Reproduction, olfaction and dominance behaviour in Senegalese sole (Solea senegalensis)

Tesis por compendioSenegalese sole is a flatfish species with increasing importance for the aquaculture industry due to its good performance (survival and growth) in captivity and high market price.However,one of the principal bottlenecks to the expansion of the species culture is the reproductive behavioural dysfunction in cultured males,which complicates establishing a closed cycle in captivity.The reproductive behavioural dysfunction is exhibited by the cultured males that do not participate in the courtship.Therefore, Senegalese sole production relies on wild males,which is unsustainable in the long term.With the aim to understand and look for solutions to this bottleneck,the present thesis focuses on different aspects of behaviour related to reproduction and the olfactory system. The effect of the presence of spawning wild Senegalese sole breeders on the reproductive behaviour and reproductive success of cohabiting cultured breeders was examined.Three groups were established,1 formed by cultures sole and 2 mixed groups (cultured and wild).Fertilised spawns were not obtained from the control group.However,fertilised spawns were obtained from the mixed-origin groups.Cultured males were observed to participate increasingly in the courtship and one cultured male fertilised 2 spawns.These results suggested a learning process in this species for behaviours associated to reproduction.With the olfactory analysis,the structure of the olfactory rosettes of 2 groups from different origin of Senegalese sole juveniles were compared.No significant differences in tissue structure,cell types and cellular distribution pattern were observed between origin specimens,however,differences were found between rosettes in number of lamellae and amount of goblet cells in the ridge region of the lamella,which were more frequent in the lower rosette.Related to the comparison of transcriptomic profiles (RNA-seq) of the upper rosette in cultured and wild sole mature males,the difference in transcripts of some olfactory receptors and other transcripts associated with the control of reproduction demonstrated clear differences in functionalities between origins.Related to the olfactory sensitivity of cultured sole, juveniles and adults to urine from mature conspecific (wild and cultured) was evaluated using EOG.Urine was confirmed to be a potent olfactory stimulus for juvenile and adult.In addition to this, urine from mature females evoked a small, but significant increase in plasmas levels of LH in mature males,further demonstrating that urine play a role in reproduction in this species.Related to dominance in Senegalese sole juveniles were used to conduct 2 dyadic tests (feeding response and territory) and group tests.This was the first study related to dominance behaviour in this species, which could be very relevant to the low participation in the parental contribution during the spawning season.Three parameters related to feeding response, 2 variables related to territory and 2 index in the group test were extracted to differentiate between dominant and subordinate sole.Moreover,2transcripts related to neurogenesis and neuroplasticity were differentially expressed between dominant and subordinate.In the present study three stress coping styles categories were found (proactive,intermediate and reactive) in Senegalese sole juveniles.Moreover,there were four transcripts related to metabolism and feeding behaviour which linked behavioural stress coping categories with brain gene expression.All together these advances strengthen the importance of these research lines as areas that can give a solution to the reproductive dysfunction that can enable the aquaculture industry to close the species life cycle in captivity to make sole culture sustainable.El lenguado Senegalés es una especie de pez plano que está en constante crecimiento de producción dentro de la industria de la acuicultura debido a su buena capacidad de supervivencia y crecimiento en cautividad,además de poseer un elevado precio en el mercado.En cambio,uno de los principales problemas de expansión del cultivo del lenguado es la disfunción reproductiva asociada al comportamiento complicando el cierre del ciclo de vida de esta especie en cautividad.La disfunción reproductiva se ve en los machos de cultivo que no participan en el cortejo.Consecuentemente,la producción del lenguado recae en los machos salvajes siendo insostenible a largo plazo.Esta tesis tiene como objetivo principal entender y buscar posibles soluciones a este problema enfocándose en diferentes aspectos relacionados con la reproducción y el sistema olfativo.Primero se realizó un análisis de efecto de la presencia de lenguados salvajes que se reproducen,en el cortejo y el éxito reproductivo de los lenguados de cultivo,que no se reproducen.Se formaron 3 grupos,1 de cultivo (control) y 2 mezcla (cultivo y salvajes).No se obtuvo puestas fecundadas del grupo control,en cabio si se obtuvo de los grupos mezcla,viendose por primera vez un aumento de participación de los machos de cultivo,donde un macho de cultivo fertilizó 2 puestas.Estos resultados sugieren un posible proceso de aprendizaje del cortejo por parte de los machos de cultivo.Con el análisis del olfato,primero se realizó una descripción y comparación histológica de la estructura de ambas rosetas olfativas de dos grupos de lenguados juveniles (cultivo y salvaje).No se hallaron diferencias significativas en la estructura tisular, tipos de células y distribución celular entre lenguados de diferente origen, en cambio, se encontraron diferencias entre las rosetas en número total de lamelas y en cantidad de células goblet concentradas en la parte apical de las lamelas siendo más frecuente en