We thank the Mississippi Department of Wildlife, Fisheries and Parks (MDWFP) for financial support using resources from the Federal Aid in Wildlife Restoration Act (W-48-61). We thank MDWFP biologists W. McKinley, A. Blaylock, A. Gary, and L. Wilf for their extensive involvement in data collection. We also thank S. Tucker as facility coordinator and multiple graduate students and technicians for their help collecting data. This manuscript is contribution WFA427 of the Mississippi State University Forest and Wildlife Research Center.

Ethics Statement: The Mississippi State University Institutional Animal Care and Use Committee approved all capture, handling, and marking techniques under protocols 04-068, 07-036, 10-033 and 13-034.
1. Establish Capture Sites, Immobilize and Transport wild White-tailed Deer
<ol>
	<li>Identify public and private properties that are enrolled in the Deer Management Assistance Program22 and establish &#8805;29 capture sites throughout three source regions in Mississippi, USA.
	<ol>
		<li>Identify several capture locations within each source region to ensure that the range of genetic variation present in the regional population is captured.</li>
		<li>Note: Here, source regions included the Delta, which comprises almost 17% of total land area in Mississippi, USA, and is considered a high-quality soil region with agriculture as the primary land use23,24. All study animals were captured from this region within the distribution of O. v. virginianus25. Other regions included the Thin Loess region (upper and lower Thin Loess combined), which comprises almost 14% of total land area in Mississippi, USA and is considered a medium quality soil region. Agriculture is also a primary land use in the Thin Loess region, though not as prevalent as in the Delta23,24. All study animals were captured from this region within the distribution of O. v. virginianus21. Lastly, the Lower Coastal Plain (LCP) soil region comprises nearly 22% of Mississippi and is classified as a low quality soil region. Primary land uses in the LCP are pine timber production and livestock grazing23,24. The LCP also overlaps the geographical distribution of O. v. osceola; four of the six source populations were within 21 km of this distribution25. This subspecies was described as smaller than O. v. virginianus26.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56059/56059fig1.jpg" /> 
Figure 1: Source Populations. Physiographic regions where pregnant dams and fawns were caught in Mississippi, USA. This figure has been modified from reference31. <a href="//cloudfront.jove.com/files/ftp_upload/56059/56059fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Select potential capture sites that meet the following criteria; habitat characteristics conducive to deer movement, proximity to roads for access, and distribution across the study area. 
	NOTE: Capture sites must allow for concealment of the capture technician.
	<ol>
		<li>Bait sites with about 10 kg of shelled corn to entice deer to visit and evaluate use based on bait consumption and deer photographed by motion-sensitive cameras. Relocate to alternate sites if deer do not attend baited sites within 5-7 days.</li>
	</ol>
	</li>
	<li>During capture events, sit in a concealed &#34;stand.&#34;
	<ol>
		<li>Place stand about 20 m downwind from the bait pile, taking the prevailing wind direction into consideration so that deer approaching the bait are less able to smell the capture technician. 
		NOTE: Elevated stands are strongly preferred, and safety harnesses are required. There are several variations of stands with a variety of commercial sources and use varies by personal preference. For example, a lock-on stand would include seating with a ladder for access attached to a tree with straps. Portable climbing stands can be carried in by the capture technician and allow for increased mobility as the technician can choose a specific tree once they arrive at the capture site. Portable climbing stands are limited to use in straight trees without branches up the chosen height.</li>
	</ol>
	</li>
	<li>Use a dart gun coupled with a 3 cc radio-telemetry dart to deliver a mixture of teletamine HCl (4.4 mg/kg) and xylazine HCl (2.2 mg/kg).
	<ol>
		<li>Schedule capture efforts to coincide with the typical crepuscular activity cycle of deer27. Begin each capture attempt 2-3 h prior to sunset.</li>
		<li>Continue capture events for 2-3 h after sunset using night-vision goggles and a red dot laser for shot placement if deer are not captured during daylight hours.</li>
		<li>Take shots at deer when they are broadside and stationary. 
		NOTE: The hind quarter of the deer is the target because it has significant muscle tissue and is located away from the heart and lungs.</li>
