Guidelines for the Use of Fishes in Research

Background


Inhibition of food intake in obese subjects by peptide YY An introduction by Herbert B. Morbidity and mortality before and after bariatric surgery for morbid obesity compared with the general population. Fishes are worthy of experimental and observational research: The extended calendar for the — school year will be posted in a few days. Franklin Author , J. A bulletin highlighting protected marine or anadromous fishes http:

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With such widespread use of supplements in the general population and with the specific focus of athletes on achieving peak performance, it is not surprising that a high prevalence of supplement use is reported in most surveys of athletes.

Some supplements may be used for multiple functions. Zinc, for example, may be taken with the aim of promoting wound healing and tissue repair, 6 or reducing the severity and duration of the symptoms of an upper respiratory tract infection. Supplements target a range of scenarios of use, so different approaches are needed to assess their effectiveness.

Supplements aimed at correcting nutrient deficiencies need to be judged on their ability to prevent or treat suboptimal nutrient status, with the benefit accruing from the removal of the associated impairment of health, training capacity or performance. The effectiveness of sports foods might be hard to isolate when they are used within the general diet to meet everyday energy needs and nutrient targets.

However, benefits may be more easily detected when they are specifically consumed before, during or after an event or training session to provide nutrients that are limiting for performance eg, to provide fuel for the muscle or brain or to defend homeostasis eg, by replacing water and salt losses. Performance-enhancing supplements, which are claimed to achieve direct or indirect benefits, pose a greater challenge in terms of a sound evidence base.

With only a few exceptions, there is a scarcity of research, and many of the available studies are not of sufficient quality to warrant their application to elite athletes. Substantiating the claims made about performance supplements and sports foods is difficult. Figure 1 provides a proposed hierarchical model of the relative strength of the evidence provided by different information sources.

However, most of the information around supplement efficacy in sport comes from models with the lowest rigour: Systematic reviews and meta-analyses, which synthesise the outputs of many studies to yield a conclusive statement of efficacy in a broad sense, are at the top of the evidence hierarchy. While these summaries help to provide information about the general use of performance supplements, scientific trials that are properly controlled and well-conducted provide the basis for these reviews as well as an opportunity to address more specific questions about supplement applications.

Thus, meta-analyses are a reflection only of the quality and quantity of the studies that are available for review, and may also be influenced by the inclusion and exclusion criteria applied to the available data. Hierarchy of evidence used to establish good practice focused on the issue of nutritional supplements.

Practical issues may cause some variations to ideal design, but sports scientists are encouraged, if they wish their results to be applicable to athletes in competition, to ensure that their studies include the following:. Given the specificity of the information that is required by some athletes and their support staff to assess the effectiveness of a supplement eg, related to a targeted event and its conditions, the specific individual, the combination with other performance strategies , it is unreasonable to expect that definitive evidence will always be available.

Scenarios that fall outside the scope of the available literature or practical research design may need to be interrogated by individual or small group case studies. Recommended methodologies for these studies include repeated baseline performances before the introduction of the supplement, or an alternating series of presentation and absence of the supplement.

For the purposes of this overview, we rely primarily on studies of healthy adults that are relevant to athletes. We recognise that data from studies of elite athletes are almost entirely absent. We also recognise that mechanistic studies on animal and cell culture models are useful in identifying mechanisms, but a mechanism is not necessary to demonstrate an effect that may be meaningful to an athlete: While the variation in the genome between individuals is less than 0.

Many micronutrients play an important role in the regulation of processes that underpin sports performance, ranging from energy production to the manufacture of new cells and proteins. When suboptimal nutritional status is diagnosed, the use of a nutrient supplement to reverse or prevent further deficiencies can contribute to the overall treatment plan. Nutritional assessment of an athlete involves systematic protocols that obtain, verify and interpret evidence of nutrition-related problems, as well as their causes and significance.

A complete assessment should ideally include a detailed medical and nutritional history, diet evaluation, anthropometry and body composition analysis, and biochemical testing. Nutrients that often need to be supplemented under these circumstances include iron, calcium and vitamin D table 1. Iodine for those living in areas with low levels of iodine in foods or not using iodised salt , folate for women who might become pregnant and vitamin B 12 for those following a vegan or near-vegan diet supplementation may be warranted in these population groups, but these considerations do not apply specifically to athletes.

Examples of micronutrients often requiring supplementation in athletes see Larson-Meyer et al 18 for additional information. Sports nutrition guidelines provide clear recommendations for targeted intake of energy and nutrients in a variety of contexts. In these cases, sports foods can provide a convenient, although usually more expensive, alternative option for meeting these nutrient goals.

Table 2 provides an overview of products that fit this description and their more common evidence-based uses. Summary of common sports foods and functional foods used by athletes. A few performance-enhancing supplements might, at the present time, be considered to have an adequate level of support to suggest that marginal performance gains may be possible.

These supplements include caffeine, creatine in the form of creatine monohydrate , nitrate, sodium bicarbonate and possibly also Beta-alanine. Whenever possible, supplements should be trialled thoroughly by the athlete in training that mimics the competition milieu as closely as possible before committing to use in a competition setting. Athletes should do a careful risk analysis to see if the marginal gains would outweigh the risk of inadvertent doping due to contamination.

Supplements with good to strong evidence of achieving benefits to performance when used in specific scenarios. Illness is a major problem for athletes if it interrupts training or occurs at a critical time, such as during a selection event or a major competition.

Vitamin C during periods of heavy exertion and zinc lozenges at the onset of symptoms may be useful, but high doses of single antioxidants, particularly vitamins C and E, may blunt exercise-induced training adaptations.

Cochrane reviews have noted the low quality of many studies on nutritional supplements that are claimed to support immunity; specifically, small samples, poor controls and unclear procedures for randomisation and blinding were commonplace. Nutritional supplements for immune health in athletes: Many products claim to provide benefits of this nature; table 5 summarises the evidence for some of the most popular compounds.

Finally, the manipulation of body composition, including gaining lean muscle mass and reducing body fat levels, can contribute to performance in many events. Protein is considered to be the premier ingredient in weight gain-promoting supplements, and evidence-based reviews conclude that protein is effective at promoting lean mass gain when combined with resistive exercise. Table 6 summarises the evidence for some of the most common ingredients or products of this type.

