Is the use of recombinant cGnRH may be a future alternative to control the fish spawning? Let us go with the goldfish example.

The use of recombinant gonadotropin-releasing hormone (rGnRH) has very rarely been tested in fish to promote spawning. This study evaluated the impact of recombinant chicken gonadotropin-releasing hormone (rcGnRH) with metoclopramide on the release of sex steroids and final maturation induction in goldfish (Carassius auratus) broodstock. For this purpose, goldfish broodstock was divided into four groups and treated with 0.9% NaCl with 20 mg/kg metoclopramide (Met) (C); 10 µg/kg body weight (BW) rcGnRH with 20 mg/kg metoclopramide (rcGn10); 15 µg/kg BW rcGnRH with 20 mg/kg metoclopramide (rcGn15); and 20 µg/kg BW rcGnRH with 20 mg/kg metoclopramide (rcGn20). The capability of the rcGnRH for eliciting biological response was tested in vivo by evaluating the changes of 17ß estradiol (E2), testosterone (T), and 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) and the induced spawning. Blood samples were obtained at 0 h, 12 h, and 24 h after injection. The rcGn10, rcGn15, and rcGn20 treatments induced lower E2 concentration, especially 24 h post-injection. T levels were significantly higher in rcGn10, rcGn15, and rcGn20 treatments 12 h post-injection than at 0 h and then decreased at 24 h post-injection. Furthermore, the rcGnRH tested significantly enhanced DHP secretion in rcGn10, rcGn15, and rcGn20 treatments 12 h post-injection before a decline at 24 h post-injection. No significant difference between the sampling times was found in the C treatment for the 3 sex steroids tested. The results also displayed that rcGnRH at 10-20 µg/kg of body weight can trigger spawning with the highest speed and efficiency of spawning at 20 µg/kg. The obtained results represent a possible strategy for enhancing the artificial reproduction and ovulation of broodstock fish by rGnRH and further support the use of recombinant hormones to promote reproduction in aquaculture.

In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis.

In European aquaculture, Eurasian perch, Perca fluviatilis L., is perceived as one of the most highly valuable freshwater fish species and a strong candidate for the development of freshwater aquaculture. In the pursuit of improving the quality of reproduction in this domesticated species, investigating the hormones mediating the final oocyte maturation (FOM) is therefore indispensable. But, the exact nature of the maturation-inducing hormone (MIH) in Eurasian perch is unknown. To further validate the existence of a maturation-inducing activity behind potential hormonal candidates in this species, we in vitro tested a group of nine hormones: cortisol (Co), 11-deoxycortisol (11-D), corticosterone (coS), 11-deoxycorticosterone (DOC), 17α,20ßdihydroxy-4-pregnen-3-one (DHP) and 17α,20ß,21 trihydroxy-4-pregnen-3-one (THP), prostaglandin E2 (PGE2), estradiol-17ß (E2) and testosterone (T), in their ability to trigger FOM advancement and the production of sex steroids potentially involved in FOM. Using mature female perch, two in vitro experiments were conducted with oocytes at the start of the FOM. The follicles were incubated for 62 h in Cortland media with and without human chorionic gonadotropin (hCG). By the end of the incubation, only DHP and THP triggered the full advancement in FOM even at low doses with the effect of DHP being in vivo validated. However, the de novo productions of E2 and DHP were not shown to be regulated by either of the MIH candidates. Progestagens are hence more credible candidates as MIH than corticosteroids in Eurasian perch. Our in vitro study also revealed that both PGE2 and DHP are strongly associated with ovulation and that PGE2 might have slightly contributed to such DHP activity.

Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging.

The aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning, and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing that regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species.

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish.

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Constant long photoperiod inhibits the onset of the reproductive cycle in roach females and males.

