AN EVALUATION OF PREPREPARATION STORAGE OF FROZEN ZOOLOGICAL SPECIMENS Open Access

Natural history collections represent invaluable sources of knowledge about the natural world (Suarez and Tsutsui 2004). They provide a physical record of the biodiversity unique to certain regions at varying times through history and represent an important aspect of a nation’s natural heritage (MacDonald and Ashby 2011, Nelson and Ellis 2019). Each specimen in a natural history collection is unique and important, and some are irreplaceable, such as those of rare, endangered, or extinct species (Albano et al. 2014). Therefore, any damage to a specimen that restricts the potential for research or education is a significant loss.

Vertebrate specimens that are collected or salvaged from the wild are often prepared as study skins or skeletons soon after acquisition (Winker 2000). This ensures fast and adequate preservation, which is especially important when collecting in remote areas where refrigeration facilities are inaccessible or limited. When a specimen cannot be prepared immediately and a freezer is available, freezing is the most common preservation method used for short-term preservation (Winker 2000). Specimens collected from the wild are generally placed in plastic bags in a cooler with ice and upon return from the field, should be placed in a freezer set at −20°C or colder (Hendry 1999). At this temperature or lower, it is possible to store an unprepared specimen for a matter of years with very little damage that would limit choice of method of preparation (Winker 2000).

The disadvantage of this preservation method, however, is that specimens are vulnerable to desiccation during extended periods of storage in a freezer (Martínez-Vargas et al. 2021). This drying occurs via the sublimation of water, which is drawn from the tissues, condenses in the air trapped within the bag, and refreezes as ice (Winker 2000). The sublimation front moves from the outside of the animal to the inside, affecting the tissues at the body surface first (Florian 1990). In particular, the head and limbs are most severely affected first (Winker 2000), which causes these parts to be the most difficult parts to prepare (Martínez-Vargas et al. 2021). This dehydrating effect leaves the skin of the specimen dry and brittle, making proper skin preparation increasingly difficult the longer a specimen is stored in a freezer (Martínez-Vargas et al. 2021). Increased time in a freezer could also have a negative effect on anatomical measurements because dried specimens shrink (Winker 1993).

Few studies have looked at the best way to preserve frozen zoological specimens. Winker (2000) recommended exposing the specimen to the least amount of air possible in the storage bag, and reducing freezer storage time and temperature fluctuations in order to minimize dehydration. One study found that birds that have undergone a necropsy or are damaged (e.g., roadkill), then stored in the freezer have less tear-resistant skin, making them more difficult to prepare (Martínez-Vargas et al. 2021). Larger specimens (body mass of 160 g or more) have smaller surface-area-to-volume ratios and thus are less susceptible to dehydration than smaller specimens (Shoffner and Brittingham 2013), which allows them to be stored effectively in freezers for longer times. This places less urgency on preparing larger, more time-consuming species, and allows for more strategic use of time in preparing smaller or damaged specimens.

Specimens that have become too dry to be prepared as skins are typically prepared as skeletons if they still represent an important addition to the collection. This eliminates the difficulty of removing fragile skin in one piece, but presents several new problems associated with long-term storage in a frozen state. Storage of zoological specimens in freezers over long periods can cause marrow in the bones to harden, making it much more difficult to remove, which could result in greater quantities of material being left in the bones (Martínez-Vargas et al. 2021). Improperly cleaned bones attract pests, which are drawn to the lipids left in bones (MacDonald 2006). These lipids begin to oxidize when specimens are stored above −70°C (Laitinen et al. 2006), resulting in various secondary reactions and future biochemical instability of the bones (Goodridge et al. 2003) if all the lipids are not removed during preparation. To facilitate efficient and effective preparation of zoological specimens for osteology collections and ensure stable bone chemistry, specimens chosen for this purpose should be stored at temperatures below −70°C (Mularchuk and Boskey 1990, Laitinen et al. 2006). Unfortunately, most natural history museums cannot afford this type of equipment, highlighting the importance of identifying less expensive variables (i.e., wrapping) that increase preservation of frozen specimens.

Our objective in this study was to determine the effects of air exposure, storage duration, and freezer environmental conditions on the amount of drying of frozen zoological specimens. Winker (2000) suggests using vacuum-sealed bags to store frozen zoological specimens, and we predict specimens wrapped in vacuum-sealed bags will be the least desiccated because the air is pumped out of the bag just prior to sealing. We further predict that specimens that are not wrapped will lose the most amount of mass because there is no barrier to sublimated water leaving the specimen.