		<li>Wait about 15 min for darted target animals (six-month-old fawns of either sex or pregnant adult females) to become fully immobilized before locating it with directional radio-telemetry equipment.</li>
		<li>Confirm individuals are sedated by checking for eye reflexes (blinking). Then apply ophthalmic ointment to the eyes and blindfold deer to reduce stress. 
		NOTE: Loss of thermoregulation is a consequence of immobilization.</li>
		<li>Use a rectal thermometer to assess body temperature after recovery. Warm deer with heated blankets if the animal&#39;s temperature is below 37.7 &#176;C. Cool deer with ice packs if the animal&#39;s temperature is above 40.0 &#176;C.</li>
		<li>Place deer in a sternal position on a military style gurney and transport deer from the capture location to an enclosed trailer.</li>
		<li>After placing the deer into the trailer, reverse the effects of xylazine HCl with 0.125 mg/kg yohimbine HCl28.</li>
		<li>Transport all captured deer to the desired captive facility (e.g., Mississippi State University Rusty Dawkins Memorial Deer Unit; MSU Deer Unit) and keep them separated by source region.</li>
	</ol>
	</li>
</ol>
2. Captive Facilities and General Husbandry Practices of Research Animals
NOTE: The MSU Deer Unit is subdivided into five 0.4 to 0.8 ha pens.
<ol>
	<li>Cover every side of each pen with shade cloth to act as a visual and physical barrier between pens. Shade cloth helps reduce injuries and provides shade during summer months.</li>
	<li>Place 1-2 elevated box blinds at one end of each pen to facilitate darting events during data collection.</li>
	<li>Place two trough style feeders at separate ends of each pen to reduce competition for food among deer. Also provide a water trough in each pen.</li>
	<li>Provide deer with a high-quality diet (20% crude protein deer pellets) ad libitum. 
	NOTE: Here, additional available forages within pens included (Trifolium spp) and fescue (Festuca spp) along with volunteer grasses and forbs.</li>
	<li>If present, maintain available forages within pens using a mixture of herbicides to control broadleaf weeds and grasses using mixture rates found on respective labels. 
	NOTE: Using off-campus facilities to house &#8805;5.5 month old males will likely be needed. These facilities consisted of two 0.7 ha pens on each of three properties with husbandry practices similar to the MSU Deer Unit.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56059/56059fig2.jpg" /> 
Figure 2: Captive Facility Locations. Study area where satellite facilities and the Mississippi State University (MSU) Deer Unit were located. Shaded areas indicate Oktibbeha (A), Noxubee (B), Attala (C), Scott (D), and Copiah (E), counties, Mississippi, USA.This figure has been modified from reference34. <a href="//cloudfront.jove.com/files/ftp_upload/56059/56059fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Parasite and Disease Control
<ol>
	<li>Monitor research animals for roundworm parasites (Strongyloides spp) using fecal floatation with parasites measured as eggs per gram (EPG).
	<ol>
		<li>If present at high levels, provide parasite control by administering a pelleted wormer (active ingredient fenbendazole) at a rate of about 0.77 kg of pelleted wormer per 22.7 kg of feed during the month of May.</li>
		<li>If parasite levels remain high after initial treatment, use an ivermectin pour-on treatment (5 mg/mL)29, mixed at a rate of 2 mL/0.45 kg and administer to animals at a rate of 0.45 kg of treated feed per 45.4 kg of animal mass. 
		NOTE: Epizootic hemorrhagic disease is sometimes lethal viral disease spread by a biting midge (Culicoides spp) during summer and fall months. If present, treat the research facility with insecticide (5% ultra-low volume insecticide) 2-3 times per week from July 1 to October 1 to decrease prevalence of the vectors among research animals. Spray this insecticide within each pen and around the perimeter of the facility about 90 min before official sunset via fogger. Preferred methods to control for parasites and diseases are unique to each captive facility. Veterinarians must be consulted during any captive wildlife research to ensure animal health and safety.</li>
	</ol>
	</li>
</ol>
4. Data Collection
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56059/56059fig3.jpg" /> 
Figure 3: Data Collection of Newborn Fawns. Measuring hind foot length from a new born fawn at the Mississippi State University Rusty Dawkins Memorial Deer Unit in Oktibbeha County, Mississippi, USA. <a href="//cloudfront.jove.com/files/ftp_upload/56059/56059fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Search the captive facility daily for fawns during the parturition season.
	<ol>
		<li>Uniquely mark newborn fawns with medium plastic ear tags using an ear tagger with antibiotic applied to the male end of the tag to prevent potential infection. Place ear tag about the center of the fawn&#39;s ear.</li>
