Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Effect of temperature decrease on oocyte development,sex steroids,and gonadotropin β-subunit mRNA expression levels in female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17β (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin β-subunit [follicle-stimulating hormone (FSHβ), luteinizing hormone (LHβ)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25°C to 15°C over 39 days, while the control group was maintained at 25°C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSHβ and LHβ mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.     

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.     

Immunohistochemical investigation of the pituitary of the sturgeon (Acipenser baeri,Chondrostei)

An immunohistochemical study of the sturgeon (Acipenser baeri) pituitary was undertaken using antisera directed against hormones from various classes of vertebrates, including the only pituitary hormone available from sturgeon, gonadotrophin. A positive reaction was obtained after application of antisera towards the following hormones 1–24 synthetic ACTH (1-24 ACTH), melanophore stimulating hormone (MSH), ovine prolactin (oPRL), ovine growth hormone (oGH), salmon growth hormone (sGH), carp gonadotrophin (cGTH) and its beta subunit (cGTH), sturgeon gonadotrophin (aciGTH), carp thyrotrophin (cTSH) and subunit of the human thyrotrophin (hTSH). The results demonstrate that, in general, the sturgeon pituitary resembles that of teleosts as regards the distribution of the different cell types: ACTH and PRL cells in the rostral pars distalis, GTH, TSH and GH cells in the proximal pars distalis and MSH and PAS-cells in pars intermedia. In addition to the topographical organization of the sturgeon pituitary, this study provides data on the immunological relationships between sturgeon pituitary hormones and those of other vertebrates.     

Lack of growth hormone-releasing peptide-6 action on in vivo and in vitro growth hormone secretion in sexually immature grass carp (Ctenopharyngodon idellus)

Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. In the present study, GHRP-6 action on GH secretion was examined in vivo and in vitro in sexually immature grass carp. GHRP-6 injected intraperitoneally had no influences on serum GH levels in juvenile grass carp. Following intraperitonal injection of GHRP-6 and dopamine (DA) or cysteamine hydrochloride (CSH), alone and in combination into juvenile grass carp, DA and CSH were effective in elevating serum GH levels, but GHRP-6 was not effective in this respect; in addition, the synergistic action of GHRP-6 and DA or CSH on GH secretion was not seen. In this work, we had adapted and validated a perifusion system and a culture system for GH regulation studies. In a perifusion system, GHRP-6 (1000 to 0.1 nM), GHRP-6 (0.1 to 1000 nM), GHRP-6 (1 μM), and Hexarelin (an analog of GHRP, 1 μM) had no action on GH release from juvenile grass carp pituitary fragments or cells. Under static incubation conditions, GHRP-6 was inactive on GH release from juvenile grass carp pituitary fragments after 1 h and 6 h incubation, but human growth hormone-releasing hormone (hGHRH; 1 to 100 nM) as positive control could stimulate GH release in a dose-dependent manner. Furthermore, when GHRP-6 (100 nM) in combination static incubation with neuropeptides [e.g., hGHRH (100 nM), salmon gonadotropin-releasing hormone analogue (sGnRH-A) (100 nM), or D-Ala⁶,Pro⁹-NEt-luteinizing hormone-releasing hormone (D-Ala⁶,Pro⁹-NEt-LHRH, LHRH-A) (100nM)], GHRP-6 did not strengthen GH secretion actions of neuropeptides, and at the same time neuropeptides also did not modify the effects of GHRP-6 on GH secretion. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies show that GHRP-6 does not function as a GH-releasing factor in juvenile grass carp as it does in tilapia, amphibians, chickens, and mammals.     

Gonadotropin-induced changes in steroid production by ovaries of the common careCyprinus carnio L. around the time of ovulation

Ovarian fragments from both primed (gonadotrophin treated) and unprimed female carp were incubated either with or without carp hypophysial homogenate and steroid hormone production measured. In incubations without hypophysial homogenate, production of all the steroids measured was either very low or nondetectable and there was no significant difference between tissue from primed and unprimed fish. In the presence of carp hypophysial homogenate a very significant increase in production of testosterone, 17-hydroxyprogesterone and testosterone glucuronide was observed, but there was no significant difference between primed and unprimed fish. 17,20-Dihydroxy-4-pregnen-3-one (17,20P) was not stimulated by carp hypophysial homogenatein vitro in ovaries from unprimed fish, but a very significant increase in production of this hormone was observed in tissue from fish which had received a priming dose of pituitary hormone. It is suggested that the priming dose of pituitary extract used in the normal hypophysation procedure to induce ovulation in teleosts initiates the potential for synthesis of 17,20P in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.A preliminary account of these results was presented at the Fish Culture Conference, Barcelona, August 1985 (Kime and Bieniarz 1985).     

