Determination of Plasma Membrane Characteristics of Boar Spermatozoa and their Relevance to Cryopreservation1

Abstract

The osmotic tolerance limits for boar spermatozoa were determined at 22 degrees C. These cells can swell to within 1.02 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. In the presence of an extender, cells can swell to within 1.1 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. Plasma membrane permeability coefficients were determined in the presence of 1 M dimethyl sulfoxide (DMSO), 1 M glycerol, and 2 M ethylene glycol (EG) at 22 degrees C. Hydraulic conductivity (Lp) was estimated to be 0.120+/-0.016 (mean+/-SEM), 0.138+/-0.006, and 0.204 +/-0.021 microm/min/atm in the presence of DMSO, glycerol, and EG, respectively, at 22 degrees C. Solute permeability (P[CPA]) was determined to be 0.930+/-0.118, 0.481+/-0.045, and 1.98+/-0.106 x 10(-3) cm/min, for DMSO, glycerol, and EG, respectively. Subsequent experiments were performed at 8 degrees C and 0 degrees C. Activation energies were calculated for Lp in the presence of glycerol and EG to be 7.20 and 11.51 Kcal/mol, respectively. The activation energies for P(CPA) were 4.06 and 7.48 Kcal/mol for glycerol and EG permeability, respectively. These membrane characteristics were used to calculate volume flux during addition and removal of cryoprotectant agents as well as during cooling and warming. In addition, the potential for intracellular ice formation during cooling and warming was calculated.

Osmotic Effects on Feline Spermatozoa from Normospermic versus Teratospermic Donors

“…Similar findings have been reported in fowl (35) and human spermatozoa (2,6). In contrast, spermatozoa from our teratospermic cat donors experienced a steep decline in intact membranes following exposure to hypotonie conditions similar to ram, bull, and boar spermatozoa (2,10). These observations clearly highlight the value of our domestic cat model that suggests that differences in membrane sensitivity may exist even between genotypes within a species.…”

“…Current methods to cryopreserve feline sperm were developed empirically, rather than by measuring biophysical properties of the specific gametes to be cryopreserved. Although such an empirical approach eventually may yield success, an increasing number of reports focus on understanding the cryobiological properties of cells to be cryopreserved (2,3,9,10,21,22). This strategy relies on developing a database first on crucial information, ranging from the membrane permeability to ionic impacts on the ability of cells to survive cryopreservation.…”

An improved cryopreservation method for a mouse embryonic stem cell line

“…Warming rates up to 1 × 10 4°C /min were simulated to verify that damage due to volume changes would not occur. An optimal warming rate is that which is fast enough to prevent devitrification, yet not too fast that after warming, the influx of water causes the cell to exceed osmotic tolerance limits (please see [12] for a discussion on the avoidance of devitrification using rapid warming rates, and [35] for a discussion of the damaging effects of volume change from overly rapid warming rates). Since the fastest practical method for warming is placing a straw in room temperature or 37°C water (the difference in cooling rates is reasonably negligible) it remains only to verify that cell volume excursions are within the osmotic tolerance limits.…”

Effect of the primary cooling rate on the motility and fertility of frozen-thawed rabbit spermatozoa

“…In view of the many potential future uses for cryopreservation of rabbit spermatozoa, it is essential to establish species-specifi c standard protocols. In general, rapid cooling and freezing of semen (cold shock) influences acrosome morphology and the percentage of post-thaw live and motile sperm (Blackshow, 1954;Gilmore et al, 1998;Watson, 2000). To avoid this severe irreversible damage, freezing semen in domestic animals requires a 2-step protocol (primary cooling and freezing) in which samples are suspended in an appropriate extender and gradually cooled from room temperature to 5°C, followed by freezing (Mocé and Vicente, 2002;Barbas and Mascarenhas, 2009).…”