		<li>Measure body mass (nearest 0.01 kg) with a digital hanging scale and measure hind foot length and total body length (nearest mm).</li>
		<li>Collect hair samples and send them to a remote site for parentage assignment (see the Table of Materials). 
		NOTE: Parentage assignment was made using DNA based on a proprietary, non-statistical custom structured query language database. In the pairwise allele comparison, the remote parentage assignment site assigned parentage when they excluded all but one sire and one dam based upon a shared allele from each parent at all loci tested (B. G. Cassidy, personal communication). This method of parentage assignment was also used in previous research conducted on captive white-tailed deer30,31.</li>
		<li>Administer 2 cc of Clostridium perfringens types C and D toxoid and Clostridium perfringens types C and D antitoxin subcutaneously and administer 0.3 cc/kg of ivermectin in propylene glycol (Mississippi State University Veterinarian School, Mississippi, USA) orally to each fawn.</li>
	</ol>
	</li>
	<li>Chemically immobilize adult males (&#8805;1.5 years-old) during October and November for data collection.
	<ol>
		<li>Immobilize penned adults using the same combination of teletamine HCl and xylazine HCl used for capture of wild animals (step 1.4).</li>
		<li>During sedation events, walk the technician who will be darting to the end of the pen where the elevated blinds are located. Have a single technician in each of two blinds.</li>
		<li>Have the individual who walked the technician to the blind walk back to the opposite end of the pen. 
		NOTE: Deer move away from these technicians and locate themselves in front of the blinds where technicians are in position to take ethical shots on each deer.</li>
		<li>Take shots at deer when they are broadside and stationary (section 1.4).</li>
		<li>Wait about 15 min for darted animals to become fully immobilized before approaching it.</li>
		<li>Confirm individuals are sedated by checking for eye reflexes (blinking). Apply ophthalmic ointment to the eyes and blindfold deer to reduce stress.</li>
		<li>After the darters successfully sedate an individual deer, monitor the deer&#39;s vital rates.
		<ol>
			<li>Use a rectal thermometer to assess body temperature after recovery. Warm deer with heated blankets if the animal&#39;s temperature is below 37.7 &#176;C. Cool deer with ice packs if the animal&#39;s temperature is above 40.0 &#176;C.</li>
		</ol>
		</li>
		<li>Load the deer on a military-style gurney, and transport it via utility task vehicle to a predetermined data collection area.</li>
		<li>Once transported, record the same morphometric measurements recorded at birth (step 4.1).
		<ol>
			<li>Measure body mass (nearest 0.01 kg) with a digital hanging scale and measure hind foot length and total body length (nearest mm). 
			NOTE: Individual deer react differently to the combination of drugs used during sedation events so administer about 0.1-0.3 cc of the teletamine HCl and xylazine HCl mixture (depending on body mass of an individual deer) if an individual comes out of sedation before data collection is completed.</li>
		</ol>
		</li>
		<li>Administer size-appropriate amounts of antibiotic, ivermectin, a clostidrial vaccine, and a leptospirosis vaccine to all deer after they are transported to the data collection area (see sections 1 and 3).</li>
	</ol>
	</li>
	<li>Take three antler measurements from adult males using an antler measuring tape while the animal is sedated.
	<ol>
		<li>Measure the inside spread (widest distance between main beams), basal circumference (smallest diameter located between the burr and G1 tine), and main beam length (distance from antler base to the tip of the main beam) of antlers prior to antler removal.</li>
		<li>Remove antlers about 3 cm above the burr using a reciprocating saw. Do not remove antlers less than 3 cm.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56059/56059fig4.jpg" /> 
Figure 4: Data Collection of Adult Males. Antler removal via reciprocating saw from a captive adult male white-tailed deer. <a href="//cloudfront.jove.com/files/ftp_upload/56059/56059fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>After all data is collected from the sedated individual, place the deer into the appropriate pen and administer either 0.125 mg/kg yohimbine HCl28 or 4.0 mg/kg tolazoline HCl32 to reverse the effects of xylazine HCl. Monitor individuals to ensure they remain in a sternal position until they come out of sedation and are fully alert. 
	NOTE: If complications occur and animals must be euthanized, then euthanasia by cerebral dislocation via bolt stunner and severing of the jugular vein are ethical means to dispatch the animal.</li>
	<li>Bring the antlers to a designated area to finish measuring antler size.
	<ol>
		<li>Measure each individual tine protruding from the main beam (G1, G2, G3, etc.) and additional abnormal points by aid of wire. 
