Serum Clinical Biochemical and Hematologic Reference Ranges of Laboratory-Reared and Wild-Caught Xenopus laevis

Abstract

The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis).

The South African clawed frog, Xenopus laevis, has a long history in biomedical research. The complete X. tropicalis genome has recently been sequenced, and similar efforts are underway for the larger bodied X. laevis.¹¹ Therefore, the establishment of normal serum clinical biochemical and hematological reference ranges is essential for phenotyping future transgenic frogs. In addition, these values will assist in disease surveillance and the diagnosis of clinical illnesses and conditions. Previously published hematograms have been reported on a very limited number of laboratory Xenopus: 10 normal adult frogs (sex and source not described)⁹ and 7 normal adult, female laboratory Xenopus;⁸ however, the number of animals studied were insufficient to establish reference ranges. Serum biochemical reference intervals have not previously been reported.

Here we report the normal reference range values for hematologic and serum clinical chemistry profiles from 207 laboratory-reared and 43 wild-caught Xenopus laevis. These values were compared with those from other amphibians, fish, and chickens because the X. tropicalis genome is strikingly similar to that of chickens.¹¹ Comparisons with mouse and domestic dog values are described to highlight differences. The reference intervals provided here will assist clinicians and researchers in their clinical and phenotypic assessments of Xenopus laevis.

Materials and Methods

Animals.

All animal procedures were conducted in accordance with a protocol reviewed by Stanford University's Administrative Panel on Laboratory Animal Care, the University's IACUC panel. Blood samples were collected from wild-caught, feral Xenopus laevis (n = 43) captured from a local pond (San Francisco, CA) and from frogs from 2 different commercial suppliers, sources A (n = 166; NASCO, Fort Atkinson, WI) and B (n = 109; Xenopus Express, Brooksville, FL). Wild-caught Xenopus were sexually mature male and female frogs with an estimated age between 2 to 4 y of age based on snout-to-vent length measurement; all of the animals in the vendor cohorts were sexually mature approximately 3- to 6-y-old female frogs. Prior to blood collection, laboratory-reared frogs had been housed for at least 1 y under similar conditions of water temperature (16 to 22 °C), room lighting (12:12-h light:dark cycle), and diet (Frog Brittle, NASCO). All water-quality parameters tracked for each cohort are provided in Table 1.

Table 1.

Water-quality parameters (mean ± SEM)

	Source A	Wild-caught
Adjusted conductivity (µOhm)	428.13 ± 29.10	567.50 ± 7.50
Salinity (mS)	ND	ND
Alkalinity (mg/L as CaCO3)	79.68 ± 4.57	ND
Ammonia (mg/L)	1.41 ± 0.25	0.56 ± 0.14
Copper (mg/L)	0.11 ± 0.02	0.43 ± 0.11
Dissolved oxygen (mg/L)	8.68 ± 0.17	7.71 ± 0.95
Free chlorine (mg/L)	0.08 ± 0.01	0.17 ± 0.04
Hardness (mg/L CaCO3)	105.53 ± 3.43	ND
Monochloramine (mg/L)	0.14 ± 0.01	0.12 ± 0.02
Nitrate (mg/L)	4.71 ± 0.50	1.02 ± 0.16
Nitrite (mg/L)	0.11 ± 0.03	0.01 ± 0.01
pH	7.37 ± 0.05	7.05 ± 0.16
Temperature (°C)	20.61 ± 0.17	15.5
Total chlorine (mg/L)	0.06 ± 0.01	0.16 ± 0.04

ND, not determined.

For laboratory-reared frogs, values were averaged over 12 mo; values for wild-caught frogs represent June through July.

The only values reported for source B were salinity (1.6 to 2.0 mS), pH (7.4 to 7.6), and temperature (18 °C).

Water quality.

Because water quality can affect blood and serum biochemical in aquatic amphibians, water-quality test values from the laboratory-frog's housing water or wild-caught frog's pond water (collected at the time the frogs were collected) are described in Table 1. Values for pH and conductivity were obtained by using a pH meter (Accumet Excel XL60, Fisher Scientific, Pittsburg, PA). Remaining water-quality values were obtained by using a spectrophotometer (D4/4000V, Loveland, CO) with Test n’ Tube reagent sets (Hach) according to the manufacturer's instructions.

Blood sample collection.

