Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Broodstock nutritional programming differentially affects the hepatic transcriptome and genome-wide DNA methylome of farmed gilthead sea bream (Sparus aurata) depending on genetic background.

BACKGROUND: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program. RESULTS: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2). CONCLUSIONS: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism.

Transcriptomic profiling of Gh/Igf system reveals a prompted tissue-specific differentiation and novel hypoxia responsive genes in gilthead sea bream.

A customized PCR-array was used for the simultaneous gene expression of the Gh/Igf system and related markers of muscle growth, and lipid and energy metabolism during early life stages of gilthead sea bream (60-127 days posthatching). Also, transcriptional reprogramming by mild hypoxia was assessed in fingerling fish with different history trajectories on O2 availability during the same time window. In normoxic fish, the expression of almost all the genes in the array varied over time with a prompted liver and muscle tissue-specific differentiation, which also revealed temporal changes in the relative expression of markers of the full gilthead sea bream repertoire of Gh receptors, Igfs and Igf-binding proteins. Results supported a different contribution through development of ghr and igf subtypes on the type of action of GH via systemic or direct effects at the local tissue level. This was extensive to Igfbp1/2/4 and Igfbp3/5/6 clades that clearly evolved through development as hepatic and muscle Igfbp subtypes, respectively. This trade-off is however very plastic to cope changes in the environment, and ghr1 and igfbp1/3/4/5 emerged as hypoxic imprinting genes during critical early developmental windows leading to recognize individuals with different history trajectories of oxygen availability and metabolic capabilities later in life.

The circadian transcriptome of marine fish (Sparus aurata) larvae reveals highly synchronized biological processes at the whole organism level.

The regulation of circadian gene expression remains largely unknown in farmed fish larvae. In this study, a high-density oligonucleotide microarray was used to examine the daily expression of 13,939 unique genes in whole gilthead sea bream (Sparus aurata) larvae with fast growth potentiality. Up to 2,229 genes were differentially expressed, and the first two components of Principal Component Analysis explained more than 81% of the total variance. Clustering analysis of differentially expressed genes identified 4 major clusters that were triggered sequentially, with a maximum expression at 0 h, 3 h, 9-15 h and 18-21 h zeitgeber time. Various core clock genes (per1, per2, per3, bmal1, cry1, cry2, clock) were identified in clusters 1-3, and their expression was significantly correlated with several genes in each cluster. Functional analysis revealed a daily consecutive activation of canonical pathways related to phototransduction, intermediary metabolism, development, chromatin remodeling, and cell cycle regulation. This daily transcriptome of whole larvae resembles a cell cycle (G1/S, G2/M, and M/G1 transitions) in synchronization with multicellular processes, such as neuromuscular development. This study supports that the actively feeding fish larval transcriptome is temporally organized in a 24-h cycle, likely for maximizing growth and development.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: a proteomic approach.

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.

Expression and characterization of European sea bass (Dicentrarchus labrax) somatolactin: assessment of in vivo metabolic effects.

The complementary DNA coding for European sea bass somatolactin was expressed in the pET-3a bacteria expression vector. The recombinant somatolactin (rbSL) was purified by size exclusion chromatography, and 95% of the protein remained in the oxidized form with negligible aggregation over prolonged cold storage. The identity of the recombinant protein was demonstrated by Western blotting with a rabbit polyclonal antibody against gilthead sea bream somatolactin. The same antibody was utilized in a radioimmunoassay procedure, using rbSL as standard and radioiodinated tracer. Curve displacements of pituitary and plasma samples paralleled the rbSL standard, and the midrange of the assay (8 ng/ml) was low enough to measure in a consistent manner the circulating SL concentration. To assess biological activity a single dose of rbSL (0.1 microg/g of body mass) was administered to juvenile gilthead sea bream by intraperitioneal injection. In comparison with saline-treated fish, rbSL did not modify the circulating amount of insulin-like growth factor I, whereas a 50% increase was found with the same dose of recombinant trout growth hormone (rtGH). Hormone treatment did not modify nitrogen-ammonia excretion, but both rbSL and rtGH increased carbon dioxide output and oxygen uptake, which in turn decreased the respiratory quotient (CO2 output per O2 uptake). This pattern of gas exchange suggests the enhancement of lipid catabolism, which is consistent with the observation that both hormones were able to inhibit the hepatic activity of acetyl-coenzyme A carboxylase. These new insights provide direct evidence for the involvement of fish somatolactin in energy homeostasis, which may serve to maintain the lipolytic tonus in different physiologic states.

