Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.

No abstract

The objective of the present study was to investigate the influence of different sucrose-based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra-rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate-buffered saline-BSA1%-based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m). After ultra-rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose-supplemented groups than in the sucrose-free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra-rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra-rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.

Vitrified horse and donkey embryos did not show higher susceptibility to cell damage than those preserved by slow freezing, whether using straws or Fibreplug. However, Fibreplug with EG 7 mol/L resulted in fewer nonviable and apoptotic cells in donkey embryos. Donkey embryos showed lower susceptibility to vitrification than horse embryos. THE SUMMARY IS AVAILABLE IN SPANISH - SEE SUPPORTING INFORMATION.

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS-egg yolk-based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25-ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0- and 6-h post-thaw. Experiment 1 compared the effect of three non-feline commercial extenders - based on TRIS-egg yolk (Triladyl), egg-yolk-free medium (AndroMed) and skimmed milk-egg yolk (Gent) - on the quality of frozen-thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen-thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS-egg yolk-based extenders, one non-feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing-thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing-thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS-egg yolk-based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.