Cardiocentesis was performed on anesthetized frogs as previously described.⁷ Briefly, frogs were immersed in approximately 5 gm/L MS222 (Finquel, Argent Chemical Laboratories, Redmond, WA) buffered to a neutral pH with sodium bicarbonate (Sigma Aldrich, St Louis, MI) until animals were fully anesthetized (determined by loss of the righting reflex and a lack of response to toe pinch). Anesthetized frogs were incised from pubis to sternum, and the coelomic and thoracic cavity opened to allow direct viewing of the heart. Whole blood (1 to 3 mL) was collected from the ventricle by using a 3-mL syringe (Kendall Monoject syringe, Covidien, Mansfield, MA) and a 22- to 23-gauge needle (Benton Dickson, Franklin Lakes, NJ) with approximately 0.1 mL heparinized saline (Sigma Aldrich) drawn up prior to blood collection. A 0.5-mL aliquot of blood was placed in a microtainer tube containing EDTA (Benton Dickson), and the remainder was placed in 1.5-mL microfuge tubes (Fisher). After blood collection, the heart was removed to ensure that frogs would not recover. For commercially obtained frogs, blood was collected from groups of 15 to 20 animals over 3 h per group. For wild-caught frogs, blood was collected from groups of 2 to 5 animals within 24 h of field collection.

Hematologic analysis.

Total RBC and WBC counts were obtained by hemocytometer methodology using Natt and Herrick stain, as previously described for species with nucleated red blood cells.^1,2,5,7,22 The hematocrit was determined in duplicate (Autocrit Ultra3 centrifuge, Benton Dickson) and centrifuging for 3.5 min at 1247 × g before reading the capillary's cell pack. Hemoglobin was determined by using an automated hemocytometry machine (Cell-Dyn 3500, Abbott, Chicago, IL). Differential cell counts are not reported here because amphibian white cell types have not been functionally characterized to date. Our laboratory is currently optimizing staining techniques to ensure consistent identification of granulocytes, which subsequently can be used to determine accurate differential cell counts.

Values were compared by t tests between cohorts (wild-caught by sex and season, source A, source B). Because wild-caught frog cohorts were not significantly different, their values were combined for presentation. No data is presented from male frogs for hematology because clotting of samples precluded accurate data collection.

Biochemical analysis.

Blood collected in microfuge tubes was allowed to clot at room temperature for approximately 1 h and centrifuged for 6.5 min at 18187 × g. Serum was pipetted to a fresh tube (Eppendorf Centrifuge 5415R, Hamsburg, Germany) and centrifuged again for 3 min at 18187 × g. After centrifugation, serum was pipetted into a 1.5-mL microfuge tube (Fisher). Lipemia and icterus can interfere with analysis of serum; however, no serum collected in this study appeared icteric or lipemic. Serum analyte values were determined (Dimension Xpand Plus Integrated Chemistry System, Siemens, New York City, NY), and values were compared between cohorts (wild-caught by season and sex, source B) by t test.

Statistical analysis.

Statistical Analysis Software (version 9.2, SAS Institute, Cary, NC) was used to compute reference intervals. By using SAS PROC GLM, variation between cohorts was obtained for hematologic data (source A, source B, and wild-caught frogs by month) and for serum biochemical data (source B and wild-caught frogs by month and sex). Cohorts were compared by using SAS PROC GLM with the model analyte = cohort and the contrast command to show t tests between each cohort. When the P value for the t test between 2 cohorts did not indicate significant difference (P > 0.01), the cohorts were combined and t tests were rerun to compare the combined cohorts with the remaining cohort to confirm that the combination was statistically valid. Once each analyte was grouped based on the t test results, the mean and SE of each cohort was obtained by using SAS PROC MEAN. By using the SAS macro ‘Reference Intervals,’¹² cohorts were transformed to maximum normality by using a Box–Cox transformation, outliers were removed, and robust distribution limits were calculated for each cohort. For serum biochemistry values, the wild-caught female frog cohorts had small sample numbers; therefore when fewer than 10 independent measurements were obtained, no interval is presented for that cohort. Because only 8 wild-caught male frogs were available, the cohort was included only when data was obtained from all 8 frogs. For anion gap, a linear transformation (n′ = n + 100) was performed in SAS prior to reference interval calculation to allow inclusion of the negative range. Concentrations of troponin I and C-reactive protein were screened but never detected (limits of detection, less than 1 ng/mL and less than 1 mg/dL, respectively); therefore, these parameters were omitted.

Species comparisons.

