Search

Northern hemisphere freshwater ecosystems are projected to experience significant warming and shortening of winter duration in this century. This change coupled with depletion of oxygen (hypoxia) will result in a shift toward fish species with higher optimal temperatures for growth and reproduction that can mitigate hypoxic stress. Here, we tested the assumption that reproduction between two distant species, i.e. anoxic-intolerant common carp (Cyprinus carpio) and anoxic-tolerant goldfish (Carassius auratus), results in the expression of genes responsible for ethanol synthesis (alcohol dehydrogenase and pyruvate dehydrogenase subunit E1β2). The expression of this ethanol-producing pyruvate decarboxylase pathway may transform the biochemical characteristics of progeny into anoxic-tolerant hybrids, expanding their suitable environmental range and potentially increasing invasiveness. Concurrently, a genetic strategy for improving fish tolerance to oxygen-depleted environments will be a valuable physiological trait in fish culture. Differential quantification of gene expression by analyzing mRNA revealed that, compared with koi×koi, koi female×goldfish male (F1 hybrid) possessed the pyruvate dehydrogenase subunit E1β2 gene construct, which was expressed at significantly greater levels in red muscle. The potential of this hybrid to both survive in extreme anoxic conditions and grow at elevated water temperatures would likely contribute to their ecological success., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Interspecies hybrids have long been studied to further understanding of speciation. Reciprocal crosses sometimes have asymmetric viability, a phenomenon termed 'Darwin's corollary to Haldane's rule'. It has been proposed that this asymmetry is caused by Dobzhansky-Muller incompatibilities between nuclear genes and cytoplasmic factors (e.g., maternal transcripts, mitochondrial genome). The molecular basis of this hypothesis has received little empirical investigation, presumably due to the lack of an appropriate model system. We report a case of extreme asymmetry in viability between reciprocal hybrids of zebrafish Danio rerio and pearl danio Danio albolineatus. Hybrids from D. rerio females × D. albolineatus males (n = 4 crosses) were viable, with 83.2 ± 9.6% surviving from fertilization to 5 days post-fertilization (dpf) and 80.1 ± 14.4% surviving from 5 to 21 dpf. Hybrids from D. albolineatus females × D. rerio males (n = 6 crosses) were inviable after embryonic development. These hybrids developed pericardial oedema at 1 dpf and only 37.2 ± 18.0% survived from fertilization to 5 dpf. Of the 595 larvae alive at 5 dpf, only one juvenile with stunted growth survived to 21 dpf. We propose that given the resources available for the D. rerio model system and the strong asymmetry in viability between reciprocal crosses, these hybrids will allow investigation of the molecular basis for Darwin's corollary to Haldane's rule., (© 2021 Fisheries Society of the British Isles.)

In the past two decades, advances in sequencing technology and analysis of the human and mouse genome have led to the discovery of many non-protein-coding RNAs (ncRNAs) including: microRNA, small-interfering RNAs, piwi-associated small RNAs, transfer RNA-derived small RNAs, long-non-coding RNAs and circular RNAs. Compared with healthy controls, levels of some ncRNAs are significantly altered in the central nervous system and blood of patients affected by neurodegenerative disorders like Alzheimer's disease (AD). Although the mechanisms are still not fully elucidated, studies have revealed that these highly conserved ncRNAs are important modulators of gene expression, amyloid-β production, tau phosphorylation, inflammation, synaptic plasticity and neuronal survival, all features considered central to AD pathogenesis. Despite considerable difficulties due to their large heterogeneity, and the complexity of their regulatory pathways, research in this rapidly growing field suggests that ncRNAs hold great potential as biomarkers and therapeutic targets against AD. Herein, we summarize the current knowledge regarding the neurobiology of ncRNA in the context of AD pathophysiology., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Bighead carps  (Hypophthalmichthys nobilis) and silver carps (Hypophthalmichthys molitrix) represent an important component of freshwater ichthiofauna in its native range, though they might become mass propagation in other systems (North America) and the reason of concern for fisheries management. Therefore, understanding their reproductive traits and particularly in the context of hybridization with other cyprinids was of value to explain their rapid propagation as well as potential benefits for aquaculture due to their unique diet, behavior, growth potential, and tolerance to deteriorating environmental conditions in freshwater ecosystems. Distant hybridization is an effective tool to create different ploidy offspring with changed phenotypes and genotypes. In this study, we reported distant hybridization of female koi carp (Cyprinus carpio haematopterus, KOC, 2n = 100) × male bighead carp (Hypophthalmichthys nobilis, BIC, 2n = 48) and the spontaneous occurrence of two new "crucian" carp-like homodiploid fish (2nGCC-L; 2nCCC-L; 2n = 100), a new type of triploid hybrid (3nKB, 3n = 124), and a new type of tetraploid hybrid (4nKB, 4n = 148). The body color of 2nGCC-L and 2nCCC-L were gray and multicolor, respectively. Both phenotypes were similar to the crucian carp (Carassius auratus). The difference was that their heads were rounder than those of the crucian carp and they had higher backs. Compared with the KOC with two pairs of barbels and BIC without barbel, 2nGCC-L, 2nCCC-L, and 4nKB had no barbel, but 3nKB had one pair of barbels. Microsatellite patterns and 5S rDNA sequences confirmed that 2nGCC-L, 2nCCC-L, and 3nKB were of hybrid origin. In regard to feeding, KOC was omnivorous and BIC was a typical filter-feeder. However, the 2nGCC-L, 2nCCC-L, and 3nKB were omnivorous. The formation of four kinds of new offspring is a groundbreaking finding in fish genetic breeding and evolutionary biology., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The disposal of acid whey (Aw), a by-product from fermented products, is a problem for the dairy industry. The fishery industry faces a similar dilemma, disposing of nearly 50% of fish processed for human consumption. Economically feasible and science-based alternatives are needed to overcome this problem. One possible solution is to add value to the remaining nutrients from these by-products. This study focuses on the breakdown of nutrients in controlled fermentations of Aw, fish waste (F), molasses (M), and a lactic acid bacteria (LAB) strain (Lr). The aim was to assess the dynamic variations in microbial diversity and the biochemical changes that occur during fermentation. Four treatments were compared (AwF, AwFM, AwFLr, and AwFMLr), and the fermentation lasted 14 days at 22.5 °C. Samples were taken every other day. Colorimetric tests for peptide concentrations, pH, and microbial ecology by 16S-v4 rRNA amplicon using Illumina MiSeq were conducted. The results of the microbial ecology showed elevated levels of alpha and beta diversity in the samples at day zero. By day 2 of fermentation, pH dropped, and the availability of a different set of nutrients was reflected in the microbial diversity. The fermentation started to stabilize and was driven by the Firmicutes phylum, which dominated the microbial community by day 14. Moreover, there was a significant increase (3.6 times) in peptides when comparing day 0 with day 14, making this treatment practical and feasible for protein hydrolysis. This study valorizes two nutrient-dense by-products and provides an alternative to the current handling of these materials.

