The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: a Comparative Analysis

February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H.

The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis

Emily S. Tarbet Hust1, Meradeth H Snow2 1SNA International, 2University of Montana

Abstract Sus scrofa domesticus limbs were obtained as a human proxy to study the effects of five distinct materials used in published methods of flesh removal: Dermestes lardarius beetles (also referred to as dermestids), distilled-water boil, bleach boil, enzyme-based detergent simmer, and ammonia simmer. Each method was evaluated based on a set of specific criteria, focusing on time efficiency, macroscopic damage, and the effects on DNA preservation and potential for future analysis. While the dermestid beetles had the longest time-expectancy and were the most labor-intensive method, they caused minimal damage to the bone surface and did not appear to affect the DNA preservation. Heated maceration methods sped up the process considerably, but that often led to decreased DNA quantity and minimal to severe amounts of macroscopic damage. The ammonia simmer method was the only method tested that was found in zoological literature but did not appear to have any published use within the forensic field, operating occasionally instead as a degreasing agent. While the ammonia method required the most safety precautions, the method was efficient both in time and tissue removal, and left amplifiable DNA, perhaps indicating a potential future in more forensic contexts. In contrast, the enzyme-based detergent method, often praised in published literature, performed poorly in multiple categories of evaluation. Each method proved to have different advantages and disadvantages, with no method performing the best or worst in every evaluated criterion. The results of this research highlight how differently each method performs and how easily skeletal material, and the DNA within, can be affected by maceration techniques. Method selection can severely impact later analysis and the choice should be made with consideration and awareness of the potential risks and desired results.

Keywords: Forensic Anthropology; Forensic Science; Maceration; Defleshing; mtDNA

Introduction nally, forensic anthropologists also use Maceration, or the removal of soft these techniques during casework to re- tissue from skeletal remains, is a process duce fleshed or decomposing individuals familiar to members of a wide array of to skeletal remains for analysis. Due to scientific fields and contexts. Flesh re- the wide array of fields and goals behind moval is used in museums to prepare the removal of soft tissue from skeletal bones for display and for comparative re- remains, there are an equally wide array search, hunters may prepare remains for of developed methods to perform the showcasing, and body donation research task. facilities use recently skeletonized re- However, no consistent standard mains of modern individuals to refine and exists for forensic applications of tissue update the markers used in biological removal, which leads to a wide range of profile construction and analysis. Additio- protocols and varied guidance in the am-

Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 30 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. ount of additives, temperatures, and tim- scored and documented in detail. The re- ing. While the interest in method efficacy sults in this study were then used to cre- has increased in recent years (Ajayi et ate a method selection flowchart, empha al., 2016; Ecklund, 2007; Frank et al., sizing the different advantages and dis- 2015; Mann & Berryman, 2012), there re- advantages of the tested methods. Be- mains a lack of cohesive agreement on cause the goals and intentions behind which additives may perform best in sim- soft tissue removal tend to vary widely ilar scenarios or which methods have the between fields and contexts, the results most negative impact on skeletal remains are designed with a specific emphasis on and DNA viability. While many of these the viability and practicality of the method methods and additives are used outside within a forensic context, but the general of the forensic sciences, safety and con- information can be relevant to any field sistency in forensic analysis is of the ut- familiar with the task. most importance. The bulk of the literature reflects primarily on macroscopic Materials and Methods damage that affects trauma analysis, as Research Sample well as methods that may complicate The research sample consisted of DNA extraction (Ecklund, 2007; Fenton 17 domesticated pig (Sus scrofa domes- et al., 2003; Frank et al., 2015; King & ticus) limbs from the North Dakota State Birch, 2015; Lynn & Fairgrieve, 2009). University agricultural program. The pig Heated maceration methods have limbs were primarily the hock portion of long been the preferred and more inves- the leg and contained fragments of two tigated method due to their shorter dura- long bones, either the tibia and fibula or tion and less manually laborious nature radius and ulna depending on whether it than unheated methods. Simmering and was a hind or front limb. The pigs were all boiling techniques utilizing a variety of from the same environment and were additives have become common practice butchered in the same manner at approx- (Couse & Connor, 2015). The additives imately the same age, all being just under range from household cleaning products a year old. such as dish and laundry detergent to di- The 17 pig hocks were assigned a rectly adding chemicals such as sodium maceration method at random, with three perborate or carbonate, while still other hocks being assigned to each category methods utilize heated water with no ad- and the remaining two being reserved for ditional additives (Lee et al., 2010; Uhre manual soft tissue removal to allow for a et al., 2015). DNA comparison with samples that did The following research focuses on not undergo one of the tested methods. several commonly used soft tissue re- Control DNA samples were not taken moval methods, involving four heated prior to the maceration of the pig hocks to maceration methods and one method uti- avoid additional variables and considera- lizing Dermestes lardarius beetles, here- tions in regard to specific element and re- after referred to as dermestids. The gion of bone (Antinick & Foran, 2018). In amount of additives used, the duration to lieu of a true control sample, consistency completion each maceration method was attempted by ensuring all pigs were took, and the effects the method had on the same age from the same environ- the resulting skeletonized material were ment and butchered at the same time

Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 31 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. with the same technique to limit back- in skeletal preparation within a zoological ground variation as much as possible museum. In forensic uses, ammonia (Arismendi et al., 2004). While this is not tends to be used as a degreaser after the a true control sample, whereby samples application of a primary method (Lee et from each pig hock would have been col- al., 2010), but in zoological preparation lected prior to treatment, the two un- solutions of up to 50% ammonia can be treated pig hocks can be used for com- used in a simmer for the entire process parison by providing an estimation and (Hoffmeister and Lee, 1963). The re- expectation of the DNA preservation and searcher’s own experience following zo- degradation resulting from the tested ological museum protocols involved methods. soaking delicate remains in a room temperature ammonia and water mixture for Tested Maceration Methods several days to remove residual tissue Five soft tissue removal methods, and grease that the dermestid colony consisting of four heated maceration leaves behind. Personal experience with methods and one method utilizing a col- use of ammonia and published uses in ony of dermestids, were selected for zoological contexts were combined to study based on methods found in previ- create the method performed in this ously published literature and researcher study. experience (Table 1). All the heated Two methods, distilled water and maceration method materials, including 6.25% bleach solution, were kept at a low start-up equipment, are widely accessi- boil, while the 12.25% household ammo- ble and relatively low cost. Cost could be nia and the 10% enzyme-based deter- additionally lowered if tap water, as op- gent methods were kept at a lower sim- posed to the distilled water used in these mering temperature for the duration of trials, and off-brand additives were used, the test. A laundry detergent with an ac- and materials were purchased in bulk cessible ingredient list was utilized to quantities. confirm that both lipase and protease en- All heated maceration methods zymes were present in the selected de- were performed in a small, ventilated tergent (Uhre et al., 2015). Method tem- room with as many variables kept con- peratures were based on those used in stant across methods as possible. Pig selected references (Table 1). The tests hocks subject to heated maceration were monitored at half-hour checkpoints methods did not undergo any manual for temperature checks and photos to en- processing (Couse & Connor, 2015). The sure consistency of temperature through- heated methods were performed using out the duration of the method and to an 18qt stockpot containing a two-gallon document the bone surface. The solution made from distilled water and defleshed bone was then dried in a fume the specific additive. The amount varies hood for 48-72 hours before being placed based on the documented amounts in within the dermestid tank, in accordance previously published literature (Table 1). with the museum protocol where the col- The household ammonia method was ony was housed (Schroeder et al., 2002). adapted and modified by the researcher The pig hocks were exposed to the der- from previous published use in a zoolog- mestid colony one at a time, but because ical context (Hoffmeister and Lee, 1963) the primary purpose of the colony was for as well as personal experience working museum specimen preparation, there Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 32 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H.

Table 1: Table showing the different additives, amounts, and references of the four heated maceration methods attempted, as well as literature references for the dermestid beetle method and manual soft tissue removal.