		NOTE: Points that do not have a matching counterpart on the opposite main beam or are not consistent with the definition of a typical antler set are defined by the Boone and Crockett Club33.</li>
		<li>Wrap the wire around where the tine intersects the main beam and mark that point for reference.</li>
		<li>Measure from this reference point to the tip of the tine and repeat for each tine.</li>
		<li>Collect remaining circumference measurements by identifying the smallest point between the G1 and G2 tines (H2 circumference), the G2 and G3 tines (H3 circumference), and the G3 and G4 tine if present (H4 circumference).</li>
		<li>If the G4 tine is not present, measure the distance between the midpoint of the G3 tine and the end of the main beam and measure the H4 circumference at the midway point.</li>
		<li>Measure less than four circumferences when antlers contain less than three tines. 
		NOTE: For example, a main beam with two typical points include only three circumference measurements. Individuals may use other guidelines (Safari Club International) for calculating antler size; however, consistent methods must be used for each animal for valid comparison.</li>
		<li>After making all measurements, calculate an antler score similar to the gross nontypical Boone and Crockett score33.</li>
		<li>Weigh antlers to the nearest 0.1 g using a scientific digital scale and assign a minimal critical antler mass of 1 g for first-year animals with antlers shorter than 3 cm.</li>
	</ol>
	</li>
	<li>Chemically immobilize penned juveniles at approximately 5.5 months of age using the same methods for adults (section 4.2) and mark juveniles with a large plastic ear tag (step 4.1.1).
	<ol>
		<li>Use the same drug mixture rates to immobilize captive adults as used for immobilizing wild deer (section 1).</li>
		<li>Collect the same measurements collected at birth (step 4.1.2) and administer the same prophylactics as adults (section 4.2). 
		NOTE: After all data is collected, transport each juvenile male to its randomly assigned satellite facility via trailer.</li>
	</ol>
	</li>
</ol>
5. Producing First- and Second-generation Offspring
<ol>
	<li>Classify six-month-old wild-caught fawns and offspring born at the captive facility from wild-caught mothers as first-generation individuals.</li>
	<li>During the breeding season, place two males with 7-16 females for an average breeding sex ratio of one male to eight females. 
	NOTE: Select breeding males from satellite facilities based on physical appearance, because the healthiest males (largest antlers and body size) are most likely to service females for the entirety of the breeding season without suffering from injury due to the aggressive nature of males during the breeding season.
	<ol>
		<li>Only allow deer to breed with other individuals from the same source region (e.g., Delta males breed Delta females, Thin Loess males breed Thin Loess females, and LCP males breed LCP males).</li>
	</ol>
	</li>
	<li>Classify deer conceived by first-generation parents as second-generation offspring. Raise these individuals in captivity from birth and feed the same high-quality diet as their parents. 
	NOTE: Females may produce offspring multiple years but typically with different sires each year. Collect the same data on second-generation offspring as collected on first-generation and wild-caught individuals.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+management+of+white-tailed+deer+on+private+lands+in+Mississippi.">Cooperative management of white-tailed deer on private lands in Mississippi.</a> Wildl Soc Bull. 11 (3), 211-214 (1983).</li><li>Pettry, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+resource+areas+of+Mississippi.">Soil resource areas of Mississippi.</a> Mississippi Agricultural and Forestry Experiment Station. , (1977).</li><li>Snipes, C. E., Nichols, S. P., Poston, D. H., Walker, T. W., Evans, L. P., Robinson, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+agricultural+practices+of+the+Mississippi+Delta.">Current agricultural practices of the Mississippi Delta.</a> Office of Agricultural Communications. , (2005).</li><li>Baker, R. H., Halls, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+classification,+and+distribution+of+the+white-tailed+deer.">Origin, classification, and distribution of the white-tailed deer.</a> White-tailed deer: ecology and management. , 1-18 (1984).</li><li>Barbour, T., Allen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+white-tailed+deer+of+eastern+United+States).">The white-tailed deer of eastern United States).</a> J Mammal. 3 (2), 65-80 (1922).</li><li>Rouleau, I., Cr&#234;te, M., Ouellet, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+the+summer+ecology+of+white-taileddeer+inhabiting+a+forested+and+an+agricultural+landscape.">Contrasting the summer ecology of white-taileddeer inhabiting a forested and an agricultural landscape.