Photoperiod and temperature are commonly accepted as the determinant factors for the control of the reproductive cycle in freshwater fishes. However, this determining effect is dependent on fish species. While applying a constant long photoperiod has an inhibitory effect in some species, the same photoperiodic manipulation has a stimulating effect in others. In cyprinids, a decrease in temperature or photoperiod can induce the gonad recrudescence. However, in roach Rutilus rutilus an early spring spawner cyprinid, there is little knowledge about the cueing role of each environmental factor. The aim of this work was to study the effect of a constant long photoperiod on the gametogenesis in roach. Fish were kept under either naturally simulated photoperiod or artificial constant long photoperiod and sampled at three times: at the beginning of photoperiod decrease, at the beginning of temperature decrease, and at the end of temperature decrease. Morphological parameters (gonado-somatic, hepato-somatic, and viscera-somatic indexes), plasma sexual steroids, and proportion of gametogenesis stages were estimated at each sampling time. The results showed that a constant, long photoperiod exerted inhibitory effects on gametogenesis advancement in both females and males that could stem from decrease of sex steroid production. Roach displayed a similar response to photoperiodic manipulations to other early spring spawners like percids, such as European perch, yellow perch and pikeperch. These results clearly showed the cueing role of the photoperiod in the induction of the reproductive cycle in roach.

Influence of waterborne gallic and pelargonic acid exposures on biochemical and reproductive parameters in the zebrafish (Danio rerio).

Gallic and pelargonic acids are biologically derived substances receiving a growing interest as eco-friendly biocides with potential applications in freshwater system management. However, some data gaps remain to address their chronic ecotoxicity issue, particularly for fish. This work aimed at investigating the sublethal effects of a long-term waterborne exposure of zebrafish to these compounds. Mature fish were exposed to gallic or pelargonic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during one month under semi-static conditions. Fecundity, hatching rate and median hatching time were regularly evaluated. Circulating sex hormone levels (11 ketotestosterone -11 KT, 17 ßestradiol -E2-), plasma vitellogenin (Vtg), and gonad histology were monitored in males and females after exposure. Lactate dehydrogenase (LDH), total glutathione peroxydase (GPx) and glutathione-S transferase (GST) activities were assessed as enzymatic biomarkers of exposure in fish liver. Significant increases of GPx activity were reported in females exposed to both type of chemicals regardless the contamination level. Moreover, 5 mg/L gallic acid induced a decrease in 11-KT levels for males. For fish exposed to pelargonic acid, decreases in circulating hormone levels were reported respectively at 0.05 and 5 mg/L for 11-KT in males, and at 0.5 mg/L for E2 in females. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic or pelargonic acid. Additional investigations concerning the mode of application and the environmental fate of these substances may warrant their further use in freshwater systems at concentrations compatible with biocidal/allelochemical effects. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 227-240, 2017.

In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11-deoxycorticosterone.

In fish, the endocrine system, especially corticosteroids pathway, strongly interacts with immune system. On the other hand, in vivo co-stimulation of both systems is not well documented. To better understand this interaction, we decided to evaluate the in vivo effects of both stimulation of the immune system and co-stimulation of both systems in Eurasian perch juveniles. Fish were injected either with 10mgkg(-1) LPS, or with a combination of LPS and 0.8mgkg(-1) cortisol or LPS and 0.08mgkg(-1) 11-deoxycorticosterone (DOC) and sampled 1, 3 or 7days after injection. LPS affected the immune system by increasing plasma lysozyme activity and blood neutrophils populations. During the same time-course, LPS decreased the proportion of a mixture of lymphocytes and thrombocytes in blood and TNF-α expression in spleen. Cortisol modulated the LPS-mediated response in TNF-α mRNA expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated the abundance of complement component 3 (C3) mRNA in spleen. On the other hand, LPS altered the corticotropic axis by decreasing mRNA expression levels of all corticosteroid receptors and of 11ß-HSD-2 in spleen. Both corticosteroids injected were not able to balance these LPS-induced suppressive effects on corticosteroid receptors and 11ß-HSD-2 expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated GR-1b expression in gills. These results indicated that LPS is a strong modulator of the corticosteroid receptors expression in spleen. Furthermore, we report for the first time a LPS-induced decrease of the mineralocorticoid receptor expression. Finally, corticosteroids were able to modulate the LPS-mediated response at the transcriptional level.

Impact of absolute food deprivation on the reproductive system in male goldfish exposed to sex steroids.

