Effects of Temperature and Restoration of Osmotic Equilibrium during Thawing on the Induction of Plasma Membrane Damage in Cryopreserved Ram Spermatozoa1

Abstract

The objective of this investigation was to examine the nature of freeze/thaw-induced plasma membrane damage in an effort to validate hypotheses about cryoinjury in ram spermatozoa. Spermatozoa were loaded with fluorescein diacetate (FDA), a marker for plasma membrane integrity, and cooled (15 degrees C/min) to temperatures between -10 degrees C and -30 degrees C on a cryomicroscope stage. Post-thaw fluorescence intensity measurements of individual cells indicated that freezing to temperatures between -10 degrees C and -15 degrees C did not induce significant membrane permeabilization. However, freezing below -15 degrees C was followed by membrane permeabilization immediately after thawing. A majority (> 60%) of flagellar plasma membranes of cells frozen to -10 degrees C remained ultrastructurally intact during thawing; principal-piece membranes were more robust than middle piece membranes (p = 0.001). Significant middle-piece membrane breakage was, however, induced as the post-thaw temperature increased from +10 degrees C to +30 degrees C (10 degrees C, 64 +/- 12.3% intact membranes [mean +/- SEM]; 30 degrees C, 43 +/- 12.5% intact membranes [mean +/- SEM]; p = 0.0085). Cells frozen to -30 degrees C did not exhibit this thawing effect, although the distinction between middle-piece and principal-piece plasma membranes was evident (p = 0.002). All sperm head plasma membranes were damaged by freezing and thawing to any combination of temperatures. Although acrosomes became swollen after freezing and thawing, the incidence of outer acrosomal membrane vesiculation remained at control (unfrozen) levels with all treatments used. Experimental exposure to the hyperosmotic conditions generated during freezing induced little flagellar membrane permeabilization, but significant damage was caused by restoration of osmotic equilibrium. It is suggested that membranes are initially destabilized during the freezing process, both by low temperature effects and by exposure to high salt concentrations. The resultant post-thaw degeneration of the plasma membrane is caused by a combination of temperature and osmotic effects.

Viabilidad in vitro e in vivo de los espermatozoides congelados/descongelados del conducto deferente de Alpacas (vicugna pacos)

“…Las funciones del epidídimo son t r a n s p o r t e , a l m a c e n a j e y m a d u r a c i ó n d e espermatozoides, a través de proteínas de bajo peso molecular y enzimas que protegen a los espermatozoides de efectos tóxicos, químicos y stress oxidatívo, pero también solutos orgánicos e iones que se encuentran dentro el fluido que tienen el rol de proteger a los espermatozoides (Acott et al 1983). D u r a n t e e l p r o c e s a m i e n t o d e s e m e n l o s espermatozoides necesitan protección contra los estreses de la congelación, shock frio, shock caliente, formación de núcleos de hielo, acción de los crioprotectores, stress osmótico, toxicidad de los solutos, para evitar el daño en su integridad del espermatozoide y posterior función (Holt and North 1994;Watson 1995).…”

“…En el presente estudio los espermatozoides procedente de los conductos deferentes soportaron el estrés de la congelación/descongelación y posterior a la descongelación se obtuvo un índice de recuperación de espermatozoides en el reproductor 1 el 24.51% y en el reproductor 2 el 33.18% (Tabla 1) de espermatozoides motiles, esta prueba de recuperación de la motilidad posterior a la descongelación es utilizada para predecir la fertilidad futura del semen congelado/descongelado en otros rumiantes como el vacuno, ovino, caprino entre otras especies donde la tecnología de inseminación artificial se utiliza como indican Foote (1970a), Foote (1970b), Foote (1972), Chen et al (1993), Rodríguez y col. (1993) y recomiendan que motilidad a la descongelación en vacunos y ovinos deben ser del 45 al 60% para lograr gestaciones satisfactorias y estos resultados comparados con el 24.51% y el 33.18% de motilidad para el reproductor 1 y 2 son menores esto debido a que los espermatozoides del conducto deferente están libres del plasma seminal, que juega un rol importante como lo aseveran (Holt and North 1994;Watson 1995). De la misma forma comparando con material espermático colectado de los epidídimos de animales post mortem, como es del ovino, vacuno y ciervo ibérico, los investigadores reportan motilidades a la descongelación del 35 al 70% (Kaabi et al 2003;Ehling et al 2006;Herold et al 2004;Martins et al 2007;Garde y col. 1998;Soler et al 2003;Soler et al 2005;Fernandez-Santos et al 2006), estas motilidades totales son superiores, comparados con las motilidades del reproductor 1 de 24.68% y para el reproductor 2 de 27.31%, encontrados en el presente estudio.…”

“…Los espermatozoides del conducto deferente al proceso de congelación/descongelación resisten y este hallazgo esta de acorde con los reportes que indican que los espermatozoides del epidídimo soportan el stress de congelación (Holt and North 1994;Watson 1995), debido a que también estos espermatozoides están protegidos por las proteínas y demás componentes que se secretan en el epidídimo y conducto deferente de las alpacas.…”

Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice

“…In ejaculated spermatozoa, studies have shown that temperature changes during freezing and thawing may result in sperm plasma membrane damage [9,10], whereas heating of spermatozoa to 58ºC for 30 min denatures sperm proteins, with an exposure to 100ºC resulting in sperm DNA fragmentation [11]. Sperm plasmalemma defects, mitochondrial damage and/ or DNA fragmentation are all characteristic features of apoptosis [12,13], but whether whole-body heat exposure produces such adverse effects on the sperm population residing in the epididymis is not known.…”

Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen

“…It has long been known that major local osmotic gradients across the sperm's membranes are generated during the freeze-thaw cycle, and a two-factor aspect of cellular damage during cryopreservation occurs (osmotic effects versus intracellular ice formation, Henry et al 1993). However, studies by Gao et al (1993) and Holt and North (1994) have shown that cell death occurs mainly during the thawing process, when the dehydrated sperm after undergoing primary hypertonic shock would be exposed to severely hypo-osmotic conditions (Gilmore et al 1996). Therefore, the inability of spermatozoa to regulate the cell volume already within physiological gradients may result in poor freezing ability of the ejaculate, and monitoring the volume regulation in frozen-thawed samples could help to identify the samples with compromised fertilising potential.…”