Fundamental cryobiology of mammalian spermatozoa

Applications for the use of many cells and tissues ex vivo have been developed over the past several decades. Among reproductive cells and tissues, utilization of spermatozoa for artificial insemination (AI) has the longest history and is the most widely used. Historically, development of cryopreservation methods for mammalian spermatozoa were essential for the widespread application of AI. These efforts were significantly enhanced in 1952 with the application of a cryoprotective agent (CPA), glycerol. Since this time, spermatozoa from many mammalian species, especially bovine and human, have been cryopreserved effectively enough to be practical for AI and other assisted reproductive technologies. However, spermatozoa from other species, such as the porcine, have not been effectively cryopreserved. Empirically derived methods of cryopreservation developed in the 1950\u27s are still those used today. These methods often result in relatively low cryosurvival ($\u3c$50% post-thaw motility) for most species. The purpose of the present study was to develop improved cryopreservation methods of mammalian spermatozoa based upon an understanding of fundamental cryobiology. The fundamental cryobiological parameters which are required for this process include: (1) cell surface area, (2) cell volume, (3) plasma membrane permeability coefficients to: (a) water (L$\rm\sb{p}),$ (b) cryoprotectant solute (P$\rm\sb{CPA}),$ and (4) the temperature dependence (activation energy) for L$\rm\sb{p}$ and P$\rm\sb{CPA}.$ These data were collected for the human and porcine. Computer simulations were performed using the derived data, and theoretical predictions were made which define: (1) optimal CPAs for human and porcine spermatozoa cryopreservation and methods for their addition and removal which minimize cell volume excursions, and (2) optimal rates for cooling and warming human and porcine spermatozoa during cryopreservation which preclude intracellular ice formation