With increasing levels of fish meal (FM) protein in aquafeeds being replaced with soybean meal (SBM) protein, understanding the molecular mechanisms involved in response to alternative diets has become a critical concern. Thus, the goal of this study was to examine transcriptional differences in the intestine of juvenile yellow perch through RNA-sequencing (RNA-seq), after their initial introduction to a formulated diet with 75% SBM protein inclusion for 61 days, compared to those fed a traditional FM-based diet. Transcriptomic analysis revealed a concise set of differentially expressed genes in juveniles fed the SBM-based diet, the majority of which were intrinsic to the cholesterol biosynthesis pathway. Analysis of total body lipid and cholesterol levels were also investigated, with no between-treatment differences detected. Results of this study demonstrate that in response to SBM-based diets, yellow perch juveniles up-regulate the cholesterol biosynthesis pathway in order to maintain homeostasis. These findings suggest that the upregulation of the cholesterol biosynthesis pathway may negatively impact fish growth due to its large energy expenditure, and future studies are warranted.

Increased temperature in the epilimnion and hypoxia in the metalimnion of a lake would result in an increase of positive-size-selective fish predation on zooplankton and in turn in a decrease of mean body size in zooplankton populations and communities. We tested this hypothesis in four types of experiments with juvenile rudd ( Scardinius erythrophthalmus ) foraging on Daphnia longispina in an indoor twin column tank system. In each experiment of the first three types, one column contained one of three types of experimental treatments differing from the control treatment (in the other column) by the following: (i) elevated temperature in the epilimnion, (ii) hypoxia in the metalimnion and (iii) simultaneous elevated temperature in the epilimnion and hypoxia in the metalimnion. In the fourth type of experiment, the gradients of temperature and oxygen concentration in both columns were the same, but prior to the experiments, Daphnia and fish in the control treatment were acclimated to normoxia and, in the experimental treatment, to hypoxia. The results confirmed our hypothesis, since the predation rate of fish was greater in each of the first three experimental treatments than in the control. We did not detect an effect of the acclimation to hypoxia on the predation rate of the fish., (© The Author(s) 2019. Published by Oxford University Press.)

Gynogenetic zebrafish Danio rerio were obtained by activating D. rerio oocytes with UV irradiated common carp Cyprinus carpio sperm and then applying one of four different shocks [two (early) meiotic and two (late) mitotic shocks]. Gynogens produced by three of the shocks survived to maturity. All adult gynogens (n = 52) except one were found to be male. There was no difference in growth rate between the biparental controls and gynogens produced through the most effective shock, thereby eliminating growth rate as a possible cause of the skewed sex ratio. Gynogen males had reduced fertility compared to biparental controls, with about half of gynogens being unable to reproduce through natural spawning (all controls reproduced successfully). Gynogen males that did reproduce gave lower fertilization rates compared with controls. This demonstrates the negative effects of increased homozygosity on male reproductive function. Families sired by meiotic gynogen males were more likely to be female biased (33% of families) compared with families sired by biparental control males (11%). In addition to confirming the polygenic nature of sex determination in D. rerio, these observations suggest that recessive or over-dominant male-determining alleles are present in domesticated D. rerio populations., (© 2018 The Fisheries Society of the British Isles.)

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal., (© 2018 Wiley Periodicals, Inc.)

The current body of work on rearing larval/juvenile zebrafish is based on (1) utilization of freshwater and (2) diurnal light/dark cycle, (3) provision of live feed at modest density, and (4) culture in high visibility environment. We challenged these rearing approaches by maintaining zebrafish under constant light for 46-48 days (days postfertilization [dpf]), while securing continuous feeding in high turbidity and saline (1.8-2.1 parts per thousand) environment for the experiment's duration, allowing 24 h feeding/growth of fish from first exogenous feeding to maturation. There was no evidence of negative effects on zebrafish larvae behavior, growth, survival, and life cycle duration at constant illumination when food was continuously available. Zebrafish were stocked at high initial density (100 larvae/L) in a static system and fed high densities of rotifers (Brachionus plicatilis) (200-400/mL) from 6 to 12 dpf. Fish density was then reduced by 50% and two diet treatments, live rotifers or brine shrimp (Artemia) nauplii (10/mL), followed. Fish were reared on these two diets until first maturation. Performance of adult zebrafish fed live rotifer followed by Artemia nauplii diet was the highest recorded in the literature after 42 dpf, 250 ± 29 (males) and 430 ± 5 mg (females). Use of these rearing conditions, during the entire life cycle, until reproduction, resulted in the shortest ever recorded generation time (from egg to egg) of 43-45 dpf and fertilization rate (1 dpf) of 80.3%-94%.

Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ± 0.5 °C until 5 days post-fertilization (dpf) - the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ± 0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ± 0.3 mm and 20.5 ± 1.5 mg for the 23 °C treatment and 18.5 ± 0.4 mm and 67.3 ± 3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ± 0.7 mm and 76.4 ± 6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hypoxia can affect various fish populations, including yellow perch Perca flavescens, which is an economically and ecologically important species in Lake Erie, a freshwater system that often experiences hypoxia in the hypolimnetic part of the lake. Fish, similarly to mammals, possess molecular oxygen sensor-hypoxia-inducible factor-1 (HIF-1), a transcription factor that can affect expression of many downstream genes related to animal growth and locomotion, protein synthesis, as well as ATP and amino acid metabolism. HIF-1 is a heterodimer, which consists of two subunits: oxygen-sensitive and oxygen-insensitive subunits, α and β, respectively. In this study, we report first on the molecular cloning and sequencing of P. flavescens HIF-1α. The full-length complementary DNA (cDNA) was isolated and submitted to the GenBank with accession number KT783483. It consists of 3529 base pairs (bp) carrying a single open-reading frame that encompasses 2250 bp of the coding region, 247 bp of the 5' untranslated region (UTR), and 1032 bp of the 3' UTR. The "de novo" prediction of the 3D structure of HIF-1α protein, which consists of 749 amino acids, is presented, too. We then utilized One-Step Taqman® real-time RT-PCR technology to monitor changes in HIF-1α messenger RNA (mRNA) copies in response to chronic hypoxic stress. An experiment was conducted using 14-day post-swim-up stage yellow perch larvae with uninflated swim bladders. This experiment included three treatment groups: hypoxia, mid-hypoxia, and normoxia, in four replicates (four tanks per treatment) with the following dissolved oxygen levels: 3, 4, and >7 mg O 2 /L, respectively. At the end (2 weeks) and in the middle (1 week) of the experiment, fish from each tank were sampled for body measurements and molecular biology analysis. The results showed no differences in survival (∼90%) between treatment groups. Oxygen concentration was lowered to 3.02 ± 0.15 (mean ± SE) mg O 2 /L with no adverse effect on fish survival. The highest growth rate was observed in the normoxic group. A similar trend was observed with fish body length. The growth rate of fish declined with decreasing water-dissolved oxygen. The number of HIF-1α mRNA copies was not significantly different between hypoxic, mid-hypoxic, and normoxic conditions, and this was true for fish obtained in the middle and at the end of the experiment. Graphical abstract.

To evaluate the effects of thiamine on physiological changes and spawning performance of Sterlet sturgeon Acipenser ruthenus, 45 farmed female fish (698.6±8.9g) were randomly distributed in 9 tanks (1000L) and fed a diet with 1g/kg of an anti-thiamine drug. This was provided for 5 months prior to spawning. Thiamine hydrochloride was intraperitoneally injected to fish at three different doses: 0 (T 0 , as control), 5 (T 5 ) and 50 (T 50 ) mg/kg body weight at days 30, 90 and 150 after the experiment started. After five months, the results showed no significant differences in weight gain and hemoglobin level, but hematocrit significantly increased in T 5 group. There was no significant difference in glucose-6-phosphate dehydrogenase and estradiol-17β, but testosterone was significantly increased in the T 50 group. Total thiamine concentration in the eggs was significantly higher in T 50 than that detected in the control group. Fecundity and larval mortality at 6day post hatch (dph) showed no significant differences among treatments, while the number of eggs per gram was significantly lower in T 0 than that observed in T 50 . Larval weights at 1 (11.6mg) and 6 (23.1mg) dph and larval lengths at 6 (15.6mm) dph were significantly affected by the treatment with the highest level of thiamine injection (T 50 ). Diseases symptoms such as yolk sac deformation, erratic pattern of swimming, and loss of equilibrium were observed at 4 dph in T 0 and T 5 groups. The overall results revealed that thiamine injection has positive effects on reproductive performance in the sturgeon and the negative impacts of anti-thiamine in the offspring can be reduced by the injection of this vitamin to the broodstock., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Haploid gynogenetic screens increase the efficiency of forward genetic screens and linkage analysis in fish. Typically, UV-irradiated zebrafish sperm is used to activate zebrafish oocytes for haploid screens. We describe the use of UV-irradiated common carp sperm to activate haploid gynogenetic zebrafish development. Carp × zebrafish hybrids are shown to have a characteristic set of features during embryonic development and exhibit functional development of several tissues (muscle, heart and nervous system). Hybrids become inviable past the embryonic stages. This technique eliminates the possibility of incompletely irradiated zebrafish spermatozoa contaminating haploid progenies. While developing this protocol, one unique zebrafish female was identified which, upon insemination with UV-irradiated carp spermatozoa, repeatedly displayed spontaneous diploidization of the maternal chromosomes in her offspring., (© 2016 The Author(s).)

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals., (© 2016 Wiley Periodicals, Inc.)

Although climate warming is expected to benefit temperate ectotherms by lengthening the summer growing season, declines in reproductive success following short, warm winters may counter such positive effects. Here we present long-term (1973-2010) field patterns for Lake Erie yellow perch, Perca flavescens, which show that failed annual recruitment events followed short, warm winters. Subsequent laboratory experimentation and field investigations revealed how reduced reproductive success following short, warm winters underlie these observed field patterns. Following short winters, females spawn at warmer temperatures and produce smaller eggs that both hatch at lower rates and produce smaller larvae than females exposed to long winters. Our research suggests that continued climate warming can lead to unanticipated, negative effects on temperate fish populations.

Traditionally, haloperidol has been the recommended antipsychotic drug for pharmacological treatment of delirium, which is a frequent complication in the critical care setting. Due to a less frequent occurrence of extrapyramidal adverse effects, second-generation antipsychotic drugs have been evaluated. In the present paper we review the current randomized prospective studies of second-generation antipsychotics as treatment for delirium in hospitalized patients and conclude that so far the evidence in favour of these drugs compared with haloperidol is still sparse.

Traditionally, haloperidol has been the recommended antipsychotic drug for pharmacological treatment of delirium, which is a frequent complication in the critical care setting. Due to a less frequent occurrence of extrapyramidal adverse effects, second-generation antipsychotic drugs have been evaluated. In the present paper we review the current randomized prospective studies of second-generation antipsychotics as treatment for delirium in hospitalized patients and conclude that so far the evidence in favour of these drugs compared with haloperidol is still sparse.

Introduction: The aim of this study was to assess the knowledge of the inhabitants of the West Pomeranian region in terms of physiotherapy, and to highlight the need for action to expand public awareness of this topic., Material and Methods: A questionnaire survey involved a group of 267 adult men and women aged 18 to 78 years. All subjects were residents of West Pomerania. The questionnaire consists of 15 open and closed questions. Respondents provided answers to questions about physiotherapist competence, learning opportunities, and the availability of physiotherapy services., Conclusions: Knowledge about physiotherapy is insufficient. There is a great need to disseminate information about the profession of the physiotherapist. It is necessary to undertake further studies on a larger group of participants.

