This study was funded by the U.S. Department of Energy (DOE) Water Power Technologies Office. The laboratory studies were conducted at PNNL, which is operated by Battelle for the DOE under Contract DE-AC05-76RL01830. The authors would like to thank Dana McCoskey and Tim Welch of DOE, Eric Francavilla, Ryan Harnish, Huidong Li, Stephanie Liss, Brian Mason, Megan Nims, Brett Pflugrath, and Ashlynn Tate of PNNL for their assistance with the study and the manuscript, and U.S. Army Corps of Engineers and Pacific States Marine Fisheries Commission for their help in collecting the juvenile shad.

PNNL is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. American shad were handled in accordance with federal guidelines for the care and use of laboratory animals11, and the protocols for our study were conducted in compliance with and approved by PNNL&#39;s Institutional Animal Care and Use Committee.
1. Preparation of a post-tagging recovery tank
<ol>
	<li>When brackish saltwater (7.5 ppt) is not readily available, use a static system for holding shad with proper aeration and circulation for 1-2 days before the release (Figure 1).</li>
	<li>In a static, circular tank, install an airlift system to provide aeration. Connect a PVC pipe to the side of the tank such that a tee is located at the top, another tee is near the middle, and a screened fitting is threaded onto the bottom of the pipe.</li>
	<li>Next, connect an air stone to a compressed air line and place the air stone at the bottom of the pipe near the screen. 
	NOTE: The screen prevents fish from swimming into the airlift system.</li>
	<li>Fill the tank with fresh water until the exit port (middle PVC tee) of the airlift is approximately halfway submerged. Then, turn off the water.
	<ol>
		<li>Next, turn on and increase the air supply until the aerated water exiting the port creates a directional flow for the fish to orient.</li>
	</ol>
	</li>
	<li>Add commercial sea salt to make a 7.5 ppt brackish saltwater solution and stir until dissolved.</li>
</ol>
2. Preparation of saltwater fish source bucket and saltwater anesthetic solution
<ol>
	<li>Measure 7.5 g of sea salt for every 0.5 L of water and dissolve in a bucket. 
	NOTE: In step 3.5, an equal volume of freshwater containing the shad is added to create a final concentration of 7.5 ppt.</li>
	<li>In another bucket, measure and dissolve 7.5 g of sea salt for every liter of water.
	<ol>
		<li>Add 120 mg of tricaine methanesulfonate (MS-222) buffered with 120 mg of sodium bicarbonate per liter of saltwater.</li>
		<li>Add supplemental air to the anesthetic buckets.</li>
	</ol>
	</li>
</ol>
3. Collection of shad with a water-to-water transfer into brackish saltwater
<ol>
	<li>Partially fill another bucket with fresh water and place it sideways into the pre-tagging source tank.</li>
	<li>Use a net or hand to gently guide shad to swim into the bucket.</li>
	<li>Once an appropriate number of fish are in the bucket, turn the bucket upright, and secure it with a perforated lid.</li>
	<li>Pour out any excess water through the lid, keeping the fish contained in the target amount of fresh water (i.e., half the volume of the final 7.5 ppt saltwater source bucket).</li>
	<li>Gently pour the shad and the freshwater into the saltwater source bucket prepared in step 2.1. 
	NOTE: The final salinity will be 7.5 ppt.</li>
	<li>Provide supplemental air to the pre-tag fish source bucket using an aquarium air pump and air stone to maintain dissolved oxygen at an acceptable level (&#62;90% saturation is ideal).</li>
</ol>
4. Implantation of an acoustic transmitter in a shad
<ol>
	<li>Disinfect transmitters in 70% ethanol for 20 min and rinse with sterile water before use.</li>
	<li>Using a dip net with smooth, extra-fine mesh (~0.4 mm), net a fish from the source bucket and into the anesthetic bucket. Shad should lose equilibrium and spinal reflexes in ~2-3 min, depending on water temperature and other water quality parameters.</li>
	<li>Once fully anesthetized to stage four12, use a gloved hand to gently place the fish on a wet measuring board to obtain its length.</li>
	<li>Move the fish into a water-filled weigh boat on a tared scale to obtain its weight.</li>
	<li>Record length and weight, acoustic tag code, and any comments on the fish&#39;s condition prior to tagging, such as scale loss or hemorrhaging.</li>
	<li>Place the fish in a transfer container filled with anesthetic saltwater and deliver it, along with the acoustic transmitter, to the fish surgeon.</li>
	<li>Place the fish, left side facing down, on a wet, water-resistant foam pad prepared with a v-groove (Figure 2A). 
	NOTE: The v-groove keeps the fish from sliding too much during the procedure and allows water to pool around the fish&#39;s mouth so it can actively draw water over its gills.</li>
	<li>Supply fresh water to the fish&#39;s mouth via tubing attached to a gravity-fed water reservoir.</li>
	<li>Using a disinfected or brand new #11 stainless steel surgical blade, make a 3 mm-long incision vertically between the myomeres near the distal end of the pectoral fin.</li>
	<li>If needed, remove a scale at the blade tip if it is obstructing the fish&#39;s skin.
	<ol>
		<li>Carefully insert the transmitter into the incision and push it posteriorly until the entire tag rests inside the body cavity (Figure 2B).</li>
		<li>If needed, use the blunt end of the scalpel (or fine-tipped forceps) to carefully insert the tag fully.</li>
	</ol>
	</li>
	<li>Place the tagged fish in a small container of 7.5 ppt saltwater with aeration to allow the fish to recover from anesthesia.</li>
	<li>Once the fish has regained equilibrium, make a water-to-water transfer from the recovery container to the post-tagging holding tank containing 7.5 ppt saltwater.</li>
	<li>Allow the tagged fish to recover in saltwater for 1-2 days prior to the release.</li>
</ol>