Percentage of Amount of Ad- Approximate Reference Additive Within ditive Starting Solution Temp Manual Soft N/A N/A Room Tem- Couse & Connor, Tissue Re- perature 2015 moval Dermestes N/A N/A Room Tem- Ajayi et al., 2016; lardarius perature Charabidze et al., Colony Expo- 2014; Schroeder et sure al., 2002 Distilled Water 0% None 99.0° C Lee et al., 2010; Rennick et al., 2005;

Uhre et al., 2015 Enzyme-based 10.0% 1.6 cups per 75.0° C Eklund, 2007; Lee et Laundry Deter- gallon al., 2010; Mooney et gent al., 1982; Nawrocki, 1997 Bleach 6.25% 1 cup per gal- 99.0° C Eklund, 2007; Na- lon wrocki, 1997; Ren- nick et al., 2005 Household 12.25% 2 cups per gal- 90.0° C Modified based on Ammonia lon Hoffmeister & Lee 1963; National Park Service 2006 were other materials present throughout & Birch, 2015; Lee et al., 2010; Stead- the duration. The dermestid beetle man et al., 2006; Table 2). Criteria were method had a much longer timeframe ex- selected and scored based on previous pectancy and therefore the exposed pig studies as well as the necessary require- hocks were checked only once a day. ments of bone preparation techniques The methods were evaluated on within forensic usages (Couse & Connor, criteria including time efficiency, DNA 2015; King & Birch, 2015; Lee et al., concentration quantity, ease of applica- 2010; Steadman et al., 2006). tion, macroscopic damage, and effective- Time efficiency was considered to ness, with scoring tables based on those be the time each specimen was directly used in previous studies for the qualita- exposed to the specified method. Any tive criteria (Couse & Connor, 2015; King pre- or post-treatments required by the

Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 33 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. method were not included in the time ef- aliquoted into the 0.2mL tubes and had ficiency calculation. Effectiveness and 1.5 µl of the associated sample’s ex- macroscopic damage were qualitatively tracted DNA added. The primers used in assessed after each method was com- the PCR phase targeted a 212 base pair pleted. fragment of the Sus scrofa mitochondrial Cytochrome c Oxidase subunit II gene; DNA Extraction and Quantification CO2susF2(5’GCCTAAATCTCCCCTCA After each method trial was com- ATGGTA -3’) and CO2susR2 (5’AGAAA- pleted, the macerated pig hock remains GAGGCA-AATAGATTTTCG-3’) (Lahiff were labeled, dried for a 24-hour period, et al., 2001; Pangallo et al., 2010). PCR and frozen (-20ºC) until the DNA extrac- was completed for sixty cycles with a tion process could be completed. Each 58ºC touchdown annealing temperature. treated pig hock contained fragments Two 2% agarose electrophoresis gels from two long bones, either the tibia and were run to confirm that amplified DNA fibula or the radius and ulna. Each of was present in all post-PCR reaction these bone fragments were lightly sand- product samples before any sequencing papered to remove any potential surface or quantification was done. If a clear contamination and then drilled with a 3/8” band of the expected size (in comparison brad-point drill-bit to collect 0.50g of bone with a 100bp ladder) was shown in the powder. The drill bits were thoroughly agarose gel for each of the 38 extractions rinsed and soaked in bleach for ~30 (two DNA extractions from each of the 15 minutes before being allowed to dry be- method-tested pig hocks and four from tween uses. Bone powder was collected each of the two physically macerated pig into new, sterile, labeled 1.5ml microcen- hocks), the samples from the PCR prod- trifuge tubes and sealed and stored at uct were prepared for sequencing to en- room temperature until DNA extraction sure the amplified DNA present in the was performed approximately one week samples was the targeted Sus scrofa mi- later. All surfaces were thoroughly tochondrial DNA (Silverman, 2018). cleaned with bleach between each sam- Extra care was taken with the ple (Silverman, 2018). DNA analysis, following standards com- DNA extraction was done follow- monly found in ancient DNA laboratories, ing a protocol provided with the pur- to enable the accessibility of even minute chased QIAamp DNA Micro Kit (QI- amounts of DNA. This included extra cy- AGEN, 2010) acting solely as a proxy in cles in PCR, gel analysis, and targeting place of more traditional forensic meth- mitochondrial DNA due to its high copy ods in an attempt to estimate preserva- number. Because of the potential for tion and degradation of DNA within some of the samples to yield very low treated samples. The extracted DNA in- quantities of DNA, it was necessary to cluded two samples per pig hock, one for ensure that even if the amounts were each long bone. The extracted DNA sam- very small, it would be possible to detect ples were amplified with the PCR proto- any and all potential DNA that could be col: 8.58 µl of H2O, 2.4 µl dNTP, 0.18 µl useful in forensic or other analyses. forward primer, 0.18 µl reverse primer, Of the 38 DNA extractions that 1.5 µl of 10X PCR MgCl2 Buffer, 0.45 µl were sent for sequencing at UM’s Mur- MgCl2, and 0.08 µl of platinum Taq. A to- doch Sequencing Core, 36 came back tal of 13.37 µl of the prepared mix was with DNA sequences that were then up- Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 34 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. Table 2: Description of the qualitative criteria and score values used to analyze each method. Scoring tables were modified from those used in previously published literature (Couse & Connor 2015; Lee et al., 2010; Silverman, 2015; Steadman et al., 2006).

