Brian C. Small

Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulfonate, quinaldine and clove oil anesthetized channel catfish Ictalurus punctatus

The present experiments were designed to determine the efficacy of metomidate hydrochloride as an alternative anesthetic with potential cortisol blocking properties for channel catfish Ictalurus punctatus. Channel catfish (75 g) were exposed to concentrations of metomidate ranging from 0.5 to 16 ppm for a period of 60 min. At 16-ppm metomidate, mortality occurred in 65% of the catfish. No mortalities were observed at concentrations of 8 ppm or less. The minimum concentration of metomidate producing desirable anesthetic properties was 6 ppm. At this concentration, acceptable induction and recovery times were observed in catfish ranging from 3 to 810 g average body weight. Plasma cortisol levels during metomidate anesthesia (6 ppm) were compared to fish anesthetized with tricaine methanesulfonate (100 ppm), quinaldine (30 ppm) and clove oil (100 ppm). Cortisol levels of catfish treated with metomidate and clove oil remained at baseline levels during 30 min of anesthesia (P>0.05). Plasma cortisol levels of tricaine methanesulfonate and quinaldine anesthetized catfish peaked approximately eight- and fourfold higher (P<0.05), respectively, than fish treated with metomidate. These results suggest that the physiological disturbance of channel catfish during routine-handling procedures and stress-related research could be reduced through the use of metomidate as an anesthetic.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

BackgroundThrough the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energys Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.ResultsA total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.ConclusionsThis project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions

Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the assay that can affect data interpretation. Most notably, the selection of an appropriate internal control for normalization is essential for expression data interpretation. In this study we investigated the suitability of seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and RNA polymerase II polypeptide B (POLR2B)] as potential quantitative references for normalizing real-time PCR data generated in the study of channel catfish physiology. Gene expression and stability were evaluated among 15 channel catfish tissues and within physiologically-relevant tissues in response to experimental manipulation (i.e. LHRH injection, fasting, and acute stress). Expression of the seven candidate reference genes varied across all tissue types tested, indicating that none of the genes could suitably serve as reference genes for cross tissue comparisons. Experimentally altering the physiological state of the fish differentially affected expression of the various reference genes depending on experimental design and tissue type, with 18S unaffected by the experimental treatment in all tissues examined. For example, the selection of a differentially expressed gene, GAPDH, as opposed to 18S, to normalize hepatic growth hormone receptor during fasting resulted in misinterpretation of the data. These results reveal the importance of providing comprehensive details of reference gene validation when publishing real-time PCR results, with this manuscript serving as a basic guideline for reference gene selection in channel catfish research.

Using Portable Lactate and Glucose Meters for Catfish Research: Acceptable Alternatives to Established Laboratory Methods?

Abstract Simple and portable methods for assessing the physiological state of channel catfish Ictalurus punctatus would be valuable tools in field situations where problems with blood storage and transportation occur. This study compared the use of handheld lactate and glucose meters with established laboratory methods in stressed (fatigued) and unstressed (control) channel catfish fingerlings. The results obtained from the Accutrend (Roche Diagnostics Corp.) lactate meter and the Accu-Chek Advantage (Roche Diagnostics) glucose meter were consistently lower (P < 0.05) than those obtained with the laboratory reference method. However, significant differences (P < 0.0001) were found between the control and fatigued fish for both lactate and glucose, regardless of the method of analysis. Both handheld meters were found to be reliable and suitable for use in field or laboratory situations where relative measurements are acceptable. The costs associated with using the handheld meters were higher than those ass...

Pre- and postprandial changes in orexigenic and anorexigenic factors in channel catfish (Ictalurus punctatus)

Ghrelin (GRLN), cocaine and amphetamine regulated transcript (CART), neuropeptide Y (NPY), and cholecystokinin (CCK) are neuropeptides involved in the regulation of appetite and feeding in vertebrates. We examined pre- and postprandial changes in the expression of plasma GHRL and mRNAs encoding GRLN, CART, NPY, and CCK in channel catfish. Fish were entrained to eat at 0900 h for 2 weeks. Fish were then sampled at 0700, 0800, and 0900 h. Remaining fish were either offered feed at 0900 h (Fed) or fasted (Unfed). Fish sampling continued at 0.5, 1, 2, and 4 h post feeding. Feeding increased abundance of whole brain CART mRNA out to 4 h with no effect observed in unfed fish. Whole brain NPY expression peaked at 0.5 h in both treatments. NPY expression then declined in fed fish but remained elevated in unfed fish. No differences in plasma or stomach GRLN expression were observed. Two separate cDNAs for CCK were identified. Brain CCKa and CCKb expression increased after feeding. These results suggest CART, NPY, and CCK play roles in the regulation of channel catfish feeding. Taken together, these results provide new insights into the neural and gastroenteric mechanisms regulating appetite in channel catfish.

