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Summary In order to study different storage conditions, dilution ratios and additives for fresh European eel sperm were assayed to determinate the optimum conditions for short-term storage. Two experiments were conducted, analysing the sperm motility and its viability at different times of incubation (1, 4, 7, 11 and 14 days). In the first part of the work, 19 pooled samples were used, and seven in the second experiment. Firstly, exposure to atmospheric and air-limited conditions were compared, registering the highest motility under air-limited conditions, showing no differences to the performance of fresh sperm samples until 96 h of incubation. Also, two different dilutions were checked, with ratios 1 : 10 and 1 : 50, with the latter obtaining better results. A second experiment was performed trying to improve the results obtained in the first experiment. Using air-limited condition and 1 : 50 dilution ratio, the effect of different additives (foetal bovine serum, FBS; N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic Acid, TAPS; bovine serum albumin, BSA; Glucose) were evaluated. The sperm diluted in the media containing glucose provided motilities without difference in comparison to fresh samples during first week of incubation. At 7 days of storage, the medium containing 300 mm glucose was the only one causing higher motility (77.9 ± 2.5%) in comparison with control medium (50.1 ± 11.5%), demonstrating that the sugar addition was positive to preserve the sperm movement. In conclusion, a new method of eel sperm preservation has been developed, using air-limited conditions, 1 : 50 dilution ratio and glucose as an additive, which provided motilities without significant differences until 7 days of incubation.

Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4–15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions.

Summary As no studies have been carried out to investigate the role of fatty acids in male European eel (Anguilla anguilla) maturation, the main aim of this work was to determine which fatty acids could play a central role in sexual maturation. To this end, European eel males were hormonally induced to sexual maturation and variations in fatty acid composition in muscle, liver, testis and milt were studied. Eels were injected weekly with hCG (Human Chorionic Gonadotropin) for 13 weeks and 10 eels per week were sacrificed. Histological analysis was performed to determine the spermatogenic stage of each male. In muscle, no variations or preferential utilization of fatty acids were detected. In liver, MUFAs (Monounsaturated Fatty Acids) were the most abundant fatty acids in the first weeks, while in the last week PUFAs (Polyunsaturated Fatty Acids) showed the highest concentrations. In testes, PUFAs content increased due to DHA (Docosahexaenoic acid), EPA (Eicosapentaenoic acid) and AA (Arachidonic acid) accumulation, while MUFAs diminished especially due to oleic and palmitoleic acid decrease. Linolenic (EPA and DHA precursor) and linoleic (AA precursor) acids also decreased. As evidenced by histology, main changes in testes matched the appearance of spermatids and first spermatozoa, at the end of meiotic divisions. In milt, no variations were appreciated and PUFAs were the most abundant fatty acids, close to 50%. Due to their increase in testes and the high concentration in milt, PUFAs probably play an important role in the sexual maturation of European eel. Further research is necessary to determine the relationship between feeding, fatty acids and sperm quality.

Summary The aim of this study was to evaluate the effects of different extenders on sea bass (Dicentrarchus labrax) spermatozoa motility and morphology. Six sperm extenders based on inactivator media, DI1 (here named SAN) and Non-Activating Medium (NAM) were tested with European sea bass spermatozoa. The best results were obtained with NAM medium (59.83 mm NaCl, 12.91 mm MgCl2, 1.47 mm KCl, 3.51 mm CaCl2, 20 mm NaHCO3, 0.44 mm glucose) plus 1 and 2% of BSA (NAM1 and NAM2, respectively). The motility of the spermatozoa incubated in those media was similar to the fresh sperm until 48 h (NAM1: 74.3 ± 5.4; NAM2: 78.8 ± 5.8%, and higher than undiluted sperm, 19.1 ± 7.8). We also checked the spermatozoa motility and morphology reactions with some of the best extenders, NAM2 and SAN, and combined them with different concentrations (2, 5, 10%) of three cryoprotectants: methanol, glycerol and dimethyl sulphoxide (DMSO). Glycerol + SAN or NAM2 caused activation of spermatozoa motility, which was lost 5 min later. Methanol and DMSO plus NAM2 extenders resulted in a low activation level and high motility 5 min after incubation, identifying these combinations as good candidates to be used in the cryopreservation of the European sea bass spermatozoa.

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−1 fish (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 different 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 significant differences 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.