</a> Ecoscience. 9 (4), 459-469 (2002).</li><li>Kreeger, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of wildlife chemical immobilization. , (1996).</li><li>Pound, J. M., Miller, J. A., Oethler, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+rates+of+injected+and+ingested+Ivermectin+from+blood+serum+of+penned+white-tailed+deer,+Odocoileus+virginianus+(Zimmermann)+(Artiodactyla:+Cervidae).">Depletion rates of injected and ingested Ivermectin from blood serum of penned white-tailed deer, Odocoileus virginianus (Zimmermann) (Artiodactyla: Cervidae).</a> J Medl Entomol. 41 (1), 65-68 (2004).</li><li>Jones, P. D., Demarais, S., Strickland, B. K., DeYoung, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inconsistent+relation+of+male+body+mass+with+breeding+success+in+captive+white-tailed+deer.">Inconsistent relation of male body mass with breeding success in captive white-tailed deer.</a> J Mammal. 92 (3), 527-533 (2011).</li><li>Michel, E. S., Flinn, E. B., Demarais, S., Strickland, B. K., Wang, G., Dacus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+nutrition+cues+switch+from+efficiency+to+luxury+phenotypes+for+a+long-lived+ungulate.">Improved nutrition cues switch from efficiency to luxury phenotypes for a long-lived ungulate.</a> Ecol Evol. 6 (20), 7276-7285 (2016).</li><li>Miller, B. F., Muller, L. I., Doherty, T., Osborn, D. A., Miller, K. V., Warren, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+antagonists+for+tiletamine-zolazepam/xylazine+immobilization+in+female+white-tailed+deer.">Effectiveness of antagonists for tiletamine-zolazepam/xylazine immobilization in female white-tailed deer.</a> J Wildl Dis. 40 (3), 533-537 (2004).</li><li>Nesbitt, W. H., Wright, P. L., Buckner, E. L., Byers, C. R., Reneau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measuring and scoring North American big game trophies. 3rd edn. , (2009).</li><li>Michel, E. S., Demarais, S., Strickland, B. K., Smith, T., Dacus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antler+characteristics+are+highly+heritable+but+influenced+by+maternal+factors.">Antler characteristics are highly heritable but influenced by maternal factors.</a> J Wildl Manage. 80 (8), 1420-1426 (2016).</li><li>Severinghaus, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+development+and+wear+as+criteria+of+age+in+white-tailed+deer.">Tooth development and wear as criteria of age in white-tailed deer.</a> J Wildl Manage. 13 (2), 195-216 (1949).</li><li>Gee, K. L., Webb, S. L., Holman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+implications+of+visually+estimating+age+of+male+white-tailed+deer+using+physical+characteristics+from+photographs.">Accuracy and implications of visually estimating age of male white-tailed deer using physical characteristics from photographs.</a> Wild Soc Bull. 38, 96-102 (2014).</li><li>Storm, D. J., Samuel, M. D., Rolley, R. E., Beissel, T., Richards, B. J., Van Deelen, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+ages+of+white-tailed+deer:+Age+and+sex+patterns+of+error+using+tooth+wear-and-replacement+and+consistency+of+cementum+annuli.">Estimating ages of white-tailed deer: Age and sex patterns of error using tooth wear-and-replacement and consistency of cementum annuli.</a> Wild Soc Bull. 38 (1), 849-865 (2014).</li><li>Montero, D., Izquierdo, M. S., Tort, L., Robaina, L., Vergara, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+stocking+density+produces+crowding+stress+altering+some+physiological+and+biochemical+parameters+in+gilthead+seabream,+Sparus+aurata.,+juveniles.">High stocking density produces crowding stress altering some physiological and biochemical parameters in gilthead seabream, Sparus aurata., juveniles.</a> Fish Physiol Biochem. 20 (1), 53-60 (1999).</li><li>Charbonnel, N., 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=Stress+demographic+decline:+a+potential+effect+mediated+by+impairment+of+reproduction+and+immune+function+in+cyclic+vole+populations.">Stress demographic decline: a potential effect mediated by impairment of reproduction and immune function in cyclic vole populations.</a> Physiol Biochem Zool. 81 (1), 63-73 (2008).</li><li>Crews, D., Gillette, R., Scarpino, S. V., Manikkam, M., Savenkova, M. I., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+transgenerational+inheritance+of+altered+stress+responses.">Epigenetic transgenerational inheritance of altered stress responses.</a> Proc Natl Acad Sci. 109 (23), 9143-9148 (2012).</li><li>Maher, J. M., Werner, E. E., Denver, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+hormones+mediate+predator-induced+phenotypic+plasticity+in+amphibian+tadpoles.">Stress hormones mediate predator-induced phenotypic plasticity in amphibian tadpoles.</a> Proc R Soc Lond B Biol Sci. 280 (1758), 20123075 (2013).</li></ol>