Swim bladders and lungs are homologous structures. Phylogenetically ancient actinopterygian fish such as Cladistians (Polypteriformes), Ginglymods (Lepisosteids) and lungfish have primitive lungs that have evolved in the Paleozoic freshwater earliest gnathostomes as an adaptation to hypoxic stress. Here we investigated the structure and the role of autonomic nerves in the physostome swim bladder of the cyprinid goldfish (Carassius auratus) and the respiratory bladder of lepisosteids: the longnose gar and the spotted gar (Lepisosteus osseus and L. oculatus) to demonstrate that these organs have different innervation patterns that are responsible for controlling different functional aspects. The goldfish swim bladder is a richly innervated organ mainly controlled by cholinergic and adrenergic innervation also involving the presence of non-adrenergic non-cholinergic (NANC) neurotransmitters (nNOS, VIP, 5-HT and SP), suggesting a simple model for the regulation of the swim bladder system. The pattern of the autonomic innervation of the trabecular muscle of the Lepisosteus respiratory bladder is basically similar to that of the tetrapod lung with overlapping of both muscle architecture and control nerve patterns. These autonomic control elements do not exist in the bladders of the two species studied since they have very different physiological roles. The ontogenetic origin of the pulmonoid swim bladder (PSB) of garfishes may help understand how the expression of these autonomic control substances in the trabecular muscle is regulated including their interaction with the corpuscular cells in the respiratory epithelium of this bimodal air-breathing fish., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Anatomical and functional studies on the autonomic innervation as well as the location of airway receptors in the air-bladder of lepisosteids are very fragmentary. These water-breathing fishes share in common with the bichirs the presence of a glottis (not a ductus pneumaticus) opening into the esophagus. In contrast to a high concentration of neuroepithelial cells (NECs) contained in the furrowed epithelium in the lung of Polypterus, these cells are scattered as solitary cells in the glottal epithelium, and grouped to form neuroepithelial bodies (NEBs) in the mucociliated epithelium investing the main trabeculae in the air-bladder of Lepisosteus osseus and L. oculatus. The present immunohistochemical studies also demonstrated the presence of nerve fibers in the trabecular striated musculature and a possible relation to NEBs in these species, and identified immunoreactive elements of this innervation. Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), 5-HT and neuropeptide immunoreactivities were detected in the intramural nerve fibers. 5-HT and VIP immunopositive nerve fibers are apparently associated with NEBs. TH, VIP and SP immunoreactivities are also present in nerve fibers coursing in the radially arranged striated muscle surrounding the glottis and its submucosa. 5-HT positive neurons are also found in submucosal and the muscle layers of the glottis. The physiological function of the adrenergic and inhibitory innervation of the striated muscle as well as the neurochemical coding and morphology of the innervation of the NEBs are not known. Future studies are needed to provide evidence for these receptors with the capacity of chemoreceptors and/or mechanoreceptors., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

An interaction of two essential nutrients, thiamine and magnesium (Mg) has been documented in in vitro and in vivo studies in mammalian metabolism. However, the role of this association in poikilothermic vertebrates, such as fish, remains elusive. The purpose of this study was first to investigate the effects of dietary thiamine and Mg, and their interaction in lake trout and second to better understand the mechanism leading to early mortality syndrome (EMS), which is caused by a low thiamine level in embryos of many species of salmonids in the wild. Semi-purified diets (SPD) were prepared to accomplish 2 × 2 factorial design that were either devoid of or supplemented with thiamine mononitrate (20 mg/kg diet), magnesium oxide (700 mg/kg diet), or both. Lake trout alevins at the swim-up stage were fed for 10 wk one of the SPD diets or a commercial diet at the same rate (2.0-1.5%) based on recorded biomass. Our results showed that the concentrations of thiamine in the trunk muscle and Mg of whole body were closely associated with the dietary level of two nutrients. The interaction of low dietary Mg and thiamine resulted in apparently worsened overt symptoms of thiamine deficiency in lake trout leading to a higher mortality of fish during the seven week long trial (P<0.05). The fish fed a thiamine-devoid and Mg-supplemented diet were presumed to survive longer (10 wk) than the fish fed diets devoid of both nutrients (discontinued after 7th wk due to high mortality). However, we did not observe histopathological changes in the brain and liver corresponding to thiamine concentrations in tissues. These data suggest that Mg enhanced utilization of the thiamine remaining in the fish body and its interdependence was consistent with observations in mammals. EMS severity might be worsened when Mg is deficient in parental diets (and consequently in yolk sac) and/or first feed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Individual variation in fatty-acid and thiamine concentrations were determined in lake trout Salvelinus namaycush eggs collected at two spawning grounds in Lake Michigan. A suite of predictor variables, including spawning location, egg fatty-acid and thiamine concentrations, were used to attempt to explain cause-and-effect in early life stage mortality among S. namaycush families. Lipid and fatty-acid composition of S. namaycush eggs differed between spawning locations. Salvelinus namaycush offspring from south-western Lake Michigan were affected by a high occurrence of yolk oedema, whereas a higher frequency of early mortality syndrome (EMS) was observed among offspring from the north-western part of the lake. Random-forest regressions revealed location as the most influential predictor of yolk oedema mortality, whereas thiamine level in eggs was the strongest predictor of EMS-related mortality. Several polyunsaturated fatty acids were also found to be predictors of both mortalities. There is evidence of spatial variability in egg fatty-acid concentration among S. namaycush in Lake Michigan that, together with diminished thiamine concentration, contribute to low survival of S. namaycush progeny., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

Anatomical and functional studies of the autonomic innervation in the conus arteriosus of the garfishes are lacking. This study reveals that the conus arteriosus of the longnose gar is primarily myocardial in nature, but additionally, large numbers of smooth muscle cells are present in the subendocardium. A well-developed system of adrenergic, cholinergic, substance P (SP) and neuronal nitric oxide synthase (nNOS) positive nerve terminals are found in the wall of the conus arteriosus. Coronary blood vessels running in the adventitia receive a rich supply of nNOS positive nerve fibers, thus suggesting their importance in the nitrergic control of blood flow in the conus arteriosus. The present data show that the patterns of autonomic innervation of the garfish conus arteriosus are more complex than previously appreciated., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Protein digestion products are transported from the intestinal lumen into the enterocyte both in the form of free amino acids (AAs), by a large variety of brush border membrane AA transporters, and in the form of di/tripeptides, by a single brush border membrane transporter known as PEPtide Transporter 1 (PEPT1). Recent data indicate that, at least in teleost fish, PEPT1 plays a significant role in animal growth by operating, at the gastrointestinal level, as part of an integrated response network to food availability that directly supports body weight. Notably, PEPT1 responds to both fasting and refeeding and is involved in a phenomenon known as compensatory growth (a phase of accelerated growth when food levels are restored after a period of growth depression). In particular, PEPT1 expression decreases during fasting and increases during refeeding, which is the opposite of what observed so far in mammals and birds. These findings in teleost fish document, to our knowledge, for the first time in a vertebrate model, a direct correlation between the expression of an intestinal transporter, such as PEPT1, primarily involved in the uptake of dietary protein degradation products and animal growth., (This journal is © 2011 The Royal Society)

Common carp (Cyprinus carpio) of average body mass 0.07+/-0.02 g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys-Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4 weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4 weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys-Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON)., (2010 Elsevier Inc. All rights reserved.)

