Male Sea Bass Research Articles

Vitamin E in early stages of sea bass ( Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass ( Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.

Effects of photoperiod on pituitary levels of three forms of GnRH and reproductive hormones in the male European sea bass ( Dicentrarchus labrax, L.) during testicular differentiation and first testicular recrudescence

Photoperiod is one of the main external cues in the control of different stages of fish development. In the present work, the effect of several photoperiods, known to delay spawning in adult fish, was investigated in juvenile male sea bass ( Dicentrarchus labrax) during testicular differentiation and first testicular recrudescence. Fish were exposed to constant long (LO), expanded (EX), constant short photoperiod with long photoperiod in October (SLoct) and constant short photoperiod with long photoperiod in December (SLdec) during two consecutive years. Pituitary levels of three forms of gonadotropin-releasing hormone (GnRH) [sea bream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH)] were analysed and plasma levels of gonadotropin (LH), testosterone (T), and 11-ketotestosterone (11-KT) were determined in the same periods. During both periods, pituitary levels of sbGnRH were higher than cGnRH-II and sGnRH in all groups. The three GnRHs had significantly lower values in November 1995 in groups exposed to artificial photoperiods during the testicular differentiation and growth period compared to the control group. In addition, a significant effect of LO, EX, and SLoct photoperiods was observed in the LH plasma profile, showing a significant delay in the peak of LH compared to the control group. However, no significant effect of the artificial photoperiods on the pituitary content of the three GnRHs has been observed during the first testicular recrudescence. Deeper studies are needed to understand the effect of artificial photoperiods on the endocrine events occurring during both histological testicular differentiation and growth and first testicular maturation in the sea bass.

Effect of photoperiod manipulation on the daily rhythms of melatonin and reproductive hormones in caged European sea bass ( Dicentrarchus labrax)

Reproduction in fish is cyclical and timed to guarantee the survival of the offspring. Seasonal variations in reproductive hormones of fish have been deeply investigated in fish over the last years. However, there are few studies regarding the daily changes in reproductive hormone profiles in teleosts. The aim of the present research was to investigate the effects of photoperiod manipulation on melatonin and reproductive hormones (pituitary sbGnRH, pituitary LH and plasma LH, testosterone [T], and 11-ketotestosterone [11KT]) daily rhythms in male sea bass, kept in net cages under farming conditions in winter (9L:15D). Fish were distributed in two groups, one under constant long photoperiod (18L:6D) and the other under natural photoperiod. The photoperiod strongly influenced the daily melatonin profile, so that the duration of the nocturnal melatonin rise was longer in the control group than in the group exposed to the artificial photoperiod (18L:6D). A daily rhythm was observed in the pituitary sbGnRH profile in both groups, showing the lowest levels during the dark period. A daily rhythm of pituitary LH was detected in the control group, which was suppressed in the group under long photoperiod. Daily variations in plasma LH were observed, the highest levels being found in the dark phase in both groups, although this profile was significantly altered by artificial light, maintaining a fixed relationship between the first nocturnal rise of melatonin and the nocturnal peaks of plasma LH in both groups. Plasma T levels showed significant fluctuations in their daily cycle following a sinusoidal pattern with an acrophase around sunrise in both groups, without any influence of light regime. No significant daily variations in plasma levels of 11-KT were observed in none of the groups. Our results provide the first evidence of the presence of daily variations in pituitary sbGnRH content, pituitary and plasma LH, and plasma T in sea bass. Artificial lights suppressed the circulating melatonin and significantly affected the daily rhythm of LH storage and release.

Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass ( Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain–pituitary cDNA library. The nucleotide sequences of the common α, the FSHβ, and the LHβ subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHβ is more similar to higher vertebrate FSHβ’s (35–37%) than to LHβ’s (26–30%), whereas sea bass LHβ is more similar to LHβ’s (40–53%) than to FSHβ’s (26–41%). Phylogenetic analysis of fish GTH sequences grouped the β subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.

