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From dysbiosis to resilience: Microbiome engineering for sustainable shrimp aquaculture.

The intensification of shrimp aquaculture has increased exposure to disease, environmental perturbations, and antimicrobial pressure, making microbial stability increasingly relevant to sustainable production. Microbiome stability-encompassing resistance to disturbance and resilience of functional recovery-provides an ecological framework for understanding how shrimp and culture-environment microbial communities respond to intensive farming. This review examines the transition from microbial homeostasis to dysbiosis and evaluates how microbiome engineering could redirect disrupted communities towards resilient states. Evidence is integrated across the intestine, hepatopancreas, rearing water, sediment and biofloc to assess how host genetics, ontogeny, diet, culture conditions, antibiotics and pollutants shape microbiome assembly and destabilization. Disease-associated changes in acute hepatopancreatic necrosis disease, white faeces syndrome, Enterocytozoon hepatopenaei infection, and white spot syndrome virus infection are critically evaluated, with explicit separation of associations, pathogen-induced dysbiosis, and community-level causality. Established and emerging interventions-including probiotics, prebiotics, synbiotics, functional diets, biofloc management, phages, postbiotics, microbiota transplantation and synthetic microbial communities-are assessed according to their capacity to modify microbial function, persistence and recovery rather than taxonomic change alone. We further examine how multi-omics, microbiome-informed breeding, and environmental monitoring could support biomarker development, predictive decision support and context-specific intervention. We argue that progress requires a shift from taxonomic description to function-guided engineering, from endpoint comparisons to direct measurement of resilience, and from laboratory efficacy to reproducible farm-scale validation. Overall, microbiome management may contribute to more disease-resilient and sustainable shrimp production, provided that its effectiveness can be validated under commercial farming conditions.

Dysbiosis↗

Inheritance of the epigenetic signature and reduced intermuscular bone phenotype acquired via DNA methylation editing of the runx2 b promoter in zebrafish.

The presence of intermuscular bones (IBs) can directly affect the economic value of aquaculture fish. Although genome editing can create IB-free fish by knocking out key IB-related genes, such as runx2b, the associated DNA sequence alterations raise food safety and health concerns, limiting its breeding applications. In this study, we used CRISPR/dCas9-mediated epigenome-editing technology targeting the runx2 b promoter in zebrafish to alter DNA methylation patterns without changing the DNA sequence. Our results showed that higher runx2 b promoter methylation patterns significantly inhibited eGFP mRNA expression levels in the recombinant plasmid. Using the CRISPR/dCas9-Dnmt7 system to enhance methylation of the zebrafish runx2b promoter, we observed a significant decrease in runx2 b mRNA expression levels in the F0 generation. The IBs in the 11 th-16 th muscle segments of the adult F0 fish were significantly shorter compared with the controls. Inbreeding of fish was used to produce F1 and F2 offspring that retained these high promoter methylation levels, along with persistent runx2b expression suppression and IB development inhibition. Transcriptome sequencing analysis suggested that increasing runx2 b promoter methylation levels may synergistically induce additional epigenetic modifications, potentially affecting the PPAR signaling pathway and FoxO transcription factor regulation, which appears to inhibit osteoblast proliferation and differentiation. Overall, this study demonstrates an innovative application of epigenetic editing technology for aquaculture breeding. By precisely regulating the expression patterns of key genes for economically important traits while preserving genomic DNA integrity, this approach provides a theoretical foundation and technical support for improving fish economic traits.

Animals↗

Multi-residue screening of chlorinated and brominated compounds from aquaculture samples using matrix solid-phase dispersion--gas chromatography-mass spectrometry.

An effective multiresidual method for the trace analysis of fifteen compounds from a diverse group of pesticides, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyl (PCBs) and polybrominated biphenyl (PBBs) in aquaculture feed is described. The analytical procedure is based on the matrix solid-phase dispersion (MSPD) of feed sample and subsequent elution with hexane. The MSPD process was evaluated using an asymmetrical experimental design 2(3)3(2)//9. Factors such as C18 sorbent amount, kind of adsorbents, solvent volume and elution mode were considered. The results suggest that the operational MSPD conditions are elution with pressure, 1 g of C18, basic alumina as adsorbent and 30 mL of hexane. The overall method including MSPD procedure and GC coupled to mass spectrometry (MS/MS) has been applied to several samples of aquaculture feed and marine species. Precision and accuracy of the analytical method were determined using the reference material from the International Atomic Energy Agency (IAEA-406), showing a good agreement to the referenced values.

Aquaculture↗

A morphological study of the brain of Solea senegalensis. I. The telencephalon.