Accessing a freezer (opening the door) allows cold air to flow out and warm air to flow in the freezer. This will disrupt the stable temperatures that existed in the freezer prior to the door opening and will cause temperatures to fluctuate greatly if the door is opened repeatedly. Therefore, we predict that increased access (door openings) to the freezer will cause fluctuations in temperature that will increase desiccation of the specimens. Maintaining high relative humidity (>95%) in a freezer is suggested to keep zoological material stable while it is stored in a frozen state (Cano-Muñoz 1991). This is because under dry ambient conditions, moist air from the specimen will diffuse towards the lower humidity of the freezer. High humidity in the freezer will reduce the humidity gradient between the freezer environment and the specimen and will reduce diffusion of humidity away from the specimen. We predict specimens stored in freezers with higher relative humidity will have less mass lost because of desiccation.

We also tested whether wrapping a paper towel around the specimen prior to freezing contributes to dehydration of the skin. Zoological specimens collected by ornithology staff at the Royal Alberta Museum are tagged, wrapped in paper towel, placed in a bag, and set on a block of dry ice in a cooler. We suspect that placing an absorbent material around a specimen will exacerbate water loss from the tissues by absorbing and holding moisture sublimated from the specimen. The paper towel is used to prevent biological fluids from the specimen staining feathers or fur, because washing and drying a specimen during skin preparation is time consuming (Winker 2000). Any benefits gained from keeping fluids off the plumage or pelage may be outweighed by the difficulties caused by increased desiccation of the specimen.

We predict specimens stored in the coldest freezer with the highest relative humidity, fewest door openings, and wrapped in vacuum-sealed bags without paper towels will lose the least amount of mass in the coldest freezer.

In 2015, we used 125 plucked chicken wings to test the efficacy of five methods of wrapping (Fig. 1) and five freezers (Table 1) on preventing desiccation of zoological specimens during storage. This experiment took place from June 2015 to February 2016 at the Royal Alberta Museum in Edmonton, Alberta, Canada. Chicken wings, composed of the entire pectoral limb bones and associated musculature of the domestic chicken (Gallus gallus domesticus), were selected as suitable analogues for whole zoological specimens because of their size and availability. Samples were acquired unfrozen from a local supermarket and frozen on the day acquired.

Five chicken wings (hereafter “specimens”) were randomly selected for each treatment group for each freezer. The treatments were as follows: 1) specimens were not wrapped prior to freezing and were fully exposed to the freezer’s ambient conditions, 2) specimens were placed in a 2-mil plastic bag with no paper towel wrapping and sealed with a twist tie, 3) same treatment as 2), but the specimens were wrapped in paper towel before being placed in the plastic bag and sealed with a twist tie, 4) specimens were vacuum sealed in 4-mil bags, with no paper towel wrapping, 5) same treatment as 4), but specimens were wrapped in paper towel before vacuum sealing. We used Cabela’s Commercial Grade Vacuum Sealer (Model 54-1102) and cut bags from a roll of Cabela’s 4-mil vacuum-bag roll. As much air as possible was removed from all wrapped specimens prior to sealing.

Samples of each treatment were divided equally amongst five cardboard storage boxes, each of which had been divided into equal sections with borders to reduce air circulation between compartments. The boxes were each placed into different freezers (labeled A–E) to test for the presence of desiccation at different temperature, relative humidity, and door opening frequency and duration. For this experiment we utilized two freezers generally used for long-term storage of zoological tissue samples (Freezers A and B). We also utilized a walk-in freezer (Freezer C) and a chest freezer (Freezer D) that had commonly been used to store whole zoological specimens awaiting preparation. In addition to this, we used a freezer above a fridge (Freezer E), which is a typical freezer to which the public has access and was the only frost-free freezer used in our experiment. Temperature (Accutemp indoor/outdoor thermometer) and humidity (Accutemp Humidiguide hygrometer) levels were measured weekly in each freezer and recorded. A trail camera (Bushnell Trophy Cam HD) was setup to capture the frequency and duration of door openings over a 3-week period. These data were then used to calculate the average daily door opening frequency and average time the door was open each day.