<ol>
	<li>Dadswell, M. J., Rulifson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrotidal+estuaries:+A+region+of+collision+between+migratory+marine+animals+and+tidal+power+development.">Macrotidal estuaries: A region of collision between migratory marine animals and tidal power development.</a> Biological Journal of the Linnean Society. 51, 93-113 (1994).</li><li>Limburg, K. E., Waldman, 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=Dramatic+declines+in+North+Atlantic+diadromous+fishes.">Dramatic declines in North Atlantic diadromous fishes.</a> BioScience. 59, 955-965 (2009).</li><li>Castro-Santos, T., Mulligan, K. B., Kieffer, M., Haro, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+plunge+pool+configuration+on+downstream+passage+survival+of+juvenile+blueback+herring.">Effects of plunge pool configuration on downstream passage survival of juvenile blueback herring.</a> Aquaculture and Fisheries. 6 (2), 135-143 (2021).</li><li>Dubois, R. B., Gloss, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+of+juvenile+American+shad+and+striped+bass+passed+through+Ossberger+crossflow+turbines+at+a+small-scale+hydroelectric+site.">Mortality of juvenile American shad and striped bass passed through Ossberger crossflow turbines at a small-scale hydroelectric site.</a> North American Journal of Fisheries Management. 13 (1), 178-185 (1993).</li><li>Pflugrath, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+susceptibility+of+Juvenile+American+shad+to+rapid+decompression+and+fluid+shear+exposure+associated+with+simulated+hydroturbine+passage.">The susceptibility of Juvenile American shad to rapid decompression and fluid shear exposure associated with simulated hydroturbine passage.</a> Water. 12 (2), 586 (2020).</li><li>Brown, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+maximum+tag+burden+for+implantation+of+acoustic+transmitters+in+juvenile+Chinook+salmon.">An evaluation of the maximum tag burden for implantation of acoustic transmitters in juvenile Chinook salmon.</a> North American Journal of Fisheries Management. 30 (2), 499-505 (2010).</li><li>Skalski, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+after+5+years+of+survival+compliance+testing+in+the+Federal+Columbia+River+Power+System+(FCRPS).">Status after 5 years of survival compliance testing in the Federal Columbia River Power System (FCRPS).</a> North American Journal of Fisheries Management. 36 (4), 720-730 (2016).</li><li>Raquel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+handling+and+trucking+on+chinook+salmon,+striped+bass,+American+shad,+steelhead+trout,+threadfin+shad,+and+white+catfish+salvaged+at+the+John+E.+Skinner+delta+fish+protective+facility+(Vol.+19).">Effects of handling and trucking on chinook salmon, striped bass, American shad, steelhead trout, threadfin shad, and white catfish salvaged at the John E. Skinner delta fish protective facility (Vol. 19).</a> Interagency Ecological Study Program for the Sacramento-San Joaquin Estuary. , (1989).</li><li>Kleinschmidt, G., Sullivan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relicensing+Study+3.3.3.+Evaluate+downstream+passage+of+juvenile+American+Shad.+Interim+Study+Report:+Northfield+Mountain+Pumped+Storage+Project+(No.+2485)+and+Turners+Falls+Hydroelectric+Project+(No.+2485)+and+Turners+Falls+Hydroelectric+Project+(No.+1889).">Relicensing Study 3.3.3. Evaluate downstream passage of juvenile American Shad. Interim Study Report: Northfield Mountain Pumped Storage Project (No. 2485) and Turners Falls Hydroelectric Project (No. 2485) and Turners Falls Hydroelectric Project (No. 1889).</a> FirstLight. , (2016).</li><li>Heisey, P. G., Mathur, D., Rineer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+tag-recapture+technique+for+estimating+turbine+passage+survival:+application+to+young-of-the-year+American+shad+(Alosa+sapidissima).">A reliable tag-recapture technique for estimating turbine passage survival: application to young-of-the-year American shad (Alosa sapidissima).</a> Canadian Journal of Fisheries and Aquatic Sciences. 49, 1826-1834 (1992).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals.: Eighth Edition. , (2011).</li><li>Summerfelt, R. C., Smith, L. C., Schreck, C. B., Moyle, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia,+surgery,+and+related+techniques.">Anesthesia, surgery, and related techniques.</a> Methods for Fish Biology. , 213-263 (1990).</li><li>Bailey, M. M., Isely, J. J., Bridges, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+and+population+size+of+American+shad+near+a+low-head+lock+and+dam.">Movement and population size of American shad near a low-head lock and dam.</a> Transactions of the American Fisheries Society. 133 (2), 300-308 (2004).</li><li>Grote, A. B., Bailey, M. M., Zydlewski, 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=Movements+and+demography+of+spawning+American+Shad+in+the+Penobscot+River,+Maine,+prior+to+dam+removal.">Movements and demography of spawning American Shad in the Penobscot River, Maine, prior to dam removal.</a> Transactions of the American Fisheries Society. 143 (2), 552-563 (2014).</li><li>Harris, J. E., Hightower, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+patterns+of+American+shad+transported+upstream+of+dams+on+the+Roanoke+River,+North+Carolina+and+Virginia.">Movement patterns of American shad transported upstream of dams on the Roanoke River, North Carolina and Virginia.</a> North American Journal of Fisheries Management. 31 (2), 240-256 (2011).</li><li>Bolland, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+acoustic-tagging+protocol+for+twaite+shad+Alosa+fallax+(Lac&#233;p&#232;de),+a+species+sensitive+to+handling+and+sedation.">Refinement of acoustic-tagging protocol for twaite shad Alosa fallax (Lac&#233;p&#232;de), a species sensitive to handling and sedation.</a> Fisheries Research. 212, 183-187 (2019).</li><li>Gahagan, B. I., Bailey, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+acoustic+tags+in+American+Shad+to+resolve+riverine+and+marine+restoration+challenges.">Surgical implantation of acoustic tags in American Shad to resolve riverine and marine restoration challenges.</a> Marine and Coastal Fisheries. 12 (5), 272-289 (2020).</li></ol>

