that cooling to +5O C significantly affected the permeability of the plasmalemma. Supercooling (-6' C) during the freezing program did not further deteriorate the integrity of the spermatozoa1membrane. The thawing procedure however, dramatically increased the frequency of spermatozoawith damaged plasma membranes. Thus particular attention must be paid on designing a better thawing proce- dure when dealing with boar semen frozen in plastic bags. Introduction Procedures are currently available for freezing and thawing bull semen without an unacceptable reduction in viability. Thus the preservation of semen is commonly prac- tised in the artificial insemination of cattle. Attempts to freeze boar semen have been less successful, and so far, no procedure has proved reliable enough. Several cryoprotective agents and freezing diluents, as well as procedures for freezing, thawing, packaging and storage have been developed and improved over the years to overcome the problem of the large ejaculate in boars (Rev. by BWANGA, 1991). At our laboratory, attempts have been made to develop alternative packaging methods and obtain acceptable post-thaw fertility (LARSSON et al., 1976; BWANGA et al., 1991c; MWANZA and RODRIGUEZ-MARTI- NEZ, 1993; KAROSAS and RODRIGUEZ-MARTINEZ, 1993). Nevertheless, the physical stress associated with cryopreservation substantially diminished the survival and fertilizing ability of the thawed spermatozoa (MWANZA and RODRIGUEZ-MARTINEZ, 1993). Boar spermatozoa are particularly sensitive to rapid cooling and freezing, as well as to slow cooling below temperatures of +15" C, which results in damage, both at the plasma- lemma1 and acrosome levels. The critical temperature zone appears to be -15 to -25" C (HESSet al., 1960), with the greatest cellular damage occurring after crystallization (NIWA and TAGUCHI, 1981). However, only 15-25 % of the spermatozoa damage occurs between U.S. Copyright Clearance Center Code Statement: 0931 - 184X/94/4101 - 0037$10.00/0