Effectiveness of Method Macroscopic Damage Ease of Application Score Description Score Description Score Description 1 Bones were cleaned completely 1 Bones show no sign of mac- 1 Application of method is easy to follow, requires only by the method tested with no roscopic damage or altera- no past experience, and needs no supervision. presence of grease. tion.

2 Bones were mostly cleaned, may 2 Slight alterations such as a 2 Application of method is simple to follow and re- have involved some additional ef- single crack or spot of visi- quires no past experience. It may require limited fort by the researcher. Little to no ble water staining on the prior knowledge of the method or minimal grease present. cortical bone are present. amounts of supervision. 3 Some cartilage and a minimal 3 Mild alterations on bone are 3 Application of method requires some knowledge amount of grease may still be present, such as multiple of or experience with method or specific guide- present, but still mostly cleaned. cracks and visible water lines to follow. May require intermittent supervi- staining on multiple areas of sion or minimal labor. the bone. Exterior of the bone may feel dried out. 4 Cartilage remains on bone, 4 Significant alterations pre- 4 Method application requires knowledge or expe- grease may still be present, inte- sent such as large cracks, rience with the method. Requires some amount rior of bone may still have some dried out and rough exterior, of consistent supervision or moderate labor. bone marrow present. slight porosity, and visible water staining. 5 Cartilage and tissue still present 5 Severe water damage or tis- 5 Method application requires prior knowledge on bones. Material is not com- sue staining present, chip- and experience with the method and time-con- pletely cleaned. ping or severe cracking visi- suming labor and supervision before, during, or ble. Bones feel dry and after the method. rough. Increased porosity. Bone structure is compromised, and water staining spots are saturated and soft.