The authors have nothing to disclose.

The need to study juvenile American shad movements around hydropower facilities has prompted the development of a handling and tagging protocol to improve the survival of tagged shad. At PNNL, initial attempts to implant juvenile shad with a novel acoustic transmitter, without the use of saltwater resulted in 100% mortality within 24 hours. The subsequent, carefully developed handling and tagging protocol demonstrated that American shad can be implanted with an acoustic microtransmitter and held long-term in a laboratory setting with a high survival rate (81.5%). Minimizing out-of-water handling and the use of brackish saltwater before and after tagging was essential to the success of tagging juvenile American shad.
In the preliminary evaluation, shad as small as 50 mm were tagged with a dummy transmitter using four implantation methods. Gastric tagging, one of the most common methods for tagging adult shad13,14,15 had promising results during pilot testing but had a high incidence of tag loss during the preliminary evaluation. Implantation through a pelvic incision has been used successfully to study adult Twaite shad movements16 and dorsal attachments have been used for short-term monitoring of juvenile American shad10. More recently, tag implantation through a pectoral incision was used to study longer-term movements of adult shad in both riverine and marine environments17. In the preliminary evaluation at PNNL, the pectoral incision location performed better than the three other locations evaluated, and the 7-d post-tagging survival was over 90%.
Overall, the results from these evaluations showed that the survival of tagged shad was comparable to the survival of untagged shad beyond the duration of the acoustic transmitter&#39;s battery life, which is expected to be ~30 days with an acoustic signal transmitted every 5 seconds.
This transmitter design and tagging protocol show great promise for studying small, sensitive, and threatened fish species like American shad in field applications, allowing researchers to gain valuable information on fish movements near hydroelectric facilities. For example, this tagging technique will be used in an upcoming field application to study the behavior of acoustic-tagged juvenile shad as they approach the spillway and powerhouse of a hydroelectric dam. The results gained from in-river studies can better inform management decisions at these facilities and can help conserve the species throughout their juvenile life stage. Future studies should evaluate the effectiveness of this procedure for tagging and tracking run-of-river fish in field conditions. In addition, these techniques are easily transferable to implanting shad or other sensitive species with Passive Integrated Transponder (PIT) tags, which can provide long-term monitoring throughout their life history.

American shad (Alosa sapidissima) is an anadromous fish species that is native to the East Coast of the United States. Reduced habitat availability and increased development of hydroelectric dams have resulted in population declines of shad across their native range1,2. Juvenile shad and other alosines, on their out-migration to the ocean, may be especially susceptible to injury and mortality when passing through hydroelectric structures3,4,5. Understanding the passage and survival rates of juvenile shad at hydropower dams is critical for informing the relicensing of these facilities as well as restoration efforts for the species. However, successful tagging techniques to assess the passage and survival rates of American shad as they migrate to the ocean are lacking. Shad tagged with transmitters for telemetry studies should be representative of the untagged population of inference and should not be negatively affected by the tag or the tagging process6,7.
To help improve the ability to track juvenile shad, Pacific Northwest National Laboratory (PNNL) developed a new acoustic micro transmitter for studying American shad and other fish species with similar compressiform body types. One of the common challenges of studying American shad and other alosines is their sensitivity to handling, transport, and tagging compared to other species. For example, Raquel et al.8 found that handling and trucking mortalities were consistently higher for juvenile American shad than for the other five species of juvenile fish in their study. Of the few published studies that have evaluated efforts to tag juvenile American shad, a wide range of survival has been reported, from as low as 2% after 7 days9 and up to 100% after 48 hours post-tagging10 and very little information on longer-term survival and transmitter retention for tagged juvenile shad is available.
The challenges in successfully handling and tagging sensitive species such as American shad have shed light on the knowledge gaps about their migration, behavior, and habitat use. The ability to track movement through hydropower dams would greatly advance the understanding of passage and survival rates for American shad. It would help inform management decisions for existing hydroelectric facilities and novel designs for systems that minimize effects on fish species and life stages not previously studied. As new transmitter technology is developed, understanding the effects of the transmitter and the tagging process is imperative to minimize bias and accurately assess passage and survival. The objectives of this study were to evaluate the 60-day survival of juvenile American shad tagged with a new acoustic micro transmitter and to provide a handling and tagging protocol that would reduce the negative effects of tagging on shad, thereby making them more comparable to their untagged counterparts.