The authors have nothing to disclose.

There are several steps associated with our protocol; however, there are four critical steps that must be taken to ensure success with this protocol. First, during capture of wild deer, there must be several capture locations throughout a single source region (step 1.1.1). Having multiple capture locations ensures that any genetic variability associated with the source region will be represented among deer. Second, deer must be kept separated by source region during breeding season (steps 1.4.9 and 5.2.1). Ensuring anima...

Environmental factors a mother experiences during gestation and lactation may influence her offspring&#39;s phenotype, independent of genotype1,2,3. Mothers who inhabit high-quality environments likely will produce offspring that exhibit a luxury phenotype (large antler and body size4), whereas mothers who inhabit a low-quality environment may produce offspring that exhibit an efficiency phenotype (small antler and body size4). Therefore, persisting in a high-quality environment may allow a mother to produce male offspring with large phenotypic characteristics, which may directly influence the offspring&#39;s reproductive opportunities5,6,7,8 and indirectly influence the mother&#39;s inclusive fitness.
Although nutrition directly influences phenotypic characteristics across taxa (Ursus americanus, Ursus arctos9; Liasis fuscusI10; Larus michahellis11), several factors may affect white-tailed deer phenotypes in Mississippi, USA. Antler and body size are about one-third larger for some populations compared to others12. This variation is strongly correlated with forage quality13,14; the largest males are found in areas with the greatest quality of forage. However, historic restoration efforts of white-tailed deer in Mississippi may have led to genetic bottlenecks and/or founder effects15,16, which may also partially explain some of the observed regional variation in white-tailed deer phenotype.
We provide the protocol we used to control nutritional quality of wild-caught white-tailed deer, which allowed us to assess whether male phenotype is restricted by population-level genetics. This protocol also allowed us to assess whether lagging maternal effects were present in our populations. Our controlled design is preferential to studies conducted on free ranging populations which are limited to using environmental variables as a proxy for nutritional restriction3,17. Our controlled design also allows for other variables such as potential chronic stress related to social interactions to be held constant as all individuals are subjected to similar housing and husbandry practices. Additionally, because nutrition directly influences other life history aspects ranging from reproduction to survival18,19, controlling nutrition allows for investigators to assess other variables that affect mammalian life history aspects. Similar protocols have been described to assess questions related to life history aspects for other ungulates across North America (e.g., 20,21).