la roseta inferior.Con referencia a la comparación transcriptómica (RNA-seq) de la roseta superior en machos maduros salvajes y de cultivo,hubo diferencias significativas en transcritos asociados a receptores olfativos y de reproducción mostrando claras diferencias en la funcionalidad de la roseta en lenguados de diferente origen.En referencia a la sensibilidad olfativa a la orina del lenguados maduros medida a través del EOG,se demostró que la orina es un potente estimulante olfativo en lenguados juveniles y adultos de cultivo y además el incremento de LH en plasma de los machos adultos utilizados en este estudio demostró que la orina juega un papel importante en la reproducción de dicha especie.En referencia a los estudios de dominancia en lenguados juveniles,se utilizaron 2 pruebas diádicas(alimentación y territorio) y una grupal extrajeron 3 parámetros de dominancia asociados a la alimentación,2 variables asociadas al territorio y 2 índices en el test grupal los cuales diferenciaron entre dominantes y subordinados.Además hubo dos transcritos relacionados con la neurogenesis y la neuroplasticidad que se expresaron diferencialmente entre dominantes y subordinados.Este estudio ha sido el primero en reportar la dominancia como comportamiento en esta especie, la cual podría ser relevante debido a la baja contribución parental durante la época de puesta.Así mismo,se determinaron 3 categorias de estilos de afrontamiento al estrés (proactivos,intermedios y reactivos) en lenguados juveniles.Se encontraron 4 transcritos asociados al metabolismo y comportamiento de alimentación que relacionó los prfiles de comportamiento con la expresión génica cerebral en lenguados juveniles siendo genes importantes a nivel biolígico y funcional. Conjuntamente estos avances fortalecen la importancia de estas líneas de investigación como áreas que pueden dar solución a la disfunción reproductiva pudiendo cerrar el ciclo de vida del lenguado en cautividad para hacer el cultivo sostEl llenguado Senegalès es una espècie de peix pla que està en constant creixement de producció dins de la industria de l'aqüicultura degut a la seua capacitat de supervivència i creixement en captivitat,a més de posseir un elevat preu al mercat.En canvi,un dels principals problemes d'expansió del cultiu d'aquesta espècie és la disfunció reproductiva associada al comportament que complica tancar el cicle de vida del llenguado en captivitat.La disfunció reproductiva s'observa als mascles de cultiu que no participen al seguici.Conseqüentment,la producció del llenguado Senegalès depèn dels mascles salvatges el qual es insostenible a llarg termini.Esta tesi té com objectiu principal entendre i buscar possibles solucions a aquest problema enfocant-se en diferents aspectes relacionats amb la reproducció i el sistema olfactiu.Primer es realitzà un anàlisi d'efecte de la presència de llenguados salvatges que es reprodueixen en el seguici i l'èxit reproductiu dels llenguados de cultiu que no es reprodueixen.Es formà 3 grups,un de cultiu (control) i 2 mescla (cultiu i salvatges).No s'obtingué posts fecundades del grup control,en canvi si s'obtingueren dels grups mescla,veient un augment de participació dels mascles de cultiu per primera volta,on un mascle de cultiu va fertilitzar 2 postes.Aquests resultats suggereixen un procés d'aprenentatge del seguici per part dels mascles de cultiu.Amb l'anàlisi de l'olfacte,primer es realitzà una descripció i comparació estructural histològica de ambdues rosetes olfactives de 2 grups de llenguados juvenils dels dos orígens.No es trobà diferències en l'estructura tissular,tipus de cèl·lules i distribució entre llenguados de diferent origen,en canvi,es trobà diferències entre rosetes en nombre total de lamel·les i quantitat de cèl·lules goblet concentrades a la part apical de la lamel·la més freqüents a la roseta inferior.En referència a la comparació transcriptómica (RNA-seq) de la roseta superior en mascles madurs salvatges i de cultiu,es trobà diferències en transcrits associats a receptors olfactius i de reproducció mostrant clares diferències en la funcionalitat de la roseta en llenguados de diferent origen.En referència a la sensibilitat olfactiva de l'orina de llenguados madurs mesurada amb EOG,es demostrà que l'orina es un potent estimulant olfactiu en llenguados juvenils i adults de cultiu i l'increment de LH en sang dels mascles adults utilitzats en aquest estudi demostrà que l'orina juga un paper important en la reproducció d'aquesta espècie.En referència als estudis de dominància de llenguados juvenils utilitzant 2 probes diàdiques (alimentació i territori) i 1 grupal,van extraure 3 paràmetres de dominància associats a l'alimentació,2 variables associades amb el territori i 2 índex en el test grupal els quals diferencià entre dominants i subordinats.Aquest etudi ha sigut el primer en reportar la dominància com a comportament en aquesta espècie,la qual podria ser important degut a la baixa participació parental durant l'època de posta.Així mateix,es determinà 3 categories d'estil d'afrontament a l'estrès (proactius,entremitjos i reactius) en llenguados juvenils.Es van trobar 4 transcrits associats al metabolisme i comportament d'alimentació sent tots ells gens d'importància biològica i funcional que s'expressaren diferencialment entre perfils de comportament.Conjuntament aquests avanços enforteixen la importància d'aquestes línies d'investigació com àrees que puguin donar solució a la disfunció reproductiva podent tancar el cicle de vida del llenguado en captivitat per a fer la producció d'aquesta espècie sostenible en l'industria de l'aqüicultura.Fatsini Fernández, E. (2017). Reproduction, olfaction and dominance behaviour in Senegalese sole (Solea senegalensis) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/81550TESISCompendi