Supplements that may assist with training capacity, recovery, muscle soreness and injury management. Supplements promoted to assist with physique changes: Adverse effects from the use of supplements may arise from a number of factors, including the safety and composition of the product per se and inappropriate patterns of use by athletes. Poor practices by athletes include the indiscriminate mixing and matching of many products without regard to total doses of some ingredients or problematic interactions between ingredients.

For example, iron supplementation in those with already adequate iron stores can result in symptoms that may begin with vomiting, diarrhoea and abdominal pain, and develop to haemochromatosis and liver failure.

These incidents were due to errors in the dose calculation: Athletes and members of their support team should be aware of the regulations that govern the manufacture and marketing of supplements.

Similar regulations apply in most other countries, where supplements are regulated in the same way as food ingredients and are therefore not subject to the stringent regulations that are applied to the pharmaceutical industry. This means that there is no requirement to prove claimed benefits, no requirement to show safety with acute or chronic administration, no quality assurance of content, and liberal labelling requirements.

It is well-recognised that there are problems with some of the dietary supplements on sale, but the options open to those responsible for food safety are limited by the legislation that applies. The FDA regularly uses its powers to recall products in breach of the regulations, although they fully admit that their resources are insufficient for comprehensive monitoring, and recalls generally occur only after many people are harmed https: Examples of product complaints have included the presence of impurities, including lead, broken glass and metal fragments, because of the failure of the producers to observe good manufacturing practice.

The risk of gastrointestinal upset because of poor hygiene during the production and storage of products is also of concern. It should be noted, though, that all of these problems are also regularly reported in normal foods.

Some supplements may actually cause harm to health, but these can be difficult to identify, and products are usually withdrawn only after a significant number of adverse events have occurred.

For example, a range of products containing hydroxycitric acid were withdrawn from sale, but only after they were linked with the death of one consumer and with a substantial number of other cases of liver toxicity, cardiovascular problems and seizures https: The biggest concern for athletes who compete under an antidoping code usually the World Anti-Doping Code, as published by WADA is that supplements can contain prohibited substances that result in an antidoping rule violation ADRV. Athletes—and their support teams—may be at risk for an ADRV if there is evidence that they have used or attempted to use products containing ingredients on the Prohibited List www.

For these athletes in particular, even if the ingestion of the prohibited substance was unintentional, the rules of strict liability within the World Anti-Doping Code mean that an AAF will be recorded, and may mean the loss of medals won or records set, and financial sanctions as well as temporary or permanent suspension from competition. Where there has been deliberate cheating or benefit accrued from the use of a prohibited substance, these penalties seem entirely appropriate, but it is undoubtedly true that some ADRVs can be attributed to the innocent ingestion of prohibited substances in dietary supplements, with catastrophic results for the athlete.

Other athletes are confused by the number of chemical names for some prohibited substances and thus fail to recognise them on the product label. However, the most worrying cause of an inadvertent AAF is the use of supplements that contain prohibited substances as an undeclared ingredient or contaminant. Since the publication of the seminal study on the presence of undeclared prohibited substances in supplements, 32 there have been numerous reports of supplement contamination.

It is difficult to gain a perspective of the true prevalence of supplement contamination. Some individual products or categories of products can be considered inherently more at risk of contamination due to the country of origin, the manufacturer, the type of product and the range of declared ingredients https: Nevertheless, it should also be recognised that common supplements, including vitamin C, multivitamins and minerals, have also been found, although rarely, to contain prohibited substances.

In some cases, the amount of the prohibited substance in a supplement may be high, even higher than the normal therapeutic dose. This amount would certainly have a potent anabolic effect, but would likely produce serious side effects, including psychiatric and behavioural effects, and significant damage to a range of body systems including the liver. Most reports of adverse health outcomes resulting from supplement use have focused on liver problems of varying degrees of severity, but other organs are also affected.

One epidemiological case—control study 38 examined the association between use of muscle-building supplements and testicular germ cell cancer TGCC risk, with TGCC cases and controls from eastern USA. Ironically, supplements that are contaminated with extremely small amounts of prohibited substances—too low to have any physiological effect—may still cause a positive doping outcome.

For instance, ingestion of norandrostenedione, a precursor of nandrolone, will result in the appearance in the urine of norandrosterone, the diagnostic metabolite for nandrolone. There can be no absolute guarantee that any product is entirely safe, but these schemes do help the athlete to manage the risk. Athletes contemplating the use of dietary supplements should consider very carefully whether the possible benefits outweigh the risks of a doping offence that might end their career.

Dietary supplements are an established part of the landscape of modern sport and are likely to remain so. Athletes who take supplements often have no clear understanding of the potential effects of supplements they are using, but supplements should be used only after a careful cost-benefit analysis has been conducted. On the other side lie the costs, the possibility of using an ineffective supplement, the possible risks to health and the potential for an ADRV. A flow of questions that could be posed in reaching an informed decision is shown in figures 2 and 3.

Flow chart to guide informed decision making and reducing risk of antidoping rule violation during nutritional supplement use. Flow chart to guide informed decision making and reducing risk of antidoping rule violation during ergogenic supplement use. In deciding whether to use a supplement, athletes should consider all aspects of their maturation in, and preparation for, their event to ensure that the supplement under consideration provides an advantage that no other strategy can address.

Whether the supplement is practical to use should also be assessed: Athletes who do not have regular access to such a network should consider decisions around supplement use as an important reason to consult an independent sports nutrition expert as well as a physician.

Analysis of the evidence around the effectiveness of supplements and their safety is often difficult. A complete nutritional assessment may provide an appropriate justification for the specific use of nutritional supplements and sports foods. For a small number of sports supplements, there is good evidence of a performance effect or indirect benefit for some athletes in some specific situations with little or no risk of adverse outcomes.

Furthermore, the situation in which the athlete wishes to use the supplement may differ in important ways from its substantiated use. Repeated trials may be necessary to establish whether a true effect, rather than just random variation, is seen in response to use of any novel intervention.

Some trial and error may also be involved in fine-tuning the supplement protocol to suit the needs of the specific situation of use or the individual athlete.