<table><tbody><tr><td>Shelled Corn</td><td></td><td></td><td></td></tr><tr><td>Elevated Stand</td><td></td><td></td><td></td></tr><tr><td>Safety Harness</td><td></td><td></td><td></td></tr><tr><td>Ground Blind</td><td></td><td></td><td></td></tr><tr><td>Model 196 Projector</td><td>Pneu-Dart, Pennsylvania, USA</td><td></td><td></td></tr><tr><td>3cc Radio-Telemetry Darts</td><td>(Pneu-Dart, Pennsylvania, USA)</td><td></td><td></td></tr><tr><td>Various Sized Darts&amp;nbsp;</td><td>(Pneu-Dart, Pennsylvania, USA)</td><td></td><td></td></tr><tr><td>Teletamine HCl&amp;nbsp;</td><td>(Telazol, Fort Dodge Animal Health, Iowa, USA)</td><td></td><td></td></tr><tr><td>Xylazine HCl&amp;nbsp;</td><td>(West Texas Rx Pharmacy, Amarillo, Texas, USA)</td><td></td><td></td></tr><tr><td>Yhoimbine HCl</td><td></td><td></td><td></td></tr><tr><td>Tolazoline HCl</td><td></td><td></td><td></td></tr><tr><td>Military Style Gurney</td><td></td><td></td><td></td></tr><tr><td>Rectal Thermometer</td><td></td><td></td><td></td></tr><tr><td>Shade Cloth</td><td></td><td></td><td></td></tr><tr><td>20% Crude Protein Deer Pellets&amp;nbsp;</td><td>(Purina AntlerMax Professional High Energy Breeder 59UB, Purina, Missouri, USA)</td><td></td><td></td></tr><tr><td>Trough Style Feeders</td><td></td><td></td><td></td></tr><tr><td>Commercial Clover&amp;nbsp;</td><td>(Durana Clover, Pennington Seed Co., Georgia, USA)</td><td></td><td></td></tr><tr><td>Commercial Fescue&amp;nbsp;</td><td>(Max-Q Fescue, Pennington Seed Co., Georgia, USA)</td><td></td><td></td></tr><tr><td>Blankets</td><td></td><td></td><td></td></tr><tr><td>Ice Packs</td><td></td><td></td><td></td></tr><tr><td>Broadleaf Weed Control (2, 4-DB Herbacide, Butyrac 200)</td><td></td><td></td><td></td></tr><tr><td>Grass Control&amp;nbsp;</td><td>(Poast Herbacide, BASF Co.)</td><td></td><td></td></tr><tr><td>Pelleted Wormer</td><td>Safeguard Co.,&amp;nbsp;</td><td></td><td>active ingredient fenbendazole</td></tr><tr><td>Parasite Pour-on Treatment&amp;nbsp;</td><td>(Ivomec, Merial Co.)</td><td></td><td></td></tr><tr><td>Insecticide</td><td>Riptide, McLaughlin Gormley King Co.)&amp;nbsp;</td><td></td><td></td></tr><tr><td>Medium and Large Plastic Ear Tags&amp;nbsp;</td><td>(Allflex, Texas, USA)</td><td></td><td></td></tr><tr><td>Remote site that assigned parentage</td><td>DNA Solutions Animal Solutions Manager (DNA Solutions, Oklahoma, USA)</td><td></td><td></td></tr><tr><td>Digital Hanging Scale&amp;nbsp;</td><td>(Moultrie, EBSCO Industries, Inc.)&amp;nbsp;</td><td></td><td></td></tr><tr><td>Tape Measure</td><td></td><td></td><td></td></tr><tr><td>Clostridium Perfringens Types C and D Toxoid Essential 3&amp;nbsp;</td><td>(Colorado Serum Co.)</td><td></td><td></td></tr><tr><td>Clostridium Perfringens Types C and D Antitoxin Equine Origin</td><td>(Colorado Serum Co.)</td><td></td><td></td></tr><tr><td>Ivermectin in propylene glycol</td><td></td><td></td><td></td></tr><tr><td>Antibiotic</td><td>(Nuflor, Schuering-Plough Animal Health Corp., New Jersey, USA)</td><td></td><td></td></tr><tr><td>Ivermectin&amp;nbsp;</td><td>(Norbrook Labratories, LTD., Down, Northern Ireland, UK)</td><td></td><td></td></tr><tr><td>Clostidrial vaccine</td><td>(Vision 7 with SPUR, Ivesco LLC, Iowa, USA)</td><td></td><td></td></tr><tr><td>Leptospirosis vaccine&amp;nbsp;</td><td>(Leptoferm-5, Pfizer, Inc., New York, USA)</td><td></td><td></td></tr><tr><td>Trailer for transport</td><td></td><td></td><td></td></tr><tr><td>Reciprocating saw&amp;nbsp;</td><td>(DEWALT, Maryland, USA)</td><td></td><td></td></tr><tr><td>Scientific Digital Scale&amp;nbsp;</td><td>(Global Industrail, Global Equipment Company Inc)</td><td></td><td></td></tr><tr><td>Antler Measuring Tape</td><td></td><td></td><td></td></tr><tr><td>Fogger</td><td></td><td></td><td></td></tr><tr><td>Plastic Ear Tags&amp;nbsp;</td><td>(Allflex, Texas, USA)</td><td></td><td></td></tr><tr><td>Plastic Ear Tagger</td><td>(Allflex, Texas, USA)</td><td></td><td></td></tr></tbody></table>

