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Because of its complex life cycle and due to multiple stress factors, the European eel is suffering a dramatic decline and has been declared Critically Endangered by the IUCN. A possible way to address this problem could be relieve the pressure on natural stocks by implementing its mass production by artificial breeding protocols. A previous study based on direct observation and parentage assignment underlined the presence of an allegedly hierarchic structure among European eel males in semi-natural mating conditions, with a consequent bias in F1 proportion assigned to each one. The aim of the present study is to attest if a different fertilization protocol based on the artificial mixing of female eggs with different males’ milt in equal volume (1 F:4 M) could represent a solution to the disproportion observed in semi-natural conditions. For this purpose, six families of European eels were generated, and 10 species-specific microsatellite loci were used to infer offspring composition by paternity assignment on 280 samples. Due to the asynchronous ovarian development of female European eels, the percentage of fertilized eggs for each female showed a great variability, ranging from 4.70% to 94.50%. A proportion of 94.02% of genotyped offspring were assigned with high confidence to their true parents. As regard males’ fertilization pattern, no substantial differences from natural mating were observed: a single male accounted for most offspring, which was just mostly composed of full sibs. Concluding, the obtained results suggest that the admixture of an equal volume of different males’ milt seems to contribute to the single-locus genetic variability (observed heterozygosity higher than expected in 7 out 9 loci), but it is not sufficient to ensure all the males the same chance to transmit their gene pool, and new fertilization strategies must be developed.

European eel Anguilla anguilla is among the highly valued species for aquaculture. Since its peculiar biology, it is not yet possible to complete the whole life cycle in artificial conditions and its supply depends entirely on wild catches. In the last 50 years this species has suffered a population reduction of 99 % mainly due to overfishing. In a conservation perspective, it is of fundamental importance to improve the aquaculture production of European eel, to avoid the extinction of this species and preserve its residual genetic variability, allowing at the same time the fulfilling of costumers request without increasing its harvesting pressure.In this study we aimed to deepen the knowledge about the mechanisms at the basis of reproduction of the European eel in semi-natural conditions, through direct observation of spawning behaviour and through the paternity assignment using microsatellite markers. The systematic and prolonged observation of the reproductive behaviour of European eel and the contextual parentage analyses we carried out for the very first time in this species on 39 adults and 432 F1 randomly collected. We contributed to unravel the sexual behaviour of this species in the most common artificial reproduction conditions (polyandry), and define the precise courtship sequence until the release of gametes, and the male-male hierarchy in courtship. We characterized for the first time three main types of male: dominant (the first who starts the courtship, and the one with the majority of F1 assigned), subordinate (which starts the courtship only in a second time and with a minor percentage of F1 ascribed) and ineffective (which sometime appears totally disinterested to courtship and has few F1 or none).The evidences here produced represent an important attempt for developing good reproduction practices of the critically endangered European eel, providing a good starting point for its future aquaculture production. Keywords: Anguilla anguilla, Aquaculture, Artificial breeding, Kinship analysis, Parentage assignment