The author thanks Barbara Berli for providing example data and the gracious funding of the Canadian Wildlife Federation.

1. Capture and Acclimation
<ol>
	<li>Using a knotless net, collect individual fish from their holding tank. Minimize capture time, avoid collecting multiple fish and do not hold fish for more than a few seconds in the net as these factors can increase stress. Stress can affect swimming performance.</li>
	<li>Place fish either in a transfer tank with anesthesia or directly to the swim tunnel respirometer ('swim tunnel'). If fish are to be anesthetized, either tricaine methanesulfonate (MS222) or clove oil can be used. Acceptable concentrations will depend on many factors, including species sensitivity and temperature, but typically range from 10-100 mg/L for either anesthetic1,2. The anesthetic concentration should induce stage 3 anesthesia (i.e. unresponsive to external stimuli) and permit rapid recovery (i.e. seconds). When using MS222 in freshwater, sodium bicarbonate should be used at equal parts (by mass) as a buffer. Without buffer, MS222 will greatly reduce pH, and low pH may harm gill tissue. Buffering is not necessary in seawater as it contains sufficient buffering capacity.</li>
	<li>Anesthetized fish can be sexed, measured for mass and length, and given a cursory external exam of their condition. Length can be taken as total length (TL), fork length (FL) or body length (BL). These different metrics are typically applied according to standard practice, species morphology (e.g. no forked fin) or fish condition (e.g. caudal fin worn away).</li>
	<li>Both anesthetized and non-anesthetized fish should be introduced in the swim tunnel as rapidly as possible and into flowing water as this will facilitate recovery. The flow rate is typically set such that fish will be able to rest on the bottom while barely swimming. An example rate for adult sockeye salmon is 0.3 BL/s.</li>
	<li>Fish need to be left in the swim tunnel to acclimate. Acclimation period can be set to traditional periods (e.g. 12 hr), or to those used in most current studies which can be as short as 30 min. To determine an ideal acclimation period for a given fish, the time taken to reach a stable, ideally low, metabolic rate can be used (detailed next section).</li>
</ol>
2. Measuring Metabolic Rates
<ol>
	<li>Changes in oxygen within the swim tunnel should be recorded over the duration of the swim test(s). Oxygen probes can be sealed directly into the swim tunnel; however, it must first be determined whether water flow over the probe affects the function of the probe. If the probe is affected by water speeds that may occur during the test, the probe will need to be placed in its own chamber outside the tunnel and supplied with tunnel water by a pump.</li>
	<li>To replenish oxygen and mitigate waste accumulation, the swim tunnel will likely need to be flushed periodically. Oxygen saturation should not be permitted to fall lower than 70% for most species as swimming performance may be reduced or there may be a shift to anaerobic metabolism. Flushing tunnel water may also help reduce any increases in water temperature; however, water temperature should be held constant by coupling the swim tunnel to a chiller. Changes in temperature will affect metabolic rate.</li>
	<li>Routine and maximum metabolic rates, abbreviated 'RMR' and 'MMR', are typically determined (Figure 1A). These rates can be expressed in volume (Vo2) or mass (Mo2) of oxygen per unit mass, per unit time, e.g. mg O2 / kg / hr. The difference between the two rates can be taken to get the 'scope for activity'. The 'oxygen debt' following exercise, referred to as 'excess post-exercise oxygen consumption' (EPOC), can also be determined and is equal to the oxygen required during the post-exercise return to RMR.</li>
</ol>
<ol>
	<li>
		Note: many oxygen probes and their output will automatically provide temperature-corrected changes in dissolved oxygen. Those that do not will need to be corrected. Additionally, some values will need to be corrected if measurements were conducted in saline conditions.</li>
</ol>
Swimming performance measurement
A variety of swimming performance abilities can be assessed in a swim tunnel. The most common way to characterize swimming performance is by the duration for which it can be maintained3. The slower the speed, the longer it can be maintained and vice versa. Speeds are generally referred to as 'burst', 'prolonged' or 'sustained', which correspond to durations of seconds, minutes to hours (max = 200 min), and hours and beyond (&gt;200 min), respectively. To determine these speeds, fish are subjected to user defined 'steps', consisting of set 'height', i.e. water flow speed increase, and 'length', i.e. step duration. Maximizing step height and minimizing step length will capture the greatest burst speed, while doing the reverse will capture the greatest sustained speed. In step tests, the traditional way to report the maximum speed achieved is equal to the speed of the last fully completed step plus the temporal portion of the final, unfinished step (Figure 1A).
