Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.

31Since 1960, the European eel (Anguilla anguilla) has suffered a dramatic reduction in 32 natural stocks. Breeding in captivity is considered an alternative, but obtaining high 33 quality sperm seems basic on this regard. The main objective of this study was to assess 34 the effects of three thermal regimes (two of them variable: T10 and T15; and one of 35 them constant: T20) and three hormonal treatments with different hormones (hCG, 36 hCG rec and PSMG) on the induction of maturation in European eel males. 37In the case of the thermal regimes, our results demonstrated that the onset and 38 progression of spermiation are strongly influenced, and perhaps closely regulated, by 39 water temperature. T20 demonstrated the best results in all the sperm parameters 40 (volume, density, motility, kinetic features, etc) throughout most weeks of treatment, 41 becoming a reliable and productive method for inducing spermiation in this species. In 42 the case of hormonal treatments, the onset and progression of spermiation in European 43 eel males were influenced by the hormone used. In this respect, hCG rec produced the 44 best results in all the sperm parameters including volume, density, motility, kinetic 45 features, etc., throughout most weeks of treatment, thus becoming an effective 46 alternative treatment to the standard hCG treatment used to induce spermiation in eel 47 species. Moreover, hCG rec gave rise to the best economical profitability, making it 48 possible to obtain good quality sperm samples at a lower price than by using the other 49 two hormonal treatments. 50 51

No abstract

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.