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Trabajo presentado en el Seminario-Programa i-COOP, celebrado en Santa Marta (Colombia) del 10 al 18 de diciembre de 2022.

Trabajo presentado en el Seminario-Programa i-COOP, celebrado en Santa Marta (Colombia) de 10 al 18 de diciembre de 2022.

Resumen del trabajo presentado en el 8th International Workshop on the Biology of Fish Gametes, celebrado en Gdansk (Polonia) del 20 al 23 de septiembre de 2022., Our study aimed to optimize the sperm cryopreservation protocol of sea bass (Dicentrarchus labrax) and evaluate the post-thawing sperm dilution in species of interest for Mediterranean aquaculture. Sea bass sperm samples (n=6) were cryopreserved using a modified Non-Activating Media (NAM) extender with or without 5% Glucose (Glu), and 10% of Me2SO or MeOH as cryoprotectant. In a second experiment, sea bass sperm samples (n=7) were cryopreserved using a modified NAM extender and Me2SO. In both experiments, an aliquot of post-thawed sperm was diluted in NAM or NAM + Bovine Serum Albumin (BSA), or not diluted (control). Senegalese sole (Solea senegalensis) sperm samples (n=13) were cryopreserved. The post-thawed sperm was diluted in Mounib or NAM media, or not diluted (control). European eel (Anguilla anguilla) sperm samples (n=12) were cryopreserved. An aliquot of post-thaw sperm was diluted in P1 or P1 + BSA media, or not diluted (control). Kinetic parameters were checked after thawing: sea bass (0, 2.5 and 6 h), Senegalese sole (0 and 3 h), and European eel (0, 24, 48, 72 and 96 h). The total motility (MOT - %), progressive motility (MOTp - %), the velocities curvilinear (VCL - µm/s), straight line (VSL - µm/s) and average path (VAP - µm/s), were evaluated using a CASA-Mot software. The highest post-thawing kinetic parameters of sea bass sperm were observed when Me2SO or Me2SO+Glu were used and post-thawed sperm was diluted in NAM+BSA. The post-thaw sperm dilution increased kinetic parameters and prolonged sperm capacity compared to undiluted (control) samples. The post-thawing dilution did not increase the quality of the Senegalese sole sperm at 0 h. However, after 3 h, the spermatozoa diluted in NAM maintained the VCL. The European eel post-thaw sperm diluted in P1 maintained the MOT at 48 h and the velocities at 96 h after thawing. The combination of Me2SO+Glu can be used to cryopreserve sea bass sperm, and MeOH should be avoided. Sea bass post-thawing sperm dilution increases sperm kinetic parameters and prolongs its use at 2.5 h. Senegalese sole post-thaw sperm dilution did not affect the sperm kinetic parameters. European eel post-thaw diluted in NAM can be used without losing quality for 48 h after thawing., Supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/012) to SEASPERM. WAG has a Margarita Salas postdoctoral contract (RD 289/2021. UAB) by the Spanish Ministry of Universities. TSF and MPS have fellowships from Brazilian institutions: CNPq (200285/2021-1), and CAPES (Finance code 001), respectively.

Resumen del trabajo presentado en el 8th International Workshop on the Biology of Fish Gametes, celebrado en Gdansk (Polonia) del 20 al 23 de septiembre de 2022., The climate change includes a decrease in seawater pH, and an increase in its temperature. It is possible that the marine fish sperm cells, which are released to the sea at spawning, can be affected by these expected changes on the water, and become unable of fulfill its role on fertilization. For that reason, different seawater pH has been tested in 3 aquaculture marine species: the European eel (Anguilla anguilla), the European seabass (Dicentrarchus labrax), and the Senegalese sole (Solea senegalensis), and their sperm motility parameters analyzed by a CASA-mot system. In the European eel, tested seawater pH, from 6.5 to 9.5, affected sperm motility and other kinetic parameters like MP, FA, VCL, VAP, LIN, STR, WOB, ALH and BFC. pH values lower than 7.8 or higher than 8.2 caused lower values of motility and the rest of kinetic parameters. The longevity was not affected by pH from 7.6 to 8.2. In other experiment a seawater pH of 7.8 caused lower motility, FA, VSL, VAP and LIN than a seawater with pH 8.2. The effect of the water temperature was tested, by comparing activation with seawater at 4 °C (our control) and at 23-24 °C. The spermatozoa beating cross frequency (BCF) was the only parameter significantly affected, being lower at 23 °C than at 4 °C. In other experiment where we combined 2 temperatures (4, 24 °C) and two pH (7.8, 8.2), a significant interaction was observed, and in general the worst results were obtained with seawater at 4 °C and at pH 7.8. Considering all these data, it seems that the seawater pH has a deeper effect on the eel sperm motility than a high temperature. Regarding European sea bass and Senegalese sole, motility and other sperm parameters were not affected by seawater temperature in the pH range from 6.5 to 9.5, thus indicating that ocean acidification would not affect their behavior. The differences found between these species, evolutively and ecologically differents, could reflect different activation mechanisms of the spermatozoa motility., Supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/012) to SEASPERM. MM has a postdoctoral contract by Generalitat Valenciana (APOSTD/2020/053). WAG has a Margarita Salas postdoctoral contract (RD 289/2021. UAB) by the Spanish Ministry of Universities. TSF and MPS have fellowships from Brazilian institutions: CNPq (200285/2021-1), and CAPES (Finance code 001), respectively.