There is a link between metabolism and reproduction as metabolic hormones affect hypothalamus-pituitary-testis (HPT) hormonal functions and vice versa. The aim of the present study was to investigate the effects of negative energy balance on the reproductive system in male goldfish exposed to testosterone (T) and 17ß-estradiol (E2). Following 7 days of food deprivation (FD), ANOVA models showed significant FD × sex steroid interactions on sperm quality and circulating sex steroid levels. When FD effects were investigated, 11-ketotestosterone (11-KT) level and sperm motility and velocity decreased in food-deprived goldfish in the control group. In E2-exposed goldfish, FD decreased sperm production in addition to sperm motility and velocity that coincided with an elevation of circulating E2 level. However, FD did not significantly impact sex steroids and sperm quality in T-exposed goldfish. ANOVA models showed non-significant FD × sex steroid interactions for HSI, GSI, circulating luteinizing hormone (Lh) level, and metabolic (preproghrelin, goat and nucb2) and reproductive (kiss1, gpr54 and gnrh3) mRNAs. Furthermore, results showed that FD decreased HSI, and increased Lh levels and testicular preproghrelin and goat mRNAs, while sex steroids increased mid-brain nucb2, kiss1 and gpr54 mRNAs. Together, our results suggest that FD-induced inhibition of androgenesis resulted in diminished sperm quality associated with activation of the testicular ghrelinergic system, and negative feedback of 11-KT increased Lh level. The FD-induced testicular metabolic and hormonal system was impacted in goldfish exposed to sex steroids. However, the negative effects of FD on sperm quality were accelerated in E2-exposed goldfish due to estrogenic activity. This study provides novel information to better understand metabolic-associated reproductive disorders in fish.

In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus).

Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20-25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p<0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.

First evidence of the possible implication of the 11-deoxycorticosterone (DOC) in immune activity of Eurasian perch (Perca fluviatilis, L.): comparison with cortisol.

Cortisol, the main corticosteroid in fish, is frequently described as a modulator of fish immune system. Moreover, 11-deoxycorticosterone (DOC) was shown to bind and transcriptionally activate the mineralocorticoid receptor and may act as a mineralocorticoid in fish. Immune modulations induced by intraperitoneal injections of these two corticosteroids were assessed in Eurasian perch juveniles. Cortisol and DOC were injected at 0.8 mg kg(-1) and 0.08 mg kg(-1) body weight respectively. Cortisol increased plasma lysozyme activity 72 h post-injection, C-type lysozyme expression in spleen from 1 to 72 h post-injection, and favoured blood neutrophils at the expense of a mixture of lymphocytes and thrombocytes. Moreover, 6 h after injection, cortisol reduced expression levels of the pro-inflammatory cytokine TNF-α in spleen. DOC had no effects on the immune variables measured in plasma, but increased expression levels of C-type lysozyme and apolipoprotein A1 mRNA in both gills and spleen. Meanwhile, DOC stimulated its putative signalling pathway by increasing expression of mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase-2 in spleen. These results confirmed the role of cortisol as an innate, short term immune stimulator. For the first time, DOC is described as a possible immune stimulator in fish.

Corticosteroid plasma kinetics and gonadal receptor gene expression during the reproductive cycle in female Eurasian Perch: Investigation of the roles of corticosteroids in vitellogenesis.

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. In vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated.

Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication--clues to physiological acclimation and humoral immune modulations.

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.

Evidence that elevated water temperature affects the reproductive physiology of the European bullhead Cottus gobio.

Climate change is predicted to increase the average water temperature and alter the ecology and physiology of several organisms including fish species. To examine the effects of increased water temperature on freshwater fish reproduction, adult European bullhead Cottus gobio of both genders were maintained under three temperature regimes (T1: 6-10, T2: 10-14 and T3: 14-18°C) and assessed for gonad development (gonadosomatic index-GSI and gonad histology), sex steroids (testosterone-T, 17ß-estradiol-E2 and 11-ketotestosterone-11-KT) and vitellogenin (alkali-labile phosphoprotein phosphorus-ALP) dynamics in December, January, February and March. The results indicate that a 8°C rise in water temperature (T3) deeply disrupted the gonadal maturation in both genders. This observation was associated with the absence of GSI peak from January to March, and low levels of plasma sex steroids compared with T1-exposed fish. Nevertheless, exposure to an increasing temperature of 4°C (T2) appeared to accelerate oogenesis with an early peak value in GSI and level of plasma T recorded in January relative to T1-exposed females. In males, the low GSI, reduced level of plasma 11-KT and the absence of GSI increase from January to March support the deleterious effects of increasing water temperature on spermatogenesis. The findings of the present study suggest that exposure to elevated temperatures within the context of climate warming might affect the reproductive success of C. gobio. Specifically, a 4°C rise in water temperature affects gametogenesis by advancing the spawning, and a complete reproductive failure is observed at an elevated temperature of 8°C.

