Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report

Abstract

The aims of this investigation were to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland(®) medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: CM + 1% BSA + 40% seminal plasma + 0.125 m sucrose. For cooling, 20-μl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM + 1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR-14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM + 1% BSA + 40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA + 40% seminal plasma.

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

“…The lipophilic compound JC‐1 has been used to evaluate the depolarisation of the mitochondrial membrane in the spermatozoa of several species of mammals and fishes. The use of this technique on the mitochondria of S. salar spermatozoa, however, has been little reported and could be considered a test of the effectiveness of the freezing protocol (Labbé et al ., ; Aitken et al ., ; Merino et al ., ). In the current experiments, there was a positive correlation between the mitochondrial‐membrane potential and the fertilization rate, which agrees with the findings of Figueroa et al .…”

Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species

“…This method can be used by reducing the temperature of the material either slowly (traditional method) or rapidly (vitrification) (Merino et al . ; Cuevas‐Uribe et al . ; Figueroa et al .…”

“…The latter can be done either by slow freezing or by a very rapid process called vitrification (Merino et al . ; Cuevas‐Uribe et al . ; Figueroa et al .…”

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.…”