The mass of each specimen was recorded prior to wrapping and freezing. All of the unwrapped samples were weighed each week after freezing for 6 weeks, then once per month until the end of the experiment. Percent mass lost between each measurement period was calculated for each unwrapped specimen. All samples were removed from wrappings (bags and paper towels), weighed and photographed 240 days (8 months) after the experiment began and percent mass lost was calculated for each. Extreme care was taken to ensure specimens did not begin to thaw when they were removed from the freezer to be weighed. All masses were measured with a Sartorius bench scale (model E12000S).

A mixed-effect linear regression with treatment group as the random variable was used to examine the influence of temperature, humidity, frequency of freezer openings, duration of freezer openings, type of plastic wrapping (twist-tied 2-mil bag versus vacuum-sealed bag) and paper towels (presence/absence) on the percent mass lost from all specimens (StataCorp 2009). Graphical examination of data prior to analysis found that temperature was nonnormal and was therefore log transformed. Type and presence of wrapping and presence of paper towel were modeled as categorical variables.

Separately, we analyzed the unwrapped specimens with a mixed-effect linear regression model with treatment group as the random variable (StataCorp 2009). We examined the unwrapped specimens separately because desiccation of “open” (necropsied) specimens is greater than “sealed” (nonnecropsied) specimens (Martínez-Vargas et al. 2021). We looked at the influence of temperature, humidity, and frequency of freezer openings on the percent mass lost from unwrapped specimens. The average duration of each freezer door opening each day was highly correlated with the average daily door opening frequency (R = 0.99), so the former was removed from the analysis to avoid collinearity effects.

We calculated the average mass lost from the unwrapped specimens from each freezer for each of the 12 measurements. This gives the ability to see amount of mass lost over time. The cumulative mass lost at each measurement was then calculated for each freezer. The average cumulative mass lost from unwrapped specimens over the duration of the experiment was analyzed with a nonlinear least-squares regression with a logistic function (StataCorp 2009).

We found that specimens that were wrapped or stored in the coldest freezer lost significantly less mass (Table 2). None of the other variables examined had a significant effect on the amount of mass lost over the 8 months (Table 1). Unwrapped specimens (X = 19.50; SE = 2.87) lost six times more mass than wrapped specimens (X = 3.12; SE = 1.30; Fig. 2).

When we examined only the unwrapped specimens, percent mass lost decreased as temperature decreased (β = −16.1, SE = 0.60, P ≤ 0.01), relative humidity increased (β = −0.12, SE = 0.05, P = 0.03), and door opening frequency decreased (β = 2.14, SE = 0.28, P ≤ 0.01). Our model shows that over one quarter of the mass of unwrapped specimens is lost over an 8-month period when freezer temperature is greater than −15°C (Fig. 3). The relative humidity in the freezers examined was between 60% and 80% (Table 1). Although statistically significant, there was only a 2.5% difference in mass between the specimens in the freezer with the lowest humidity versus the one with the highest humidity. Two percent more mass was lost from the unwrapped chicken wings in 8 months for every increase of one average freezer door opening per day (Fig. 4).

The five nonlinear least-squares regression models (one for each freezer) with logistic functions looking at the relationship between the cumulative mass lost from unwrapped specimens and number of days fit the data well (adjusted R² > 0.98). Unwrapped specimens in freezers −40°C and colder lost around two percent of their mass in the first week, whereas specimens in the three warmer freezers lost around six percent during the first week (Fig. 5). The unwrapped specimens in the coldest freezer (A) lost 1.8% of their mass in the first week, but then remained relatively stable for the duration of the experiment, not losing more than 2.0% over the 8 months (Fig. 5).

Zoological specimens awaiting preparation in museums and taxidermist workshops are stored in freezers, often for long periods (Cambell and Baars 2019, Martínez-Vargas et al. 2021). The tissues from these specimens dry out over time, which can lead to increased difficulty in preparing the specimen as a study skin or mount (Martínez-Vargas et al. 2021). This experiment aimed to identify factors that reduce the rate of desiccation of zoological specimens stored in freezers. Although the duration of our experiment was only 8 months and many zoological specimens are often stored for years in freezers, our results identify the factors that would have the greatest effect on specimen desiccation over longer periods of time.

Specimens wrapped in plastic lost less mass through the duration of the experiment. Vacuum-sealed bags may be preferred over conventional bags because vacuum-sealed bags contain minimal air, allowing less room for sublimated water to move away from the skin of the specimen. Counter to our predictions, however, there was no significant difference between storing the specimens in a 2-mil plastic bag with a twist tie and a vacuum-sealed bag (Table 2). Although Winker (2000) suggests storing zoological specimens in a vacuum-sealed bag, we found that this may not be necessary, because the same amount of desiccation occurred when the specimens were stored in either bag type. However, extending the duration of this experiment might have yielded a noticeable advantage to vacuum-sealed bags over time.