In this paper we present an anatomical description of the telencephalon of Solea senegalensis based on cresyl violet and haematoxilin-eosin-stained serial transverse sections. This work was conducted as a basis for the precise localization of neuroendocrine territories in the brain of a species with growing interest in marine aquaculture. The external asymmetric morphology of Senegalese sole is correlated with the asymmetry of the forebrain. The right olfactory nerve and bulb are larger than the contralateral ones and this asymmetry is also extended to the cerebral hemispheres. The olfactory bulb comprises an outer olfactory nerve fiber layer, a glomerular layer, an external cellular layer, a secondary olfactory fiber layer and an internal cellular layer. The telencephalic hemispheres can be divided in area dorsalis and area ventralis, consisting of eleven and eight cell masses, respectively. The area dorsalis comprises five subareas: a pars medialis (Dm), subdivided into four nuclei termed Dml to Dm4; a pars dorsalis (Dd); a pars lateralis (D1), which consists of dorsal (Dld), ventral (Dlv) and posterior (Dlp) subdivisions; a pars centralis (Dc); and more caudally, a pars posterioris (Dp), which is very prominent in this species. A nucleus taenia (NT) was observed in the transitional region between area dorsalis and area ventralis. The area ventralis consists of pars dorsalis (Vd), pars ventralis (Vv), pars supracommissuralis (Vs), pars postcommissuralis (Vp), pars lateralis (V1), pars centralis (Vc), pars intermedia (Vi) and nucleus entopeduncularis (E). A periventricular organ, that we have termed lateral septal organ (LSO), was observed in the ventral telencephalon, medial to Vv.

Animals↗

Metabolic requirements of red drum, Sciaenops ocellatus, for protein and energy based on weight gain and body composition.

Precise formulation of diets that meet but do not greatly exceed nutritional requirements should assist in lowering feed costs for commercial aquaculture of red drum, Sciaenops ocellatus. In this study, protein and energy requirements of red drum for maintenance and maximum gain were determined by feeding a diet containing digestible protein (DP) at 36.5% and 14.2 kJ digestible energy (DE) per gram at various rates for 8 wk in two separate experiments. Changes in weight and whole-body energy and protein were measured and regressed against protein or energy fed using a nonlinear procedure. In the first experiment, juvenile fish [ approximately 3. 4 g initial body weight (BW)] were either starved or fed at one of the following g/(100 g BW.d): 0.5, 1, 2, 4, 6, or 8. The second experiment utilized larger red drum (approximately 5.5 g initial weight), fed 0.75, 1.5, 3, 5, 5.5, 6, 6.5 or 7 g/(100 g BW.d) to confirm and refine results from the first experiment. Based on maintenance of body weight in both experiments, red drum had a protein maintenance requirement of 1.5 and 2.5 g DP/(kg BW.d) whereas estimates based on maintenance of whole-body protein were 0.5 and 2.2 g DP/(kg BW.d). Energy requirements for maintenance of weight and body energy ranged from 58 to 93 and 92 to 97 kJ DE/(kg BW.d), respectively. Protein requirements for maximum weight gain and change in body protein ranged from 20 to 25 g DP/(kg BW.d), whereas energy requirements for maximum weight gain and whole-body deposition ranged from 776 to 958 and 914 to 985 kJ DE/(kg BW.d), respectively. These requirements for maintenance and maximum gain of red drum should assist in formulation of diets for a variety of desired feeding strategies.

Animal Nutritional Physiological Phenomena↗

Ecological data in Integrated Coastal Zone Management: case study of Posidonia oceanica meadows along the Corsican coastline (Mediterranean Sea).

Integrated Coastal Zone Management (ICZM) contributes towards maximizing the benefits provided by the coastal zone and minimizing conflicts and the harmful effects of activities upon each other. The coastal zone includes highly productive and biologically diverse ecosystems, but ecological data (including structure and processes) seem to be neglected. The purpose of this article is to present a case study of Posidonia oceanica meadows (seagrass beds) along the Corsican coastline (Mediterranean Sea) in order to exemplify the usefulness of ecological data to Integrated Costal Management programs. We will try to determine how the use of organisms could be enhanced. These investigations show the undoubted success of the Corsican Posidonia oceanica protection program, with a detailed description of the ICZM that precisely presents each component (e.g., mapping, assessment of water quality, implementation of a system to aid decision-making concerning the installation of new aquaculture units). This experience on the Corsican coasts could be used as an example in order to transfer to other locations in the Mediterranean Sea and/or to other target species.

Alismatales↗

Intra- and interlaboratory performance of antibiotic disk-diffusion-susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments.

In the course of an international research project on hazard analysis of antimicrobial resistance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquaculture environments was sent by the central laboratory to the other participating laboratories. In each laboratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were performed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of subcultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global averaging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indicated that the use of either ISA or MH medium in DD testing has a limited impact on the method's precision among different laboratories. In conclusion, the current study has provided a validated SOP to promote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive breakpoints should be specifically designed and validated for aquaculture drugs and organisms.