The use of sand substrate modulates dominance behaviour and brain gene expression in a Flatfish Species

Physical complexity adds physical enrichment to rearing conditions. This enrichment promotes fish welfare and reduces detrimental characteristics that fish develop in captivity. Senegalese sole (Solea senegalensis) is an important species for European aquaculture, where it is reared in intensive conditions using fibreglass tanks. However, reproductive dysfunctions present in this species do not allow it to complete its life cycle in captivity. Recently, dominance behaviour has been studied to try to solve this problem. The present study aimed to assess the effect of sand as environmental enrichment in the dominance behaviour and brain mRNA abundance of Senegalese sole juveniles. Four tanks of sole (n = 48 fish in total) were established in two different environments (with and without sand). Juveniles were subjected to dominance tests of feeding and territoriality. Behaviours analysed by video recordings related to the distance from the food delivered and harassment behaviour towards other individuals (e.g., resting of the head on another individual). In both environments, dominant sole were the first to feed, displayed more head-resting behaviour and dominated the area close to the feeding point, where the events were reduced in fish maintained in the sand. mRNA expression related to differentiation of dopamine neurons (nr4a2) and regulation of maturation (fshra) were significantly upregulated in dominant fish in the sand environment compared to dominants maintained without sand. The use of an enriched environment may affect Senegalese sole dominance, enhance welfare and possibly advance future maturation.Programa Operacional Mar2020, MAR-16-02-01-FMP-0059; d CONDISOLE (CeiMar funds, CEIJ-005); LA/P0101/2020;info:eu-repo/semantics/publishedVersio

Dominance behaviour in a non-aggressive flatfish, Senegalese sole (Solea senegalensis) and brain mRNA abundance of selected transcripts