Evidence to support the effectiveness and safety of many of the supplements targeted at athletes, however, is largely absent. There seems to be little incentive for those selling supplements to invest the substantial sums needed to undertake detailed scientific evaluation of their products. Even where some evidence does exist, it may not be relevant to the high-performance athlete because of limitations in the study design such as the specificity of the exercise tests , the study population or the context of use.

Failure to verify the composition of the supplements used may also give misleading results. It seems sensible to exercise caution when using supplements, as any compound that has the potential to enhance health or exercise performance by altering physiological function must also have the potential for adverse effects in some individuals.

Athletes should see good evidence of a performance or other benefit, and should be confident that it will not be harmful to health, before accepting the financial cost and the health or performance risks associated with any supplement. Finally, the athlete should be sure, if supplements or sports foods are to be used, that they have undertaken due diligence to source products that are at low risk of containing prohibited substances. A few supplements can directly enhance competition performance.

A strict risk-benefit analysis involving a decision tree approach to the effectiveness, safety and risks should identify the small number of products that may benefit the athlete. Such an analysis requires the input of a well-informed sports nutrition professional. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Provenance and peer review Not commissioned; externally peer reviewed. You will be able to get a quick price and instant permission to reuse the content in many different ways.

Skip to main content. We use cookies to improve our service and to tailor our content and advertising to you. More info You can manage your cookie settings via your browser at any time. To learn more about how we use cookies, please see our cookies policy. Log in via OpenAthens. Log in using your username and password For personal accounts OR managers of institutional accounts.

Forgot your log in details? Register a new account? Forgot your user name or password? Search for this keyword. Latest content Current issue Archive Authors About. Log in via Institution. Abstract Nutrition usually makes a small but potentially valuable contribution to successful performance in elite athletes, and dietary supplements can make a minor contribution to this nutrition programme.

What is a supplement? For the purposes of this overview, we define a dietary supplement as the following: Prevalence of, and rationale for, use by athletes With such widespread use of supplements in the general population and with the specific focus of athletes on achieving peak performance, it is not surprising that a high prevalence of supplement use is reported in most surveys of athletes.

Assessing the evidence base for supplement use Supplements target a range of scenarios of use, so different approaches are needed to assess their effectiveness. Figure 1 Hierarchy of evidence used to establish good practice focused on the issue of nutritional supplements. Supplements used to prevent or treat nutrient deficiencies Many micronutrients play an important role in the regulation of processes that underpin sports performance, ranging from energy production to the manufacture of new cells and proteins.

View inline View popup. Table 1 Examples of micronutrients often requiring supplementation in athletes see Larson-Meyer et al 18 for additional information. Supplements sports foods used to provide a practical form of energy and nutrients Sports nutrition guidelines provide clear recommendations for targeted intake of energy and nutrients in a variety of contexts.

Table 2 Summary of common sports foods and functional foods used by athletes. Supplements that directly improve sports performance A few performance-enhancing supplements might, at the present time, be considered to have an adequate level of support to suggest that marginal performance gains may be possible. Table 3 Supplements with good to strong evidence of achieving benefits to performance when used in specific scenarios.

Table 4 Nutritional supplements for immune health in athletes: Table 5 Supplements that may assist with training capacity, recovery, muscle soreness and injury management. Table 6 Supplements promoted to assist with physique changes: Adverse effects Adverse effects from the use of supplements may arise from a number of factors, including the safety and composition of the product per se and inappropriate patterns of use by athletes.

Practical implications and decision tree Dietary supplements are an established part of the landscape of modern sport and are likely to remain so. Study objectives will determine the proper selection of type, volume, and concentration of anticoagulant, if needed.

Three main techniques have been devised for collecting blood from fishes: The tail is the preferred site for blood sampling. The vessels running beneath the vertebrae of the fish can be sampled by using a lateral or ventral approach. Cardiac punctures from the ventral side are sometimes used in fusiform fishes or through the operculum in laterally compressed species.

For repeated sampling, cannulae may be implanted in the dorsal aorta through the buccal cavity. Blood from the caudal vessels may be collected directly into collection tubes by cutting off the tails of sedated fish that will be euthanized following the procedure.

However, extraneous fluids and proteins that may influence cell quality often co-occur with this procedure. Caution must be exercised to ensure that the method of sedation will not interfere with subsequent analyses. Additional information on sampling methods for the collection of blood from fishes has been described by Klontz and Smith , Smith et al.

Additional tissues that are useful for collection include otoliths, gills, kidney, thyroid, spleen, testes, ovaries, liver, heart, brain, and muscle.

Collection of internal tissues typically requires sacrifice of the subject animals and must be preceded by appropriate anesthesia or euthanasia see section 8.

These tissues can also be used for such purposes as contaminants analyses see section 5. Tissues may be used fresh or frozen, or placed in a fixation or preserving medium such as buffered formalin, ethanol, or methanol and then histologically processed Luna ; Presnell et al. The purposes of some studies may be served by collections of scales, spines, or small pieces of fin, which can be accomplished with minimal effects on live fish and may be considered non-invasive sampling.

This is important when working with imperiled species and small populations see section 5. When transporting live tissues, the medium must have appropriate ionic and osmotic concentrations and may contain a sugar as an energy source.

Noncytotoxic antibiotics or antimycotic agents may be included to prevent the growth of bacterial and fungal organisms Jenkins a; Jenkins et al. Certain cell and nucleic acid stabilizers can make sampling of fish possible from remote locations for later tissue analysis in the laboratory Olivier and Jenkins, in press. Tagged and marked animals have been studied to obtain information on their behavior, population dynamics, and ecology, all of which are essential for developing conservation and management strategies.

Investigators can use both intrinsic and extrinsic identification systems, allowing the nature of the study to dictate the type of tag or mark employed. Integrated use of more than one tagging or marking technique helps ensure fish identification and is helpful in estimating tag loss rates.

Basic considerations for selecting a particular type of tag or mark in the context of the study objectives include potential effects on animal survival, behavior, and growth; tag permanency and recognition; number and size of the animals; stress of capture, handling, and marking; total costs; recovery of the marked fishes; and any required coordination among agencies, states, provinces, or countries Pine et al.

Investigators should also determine if the animal will be at greater than normal risk to predation, if its desirability as a mate will be reduced, and if a risk of infection is increased substantially, as well as other potential impacts ASIH et al. Because techniques for tagging and marking fishes have been extensively reviewed and are constantly evolving, literature reviews should inform the researcher McFarlane et al. The effects of marking on fishes depend on the physical condition of the fish at the time of release.

Occurrence of injury is species and size specific, and smaller fishes may be more susceptible. Minor wounds caused by most tagging and marking procedures typically heal satisfactorily without treatment with antibiotics.

All sedatives or antibiotics administered must be used in a manner consistent with regulatory requirements. The use of external tags and marks has evolved over a long period of time McFarlane et al. Both natural marks and artificial tags or marks are in common use in fisheries research, and each type offers different capabilities, as well as limitations. Natural, external marks include meristic characteristics, pigmentation, morphometric measures, and scale characteristics, but natural marks are subject to environmental and genetic influences.

Fish scale shape and size, as well as circulus spacing, are frequently used. The effective use of natural marks requires being well informed on fish life history.

Multiple methods are available for generating artificial external marks on fishes. Alteration of fins or other body parts, in practice for over years, can be accomplished by clipping or hole punching.

The selection of fins for clipping or removal is dependent upon the species under study; for example, clipping the anal fin of poeciliid males would be inappropriate because it functions as a copulatory organ, yet removal of the adipose fin of a salmonid would have negligible impacts ASIH et al. Hot or cold branding, the process of marking by placing an apparatus e. Fishes should be anesthetized prior to branding. External colorants for marking fishes include dyes, stains, inks, paints, liquid latex, visible implant elastomers, and plastics that are administered by immersion, spraying, injection, or tattooing.

Care is needed for distinguishing external colorant marks of similar tones Curtis External tags are conspicuous by their color, shape, size, or location of attachment and are composed of various materials. Designed for hydroturbine passage survival studies, an external transmitter that is molded to the fish has shown utility Deng et al. External tags commonly applied to fishes include dart nd t-bar anchor tags, disc tags, Carlin tags, and spaghetti or loop tags Guy et al.

Dart and anchor tags are the most frequently used external tags Nielsen , but a high loss rate has been reported in some species Guy et al. Proper insertion technique and use of small tags relative to fish size can reduce the potential for fish injury and tag loss Guy et al. Implanted coded wire tags, radio and acoustic telemetry transmitters, archival biologgers, passive integrated transponder PIT tags, visible implanted alpha numeric tags, otolith marks, and natural parasites are internal marking systems used to identify fish Prentice et al.

The use of a coded wire tag identification system has been tested for management and research applications with multiple genera of fishes Buckley and Blankenship including juvenile salmonids Liedtke et al. The coded wire tag is normally injected into cartilage, connective tissue, or muscle and is detected electronically later with a handheld device. Each PIT tag carries a unique code that is relayed to a handheld or stationary reading device when the tag is within range.

Advantages of PIT tags include a long lifespan and generally a high retention rate Freeland ; Guy et al. The PIT tag data can be read through soft and hard fish tissue; in seawater and freshwater; through glass, plastic, and metal containers; and when fishes are moving at some velocity.

Above certain fish size thresholds Tatara , they have little or no effect on fish growth, survival, or behavior Prentice et al. Various tags and methods are available for the PIT tagging procedures; information can be found at state websites e. Visible implanted tags are alphanumerically coded and made of polyester film. They are inserted subcutaneously into transparent tissue so that they remain externally visible Haw et al.

Common tagging locations include transparent tissues posterior to the eye, in the lower jaw, or in fin membranes. Tag retention varies by species, tag location within the body, and fish size; very small fishes may have insufficient transparent tissue to accommodate the size of the tag Griffiths Manipulating environmental temperature, feeding rates, photoperiod, external chemical baths, or labeled feeds can induce specific marks in fish otoliths.

Fishes being propagated under controlled conditions are ideal for such manipulations. Otolith microstructural features and induced marks are permanent and can be viewed and analyzed in fish of any age. Tetracycline and other fluorescent compounds e. Fish size, compound dosage and uptake method, and water chemistry can influence marking success with fluorescent compounds Beckman et al. Marking success is highest during times when fish growth is rapid Conover and Sheehan Otoliths and other calcified structures can also be marked with alkaline earth and rare earth elements Behrens Yamada and Mulligan or isotopically labeled compounds Munro et al.

Fisheries that require stock definitions and assessment of stocking success or dispersal of early life stages are well suited to otolith-marking techniques. Several taxonomic groups of fish parasites have been used as biological tags, and this method is best suited to the separation of relatively self-contained stocks of fishes MacKenzie Recovery of internal parasites used as biological tags is enhanced if parasites are associated with a specific anatomical site on the fish.

The decision to use a parasite as a natural mark on fish is determined by calculating the ratio of incidence of that parasite in one fish population to its incidence in another Wydoski and Emery Underwater biotelemetry involves attaching a device that relays biological information via ultrasonic or radio signals from a fish to a remote receiving system Cooke et al.

Radio transmission is practical only in freshwater at relatively shallow depths ASIH et al. The selection of a tag or transmitter and the method and site of attachment or implantation is to be appropriate for the species and size of fish and performed by trained personnel.

Surgical implantation of transmitters into the coelom is common with free-ranging fishes. Use of the smallest and lightest transmitter that provides the desired signal type, strength, and battery lifespan will minimize tag loss and potential effects of transmitter attachment on fish survival, growth, and behavior.

External, neutrally buoyant transmitters have been developed for turbine-passage studies with juvenile salmonids at hydroelectric facilities Deng et al.

With fish exposed to rapid pressure changes, external transmitters may decrease the likelihood of injury or death compared to surgically implanted transmitters Brown et al.

Techniques to minimize skin irritation should be used following attachment of external transmitters Crook see section 7. The development of techniques employing markers based on chromosome and nuclear DNA polymorphisms has been rapid and continues to evolve. Benefits have emerged for using DNA marks in selective breeding programs, in evaluating the contribution and effects of stocked species, and in delineating specific habitat requirements for hatchery-produced fish Purdom For managing natural populations, knowing whether the fish species exists as a single genetic unit or relatively genetically distinct groups is critical Beaumont and Hoare An additional incentive for the use of genetic tagging is that adequate tissue samples can be obtained nonlethally e.

Genetic tags are permanent and exist in all individuals, thus representing a good alternative to traditional tags. Prior to the development of DNA techniques for differentiating fish populations, investigators studied allozymes—variant enzyme forms that are coded by different alleles at the same locus or DNA sequence. This type of genetic analysis sometimes required sacrificing fish to obtain appropriate samples, and with karyotype analysis, the examination of dividing cells was required.

Small laboratory fishes such as Japanese Medaka and Zebrafish were used extensively as models for studies in vertebrate developmental genetics and for transgenic investigations Ozato and Wakamatsu Next-generation sequencing technologies rapidly obtain short DNA sequences at thousands of loci, providing a depth of potential for gathering genomic information Mardis Fisheries scientists dealing with such questions will need to update their knowledge of the appropriate, scientifically accepted genetic identification systems for their potential applications Lincoln ; Poompuang and Hallerman Stable isotopes are nonradioactive, naturally occurring forms of chemical elements that do not decay spontaneously and are generally energetically stable.

Stable isotopes of a particular chemical element differ in mass but otherwise have equivalent chemical properties. In contrast to radioisotopes, which are tightly regulated, the use of stable isotopes does not require specially approved facilities and permits. Isotope fractionation has been studied for many years in natural systems, and stable isotope ratios are now used with relative frequency for fish marking. Stable isotopes can inform studies on trophic food-web structures, feed efficiencies, fish migration and places of origin, contaminant bioaccumulation, and other physiological and ecological processes.

A variety of elements e. For obtaining fish tissues, sedation may be required see section 7. Sedatives and Related Chemicals or sacrificing may be necessary. Depending upon the objectives of the research, nonlethal sampling may be possible by using scales, sectioned fin rays or spines, fin clips, or muscle tissue samples obtained with a small biopsy punch for stable isotope analyses.

Sampling of otoliths as metabolically inert structures is also common. Different types of metabolically active tissues have different elemental turnover rates; therefore, each investigator must determine which tissues may provide materials needed to satisfy the requirements of the studies.

Representative information on the use of stable isotopes in animal ecology has been provided by Fry and Rubenstein and Hobson In a manner similar to stable isotopes, fatty acids can be used as biomarkers to identify nutrient pathways in food webs, predator-prey relationships, and the relative contributions of allochthonous remote versus autothonous local inputs.

The use of fatty acids as biomarkers is based on the principle that fishes and many other aquatic organisms are composed of what they have eaten. Once consumed, fatty acids may be catabolized for energy or biotransformed, so the fatty acid profiles within tissues tend to reflect the dietary fatty acid profile.

Some fatty acids cannot be synthesized by vertebrates i. As mentioned for stable isotopes, various tissues have different metabolic turnover rates; thus, to be accurate, efforts linking tissue fatty acid profiles with chronological records of feeding behavior involve validation studies to account for establishing rates of profile change. For example, phospholipid profiles tend to include certain saturated fatty acids e. Traditionally, muscle and liver tissues have been used for analyses, and sacrificing the animal has been necessary.

However, adipose fin clips have shown utility for such analyses M. For more information on the role of fatty acids in aquatic ecosystems and the use of fatty acids as biomarkers, see Arts et al. Working with live fishes under laboratory conditions requires attention to many details concerning the requirements for, and limits of tolerance of, the particular species under study.

Acceptable physical facilities and an adequate supply of water with good quality must be provided, even if the fishes are to be held for only short periods of time. Although fish may tolerate marginal facilities and conditions for a few hours or even several days, holding them under less than optimal conditions will affect the results of the research.

Standards for humane treatment of animals must also be maintained, regardless of the length of time that the fishes are held. The reader should note that some content of section 7 is not restricted to laboratory activities, but may be applicable to field situations, as well. Prior to bringing fishes into a laboratory, facilities and plans should be in place to ensure that the fish cannot escape, especially species not native to the watershed, and that the introduced fishes can be isolated physically from fishes already present.

Each holding unit should have its own set of nets and other equipment. If the introduced fishes may carry disease agents, especially pathogens or parasites that are not endemic to the area, quarantine-level facilities should be used. The level of quarantine required will vary with the seriousness of the known or suspected disease agent see section 2. Control of Pathogens and Parasites. Individual fish with suspected ill health should be quarantined from the others so as to negate the potential for spread of potential disease agents.

Such fish should be evaluated by an individual with expertise in fish diseases fish pathologist or veterinarian , and the proper therapeutant should be applied as directed. Providing guidance for the treatment of specific diseases is beyond the scope of this document. The investigator is strongly urged to establish a working relationship with individuals with expertise in fish health with whom they may consult.

Experimentation with nonindigenous fishes, transgenic fishes, or other genetically modified fishes is a special situation that requires additional precautions to preclude their escape. Permitting with site visits by state wildlife agencies may be required for holding nonindigenous species see section 3.

The specific barriers may be similar to those used to prevent the escape of disease agents but must be developed to fit the physical characteristics of the laboratory or experimental facility. The USDA has developed specifications for its own facilities and published voluntary guidelines USDA a, b intended to ensure appropriate consideration of the potential genetic and ecological effects of research activities.

These USDA guidelines a, b assist in determining appropriate procedures and safeguards so that research can be conducted without causing potentially adverse effects on the environment. Suggestions are provided for developing facility inspection guidelines and risk management procedures, appropriate locations, construction of containment structures, and nonstructural containment strategies. Institutional guidelines for working with transgenic or other genetically modified animals must be variable enough to adapt to site-specific and study-specific goals but should be sufficient to ensure that accidental release cannot occur during floods or other natural disasters or during equipment failures.

Ultimately, individual scientists are responsible for ensuring the containment of animals. Effluents from units used to hold newly introduced fishes should be treated.

At a minimum, effluents should pass through screens with openings sufficiently small to retain any escaped fishes and, in turn, chemical or other treatments should be applied to kill all pathogens and parasites if they are expected to be present. Facilities conducting research on controlled disease agents see OIE lists at http: In addition, physical barriers must be in place with sufficient capacity to prevent outflow of any water in the event that all holding units are emptied USDA a, b.