Burst swimming performance assessment
<ol>
	<li>To capture burst swimming or sprint ability, following acclimation, bring the flow speed to an estimated maximum (e.g. twice Ucrit; see below), and motivate the fish to swim. Fish should be resting at the rear of the chamber as the flow speed is rapidly increased. Fish may need to be motivated to swim. Motivation can be mechanical (e.g. touch) or electrical (e.g. a small charge applied to a shock grid at the back of the swim tunnel). Although not specifically a test of burst ability, fish can also be given a 'constant acceleration test' (CAT or U&Delta;V) consisting of several steps of short duration (e.g. 1 min)4,5.</li>
	<li>Record the duration of swimming. The number of individual burst and their distance can also be recorded6. Speed can be given as the maximum achieved or calculated according to the equation in Figure 1A. Prolonged and sustained swimming performance assessment The most frequent measure used to estimate prolonged or sustained swimming ability is referred to as 'critical swimming performance' (Ucrit). Step length for Ucrit has traditionally been set at 20 min, although shorter times such as 10 min have also been used. A general rule is that at least ten steps should be taken prior to fatigue, which can result in lengthy tests, e.g. &gt;3 hrs/fish. To shorten the Ucrit test, the initial seven steps can be abbreviated to 5 min (the test is then termed 'ramp- Ucrit').</li>
</ol>
<ol>
	<li>Following acclimation and/or a practice swim, increase flow speed according to step size.</li>
	<li>To set the appropriate step height for Ucrit, it will likely be necessary to estimate Ucrit for a sample of the fish to be tested. This can be accomplished by using literature values. Alternately, fish can be given a 'practice swim' after introduction to the tunnel. For this, give fish short, user-defined steps, until fatigue. Use the final speed reached as an early estimate of Ucrit. Adjust step size (height and length) for each fish.</li>
	<li>To test recovery ability or determine individual variation in performance, fish can be given a second swimming test following a recovery period7-9.</li>
	<li>Fish can also be tested in schools, although fish tend to fatigue at different times during the tests which can be problematic for removing them from the tunnel and eliminate the ability to measure oxygen uptake.</li>
	<li>Once fatigued, fish should be allowed to recover or removed immediately for tissue sampling. Several performance metrics related to swimming can be collected during the tests or after using video analysis (Figure 1 C,D).</li>
 <li>To measure ventilation rate and tail beat frequency, the opercular pumping rate and number of tail beats should be sampled over periods of several seconds (Figure 1B)10,11.</li>
	<li>To measure tail beat amplitude, the maximum deflection of the caudal fin can be measured (Figure 1B)12.</li>
	<li>Following testing, fish can be euthanized, given a post-mortem exam and tissues collected to determine hematocrit (packed red cell volume), stress hormone concentrations, muscle energy stores (e.g. phosphocreatine, glycogen), enzyme activities (e.g. lactate dehydrogenase, acetylcholine esterase), or other physiological parameters.</li>
</ol>
3. Representative Results
<img src="/files/ftp_upload/2572/2572fig1.jpg" alt="Figure 1" /> Figure 1. A) An example ramp-Ucrit test given to a juvenile salmonid, a hatchery raised rainbow trout (Oncorhynchus mykiss), 6.3 cm in body length (BL) and 3.5 g in mass. The test was carried out in a 12 L modified Brett-type respirometer (Loligo Systems, Denmark; <a href="http://www.loligosystems.com/">www.loligosystems.com</a>). Routine and maximum metabolic rates were calculated as slope (mg/hr) &times; volume (L) / fish mass (kg), and were 198 and 961 mg O2/kg/hr, respectively (scope for activity = 4.84). These rates are in keeping with those reported by Brett for juvenile sockeye salmon (O. nerka)13. Note: for oxygen uptake calculations, the volume of water is equal to the respirometer volume less fish volume. The cross indicates the point where the fish refused to swim further and where Ucrit was calculated. B) Tail beat frequency values were recorded over 10 s six times; tail beat amplitude values were recorded three times; values shown are mean &plusmn; SEM. C) An enlarged side view and D) top view of the fish swimming within the swim chamber. D) Shows the fish tracked by EthoVision software (Noldus, Netherlands; <a href="http://www.noldus.com/">www.noldus.com</a>). Abbreviations: Ucrit = critical swimming velocity; Uf = speed of the last fully completed step, Us = step speed, tf = time spent on last step, ts = step time.

