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V! ~ ~ Records ant' Information Control A. IDENTIFICATION OF INDIVIDUALS 1. Numbering The identification of individual animals-often necessary for the manage- ment of experimental animals-is mandatory in breeding and genetic pro- cedures. Several techniques can be used to identify individual amphibians. Selection depends on the species and the developmental stage. Ideally, the system of choice would allow identification of individual animals at all stages of development from the time of fertilization through larval and postmetamorphic development. However, aside from isolation in indivi- dual enclosures, no system adequate to accomplish this objective is avail- able. In practice, a clutch of eggs or a shipment of animals may be given a serial identification number such as 35,000 with subsequent numbers in the series reserved in proportion to the number of clutch or shipment members to which individual numbers will be assigned. As animals are assigned for specific experimental purposes, the experimental group or individual may be given numbers from among those reserved. Thus, 35,XXX identifies the clutch or shipment; the hundred's digit may be used to identify groups and the ten's and unit's digits to identify individuals. Serial numbers that do not include letters are recommended to facilitate adaptation to computer techniques, should these be found useful (see Sec- tion B below). Identification numbers may be written in waterproof ink on waterproof labels that, when appropriate, may be easily transferred from one enclosure to another. In the Amphibian Facility of the University of Michigan, it has been found useful to assign unique group numbers to members of a clutch or shipment that share an enclosure. This permits 108

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109 tracing enclosure mates and identifying individuals with common environ- mental experiences. 2. Tattooing Tatooing by ink injection techniques has proved successful. It is particu- larly useful for larvae with well-developed tail fins, although its use is not limited to them. in this technique the animal is lightly anesthetized (see Chapter IX, Section F) and a fine needle attached to a hypodermic syringe containing tattoo ink is inserted into the tail fin or into the skin. The ink is injected as the needle is withdrawn, leaving a "bar" of ink. By using inks of several colors and forming a series of parallel bars, unique identification codes may be constructed. Recently, several useful variations on this prin- ciple have been published (Woolley, 1973~. Tattooing by the use of vibrating needles, as used in human tattooing, cannot be applied to larvae or small postmetamorphic animals; in fact, this method can only be used for larger postmetamorphic animals and even then must be renewed frequently. R. pipiens and bullfrogs retain such a tattoo for only 3-4 months. Because these and other amphibians shed their skin, it is difficult to assure that the needle-driven tattoo pene- trates into skin layers that are not lost. 3. Branding Wolf and Hedrick (1971) describe "chemical branding" as a procedure for marking Xenopus; these are permanent for at least a year. Using a cotton swab dipped in a solution of 0.5 percent amido Schwartz in 7 percent acetic acid, label figures are formed on the back of an animal after mucous secretions are removed by repeated wiping with paper tissues. After 1 min of contact the animals are returned to an enclosure. The dye provides a temporary identification until scar tissue is formed a few days later in response to the acid treatment. The cold branding technique (Farrell and Johnson, 1973) has proved satisfactory for adult R. pipiens and R. catesbeiana and may be applied to the dorsal surface where the label can be easily read. The advantage of this technique over tattooing is the longer period of label retention. How- ever, cold branding also disappears with time and cannot be applied to juvenile animals. Tattooing and cold branding are preferable to heat brand- ing because they result in less trauma to the subject. Urodeles can be tattooed or branded at relatively early stages if it is done under light anesthesia (see Chapter IX, Section F). Such markings are lost, however, as the skin is shed and must be renewed periodically.

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110 4. Toe Clipping Toe clipping is an effective technique for amphibian identification and is appropriate for most anurans that do not readily regenerate lost digits. However, it is inadequate for urodeles and Xenopus in which the regen- eration process replaces the removed digits, unless the regeneration is in- hibited by treatment with berilium nitrate (Heatwole, 1961~. Ranidae may be toe clipped shortly after metamorphosis using light anesthesia and cuticle scissors. Although older R. pipiens may be toe clipped with scissors, mature R. catesbeiana, because of their size, may bleed excessively and danger of local infection occurs when toes are clipped in this manner. However, toe clipping large anurans may be ac- complished by using an appropriate cautery knife or loop to seal the wound at the time of amputation. A coding system for toe clipping based on the system for punching edge coded cards and that allows 9,999 in- dividuals to be distinguished with only two amputations per foot is illustrated in Figure 24. 5. Other Marking Systems Various systems for labeling amphibians by the insertion of plastic rings and other devices have been attempted but without notable success. 4 7~' 1, 000's Frog's Left ADPLaNTAR VIEW (Dorsal ) FORE 4 CLIP TO ~J DOTTED LINE ~/ Frog's Right IO's /\20,000 10,000l?(! ~ Tr9 ~'/1 A \ HIND PLANTaR VIEW (Ventral) units 30,000 BOTH 20,000 AND 10,000 CLIPPED ABOVE 39,999, TREAT AS FROM I TO 39,999 FIGURE 24 Toe clip conventions (Nace et a/., 1973; reproduced with permission from the American Zoo/ogist).

