Freeze-branding to permanentlymark bats

Richard E. Sherwin, Shauna Haymond, Dave Strick lan,and Rebeccah Olsen

Abstract We tested the effectiveness of freeze-branding as a permanent marking technique on 4species of bats: Brazilian free-tailed bat (Tadarida brasiliensis), Townsend's big-eared bat(Corynorhinus townsendil), big brown bat (Eptesicus fuscus), and western small-footedmyotis (Myotis ciliolabrum). Small copper branding irons (9.3 g and 15.6 g) were cooledin a mixture of dry ice and ethyl alcohol and applied to the dorsal skin for varying lengthsof time (range=1.5-7.0 seconds). Branding was successful for all species when appliedwith brand temperatures ranging from -56.70 to -67.7°C. Branded areas grew white hairin an average of 36 days (range=22-60 days). Branded bats were identified easily whileroosting, thus eliminating the need to handle them to identify individuals. No adverseeffects were observed during or after branding. Results of this study indicate that freeze-branding is a safe and effective method to permanently mark bats.

Key words Corynorhinus townsendii/ Eptesicus fuscus/ marking techniques, Myotis ciliolabrum/Tadaridabrasiliensis,wildlifemarking

Studies investigating movement patterns, popula-tion dynamics, or behaviors of animals require indi-viduals or groups of individuals to be marked sothat researchers can identify them over time (Niet-feld et al. 1994). Such markings should be readilyidentifiable from a distance, thus eliminating stressof recapture (Nietfeld et al. 1994). For bats, themost common method of marking is attachment ofaluminum or plastic wing bands to the forearm ofthe animal. This method has provided invaluableinformation on many aspects of the natural historyof bats (Davis 1966, Baker 1978,Tuttle and Steven-son 1982). However, chaffIng, inflammation, andovergrowth of flesh are common injuries associat-ed with wing bands (Phillips 1985). High infectionrates and mortality have been described inTownsend's big-eared bats (Corynorhinustownsendit) as a direct result of wing banding

(pierson and Fellers 1994), and wing bands mayadversely affect foraging success of banded individ-uals (Norman et al. 1999).

Another semi-permanent marking technique forbats is punch-mark tattooing, which creates smallholes in the wing membrane in the form of num-bers for individual identification (Bonaccorso and

Smythe 1972). This technique is easy to perform,very legible, and safe for the aiilinal. However, themark becomes illegible after 5 months, with notrace remaining after 6 months, and the animalmust be recaptured to be identified (Bonacccorsoet al. 1976).

Freeze-branding is a common technique to mark

large mammals, particularly livestock (Farrell et al.1978, Newton 1978), and has been used successful-ly on small mammals such as house mice (Musmusculus) and hooded rats (Rattus norvegicus,

Address for Richard E. Sherwin and Shauna Haymond: Department of Zoology, Brigham Young University, Provo, UT 84602, USA;

present address for Sherwin: Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA; e-mail: rsherwin@unm.edu; present address for Haymond: Holistic Wildlife Services Biological Consulting, Rio Rancho, NM, 87124, USA.Address for Dave Stricklan: USDA Forest Service, Rocky Mountain Research Station, Shrub Sciences Lab, Provo, UT 84606, USA;present address: Department of Biology, Brigham Young University - Idaho, Rexburg, ID 83460, USA. Address for RebeccahOlsen: 30 E 350 N, Orem, UT 84057, USA; present address: 922 N Maura Place, Meridian, ID 83642, USA.

Lazarus and Rowe 1975), squirrels (Sciurus spp.,Hadow 1972), brown lemurs (Lemur fulvus, Milleret al. 1983), and dwarf mongoose (Helogale parvu-la, Rood and Nellis 1980). Barclay and Bell (1988)indicated the potential of freeze-branding to per-manently mark bats, but it had not been tested. Themark is achieved by applying a super-cooled brand-ing tool to the skin of the animal, or by spraying arefrigerant (e.g., Freon 12, dichlorodifluoro-methane) directly onto the skin, which destroys themelanocytes in the hair follicles. Subsequent hairgrowth in the branded area is permanently coloredwhite.

Our objective was to determine whether freeze-branding was an effective and safe marking tech-nique for temperate species of bats and to recom-mend a standardized marking technique.