Trabajo presentado en el 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.

Trabajo presentado en el 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.

Trabajo presentado en 6th Genomics in Aquaculture Symposium, celebrado en Granada (España) del 04 al 06 de mayo de 2022.

Trabajo presentado en el Ciclo de Conferencias Red de Acuicultura, celebrado en Santiago de Chile (Chile) el 06 de abril de 2022.

XIII Congress of the Iberian Association of Comparative Endocrinology - XIII Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), 16-17 September 2021, Epigenetics concerns mechanisms modifying gene expression that do not imply changes to the DNA sequences. Epigenetic marks, also to be maintained during mitotic division, can be transmitted from parental germ cells to offspring. DNA methylation, one of the most studied epigenetic modifications, is commonly associated with gene expression silencing. In vertebrates, it mostly occurs in CpG sites. Promoters and other regulatory regions are usually enriched with CpGs. DNA methylation has important roles in vertebrates, being associated with phenomena such as genome imprinting and reprogramming, the latter playing an important role in embryonic development. Reprogramming during early development is well studied in mammals. However, much is still to be done in fish. Here we review the situation concerning epigenetic reprogramming events that occur during embryo development across vertebrates. In mammals is well demonstrated that reprogramming occurs during primordial germ cell development and early embryogenesis. In the second process, paternal and maternal methylomes experience active demethylation during the early cleavage stages, reaching the lowest level upon specification to the inner cell mass. Nevertheless, in fish, information is available only for a couple of species. In zebrafish (Danio rerio) methylation changes were mostly detected in oocyte-specific CpGs, to resemble paternal methylome before zygotic genome activation. In contrast, in medaka (Oryzias latipes), strategies similar to mammals seem to be adopted, since in both parental genomes methylation levels were decreased up to 20%, earlier, by the 1st cell cycle. This scarce data seems to suggest that epigenome reprogramming could be species-specific in fish. Consequently, our objective is to determine what is the situation during European sea bass (Dicentrarchus labrax) embryo development, firstly because it is phylogenetically distant from medaka (Cyprinodontiforme) and zebrafish (Cyprinid), and second, to improve our knowledge about epigenetic inheritance to devise breeding strategies able to maintain certain favorable traits in cultured species, Research funded by the Ministry of Science and Innovation “EPIPURE” (PID2019-108888RB-I00) grant to FP

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., Gilthead sea bream is a highly cultured marine fish in all the Mediterranean area, but new and strict criteria of welfare are needed to assure that the intensification of production has no negative effects in animal farming. Most welfare indicators are specific of a given phase of the production cycle, but others such as the timing of puberty and/or sex reversal are of retrospective value. This is of particular relevance in the protandrous gilthead sea bream, in which the sex ratio is highly regulated at the nutritional level. Social and environmental factors (e.g. contaminant loads) also alter sex ratio, but the contribution of genetic component remains unclear. To assess this complex issue, five sea bream families representative of slow/intermediate/fast growth were grown out with control or a plantbased diet in a common garden system from early life to completion of their sexual maturity in threeyear old fish. The plant-based diet enhanced the male-to-female sex reversal by more than 50% in average. This occurred in parallel with the progressive impairment of growth performance, which was indicative of changes in nutrient requirements as the result of the different energy demand for growth and reproduction through development. The effect of a different nutritional and genetic background on the reproductive performance was also assessed by measurements of circulating levels of sex steroids during two consecutive spawning seasons, varying plasma levels of 17β-estradiol (E2) and 11-ketotestosterone (11-KT) with age, gender, diet and genetic background. Principal component analysis of three-year-old fish displayed a gradual increase of the E2/11-KT ratio from males to females with the improvement of nutritional/genetic background. Altogether, these results support the use of a reproductive tract scoring system for leading farmed fish towards their optimum welfare condition, contributing to improve the productivity of the current gilthead sea bream livestocks., Bream-AquaINTECH (RTI2018-094128-B-I00) project