Effects of bovine growth hormone (Posilac®) on growth performance, body composition, and IGFBPS in two strains of channel catfish

The effects of recombinant bovine growth hormone (rbGH; Posilac®) on growth rate, feed efficiency, body composition, and insulin-like growth factor binding proteins (IGFBPs) were investigated in Norris and NWAC103 strains of channel catfish. Three hundred and twenty fish from each strain were assigned randomly to four treatments with four replicates each. The treatments were (1) Sham-injected control (needle puncture per 3 weeks); (2) Low (30 μg g−1 BW per 3 weeks, Posilac®); (3) Medium (60 μg g−1 BW per 3 weeks, Posilac®); and (4) High (120 μg g−1 BW per 3 weeks, Posilac®). Fish were reared in 76-l tanks supplied with 26.0 °C flow-through well water for 9 weeks. Fish were fed a 36% CP commercial diet twice each day to apparent satiation. Feed consumption increased (P<0.05) 15% with rbGH treatment in the Norris strain while no significant increase in feed consumption was observed in rbGH-treated NWAC103 catfish. Compared to sham controls, all treatments (Low, Medium, and High) increased final weights (P<0.05) (168±13.2 vs. 144±10.0 g) (average weight of the three treatments), but no overall improvement in feed conversion ratio (FCR) was observed in NWAC103 fish. In the Norris strain, the High treatment increased (P<0.05) final weight (135±6.2 vs. 106±8.7 g) but no improvement in FCR was observed when compared to sham controls. rbGH treatments increased (P<0.05) total length in both strains, but no difference in condition factor (CF), hepatosomatic index (HSI), or body composition was observed. On day 63, levels of a 45-kDa IGFBP (catfish-IGFBP-3) were similar between treated and untreated fish in both strains. Results of this study indicate the Low treatment was as effective in promoting growth as the High treatment in the NWAC103 strain. Results of the body composition analysis suggest that the increase in weight gain was not due to an increase in fat deposition. The observed increase in length suggests rbGH enhances linear growth in channel catfish. Similar levels of cf-IGFBP-3 between treated and untreated fish may reflect “steady state” levels of cf-IGFBP-3 in growing fish. Identifying other endogenous growth factor(s) responsible for the observed increase in growth rate will be crucial in our understanding of improving growth in cultured channel catfish.

Routine Measures of Stress Are Reduced in Mature Channel Catfish during and after AQUI-S Anesthesia and Recovery

Abstract Mature channel catfish Ictalurus punctatus were exposed to water containing three different concentrations (20, 40, and 60 mg/L) of AQUI-S (50% isoeugenol) during routine handling procedures at a commercial catfish facility. Anesthetic efficacy, recovery time, and the effects of AQUI-S on routine measures of stress were compared with similar measures in a group of fish sampled prior to anesthesia (preanesthesia [PA] group) and a group anesthetized with 100 mg tricaine methanesulfonate (TMS)/L. On average, all the fish lost equilibrium at 8.0, 3.9, and 3.7 min when anesthetized in 20, 40, and 60 mg AQUI-S/L, respectively. Fish anesthetized with TMS lost equilibrium at 4.5 min. Recovery time in freshwater was 2.1, 2.8, and 5.3 min for fish anesthetized in 20, 40, and 60 mg/L AQUI-S, respectively. Recovery time after TMS anesthesia was 1.7 min. Short-term (24-h) survival was 100% for all treatments, and long-term (21-d) survival ranged from 87.5% for TMS-anesthetized fish to 98.8% for fish anestheti...

Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration on target tissue mRNA expression. Analysis of sequence similarities indicated two genes putatively encoding GHS-R1 and GHS-R2, respectively, which have been known to be present in zebrafish. Organization and tissue expression of the GHS-R1 gene was similar to that reported for other species, and likewise yielded two detectable mRNA products as a result of alternative splicing. Expression of both full-length, GHS-R1a, and splice variant, GHS-R1b, mRNA was highest in the pituitary. Gene organization of GHS-R2 was similar to GHS-R1, but no splice variant was identified. Expression of GHS-R2a mRNA was highest in the Brockmann bodies. GHS-R1a mRNA was detected in unfertilized eggs and throughout embryogenesis, whereas GHR-R2a mRNA was not expressed in unfertilized eggs or early developing embryos and was the highest at the time of hatching. Catfish intraperitoneally injected with catfish ghrelin-Gly had greater mRNA expression of GHS-R1a in pituitaries at 2 h and Brockmann bodies at 4 h, and of GHS-R2a in Brockmann bodies at 6 h post injection. Amidated catfish ghrelin (ghrelin-amide) had no observable effect on expression of either pituitary receptor; however, GHS-R1a and GHS-R2a mRNA expression levels were increased 4 h post injection of ghrelin-amide in Brockmann bodies. This is the first characterization of GHS-R2a and suggests regulatory and functional differences between the two catfish receptors.

Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels

We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0ng/ml and the ED(50) value (standard curve range 150-0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose-response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 microg/g body weight, 3 injections every 5 days) with either bovine GHRH(1-29)-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH(2)) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly (p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly (p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.

Effect of Fasting on Pituitary Growth Hormone Expression and Circulating Growth Hormone Levels in Striped Bass

Abstract The mechanisms controlling the circulating levels of growth hormone (GH) during periods of starvation in fish are not well defined. In this study, the effect of fasting on GH release and pituitary messenger RNA (mRNA) expression in striped bass Morone saxatilis was examined. Over a 4-week period, the body mass of striped bass fed to satiety increased 22.9%, while that of fasted fish decreased 17.3%. The plasma GH concentrations of fed fish remained low throughout the experiment, but those of fasted fish increased significantly (P < 0.001) after 2 and 4 weeks. The striped bass that were fasted for 2 weeks demonstrated a 33% increase (P = 0.10) in GH mRNA expression compared with those fed to satiety. These data confirm that short-term fasting in striped bass increases their circulating levels of GH in a manner similar to that experienced in other species of fish. This study also demonstrates—for the first time in a fish species—that pituitary GH mRNA expression increases during fasting, which sugg...