<ol>
	<li>Anderson, W. G., McKinley, R. S., Colavecchia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+clove+oil+as+an+anesthetic+for+rainbow+trout+and+its+effects+on+swimming+performance.">The use of clove oil as an anesthetic for rainbow trout and its effects on swimming performance.</a> North Am. J. Fish Manage. 17, 301-307 (1997).</li><li>Cho, G. K., Heath, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+tricaine+methanesulphonate+(MS222)+and+clove+oil+anaesthesia+effects+on+the+physiology+of+juvenile+chinook+salmon+Oncorhynchus+tshawytscha+(Walbaum).">Comparison of tricaine methanesulphonate (MS222) and clove oil anaesthesia effects on the physiology of juvenile chinook salmon Oncorhynchus tshawytscha (Walbaum).</a> Aquacult. Res. 31, 537-546 (2000).</li><li>Beamish, F. W. H., Hoar, W. S., Randall, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swimming capacity in Fish Physiology. , 101-187 (1978).</li><li>Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+swimming+performance+in+rainbow+trout+using+constant+acceleration+and+critical+swimming+speed+tests.">Comparisons of swimming performance in rainbow trout using constant acceleration and critical swimming speed tests.</a> J. Fish Biol. 72, 693-710 (2008).</li><li>Tierney, K. B., Casselman, M., Takeda, S., Farrell, A. P., Kennedy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+cholinesterase+inhibition+and+two+types+of+swimming+performance+in+chlorpyrifos-exposed+coho+salmon+(Oncorhynchus+kisutch).">The relationship between cholinesterase inhibition and two types of swimming performance in chlorpyrifos-exposed coho salmon (Oncorhynchus kisutch).</a> Environ. Toxicol. Chem. 26, 998-1004 (2007).</li><li>Martinez, M., Bedard, M., Dutil, J. -. D., Guderley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+condition+of+Atlantic+cod+(Gadus+morhua)+have+a+greater+impact+upon+swimming+performance+at+Ucrit+or+sprint+speeds.">Does condition of Atlantic cod (Gadus morhua) have a greater impact upon swimming performance at Ucrit or sprint speeds.</a> J. Exp. Biol. 207, 2979-2990 (2004).</li><li>Marras, S., Claireaux, G., McKenzie, D. J., Nelson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+and+repeatability+in+aerobic+and+anaerobic+swimming+performance+of+European+sea+bass,+Dicentrarchus+labrax.">Individual variation and repeatability in aerobic and anaerobic swimming performance of European sea bass, Dicentrarchus labrax.</a> J. Exp. Biol. 213, 26-32 (2010).</li><li>Kolok, A. S., Plaisance, E. P., Abdelghani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+in+the+swimming+performance+of+fishes:+an+overlooked+source+of+variation+in+toxicity+studies.">Individual variation in the swimming performance of fishes: an overlooked source of variation in toxicity studies.</a> Environ. Toxicol. Chem. 17, 282-285 (1998).</li><li>Tierney, K. B., Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationships+between+fish+health,+metabolic+rate,+swimming+performance+and+recovery+in+return-run+sockeye+salmon,+Oncorhynchus+nerka+(Walbaum).">The relationships between fish health, metabolic rate, swimming performance and recovery in return-run sockeye salmon, Oncorhynchus nerka (Walbaum).</a> J. Fish Dis. 27, 663-671 (2004).</li><li>Webber, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upstream+swimming+performance+of+adult+white+sturgeon:+Effects+of+partial+baffles+and+a+ramp.">Upstream swimming performance of adult white sturgeon: Effects of partial baffles and a ramp.</a> Trans. Am. Fish. Soc. 136, 402-408 (2007).</li><li>Farrell, A. P., Gamperl, A. K., Birtwell, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+swimming,+recovery+and+repeat+swimming+performance+of+mature+sockeye+salmon+Oncorhynchus+nerka+exposed+to+moderate+hypoxia+and+pentachlorophenol.">Prolonged swimming, recovery and repeat swimming performance of mature sockeye salmon Oncorhynchus nerka exposed to moderate hypoxia and pentachlorophenol.</a> J. Exp. Biol. 201, 2183-2193 (1998).</li><li>Flammang, B. E., Lauder, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speed-dependent+intrinsic+caudal+fin+muscle+recruitment+during+steady+swimming+in+bluegill+sunfish,+Lepomis+macrochirus.">Speed-dependent intrinsic caudal fin muscle recruitment during steady swimming in bluegill sunfish, Lepomis macrochirus.</a> J. Exp. Biol. 211, 587-598 (2008).</li><li>Brett, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+metabolism+and+swimming+performance+of+young+sockeye.">The respiratory metabolism and swimming performance of young sockeye.</a> J. Fish. Res. Board Can. 21, 1183-1226 (1964).</li><li>Bla&#382;ka, P., Volf, M., &#268;epela, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+type+of+respirometer+for+the+determination+of+the+metabolism+of+fish+in+an+active+state.">A new type of respirometer for the determination of the metabolism of fish in an active state.</a> Physiol. Bohemoslov. 9, 553-558 (1960).</li><li>Peake, S. J., Farrell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatigue+is+a+behavioural+response+in+respirometer+confined+smallmouth+bass.">Fatigue is a behavioural response in respirometer confined smallmouth bass.</a> J. Fish Biol. 68, 1742-1755 (2006).</li></ol>