loaded into Sequencher 5.4.6 for editing their time efficiency in a Pearson’s R cor- and analysis. Two extractions failed to relation test to determine if weight of the produce sequence data, but other DNA specimen had an impact on the time effi- extractions from the same samples did ciency of each method. DNA concentra- sequence and the failed sequences were tion values were subjected to a Kruskal- considered to be caused by error. All the Wallis test to determine if the concentra- finalized sequences were run through tion values from the tested methods were Basic Local Alignment Search Tool significantly different from the samples (BLAST) registered to the National Cen- that only underwent manual soft tissue ter for Biotechnology Information (NCBI) removal. GenBank to search for a corresponding sample based on nucleotide matches. Results DNA concentration quantification was Initial Weight Distribution done using a Qubit® dsDNA BR Assay The weight of the 17 pig hocks Kit (ThermoFisher). Extracted DNA from ranged from 340.00g – 586.00g (x̅ = all 38 of the DNA samples were quanti- 432.88g, s = 16.84). A one-way single fied for initial DNA concentration values. factor ANOVA showed no statistically Thin walled 0.5mL PCR tubes were used significant difference in the start weights for each of the samples as well as the two of the pig hocks across the different soft standards provided with the kit. A Qubit® tissue removal methods (F(5,11) = dsDNA BR Buffer was added to each of 0.7768, p = 0.5836). When the pig hocks the tubes so that each tube contained a were calculated against their completion total of 200µL. DNA samples contained times within their assignment method 195µL of the buffer and 5µL of the ex- group in a Pearson’s R correlation test, tracted DNA and the standards each had all methods individually showed minimal 10µL of the standard and 190µL of the to no correlation between initial weight buffer. If a sample was too low to provide and completion time. a readable value, they were tested a sec- ond time with a new buffer mix to ensure DNA Yields and Sequencing it was not user error. If the sample still The extracted DNA concentra- failed to provide a readable result, they tions calculated by the Qubit are shown were tested on a later day using a high in Figure 1. Averages were calculated sensitivity assay (Qubit® dsDNA 1X HS using both the broad range and high sen- Assay Kit) to read the lower concentra- sitivity assay concentration values. All tion value (Silverman, 2018). samples produced a significantly different DNA concentration when compared Statistical Analysis with the samples that had the soft tissue The starting weights of each hock manually removed, except for the der- portion were used in a one-way single mestid samples (χ2 = 0.15, p = 0.69854). factor ANOVA statistical test sorted by This demonstrated the DNA quality was their assigned methods to ensure there not significantly impacted by the dermes- was no significant difference in weight tid method in comparison to the samples between sample groups. Additionally, the that were not exposed to a tested treat- start weights were compared against ment method, as opposed to the heated

Figure 1: Scatter graph showing the average extracted DNA concentration in μg/μL of each method tested. Averages are shown by solid black line; black circles represent individual specimen results.

maceration methods that all showed sig- produced results using the high sensitiv- nificantly lower yields (p < .05; Figure 1). ity assay. These results are summarized The bleach samples failed to pro- in the below table (Table 3). The samples duce any readable results using the with readable DNA concentration yields broad range assay and the enzyme- using a broad range assay were consid- based detergent method only produced ered to be less significantly impacted by readable results for two of the six sam- the method. No high sensitivity assay re- ples. The distilled water boil and the am- sult exceeded 0.0003 μg/μL. monia samples both produced readable Although the Qubit calculated results for most of the samples tested, concentration values for the samples var- although the concentration yields were ied, with some readings as low as 0.0001 significantly lower than that of the physi- μg/μL, all but two of the samples were cally macerated samples. In total, twelve able to be amplified and sequenced by samples failed to produce readable re- the University of Montana Genomics sults using the broad range assay, but all Core. All sequences queried 99% for

Susscrofa mitochondrial DNA, demon- failed to remove cartilage, and all long strating that even the methods that had a bones treated with this method failed to severe impact on the DNA yields, such separate from adjacent bones. The dis- as the bleach boil methods, were still tilled water boil method also had cartilage able to produce DNA extractions that remaining on all three pig hocks tested could be amplified and sequenced for at with the method, but the amounts were least mtDNA. less than that of the enzyme method, and the bones present separated from one Scored Criteria Analysis another. The dermestid specimens were The methods were all scored left with minimal amounts of remaining based on the criteria descriptions listed in tissue that was mostly located on the in- Table 2 and averaged for comparison terior of the bone. The other heated mac- (Table 4). Time efficiency is specifically eration methods (bleach and ammonia) documented in Figure 2. Even excluding were completed with little to no visible tis- preparation time, the dermestid method sue and with minimal amounts of carti- took the longest to complete, ranging lage remaining on several of the treated from three to eight days. The heated bones. Specifically, the ammonia simmer maceration methods were all completed and the bleach boil method each had one in less than a single day, with the bleach pig hock of the three retain some remain- and ammonia averaging under four hours ing remains from a majority of the tested per sample (Figure 2). On average, the methods did not have a greasy texture or distilled water boil method took an hour a lingering odor. The bleach and ammo- longer than the ammonia and bleach, nia methods both resulted in no detecta- while the enzyme-based laundry deter- ble grease, either visually or to the touch, gent simmer method took over twice as cartilage on the epiphyses. The resulting long as the bleach boil method. remains from a majority of the tested Overall, all methods performed methods did not have a greasy texture or effectively, with a majority of the tissue a lingering odor. The bleach and ammo- left behind being cartilage between epi- nia methods both resulted in no detecta- physeal plates and joints. The enzyme- ble grease, either visually or to the touch, based detergent method in particular nor any noticeable odor. The pig hocks