Many common fish pathogens are opportunistic and are present in virtually all environments. Some are difficult to avoid e. While the investigator can reduce problems from opportunistic pathogens by using good husbandry, the obligate pathogens must be avoided.

This can be typically done by establishing an integrated fish health management plan with regular fish health inspections by appropriately qualified fish health professionals. The investigator must be aware of certain diseases and agents that are problematic in the specific geographic region where work is conducted e.

Regardless, implementing biosecurity protocols is an effective strategy to minimize, if not eliminate, the risk of spreading localized or ubiquitous pathogens and invasive species. Thus, biosecurity see section 3. Comprehensive biosecurity plans can go well beyond simple disinfection procedures to include information on a wide variety of topics such as holding facility layout and design, fish sourcing and quarantine, and record keeping see section 3.

What constitutes effective biosecurity will vary from one setting or research scenario to another. Included in this document is a table summarizing the various disinfectants e. Before studies begin, fishes should be given time to acclimate to new environments see section 5. Slow acclimation to change is often critical Casebolt et al.

It is not uncommon for fishes to exhibit acute health problems 48—72 hours following transfer. The time used for acclimation within and between experiments should be standard and specific for a species. Preliminary studies may be needed to establish the most appropriate time to be used during individual studies. A commonly used acclimation period is 1—2 weeks. Formalin increases the chemical oxygen demand during decomposition over the 12—24 hours after administration.

The formalin bath is commonly repeated at weekly intervals for 3 weeks during the initial fish holding period. Salt is another compound that can be used. If eggs or eyed eggs are brought from the field into the lab, other disinfectants such as iodine, hydrogen peroxide, or formalin can be used.

Investigators should note that laboratory holding conditions may cause physiological changes in animals brought from the field. Even though no visual signs of stress may be present, immunosuppression Miller and Tripp or loss of tidal or diel rhythmicity may occur. Laboratory culture systems are based upon a variety of designs, ranging from a few aquaria to large systems with a full complement of aquaria, raceways, and ponds.

The numerous fish species have a variety of requirements; therefore, the laboratory should be designed to be flexible and to accommodate all species of potential interest. Culture systems will vary according to the physical size of the lab, the availability of water, the fish species, the number and density of test animals see section 7. Fishes can be raised and maintained successfully in many types of systems, but there are optimum conditions for each species see section 5.

In the design, minimizing stress should be a factor paramount for ensuring quality research animals. Adequate water flow providing both volume and flow patterns will deliver adequate dissolved oxygen and flush metabolic waste products Piper et al.

Consideration should also be given to eliminating, or at least reducing, the potential spread of disease agents within a system.

Not only should items such as nets and other laboratory equipment be suspect as vehicles for pathogen transmission, but airborne movements of aerosols containing pathogens are also important means by which fish pathogens may spread Wooster and Bowser ; Bishop et al. Implementing pathogen control measures is an emphasis for fish biomedical research facilities because underlying disease or chronic infections can impact host physiology and other research endpoints Lawrence et al.

Facilities that are poorly designed and constructed can hinder research activities because they cannot maintain the required quality, number, sizes, or species of fish. Water of excellent quality and quantity may be rendered useless for fish if pipes and valves release heavy metals or other contaminants into the water Brauhn and Schoettger ; also see section 7. Suboptimal water quality and contaminating compounds can influence physiological parameters and behavior.

Floor drains should be numerous and appropriately spaced; floors need not be impervious to water but should be slip-resistant. Ground-fault interrupted electrical connections will assure animal and personnel safety. Log books for equipment checks and maintenance, and for animal feeding and record-keeping are typically in use in housing facilities. Construction materials are available that minimize contact with potentially toxic substances.

Appropriate construction materials for the holding system components e. Army Corps of Engineers Brass, copper, lead, zinc, and rubber should be avoided ASTM , as should corrodible substances Hawkins Regular monitoring of water quality is essential see section 7. Systems designed for saltwater fishes will require additional attention to factors related to salinity and potential effects of corrosion, but the same general design considerations discussed above are applicable.

The density of fish that can be held in an experimental unit depends on a series of environmental factors. The most immediate issues are maintaining a supply of dissolved oxygen and the water temperature and elevation Piper et al. Accumulation of waste products, especially ammonia, is generally the next factor limiting density Piper et al.

Oxygen demand and excretion of ammonia are directly related to the amount of feed supplied to the fishes. The amount of feed is in turn determined by the number and the size of the fish in the unit. In general, flow-through systems can sustain a greater density of fish than static units because of continual replenishment of dissolved oxygen and removal of ammonia. However, bead filter technologies used in recirculation systems have increased fish densities that can be maintained Malone and Beecher ; see section 7.

Static units must be equipped with aeration and charcoal filtration equipment if the density of fishes is greater than the minimum levels that can be sustained through direct atmospheric exchange. Ammonia concentrations should be near zero, especially at higher pH levels see section 7. Physiological stress, susceptibility to disease agents, and transmission of disease agents are additional factors that must be considered when density levels are established. Fish vary from species to species, and even within a species, as to the degree of crowding that they will tolerate before behavioral patterns are disrupted.

No specific guidelines can be provided, but the potential effects of crowding should be included in each research design Piper et al. Generally, practical density is determined by the water treatment and feed delivery systems and reaches its maximum at that density as determined by social interactions. Investigators and IACUCs are cautioned to recognize the variability in appropriate densities for various species and specific studies.

No standard, preferred density applies to all species. Although most species of adult fishes can survive several weeks without food especially at lower temperatures , they must be provided with food that is palatable and meets basic nutritional requirements in order to remain in satisfactory condition as research subjects.

Migratory fishes or other fishes which exhibit fasting as a normal part of their life cycle or behavior may be exceptions to this general rule. A review of a species life history can provide guidance regarding feeding requirements. If the nutritional requirements and life stage are not known, a balanced mix of items found in the diet of free-ranging individuals of the species should provide adequate nutrition.

