Saragusty, J., Institute of Animal Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel, Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel, Department of Reproductive Management, Leibniz Institute for Zoo and Wildlife Research, Alfred Kowalke Str. 17, 10315 Berlin, Germany
Gacitua, H., Institute of Animal Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
Rozenboim, I., Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel
Arav, A., Institute of Animal Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component - mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 μm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37°C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing. © 2009 Wiley Periodicals, Inc.