Table 3: Table showing how many samples from each method required the high sensitivity assay to produce readable DNA concentration yields and how many samples were able to be sequenced. Manual Dermestes Distilled Enzyme- Bleach Ammonia

Soft Tissue lardarius Water Based Deter- Boil Simmer Removal Colony Expo- Boil gent Simmer sure Readable yields 8/8 6/6 6/6 2/6 0/6 4/6 with broad range assay Readable yields N/A N/A N/A 4/4 6/6 2/2 with high sensitivity assay Sequenced 7/8 5/6 6/6 6/6 6/6 6/6

Table 4: Starting and final weight ranges and averages, the time to completion ranges and averages, the DNA concentration value ranges and averages, and the average score values of all the samples within a method for each of the criteria in Table 2.

Starting Final Time in DNA Con- Effective- Macro- Ease of Weight Weight Hours centration ness scopic Applica- (Aver- (Aver- (Aver- (Average)* Damage tion age) age) age) Der- 424- 38-51g 84.73- 2.98-6.04 3 1 5 mestes 540g (43.5g) 182.25h μg/μL (4.31 lardarius (473g) (136.92h) μg/μL) p = 0.699 Distilled 340- 45-54g 4.72- 0.46-3.04 2 4 2 Water 439g (50g) 5.27h μg/μL (1.311 Boil (396g) (5.00h) μg/μL) p = 0.020 Enzyme- 378- 58-87g 5.83- 0.00-0.66 4 5 3 Based 424g (68g) 6.75h μg/μL (0.21 Simmer (401g) (6.38h) μg/μL) p = 0.002 Bleach 358- 33-80g 2.52- 0.00-0.00 1 1 3 Boil 494g (49g) 3.57h μg/μL (0.00 (406g) (3.12h) μg/μL) p = 0.002 Ammonia 342- 31-99g 2.78- 0.00-0.54 1 2 3 Simmer 586g (73g) 4.05h μg/μL (0.33 (472g) (3.46h) μg/μL) p = 0.002 *Samples with soft tissue manually removed had DNA concentrations ranging from 0.96- 6.12 μg/μL. P-values reported with DNA concentration values are Kruskal-Wallis significance test results in comparison with the samples that were not exposed to a tested treatment.

Figure 2: Scatter graph showing time efficiency of each heated maceration method. Av- erages are shown by a solid black line; black circles represent individual specimen results.