Xenopus laevis hematologic and limited serum biochemical reference intervals were compared with published reference intervals for the following amphibians: the Eastern hellbender (Cryptobranchus alleganiensis) ¹⁹ and the American bullfrog (Rana catesbeiana³; Figure 1). Because of the similarity of the X. tropicalis genome to that of chicken (Gallus gallus domesticus),¹¹ hematologic and biochemical comparisons were assessed between Xenopus and selected species representing other familiar animal classes: dogs (Canis familiaris),¹⁷ mice (Mus musculus),^16,18 birds (chickens),^4,6,14 and boney fish (tilapia; Oreochromis hybrids;¹³ Figure 2). The low endpoint and width of the intervals for each analyte were normalized by dividing all values by the largest interval endpoint among the species being compared; the normalized values then were plotted together to allow rough comparison. The normalized intervals for Xenopus laevis, American bullfrogs (R. catesbeiana), and Eastern hellbenders (C. alleganiensis) are plotted in Figure 1. The normalized intervals for chickens, dogs, mice, tilapia, and X. laevis are plotted in Figure 2. These plots are intended for rough comparisons only; normalized values are not an appropriate clinical tool.

Figure 1.

Overlap of normalized reference intervals for the Eastern hellbender (Cryptobranchus alleganiensis; reference 19), Xenopus laevis, and American bullfrog (Rana catesbeiana; reference 3). Values are normalized to the range of each analyte; X. laevis intervals represent frogs from commercial source A.

Figure 2.

Overlap of hematologic reference intervals for chicken,^4,14 tilapia,¹³ mouse,^16,18 dog,¹⁷ and Xenopus laevis. Values are normalized to the range of each analyte; only analytes for which all species had an interval reported are presented. X. laevis intervals represent frogs from commercial source A.

Results

Normal reference ranges for hematologic values from 3 distinct populations of X. laevis—wild-caught frogs (n = 43) and those from 2 commercial sources (A, n = 166; B, n = 109)—are shown in Table 2. For serum biochemistry analytes, we determined normal reference ranges categorized by sex and season for frogs from commercial source A (n = 166) and wild-caught frogs (Table 3). Significant differences (t test, P < 0.01) across populations were found in WBC and RBC counts, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences (t test, P < 0.01) were found across albumin, globulin, albumin:globulin ratio, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase, creatine phosphokinase, indirect bilirubin, direct bilirubin, total bilirubin, cholesterol, low-density lipoprotein lipase, carbon dioxide glucose, lactacte dehydrogenase, calcium, chloride, sodium, and anion gap values. Figure 1 displays the similarity between the intervals for X. laevis and another fully aquatic amphibian, the eastern hellbender (C. alleganiensis), and the contrast between the fully aquatic species and the semiaquatic American bullfrog (R. catesbeiana). Figure 2 shows the inherent differences between the hematologic and biochemical parameters of X. laevis, mice, dogs, chickens, and finfish.

Table 2.

Hematologic reference intervals by cohort

	Source A (n = 166)		Source B (n = 109)		Wild–caught (n = 20)
	Mean ± SE	Reference interval	Mean ± SE	Reference interval	Mean ± SE	Reference interval
WBC (x103/μL)	4.35 ± 0.32	0.64–9.56	3.14 ± 0.16	1.33–6.61	10.78 ± 1.07	3.291–17.07
RBC (x106/μL)	0.76 ± 0.02	0.80–1.48	1.10 ± 0.02	0.49–1.08	0.67 ± 0.03	(0–0.63)–0.93a
Hemoglobin (g/dL)	9.64 ± 0.30	6.06–15.19	14.62 ± 0.31	8.22–21.68	10.94 ± 1.01	5.52–20.46
Hematocrit (%)	36.9 ± 0.7	23.3–47.0	49.4 ± 0.5	41.9–56.2	41.2 ± 1.9	(0–33.1)–54.7a
Mean corpuscular hemoglobin (pg)	15.4 ± 0.3	6.9–22.1	15.4 ± 0.3	6.9–22.1	15.4 ± 0.3	6.9–22.1
Mean corpuscular hemoglobin concentration (g/dL)	25.7 ± 0.5	19.3–32.3	29.5 ± 0.5	19.0–40.5	ND	ND
Mean corpuscular volume (fL)	50.7 ± 1.4	31.6–62.8	46.5 ± 0.8	29.9–76.3	ND	ND

ND, not determined.