Cortisol is responsible for positive and negative effects in the ovarian maturation induced by the exposure to acute stressors in Nile tilapia, Oreochromis niloticus.

The aim of the present study was to evaluate the effect of acute stress and cortisol injection on oocyte final maturation process in female Nile tilapia (Oreochromis niloticus). Handling followed by a prophylactic treatment (0.3 mL L(-1) H(2)O(2), 5 g L(-1) NaCl solution during 30 min) and an environmental change (transfer from a 2 m(3) fibreglass square tank to 50 L aquaria) were used as acute stressors and compared to a single cortisol injection (0.5 or 5 mg kg(-1) body weight). For both acute stress and cortisol injection (0.5 mg kg(-1) body weight), serum cortisol level was significantly increased from 2.3 to 134.1 ng mL(-1) 1 h post-stress/injection and returned to a resting basal value 24 h after the stress/injection. In fish injected with 5 mg kg(-1) body weight cortisol, mean serum cortisol level reached a peak up to 2500 ng mL(-1) 1 h after injection. 63 % of the females (mean body weight: 242 ± 4 g) submitted to the acute stress ovulated within 72 h after the stress. In the same way, cortisol injection (5 mg kg(-1) body weight) at the 10th day of the maturation cycle led to a twofold reduction of the time before ovulation compared to vehicle injected control fish. Relative and total fecundity were significantly decreased in females submitted to an acute stress or cortisol injected at 5 mg kg(-1) body weight, but not fertilization or hatching rates. In conclusion, acute stress and cortisol induction exert both positive and negative effects on the final reproductive process in O. niloticus, and cortisol is the endocrine mediator causing these changes.

Spawning Performance and Sex Steroid Levels in Female Pikeperch Sander lucioperca Treated with Poly(lactic-co-glycolic acid) Microparticles.

Pikeperch Sander lucioperca is a piscivorous species considered a promising candidate for the diversification of intensive aquaculture. This study aimed to determine the effect of a sustained-release delivery system incorporating mammalian gonadotropin-releasing hormone agonist (mGnRHa) into poly(lactic-co-glycolic acid) (PLGA) microparticles on the sex steroid levels and aspects of artificial reproduction of pikeperch. Fish were divided into four groups and injected with 20 µg mGnRHa/kg, 5-day release microparticles encapsulated with 5 µg GnRHa/kg BW (PLGA 5), 20 µg GnRHa/kg (PLGA 20), or 1 mL/kg 0.9% NaCl (control). Cumulative percentage ovulation was 100% in the PLGA 5 group, significantly higher than in other tested groups. No differences among groups were observed in latency or fecundity. The level of 11-ketotestosterone (11-KT) peaked at 40 h post-injection, and was sustained during ovulation, in all treated groups. The 17ß-estradiol (E2) concentration increased in the mGnRHa-only group immediately after hormone injection, while both PLGA groups showed a reduction in E2 after injection, continuing to decrease until ovulation. A low dose of mGnRHa in PLGA microparticles significantly improves induction of ovulation and results in acceptable reproductive performance, which may positively affect pikeperch production under controlled conditions.

The effect of two different experimental rearing temperatures on the quality and the large-scale cryopreservation of Eurasian perch (Perca fluviatilis) sperm.

