Measurement of the Water Permeability of the Membranes of Boar, Ram, and Rabbit Spermatozoa Using Concentration-Dependent Self-Quenching of an Entrapped Fluorophore

• Published in: Cryobiology Vol. 41; no. 2; pp. 167 - 173
• Main Authors: Curry, M.R., Kleinhans, F.W., Watson, P.F.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.09.2000; Elsevier
• Subjects: Animals; Biological and medical sciences; carboxyfluorescein; Cell Membrane Permeability; Cell Size; Cryopreservation; Fluoresceins - metabolism; Fluorescent Dyes - metabolism; Fluorometry - methods; fluorophores; Fundamental and applied biological sciences. Psychology; Male; Mammalian male genital system; Models, Theoretical; Morphology. Physiology; Osmolar Concentration; Rabbits - physiology; Semen Preservation; Sheep - physiology; Species Specificity; Spermatozoa - metabolism; Spermatozoa - ultrastructure; Swine - physiology; Vertebrates: reproduction; Water - metabolism; Spermatozoa; Semen; Rabbit; Male; Concentration; Germinal cell; Lagomorpha; Water permeability; Pig; Vertebrata; Mammalia; Fluorophore; Animal; Plasma membrane; Self quenching; Sheep; Artiodactyla; Ungulata
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1006/cryo.2000.2277

Published values for sperm membrane water permeability (Lp) obtained using a time-to-lysis methodology have produced anomalous results when used to model optimal cooling rates for cryopreservation of spermatozoa. As the lysis method is dependent on potentially questionable assumptions, we describe an alternative method for measuring sperm Lp. Spermatozoa were exposed to hypo- and hyperosmotic conditions using a stopped-flow apparatus and the time course of resulting volume changes was measured using concentration-dependent self-quenching of the entrapped fluorophore, carboxyfluorescein (CF). Lp was measured for boar, rabbit, and ram spermatozoa using a range of osmotic stresses (±50–100 mOsm). Values for exosmotic and endosmotic flow showed no evidence of rectification. Mean Lp values were 0.84 μm/min/atm (boar), 0.28 μm/min/atm (rabbit), and 2.79 μm/min/atm (ram). These values are lower than the lysis method estimates, with the ram value reduced by approximately two-thirds using the current methodology. The value for boar spermatozoa showed good agreement with published values obtained using an electronic cell-sizing technique. Substitution of the revised values for Lp into the model for optimal cooling rates brings the calculated optimal rate closer to the lower empirically observed value but does not fully account for the previously reported discrepancies.