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111 6. Drawings and Photographs The most foolproof method of identifying individual amphibians is the use of photographs that record the details of natural disruptive patterning. Figures 25, 26, and 27 illustrate disruptive patterning in R. pipiens and how these patterns are coded for identification purposes in the Amphibian Facility of the University of Michigan (Nace et al., 1973~. Similar classifi- cation of the patterns of other amphibians is possible, but has not as yet been completed. In laboratories with small numbers of animals, it is adequate to prepare duplicated outline drawings of the amphibian and to fill in these outlines with drawings of the disruptive patterning. This technique, however, is too time-consuming for a large colony of animals; thus, photographs are rec- ommended. Using the classification system, it is possible to identify quickly the class of patterns to which an animal belongs and then, by comparison with the appropriate specific photographs or drawings, to identify the individual animal. When the pattern classification and recorded pattern are used in conjunction with other characteristics-such as sex, last recorded snout-vent length, and other-unique characteristics-a very large number of individuals may be identified. B. INFORMATION CONTROL SYSTEMS The management of animal colonies or of data collected in even small col- onies becomes increasingly complex as the numbers of animals or the his" tory of the colony increases. This problem can be greatly alleviated by the adoption of computer-based techniques. Recent improvements in available computer programs and time-sharing techniques, even over long-distance telephonic connections, permit even those inexperienced in computer use to adopt these procedures readily. Although they may be adopted at any" time in the history of a colony, the earlier they are used, the greater the economy that is realized. Current technology is sufficiently advanced that standardization of the data base or of the software is not necessary. It is sufficient to record the available data in machine-readable form; the com- puter itself can restructure the data in accordance with the requirements of specific current or future software. A computer-based system currently in use for the management of am- phibians is described in some detail in Nace et al. (19731. This system is in the public domain and accessible to long-distance users. Other systems that may be adaptable to users of amphibians include the ARE system (Laboratory Research Enterprise, Inc., Kalamazoo, Mich.) for Beagles and a system used for microorganisms (Bachmann et al., 1973~.

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112 NO SPOTS ON ~ IDLI NE 6-10 NO. OF SPOTS HEAD PATTERNS r ~ !1 ~ i I ,0_ NO ~ ~SPOT ON if ~\ SPOTS ~SNOUT ONLY I , i r ' <2 Eye Spots Unequol byFoctorof Two A _ I SPOT ON SPOT ON | I `~\ SNOUT | SNOUT EYE RIGHT LEFT t3OTH SPOTS NO SPOT ~5 ^6 -7 `~. ~ON SNOUT EYE RIGHT LEFT BOTH SPOTS ~ 8 ~ 9 ^10 SPOT ON p~t [ ~{~\ SNOUT OTHER EYE SPOT SNOUT SPOT _ 15; ~3/4 of spot 3/4 of spot between Em . ~on eye or posterior to nostrils _ ^~. ond anterior to eve BODY PATTERN SPOTS ON MIDLINE 134 [' .i' - N ~ i \d REGU LAR it' lb.' ,' L'' at. I PREGULAR BODY PATTERN SPOTS BETWEEN PLICAE POSTERIOR TO EYE ANTERIOR TO VENT 15 OTH ER (NO EXAMPLE) FIGURE 25 Fiana pipiens head and body pattern code characteristics. The num- bers appearing by each pattern in Figures 25 and 26 are used to write an identifica- tion formula. Thus, 7~24 identifies the animal shown with body pattern "6" Mace eta/., 1973; reproduced with permission from the American Zoologist).

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113 NO MARKING tiP BACKGROUND PaTTERNS `-' 1 Hi'' ir~.'lii i,,,' FEW,COUNTABLE (~100) ,PINPOIN~ [,..' ~ ~ text MANY (~100) PINPOINT, DISCRETE OR FUSED l bit lo. A ,~ ~ COMPLEX COMPLEX REGULAR IRREGULAR SCULPTI NG SCU LPTI NO (os Kandlyoh i ) OTHER PATTERNS -_ 3 - 1 t~ 1 ~ an. *~ ~ Ski ~ ~ ~ I ~ NO SPOTS ON ARMS NONE ON | ON LEGS LEGS I L:l i. he F If Or SPOTS ON ARMS NONE ON | ON LEGS LEGS ~ FIGURE 26 Background and other patterns (Nace et a/., 1973; reproduced with permission from the American Zoologist).

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114 SPOT: DETERMINED BY CHARACTER OF SURROUNDING SPOTS FUSED SPOT = ONE SPOT - IRREGULAR SPOTONMIDLINE: WHEN 1/3 OR MORE OF SPOT OVER MIDLINE Background y'< I.~! REGU LAR: APPROXI MATES A SOUARE'OVALOR CIRCLE IRREGULAR: NOT SYMMETRICaL I N DENTED OR LONG LONG SPOTS 3 x (or more) AS LONG AS AVERAGE WIDTH i., Irregular One Spot ill: I-, lo; in, Hi\\ Irregular my' Reg ul tar ~ ~ l irte FIGURE 27 Conventions used to characterize spots (Nace et a/., 1973; reproduced with permission from the American Zoologist).