Name: protocol for assessing the relative effects of environment and genetics on antler and body growth for a long-lived cervid
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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
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	<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.</stron...

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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).

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			<td height="20" style="width:416px;height:20px;">Shelled Corn</td>
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			<td style="width:64px;"></td>
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			<td height="20" style="height:20px;">Elevated Stand</td>
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			<td height="20" style="height:20px;">Safety Harness</td>
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			<td height="20" style="height:20px;">Ground Blind</td>
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			<td height="20" style="height:20px;">Model 196 Projector</td>
			<td style="width:316px;">Pneu-Dart, Pennsylvania, USA</td>
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			<td height="20" style="height:20px;">3cc Radio-Telemetry Darts</td>
			<td style="width:316px;">(Pneu-Dart, Pennsylvania, USA)</td>
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			<td height="20" style="height:20px;">Various Sized Darts&#160;</td>
			<td style="width:316px;">(Pneu-Dart, Pennsylvania, USA)</td>
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			<td height="20" style="height:20px;">Teletamine HCl&#160;</td>
			<td style="width:316px;">(Telazol, Fort Dodge Animal Health, Iowa, USA)</td>
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			<td height="20" style="height:20px;">Xylazine HCl&#160;</td>
			<td style="width:316px;">(West Texas Rx Pharmacy, Amarillo, Texas, USA)</td>
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			<td height="20" style="height:20px;">Yhoimbine HCl</td>
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			<td height="20" style="height:20px;">Tolazoline HCl</td>
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			<td height="20" style="height:20px;">Military Style Gurney</td>
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			<td height="20" style="height:20px;">Rectal Thermometer</td>
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			<td height="20" style="height:20px;">Shade Cloth</td>
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		<tr height="20">
			<td height="20" style="height:20px;">20% Crude Protein Deer Pellets&#160;</td>
			<td style="width:380px;">(Purina AntlerMax Professional High Energy Breeder 59UB, Purina, Missouri, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trough Style Feeders</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial Clover&#160;</td>
			<td style="width:380px;">(Durana Clover, Pennington Seed Co., Georgia, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial Fescue&#160;</td>
			<td style="width:380px;">(Max-Q Fescue, Pennington Seed Co., Georgia, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blankets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice Packs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Broadleaf Weed Control (2, 4-DB Herbacide, Butyrac 200)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grass Control&#160;</td>
			<td style="width:316px;">(Poast Herbacide, BASF Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pelleted Wormer</td>
			<td>Safeguard Co.,&#160;</td>
			<td></td>
			<td style="width:227px;">active ingredient fenbendazole</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parasite Pour-on Treatment&#160;</td>
			<td>(Ivomec, Merial Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insecticide</td>
			<td style="width:316px;">Riptide, McLaughlin Gormley King Co.)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medium and Large Plastic Ear Tags&#160;</td>
			<td>(Allflex, Texas, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Remote site that assigned parentage</td>
			<td style="width:380px;">DNA Solutions Animal Solutions Manager (DNA Solutions, Oklahoma, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hanging Scale&#160;</td>
			<td style="width:316px;">(Moultrie, EBSCO Industries, Inc.)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tape Measure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostridium Perfringens Types C and D Toxoid Essential 3&#160;</td>
			<td>(Colorado Serum Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostridium Perfringens Types C and D Antitoxin Equine Origin</td>
			<td>(Colorado Serum Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ivermectin in propylene glycol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic</td>
			<td style="width:380px;">(Nuflor, Schuering-Plough Animal Health Corp., New Jersey, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ivermectin&#160;</td>
			<td style="width:380px;">(Norbrook Labratories, LTD., Down, Northern Ireland, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostidrial vaccine</td>
			<td style="width:316px;">(Vision 7 with SPUR, Ivesco LLC, Iowa, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leptospirosis vaccine&#160;</td>
			<td style="width:316px;">(Leptoferm-5, Pfizer, Inc., New York, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trailer for transport</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reciprocating saw&#160;</td>
			<td style="width:316px;">(DEWALT, Maryland, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scientific Digital Scale&#160;</td>
			<td style="width:316px;">(Global Industrail, Global Equipment Company Inc)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antler Measuring Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fogger</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Ear Tags&#160;</td>
			<td>(Allflex, Texas, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

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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