<table><tbody><tr><td>#11 stainless steel surgical blade</td><td>Exel</td><td>29502</td><td>purchased from Med-Vet International; no real preference on blade vendor</td></tr><tr><td>#11 stainless steel surgical blade</td><td>Miltex</td><td>MIL4-311</td><td>purchased from Med-Vet International; no real preference on blade vendor</td></tr><tr><td>2 gallon bucket</td><td>Leaktite</td><td>#2GL White Pail</td><td></td></tr><tr><td>acoustic transmitter for American shad</td><td>Pacific Northest National Laboratory</td><td>Patent-Pending</td><td>BattelleIPID: 32500</td></tr><tr><td>air stone</td><td>Pentair</td><td>AS3</td><td></td></tr><tr><td>aquarium air pump</td><td>Tetra</td><td>Whisper</td><td></td></tr><tr><td>dissolved oxygen meter</td><td>YSI</td><td>ProODO or ProSolo</td><td></td></tr><tr><td>ethanol</td><td>Decon Laboratories</td><td>2805HC</td><td></td></tr><tr><td>fine mesh net</td><td>Blue Ribbon</td><td>ABLEC8</td><td></td></tr><tr><td>fish holding tank</td><td>Reiff Manufacturing</td><td>NA</td><td>round aquaculture tank</td></tr><tr><td>foam garden kneeler</td><td>Tommyco</td><td>12003</td><td></td></tr><tr><td>plastic storage container</td><td>Ziploc</td><td>discontinued; 8oz container with lid</td><td></td></tr><tr><td>PVC cement</td><td>Oatey</td><td>30821</td><td></td></tr><tr><td>PVC pipe</td><td>Charlotte Pipe</td><td>NA</td><td>PVC Schedule 40 2&quot; diameter</td></tr><tr><td>PVC primer</td><td>Oatey</td><td>30757</td><td></td></tr><tr><td>PVC tee</td><td>Charlotte Pipe</td><td>NA</td><td>2&quot; PVC Schedule 40 S x S x S Tee</td></tr><tr><td>sea salt</td><td>InstantOcean</td><td>SS15-10</td><td></td></tr><tr><td>silicone tubing 3/16&quot;</td><td>Pentair</td><td>tp30s</td><td>tubing to supply water during tagging</td></tr><tr><td>sodium bicarbonate</td><td>Fisher Chemical</td><td>S233-500</td><td></td></tr><tr><td>sterile water</td><td>NA</td><td>NA</td><td>water is sterilized using an autoclave</td></tr><tr><td>tricaine methanesulfonate</td><td>Syndel USA</td><td>15650</td><td></td></tr><tr><td>tubing for airline</td><td>Hydromaxx</td><td>1403038050</td><td></td></tr></tbody></table>

handling and tagging techniques for implanting juvenile american shad with a new acoustic microtransmitter

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through hydropower systems, has been challenging because shad are widely known to be particularly sensitive to handling. The goal of this study was to develop a tagging protocol using a new, acoustic micro transmitter that minimizes the detrimental effects of the tagging process and maximizes post-tagging survival of juvenile American shad. Limiting out-of-water handling and the use of brackish saltwater (7.5 parts per thousand) before and after tagging improved survival for shad tagged using a simple pectoral implantation method. This protocol provides a detailed, step-by-step procedure for tagging juvenile shad with acoustic transmitters. Fish tagged using this procedure and held in the laboratory for 60 days had an 81.5% survival rate, compared to 70% for their untagged counterparts. The successful tagging and handling practices developed in this study could be applied to field telemetry studies of juvenile shad and other sensitive species.