When values in the same row are the same, the cohorts are statistically equivalent.

^a90% confidence interval of limit is shown in parentheses because otherwise the limit would be 0.

Table 3.

Serum biochemical reference intervals of X. laevis by cohort

			Wild–caught males,		Wild-caught females
	Source A (n = 166)		summer (n = 8)		February-May (n = 16)		June-July (n = 16)		August-September (n = 12)
	Mean ± SE	Reference interval	Mean ± SE	Reference interval	Mean ± SE	Reference interval	Mean ± SE	Reference interval	Mean ± SE	Reference interval
Albumin (g/dL)	1.0 ± 0.1	0.1–2.3	1.0 ± 0.1	0.1–2.3	1.8 ± 0.2	0.7–4.8	1.0 ± 0.1	0.1–2.3	1.0 ± 0.1	0.1–2.3
Globulin (g/dL)	2.3 ± 0.1	1.1–4.1	1.2 ± 0.1	0.3–2.7	1.2 ± 0.1	0.3–2.7	1.2 ± 0.1	0.3–2.7	1.2 ± 0.1	0.3–2.7
Albumin:globulin ratio	0.7 ± 0.1	0.1–5.1	2.5 ± 0.3	0.1–8.6	2.5 ± 0.3	0.1–8.6	2.5 ± 0.3	0.1–8.6	2.5 ± 0.3	0.1–8.6
Total protein (g/dL)	3.3 ± 0.1	2.0–4.6	2.0 ± 0.3	0.8–4.6	2.9 ± 0.2	1.1–5.5	2.1 ± 0.2	0.7–3.1	2.9 ± 0.2	1.1–5.5
Amylase (I/L)	270 ± 16	43–617	ND	ND	ND	ND	ND	ND	270 ± 16	43–617
Lipase (U/L)	98 ± 4	58–163	ND	ND	ND	ND	ND	ND	156 ± 38	19–366
Alkaline phosphatase (IU/L)	148 ± 5	59–282	148 ± 5	59–282	148 ± 5	59–282	148 ± 5	59–282	148 ± 5	59–282
ALT (U/L)	21 ± 1	10–39	9 ± 1	1–20	9 ± 1	1–20	9 ± 1	1–20	9 ± 1	1–20
AST (U/L)	453 ± 44	27–1774	453 ± 44	27–1774	453 ± 44	27–1774	453 ± 44	27–1774	453 ± 44	27–1774
GGT (U/L)	4 ± 1	1–19	17 ± 2	2–50	17 ± 2	2–50	17 ± 2	2–50	4 ± 1	1–19
CPK (U/L)	1658 ± 188	10–5400	3237 ± 338	425–10339	3237 ± 338	425–10339	3237 ± 338	425–10339	3237 ± 338	425–10339
BUN (mg/dL)	5 ± 1	2–10	ND	ND	5 ± 1	2–10	5 ± 1	2–10	5 ± 1	2–10
BUN:creatinine ratio	16.5 ± 4.9	3.0–36.0	ND	ND	16.5 ± 4.9	3.0–36.0	ND	ND	16.5 ± 4.9	3.0–36.0
Creatinine (mg/dL)	0.4 ± 0.1	0.1–1.1	ND	ND	0.4 ± 0.1	0.1–1.1	ND	ND	0.4 ± 0.1	0.1–1.1
Uric acid (mg/dL)	0.2 ± 0.1	0.1–0.4	ND	ND	ND	ND	ND	ND	0.2 ± 0.1	0.1–0.4
Indirect bilirubin (mg/dL)	0.05 ± 0.01	0.02–0.80	0.01 ± 0.01	0–0.01	0.01 ± 0.01	0–0.01	0.01 ± 0.01	0–0.01	0.01 ± 0.01	0–0.01
Direct bilirubin (mg/dL)	0.02 ± 0.01	0.01–0.04	0	0	0	0	0	0	0	0
Total bilirubin (mg/dL)	0.07 ± 0.01	0.01–0.26	0.01 ± 0.01	0.00–0.1	0.01 ± 0.01	0.00–0.1	0.01 ± 0.01	0.00–0.1	0.01 ± 0.01	0.00–0.1
Cholesterol (mg/dL)	232 ± 12	56–563	205 ± 10	39–299	205 ± 10	39–299	205 ± 10	39–299	205 ± 10	39–299
HDL (mg/dL)	36 ± 1	14–63	ND	ND	ND	ND	ND	ND	36 ± 1	14–63
LDL lipase (mg/dL)	114 ± 6	21–240	ND	ND	ND	ND	ND	ND	26 ± 5	3–62
Triglycerides (mg/dL)	117 ± 7	57–555	ND	ND	ND	ND	ND	ND	117 ± 7	57–555
Carbon dioxide (mmol/L)	20.7 ± 0.6	8.4–34.3	ND	ND	45.3 ± 2.7	37.0–59.3	28.9 ± 2.1	16.3–48.0	25.7 ± 3.7	8.5–64.3
Glucose (mg/dL)	53 ± 2	18–111	42 ± 3	13–91	42 ± 3	13–91	34 ± 2	15–56	42 ± 3	13–91
LDH (U/L)	1809 ± 108	264–3893	ND	ND	ND	ND	ND	ND	1025 ± 89	0–1423
Calcium (mg/dL)	8.9 ± 0.2	5.2–12.3	5.4 ± 0.8	0–9.6	8.9 ± 0.2	5.2–12.3	8.9 ± 0.2	5.2–12.3	8.9 ± 0.2	5.2–12.3
Chloride (mmol/L)	82.5 ± 0.6	72.7–92.7	ND	ND	75.6 ± 1.2	67.6–85.0	ND	ND	82.5 ± 0.6	72.7–92.7
Phosphorus (mg/dL)	7.4 ± 0.2	3.5–11.6	7.4 ± 0.2	3.5–11.6	7.4 ± 0.2	3.5–11.6	7.4 ± 0.2	3.5–11.6	7.4 ± 0.2	3.5–11.6
Potassium (mmol/L)	4.0 ± 0.1	2.3–7.3	ND	ND	4.0 ± 0.1	2.3–7.3	ND	ND	4.0 ± 0.1	2.3–7.3
Sodium (mmol/L)	123 ± 1	111–134	ND	ND	114 ± 2	ND	ND	ND	123 ± 1	111–134
Anion gap (mmol/L)	23.6 ± 0.7	13.1–36.1	ND	ND	−0.5 ± 2.8	−16.5–53.8	ND	ND	23.6 ± 0.8	13.1–36.1
Calcium:phosphorus ratio	1.3 ± 0.1	0.7–2.0	1.3 ± 0.1	0.7–2.0	1.3 ± 0.1	0.7–2.0	1.3 ± 0.1	0.7–2.0	1.3 ± 0.1	0.7–2.0
Sodium:potassium ratio	33.6 ± 0.7	14.7–53.8	ND	ND	33.6 ± 0.7	14.7–53.8	ND	ND	33.6 ± 0.7	14.7–53.8