Trabajo presentado en el XVII Congreso Nacional de Acuicultura, celebrado en Cartagena (España) del 7 al 10 de mayo de 2019, [ES] La precocidad en machos de lubina (Dicentrarchus labrax L.) está bien documentada en la literatura y se conocen los efectos negativos que esta situación trae consigo para el sector acuícola. Sin embargo, la incidencia en hembras es menos conocida y el impacto que esta situación puede acarrear a nivel productivo no se ha evaluado. Por ello, en el presente trabajo se analizó el porcentaje de hembras prepúberes que, bajo condiciones de cultivo, son capaces de alcanzar un estadio de vitelogénesis avanzada (VTGa) a los dos años de edad. Un hecho que puede considerarse como un proceso de maduración gonadal adelantada en hembras de esta especie. Para ello, se establecieron tres lotes de lubinas en los que periódicamente se evaluó el crecimiento, los valores de ciertos índices biométricos y los niveles plasmáticos de hormonas clave en reproducción. La evaluación del estado gonadal se llevó a cabo mediante técnicas histológicas. Los resultados obtenidos indican que existen hembras prepúberes de dos años de edad que poseen ovocitos en estadio de VTGa y que este porcentaje puede llegar a alcanzar valores hasta del 25-28%. Estas hembras son un 17% más grandes en peso que aquéllas que poseen ovocitos inmaduros (crecimiento primario y/o vitelogénesis incipiente). Asimismo, estas hembras en VTGa poseen un índice gonadosomático mayor que el de las hembras inmaduras (i.e., 3,05% vs 0,49%) que se correlaciona con valores bajos de sus índices viscerosomáticos. A nivel hormonal, los valores plasmáticos de vitelogenina muestran diferencias significativas asociadas con el estado de desarrollo gonadal de las hembras., [EN] Precocious puberty in sea bass males (Dicentrarchus labrax L.) has been widely reported in the literature and the negative effects that it has in aquaculture are known. However, the incidence in females is little known and its impact in the sector has not been evaluated. In the present work, we analysed the percentage of prepuber females that, under rearing conditions, are able to develop oocytes in advanced vitellogenic stage (VTGa) at two years of age. This could be considered as females with an early gonadal development in this species. Three different trials were established and fish were periodically measured for somatic growth and biometric indexes, while plasmatic levels of reproductive hormones were also evaluated. The gonadal development was performed by histological analysis. Results demonstrated that prepuber females are able to exhbit VTGa up to 25-28% at 2 years old. These females are 17% heavier in weight than those with immature oocytes (i.e., primary oocytes and early vitelogenesis). Females in advanced vitellogenic stage have a gondosomatic index higher than that of immature females (i.e., 3.05% vs 0.49%) in correlation with the low values of their viscersomatic indexes. Plasmatic levels of vitellogenin showed significant differences according to ovarian stage of female fish.