Two series of tagging evaluations were conducted to address the efficacy of tagging juvenile shad - preliminary trials in 2020 and a long-term holding study in 2021. Preliminary laboratory evaluations were conducted at PNNL in November 2020 to determine a preferred method for implanting American shad with a novel acoustic microtransmitter. Prototype transmitter designs (n = 4, P1-P4) were paired with different tagging locations (gastric, pectoral, pelvic, and dorsal) for a combined total of 4 unique acoustic transmitter-tagging location treatments (n = 40 fish per treatment, Table 1). All test fish were randomly assigned to a treatment and holding tank. Test fish were held in 2 holding tanks with equal numbers of fish from each treatment (i.e., 20 fish per treatment) per tank for 14 days. For the first 2 days of the evaluation, shad were held in brackish saltwater (7.5 ppt) and allowed to recover from tagging and handling. Then the tanks were switched to flow-through fresh water for the remainder of the evaluation period.
For the preliminary evaluation, tagged fish and fin-clipped controls ranged in size from 50-80 mm in fork length. Juvenile shad survival and tag retention were highest for fish implanted via a pectoral incision compared to the other tagging techniques (Figure 3). Additional pilot evaluations also demonstrated that handling techniques such as water-to-water transfers and holding fish in brackish saltwater before and after stressful events, such as tagging, were critical to increasing survival rates.
Using the successful tagging and handling protocols from the preliminary evaluation, a laboratory study was conducted at PNNL in 2021 to evaluate the long-term 60 d survival and tag retention of juvenile American shad implanted with an acoustic transmitter using the pectoral incision tagging method. The long-term evaluation used the dummy transmitter P5 (Figure 4), an improved prototype design similar in form and size to the P1 design used in the preliminary evaluation. The average dimensions and weight of the dummy P5 tag were 7.6 mm long &#215; 2.3 mm in diameter and a weight in air of 0.058 g (standard deviation 0.002 g), which resulted in a tag burden of &#60;1%. The prototype acoustic transmitter with functional components (Figure 4) has dimensions of 7.6 mm long x 2.0 mm in diameter and a weight in air of 0.050 g.
Juvenile American shad used in the long-term evaluation had been held in captivity for 4 months at the time of testing. While the experiment was designed to have equal numbers of treatment and control fish held in two tanks for a 60 day period, the remaining shad numbers at the time of tagging were low. Therefore, more shad were randomly assigned to the tagged treatment group than the control group to get a better understanding of the long-term efficacy of the tagging technique on shad. Each of the two tanks held 27 treatment fish and 9 or 10 control fish. However, since survival from Tank A (13.8%) was significantly worse than Tank B (78.4%; Fisher&#39;s exact test, p &#60; 0.001) and there was no difference in survival between the tagged and control groups within each tank, only the results for tank B are included here.
Shad (fork length 69-105 mm; weight 3.9-11.7 g) were either tagged with the P5 transmitter using the pectoral incision (n = 27) or assigned to the control group (n = 10). Control fish were handled using the same procedures, including being placed on the surgical pad for ~20 s, but they did not receive a fin clip or an incision nor were they implanted with a transmitter. After tagging, both treatment groups were held in brackish saltwater (7.5 ppt) for 1 day and then switched to flow-through river water for the remainder of the study. Survival at 60 days was 81.5% for the tagged group and 70% for the untagged controls (Figure 5). Survival for tagged fish in this evaluation was defined as both survival and tag retention because tag expulsion cannot be differentiated from a mortality event in a telemetry study. There was no significant difference in survival between the two groups (Fisher&#39;s exact test, P = 0.884); however, the power to detect a difference was 38.4% due to the small sample sizes. Although the power to detect a difference between the treatments was low, the results of the long-term evaluation show that this handling and tagging protocol can be used with moderate success to implant American shad with acoustic transmitters.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65694/65694fig1v2.jpg" /> 
