Abstract
Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates—and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.
Highlights
Much effort has been invested in improving and simplifying the techniques for cryopreserving the sperm of wild ungulates and facilitate their use under field conditions [1]
The cooling rate affects the extracellular ice formation; the cells and the dissolved salts are excluded from the ice and become concentrated in the unfrozen fraction remaining between the growing ice masses [9]
Displacements of microtubules were more usual in sperm samples conventionally frozen (S1 Fig). This is the first report to assess the ultrastructural damage to ibex sperm cells after cryopreservation. It is suggested by Cryo-Scanning electron microscopy (SEM) method that ultra-rapid cooling failed to induce a stable, glass-like state in the extracellular milieu, but the ice crystal morphology was different to that seen in samples cryopreserved by conventional slow cooling
Summary
Much effort has been invested in improving and simplifying the techniques for cryopreserving the sperm of wild ungulates and facilitate their use under field conditions [1]. While conventional freezing requires the use of cryoprotectants to prevent damage caused by ice crystal formation, vitrification techniques allow glass transition and forms and stable structure without the presence of ice crystals through high concentrations of permeable cryoprotective agents [11] and/or very rapid freezing rates (kinetic vitrification) [12]. Some non-permeable additives with cryoprotective activity, such as human serum albumin and sucrose, have been successfully used in the kinetic vitrification of human sperm [16] This modified method was subsequently used to successfully cryopreserve dog [17] and fish sperm [18], and more recently Iberian ibex (Capra pyrenaica) [6] and European mouflon (Ovis musimon) [19] sperm. Extracellular ice is a primary cause of sperm damage during the cryopreservation process [23]
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