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., The European eel (A. anguilla) is a valued species targeted for aquaculture production. However, it is considered a critically endangered species and its reproduction in captivity seems to be the only alternative to solve both economic and ecological problems. Although it is possible to obtain eel gametes for artificial fertilization, it requires longterm hormonal treatments. Recent studies found that cold seawater stimulates the brainpituitary-gonad axis in eel males, inducing the start of spermatogenesis. These results suggested that cold seawater could be used as a pre-treatment before the standard hormonal therapy applied to induce sexual maturation, to obtain higher sperm quantity and quality. Thus, our study evaluates the effects of cold seawater pre-treatments of different duration (1, 2 and 4-weeks) on reproductive performance of eel males. Biometric parameters from control and pre-treated fish were measured. Blood and testis samples were collected for steroid (T, 11-KT and E2) and histology analyses, respectively. Once eels were spermiating, sperm parameters were evaluated using a CASA system. Eel males pre-treated with cold temperature showed increased biometric indexes, androgens and E2 plasma levels. Males maintained at 10 ºC for 4-weeks showed a significantly higher percentage of differentiated type A spermatogonia in comparison to undifferentiated ones. However, the low sperm quality found in the pre-treated groups revealed that pretreatments followed by recombinant human chorionic gonadotropin (rechCG) treatment at high temperatures impairs spermatogenesis. Cold temperature pre-treated males had a time-dependent and fast response to rechCG treatment, but their spermiation period was shorter and sperm parameters were significantly lower. Further research is needed to study the effect of thermic pre-treatments on gene regulation controlling spermatogenesis., Funded by the Spanish MICIU (Project EELGONIA; RTI2018-096413-B-I00). LF and MM have contracts funded by Generalitat Valenciana (GRISOLIAP/2020/063 and APOSTD/2020/053). VG had a grant from the MICIU (IJCI2017-34200).

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., European sea bass (Dicentrarchus labrax L.) is a teleost fish species that presents sexual dimorphism affecting growth. Under culture condition, a substantial proportion of fish develop as males and those exhibiting larger size usually mature earlier during its first year of age (i.e., precocious males). This situation presents economic problems for producers and thus the production of highly female-biased stocks is considered as an opportunity to benefit its aquaculture. Females grow faster than males and mature later, around 3 years old, reaching a marketable size (∼400 g) after 2-year production cycle. Nevertheless, there is limited information about relationships among body weight in females and how this may affect the rate of oocyte development. The objective of this work is to determine those factors that are important links in the mechanisms by which growth influences ovarian development. To this aim, the evolution of growth and reproductive performance of prepubertal females has been determined in a total of seven batches of fertilized eggs that were reared over the first two years of age. Firstly, results show that there are significant differences of growth in females. Secondly, prepubertal females with slower growth remain immature while those fish with faster growth display advanced oocyte development. It evidences that sea bass females are able to undergo advanced vitellogenesis at 2 years of age although the rate of fish entering into advanced puberty varies among sea bass batches. Significant changes among body weight and plasma Fsh, E2, and Igf-1 levels are also observed between immature and advanced females. These findings suggest that these factors are important links that signals growth and nutritional status to the reproductive axis. The present results show that growth performance in female sea bass has indeed some interesting features that merit to be investigated to face its profitability in aquaculture., This research was financially supported by the Spanish Ministry of Science and Innovation (AGL2016-75400, PUBERTRAIT).