CDNA cloning and sequence of European sea bass (Dicentrarchus labrax) somatolactin.

Three cDNA clones encoding for European sea bass somatolactin (SL) were obtained by RT-PCR and 3' RACE of RNA of pituitary origin. Clone 1 was 582 bp in length, and included a part of the signal peptide and the 5' end of the mature protein. Clone 2 (1075 bp) included a fragment of the coding sequence and the 3' untranslated region, which was 888 bp in length and contained two putative polyadenylation signals (AATAAA) at 12-17, and 202-207 nucleotides upstream of the poly (A) tail. Clone 3 was 624 bp in length and its nucleotide sequence encoding for the entire mature protein confirmed the sequence already determined from the first two clones. The size of SL mRNA transcripts was estimated by Northern blot analysis and a single band of approximately 1.6 kb was observed with pituitary RNAs. No band was found with RNAs of brain and liver origin. Alignment of the deduced amino acid sequence revealed that European sea bass SL shared 90-84% identity with perciform, pleuronectiform and scorpaeniform fish SLs, and 77-57% with other SLs of more distant fish orders, with a strict conservation of Cys residues and the N-glycosylation site (Asn-Lys-Thr) at 121-123 amino acid positions. The reconstruction of the phylogenetic tree based on SL nucleotide sequences. and analyzed by maximum likelihood distances, showed the same clustering as the present hierarchy of fish. When comparisons were made among SL, prolactin and growth hormone of European sea bass, the overall amino identity was relatively low (22-23%). However, a high degree of amino acid homology was found at the C-terminus, which contains three of the four Cys residues strictly conserved in all the members of GH/PRL family.

Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay.

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Growth hormone as an in vitro phagocyte-activating factor in the gilthead sea bream (Sparus aurata).

The stimulatory action of growth hormone on gilthead sea bream phagocyte-enriched cultures was demonstrated in vitro for the first time in a fish species. Phagocytes consisted mainly of macrophages, with a small number of neutrophils and eosinophils. Macrophages were unequivocally identified by their esterase staining and the lack of myeloperoxidase staining. Cells primed with recombinant rainbow trout GH showed clear morphological (light- and scanning electron-microscopic) and functional differences from non-primed cells. Stimulated phagocytes exhibited numerous branched lamellipodia, abundant membrane ruffles, increased spreading, and cell size. When incubated with sheep red blood cells, the phagocytic index and phagocytic capacity was also enhanced in primed cells. A bell-shaped dose-response curve (1.5-500 nM) was obtained when the metabolic activity of growth-hormone-activated cells was measured. This finding suggests that the homodimerization of the growth hormone receptor is a characteristic feature both in mammals and fish.

Evidence for a direct action of GH on haemopoietic cells of a marine fish, the gilthead sea bream (Sparus aurata).

Receptors for GH were characterized in the head kidney of gilthead sea bream (Sparus aurata), using radioiodinated and biotinylated ligands. The specific binding of radiolabelled recombinant gilthead sea bream GH (rsbGH) to head kidney membrane preparations was dependent on membrane concentration. Salmon prolactin, salmon gonadotrophin and carp gonadotrophin did not compete for 125I-labelled rsbGH-binding sites. Unlabelled rsbGH competitively displaced 125I-labelled rsbGH bound to head kidney membranes. Scatchard plots were always linear, denoting the presence of a single class of binding sites. The binding affinity (Ka = 2.7 x 10(9) M-1) was equivalent to that found in liver membrane preparations, but the binding capacity (2.5 +/- 0.30 fmol/mg protein) was 50- to 75-fold lower. To identify the cells which express the GH receptor, head kidney smears were incubated with biotinylated rsbGH, followed by incubation with an avidin-biotin complex conjugated to alkaline phosphatase. The reaction with the new-fuchsin substrate gave a red precipitate, showing a specific and intense labelling in erythroblasts, polychromatophilic erythroblasts and myeloblasts. Noticeable binding was observed in myelocytes and immature granulocytes, tending to disappear at the latter stages of granulocyte maturation. Light but appreciable binding was also observed in monocytes, lymphocytes and acidophilic erythroblasts, whereas it was completely absent in proerythrocytes and erythrocytes. The proliferative action of rsbGH and recombinant human IGF-I on in vitro cultures of head kidney cells was demonstrated by a 5-bromo-2'-deoxy-uridine immunoassay. To our knowledge, this is the first report that provides suitable evidence for a role of GH as a haemopoietic growth and differentiation factor in lower vertebrate species.

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.