MethodsWe freeze-branded bats of 2 families and 4

species: 3 Vespertilionids (Townsend's big-earedbat, big brown bat [Eptesicus fuscus], and westernsmall-footed myotis [Myotis ciliolabrum]) and oneMolossid (Brazilian free-tailed bat [Tadarida

brasiliensis]). We captured individuals during fall1996 using standard trapping techniques describedby Kunz and Kurta (1988). We removed animals

from a residential building (which was subsequent-ly demolished) in Utah County, Utah, USA, exceptfor all C. townsendii, which we captured at a nightroost in Juab County, Utah, USA.

We maintained bats in captivity for up to onemonth prior to branding to ensure that individualswere adapted to captivity (e.g., eating well, main-taining weight, active) and for up to 6 months fol-lowing branding to monitor possible adverseeffects resulting from the marking process (e.g.,behavioral changes, scabbing, infections). Animalswere maintained in soft-sided cages (Barnard 1995)

by a licensed bat rehabilitator (R. Olsen) followingguidelines set forth by Wilson (1988) and Barnard(1995). Capture and handling methods followedprotocol approved by the Animal Care and UseCommittee (Rogers 95-1207), Department of Zool-ogy, Brigham Young University, Provo, Utah, USA,under licenses obtained from the Utah Division of

Wildlife Resources. We initially identified individu-als with celluloid forearm bands (A. C. Hughes Ltd.,Hampton Hill, Middlesex, United Kingdom). Care

was taken to ensure that bats sustained no injuriesduring banding.

We used 2 branding irons for freeze-marking. Thefirst was 9.3 g with a 5.0-mm-diameter surface thatproduced a circular brand. The second was 15.6 gwith a 1.0-mm x 3.0-mm surface that produced alinear brand. We constructed brands using woodendowels (ca. 100 mm long) with a copper washeraffixed to one end. We cooled the brands in a mix-ture of dry ice (frozen carbon dioxide) and 95%ethyl alcohol (1.0 g dry ice/1.3 ml ethyl alcohol) totemperatures between -57.2 and -67.7° C. Weselected dry ice and ethyl alcohol because theywere relatively easy to acquire, can be used safelyunder field conditions, and were reasonably inex-pensive. We placed the mixture in a glass dish tohelp maintain temperature. Prior to branding, weclipped the dorsal fur to a short length and mois-tened the area with alcohol to ensure adequatecontact between brand and skin (Farrell and John-ston 1973). We applied brands for 1.5-7.0 secondswithout pressure beyond their own mass. Webranded all bats on the dorsal side between scapu-lae as this area provided the largest and most uni-form branding surface, is corpulent, and is easilyobserved from a distance during roosting. Werecorded temperature of solution (OC), mass ofbrand (g), time of application (seconds), and behav-ioral responses of bats during and immediately fol-lowing branding.

We considered branding to be immediate1x effec-tive if the branded skin appeared frozen (stiff andwhite) following application of the brand. Anunsuccessful brand would suggest too brief anapplication time, whereas scabbing or baldnesswould indicate that the brand was applied for toolong (Fourie and Perrin 1986). Ultimately,we basedsuccess of branding on regrowth of white hair onthe brand site. Marked animals were released inlate spring 1997 at point of capture and oppor-tunistically monitored in the field.

Results arid discussionWe branded 23 bats (10 Brazilian free-tailed bats,

6 Townsend's big-eared bats, 5 big brown bats, and2 western small-footed myotis). Branding appearedimmediately effective on 9 of the animals (skinappeared frozen immediately following applicationof brand) and was ultimately successful on all indi-viduals (Table 1). Some discomfort was evidentcoincident to the application of the brand, but nolasting behavioral changes were observed followingbranding or subsequent hair regrowth.

I

Table 1. Samplesize, mass of branding iron (g), temperature of solution (OC), time of brandapplication (see), and days until white hair growth of freeze-branded bats.

4.3

4.35.04.03.83.5

teria" (i.e., the mark is per-manent, it is legible at adistance, equipment isinexpensive [costs aver-

aged $0.28/bat], the pro-cedure is humane [thecold .temperature of thebrand solution acts as a

local anesthetic, Farrelland Johnston 1973], it is a

relatively quick markingprocess, and it is easy touse [Rice and Kalk1996]).