Figure 1: Post-tagging recovery tank filled with brackish saltwater. An airlift system supplies oxygen to the static tank. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65694/65694fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65694/65694fig2v2.jpg" /> 
Figure 2:&#160;Acoustic transmitter implantation of a Juvenile American shad.&#160;Juvenile American shad (A) with a pectoral incision and (B) with the dummy P5 transmitter inserted into the incision. Note, the mouth of the shad is partially submerged in water flowing from the blue tubing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65694/65694fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65694/65694fig3v2.jpg" /> 
Figure 3:&#160;Survival percentage over a preliminary 14 d evaluation with one group of untagged controls and four tagged groups of juvenile American shad. The tagged treatments consisted of four tagging locations (gastric, pectoral, pelvic, and dorsal) each paired with a unique transmitter prototype (P1-P4). Survival of the tagged fish was defined as both survival and tag retention. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65694/65694fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65694/65694fig4v2.jpg" /> 
Figure 4:&#160;Acoustic and dummy transmitters for tagging juvenile American shad. (A) Functional acoustic microtransmitter and (B) the dummy P5 prototype transmitter, that was used in the 60 d laboratory survival study. Note the numbers 4-7 on the ruler represent centimeters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65694/65694fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65694/65694fig5v2.jpg" /> 
Figure 5:&#160;Survival percentage of American shad over a long-term 60 d holding study. Juvenile shad were either untagged (Untagged Controls; solid line) or tagged (Tagged [Pectoral P5]; dashed line) with a dummy transmitter. Survival of the tagged group was defined as both survival and tag retention. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65694/65694fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style="font-size: 12px;">
	<tbody>
		<tr>
			<td rowspan="2">Tag Type</td>
			<td rowspan="2">Tag Location</td>
			<td rowspan="2">N</td>
			<td colspan="2">Fork Length (mm)</td>
			<td rowspan="2">Mean Tag Weight (SD; g)</td>
			<td rowspan="2">Tag Burden (%)</td>
			<td rowspan="2">Survival (%)</td>
			<td rowspan="2">Mean Time to Tag/Clip (s)</td>
		</tr>
		<tr>
			<td>Range</td>
			<td>Mean (SD)&#160;</td>
		</tr>
		<tr>
			<td>P1</td>
			<td>Gastric</td>
			<td>40</td>
			<td>50 - 76</td>
			<td>60 (6.0)</td>
			<td>0.058 (0.003)</td>
			<td>1.5 - 5.2</td>
			<td>45</td>
			<td>12</td>
		</tr>
		<tr>
			<td>P2</td>
			<td>Pectoral</td>
			<td>40</td>
			<td>50 - 78</td>
			<td>60 (7.3)</td>
			<td>0.039 (0.001)</td>
			<td>1.0 - 3.2</td>
			<td>80</td>
			<td>23</td>
		</tr>
		<tr>
			<td>P3</td>
			<td>Pelvic</td>
			<td>40</td>
			<td>50 - 70</td>
			<td>58 (5.3)</td>
			<td>0.039 (0.001)</td>
			<td>1.0 - 4.1</td>
			<td>55</td>
			<td>26</td>
		</tr>
		<tr>
			<td>P4</td>
			<td>Dorsal</td>
			<td>40</td>
			<td>50 - 80</td>
			<td>61 (6.8)</td>
			<td>0.088 (0.004)</td>
			<td>0</td>
			<td>60</td>
			<td>57</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>NA (Clip)</td>
			<td>40</td>
			<td>50 - 80</td>
			<td>59 (5.7)</td>
			<td>NA</td>
			<td>0</td>
			<td>92.5</td>
			<td>14</td>
		</tr>
	</tbody>
</table>
Table 1:Tagging and survival information for American shad implanted with prototype transmitters (P1-P4) or marked with upper caudal and lower caudal fin clips (Control) as part of the preliminary evaluation. Tag location for the control group is Not Applicable (NA) as these fish only received fin clips (Clip). Note, that the tag type P4 was a neutrally buoyant design. The standard deviation (SD) of the mean is listed in parentheses.

