The characteristic call and large size of the American bullfrog (Lithobates catesbeianus) make these animals popular in zoological institutions as well as a common pet in many American households. Amphibian populations, including those of the American bullfrog, are vital to our ecosystem, and their numbers are dwindling worldwide due to anthropogenic activities, disease states, climate change, and invasive species.1,2 Captive populations serve as a safety net for wild populations, and they are known to develop neoplasia. According to a 2020 literature review for the Exotic Species Cancer Research Alliance; 50 cases of amphibian neoplasia have been published between 1954 and 2018. For 69% of these cases, no therapy was pursued.3 The importance of establishing chemotherapeutic protocols is emphasized when you consider endangered populations. A European retrospective study4 focusing on the endangered mountain chicken frog (Leptodactylus fallax) states that 42 out of 212 necropsies had evidence of neoplasia. Intestinal adenocarcinoma was the known cause of death for 31 of these frogs. Cancer in amphibians can be challenging to treat due to their unique physiological and morphological characteristics that make it difficult to define a species-specific treatment protocol without known published studies.
The majority of chemotherapeutic drugs are administered on the basis of body surface area (BSA) rather than weight.5–11 BSA measurements are proportional to metabolic rate and provide a more accurate representation of the entire animal.12 The dosing of chemotherapeutics between different members of the same species is therefore more accurate for each individual. BSA is more accurate than weight-to-dose chemotherapeutics for subjects that differ in size not shape,13 which is commonly seen in exotics including frogs. The equation used to calculate BSA6 is BSA = K X (body weight in g)2/3. K is a species-specific shape constant and is the key to determining appropriate chemotherapeutic doses. A noninvasive method to evaluate BSA in novel species can be through CT scans. This method has been used successfully in mammals such as ferrets,14 rabbits,15 human neonates,16 and human adults.17 Most recently, a CT-derived species-specific formula for BSA in a reptile, bearded dragons, has been defined.18
The following study uses a similar methodology to the bearded dragon report18 with alterations made in respect to the American bullfrog. To our knowledge, no K constant for BSA has been published for amphibians or, specifically, American bullfrogs. The objective of the following study is to use CT-derived values to calculate a species-specific shape constant (K) and BSA formula in American bullfrogs. By establishing a K constant for American bullfrogs, we aim to assist in the ability to calculate chemotherapeutic medications for this species. This study does not evaluate the efficacy, toxicity, and pharmacokinetics of chemotherapeutic drugs in American bullfrogs, and further research is indicated in these areas.
Methods
Animals
Five female and 7 male adult American bullfrogs of the same lineage housed at the North Carolina State College of Veterinary Medicine Department of Laboratory Animal Research (NC State CVM DLAR) participated in this study. The bullfrogs received physical examinations in November 2022, and inclusion criteria were determined based on health status and normal body conformation. Low-stress handling and restraint were utilized so that no sedative or anesthetic medications were required for the American bullfrogs. Researchers wore nitrile, latex-free, exam gloves to handle the frogs. This study was approved by the North Carolina State University Institutional Animal Care and Use Committee (No. 19-614-O).
Examinations
The American bullfrogs underwent thorough and complete physical examinations under the supervision of a licensed veterinarian. The examination focused on assessing normal health and included heart and respiratory rate measurements, abdominal palpation, oral cavity and ocular exam, sex determination, evaluation of normal body conformation, and weight measurements. Sex was determined through evaluation of secondary sex characteristics.19 Males had larger tympanums than their eyes as well as a defined digit known as the nuptial pad while females had tympanums comparable in size to their eyes.
The American bullfrogs were not sedated or anesthetized for the examinations or CT scans. To minimize movement, animals were placed in individual plastic boxes (Style Selections Storage Container; Belle Storage Solutions; 32.79 cm X 23.14 cm X 13.06 cm) with foam around all four sides and a lid (Figure 1). The foam had a hole carved into it that was the size of the bullfrog. Moisture was removed from the frogs with a towel before being placed in the boxes. A 64-slice helical CT scanner was used to acquire transverse images with a 0.6-mm-slice thickness at 130 kVp and 25 mA. The entire body of the frog was scanned.
American bullfrog being placed in a plastic container lined by foam with a frog-sized hole in the center. A plastic lid was placed for the CT scans.
Citation: American Journal of Veterinary Research 84, 11; 10.2460/ajvr.23.06.0131
3-D CT reconstructions
CT surface models were created following the methodology established in a previous study.18 The CT images were reconstructed in a bone reconstruction algorithm (viewed at window width, 2,000 HU; window level, 400 HU) and analyzed with dedicated CT image analysis software (Mimics Innovation Suite; Materialise NV). A minimum threshold value of −500 HU with no upper limit was defined for each frog to ensure densities outside of the frog were excluded. The gas imaged in each frog was manually removed with a tracing tool (Mimics Innovation Suite; Materialise NV) and filled in with the appropriate density to ensure that these cavities were not included in the frog's surface area. Cavities filled included the lungs, tympanum, and gastrointestinal tract. Water and mucous contamination did not occur due to the drying of the frogs dry before the CT scan. If there was residual water and mucus interference, it would be easily recognized by noticing an abnormal conformation of the frog on the resulting 3-D image. The software generated 3-D models, which were visually assessed for appropriate body contour and completeness. Additionally, the software calculated BSA for each frog, which was then used for K constant determination.
Statistical analysis
BSA determined from the CT-derived measurements and body weight data allowed us to solve for the K constant in our study with the formula BSA = K X (body weight in g)2/3. Utilizing nonlinear regression, we plotted BSA against body weight and calculated additional K constants to be used in frogs of varying sizes. The results were finalized for all animals, grouped by sex, and combined.
Results
Our study group consisted of 12 healthy adult American bullfrogs (5 females and 7 males). The mean body weight of the bullfrogs was 354 grams (range, 324 to 419 grams; median, 347.5 grams). All animals were identified by previously placed microchips, healthy, and owned by NC State CVM. No frogs had abnormal body contour conformation. The average heart rate was 60 beats/min (all frogs had the same heart rate) and the average respiratory rate was 64 breaths/min (range, 30 to 80 breaths/min; median, 70 breaths/min).
The mean BSA was 414.92 cm2 (range, 382.76 to 445.42 cm2; median, 412.87 cm2) (Figure 2). Results were displayed as BSA plotted against body weight for each American bullfrog. Results were grouped by sex and as an overall group (Figure 3). The derived K constant for the female bullfrogs was 8.07, and the derived K constant for male bullfrogs was 8.44. There was no statistical difference between the K constant for male or female bullfrogs (P = 0.5). The results for all 12 bullfrogs were considered and the K constant derived was 8.28. Based on our results, the CT-derived BSA formula for American bullfrogs is BSA = 8.28 X (body weight in grams)2/3. Results are summarized (Table 1).
Representative surface models of bullfrogs used to calculate body surface area K values.
Citation: American Journal of Veterinary Research 84, 11; 10.2460/ajvr.23.06.0131
K values in American bullfrogs in relation to body weight (g) and body surface area (BSA; cm2) grouped by sex. A—Male and female. B—Female. C—Male.
Citation: American Journal of Veterinary Research 84, 11; 10.2460/ajvr.23.06.0131
Body weight and body surface area (BSA) of American bullfrogs (Lithobates catesbeianus) for overall measurements and those specifically for male and female frogs.
| Sex | All animals (n = 12) | Male (n = 7) | Female (n = 5) |
|---|---|---|---|
| Body weight (g) | Mean: 354 | Mean: 246.14 | Mean: 362.2 |
| Median: 347.5 | Median: 334 | Median: 354 | |
| Range: 324–419 | Range: 324–388 | Range: 340-419 | |
| CT-derived BSA (cm2) | Mean: 414.92 | Mean: 418.44 | Mean: 410 |
| Median: 412.87 | Median: 416.33 | Median: 403.79 | |
| Range: 382.76–445.42 | Range: 410.32–437.68 | Range: 383.76–445.72 |
Discussion
Neoplasia affects all fields of veterinary medicine, and it does not discriminate between small animals, large animals, zoo animals, or exotic species. While published K constants exist for dogs and cats,5 10.1 and 10.0, respectively, the determination of K constants in nondomestic animals in a relatively novel endeavor. Previous studies have utilized CT-derived K constants in ferrets (9.94),14 rabbits (9.9),15 and bearded dragons (11.6).18 Interestingly, before the publication of the bearded dragon BSA formula, the K constant for reptiles was estimated as K = 10 on the basis of metabolic energy requirements.20 These results of the bearded dragon paper18 are clinically important because they suggest that higher doses of chemotherapeutic agents may be needed than previously thought. When treating mammals such as dogs with chemotherapeutic medications, BSA is typically used5; however, the use of BSA has been reported to lead to a greater risk of overdose in smaller breeds, which is why dogs less than 15 kg are dosed on the basis of body weight instead.5 However, there are some studies21 suggesting these differing K constants in dogs are more based on height and weight than weight alone. Additionally, there are other publications in cats suggesting that BSA was more accurate for animals that differ in size and not shape,13 which may be further reason to use species-specific K constant and BSA calculations rather than milligram per kilogram calculations.
The American bullfrogs in this study successfully underwent CT without the use of sedatives or anesthetics. Our hands-off approach and plastic box technique limited stress in the species and allowed us to produce clear CT images for reconstruction. Heart rate and respiratory rate tend to increase in times of stress, and in our bullfrogs the average heart rate was 60 beats/min and the average respiratory rate was 64 breaths/min, which is well within normal resting rates for American bullfrogs.22 The resulting CT images were clear and 3-D reconstruction required no alterations other than manually removing gas from body cavities. The frogs were required to be partially dried as in our pilot study of these animals we found that when the animals were wet, water would soak into the surrounding foam increasing its effective Hounsfield units. This interfered with the automatic segmentation of the frog from the surrounding foam by the reconstruction software. Ensuring that the frog was dry allowed us to get an accurate reconstruction.
Our CT-derived BSA for American bullfrogs is BSA = 8.28 X (body weight in grams)2/3. The K constant derived for females (8.07) and males (8.28) was not significantly different. On average, female American bullfrogs are typically larger; however, the growth rate of wild American bullfrogs does not differ between sex.19 The males and females in our study group were all adults, suggesting that they could be of similar body size. Our largest frog was a female weighing 419 grams, the smallest frog was a male weighing 324 grams, and the average frog size was 347.5 grams.
Evaluating American bullfrogs of the same age and same lineage was a limitation of the present study as we cannot determine if other age stages would have a similar BSA; however, adults tend to develop more cancer than juveniles, so a surface area based on the adult BSA may be more clinically important. The average lifespan of an American bullfrog is 7 to 9 years in the wild, and they can live up to 16 years of age under human care.23 Future research could be to evaluate frog postmetamorphosis to the nature of their growth rates as described above.
The usage of BSA for chemotherapeutic medication dosing is reliant on the assumption that glomerular filtration rate and metabolic rate are proportional to BSA.17,24 Amphibians have lower metabolic rates compared to mammalian species, and further research is indicated to determine if the glomerular filtration rate is proportional to BSA. While this study produced a BSA formula specific to American bullfrogs, which can be applied to chemotherapeutic dosing, it did not evaluate the pharmacokinetics or pharmacodynamics of chemotherapeutic agents in American bullfrogs. Veterinarians should be cautious when dispensing potentially toxic chemotherapeutic agents and should consider the size of the patient when determining chemotherapeutic dosages in all animals, including amphibians.
Our study defined a formula for determining BSA in American bullfrogs that is both minimally invasive and species specific. The information provided in this study could be used to calculate chemotherapeutic doses for American bullfrogs and is the first of its kind to define a K constant for an amphibian. Further research is indicated to study pharmacokinetic data, toxicity possibilities, and species-specific BSA formulas in other amphibians.
Acknowledgments
The authors would like to acknowledge the following people for helping make this study possible: Dr. Julie Balko, Daria Hinkle, the NC State Laboratory Animal Resources Staff, Jim Robey, Kent Passingham and Dr. Greg Lewbart.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding was provided from the Exotic Species Cancer Research Alliance (ESCRA).
References
- 1.↑
Densmore CL, Green DE. Diseases of amphibians. ILAR J. 2007;48(3):235–254. doi:10.1093/ilar.48.3.235
- 2.↑
Collins JP (2010) Amphibian decline and extinction: what we know and what we need to learn. Dis Aquat Org. 92:93–99. doi:10.3354/dao02307
- 3.↑
Hopewell E, Harrison SH, Posey R, Duke EG, Troan B, Harrison T. Analysis of published amphibian neoplasia case reports. J Herpetol Med Surg. 2020;30(3):148–155. doi:10.5818/19-09-212.1
- 4.↑
Ashpole IP, Steinmetz HW, Cunningham AA, et al. A retrospective review of post-metamorphic mountain chicken frog (Leptodactylus fallax) necropsy findings from European zoological collections, 1998 to 2018. J Zoo Wildl Med. 2021;52(1):133–144.
- 5.↑
Chun R, Garrett LD, Vail DM. Cancer chemotherapy. In: Withrow SJ, Vail DM, eds. Withrow and Macewen's Small Animal Clinical Oncology. 4th ed. Saunders; 2007:163–192.
- 6.↑
Du Bois D, Du Bois EF. A formula to estimate the approximate surface area if height and weight be known. Arch Intern Med. 1916;17:863–871.
- 7.
Price GS, Frazier DL. Use of body surface area (BSA)-based dosages to calculate chemotherapeutic drug dose in dogs: I. Potential problems with current BSA formulae. J Vet Intern Med. 1998;12(4):267–271. doi:10.1111/j.1939-1676.1998.tb02121.x
- 8.
Pinkel D. The use of body surface area as a criterion of drug dosage in cancer chemotherapy. Cancer Res. 1958;18:853–856.
- 9.
Grochow LB, Baraldi C, Noe D. Is dose normalization to weight or body surface area useful in adults? J Natl Cancer Inst. 1990;82(4):323–325. doi:10.1093/jnci/82.4.323
- 10.
Kaestner SA, Sewell GJ. Chemotherapy dosing part i: scientific basis for current practice and use of body surface area. Clin Oncol. 2007;19(1):23–37. doi:10.1016/j.clon.2006.10.010
- 12.↑
Girens R, Bukoski A, Maitz CA, et al. Use of computed tomography and radiation therapy planning software to develop a novel formula for body surface area calculation in dogs. J Vet Intern Med. 2019;33(2):792–799. doi:10.1111/jvim.15440
- 13.↑
Kent MS. Cats and chemotherapy: treat as ‘small dogs’ at your peril. J Feline Med Surg. 2013;15(5):419–424. doi:10.1177/1098612X13483240
- 14.↑
Jones KL, Granger LA, Kearney MT, et al. Evaluation of a ferret-specific formula for determining body surface area to improve chemotherapeutic dosing. Am J Vet Res. 2015;76(2):142–148. doi:10.2460/ajvr.76.2.142
- 15.↑
Zehnder AM, Hawkins MG, Trestrail EA, Holt RW, Kent MS. Calculation of body surface area via computed tomography-guided modeling in domestic rabbits (Oryctolagus cuniculus). Am J Vet Res. 2012;73(12):1859–1863. doi:10.2460/ajvr.73.12.1859
- 16.↑
Schloesser RL, Lauff M, Buxmann H, Veit K, Fischer D, Allendorf A. Three-dimensional body scanning: a new method to estimate body surface area in neonates. Neonatology. 2011;100(3):260–264. doi:10.1159/000327516
- 17.↑
Villa C, Primeau C, Hesse U, Hougen HP, Lynnerup N, Hesse B. Body surface area determined by whole-body CT scanning: need for new formulae? Clin Physiol Function Imaging. 2017;37(2):183–193. doi:10.1111/cpf.12284
- 18.↑
Keeney CMH, Nelson NC, Harrison TM. Use of computed tomography to determine a species-specific formula for body surface area in bearded dragons (Pogona vitticeps). Am J Vet Res. 2021;82(8):629–633. doi:10.2460/ajvr.82.8.629
- 19.↑
Asahara M, Obayashi Y, Suzuki A, Kamigaki A, Ikeda T. Sexual dimorphism in external morphology of the American bullfrog Rana (Aquarana) catesbeiana and the possibility of sex determination based on tympanic membrane/eye size ratio. J Vet Med Sci. 2020;82(8):1160–1164. doi:10.1292/jvms.20-0039
- 20.↑
Mayer J. Allometric scaling. In: Divers SJ, Scott SJ, eds. Mader's Reptile and Amphibian Medicine and Surgery. 3rd ed. Elsevier; 2010:1186–1190.
- 21.↑
Saganuwan SA. Derivation of a unique body surface area (BSA) formula for calculation of relatively safe doses of dog and human anticancer drugs. J Cancer Sci Ther. 2017;9:10. doi:10.4172/1948-5956.1000493.
- 22.↑
Cabanac A, Cabanac M. Heart rate response to gentle handling of frog and lizard. Behav Process. 2000;52(2–3):89–95. doi:10.1016/s0376-6357(00)00108-x
- 23.↑
Bruening S. 2002. Lithobates catesbeianus (Online). Animal Diversity Web. Accessed August 11, 2023. https://animaldiversity.org/accounts/Lithobates_catesbeianus/
- 24.↑
Dooley MJ, Poole SG. Poor correlation between body surface area and glomerular filtration rate. Cancer Chemother Pharmacol. 2000;46(6):523–526. doi:10.1007/PL00006751
