Frozen dog spermatozoa are negatively affected during storage at -80, -21 and -8 ºC.

Cryopreservation in liquid nitrogen (LN2) allows for semen to be stored for long periods of time while there is sustaining of sperm viability. In this study, there was assessment of effects induced by different storage temperatures on cryopreserved dog spermatozoa. After cryopreservation at -196 °C, sperm samples were transferred to storage conditions of -80, 21 or -8 °C. Sperm motility, morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation were determined in samples stored at -196 °C (evaluation time =0 h), and then after 12 h and 1, 4, 7 and 15 d of storage at 80, -21 and -8 °C. In samples stored at -80 °C, sperm morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation did not differ at successive evaluation times. Progressive motility was less (P < 0.05) after 12 h and total motility after 4 d of storage at -80 ºC as compared with that of the 0 h sample. With storage at the other temperatures (-21 and -8 ºC), there was a reduction of mean values for sperm total and progressive motility, viability and mitochondrial membrane potential after 12 h of storage at these temperatures. Results, therefore, indicate the use of ultra-freezers at -80 ºC to store frozen dog semen allows for maintenance of sperm characteristics for at least 15 d but motility is sustained for only 1 d. Neither of the -21 or -8 ºC storage temperatures were effective for storing of frozen dog sperm and retaining viability.

Vitrification induces critical subcellular damages in ram spermatozoa.

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 106 spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.