shown in small visible water stains on the method, the minimal damage produced cortical bone that in two instances from all other treatments did not appear showed saturation and compromised to obstruct any analysis of the bone or af- bone integrity, as well as several small fect long-term storage potential. cracks on two of the three tested pig hocks. The enzyme-based detergent as Discussion well as increased porosity and noticeable Results found within this study in cracking on at least one element from a majority of cases appeared to largely each pig hock. The saturation caused a parallel that of prior research. The litera- decrease in solidity of the bone composi- ture in time efficiency, while variable due tion, which caused difficulty when drilling to the difference in sample specimen and the bones for DNA. The bone surface temperatures utilized, all largely praised was left with a dried out, sandpaper-like heated maceration methods for their texture that in some instances left visible speed and specifically found higher tem- striations along the bone. Aside from the peratures and boiling, when done care- samples treated with the enzyme-based fully with adequate supervision, to be Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 40 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. more efficient than the low or no heat al- work most effectively, without causing ternatives (King & Birch, 2015; Nawrocki, damage, and why. 1997). The current study did demonstrate While many of these results paral- the expected decrease in DNA preserva- lel that found in prior studies, there were tion in the bleach tested samples, corrob- also several differences found within this orating with other similar studies (Stead- study that either differ or directly contra- man et al., 2006), but while several stud- dict that found in prior research. Most sig- ies saw decrease in bone quality or sur- nificantly, the enzyme-based detergent face alteration in samples with bleach, method tested within this study appeared the bleach-tested hocks within this study to cause serious degradation to the DNA, showed no bone alteration or surface tex- a point directly in opposition with prior at- ture changes and macroscopically ap- tempts at this method (Lee et al., 2010; peared to be in excellent condition. This Steadman et al., 2006). Additionally, the could be due in part to other studies test- pig hocks exposed to the enzyme-deter- ing the bleach solution at lower tempera- gent method were among the most mac- tures, consequently causing the ele- roscopically damaged and the bone qual- ments to have a much longer exposure ity was significantly compromised, a fac- time, versus the boiling temperature used tor not noted in any prior published us- within the present study where samples ages. While the enzyme-based method is were exposed to the bleach solution for a one that has been praised in past docu- maximum of under four hours (King & mented uses (Lee et al., 2010; Simonsen Birch, 2015; Steadman et al., 2006). et al., 2011), this research did not see the The ammonia simmer method, same level of successful results, with the while occasionally found in zoological re- method taking longer, proving less effec- search or as a degreaser after a primary tive, and causing more damage both method (Fenton et al., 2003; Lee et al., macroscopically and to the DNA quality 2010; Steadman et al., 2006), is not one than previously reported. In previous that could be located in published foren- publications utilizing this method, it has sic research and therefore the results been mentioned that a protease and li- could not be compared. The promising pase in the correct concentration and ra- performance by the ammonia within this tio are the most effective, however, laun- study opens opportunity for a new heated dry detergents do not always specify the maceration method and could benefit amount or specific enzymes included from additional research and data collec- (Uhre et al., 2015). The inability to clearly tion through repetitions of the method de- compare the detergents makes it difficult scribed in this study, as well as other var- to conclude if the failure of the method is iations with temperature and amount of due to a difference in specific enzyme ra- additive. tios present within the detergent selec- Overall, each method tested tions or the temperature reaching too within this study was able to perform the high of a degree for proper enzyme acti- task of skeletonizing the Sus scrofa limbs vation. The poor results of the method efficiently in a relatively short amount of within this study call for a reevaluation of time, but each method has advantages the detergent-based methods within the and disadvantages associated with it. field to identify what brands of detergent The bleach boil method performed

Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 41 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. quickly and efficiently but did significantly The process of soft tissue removal affect the DNA quality, and in a situation is one that varies significantly, with even where that is a concern, this method the same additive having documented should be removed from consideration. usages of very different concentrations The dermestids and ammonia simmer (King & Birch, 2015; Steadman et al., method both performed well with little 2006). There is a need to consider the damage or added concern, but with the long-term effects on the bone and dermestids taking days opposed to the whether the potential risks are worth the hours of a heated maceration method. potential benefits. While one should al- While the distilled water boil method did ways seek to limit any damage to skeletal cause more morphoscopic damage than material, all methods will have some last- several of the other methods, the integrity ing impact on the end result and should of the bone largely remained intact. The be considered as a method is selected. time efficiency and cleanliness of each method did vary moderately, but all meth- Conclusion ods could be a viable selection in most This study highlights the time ex- general situations. While all the methods, pectations, effectiveness, and DNA ex- even the bleach boil, were able to be se- traction quality of several of the more quenced for DNA, it should be kept in common methods utilized within current research as well as additionally testing a mind that the DNA analysis methods less common method of a sole ammonia used were those common in ancient DNA simmer. This study aims to be the begin- laboratories in anticipation of the reduced ning of a roadmap for choosing a method DNA quantity. The low DNA yields of the that best suits a researcher’s needs, ex- bleach boil still indicate the method to not pectations, and desires of the final prod- be appropriate in the event DNA will be uct, while introducing the ammonia sim- required from the remains after the mer method to a forensic context and method is completed. Additionally, while highlighting previously unnoted issues the enzyme-based detergent method with enzyme-based detergent macera- samples similarly were all able to be se- tion. Beyond forensics, the awareness quenced, the surface damage and satu- and consideration of the long-term ef- ration to the bone that the method fects soft tissue removal methods may caused made cutting the bone for DNA have on skeletal material is relevant across any field performing skeletoniza- very difficult and caused even further tion or storing the resulting remains. A damage to the element. This, along with majority of methods in some way have the low average DNA yield of the tested been shown to violate the skeletal prepa- samples, suggest that the enzyme-based ration standards set forth within the field, detergent method as it was performed whether they deteriorate the surface, al- here is also not a preferable method ter the material, or render the bone unfit choice when DNA viability is a factor for DNA analysis or long-term storage. needing to be considered. Figure 3 out- Similar to former studies, these results lines a basic method selection flowchart demonstrate that no single method can based on the results within this study. successfully satisfy all the requirements of an ideal skeletonization method (King Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 42 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H.

Figure 3. Method selection flowchart based on the results presented in this study.

& Birch, 2015; Scientific Working Group of decomposition and environmental pro- for Forensic Anthropology, 2011). While cesses, such as scavenging and bug ac- the method that is likely to do the least tivity. While the concerns raised in this re- amount of harm to both the skeletal ma- cent research have significant implica- terial and DNA preservation should al- tions for post-mortem interval studies, ways be the chosen method, the priori- early stages of decomposition and skele- ties of what constitutes the least amount tal composition have still been said to be of harm may vary based on the desired homogenous enough for research pur- results and intended future of the re- poses (Connor et al., 2017; Knobel et al., mains. 2018). Results and comparisons detailed With only three repetitions of each here are greatly limited by the small sam- method as done in the present study, ple size and the usage of pig hocks as a trends or patterns within the data may be human proxy. While there has been re- obstructed or overlooked. Furthermore, cent research showing dissimilarities in there are many methods of soft tissue re- decomposition processes between pig moval available and this study examined and human remains, a large bulk of this only five methods with only one concen- research has focused on the later stages tration of each additive. Different results Tarbet Hust, E.S. and M. Snow (2021) The Effects of Soft Tissue Removal Methods on Porcine Skeletal Remains: A Comparative Analysis. New Florida Journal of Anthropology 1(2), 30-46 DOI 10.32473/nfja.v1i2.124117 43 February 2021 New Florida Journal of Anthropology Tarbet Hust, E.S. and Snow, M.H. for additives could be produced by using ency and replicability. In particular, the different concentration levels, tempera- poor performance of enzyme-based de- tures, or different skeletal elements. The tergent within this study warrants further enzyme-based detergent method tested evaluation of detergents at different con- in this study did not perform to the same centrations and temperatures to further quality as other previously published us- examine the differences in detergent per- ages and may warrant a reevaluation in formance. The use of household ammo- the field. While better results may be nia, a new method in the forensic context, found in other variations of detergent is recommended to be further investi- (Ajayi et al., 2016; Lee et al., 2010; Si- gated at varying levels of concentration monsen et al., 2011) or by directly adding and temperature to document additional enzymes to the water solution (King & effects of the method. Additionally, the Birch, 2015; Uhre et al., 2015), further ex- DNA extraction method utilized in this amination of different detergents, con- study was used as a proxy to estimate centrations, and temperatures are preservation and degradation of DNA needed to identify the limitations of this within treated samples, more traditional method. forensic methods of DNA extraction Future research is recommended should be tested in order to better deter- with additional methods, additives, con- mine the viability of the methods in a fo- centrations, and criteria for further data rensic context. collection, as well as repetitions of the methods tested here to ensure consist-

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