No conflicts of interest declared.

In a natural setting, behaviors such as predator escape and migration depend on being able to swim at specific intensities for different times. In a lab setting, swim tunnel respirometers and stepped increases in speed can be used to estimate the ability of fish to perform numerous behaviours. Measures of metabolic rate and other tissue-level assessments (such as lactate production) can be included with swim tests to help determine physiological mechanisms underlying different swimming modes.
Swim tunnel design can greatly influence swimming performance. There are several types of swim tunnels used to determine swimming performance, most are characterized as either Brett13- or Blazka14-types. For either design, major considerations include the 'evenness' of the water flow (how rectilinear it is), the maximum flow speed and the length of the swim chamber. Evenness is important because fish will instinctively find and swim in low flow areas to minimize effort. High potential flow rate may be necessary to test burst swimming ability or fast swimming species (e.g. tunas). Swim chamber length can be very meaningful as in chambers of small length, i.e. fish length &gt; 10 % of chamber length, fish may be unable to utilize all swimming modes. Specifically, small tubes may restrict the ability of fish to transition from a steady to unsteady swimming gate, i.e. to burst and coast swimming. Therefore, fish may stop swimming at approximately the same speed regardless of the step length and Ucrit may be analogous to 'gate transition speed'15.
All swim tunnels need to be calibrated using devices such as flow probes or particle imaging velocimetry (PIV) equipment. Ideally swim tunnels will have rectilinear flow, but if fish are found to favor an area, their swim speed can be adjusted based on local differences in flow.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>tricaine methanesulfonate (MS222)</td>
<td>&nbsp;</td>
<td><a href="http://www.argent-labs.com">www.argent-labs.com</a></td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
<tr>
<td>Clove oil</td>
<td>&nbsp;</td>
<td>Pharmacy or <a href="http://www.sigmaaldrich.com">www.sigmaaldrich.com</a></td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
<tr>
<td>Swim tunnel respirometer</td>
<td>&nbsp;</td>
<td>Custom made or <a href="http://www.loligosystems.com">www.loligosystems.com</a></td>
<td>&nbsp;</td>
<td>Required</td>
</tr>
<tr>
<td>High speed cameras</td>
<td>&nbsp;</td>
<td>Numerous sources</td>
<td>&nbsp;</td>
<td>optional</td>
</tr>
<tr>
<td>Video capture card</td>
<td>&nbsp;</td>
<td>Numerous sources</td>
<td>&nbsp;</td>
<td>optional</td>
</tr>
<tr>
<td>Behaviour analysis software</td>
<td>&nbsp;</td>
<td><a href="http://www.noldus.com">www.noldus.com</a> <a href="http://www.cleversysinc.com">www.cleversysinc.com</a> <a href="http://www.loligosystems.com">www.loligosystems.com</a> <a hre</td>
<td>&nbsp;</td>
<td>optional</td>
</tr>		</tbody>
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swimming performance assessment in fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

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Swimming Performance Assessment in Fishes

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

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25.4K Views.  University of Alberta. The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

Video: Swimming Performance Assessment in Fishes

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Swimming Performance Assessment in Fishes

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A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

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A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

Medium-throughput Screening Assays for Assessment of Effects on Ca²⁺-Signaling and Acrosome Reaction in Human Sperm

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish