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IV Sources A. SE LECTION Guidelines for the selection of species for specific investigations are be- yond the objectives of this document. However, certain general principles should be borne in mind. In order of availability or accessibility to the investigator, one may list wild-caught, wild, laboratory-reared, and labora- tory-bred animals. Although the quality of the data may be in proportion to the degree of definition of the animals (see Chapter III), the current state of the art and of supply is such that the investigator should choose the type of animal that is most abundant, yet adequate for his investiga- tion. If simply a live frog is sufficient and if parasite or genetic control is not necessary, choose wild or wild-caught animals, at least until the supply situation has improved. Perhaps the most important criterion in a selection process is to choose a supplier whose animals are reliable. "Sources of Amphibians for Re" search" (Nace et al., 1971) lists sources, species, and geographic origins for approximately 150 species of anurans and urodeles. Most of these species are only available from other investigators and on a seasonal or "on hand" basis. The list also includes the several large dealers that sup" ply over 80 percent of the amphibians used in research. The investigator should learn to know the seasonal characteristics of his animal and the re- sources and reliability of his supplier (Emmons, 1973~. He should deter- mine whether the supplier is a collector or whether the supplier obtains his animals from a primary supplier. The adequacy of the supplier's fa- cilities must be determined. Those who expect to use many animals should visit the facilities of the suppliers. Endangered amphibian species, which are not available from commer- cial suppliers, are listed in Appendix A. Reputable users and dealers do not undertake the use or supply of endangered species. 39

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40 B. DEALER CARE An important responsibility of the dealer is to reduce the stress on the animals in his charge for the period he holds them in his establishment. This presents problems when dealing with animals throughout the year and from different geographical origins, as their physiological state is highly dependent on season and origin (see Chapters II, V, and VI). It is useful for the dealer to know the objectives of his users: For example, is the user engaged in studies involving reproduction, cellular physiology, or organ physiology? Dealers must establish those procedures in their plants that will meet the requirements for the standards of animals they choose to supply (see Chapter III). For example, hibernating northern frogs can be held in dealer's facilities if water quality (see Chapter V, Section B.2.a) and other conditions (see Chapter V, Section C.S and Chapter VI, Section B.1) are controlled. In the past, hibernating frogs have been kept by many dealers in small ponds that freeze over in winter and are supplied by untreated water from underground springs. In general, this is a poor practice; the water is often oxygen deficient and too warm for optimal hibernation conditions. Field studies have indicated that the hibernating frog in nature is extremely sensitive to its surroundings, adjusting repeatedly throughout the winter to changes in the environment. The two most significant envi- ronmental variables appear to be temperature range and oxygen availabil- ity, and it is not uncommon for the hibernating frog to move several hun- dred yards from one location in a lake or stream to another to optimize these two factors. Interest has been shown by the research community in wild-caught conditioned animals. Commercial dealers are attempting to meet this demand for R. catesbeiana and in a more limited way for R. pipiens. The latter is limited because R. pipiens is more difficult to feed artificially, and the established price structure restricts the introduction of the necessary technology. C. ORDERING, SHIPPING, AND RECEIVING 1. Ordering The user is responsible for ordering animals in such a manner that the dealer can reasonably be expected to meet his desires. The user must be specific in stating requirements as to size, geographic origin, nature of the research, and level of definition required (see Chap- ter III, Section B). Supply limitations must be recognized. For example,

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41 in dealers' pens most R. pipiens over 90 mm (3.5 in.) in body length are female. Therefore, orders for " 105 mm (4 in.) grassfrogs (half male and half female)" are frustrating to the dealer, if not impossible, to Din. The purchasing department should include the name of the user on the order and, if possible, reference to a previous order for similar animals. This permits the dealer to maintain uniformity in shipments. Order amphibians by "use date, " rather than "shipping date. " Leave the time and method of shipment to the supplier. Though dealers limit their guarantee to live arrival, reputable dealers attempt to ship under optimal circumstances to ensure healthy arrival. Do not order amphibians for delivery on Monday mornings. This yen" orally necessitates a weekend holding period. Upon receipt of the animals, it is important that they receive prompt attention as described below. 2. Shipping Shipping is the responsibility of the dealer who must meet his guarantee of live arrival. Any procedures that interfere with an amphibian's ability to regulate its body temperature will result in the loss of animals. Am- phibians, as ectotherms, regulate their temperature by selecting an appro- priate habitat or posture-processes impossible in transit. The supplier must provide the correct environment for shipping. The-most difficult times for shipping ranids are periods of temperature and humidity em tremes. Inadequate cooling in the summer results in what is known in the jargon of amphibian dealers as "hot frogs"-the state of being hyperactive to the point of convulsion. Although frogs and salamanders may be shipped in boxes containing sphagnum moss or other protective material, damage caused by improper handling en route may occur (Gibbs e! al., 1971~. Aquatic forms such as tadpoles, axolotls, and Xenopus may be shipped in thermos jugs or plastic bags in insulated containers. The numbers of animals that can be shipped per unit volume is critical and varies greatly with species and size. In the summer, the containers should include ice to minimize activity and sealed containers should also include oxygen to facilitate respiration. A given shipping container should contain animals of only one species In some cases sexes should also be separated, e.g., amplexing animals. Hibernating animals may be shipped submerged in cold water 2-4 C (36-39 F), nonhibernating animals in well~ventilated cartons. Before shipment, food should be withheld from amphibians for a sufficient time to prevent contamination of the shipping container. This is particularly true of aquatic forms such as Xenopus. Most amphibian deliveries to laboratory facilities should be either by Air Parcel Post Special Delivery or Air Express.

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42 Accompanying the shipment, suppliers should include information as follows if the amphibians are to be used for research: Simple directions for handling and holding the animals after their arrival with references to more extensive sources of information such as this document and . The standard met by the animals and the information specified by the criteria for the standard (see Chapter III). 3. Receiving The user is responsible for receiving the animals in such a manner that the specifications in his order and the efforts of the dealer are not nullified. The details of procedures to be followed upon receipt of amphibians de- pends on the species. However, certain general principles can be stated. Unless the user is prepared to develop suitable facilities and meet demand- ing personnel requirements for the long-term care of amphibians, they should not be held for more than a few days. If long-term storage or ob- servation is necessary, prepare the required facilities prior to placing the order for animals (see Chapters V and VI). It is important to realize that seasonal variations determine the hardiness and the nature of the care required by most amphibians. Thus, northern R. pipiens that have spawned are not as hardy as animals collected in the fall of the year after feeding actively throughout the summer prior to hibernation. a. Aquatic Amphibians The aquatic animals such as axolotls, Xenopus, and tadpoles should be transferred immediately from the shipping containers to previously pre- pared containers. Last minute cleaning of spare containers often results in shoddy cleaning and inadequate rinsing to remove soap or detergent! Do not discard the water in which the animals were receded. The rapid transfer of animals to water that differs radically in quality may result in severe shock or death. As a regular practice, animals should be conditioned to the water in the laboratory by slow dilution of the transport water. Strain it through gauze and use it in the clean containers. Save half of this water in case the following steps cause the animals to show discomfort. If this water in which you will place the animals is warmer than the tem- peratures specified in Chapter VI for the species in question, add ice made from dechlorinated laboratory water (see Chapter V, Section B.2.a), but do not expose the animals to temperature changes greater than about 1 C (2 F) per hour. After the animals become quiescent and give signs of

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43 adapting to the new environment, add no more than one~fourth the vol- ume of laboratory water. If no distress is obvious after several hours, more fresh water may be added. Repeat these steps until the animals have ads lusted to your water supply. If they show distress at some step, return them to the previous water mixture in which they did not show distress and prepare new water from a different source. Water quality is only one aspect of their new environment. In addition, container size, shape, and transparency must be considered. individuals with obvious stress or damage should be isolated and treated as discussed in Chapter IX, Section B.2. After acclimatization, normal care procedures may be followed. b. Terrestrial Amphibians Because of their shape and posture, terrestrial urodeles ship quite well un- less seriously overheated or dehydrated. They may be handled in much the same way as the aquatic animals, i.e., they should be moved to permanent housing through a series of gradual temperature steps. To avoid confusion and damage to terrestrial anurans, the shipping box should be emptied into an enclosure such as a large plastic garbage can con- taining 0.15-0.25 m (6-10 in.) of water in order to deny the frogs a surface from which they can jump. Wash them free of packing material and sepa- rate the apparently healthy from obviously sick or damaged animals. In the winter, the wash water should be cooled to the temperature of the animals; in the summer, water at tap water temperature may be used. Do not wash them vigorously under a full flow of water from a tap or hose. Plastic vegetable crispers with slide-on lids are good isolation containers. The con- tainers should be filled with water to about shoulder height for leopard frogs; toads need only a damp bottom. From October through March and depending on the research objectives, wild-caught nonconditioned animals from north of the ice line may be re- turned to hibernation. This may be done by taking them from shipping temperature to hibernation temperature through a series of 9-12 h at each of several intermediate temperatures with each step being 3-5 C (6.4- 10.7 F). Alternatively, if 20 or more gallons in the hibernaculum are used (see Chapter V, Sections C.5 and 6.a), the animals may be placed in the water at room temperature. They will acclimate as the water cools when the container is placed in the coldroom. This procedure should be followed within hours after receipt of the animals if they are to be used for their eggs (see Chapter It, Section B.2.e). Never use this chilling procedure with summer or Mexican frogs. Adult R. pipiens to be used at room temperature, or whose eggs are not

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44 5. 1. Connecticut Maine Massachusetts New Hampshire Vermont 2. Delaware Maryland New Jersey New York Rhode Island ~. Michigan Minnesota North Dakota South Dakota Wisconsin 4. Idaho Montana Oregon Washington Wyoming Kentucky North Carolina Tennessee Virginia 6. Illinois I ndiana Ohio Pennsylvania West Virginia 7. Colorado lows Kansas Missouri Nebraska 8. Alabama Florida Georgia Mississippi South Carolina 9. Arkansas Louisiana New Mexico Oklahoma Texas 1 0. Arizona California Nevada Utah 1 1. Alaska Hawaii JAN FED MAR APR MAY JUN Jo FIGURE 13 Amphibian closed season regulations. Hatched time spaces represent the closed seasons. Numbers in the squares refer to dates. The states are listed by tiers: northern, 1 4; central, 5-7; southern, 8-10; and other. Data on the Canadian provinces may be found in Appendix B. Data assembled with the assistance of Stafford Cox, student of environmental law, University of Michigan.

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45 needed, may be maintained at room temperature at any season (see Chap- ter V, Section C.4~. They must be fed, however, if they are to be kept more than a few days. D. Legal Aspects It is incumbent upon both dealers and users of amphibians to be aware of and abide by the laws established to regulate their use. At the federal level the Endangered Species Conservation Act provides for the conservation, protection, and propagation of any wild mammal, fish, wild bird, amphib- ian, reptile, mollusk, or crustacean threatened with extinction or likely within the future to become threatened with extinction. It further directs that, insofar as is practicable and consistent with the primary purposes of bureaus, agencies, and services, all federal departments and agencies shall exert their authorities in furtherance of this Act. The secretaries involved determine, at their discretion, the endangered species based on the best scientific and commercial data available; and after consultation with the affected states, interested persons, and organizations, the secretaries pub- lish the endangered species list in the Federal Register. Upon the petition of an interested person, the secretaries conduct a review of any listed or unlisted species proposed to be removed or added to the list. The evidence required for such a review pertains to any one of the following factors: the destruction, drastic modification, or severe curtailment of habitat; overutilization for commercial, sporting, scientific, or educational pur- poses; the effects of disease or predation; the inadequacy of existing regulatory mechanisms; or other natural or man-made factors affecting continual existence of the species in question. With respect to amphibians, the documentation and quantification of these factors is not defined. A listing of amphibians judged to be endangered is to be found in Appendix A. State regulations concerning amphibians are highly variable as seen in Appendix B. These regulations are summarized in Figure 13, which shows the closed seasons for "frogs" in each state. in this figure, the states are assembled by geographic regions. Figure 13 and Appendix B do not show that collection for scientific purposes is exempt or given special treatment by the law in some states. Caution should be exercised in interpreting Figure 13 as state regulations affecting frogs are currently under review in many states.