Anti-Bacterial Agents↗

Liquid chromatographic determination of oxytetracycline in edible fish fillets from six species of fish.

The approved use of oxytetracycline (OTC) in U.S. aquaculture is limited to specific diseases in salmonids and channel catfish. OTC may also be effective in controlling diseases in other fish species important to public aquaculture, but before approved use of OTC can be augmented, an analytical method for determining OTC in fillet tissue from multiple species of fish will be required to support residue depletion studies. The objective of this study was to develop and validate a liquid chromatographic (LC) method that is accurate, precise, and sensitive for OTC in edible fillets from multiple species of fish. Homogenized fillet tissues from walleye, Atlantic salmon, striped bass, white sturgeon, rainbow trout, and channel catfish were fortified with OTC at nominal concentrations of 10, 20, 100, 1000, and 5000 ng/g. In tissues fortified with OTC at 100, 1000, and 5000 ng/g, mean recoveries ranged from 83 to 90%, and relative standard deviations (RSDs) ranged from 0.9 to 5.8%. In all other tissues, mean recoveries ranged from 59 to 98%, and RSDs ranged from 3.3 to 20%. Method quantitation limits ranged from 6 to 22 ng/g for the 6 species. The LC parameters produced easily integratable OTC peaks without coelution of endogenous compounds. The method is accurate, precise, and sensitive for OTC in fillet tissue from 6 species of fish from 5 phylogenetically diverse groups.

Animals↗

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry.

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.

Aniline Compounds↗

Marker-assisted estimation of quantitative genetic parameters in rainbow trout, Oncorhynchus mykiss.

Estimation of quantitative genetic parameters conventionally requires known pedigree structure. However, several methods have recently been developed to circumvent this requirement by inferring relationship structure from molecular marker data. Here, two such marker-assisted methodologies were used and compared in an aquaculture population of rainbow trout (Oncorhynchus mykiss). Firstly a regression-based model employing estimates of pairwise relatedness was applied, and secondly a Markov Chain Monte Carlo (MCMC) procedure was employed to reconstruct full-sibships and hence an explicit pedigree. While both methods were effective in detecting significant components of genetic variance and covariance for size and spawning time traits, the regression model resulted in estimates that were quantitatively unreliable, having both significant bias and low precision. This result can be largely attributed to poor performance of the pairwise relatedness estimator. In contrast, genetic parameters estimated from the reconstructed pedigree showed close agreement with ideal values obtained from the true pedigree. Although not significantly biased, parameters based on the reconstructed pedigree were underestimated relative to ideal values. This was due to the complex structure of the true pedigree in which high numbers of half-sibling relationships resulted in inaccurate partitioning of full-sibships, and additional unrecognized relatedness between families.

Animals↗

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography.

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.

Amino Acids↗

Performance of a proposed determinative method for p-TSA in rainbow trout fillet tissue and bridging the proposed method with a method for total chloramine-T residues in rainbow trout fillet tissue.

Chloramine-T is an effective drug for controlling fish mortality caused by bacterial gill disease. As part of the data required for approval of chloramine-T use in aquaculture, depletion of the chloramine-T marker residue (para-toluenesulfonamide; p-TSA) from edible fillet tissue of fish must be characterized. Declaration of p-TSA as the marker residue for chloramine-T in rainbow trout was based on total residue depletion studies using a method that used time consuming and cumbersome techniques. A simple and robust method recently developed is being proposed as a determinative method for p-TSA in fish fillet tissue. The proposed determinative method was evaluated by comparing accuracy and precision data with U.S. Food and Drug Administration criteria and by bridging the method to the former method for chloramine-T residues. The method accuracy and precision fulfilled the criteria for determinative methods; accuracy was 92.6, 93.4, and 94.6% with samples fortified at 0.5X, 1X, and 2X the expected 1000 ng/g tolerance limit for p-TSA, respectively. Method precision with tissue containing incurred p-TSA at a nominal concentration of 1000 ng/g ranged from 0.80 to 8.4%. The proposed determinative method was successfully bridged with the former method. The concentrations of p-TSA developed with the proposed method were not statistically different at p < 0.05 from p-TSA concentrations developed with the former method.

Animals↗

High-performance liquid chromatography method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate.

