The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325-330 mOsm kg )1 ), pH (8.4-8.6) and the ionic composition (concentration of Na + , K + , Mg 2+ and Ca 2+ ) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K + concentration increased, while Ca 2+ and Mg 2+ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na + showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5±14.5 and 36.6±6.7%). The addition of L-a-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g À1 ¢sh (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer-assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium-velocity spermatozoa, as well as di¡erent motility parameters. Sperm samples from A-D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E-treated males showed 0% motile cells. No signi¢cant di¡erences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A^D groups.

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The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.