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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 colspan="2">discontinued; 8oz container with lid</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&#34; 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&#34; 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&#34;</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/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/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/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/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/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

Research

JoVE Journal

Behavior

2.0K Views.  Pacific Northwest National Laboratory. 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.

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

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.

Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

Infant Auditory Processing and Event-related Brain Oscillations

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral and temporal changes that are characteristic of human language. These abilities lay the foundation for effective language acquisition; allowing infants to hone in on the sounds of their native language. Invasive procedures in animals and scalp-recorded potentials from human adults suggest that simultaneous, rhythmic activity (oscillations) between and within brain regions are fundamental to sensory development; determining the resolution with which incoming stimuli are parsed. At this time, little is known about oscillatory dynamics in human infant development. However, animal neurophysiology and adult EEG data provide the basis for a strong hypothesis that rapid auditory processing in infants is mediated by oscillatory synchrony in discrete frequency bands. In order to investigate this, 128-channel, high-density EEG responses of 4-month old infants to frequency change in tone pairs, presented in two rate conditions (Rapid: 70 msec ISI and Control: 300 msec ISI) were examined. To determine the frequency band and magnitude of activity, auditory evoked response averages were first co-registered with age-appropriate brain templates. Next, the principal components of the response were identified and localized using a two-dipole model of brain activity. Single-trial analysis of oscillatory power showed a robust index of frequency change processing in bursts of Theta band (3 - 8 Hz) activity in both right and left auditory cortices, with left activation more prominent in the Rapid condition. These methods have produced data that are not only some of the first reported evoked oscillations analyses in infants, but are also, importantly, the product of a well-established method of recording and analyzing clean, meticulously collected, infant EEG and ERPs. In this article, we describe our method for infant EEG net application, recording, dynamic brain response analysis, and representative results.

High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain.

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral ...

New Variations for Strategy Set-shifting in the Rat

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.

Set-shifting, a form of behavioral flexibility, requires an attentional shift from one stimulus dimension to another. We extended an established rodent set-shifting task1 by requiring attention to different stimuli according to context. The task was combined with specific lesions to identify neuron subtypes underlying a successful shift.

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Lexical Decision Task for Studying Written Word Recognition in Adults with and without Dementia or Mild Cognitive Impairment

Older adults are slower at recognizing visual objects than younger adults. The same is true for recognizing that a letter string is a real word. People with Alzheimer&#39;s disease (AD) or Mild Cognitive Impairment (MCI) demonstrate even longer responses in written word recognition than elderly controls. Despite the general tendency towards slower recognition in aging and neurocognitive disorders, certain characteristics of words influence word recognition speed regardless of age or neuropathology (e.g., a word&#8217;s frequency of use). We present here a protocol for examining the influence of lexical characteristics on word recognition response times in a simple lexical decision experiment administered to younger and older adults and people with MCI or AD. In this experiment, participants are asked to decide as quickly and accurately as possible whether a given letter string is an actual word or not. We also describe mixed-effects models and principal components analysis that can be used to detect the influence of different types of lexical variables or individual characteristics of participants on word recognition speed.

This article describes how to implement a simple lexical decision experiment to assess written word recognition in neurologically healthy participants and in individuals with dementia and cognitive decline. We also provide a detailed description of reaction time analysis using principal components analysis (PCA) and mixed-effects modeling.

Older adults are slower at recognizing visual objects than younger adults. The same is true for recognizing that a letter string is a real word. People ...