This study presents an HPLC method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate lipid fraction. The method includes microscale saponification and extraction with n-hexane. Liposoluble vitamins and cholesterol were quantified by HPLC with UV detection (HPLC-UV), on a 25 cm x 0.46 cm SS Exil ODS 5 microm column, mobile phase 68:28:4 (v/v/v) methanol:acetonitrile:water; flow rate 1.4 ml/min; column temperature 36 degrees C. The detection was operated using two channels of a diode-array spectrophotometer, 325 nm for retinol and 208 nm for alpha-tocopherol and cholesterol. With these conditions, the overall recovery was 95.7, 100.8, and 98.0% for retinol, alpha-tocopherol, and cholesterol, respectively. The method precision (relative standard deviation) was 1.83% for retinol, 2.32% for alpha-tocopherol, and 1.98% for cholesterol. This method was used to quantify the cited analytes in the hydrolysate obtained during lactic acid fermentation of shrimp waste. This hydrolysate may be a valuable supplement of nutrients in fish production.

Animals↗

Simple confirmatory method for the determination of erythromycin residues in trout: a fast liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry.

In recent years, erythromycin has received considerable attention for its therapeutic efficacy against some bacterial kidney diseases in aquaculture and, therefore, suitable and sensitive analytical methods to monitor erythromycin residues in fish are required. A fast sample treatment followed by an LC-ESI-MS/MS method is described for the purification, identification, and quantification of erythromycin A residues in fish. After two extractions with acetonitrile, samples were defatted with n-hexane, filtered, and analyzed by tandem mass spectrometry. Three characteristic transition reactions (m/z 734 --> 716, 734 --> 576, and 734 --> 558) in multiple reaction monitoring were tested for the determination and confirmation of erythromycin A. The method was in-house validated through the determination of precision, accuracy, specificity, stability, calibration curve, decision limit (CCalpha), and detection capability (CCbeta), in accordance with European Commission Decision 657/2002. The coefficients of variation ranged from 1.8 to 9.4% and from 7.5 to 10.9% for intra- and interday repeatability, respectively. Recovery data were also satisfactory, with values varying from 85 to 97%. The method was specific, stable, and robust enough for the required purposes. The calibration curve showed a good linearity in the whole range of the tested concentrations (0-1000 microg kg(-1)) with a correlation coefficient (r2) equal to 0.9956. CCalpha and CCbeta were found to be 220 and 238 microg kg(-1), respectively.

Animals↗

Male reproductive system in Senegalese sole Solea senegalensis (Kaup): anatomy, histology and histochemistry.

Despite the fact that the Senegalese sole Solea senegalensis is a target species for the aquaculture industry in Spain and Portugal, very little is known about its gametogenesis and especially about testicular development. Therefore, the male reproductive system in adult S. senegalensis was described using a histological and histochemical approach. Mean gonadosomatic index was very low (0.094+/-0.004%) and suffered slight changes throughout the experimental period. In transverse sections, the testis presents a reniform structure with two main regions, a cortical one with seminiferous lobules where germ cells proliferate in spermatocysts (germ/Sertoli cells units), and a medullar one with spermatic ducts that collect and store the produced sperm. The germinal compartment is organized into branching lobules of the unrestricted spermatogonial type, although the majority of type A spermatogonia are located at the distal part of the lobules. Spermatogenesis seems to be semi-cystic, since spermatids are released into the lobule lumen, where they are transformed into spermatozoa. Proteins in general, especially those rich in arginine, and carboxylated mucosubstances/glyco-proteins increased from spermatogonia to spermatozoa. 3beta-Hydroxysteroid dehydrogenase enzymatic activity was exclusively observed in Leydig cells. The present study provides the first precise description of male reproductive apparatus in S. senegalensis.

Animals↗

Confirmation of gentian violet and its metabolite leucogentian violet in catfish muscle using liquid chromatography combined with atmospheric pressure ionization mass spectrometry.

Gentian violet (GV) is a triphenylmethane dye antiseptic with potential for illegal use in livestock production, especially aquaculture where the related malachite green has been widely used. This potential misuse has regulatory importance because of the observed rodent carcinogenicity of GV. This report describes the use of online LC-APCI/MS for confirmation of incurred GV residues, and those of its principal metabolite, LGV, in catfish muscle following treatment of live catfish with GV under putative use conditions. LC with APCI/MS detection provided sensitive analysis of GV and LGV with estimated detection limits of < 1 pg observed for both compounds. Fragmentation of GV and LGV via in-source CID was effected by varying the sampling cone-skimmer voltage. Ion intensity data were collected using a rapid cone voltage switching procedure that permits selected ion acquisition under optimal conditions for the parent molecule and several selected fragment ions. For GV, four ions including the ionized molecule were used and for LGV, six ions including the protonated molecule were used. The levels of GV and LGV in muscle from fish dosed with 10 micrograms/l in aquarium water were determined by LC/VIS to be 0.5 and 44 ppb, respectively. Analysis of these samples yielded ion intensity ratios that agreed precisely between injections (< 5%) and accurately with those generated by a comparable amount of authentic GV and LGV (< 10% deviation). These results show the utility of on-line LC-APCI/MS to do both sensitive confirmatory analyses of incurred drug residues for use in monitoring the food supply.

Animals↗