Cryopreservation of European eel ( Anguilla anguilla) spermatozoa: Effect of dilution ratio, foetal bovine serum supplementation, and cryoprotectants

The main aim of the present study was to investigate the effect of sperm freezing medium dilution ratio (1:1, 1:2, and 1:5 v/v), two cryoprotectants: dimethyl sulphoxide (Me 2SO) and methanol (MeOH), and the addition of foetal bovine serum (FBS) on the cryopreservation of European eel sperm. The effect of these factors was evaluated comparing post-thawing viability with fluorescent staining (Hoechst bisbenzimide 33258) and the spermatozoa head morphometry, determined with computer-assisted morphology analysis (ASMA). The 1:5 (v/v) dilution ratio resulted in a lower viability in comparison with 1:1 and 1:2 (52.8 ± 2.3% vs. 67.4 ± 2.3% and 65.1 ± 2.3%, respectively, p = 0.0001), but without effects on the head morphology. Although the viability was not significantly different between Me 2SO and MeOH (60.4 ± 1.9 vs. 63.2 ± 1.9%, respectively, p = 0.305), a decrease of spermatozoa head area and perimeter was found when spermatozoa were frozen with methanol (6.19 ± 0.01 vs. 6.36 ± 0.01 μm 2 and 17.28 ± 0.05 vs. 17.49 ± 0.05 μm, for area and perimeter and MeOH and Me 2SO, respectively, p = 0.0001). Finally, a higher viability (75.1 ± 1.7 vs. 48.5 ± 1.7, with or without FBS, respectively, p = 0.0001) and higher spermatozoa head size (6.40 ± 0.01 vs. 6.15 ± 0.01 μm 2 and 17.88 ± 0.05 vs. 16.89 ± 0.05 μm, for area and perimeter, with or without FBS, respectively, p = 0.0001) were found when cells were frozen-thawed in freezing media supplemented with FBS. Based on the above findings, dilution ratios lower than 1:5 (v/v) and the addition of serum improved the viability results after cryopreservation. Future studies are required in order to understand the spermatozoa membrane interchange mechanisms in response to the changes in spermatozoa head size caused by cryoprotectants and freezing media supplements.