Eurasian perch is an important fish species for European aquaculture diversification, but the quality of reproduction still remains one of the main limitations for further industry development. In particular, the optimal condition to obtain the best quality of sperm is poorly understood. The aim of our study was to measure the possible effects of two experimental rearing temperatures (6 °C and the conventionally used 12 °C) and of hormonal stimulation, on the motility parameters (pMOT, VCL, VSL, LIN, ALH, BCF), osmolality and fertilizing capacity of Eurasian perch sperm at the end of the reproductive cycle. A prior untested, large-scale (5 mL cryotube and Polystyrene box) cryopreservation method was implemented using fresh sperm obtained from the two above mentioned temperature groups. Males were injected with 100 µg body weight kg-1 sGnRHa. No significant difference was recorded between the two rearing temperatures and between the saline control and sGnRHa treated groups on the different features of sperm quality. A similar fertilization rate was monitored in all sGnRHa treated (6 °C: 69 ± 13%, 12 °C: 81 ± 11%) and saline control groups (6 °C: 79 ± 10%, 12 °C: 87 ± 4%). Correspondingly, no significant difference in hatching rate was observed in the sGnRHa injected (6 °C: 27 ± 9%, 12 °C: 40 ± 20%) and saline control (6 °C: 35 ± 18%, 12 °C: 36 ± 7%) males. However, a notable negative effect of freezing process on sperm movement was observed following thawing in both temperature groups. No significant difference in the motility parameters was measured between the two temperature groups following large-scale cryopreservation. Furthermore, a similar result was observed in the fertilizing capacity (6 °C: 79 ± 10%, 12 °C: 75 ± 8) of thawed sperm as well as in the hatching rate (6 °C: 52 ± 13%, 12 °C: 46 ± 19%). Our results indicate that fresh Eurasian perch sperm can tolerate a reduced rearing temperature following hormonal treatment. The adopted large-scale cryopreservation method could be used efficiently in the future for the fertilization of large amounts of Eurasian perch eggs following a precise standardization process.

The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review.

During the last decade, a number of studies have shown that, in addition to their classically described reproductive function, estrogens and androgens also regulate the immune system in teleosts. Today, several molecules are known to interfere with the sex-steroid signaling. These chemicals are often referred to as endocrine disrupting contaminants (EDCs). We review the growing evidence that these compounds interfere with the fish immune system. These studies encompass a broad range of approaches from field studies to those at the molecular level. This integrative overview improves our understanding of the various endocrine-disrupting processes triggered by these chemicals. Furthermore, the research also explains why fish that have been exposed to EDCs are more sensitive to pathogens during gametogenesis. In this review, we first discuss the primary actions of sex-steroid-like endocrine disruptors in fish and the specificity of the fish immune system in comparison to mammals. Then, we review the known interactions between the immune system and EDCs and interpret the primary effects of sex steroids (estrogens and androgens) and their related endocrine disruptors on immune modulation. The recent literature suggests that immune parameters may be used as biomarkers of contamination by EDCs. However, caution should be used in the assessment of such immunotoxicity. In particular, more attention should be paid to the specificity of these biomarkers, the external/internal factors influencing the response, and the transduction pathways induced by these molecules in fish. The use of the well-known mammalian models provides a useful guide for future research in fish.

Split it up and see: using proxies to highlight divergent inter-populational performances in aquaculture standardised conditions.

BACKGROUND: Considering wild inter-populational phenotypic differentiation can facilitate domestication and subsequent production of new species. However, comparing all populations across a species range to identify those exhibiting suitable key traits for aquaculture (KTA; i.e. important for domestication and subsequent production) expressions is not feasible. Therefore, proxies highlighting inter-populational divergences in KTA are needed. The use of such proxies would allow to identify, prior to bioassays, the wild population pairs which are likely to present differentiations in KTA expressions in aquaculture conditions. Here, we assessed the relevance of three alternative proxies: (i) genetic distance, (ii) habitat divergence, and (iii) geographic/hydrologic distances. We performed this evaluation on seven allopatric populations of Perca fluviatilis for which divergences in KTA had already been shown. RESULTS: We showed differences in the correlation degree between the alternative proxy-based and KTA-based distance matrices, with the genetic proxy being correlated to the highest number of KTA. However, no proxy was correlated to all inter-populational divergences in KTA. CONCLUSION: For future domestication trials, we suggest using a multi-proxy assessment along with a prioritisation strategy to identify population pairs which are of interest for further evaluation in bioassays.

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period.

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.