protocol for assessing the relative effects of environment and genetics on antler and body growth for a long-lived cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Individual age, nutritional quality, and genetics influence male white-tailed deer phenotype. Our study design allowed us to control the quality of nutrition deer were consuming and allowed us to identify the age of each deer for valid comparisons within year classes. By controlling nutrition and age with our study design, we were better able to understand whether population-level genetics were restricting the phenotype of males from two study populations. Improved nutrition had a positiv...

Watch this Scientific Journal Video about Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid at JoVE.com

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid | Environment | Video

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

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7.3K Views.  Mississippi State University. Fenotypische verschillen tussen de cervid bevolking kunnen worden gerelateerd aan populatieniveau genetica of voeding; comforthotel die moeilijk is in het wild. Dit protocol beschrijft hoe we ontwierpen een gecontroleerde studie waar voeding variatie werd uitgeschakeld. We vonden dat fenotypische variatie van mannelijke Witstaarthert was beperkter door voeding dan genetica.

Video: Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

Developmental Biology

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Cancer Research

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Neuroscience

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

Medicine

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Immunology and Infection

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from human infrastructure and recreational activity. Artificial light at night (ALAN) has the potential to have widespread effects on animal behavior, physiology, and fitness, which can translate into broader-scale effects on populations and communities. Understanding the effects of ALAN on free-ranging animals is non-trivial due to challenges such as measuring levels of light encountered by mobile organisms and separating the effects of ALAN from those of other anthropogenic disturbance factors. Here we describe an approach that allows us to isolate the effects of artificial light exposure on individual animals by experimentally manipulating light levels inside nest boxes. To this end, a system can be used consisting of light-emitting diode (LED) light(s) adhered to a plate and connected to a battery and timer system. The setup allows exposure of individuals inside nest boxes to varying intensities and durations of ALAN while simultaneously obtaining video recordings, which also include audio. The system has been used in studies on free-ranging great tits (Parus major) and blue tits (Cyanistes caeruleus) to gain insight into how ALAN affects sleep and activity patterns in adults and physiology and telomere dynamics in developing nestlings. The system, or an adaptation thereof, could be used to answer many other intriguing research questions, such as how ALAN interacts with other disturbance factors and affects bioenergetic balance. Furthermore, similar systems could be installed in or near the nest boxes, nests or burrows of a variety of species to manipulate levels of ALAN, evaluate biological responses, and work towards building an interspecific perspective. Especially when combined with other advanced approaches for monitoring the behavior and movement of free-living animals, this approach promises to yield ongoing contributions to our understanding of the biological implications of ALAN.

Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Artificial Rearing and Heavy Metal Tolerance in Cabbage White Butterflies

Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ecology. Cabbage whites can be easily reared in controlled conditions on an artificial diet, making them a model organism of the butterfly world. In this paper, a manipulation of heavy metal exposure is used to illustrate basic methods for rearing this species. The general protocol illustrates how butterflies can be caught in the field, induced to lay eggs in greenhouse cages, and transferred as larvae to artificial diets. The methods show how butterflies can be marked, measured, and studied for a variety of research questions. The representative results give an idea of how artificial diets that vary in components can be used to assess butterfly performance relative to a control diet. More specifically, butterflies were most tolerant to nickel and least tolerant to copper, with a tolerance of zinc somewhere in the middle. Possible explanations for these results are discussed, including nickel hyper-accumulation in some mustard host plants and recent evidence in insects that copper may be more toxic than previously appreciated. Finally, the discussion first reviews variations to the protocol and directions for troubleshooting these methods, before considering how future research might further optimize the artificial diet used in this study. Overall, by providing a detailed video overview of the rearing and measurement of cabbage whites on artificial diets, this protocol provides a resource for using this system across a wide range of studies.

Author Spotlight: Investigating the Tolerance of Cabbage Butterflies to Urban Pollutants 

This paper presents a detailed protocol for rearing the cabbage white butterfly in controlled lab conditions with an artificial diet, which allows precise manipulations of early-life nutrition and toxin exposure. The representative results show how heavy metal toxicity can be assayed with this protocol.

The cabbage white butterfly (Pieris rapae) is an important system for applied pest control research and basic research in behavioral and nutritional ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

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