We suggest that thesame technique may be appropriate for all bat

species with dark pelage. Branding is performedmost easily with 2 people, one to restrain the ani-

mal and one to apply the brand. A small brandingsurface is preferred, to ensure better contact withthe skin, yielding a more legible brand with fewerbroken lines.

While the results of this study are promising, sev-eral considerations must be made if this method isto be used. Symbols or brand patterns must be eas-ily distinguishable (see Farrell 1969) and be coordi-

nated with other researchers using this method:Brands should be made as inconspicuous as possi-ble while still remaining visible to the researcher,thus reducing increased risk of predation due toalteration of cryptic coloration. The necessity ofclipping the fur dictates that animals not be marked

during or immediately prior to periods of hiberna-tion. This technique should not be used on bats

that are federally (USA) threatened or endangereduntil long-term effects of branding are known.

. Freeze-branding bats is most effective for long-termstudies as it can take up to 2 months for the brandto appear.

Time of Days until whiteapplication (see) hair growth

x SE x SE

0.70.70.61.00.50.7

24.325.032.826.053.435.4

1.3

1.5

4.4

2.0

4.7

1.1

Mean time of white hair regrowth was 36 days(range 22-60 days) for all species combined (Table

1). Branding was successful within the range ofparameters examined (Table 1).

We observed 4 Townsend's big-eared bats inroosts 3 years following branding, and markingsremained distinct. We propose that lack of detec-tion of other individuals reflected the elimination

of their roost subsequent to capture and did notindicate negative impacts of branding. This tech-nique leaves permanent marks on other groups ofmammals; hence, based on our limited observa-tions, we are confident that freeze-branded bats willmaintain markings during their lifetime.

Freeze-branded animals were identified easilyafter branding and white hair regrowth (Figure 1).We did not detect any persistent adverse responsesduring or following branding. This marking methodappears to satisfy many of the "perfect marking cri-

Figure 1. Big brown bat following freeze-branding and whitehair regrowth. The branding iron used was 9.3 g, with a 5.0-mm-diameter copper washer as the branding surface. Photo byE. Linder.

Acknowledgments. We thank the Department ofZoology, Brigham Young University, and UnitedStates Department of Agriculture Forest Service,Rocky Mountain Research Station for support. P.M.Cryan and W L. Gannon reviewed an earlier draft of

the manuscript, D. S. Rogers provided logistical sup-port, and T. Maher provided laboratory assistance;

We also thank the assigned reviewers for providingconstructive comments on the manuscript. R.Olsen maintained bats in captivity under Utah Divi-sion of Wildlife Resources license number4REHE02356.

Mass of Temp. of

brandingsolution (°C)

Species n iron (g) x SE

Corynorhinustownsendii 3 9.3 -64.0 1.8C. townsendii 3 15.6 -65.9 1.8

Eptesicus fuscus 5 9.3 -61.3 1.1

Myotis ciliolabrum 2 15.6 -62.2 0.0

Tadarida brasiliensis 5 9.3 -62.5 1.4T. brasiliensis 5 15.6 -58.3 0.0

literature citedBAKER,R. R. 1978. The evolutionary ecology of migration.

Holmes and Meier, New York, New York, USA.

BARCLAY,R. M. R., ANDG. P BELL. 1988. Marking and observa-

tional teclUliques. Pages 59-76 in T. H. Kunz, editor. Ecolog-ical and behavioral methods for the study of bats. .Smithson-

ian Institution, Washington, D.C., USA.BARNARD,S. 1995. Bats in captivity. Wild Ones Animal Books,

Springville, California, USA.BONACCORSO,E ]., ANDN. SMYTHE.1972. Punch-marking bats: an

alternative to banding. Journal of Mammalogy 53: 389-390.

BONACCORSO,E ]., N. SMYTHE,AND S. R. HYMPHREY. 1976.

Inlproved techniques for marking bats. Journal of Mammal-ogy 57: 181-182.

DAVIS,R. 1966. Homing performance and homing ability in bats.

Ecological Monographs 36: 201-237.FARRELL,R. K. 1969. New developments in animal identification.

Washington State University Animal Health Notes 8: 5-10.FARRELL,R. K., R. 1. HOSTELTER,AND]. B. JOHNSON. 1978. Freeze

marking farm animals. Pacific Northwest Bulletin 173: 1-8.FARRELL,R. K., ANDS. D. JOHNSTON. 1973. Identification of labo-

ratory aninlals: freeze marking. Laboratory Animal Science23: 107-110.