Colder freezer conditions resulted in reduced loss of mass. We were surprised to see almost no mass lost from the unwrapped specimens stored in the −70°C freezer after the first month (Fig. 5). If available, storing vertebrate zoological material in a freezer that is −70°C or colder is recommended. Even a decrease of 12°C in a freezer can double the storage life of animal carcasses (Cano-Muñoz 1991). This will allow for longer storage times before preparation, but freezers that are capable of these temperatures are expensive to maintain, regardless of whether they are mechanical or liquid-nitrogen–based freezers. If a freezer this cold is available, priority should go to damaged or small specimens, or those with unknown histories, because they are most susceptible to drying in warmer freezers (Martínez-Vargas et al. 2021).

The unwrapped specimens in the coldest freezers lost the least amount of mass during the first week of the experiment. This likely occurred because the temperature of the specimen dropped quicker, allowing less drying to occur (Herren 2012). The unwrapped specimens in all freezers except the coldest one continued to lose mass throughout the experiment. Unless specimens are kept in a freezer with a temperature of −70°C or colder, frozen specimens will continue to degrade over time.

Contrary to our predictions, wrapping paper towels around specimens did not significantly affect mass lost. The Royal Alberta Museum will continue with the practice in the field of wrapping specimens in paper toweling before putting them in a bag to prevent punctures from claws, beaks, and teeth. Once back in the laboratory the paper towel will be replaced with plastic film, as suggested by Martínez-Vargas et al. (2021). Maintaining the integrity of the wrapping around the specimens is important because holes will allow sublimated moisture to leave the bag and result in increased drying. Wrapping specimens also helps control temperature fluctuations, another key factor that needs to be controlled to minimize desiccation of specimens (Winker 2000).

Our predictions that more mass would be lost from specimens stored in frequently opened freezers with lower relative humidity was supported. The number of times the freezer door was opened had a significant effect on the amount of mass lost from unwrapped specimens. Opening and closing the door warms the air in the freezer and circulates this warm air through changes in air pressure, exposing specimens to temperature fluctuations. This increases desiccation of zoological specimens but can be minimized by further storing specimens in a sealed container such as a cooler (Winker 2000). Although variation in relative humidity only explained 2.5% of the mass lost from unwrapped specimens in our experiment, our results support the suggestion that a high-humidity (>90%) environment is recommended to reduce desiccation (Cano-Muñoz 1991).

If this experiment were conducted again, another variable to be tested would be the presence of feathers or fur to represent the storage of a whole vertebrate specimen better. Had we included this variable, we suspect a greater mass would have been lost from all treatments because the air trapped between the feathers or fur and skin would increase the volume of air available for sublimated water away from the skin. Presence of feathers or fur may have also prevented some moisture lost from the unwrapped specimens.

Another area of future study is testing whole zoological specimens stored in solid blocks of ice to see if this extends the amount of time a specimen can be stored effectively in a freezer. Water should sublimate from the outside of the block, but frozen water immediately adjacent to the skin should prevent sublimation from the specimen and thus prevent the skin from drying. Although this would likely increase the amount of time a specimen can safely be stored in a freezer, it would also increase the amount of space a specimen occupies in the freezer, increasing the amount of freezer space needed. Freezing specimens in blocks of ice would increase the amount of time it takes to prepare a skin from the specimen because it would need to be washed and dried as part of the preparation process.

Our results indicate that zoological specimens should be stored in the coldest freezer available with high relative humidity, for the shortest period possible, wrapped in an impermeable membrane (i.e., plastic) that is further isolated (e.g., in a cooler) from temperature fluctuations caused by the freezer door opening. These are the same recommendations made by Cano-Muñoz (1991) and Winker (2000), but our evidence also suggests a vacuum-sealed bag may not be necessary and the number of times the freezer door is opened should be minimized. Additional improvements to storing frozen specimens include prioritizing specimens with high surface area to volume ratios and specimens with significantly damaged skin (e.g., roadkill, necropsied, etc.; Martínez-Vargas 2021). These results identify effective techniques for storing frozen zoological specimens, which will help ensure specimens can be best prepared when resources are available.