Dominance is defined as the preferential access to limited resources. The present study aimed to characterise dominance in a non-aggressive flatfish species, the Senegalese sole (Solea senegalensis) by 1) identifying dominance categories and associated behaviours and 2) linking dominance categories (dominant and subordinate) with the abundance of selected mRNA transcripts in the brain. Early juveniles (n= 74, 37 pairs) were subjected to a dyadic dominance test, related to feeding, and once behavioural phenotypes had been described the abundance of ten selected mRNAs related to dominance and aggressiveness was measured in the brain. Late juveniles were subjected to two dyadic dominance tests (n= 34, 17 pairs), related to feeding and territoriality and one group test (n= 24, 4 groups of 6 fish). Sole feeding first were categorized as dominant and sole feeding second or not feeding as subordinate. Three social behaviours (i. “Resting the head” on another fish, ii. “Approaching” another fish, iii. “Swimming above another” fish) were associated with dominance of feeding. Two other variables (i. Total time occupying the preferred area during the last 2 hours of the 24 h test, ii. Organisms occupying the preferred area when the test ended) were representative of dominance in the place preference test. In all tests, dominant fish compared to subordinate fish displayed a significantly higher number of the behaviours “Rest the head” and “Approaches”. Moreover, dominant sole dominated the sand at the end of the test, and in the group test dominated the area close to the feed delivery point before feed was delivered. The mRNA abundance of the selected mRNAs related to neurogenesis (nrd2) and neuroplasticity (c-fos) in dominant sole compared to subordinate were significantly different. This is the first study to characterise dominance categories with associated behaviours and mRNA abundance in Senegalese sole and provides tools to study dominance related problems in feeding and reproduction in aquaculture

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis

Cryopreservation of germ cells would facilitate the availability of cells at any time allow ing the selection of donors and maintaining quality control for further applications such as transplan tation and germline recovery. In the present study, we analyzed the efciency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In tes tes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatch ing (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 106 cells). Lipid peroxidation and DNA frag mentation were also signifcantly lower in this treat ment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, refected also in terms of DNA damage. Transplan tation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germline.LA/P/0101/2020info:eu-repo/semantics/publishedVersio

The presence of wild Senegalese sole breeders improves courtship and reproductive success in cultured conspecifics

The life cycle of Senegalese sole (Solea senegalensis) is not closed in captivity due to a reproductive dysfunction related to the lack of participation of cultured male breeders in the courtship. To discern a possible solution to this social reproductive dysfunction, the main objective of this study was to determine the effect of the presence of spawning wild Senegalese sole breeders on the courtship and reproduction of cultured breeders. Three experimental groups were formed: Control group (n = 10) formed with only cultured sole; groups M1 and M2 constituted of mixed-origin sole (10 cultured and 8 or 9 wild breeders). All cultured breeders were from the same stock, which had never successfully reproduced, whilst the wild broodstock had spawned viable eggs in captivity. All groups were held in the same captive conditions and spawning, and behaviour were recorded for each group over four spawning seasons. All spawns were collected, and the parameters registered were floating and non-floating eggs, fertilization and hatching rates. In addition, parental analysis was made of larvae from viable spawns. Behaviour was studied with video recordings to compare locomotor activity and courtship behaviours including the "Follow" behaviours where sole swim after each other in a procession. Fertilized spawns were obtained from the mixed-origin groups (M1 and M2) including spawns involving a cultured male. The cultured males also participated actively in the "Follow" behaviours with the courting wild sole and this participation of cultured males increased significantly over the four years of the experiment. Male percentage participation in the "Follow" behaviours was positively correlated (R = 0.81) to participation in spawning to indicate the importance of increasing cultured male participation in the "Follow" behaviours. A total of seven spawns were obtained from a cultured male that fertilized eggs from one cultured female and two wild females. The same cultured female also spawned with a wild male. No viable spawns were obtained from the Control group and locomotor activity and courtship behaviour counts were significantly lower than in the experimental mixedorigin groups. This is the first report of cultured male breeders participating in reproductive behaviour and spawning, which could be associated with social learning processes, mate selection and dominance where cultured males copied the behaviour and spawning of wild Senegalese sole breeders.Spanish National Institute for Agronomic Research (INIA)-FEDER [RTA201100050, RTA2014-00048]FCT - Foundation for Science and TechnologyPortuguese Foundation for Science and Technology [UID/Multi/04326/2019]info:eu-repo/semantics/submittedVersio