We have shown previously that diets in which the nitrogen portion is based on synthetic dipeptides (PP) resulted in weight gain by rainbow trout alevins when free amino acid (FAA) based diets did not. However, the protein-based diet used as a control in the previous study resulted in a significantly better performance of fish than peptide- or FAA based diet fed fish. Therefore, the objectives of our study were (i) to test how stomachless fish respond to peptide-based diets, (ii) to evaluate PP and protein-PP mixture diets and (iii) to examine if post-prandial response to FAA concentrations in the fish body can be used as an indicator of the availability of dietary amino acid sources. The first experiment was conducted with a 4-day old Koi carp (Cyprinus carpio) larvae and included groups fed a casein-gelatin (CG) based diet, a commercial diet and live Artemia nauplii. In the second experiment, fish fed live Artemia for 3 weeks (69 +/- 12 mg) were placed in glass aquaria in triplicate per dietary treatment. Four diets provided equivalent amounts of nitrogen in the form of a CG, a PP, a 50% PP and 50% CG (PP50) mix and as FAA. Fish were fed at 1 and 3 h intervals, and the survival and growth were monitored during the second and third weeks of experiments 1 and 2 respectively. Following the completion of feeding, juvenile carp were sampled prior to feeding and 3 h after a meal. Whole body FAA analyses were carried out. We observed marginal suitability of the CG diet for larval Koi carp, as earlier indicated in common carp. Juvenile Koi carp fed the CG diet achieved 236 +/- 19 mg, whereas PP50, PP and FAA diet fed fish grew to 140 +/- 37, 70 +/- 8 and 73 +/- 5 mg respectively. Free amino acids in the fish body, and in particular indispensable amino acids (IDAA), were excellent indicators of dietary availability. The present experiment shows that a dietary 1:1 ratio of protein to synthetic PPs results in better growth, survival and whole body IDAA concentrations in relation to FAA- and PP-based diets confirming earlier reports on the use of synthetic PP-based diets, although this diet does not match the performance of complete protein, CG-based diet.

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl-glycine dipeptide supplemented diet - PP, free lysine and glycine supplemented diet - AA, control diet with no lysine - CON) or commercial starter (Aller Futura - AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.

DNA fragments related to the cloudy catshark Scyliorhinus torazame L-gulono-gamma-lactone oxidase (GULO) cDNA were detected in a distant fish species. Although the Southern hybridization pattern was more distinct in species with active GULO, DNA fragments related to the GULO gene were also discovered in the common carp Cyprinus carpio. Additionally, in the common carp, inter-individual variation of the hybridization pattern was observed. Regular screening of available teleost fish gene libraries did not reveal GULO related DNA sequences.

The objectives of this study were to examine the effect of thiamine immersion of fish from a population known for compromised survival as a result of early mortality syndrome (EMS) and to investigate the cause-response relationship between thiamine concentration and lesions in tissues in swim-up-stage lake trout Salvelinus namaycush alevins. Lake trout eggs from 14 fish from Lake Michigan were artificially fertilized and the progeny divided into two groups based on the thiamine concentration (low [< 0.73 nmol/g] or high [> 0.85 nmol/g]) in the unfertilized eggs. Progeny were treated or not with a thiamine solution (2,000 mg/L for 2 h) at hatching or the swim-up stage. The survival of progeny in control groups at the swim-up stage correlated with thiamine concentration. The low thiamine-treated groups had significantly higher survival between the swim-up stage (812.0 degree-days) and 16 d after swim-up (963.3 degree-days) than the control groups; the survival of the high thiamine-treated groups did not differ between treated and control fish, regardless of the treatment at hatching and the swim-up stage. Control alevins that had low thiamine levels showed EMS, which resulted in 94.9-100% mortality 16 d after the swim-up stage. No pathological changes were observed in the brain, olfactory lobe, eye, liver, or muscle in alevins of high thiamine-treated group. Glycogen deposits in the liver of alevins from the low control group were variable, no glycogen being observed in the hepatocytes of 7 of the 24 fish. We demonstrate that thiamine treatment at swim-up enhances the survival of EMS-affected lake trout relative to treatment at hatching.

The natural reproduction of lake trout Salvelinus namaycush in Lake Michigan is thought to be compromised by nutritional deficiency associated with inadequate levels of thiamine (vitamin B1) in their eggs. However, mortality driven by thiamine deficiency (commonly referred to as early mortality syndrome [EMS]) is not the only significant cause of low lake trout survival at early life stages. In this study, we sought to better understand the combined effects of variable levels of thiamine and fatty acids in lake trout eggs on prehatch, posthatch, and swim-up-stage mortality. We sampled the eggs of 29 lake trout females from southwestern Lake Michigan. The concentrations of free thiamine and its vitamers (e.g., thiamine monophosphate [TMP] and thiamine pyrophosphate [TPP]) as well as fatty acid profiles were determined in sampled eggs. Fertilized eggs and embryos were monitored through the advanced swim-up stage (1,000 degree-days). Three distinct periods of mortality were identified: prehatch (0-400 degree-days), immediately posthatch (401-600 degree-days), and swim-up (601-1,000 degree-days). Stepwise multiple regression analysis revealed (1) that cis-7-hexadecenoic acid in both neutral lipids (NL) and phospholipids (PL) correlated with prehatch mortality, (2) that docosapentaenoic acid in PL and docosahexaenoic acid in NL correlated with posthatch mortality, and (3) that total lipids, TPP, and palmitoleic acid in NL, linoleic acid, and palmitic acid in PL correlated with the frequency of EMS. These results indicate the complexity of early life stage mortality in lake trout and suggest that inadequate levels of key fatty acids in eggs, along with variable thiamine content, contribute to the low survival of lake trout progeny in Lake Michigan.