Group-synchronous ovarian development, spawning and spermiation in the European sea bass (<i>Dicentrarchus labrax</i> L.) could be regulated by shifts in gonadal steroidogenesis

European sea bass ( Dicentrarchus labrax L.) is a species with group-synchronous ovarian development. A mechanism is required which enables maturation to occur in the first clutches of oocytes without inducing maturation in subsequent clutches. The present study examined the individual plasma variations of testosterone (T), 17,20?-dihydroxy-4-pregnen-3-one (17,20?P) and 17,20?,21-trihydroxy-4-pregnen-3-one (20?S) in both sexes, estradiol (E 2 ) and vitellogenin (VTG) in females (n=15) and 11-ketotestosterone (11KT) in males (n=21), in an effort to elucidate the hormonal control of the reproductive cycle in this species. A sample of oocytes was obtained at every sampling from each female and the stage of development of the most advanced clutch of oocytes was determined and related to the individual hormone plasma levels. Total expressible milt was collected from males at each sampling during the spawning period and variations in the sperm production were related to hormone plasma levels. Successive elevations of plasma T and E 2 levels were observed prior to peaks of progestagens, which resulted from the shift in gonadal steroidogenesis and coincided with the maturation-ovulation of the different clutches of oocytes or with increases in the sperm production. Following each progestagens wave, a new shift in gonadal steroidogenesis, resulting in a new elevation in plasma T and E 2 , was observed. This hormonal pattern was repeated several times depending on the number of ovulations per female. Results from the present study suggest a mechanism, based on shifts in gonadal steroidogenesis, which may be responsible for regulation of group-synchronous ovarian development, ovulation and spermiation in this species. In addition, evidence is presented which supports a role for both 17,20?P and 20?S as the maturation-inducing steroids (MIS) in male and female European sea bass.

Stress-induced changes of plasma antioxidants in aquacultured sea bass, Dicentrarchus labrax

Antioxidant plasma activities of ascorbate, α-tocopherol and glutathione peroxidase were analysed in adult male sea bass, Dicentrarchus labrax, in normal conditions and after hypoxia-recovery. In addition, tank measurements of temperature, pH, salinity and chlorine changes were carried out. Ascorbate and α-tocopherol were measured using a high-pressure liquid chromatography method and glutathione peroxidase activity enzymatically. Ascorbate and α-tocopherol showed a relationship with the velocity of body growing in normal and hypoxia-recovery conditions. In sea bass exposed to hypoxia, only ascorbate and α-tocopherol levels were significantly lower compared with the control group. Slope study and expression percent of antioxidants reduction after stress conditions revealed a predominant role of plasma α-tocopherol. Sea bass subjected to variations of salinity and chlorine showed a significant decrease in plasma α-tocopherol. A relationship could be suggested between antioxidant defence and fish response in aquaculture.

Changes in plasma levels of reproductive hormones during first sexual maturation in European male sea bass ( Dicentrarchus labrax L.) under artificial day lengths

Photoperiod has been considered one of the most important factors triggering puberty, as well as reproduction, in several fish species, including sea bass ( Dicentrarchus labrax L.). In the present work, the effect of expanded (EX) and compressed (CO) photoperiods on plasma levels of reproductive hormones (gonadotropin-2 (GTH-2), testosterone (T) and 11-ketotestosterone (11-KT)) and gonadal maturation (spermiation time and gonadosomatic index (GSI)) were investigated in male sea bass during the first sexual maturation (October to April). Spermiation in controls was apparent from December to February–March. In EX and CO groups, spermiation was advanced by 2 months, although the CO group displayed a bimodal pattern, and testicular growth in both experimental groups was significantly reduced with respect to the controls. Plasma GTH-2 levels in controls showed the highest value (∼30 ng/ml) in the middle of the spermiation period, while EX group displayed the maximum level 4 months earlier (35±2.7 ng/ml) than controls. The CO group presented two peaks, the first of which (15.16±5.20 ng/ml) was advanced by 3 months with respect to the control peak. T and 11-KT levels in the control displayed the highest values during the spermiation period. The EX group showed lower T levels than controls, but both peaked at the same time. However, 11-KT levels remained low and unchanged. The CO group displayed two significant increases in T levels accordingly with the spermiation pattern, while the 11-KT profile only exhibited a significant increase 2 months earlier than in controls. Results obtained indicated an involvement of GTH-2 in gonadal maturation. In addition, T is suggested to be involved in the activation of the brain–pituitary–gonad (BPG) axis during pubertal development, while 11-KT may act by stimulating spermatogenesis and/or spermiation in juvenile male sea bass. Furthermore, the profiles of these reproductive hormones were altered by both expanded and compressed photoperiods, and first sexual maturation was advanced by at least 2 months.