It cannot be assumed that supplying natural foods, especially of a single type, will meet the complete nutritional requirements of the captive fishes. Some species can be trained with relative ease to accept formulated feeds, thereby eliminating the problems inherent in providing natural foods, such as high cost, inconvenience, and inadvertent pathogen introduction. Formulated feeds can be expected to provide the nutritional requirements of the species for which those feeds were designed, especially if manufactured to the specifications of a specific list of ingredients an open formula.

Although captive fishes frequently will consume feeds designed for other species e. Commercial, formulated feeds usually are not based on a specific list of ingredients closed formulas but, rather, are designed to meet the broad nutrient requirements for protein, carbohydrates, and fats. Specific ingredients selected to fulfill the guaranteed analysis description provided with the feed can vary considerably from batch to batch, even though the proximate composition protein, carbohydrate, fat remains constant.

Thus, a feed may meet overall macronutrient demands e. Investigators must consider the possible effects of variability in ingredients on the physiology of their experimental subjects when the studies are designed Barrows and Hardy and consider consulting with a feed manufacturer or fish nutritionist when selecting diet formulations.

Switching diets can have effects on the experimental animals Gatlin ; see Southern Regional Aquaculture Center fact sheets are available at https: The amount of feed to be provided will vary with the nutrient and energy content of the food, as well as the age and size of the fish, and environmental conditions, especially water temperature. Feeding to satiation is the normal practice unless the research design or other logistics dictate reduced feeding rates.

Optimal feeding times depend on species-specific behavior but generally can be modified to accommodate reasonable schedules of the fish caretakers. If the species of fish typically feeds at night or at dusk and dawn, it is desirable to provide feed at the times when they would feed naturally.

Most formulated feeds can be dispensed by a variety of mechanical feeders or demand feeders triggered by the fish themselves. The method of feeding combined with the stocking density may differentially impact behavior and stress Attia et al. Feeding by machine has the potential to prevent habituation by the fish to the presence of feeding personnel and allows flexibility in feeding schedules.

Excess uneaten feed should be removed from a tank within a short time following feeding, as it decreases water quality and can support fungal growth. Water quality will be diminished by accumulated feed, and water-soluble nutrients will be leached from the water-soaked pellets. Providing water of appropriate physical and chemical quality is probably the single most important factor for the care and maintenance of captive fishes.

Transferring fishes into water having a temperature outside their limits of tolerance, or in excess of their capacity to adapt, can lead to death, either immediately or delayed, usually within 72 hours. Limits of tolerance and ability to tolerate temperature changes are influenced by the previous thermal histories of individual fish as well as species characteristics Carmichael et al. The presence of toxic substances in water or the absence of sufficient dissolved oxygen can cause immediate death to fishes placed in such water.

Chronic water quality problems, such as elevated nitrite levels, may not cause obvious reactions but can seriously affect the physiology of the fish and research results. Routine, periodic monitoring of temperature, dissolved oxygen, ammonia, alkalinity, nitrite, and pH should be conducted. In the case of soft water, or water that is poorly buffered, substantial changes in pH may cause adverse effects. Un-ionized ammonia is quite toxic to fishes and a cause of stress or even mortality, especially at higher pH levels.

The addition of buffering agents may be warranted in these situations. The effects of temperature and elevation on water quality parameters must be known and managed to maintain conditions within acceptable limits Boyd ; Avault ; Colt and Tomasso This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license http: Choose your preferred view mode Please select whether you prefer to view the MDPI pages with a view tailored for mobile displays or to view the MDPI pages in the normal scrollable desktop version.

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Use mobile version Use desktop version. You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled. Volume 7, Issue 9. No citations found yet 0. Create a SciFeed alert for new publications With following keywords magnesium. By following authors Uwe Gröber.

One email with all search results. One email for each search. Open Access This article is freely available re-usable Nutrients , 7 9 , ; doi: Department of Internal Medicine I, St. Anna-Hospital, Herne , Germany.

Introduction Magnesium is the eight most common element in the crust of the Earth and is mainly tied up within mineral deposits, for example as magnesite magnesium carbonate and dolomite. Functions of Magnesium Magnesium is primarily found within the cell where it acts as a counter ion for the energy-rich ATP and nuclear acids.

Functions of magnesium selection [ 6 , 7 , 8 , 9 , 10 ]. Magnesium is involved in more than essential metabolic reactions e. Cardiovascular system Economization of cardiac pump function, regulation of potassium movement in myocardial cells, protection against stress, vasodilation of the coronary and peripheral arteries, reduction of platelet aggregation. Membrane function Transmembrane electrolyte flux, active transport of potassium and calcium across cell membranes, regulation of cell adhesion and cell migration.

Structural roles Component of mineralized bone structure, microarchitecture , multiple enzyme complexes, mitochondria, proteins, polyribosomes, and nucleic acids. Nutrient metabolism Metabolic activation and utilisation of vitamin D, B-vitamins e. Deficiency signs and symptoms [ 7 ]. Anxiety, lethargy, weakness, agitation, depression, dysmenorrhea, hyperactivity, headache, irritability, dysacusis, low stress tolerance, loss of appetite, nausea, sleep disorders, impaired athletic performance.

Muscle spasm, cramps in the soles of the feet, leg cramps, facial muscles, masticatory muscles, and calves, carpopedal spasm, back aches, neck pain, urinary spasms, magnesium deficiency tetany. Nervousness, increased sensitivity of NMDA receptors to excitatory neurotransmitters, migraine, depression, nystagmus, paraesthesia, poor memory, seizures, tremor, vertigo.

Risk of arrhythmias, supraventricular or ventricular arrhythmias, hypertension, coronary spasm, decreased myocardial pump function, digitalis sensitivity, Torsade de pointes, death from heart disease.

Hypokalaemia, hypocalcaemia, retention of sodium. Dyslipoproteinemia increased blood triglycerides and cholesterol , decreased glucose tolerance, insulin resistance, increased risk of metabolic syndrome, disturbances of bone and vitamin D metabolism, resistance to PTH, low circulating levels of PTH, resistance to vitamin D, low circulating levels of 25 OH D, recurrence of calcium oxalate calculi.