This article provides a detailed procedure for optimal handling practices and implantation of an acoustic micro transmitter into juvenile American shad. The results of our laboratory study suggest that these tagging techniques could be implemented in field studies of juvenile American shad with a high probability of survival.

Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through ...

handling-tagging-techniques-for-implanting-juvenile-american-shad

Nanyang Technological University

Research

JoVE Journal

Behavior

2.2K Views.  Pacific Northwest National Laboratory. 1 Amerikaanse elft is een endogene vissoort2 afkomstig uit het oosten van de Verenigde Staten en Canada, 3 waar de aantallen afnemen als gevolg van verschillende factoren. 4 Deze vissen staan bekend als 5 een gevoelige soort voor behandeling, 6 en zeer weinig merkstudies zijn succesvol geweest 7 in het bestuderen van hun gedrag en overleving 8 zonder hoge sterftecijfers waar te nemen. 9 Ter ondersteuning bij onderzoek naar kortetermijnbewegingen 10 van kle...

Video: Hanteer- en merktechnieken voor het implanteren van juveniele Amerikaanse elft met een nieuwe akoestische microtransmitter

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Developmental Biology

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Neuroscience

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

Environment

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Micro-Drive Implant for Recording Daily Neural Activity in Juvenile Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Author Spotlight: A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice  

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Long-Term Continuous Electroencephalography Monitoring in a Rodent Pup

Source: Zayachkivsky, A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52554">Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System.</a> J. Vis. Exp. (2015)
The video demonstrates long-term continuous electroencephalography (EEG) monitoring in rodent pups using a wireless transmitter system. The transmitter contains two recording electrodes and one reference electrode extending to the brain&#39;s surface. Upon placing the pup in a recording chamber, EEG can be continuously monitored throughout the pup&#39;s development into adulthood.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter

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