Influence of daylength on the age at first maturity and somatic growth in male sea bass ( Dicentrarchus labrax, L.)

Groups of 4-month-old sea bass were each exposed over three consecutive years to a natural photoperiod (40°N) (NP), constant long photoperiod (15L:9D) (LO) and 18-month-expanded photoperiod (the natural light cycle extended to 18 months) (EX), under natural conditions of temperature (12.0–26.3°C). During the differentiation period, a significant increase in number of precocious males was found in the LO (26.8%) and EX (17.7%) groups compared to controls (5.3%) ( P<0.05). During the first reproductive season (20–25 months of age) gonadal size (GSI) in both the LO (0.82±0.16%) and the EX (0.28±0.07%) groups was significantly lower than controls (1.28±0.41%). The percentage of spermiating males during the first reproductive period was lower in LO (<60%) and EX (<35%) groups compared to controls (>80%). At the second reproductive period (32–37 months of age) gonadal maturation was advanced in EX group, whereas a significant delay was observed in LO group with respect to control. Spermiation in EX group was advanced with respect to control group. During the second reproductive period, percentage of spermiating males was significantly higher in EX group (∼80%) than in LO group (<50%). LO and EX groups displayed enhanced growth (655.52±37.21 g and 376.40±6.33 mm; 510.44±25.80 g and 350.14±5.27 mm, respectively) compared to controls (459.93±21.20 g and 343.62±4.82 mm). The present results indicate a significant beneficial effect of constant long and expanded photoperiods on reducing sea bass gonadal development and enhancing growth at the age of commercialisation, with a potential application in aquaculture.

Pituitary Levels of Three Forms of GnRH in the Male European Sea Bass (Dicentrarchus labrax, L.) during Sex Differentiation and First Spawning Season

In the present study, levels of three GnRH forms [seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH)] were analyzed in the pituitary of male sea bass during sex differentiation and the first spawning season. Plasma levels of gonadotropin (GTH-2), testosterone (T), and 11-ketotestosterone (11-KT) were determined during the same periods. All GnRH forms were present in the pituitary. sbGnRH levels were 9-fold higher than cGnRH-II and 17-fold higher than sGnRH levels. The highest GnRHs levels were detected in November 1995, when fish were 9 months old and when the gonads started to differentiate. Levels of the three forms decreased and remained low during the first spawning season, with the exception of sbGnRH, which showed a significant increase in November 1996. Plasma GTH-2 levels were lowest in November 1995, later increasing 2.5 times during the next months. During the first spawning season, plasma GTH-2 levels peaked in December 1996, 1 month after the peak of sbGnRH. During sex differentiation, plasma T levels were high in November 1995 but decreased over the next months, while levels of 11-KT remained low and unchanged. During the first spawning season, both steroids peaked in January 1997. These results suggest a possible role for all three GnRH forms in achieving gonadal differentiation, while sbGnRH may be the most relevant form in the regulation of the first spawning season in male sea bass. Moreover, GTH-2 and 11-KT may play important roles in gonadal maturation, since plasma GTH-2 and 11-KT levels were high throughout the period of spermiation.

Development and validation of an enzyme immunoassay for testosterone: effects of photoperiod on plasma testosterone levels and gonadal development in male sea bass (Dicentrarchus labrax, L.) at puberty.

A specific immunoassay was developed for the quantification of testosterone (T) in sea bass plasma. Specific primary antibody against T was produced using an antigen BSA conjugated with T. The enzyme immunoassay (EIA) had a sensitivity of 5–0.009 ng ml−1 and 6.2% intra-assay variation; inter-assay variation was 9.5% for sea bass plasma. The effects of two different accelerating photoperiod regimes, compressed photoperiod (CO; 6 months), and constant short photoperiod (9L:15D) with a long photoperiod (15L:9D) in March (SLmar), on T plasma levels and sexual maturation were examined during the onset of puberty in male sea bass. Natural photoperiod (NP) and SLmar groups exhibited the highest T values in December (8.69±1.03 and 10.85±1.04 ng ml−1, respectively). However, SLmar group showed the first significant decrease in T plasma levels in January, two months earlier than the NP group, which presented elevated T levels until February. The CO group displayed two significant T peaks, one in October (8.90±1.60 ng ml−1) and the other in January (9.60±1.10 ng ml−1). Gonadosomatic index (GSI) in the NP and SLmar groups displayed the highest values from December to February (>2.5%). However, the SLmar group showed the first significant increase in GSI in November, one month before the controls, indicating a clear advancement of gonadal development with respect to the NP group. In the CO group, a bimodal pattern was observed with two peaks, one in October–November (1.30±0.25%) and the second in March–April (0.97±0.33%) (P<0.05). In NP group, the percentage of running males was about 80% from December to March while the percentage of running males in the SLmar group (∼70%) lasted only three months (December to February) decreasing (P<0.05) in March. In the CO group, spermiation began in October (60%), decreased during the next months, and increased again in March–April (30%) (P<0.05). These results indicate the advancement of puberty by either one or two months with respect to the control group in the SLmar and CO groups, respectively, and the presence of a second reproductive surge in the CO group. Collectively, these results suggest that exposure of fish to these photoperiod regimes may affect both the time of the onset of puberty and the pattern of gonadal development in prepuberal male sea bass.

Effects of sustained administration of testosterone in pre-pubertal sea bass ( Dicentrarchus labrax L)

This study investigated the effects of testosterone (T) on the levels of immunoassayable salmon GnRH (sGnRH) in the brain and the pituitary in relation to blood plasma steroid levels and gonadal activity in juvenile male sea bass ( Dicentrarchus labrax L, <1-year of age). Gonad-intact male fishes were intraperitoneally implanted for 76 days with Silastic pellets containing no steroid (control) or testosterone (T; 100 μg/g body weight). The serum T response, the sGnRH content in whole brain and pituitary and the testicular development at 0, 15, 30 and 76 days following T implantation were assessed. Treatment with T did not significantly affect somatic growth or weight of gonads. However, gonad maturation was histologically more advanced in T-treated fishes compared to control fish. Histological examination showed that 15 days after implantation, 100% of steroid-treated fishes exhibited differentiated gonads, compared to only 50% of the controls. In addition, discrete spermiation was observed in 10% of the steroid-treated fishes 45 days after implantation while spermiation did not occur in the controls. Steroid treatment did not affect plasma oestradiol (E2) levels and all animals exhibited similar basal levels (<0.6 ng E2/ml). Fifteen days after T-implantation, the sGnRH content of brain and pituitary and the plasma levels of T were elevated. Over the following weeks a progressive decrease in all these parameters was observed. A second implant of T provoked a new and significant ( p<0.05) rise of both plasma T and whole brain sGnRH content, indicating that the GnRH system was still responsive to the additional steroid administration. These results provide evidence of a positive feedback of steroids at the brain and/or pituitary level, indicating a possible participation of sex steroids in the acceleration of gonadal differentiation and stimulation of spermatogenesis in pre-pubertal sea bass.

Effects of constant short and long photoperiod regimes on the spawning performance and sex steroid levels of female and male sea bass

Effects of constant short and long photoperiod regimes on the spawning performance and sex steroid levels of female and male sea bass

Effects of constant short and long photoperiod regimes on the spawning performance and sex steroid levels of female and male sea bass

Under constant short photoperiod, the spawning time of 2‐year‐old sea bass Dicentrarchus labrax was advanced as compared to controls, whereas spawnings were delayed under constant long photoperiod. High plasma levels of 17β‐oestradiol (E2/) and testosterone (T) in females were coincident with the appearance of vitellogenic oocytes in the ovary, while high levels of 11‐ketotestosterone (11‐KT) and T in males were coincident with the presence of spermiating males. Although plasma levels of E2 in females and 11‐KT in males were low during the remainder of the cycle, levels of T were always &gt;1 ng ml−1 in both sexes, suggesting that T could play an important role during the initial stages of gonadal development. The profiles of E2 and T in females and 11‐KT and T in males exposed to constant short days were similar to those in the control group, but fish which were maintained under constant long photoperiods showed a bimodal pattern of these steroids. The results obtained from fish exposed to constant photoperiod regimes provide further evidence that an endogenous process could be operating to control the reproduction of sea bass.