We have chosen the silver arowana (Osteoglossum bicirrhosum), a representative of the most ancient teleost family Osteoglossidae, to address the question of yolk nutrients utilization. Silver arowana have particularly large eggs (1-1.5 cm of diameter) and a unique morphology of the yolk. We present evidence that the yolk cytoplasmic zone (ycz) in the "yolksac juveniles" is a very complex structure involved in sequential processes of yolk hydrolysis, lipoprotein particles synthesis, their transport, and exocytosis. Vacuoles filled with yolk granules in different stages of digestion move from the vitellolysis zone through the ycz to be emptied into the microvillar interspace in the process of exocytosis. The area of the ycz with the abundance of the mitochondria must play an important role in providing energy for both the transport of vacuoles and the release of their contents. Therefore, we postulate that the function of yolk syncytial layer (ysl) as the "early embryonic patterning center" transforms in fish larvae or yolksac juveniles into a predominantly specialized role as the yolk trophoblastic layer (ytl) involved in yolk nutrients utilization. In addition to discovering the mechanism of transformation of the ysl function into ytl function, we suggest that the machinery involved in nutrient mobilization and exocytosis in yolk of arowana yolksac juveniles can be very attractive system for studies of regulatory processes in almost all secretory pathways in animal cells.

The objectives of this study were to examine the relationship between thiamine concentrations in unfertilized eggs and yolksac individuals of lake trout (Salvelinus namaycush), along with any associated histopathological changes in the tissues of alevins at the hatching stage. We address these questions in a lake trout population from different spawning grounds of Lake Michigan (North and South), known for compromised survival due to early mortality syndrome (EMS). However, a dichotomous forage base of lake trout spawning stocks, with a dietary thiaminase-rich alewife in the North, and dietary low-thiaminase round goby in the South, provides the basis for the assumption that different diets may lead to differences in severity of EMS between different stocks. Lake trout eggs of 18 females were collected and fertilized individually with the sperm of several males. The eggs, eyed embryos and newly-hatched alevins were sampled to examine thiamine utilization during embryogenesis. Progenies of females with low (< 0.73 nmol/g) and high (> 0.85 nmol/g) levels of thiamine were chosen for histological studies. The obtained results showed that total thiamine levels in the body and yolk of eyed embryos and alevins at hatching were influenced by thiamine levels of unfertilized eggs and it decreased during embryogenesis (to 51% in eyed embryos and 28% in newly-hatched alevins in comparison to unfertilized eggs). The survival of lake trout until hatching stage does not correlate with the thiamine level, however it was affected by collection site and was significantly higher in fish from the South site (Julian's Reef). At the hatching stage, no pathological changes were observed in the brain, olfactory lobe, retina or liver in embryos regardless of thiamine concentrations in unfertilized eggs. It has been concluded that an enhanced thiamine requirement for the fast muscle mass growth near the swim-up stage is responsible for overt and histopathological signs of EMS. Current study confirms earlier findings that lake trout suffering from EMS can be successfully treated by immersion in thiamine solution as late as at the swim-up stage.

Studies on larval rearing of silver bream (Vimba vimba), a migratory cyprinid fish have addressed on limited scale larval and juvenile rearing using commercial and semipurified diets along with live feeds, such as brine shrimp Artemia nauplii. The objectives of the present study were (1) to determine whether experimental, protein-, peptide-, free amino acid-based diets are adequate for larval silver bream, a stomachless fish, (2) to evaluate whether commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream, and (3) to examine whether free amino acid concentrations in fish body are potential indicators of availability of amino acid sources. We report here the differences in diets acceptance, fish growth and diet utilization in silver bream in comparison to other cyprinid fishes. We specifically address the response in free amino acids in the body to dietary treatments. Experimental diets included: a commercial Aglo Norse feed, casein-gelatin based diet (CG), free amino acid mixture diet (FAA), dipeptide (PP), dipeptide-protein (PP50) based diet, and dipeptide diet without arginine (NoArg). In addition, live Artemia were offered to 3 groups and "fasting" control treatment was included during 3 week long trial. Fish offered Artemia overperformed those offered formulated diets both in terms of mass (80.7+/-26.3 mg) and survival (97.2%). We also indicate that commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream. Our experiment also showed that the whole body free amino acid concentrations of 9 indispensable amino acids (IDAA) out of 10 (His, Thr, Arg, Val, Met, Ile, Leu, Trp, Lys) in the PP50 group was the highest among 7 diet treatments and the totalfree amino acid concentration, total dispensable amino acids (DAA) and total IDAA of the PP50 diet fed fish showed the same trend. This may indicate that diets based on 50% of dipeptides and 50% of protein are adequate for silver bream larvae as they result in similar growth, survival and whole body IDAA concentrations as in fish fed CG (protein-based) diet fed fish.

There is considerable controversy in the literature regarding the existence of a "longitudinal slit" versus a "duct" connecting the respiratory gas bladder with the alimentary tract in various species of garfishes (Lepisosteidae). The aim of the present work has been to address these discrepancies on the basis of our own work on longnose gar Lepisosteus osseus in conjunction with a review of the literature. We provide anatomical evidence for a better understanding of the functional advantages of the "longitudinal slit" versus the "duct" in respect to feeding and respiration in juveniles longnose gar. The anterior part of the garfish body cavity, from the oral cavity to the stomach, was used for morphological and histological analysis. It has been observed that the air bladder in the longnose gar is open to the alimentary tract through a longitudinal slit which is present in the glottis on the dorsal side of the oesophagus. The external side of the glottal ridges in the anterior oesophagus, on the side of the opening, consist of stratified squamous epithelium which then turns into the pseudostratified columnar ciliated epithelium in the posterior oesophagus. The glottal ridges are comprised of epithelia and radially arranged striated muscle fibres surrounded by connective tissue. It can be surmised that the tunica muscularis in the anterior oesophagus of garfish aids the glottis in moving air in and out because it supports the dorsal retractor muscle in propelling air through the longitudinal slit from the buccal cavity. When the buccal cavity of the garfish is contracted during holding prey, the muscles of the gas bladder wall and tunica muscularis, located inside and outside the glottal ridge, participate in exhalation.