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: The European eel (Anguilla anguilla) is a highly valued species targeted for aquaculture production. However, it is considered a critically endangered species by CITES, and reproduction in captivity seems to be the only realistic alternative to solve both economic and ecological problems (Asturiano, 2020). It is possible to obtain gametes for artificial fertilization but it requires hormonal treatment, which is a time-consuming and expensive process (Gallego et al., 2012). Moreover, the variable response to these treatments results in high mortality rates during the early stages of development, which could be caused (at least in part) by low sperm quality and uncontrolled epigenetic factors (Herráez et al., 2017). Considering the environmental conditions experienced during eels oceanic migration, the effects of low temperature combined with hormonal treatment have been used to better understand the onset of eel maturation (Peñaranda et al., 2016). Thus, the present study considers the use of low temperature as a pre-treatment before starting a standard hormonal treatment and evaluates its impact on eel maturation and male reproductive performance., [Material and Methods]: Eighty-eight male European eels were maintained for 3-4 days in freshwater at 20 ºC. Then, 6 males were sacrificed, as initial control, and the rest were changed to seawater at 10 ºC, except the control group, which was changed to seawater but maintained at 20 ºC. Low water temperature pre-treatments were applied during 1, 2 or 4 weeks to groups of 20 males. Once finished each pre-treatment, 8 fish per group were sacrificed for sampling. The temperature of the water of the remaining males was increased to 20 ºC in 3-4 days and they started receiving standard hormonal treatment (weekly administration of recombinant chorionic human gonadotropin, rhCG; Ovitrelle, Spain, 1.5 IU/g fish). From sacrificed fish (controls and pre-treated) biometric parameters were measured. Blood and testis samples were used to perform steroid (T, 11-KT and E2) analyses and histology, respectively. Once eels were spermiating, sperm volume, motility and kinetic parameters were evaluated weekly using a CASA system., [Results]: The eye, fin and hepatosomatic indexes from 1-week, 2- and 4-weeks pre-treated fish were significantly higher than those of the control group. The gonadosomatic indexes registered in the control group were unusually high and significantly differed with 1-week pre-treatment group but not with the 2- and 4-weeks groups. Androgen levels (T, 11-KT) were significantly higher after 4-weeks of pre-treatment in comparison with the control. The E2 levels did not show the same increasing profile. The control and 1-week pre-treated males showed significantly higher percentages of undifferentiated and differentiated type A spermatogonia in comparison with early spermatogonia type B. The males after 2- and 4-weeks of pre-treatment revealed a significantly increased proportion of differentiated spermatogonia type A. After the rhCG treatment, males from the 4-weeks pre-treatment group started to produce sperm earlier than males from the other groups. The sperm volume in the 4-weeks pre-treatment group increased during the first weeks of spermiation and in the control group at the end of the experiment, but no significant differences were found. Sperm motility and kinetics parameters were low in pre-treated groups throughout the whole experiment while in the control group increased significantly at the end of the hormonal treatment., [Discussion]: The onset of maturation in the European eel is controlled by environmental conditions found during their migration. The application of pre-treatments can be important to induce maturation in aquaculture eels, which do not encounter these conditions (Palstra and van den Thillart, 2009). Several factors such as temperature, salinity, forced swimming and pressure have been used as part of pre-treatments applied to eels (reviewed by Asturiano et al., 2020). Rozenfeld et al. (2019) evaluated the effects of cold seawater in European eel maturation without hormonal treatment. In eels treated at 10 ºC, the T and 11-KT levels were higher and there was an increase in the proliferation of differentiated type A spermatogonia. Our results also corroborate that low-temperature pre-treatment (4-weeks at 10 °C) induces an early sexual development, thus leading to the release of androgens which promote the proliferation of type A spermatogonia. However, the low sperm quality found in the pre-treated groups revealed that thermal pre-treatment followed by rhCG treatment at high temperatures impairs spermatogenesis. Low-temperature pre-treatments induce testis cell proliferation and synchronization but inhibit further maturation. Peñaranda et al. (2016) suggested that low seawater temperature inhibits the gene expression of enzymes responsible for the synthesis of maturation-inducing steroids (MIS) during eels oceanic migration. Thus, the premature maturation of the testis is prevented before eels arrive to the spawning areas and a temperature threshold is reached allowing the change of the steroidogenic pathway to change from androgens to MIS. In our experiment, lowtemperature pre-treated males had a time-dependent and fast response to rhCG treatment (in comparison with control males maintained at 20 °C), but their spermiation period was shorter and their sperm motility parameters were significantly lower. If this is evidencing a lower rate of gene expression in the testis, and thus limiting the final sperm maturation process, is an open question that requires further research.

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., Several studies suggest that body weight in teleost during critical periods affects the proportion of fish that reach maturation in a given reproductive season. Although there is a large variation in the age of maturity, this trait depends on genetic, physiological and environmental factors that contribute to gonadal growth. While interactions between growth and reproductive competence in male fish have been reported, much is still to be resolved in females. In the sea bass a substantial proportion of males mature precociously during the first year of life suggesting that it is only allowed in those fish which have reached a certain threshold of development or accumulated enough lipid reserves. Precocious males, which are usually larger in size, exhibit a significant increase in 11-KT presumably induced by Fsh that is able to block amh expression, a meiosis inhibiting factor, thus evoking spermatogenesis progression. Little is known about the oocyte growth in prepubertal female sea bass and its relationships with body growth; however it has been reported that advanced puberty can also affect captivereared females. Recently, we have monitored the growth history of female sea bass and the degree of advancement in oocyte development during the first two years of life. Some body indexes and circulating vitellogenin served as markers of advanced ovarian growth. Over the second year of age, females could be divided into four distinct groups: females that remained at perinucleolar stage, females with oocytes in varying degrees of cortical alveoli incorporation and no lipid droplets, females with oocytes in secondary growth including oocytes in varying degrees of lipid droplet incorporation and no yolked droplets and females with oocytes with both lipid and yolked droplets inside. Consequently, our objective is to determine changes in ovarian gene influencing the rate of oocyte development in this aquaculture finfish species., This research was financially supported by the Spanish Ministry of Science and Innovation (AGL2016-75400, PUBERTRAIT).