Canine sperm vitrification with sucrose: effect on sperm function

Abstract

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra-rapid cryopreservation in canine sperm was investigated. Swim-up selected spermatozoa of second-fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m) in proportion 1 : 1 v/v with HTF-BSA 1%. From each group, 30-μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF-BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF-BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome-reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra-rapid cryopreservation.

Effects of vitrification on ram spermatozoa using free-egg yolk extenders

“…Nawroth et al [30], who vitrified human spermatozoa using extenders with and without cryoprotectans, observed the best results when samples were vitrified with free-cryoprotectans extenders. Similarly, the removal of permeable cryoprotectans from the vitrification media caused an improvement on the cryosurvival of fish and dog spermatozoa [26,37], although Agha-Rahimi et al [1] did not found differences in the sperm quality of vitrified human spermatozoa using extenders with and without cryoprotectans.…”

“…Contrary, higher concentrations of sucrose (0.15 M and 0.25 M) and glycerol (14% and 18%) resulted in lower sperm quality. In human, canine and rabbit, 0.25 M concentrations of sucrose had not a toxic effect on spermatozoa [14,36,37]. It has been described that sugars have a protector effect against osmotic changes decreasing the ice formation and stabilizing the solute concentration inside of sperm cell during osmotic stress as well as, have the property of stabilizate the cell membrane [19,20,29].…”

“…However, many assisted reproductive techniques, as sex-sorting, are performed using samples highly diluted without lack of sperm quality adding protector substances as egg yolk or seminal plasma [24]. In our study, the B5 control was used to mimic the conditions of vitrification, since the most of authors vitrify with low concentration and volume [1,3,26,37] and in this way to reject the hypothesis of a possible damage due to dilution.…”

“…Thus, extenders based on soybean or extenders containing sugars with high molecular weight combined with permeable cryoprotectants such as glycerol has been studied for conventional sperm freezing in different species [2,6,[16][17][18]27,34]. Moreover, some investigations have been aimed to evaluate the effect of using permeable cryoprotectants [36], sucrose [37], or even a cryoprotectants-free technique joined to other preservation methods such as vitrification [11,13,36].…”

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature?

“…9). This, together with the failure of the method even for 25 μL droplets (another Isachenkos modification) to vitrify spermatozoa of polar bear and 4 raptor bird species described in the major text of this Chapter, indicates that the Isachenko method of "cryoprotectant"-free cryopreservation" works satisfactory for some species of sperm [Merino et al, 2011a;Merino et al, 2011b;Sanchez et al, 2011] but not for the others, and it is completely inapplicable to big and watery "oocytes and embryos. And without further clarification, the method of kinetic VF in "large volumes" have not been able to be repeated independently even for human and bovine sperm.…”