Abstract :
The main objective was to compare the efficiency of vitrification techniques and solutions
on the preservation of morphology, ultrastructure and viability of sheep preantral follicles
enclosed in ovarian tissue fragments. The fragments were cryopreserved by using macrotube
vitrification (MTV), solid-surface vitrification (SSV) or conventional vitrification (CV).
These techniques were combined with one of the six solutions containing 6 M ethylene
glycol (EG) and with or without sucrose (SUC) (0.25 or 0.50 M) and with or without fetal
calf serum (FCS) (10%). After one week, samples were warmed and histological analysis was
performed, showing that the percentage of normal follicles after CV (66.20
±
8.87%) using
a solution containing 6 M EG, 0.25 M SUC and 10% FCS (vitrification solution 4 – VS4) was
similar to fresh control (79.40
±
7.83%), MTV (53.40
±
10.60%) and SSV (56.75
±
15.33%), all
of them with the same vitrification solution (P < 0.05). For follicular viability evaluation,
ovarian fragments were vitrified as described above. After warming, follicles were assessed
by trypan blue dye. Controversially, the highest percentage of viable follicles was observed
in MTV (97.06%) and was similar to fresh control (92.62%) (P < 0.05), but was significantly
different from SSV (81.08%) and CV (83.81%) (P < 0.05). These results were validated by
transmission electron microscopy that showed normal follicles observed in MTV and in
fresh control. In addition, to verify the MTV with VS4 (a combination of the best technique
plus the best solution), follicle viability was evaluated after 48 h in vitro culture. The viability
assay was performed by fluorescence microscopy (calcein-AM and ethidium homodimer-1)
analysis as follows: follicles isolated from fresh tissue were forthwith analyzed or underwent
48 h in vitro culture before analysis, whereas others fragments were vitrified/warmed
and immediately analyzed or underwent 48 h in vitro culture before analysis. These results
showed that, although follicular viability after MTV/VS4 (65%) was reduced when compared
to the non-vitrified follicles at day 0 (100%), follicular viability after MTV/VS4 at day
2 (36.5%) was similar to follicles vitrified at day 0 (65%) and similar to non-vitrified follicles
at day 2 (62.5%) (P > 0.05). As the decrease of viability in non-vitrified follicles at day
2 was similar to the decrease of MTV/VS4 in the same time, follicle viability at day 2 is
not affected by MTV/VS4. In conclusion, using the experimental conditions of the present
study, an efficient solution (VS4: 6 M EG, 0.25 M SUC and 10% FCS) and technique (MTV)
were successfully used to vitrify ovine ovarian tissue.