Effects of dietary 60 g kg−1 dried distiller's grains in least‐cost practical diets on production and gut allochthonous bacterial composition of cage‐cultured fish: comparison among fish species with different natural food habits

Dried distiller's grain (DDG) is considered as an alternative ingredient of dietary feed due to its high contents of protein, fibre and fat. In this study, 60 g kg^‐1 of DDG was used to feed grass carp (Ctenopharyngodon idella), bluntnose black bream (Megalobrama amblycephala), gibel carp (Carassius gibelio) and black carp (Mylopharyngodon piceus) for 8 weeks, and its effect on fish production and gut allochthonous microbiota was investigated for the development of a suitable fish feed high in nutrients and low in cost for polyculture freshwater fish. DDG supplementation resulted in the less weight gain and higher feed conversion ratio of black carp (P < 0.05), but had no significant effects on other fish or parameters. PCR–denaturing gradient gel electrophoresis (DGGE) analysis indicated that all four fish species had some common and unique bacteria in their digestive tracts, and the gut microbiota of bluntnose black and gibel carp fed the control diet and DDG diets were very similar (Cs > 91%); of them, the total counts of intestinal bacteria studied by qPCR increased in grass carp (P < 0.05) and depressed in black carp (P < 0.05) when fed dietary DDG. Thus, we assumed that dietary DDG modulated production and gut microbiota of fish in a host‐specific way.     

Dietary nano‐selenium enhances antioxidant capacity and hypoxia tolerance of grass carp Ctenopharyngodon idella fed with high‐fat diet

To evaluate the effects of dietary nano‐selenium (Nano‐Se) on antioxidant capacity and hypoxia tolerance of grass carp fed with high‐fat diet, experimental fishes were fed Nano‐Se supplemented diets at doses of 0 (Control), 0.3, 0.6, 0.9 and 1.2 mg/kg for 10 weeks. After feeding trial, a part of the fishes were exposed to hypoxia stress. Results showed that the survival ratio of grass carp significantly increased in 0.6 and 0.9 mg/kg Nano‐Se group, and the content of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) significantly decreased in 0.6–1.2 mg/kg Nano‐Se groups compared with the control group. In addition, dietary Nano‐Se significantly enhanced glutathione peroxidase (GPX) activity and reduced the malondialdehyde (MDA) content in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. Dietary Nano‐Se significantly elevated mRNA expression of GPX1 and catalase (CAT) by promoting the mRNA expression of NF‐E2‐related nuclear factor 2 (Nrf2) in the hepatopancreas. After hypoxia stress, the GPX and superoxide dismutase (SOD) activities were significantly enhanced, and the MDA content and mortality rate consequently decreased in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. In summary, these results suggested that optimal Nano‐Se in diet enhanced the antioxidant capacity and hypoxia tolerance of grass carp.     

Determination of hormones inducing oocyte maturation in <Emphasis Type="Italic">Chalcalburnus tarichi</Emphasis> (Pallas, 1811)

Chalcalburnus tarichi is an endemic cyprinid species living in the Lake Van basin, in eastern Anatolia, Turkey. The present study was undertaken to determine which hormones induce oocyte maturation in C. tarichi. The levels of 17α,20β,21-trihydroxyprogesterone (20β-S), progesterone (P), 17α-hydroxyprogesterone (17α-HOP), 11-deoxycortisol (11-DOC), and 17α-hydroxy-20β-dihydroprogesterone (17,20β-P) were measured in fish caught from Lake Van and the Karasu River, and injected with human chorionic hormone (hCG) (1,000 and 1,500 IU/kg). Oocytes of fish caught from the lake were also incubated in vitro with different doses (50, 200, and 1,000 ng/ml) of 20β-S, 17α-HOP, 11-DOC, and 17,20β-P. 11-DOC was found to be the most effective hormone among those measured for inducing oocyte maturation in vivo and in vitro. 17,20β-P could not be determined in the plasma of any fish in vivo (P < 0.05). 1,000 IU/kg dose of hCG given by injection caused a statistically significant increase in all plasma hormone levels (P < 0.05). It was found that there was a significant decrease in the P level only at 1,500 IU/kg dose of hCG injected (P < 0.05), while the level of other hormones increased at this dose (P < 0.05). It was also determined that all the hormones were effective in germinal vesicle breakdown (GVBD) in in vitro oocyte culture (P < 0.05). However, 11-DOC was found to be the most effective hormone in GVBD at a dose of 200 ng/ml (70% GVBD). In conclusion, 11-DOC synthesized during final oocyte maturation in C. tarichi was found to be a potent inducer of GVBD, which shows that 11-DOC may be described as an oocyte maturation steroid in this species.     

