Vitrification in straws conserves motility features better than spheres in donkey sperm

Abstract

Contents Sperm vitrification as alternative to conventional freezing is increasing in popularity in many species. It has been achieved by direct exposure of diluted semen to liquid nitrogen in spheres or straws. Both techniques have been successfully developed, but they had not been compared yet in donkeys. The aim of this study was to compare these two methods of vitrification for donkey semen. Ejaculates from six Andalusian donkeys were collected and extended in Gent without glycerol supplemented with sucrose 0.1 M (Molar). Samples were slowly cooled at 5°C. For vitrification, 30 μl suspensions (spheres) were dropped directly into liquid nitrogen (LN2) or filled in covered 0.25 ml straws and then plunged into the LN2 (straws). For warming, straws and spheres were directly immersed in 3 ml of INRA‐96 at 43°C. Total (TM, %) and progressive motility (PM, %) were objectively evaluated by computer‐assisted sperm analysis and plasma membrane integrity (PMI, %) by epifluorescence microscopy. Results showed the straw method resulted in significantly higher values than spheres for: TM (54.7% ± 10.1 vs. 28.6% ± 6.5) and PM (44.2% ± 9.4 vs. 17.7% ± 6.4), but no significant differences were found between straws or spheres for PMI (31.5 ± 10.7 vs. 41.6 ± 14.3) respectively. In conclusion, donkey sperm could be vitrified in straws obtaining better sperm motility parameters after warming in comparison to the sphere method.

Human sperm vitrification: the state of the art

“…This method worked successfully with 35 vitrified human semen samples [20]. Diaz-Jimenez et al [71] cryopreserved six donkey ejaculates, which were vitrified with either 30 μl sperm solution sphere suspensions or 100 μl in 0.25 ml straws with 0.1 M sucrose without glycerol. They found the straw method resulted in higher total and progressive motility while no difference in plasma membrane integrity [71].…”

“…Diaz-Jimenez et al [71] cryopreserved six donkey ejaculates, which were vitrified with either 30 μl sperm solution sphere suspensions or 100 μl in 0.25 ml straws with 0.1 M sucrose without glycerol. They found the straw method resulted in higher total and progressive motility while no difference in plasma membrane integrity [71]. It appears that sperm vitrification does not compromise post-thaw motility when using a closed carrier.…”

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

“…Sperm vitrification was carried out following the methodology previously described [24,25]. Briefly, a styrofoam box was loaded with LN 2 and five 30 µL drops from each treatment were plunged directly into the LN 2 .…”

“…The optimal concentration of CPAs seems to be species-specific and has been proposed as a key factor for sperm vitrification success, depending on the methodology used [24]. In a preliminary research, donkey sperm vitrified in straws showed significant higher sperm motility percentages when compared to vitrification in spheres [25]. However, only sucrose was tested as cryoprotectant agent, and a fixed concentration was employed for both methods derived from previous studies in other species.…”

Low‐density lipoproteins and milk serum proteins improve the quality of stallion sperm after vitrification in straws

“…Proteins such as human serum albumin (Isachenko, Isachenko, Katkov, Montag, et al., ) and BSA (Diaz‐Jimenez et al., ; Pradiee et al., ) are used for sperm vitrification to increase the viscosity of the medium and the glass transition temperature (Isachenko, Isachenko, Katkov, Rahimi, et al., ). Serum of cow milk contains BSA as well as β‐lactoglobulin, that have also demonstrated a protective effect on stallion sperm during cooling (Batellier, Magistrini, Fauquant, & Palmer, ).…”