A variety of recombinant vaccines and vaccine delivery systems are currently under development as alternatives to vaccines produced in animals that are primarily administered by injections. These nonanimal alternatives do not transmit animal pathogens, are often rapid to develop, and can be produced on a large scale at low costs. Many of these new vaccine technologies are based on oral delivery systems and avoid the risks of disease transmission associated with the use of syringes for injectable vaccines. In addition, many of these novel systems have extended shelf life, often not requiring refrigeration and thus are applicable in developing countries or remote locations. Here we describe the development of microalgal-based immunization systems. Antigens expressed in the chloroplast or anchored to the surface of plasma membrane are shown to effectively immunize fish and rabbits. The effective oral delivery of antigens by microalgae provides a safe and inexpensive mechanism to immunize animals. The applications of microalgal vaccines are currently being investigated.

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m(-2). HS of 35+/-1 degrees C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m(-2) for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35+/-1 degrees C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation.

The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.

The premise that a dietary dipeptide approach will improve the understanding of amino acid utilization in the fastest-growing vertebrate, the teleost fish, was tested by examining the muscle free amino acid (FAA) pool and enzyme activities, in concert with growth response, when dietary amino acids were provided in free, dipeptide or protein molecular forms. We present the first evidence in fish that, in response to a synthetic dipeptide diet, muscle FAA varies as a result of both growth rate and amino acid availability of specific peptides. We demonstrate significantly diminished muscle indispensable FAA (3-10-fold) in rainbow trout alevins fed a dipeptide-based diet compared with a protein-based diet. The dipeptide-based diet did not contain proline, resulting in 10-27-fold less muscle free proline and hydroxyproline in alevins. The response of alevins fed FAA-based or peptide-based diets can be indicative of collagen turnover (Hyp/Pro ratio) and showed significant differences between dietary treatments. Pyrroline-5-carboxylate (P5C) reductase activity was detected, suggesting that P5C may ameliorate proline deficiency, but synthesis from glutamate could not maintain free proline levels in muscle. This finding will provide an impetus to test whether proline is conditionally indispensable in young fish, as in mammals and birds. This study shows that amino acids given entirely as dipeptides can sustain fish growth, result in muscle FAA and enzyme responses in line with dietary levels and identify growth-limiting amino acids. The understanding of these factors necessitates a diet formulation that will improve the accuracy of determining amino acid requirements in the early life stages of vertebrates.

In this study we sought to demonstrate that Comet assay can be applied to sea lamprey sperm DNA fragmentation and used to describe the relationship between sperm DNA damage and sperm fertilizing ability. We show that the assay can be used reliably and accurately, and unlike in the case of mammals, there is no need for additional steps related to improvement of efficacy of lysis and DNA decondensation. This agrees with the presence of histone proteins in lamprey sperm. An increase in DNA fragmentation was noted during short-term storage of milt on ice (0-4 days). We demonstrated genotoxic effects of UV radiation and oxidative stress (exposure to hydrogen peroxide) and found that oxidative damage to sperm DNA was likely repaired after fertilization in the embryo. Repairing capacity of the oocyte toward sperm DNA lesions caused by UV was restricted. Toxic effect of p,p-bis-(1-aziridinyl)-N-methylphosphinothioic acid (p,p-bis(1-aziridinyl)-N-methylphosphinothioic amide), a sea lamprey chemosterilant, could not be linked to DNA fragmentation in the in vitro tests. Its genotoxicity in vivo may possibly be associated with other mechanisms of DNA degradation (oxidation or DNA-protein and DNA-DNA cross-linking). In conclusion, this study demonstrates that Comet assay can be successfully applied to monitor effects of environmental disturbances and imposed injuries in sea lamprey spermatozoa and possibly other species of ancient fish with acrosomal sperm.

Most studies on the antioxidants, lipoic acid (LA) and ascorbic acid (AA), focused on species that, unlike teleost fish, are not scurvy-prone, and are able to synthesize AA. The antioxidant properties of LA may make it useful in aquaculture nutrition, but several effects must first be investigated, and we address here plasma free amino acids (FAA). In mammals, LA and AA in high doses were claimed to alter plasma FAA profile; to our knowledge, however, no data are available in fish. We therefore studied the effects of dietary LA and AA on plasma FAA in the South American teleost fish pacu, which is being used increasingly in aquaculture. LA treatment decreased concentrations of 18 of 23 individual FAA; specifically, dispensable and total FAA were significantly affected. Ornithine was elevated (+26%) in LA-treated fish and significantly decreased ratios of plasma [Arg]/[Orn] and other individual [FAA]/[Orn] were observed. LA and AA both affected sulfur FAA concentrations. Plasma cystine levels were significantly increased in the LA-supplemented groups. AA had little effect on most amino acids, and no interaction with LA was detected. AA supplementation did, however, significantly lower taurine (-42%) and cystathionine (-31%) levels in plasma. No effect on the branched chain:aromatic amino acid ratios was observed. The data indicate that at the dietary level studied, LA and AA independently affect selected plasma FAA in pacu, and suggest that any use of LA in particular as a dietary supplement should take into account an altered plasma FAA profile.

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.

The high cost of fish meal in tilapia diets warrants the potential use of cottonseed meal (CSM) as an alternative source of high quality protein. The effects of varying levels of CSM (0, 25, 50, 75 and 100%) as fish meal protein replacement on growth, blood parameters, spleen characteristics, free and bound gossypol in blood plasma, haemoglobin and haematocrit were determined in tilapia. Gossypol (C(30)H(30)O(8)) is a polyphenolic substance found in cottonseed that has known toxic effects in fish. Tilapias (n = 219, average weight = 11.3 +/- 3.9 g) were randomly distributed into 15, 32-L glass aquaria, representing five dietary treatments and three replicates per treatment. Each aquarium containing 13-16 fish was supplied with thermoregulated, recirculating water (27 +/- 1 degrees C) at 1 L min(-1) flow rate and photoperiod was constant (12 h L/12 h D). Fish fed 25-50% CSM protein replacement showed similar body weights and total lengths as the controls at the completion of the 16-week trial. Fish fed 75 and 100% CSM protein replacement showed a significant decline in body weight and total length. Fish fed 25-100% CSM protein replacement had significantly lower haematocrit and haemoglobin (ANOVA/LSD, P < 0.05) compared with levels in controls. The decline was most prominent in groups fed diets with 50-100% CSM protein replacement. Total and free gossypol concentrations of blood plasma significantly increased with increasing levels of CSM replacement (P < 0.05). No gossypol was found in blood plasma of fish from the control group. The occurrence of immature and abnormal erythrocytes was significantly greater among fish fed 75 and 100% CSM diets compared with fish fed 0-50% CSM diets. Spleen-somatic index (spleen weight/body weight x 100) did not differ between control fish and fish fed 50-100% CSM diets. Spleen abnormalities, such as large depositions of haemosiderin and melanin pigments and proliferation of melano-macrophage centres, lymphocytic depletion of the white pulp areas (hypocellularity), and presence of vacuoles and necrotic areas were observed among fish fed 50-100% CSM protein diets. In general, the pathological effects of gossypol in tilapia (low haemoglobin and haematocrit levels, abundance of immature red blood cells or polychromatocytes, abnormal spleen morphology) were similar to the effects of vitamin E and/or vitamin C deficiencies observed in other studies.