Identification of immunogenic proteins of Flavobacterium columnare by two‐dimensional electrophoresis immunoblotting with antibacterial sera from grass carp,Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram‐negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two‐dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti‐grass carp‐recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl‐CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose‐bisphosphate aldolase, 3‐hydroxybutyryl‐CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.     

Sample digestion for determining chloramphenicol residues in carp serum and muscle

Chloramphenicol (CAP) residues in carp (Cyprinus carpio L.) were investigated after intramuscular injection of a single-dose of 80 mg/kgbw. CAP concentrations in carp serum and muscle with and without addition of β-glucuronidase were determined by high-performance liquid chromatography (HPLC) with UV detection. The results showed that CAP-glucuronide (CAP-G), an important conjugate of CAP in the metabolic process and potentially toxic to human health, was observed after the fish were injected with CAP. CAP concentrations in serum and muscle with added β-glucuronidase were significantly (p < 0.05) higher than those without added enzyme. For example, within 24 h of injection, average CAP concentrations in serum and muscle with enzyme digestion were 30 and 48% more than those without the enzyme. In order to exactly determine the trace CAP content of fish, for evaluation of food safety, it is suggested that samples be hydrolyzed with enzyme (e.g., β-glucuronidase).     

Diet and fish choice of Eurasian otters (<Emphasis Type="Italic">Lutra lutra</Emphasis> L.) in fish wintering ponds in Hungary

The diet composition and fish preference of piscivorous Eurasian otters (Lutra lutra) were studied in two fish farm systems in Hungary using spraint (otter faeces) analysis during two wintering periods. The primary food source of otters in both fish farms was fish (97–99% of biomass). The main fish prey was small-sized, below 100 g in weight (96% in both areas), while fish prey above 500 g comprised only 0.1–0.4% of the diet. The bulk of the otters’ diet consisted of less-valued species, especially non-native Prussian carp (Carassius auratus gibelio). Consumption of commercial fish species ranged between 15 and 31% of the total diet. Otters preferred fish below 100 g in weight (Ivlev’s electivity index, E _i = 0.65–0.70), and showed a lesser preference for (or avoided) fish above 100 g in weight (E _i = −0.37–1.00). With regard to species distribution, otters preferred small (below 100 g) grass carp (Ctenopharyngodon idella), zander (Sander lucioperca), pike (Esox lucius), Prussian carp, topmouth gudgeon (Pseudorasbora parva), while they consumed common carp (Cyprinus carpio), the most important commercial species, proportionally to its abundance in the environment (E _i = −0.18–0.29).     

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.     

Identification of a protein kinase which phosphorylate α4 subunit of the 26S proteasome in goldfish oocytes

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3β (anti-goldfish 20S proteasome component 3β) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the α4 subunit of goldfish 20S proteasome (α4 _ca ) were isolated by an immuno-screening method using GC3β. Phosphatase treatment of the 26S proteasome revealed that a part of α4 _ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant α4 _ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1α (CK-1α). The result suggests that CK-1α phosphorylate α4 subunit of the 26S proteasome in immature oocyte of goldfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

Optimal dietary alpha‐linolenic acid/linoleic acid ratio improved digestive and absorptive capacities and target of rapamycin gene expression of juvenile grass carp (Ctenopharyngodon idellus)

Growth performance, digestive and absorptive capacities and target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and eIF4E‐binding protein (4E‐BP) gene expression in the hepatopancreas and intestine of juvenile grass carp (Ctenopharyngodon idellus) fed graded ratios of dietary alpha‐linolenic acid/linoleic acid (ALA/LNA) (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days were investigated. The results showed that ALA/LNA ratio of 1.03 significantly improved (i) per cent weight gain (PWG) and feed efficiency, (ii) hepatopancreatic trypsin, chymotrypsin, lipase, amylase and intestinal creatine kinase (CK) activities, (iii) hepatopancreatic trypsinogen‐2 and chymotrypsinogen mRNA levels. Meanwhile, fish fed with ALA/LNA ratio of 0.68 significantly enhanced, (iv) Na⁺/K⁺‐ATPase and γ‐glutamyl transpeptidase activities in whole intestine, and alkaline phosphatase activities in the proximal intestine (PI) and distal intestine, (v) amylase, intestinal Na⁺/K⁺‐ATPase alpha‐subunit isoform 1, Na⁺/K⁺‐ATPase alpha‐subunit isoform 8 and CK mRNA abundances, (vi) TOR and S6K1 gene expression in the hepatopancreas and intestine of juvenile grass carp. Based on the quadratic regression analysis of PWG, cholecystokinin and leptin contents in the PI, optimal dietary ALA/LNA ratio of juvenile grass carp (8.78–72.00 g) was estimated to be 1.08, 1.19 and 1.05, respectively.