The aim of this work was to characterize stress coping styles of Senegalese sole (Solea senegalensis) juveniles and breeders and to select an operational behavioural screening test (OBST) that can be used by the aquaculture industry to classify and select between behavioural phenotypes in order to improve production indicators. A total of 61 juveniles and 59 breeders were subjected to five individual behavioural tests and two grouping tests. At the end of the individual tests, all animals were blood sampled in order to measure cortisol, glucose and lactate. Three tests (restraining, new environment and confinement) characterized the stress coping style behaviour of Senegalese sole juveniles and breeders and demonstrated inter-individual consistency. Further, the tests when incorporated into a principal components analysis (PCA) (i) identified two principal axes of personality traits: ‘fearfulness-reactivity’ and ‘activity-exploration’, (ii) were representative of the physiological axis of stress coping style, and (iii) were validated by established group tests. This study proposed for the first time three individual coping style tests that reliably represented proactive and reactive personalities of Senegalese sole juveniles and breeders. In addition, the three proposed tests met some basic operational criteria (rapid testing, no special equipment and easy to apply and interpret) that could prove attractive for fish farmers to identify fish with a specific behaviour that gives advantages in the culture system and that could be used to establish selection-based breeding programmes to improve domestication and production.

The existence of daily rhythms under light/dark (LD) cycles in plasma cortisol, blood glucose and locomotor and self-feeding activities, as well as their persistence (circadian nature) under constant light (LL), was investigated in Senegalese sole (Solea senegalensis). For the cortisol and glucose rhythms study, 48 soles were equally distributed in 8 tanks and exposed to a 12:12 LD cycle and natural water temperature (experiment 1). After an acclimation period, blood was sampled every 3 h until a 24-h cycle was completed. Blood glucose levels were measured immediately after sampling, while plasma cortisol was measured later by ELISA. In experiment 2, the fish were exposed to LL for 11 days, and after this period, the same sampling procedure was repeated. For the study of locomotor and self-feeding rhythms (experiment 3), two groups of sole were used: one exposed to LD and the other to LL. Each group was distributed within 3 tanks equipped with infrared photocells for the record of locomotor activity, and self-feeders for feeding behavior characterization. The results revealed a marked oscillation in cortisol concentrations during the daily cycle under LD, with a peak (35.65 ± 3.14 ng/ml) in the afternoon (15:00 h) and very low levels during the night (5.30 ± 1.09 ng/ml). This cortisol rhythm persisted under LL conditions, with lower values (mean cortisol concentration = 7.12 ± 1.11 ng/ml) and with the peak shifted by 3 h. Both rhythms were confirmed by COSINOR analysis (p < 0.05). The synchronizing role of temperature and feeding schedule, in addition to light, is also discussed. Diel rhythms of glucose were not evident in LD or LL. As to locomotor and self-feeding activity, a very marked rhythm was observed under LD, with higher activity observed during the night, with acrophases located at 2:14 and 3:37 h, respectively. The statistical significance of daily rhythms was confirmed by COSINOR analysis. Under LL, both feeding and locomotor rhythms persisted, with an endogenous period (τ) around 22.5 h. In short, our findings described for the first time the existence of circadian cortisol and behavioral circadian rhythms in flat fish. Such results revealed the importance of taking into account the time of day when assessing stress responses and evaluating physiological indicators of stress in fish.

cies and also within some species. Mate selection and gamete release is a key factor that determines which genes or phenotypes are either passed to the next generation or lost (Berglund, 1997;Edward, 2015). Therefore, the description and understanding of a species spawning behaviour has implications for evolution and practical applications for aquaculture and conservation biology to maintain genetic pools and improve stocks. RESEARCH ARTICLE OPEN ACCESS AbstractSenegalese sole (Solea senegalensis) have a high potential for aquaculture that is hampered by reproductive behavioural problems. These problems result in limited breeder participation in spawning. The present study provided an ethogram and described mate selection and spawning of captive wild Senegalese sole. Two tanks of breeders were studied that had 29 and 25 breeders (mean weight = 1.6 ± 0.1 kg). The behaviour was studied during 20 periods of 24 hours: 10 periods where spawning events were recorded and 10 control periods without spawning events. Periods where spawning occurred had three times more locomotor activity than periods without spawning. Two distinct behaviours, termed the "following" behaviour and the "coupled swim", were only observed during periods with spawning. The courtship sequence (n=12) began with males predominantly involved in "following" behaviours, whilst females remained mainly stationary on the bottom of the tank. Males rested on the females and encouraged the females to begin swimming. When the female began to swim the male swam under the female and the pair made a "coupled swim" to the surface to release gametes. Gamete release was strictly in pairs of one male with one female. Failed "coupled swims" without gamete release were 5.6 times more frequent than successful "coupled swims". Mate selection was evident as the sole engaged in: paired spawning, males displayed to females, males encouraged females to spawn and females accepted or rejected the male's advances. The mate selection process provided the opportunity for fish to dominate the spawning and also demonstrated how fish were excluded from spawning.

One method to overcome the difficulties involved in reproducing first generation Senegalese sole that are born in captivity (F1) is to obtain gametes by stripping and then using them for artificial fertilization. The aim of this paper was to determine an appropriate protocol for hormonal induction of ovulation and artificial fertilization. To induce ovulation, two different GnRHa administration methods, weekly injections versus a single GnRHa implant, were compared. The number of spawns per female and the relative fecundity were significantly higher in females that received the hormonal therapy through repeated injection (6.3 ± 0.8 spawns per female and 574.9 ± 67.2 x 10 3 eggs kg -1 BW) than those treated with a single implant (2.7 ± 0.6 spawns per female and 134.6 ± 40.9 x 10 3 eggs kg -1 BW). The lapsed time to ovulation and the changes in fertilization and hatching rates of eggs that were retained in the ovarian cavity after ovulation were determined after GnRHa treatment. At 16°C, ovulation was detected at 41-44 h post-treatment in 70% of the females. The highest fertilization and hatching rates were obtained during the first 3 h after ovulation, and A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 they significantly decreased thereafter. The results suggested that the GnRHa injection was the most appropriate administration method for the induction of ovulation. Ovulated eggs inside the female were viable for a short time, and therefore it is important to determine the time of ovulation after hormonal treatment.

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