Sterile-male-release technique is currently used to control the sea lamprey (Petromyzon marinus) population in the Great Lakes. The chemosterilant (bisazir) used in this program is extremely hazardous; special safety measures are necessary when handling this chemical. Therefore, replacement of bisazir with safer agents is desirable. In this study, we examined the effects of low-toxicity compounds with previously described spermicidal activity (mainly against mammalian sperm) on motility and fertilizing ability of sea lamprey spermatozoa. Nonoxynol-9, benzalkonium chloride, zinc acetate, cupric chloride, cysteamine, tannic acid and propranolol were able to inhibit both sperm motility and fertilizing ability. Effective concentrations of these spermicides ranged from 0.15 to 1%. Therefore, they can be potentially used in further study directed at in vivo sterilization of male sea lampreys.

Ascorbic acid synthesis in the pig occurs at mid-pregnancy, but activity of the enzyme l-gulono-gamma-lactone oxidase (GLO) declines thereafter during gestation and remains low when the pig nurses the sow. During late gestation the ascorbic acid concentration in the fetus increases, but serum and liver ascorbic acid concentration in the sow declines without affecting the dam's liver GLO activity. It is presumed that as gestation progresses an increased amount of maternal ascorbic acid is transferred to the fetus and to the mammary gland. Colostrum and milk are rich sources of the vitamin and supply the nursing pig with ascorbic acid. The available data suggest that high amounts of ascorbic acid appear to suppress liver GLO activity in the pig. Upon weaning, when exogenous vitamin C is generally not provided, liver GLO activity and serum ascorbic acid increases. During the initial periods postweaning, some reports have indicated growth benefits of supplemental vitamin C. Body tissues differ in their concentrations of ascorbic acid, but tissues of high metabolic need generally have greater concentrations. The corpus luteum in the female, the testis in the male, and the adrenal glands in all pigs contain greater concentrations of the vitamin. Knockout genes preventing ascorbic acid synthesis in pigs have demonstrated poor skeletal and collagen formation and poor antioxidant protection. Under periods of stress ascorbic acid declines in the adrenal, but the pig rapidly recovers to its resting state once the stressor agent is removed. Although there are periods when supplemental vitamin C has been shown to promote pig performance (e.g., during high environmental stress and early postweaning), supplemental vitamin C has not been shown to routinely enhance pig performance.

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and embryos resulted in an average embryo survival rate of 71% in optimal source variants after 17 days of refrigeration (7 days oocytes+10 days embryos). The study shows that both gamete and embryo refrigeration can be successfully used as an efficient tool for prolonging availability of rainbow trout embryos in early developmental stages., (Copyright 2003 Wiley-Liss, Inc.)

In vitro studies of brush-border intestinal transport of dipeptides and cytoplasmic hydrolysis in fish suggest that these processes could be key mechanisms in the absorption and utilization of nutrients for growth. However, in vivo experimentation to study the nutritional importance of these processes was needed. We compared three dietary formulations based on free, peptide and protein sources of amino acids. Our results were the first to show that a synthetic dipeptide (PP)-based diet could support growth in the early stages of ontogenesis of a teleost fish, rainbow trout (Oncorhynchus mykiss), whereas a free amino acid (FAA)-based diet failed. We found that fish fed an FAA-based diet had an increased rate of ammonia excretion [1.78 +/- 0.19 mmol NH(3)-N/(kg body wt.h)], compared with fish fed a PP-based diet [1.25 +/- 0.07 mmol NH(3)-N/(kg body wt.h)], suggesting that deamination is involved in the metabolism of dietary FAA. Teleost fish are known to obtain a high proportion of total energy from protein, compared with higher vertebrates. However, we found that feeding trout alevins a PP-based diet increased postprandial oxygen consumption for 2 to 24 h, whereas other treatments decreased 24-h postprandial metabolism. This may indicate that peptide metabolism is less efficient than protein metabolism. Juvenile rainbow trout differed from alevins in their response to FAA- and PP-based diets. These observations strongly suggest that intestinal dietary peptide transport and hydrolysis could support protein synthesis and growth in vertebrates that respond poorly to FAA-based diets. We conclude that nutrient administration may be improved by manipulating dietary peptide composition and peptide/protein ratios, leading to better utilization of synthetic peptides, with nutritional and therapeutic implications for all vertebrates.

We evaluated how two diets with different lipid levels (4% vs. 10%) influenced nutrient expenditure in juvenile walleye Stizostedion vitreum subjected to starvation, prolonged swimming, and predator presence. We also determined how exercise and predator presence influenced stress indicators such as blood plasma cortisol and glucose. Groups of six fish were placed in triplicate rectangular wire cages per treatment and submerged randomly in three artificial stream compartments at a water temperature of 14 degrees C. Three treatments were established: (A) no water current and no predator, (B) water current (1.5 body lengths s(-1)) and no predator, (C) water current and predator. Six 1-year-old muskellunge (Esox masquinongy, 28.4+/-2.3 cm) were used as predators. They were allowed to swim freely outside walleye cages and were fed with walleye. To simulate poststocking period of fasting, walleyes were exposed to experimental treatments for 6 weeks and were deprived of food. Regardless of the prior diet, fish weight significantly declined in all treatments but no differences were found among them. Lipids declined in both dietary groups of fish; in turn, body moisture increased. Protein levels were only affected in fish fed with high-lipid diet prior to the experiment and subjected to current and predator presence. Plasma glucose gradually declined in fish fed with high-lipid diet prior to the study; whereas in the fish fed with low-lipid diet before the experiment, it remained low regardless of the treatment severity. Plasma cortisol concentrations never exceeded 30 ng ml(-1) indicating no signs of acute stress during the experiment. We concluded that fish fed with diet containing higher lipid levels prior to stocking would possess better potential of withstanding adverse conditions and better survival once released to the wild.