FOURIE,1.]., ANDM. R. PERIN. 1986. Effective methods of trap-

ping and marking rock hyrax. South African Journal ofWildlife Research 16: 58-61.

HADow, H. 1972. Freeze-branding: A permanent marking tech-

nique for pigmented manlffials. Journal of Wildlife Manage-ment 36:645-649.

KUNZ,T. H., ANDA. KURTA. 1988. Capture methods and holding

devices. Pages 1-29 in T. H. Kunz, editor. Ecological andbehavioral methods for the study of bats. Smithsonian Insti-

tution, Washington, D.C., USA.LAzARus,A. B., ANDE P ROWE. 1975. Freeze-marking rodents

with a pressurized refrigerant. Mammal Review 5: 31-34.MILLER,D. S.,]. BERGLAND,ANDM. JAY. 1983. Freeze-mark tech-

niques applied to mammals at the Santa Barbara Zoo. Zoo

Biology 2: 143-148.NEWTON,D. 1978. Freeze branding. Pages 142-144 in B. Stone-

house, editor. Animal marking. University Park, Baltimore,

Maryland, USA and MacMillan, London, United Kingdom.NIETFELD,M. T., M. W BARRETT,ANDN. SILVY.1994. Wildlife mark-

ing techniques. Pages 140-168 in T. A. Bookhout, editor.Research and management techniques for wildlife and habi-

tats. Fifth edition. The Wildlife Society, Bethesda, Maryland,USA.

NORMAN,A. P., G. JONES,ANDR. ARiETrAZ. 1999. Noctuid moths

show neural and behavioural responses to sounds made by

some bat-marking rings. Animal Behaviour 57:829-835.PRILUPS,W R. 1985. The use of bird bands for marking tree-

dwelling bats a preliminary appraisal. Macroderma 1: 17- 21.PIERSON,E. D., ANDG. M. FELLERS.1994. Injuries to Plecotus

townsendii from lipped bands. Bat Research News 34:89-91.

RICE,C. G., ANDP KALK. 1996. Identification and marking tech-

niques. Pages 56-66 in D. G. Kleiman, M. E.Allen, K. V. Thomp-

son, S. Lumpkin, and H. Harris, editors. Wild manlffials in cap-tivity. University of Chicago, Chicago, lllinois, USA.

ROOD,]. P., ANDD. W NEWS. 1980. Freeze marking mongooses.

Journal of Wildlife Management 44:500-502.TuTTI.E,M. D., ANDD. STEVENSON.1982. Growth and survival of

bats. Pages 105-150, in T. H. Kunz, editor. Ecology of bats.Plenum, New York, New York, USA.

WILSON,D. E. 1988. Maintaining bats for captive studies. Pages

247-264 in T. H. Kunz, editor. Ecological and behavioral

methods for the study of bats. Smithsonian Institution, Wash-

ington, D.C., USA

Richard (Rick) Sherwin (photo) is currently a doctoral candidatein biology at the University of New Mexico. He received hisundergraduate degree in conservation biology and his M.5. inzoology from Brigham Young University. His research interestsinclude habitat restoration and conservation. He is currentlyinvestigating relationships of various species of bats with aban-doned mines. Shauna Haymond is part owner of a biologicalconsulting company that specializes in bat-conservation andmanagement. She received her undergraduatedegree in con-servation biology and her M.S. in zoology from Brigham YoungUniversity. She studied habitat use and distribution patterns ofbats in southeastern Idaho. She is particularly interested inhabitat relationships and conservation. Dave Stricklan is a pro-fessor of biology at Brigham Young University-Idaho, Hereceived his B.S. degree and M.S. in wildlife management fromBrigham Young University. Previously he worked as a wildlifebiologist for the United States Department of Agriculture ForestService at duty stations in South Dakota, Nebraska, Utah, andIdaho. His research focuses on wildlife habitat relationships insagebrush steppe. Rebeccah (Beki) Olsen has been working asa licensed bat rehabilitator from her home since 1993. Shereceived her B.S. degree in biology (secondary education) fromBrigham Young University. She is an active member of the GemState Grotto and the Idaho Bat Working Group. Beki is partic-ularly interested in public education and awareness regardingthe importance of bats and gives presentations at public schoolsthroughout her community.

Associate editor: Miller ~