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis

[EN] Cryopreservation of germ cells would facilitate the availability of cells at any time allowing the selection of donors and maintaining quality control for further applications such as transplantation and germline recovery. In the present study, we analyzed the efficiency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In testes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialde- hyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatching (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 10 6 cells). Lipid peroxidation and DNA fragmentation were also significantly lower in this treatment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, reflected also in terms of DNA damage. Transplantation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germlineSIOpen access funding provided by FCT|FCCN (b-on). This study received Portuguese national funds from Foundation for Science and Technology (FCT) through project GERMROS (EXPL/CVT-CVT/0305/2021) and European Union’s Horizon 2020 research and innovation program under grant agreement No. 871108 (AQUAEXCEL3.0). This work was also supported by CCMAR Strategic Plan-UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020. E. Fatsini was supported by FCT contract 2020.04181.CEECIN

Linking stress coping styles with brain mRNA abundance of selected transcripts for Senegalese sole (Solea senegalensis) juveniles

In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable “Total activity” (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt-qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß, showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole.info:eu-repo/semantics/acceptedVersio

Senegalese sole (Solea senegalensis) coping styles are consistent over time: behavioural and physiological responses during ontogenesis

Individuals differ in how they cope with stressful situations along a behavioural continuum, being proactive and reactive at the extremes of this continuum. Proactive individuals are usually bold, highly active and take risks, while reactive organisms are generally shy, exhibit low activity and avoid risky situations. Definitions of stress coping styles state that proactive and reactive traits are consistent over time and across contexts. The present study evaluated the individual differences in stress coping style, physiological changes and reproductive status in Senegalese sole juveniles and breeders over three and two-years, respectively. To determine stress coping style, the fish were subjected to three individual (restraining, new environment, confinement) and one group screening test (risk taking). Both groups were tested on three occasions, juveniles were tested each year and adults were tested in the first year and twice (spring and autumn) in the second year. On the third year, a proportion of the juveniles initiated puberty and the reproductive status of all individuals was assessed and compared with their behavioural responses. Results demonstrated individual differences that were consistent with proactive and reactive traits in juveniles and breeders. Significant intra-individual repeatability and consistency of juveniles and breeder's behavioural responses were observed over time and across situations. In addition, glucocorticoid levels (cortisol) were consistent for individuals. Another result to highlight was that juveniles that past puberty and initiated gametogenesis had significant higher activity, risk predisposition and lower plasma cortisol levels compared to fish that remained immature (did not initiate puberty)

Kisspeptin influences the reproductive axis and circulating levels of microRNAs in Senegalese sole

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.Project ReproF1 (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059 to E.C.), FCT: UIDB/04326/2020/ DL 57/2016/CP1361/CT0007 and SFRH/BD/130910/2017. COST O ce (COST Action 16120: EPITRANS)info:eu-repo/semantics/publishedVersio

Exploring the relationship between stress coping styles and sex, origin and reproductive success, in Senegalese sole (Solea senegalensis) breeders in captivity

Individual animals commonly adopt different stress coping styles that have been shown to impact reproductive success and differ between sexes (female/male) and origin (wild/hatchery). Hatchery reared Senegalese sole (Solea senegalensis) exhibit a behavioural reproductive dysfunction and a complete failure to spawn viable eggs. Hence, the present study examined whether 1) reproductive success of Senegalese sole was linked to coping styles and 2) behavioural differences exist in relation to sex or origin. A total of 198 breeders held in two research institutions were submitted to three individual tests (restraining, new environment and confinement) and one grouping test (risk taking). In addition, a blood sample to quantify cortisol, glucose and lactate levels was obtained from each individual after completing the individual tests. Senegalese sole breeders showed individual differences in behaviour across the different behavioural tests that were consistent with proactive and reactive coping styles traits. However, the most striking result was that reproductive success, sex and origin of Senegalese sole was not biased to any specific coping style. Indeed, the behavioural responses were similar and consistent between fish grouped by reproductive success, sex and origin. This study presented information that contrasts with different studies on dominant aggressive species and indicated that social non-aggressive species such as Senegalese sole follow a cooperative strategy that favours equal opportunities between stress coping styles and sexes. Therefore, results suggest that maintaining both coping styles strategies are fundamental for a sustainable breeder population approach