Alterations in total protein, IgM and specific antibody activity of male and female sea bass (Dicentrarchus labraxL., 1758) sera following injection with killedVibrio anguillarum

The relationship between the levels of total proteins, immunoglobulins and antibody activity in serum of sea bass broodstock, following one or two intraperitoneal injections of heat-killedVibrio anguillarum(V1), was studied over a period of 10 months. Determination of immunoglobulin (IgM) level was carried out using a double monoclonal antibody sandwich ELISA and the specific antibody activity to V1 (AbV1) quantified by indirect ELISA. IgM level decreased during the first month post-immunisation (p.i.), but persisted at a high level from the third month until the eighth month p.i. in injected fish and until the tenth month p.i. in boosted fish. AbV1 level increased after 2 weeks and remained elevated for longer than 10 months p.i. Neither the IgM nor the AbV1 levels of control fish were changed following an injection of phosphate-buffered saline. The V1 injection did not modify the protein level, but alteration of protein levels over time differed between males and females. Moreover IgM production was significantly higher, but no significant difference was found between males and females in AbV1 production. The decrease of IgM level after injection may be explained by consumption of IgM, with paratopes close to the injected antigen epitope, which are mobilised to compensate for the lack of the specific antibodies. This early decrease is likely to be an adaptation rather than a deficiency of the immune system.

Annual and daily variations of plasma cortisol in sea bass, Dicentrarchus labrax L.

Plasma cortisol levels in 2.5-year-old male sea bass, hatched and reared in captivity, were very high (range: 10–16 μg/dl). Significant annual variations of plasma cortisol were observed, and the oscillations coincided with water temperature and photoperiod. The spermiation period, from December to April (which are the coldest months), corresponded to the lowest cortisol levels of the year. The highest cortisol levels were observed in August–September, the warmest months, in the early prespawning period. Daily variations of cortisol are analysed in male fish over 1 year old in 4 months of the year (January, April, July and September). These variations were significant in September, when the water temperature was highest, with a peak at the beginning of the night. Dependence on high water temperature seems evident.

Advancement of sexual maturation and spawning of sea bass, Lates calcarifer (Bloch), using pelleted luteinizing hormone-releasing hormone analogue and 17 α-methyltestosterone

The ability of luteinizing hormone-releasing hormone analogue [(D-Ala 6,Pro 9-NEt)LHRH], 17 α-methyltestosterone (MT), and their combination to advance gonadal maturation and spawning in captive sea bass ( Lates calcarifer Bloch) broodstock was tested. Several hormonal therapies were tested including monthly implantations of a low dose (100 μg/kg body weight) of each hormone and a high dose (200 μg/kg) implanted to sexually-quiescent sea bass at 45-day intervals. A high dose of LHRHa alone or in combination with MT induced a significant number of mature females (43–71%) in April, 45 days after a single implantation in early March. A lower dose of pelleted LHRHa and LHRHa plus MT was found effective (78–80%) only in May following three monthly implantations. Two and three monthly implantations of a low dose of MT failed to stimulate oocyte growth in April and May whereas a less frequent mode of application of a high dose of the androgen inhibited ovarian growth in April only. A low dose of MT alone significantly increased the number of mature males in April (90%) and May (100%) after two and three monthly implantations. All male sea bass which received three monthly implantations of a low dose of LHRHa and LHRHa plus MT were sexually mature in May. Sexually mature sea bass obtained from these experiments spawned in early May after a single intramuscular injection of LHRHa. These results demonstrate the potential use of pelleted LHRHa and MT to significantly advance gonadal development and spawning in sea bass earlier than the annual breeding season.