Asthma, chronic fatigue syndrome, osteoporosis, hypertension, altered glucose homeostasis. Drug-induced magnesium loss and hypomagnesemia [ 63 , 64 , 65 ]. Magnesium deficiency and diabetes [ 63 , 71 ]. Magnesium and vascular function, according to [ 52 , 95 ]. Conflicts of Interest The authors declare no conflict of interest. Anorganische Chemie ; Chemie GmbH: Lehrbuch der Anorganischen Chemie ; De Gruyter: Comparative community genomics in the Dead Sea: An increasingly extreme environment.

Magnesium intake, C-reactive protein, and the prevalence of metabolic syndrome in middle-aged and older U. Diabetes Care , 28 , — Metabolic Tuning-Prevention-Therapy , 1st ed. Stuttgart, Germany, ; pp. In Encyclopedia of Dietary Supplements , 2nd ed. In Modern Nutrition in Health and Disease , 11th ed. Magnesium metabolism and its disorders. Current state of knowledge and future research directions. Nutrients , 5 , — The clinical importance of magnesium.

The indications for supplementation and therapy. Internist , 39 , — Magnesium requirements in human nutrition. The significance of magnesium in insulin resistance, metabolic syndrome, and diabetes—Recommendations of the Association of Magnesium Research e.

Correlation of magnesium deficiency with C-reactive protein in elective cardiac surgery with cardiopulmonary bypass for ischemic heart disease. Medicina Kaunas , 51 , — Department of Agriculture, Agricultural Research Service: Short-term magnesium deficiency downregulates telomerase, upregulates neutral sphingomyelinase and induces oxidative DNA damage in cardiovascular tissues: Associations of serum and dietary magnesium with cardiovascular disease, hypertension, diabetes, insulin, and carotid arterial wall thickness: Atherosclerosis Risk in Communities Study.

Magnesium in drinking water and ischemic heart disease. Magnesium, inflammation, and obesity in chronic disease. National Health and Nutrition Examination Survey. Plasma and dietary magnesium and risk of sudden cardiac death in women.

Dietary magnesium intake and risk of stroke: A meta-analysis of prospective studies. Magnesium homeostasis and aging. Gastrointestinal absorption and excretion of Mg 28 in man. Metabolism , 9 , — An update on physiological, clinical and analytical aspects.

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Overlooked cause of low vitamin D status? Magnesium, vitamin D status and mortality: Impaired release of parathyroid hormone in magnesium deficiency. The relationship between magnesium and calciotrophic hormones. Magnesium metabolism in health and disease. Spironolactone reduced arrhythmia and maintained magnesium homeostasis in patients with congestive heart failure.

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Hypomagnesemia in patients with type 2 diabetes. Magnesium deficiency and therapy in cardiac arrhythmias: Recommendations of the German Society for Magnesium Research. Hypomagnesemia in patients in postoperative intensive care.

Chest , 95 , — Frequency of hypomagnesemia and hypermagnesemia. JAMA , , — Current recommendations of the Society for Magnesium Research. Interactions between drugs and micronutrients. Antihypertensives and magnesium—Update Magnesium and cardiovascular complications of chronic kidney disease. Oral magnesium supplementation improves glycaemic status in subjects with prediabetes and hypomagnesaemia: A double-blind placebo-controlled randomized trial. Lowered magnesium in hypertension.

Hypertension , 62 , e Serum magnesium concentration is a significant predictor of mortality in maintenance hemodialysis patients. Magnesium and cardiovascular biology: An important link between cardiovascular risk factors and atherogenesis.

Insulin increases renal magnesium excretion: A possible cause of magnesium depletion in hyperinsulinaemic states. Serum magnesium levels as an indicator of status of Diabetes Mellitus type 2. Proton pump inhibitors and hypomagnesemia: A rare but serious complication. Efficacy and safety of oral magnesium supplementation in the treatment of depression in the elderly with type 2 diabetes: A randomized, equivalent trial.

Mechanisms of diabetic complications. Magnesium intake and risk of type 2 diabetes: Magnesium intake in relation to systemic inflammation, insulin resistance, and the incidence of diabetes. Diabetes Care , 33 , — Meta-analysis of prospective cohort studies. Diabetes Care , 34 , — Dietary magnesium intake improves insulin resistance among non-diabetic individuals with metabolic syndrome participating in a dietary trial.

Higher magnesium intake reduces risk of impaired glucose and insulin metabolism and progression from prediabetes to diabetes in middle-aged americans. Diabetes Care , 37 , — Oral magnesium supplementation reduces insulin resistance in non-diabetic subjects—A double-blind, placebo-controlled, randomized trial.

Erhöhtes Risiko einer diabetischen Retinopathie bei niedrigem Serum-Magnesium. Long term magnesium supplementation influences favourably the natural evolution of neuropathy in Mg-depleted type 1 diabetic patients T1DM. The link between lower serum magnesium and kidney function in patients with diabetes mellitus type 2 deserves a closer look. Diabetes , 41 , 35— Correlation of magnesium intake with metabolic parameters, depression and physical activity in elderly type 2 diabetes patients: Hypomagnesaemia and risk for metabolic glucose disorders: A year follow-up study.

Its proven and potential clinical significance. Pulse pressure, plasma magnesium status, and antihypertensive therapy. Membrane model for the pathogenesis of primary hypertension. Alterations in calcium and magnesium content of red cell membranes in patients with primary hypertension. Magnesium deficiency produces insulin resistance and increased thromboxane synthesis.

Hypertension , 21 , — Magnesium sulfate for the treatment of eclampsia: Stroke , 40 , — The effect of magnesium supplementation on blood pressure: A meta-analysis of randomized clinical trials. Magnesium supplementation for the management of essential hypertension in adults. Effect of magnesium supplementation on blood pressure: Oral magnesium supplementation reduces ambulatory blood pressure in patients with mild hypertension.

Oral magnesium supplementation improves borderline hypertension. A pilot study on the effects of magnesium supplementation with high and low habitual dietary magnesium intake on resting and recovery from aerobic and resistance exercise and systolic blood pressure.

Urinary and plasma magnesium and risk of ischemic heart disease. Circulating and dietary magnesium and risk of cardiovascular disease: A systematic review and meta-analysis of prospective studies.

Magnesium orotate in severe congestive heart failure MACH.