ALT, alanine aminotransferase; AST, asparagine aminotransferase; BUN, blood urea nitrogen; CPK, creatine phosphokinase; GGT, γ-glutamyl transpeptidase; LDH, lactate dehydrogenase.

When values in the same row are the same, the cohorts are statistically equivalent.

Discussion

The results described here highlight the differences in important biologic parameters between healthy adult Xenopus from different sources (laboratory-bred and -reared compared with wild-caught) and between adult male and female frogs. Previously described studies of X. laevis hematology did not report adequate numbers of frogs or include extensive documentation of the animal's health, age, source, gender, diet, or other environmental factors (water quality, ambient temperature, light cycles, etc.), which are necessary to establish the reference ranges and suitability of the samples.¹² Our results show that healthy laboratory- raised, sexually mature female frogs from 2 different vendors and wild-caught frogs differed significantly in baseline hematologic and serum biochemical values. Xenopus in research often are maintained at a variety of water temperatures according to the preference of the researchers, and changes associated with cold adaptation, including hematologic effects, have been documented.^8,10 Additional factors likely to affect amphibian blood values have been described.¹⁹ Therefore, these results are applicable to frogs housed in similar conditions and of similar genetic background.

Intercohort variation can be explained in part by variation in environmental factors. For example, the significantly higher hematocrit and hemoglobin levels observed in the wild-caught frogs may be due, in part, to cold adaptation (adaptation to cooler water temperatures), which is known to increase hematocrit and hemoglobin in amphibians.^20,21 Similarly, the notable increase in WBC counts in wild-caught frogs may be due to capture-associated stress (if frogs respond to stress with the typical ‘stress neutrophilia’ that mammals show) not present in the laboratory-conditioned frogs accustomed to human handling. The marked variation in RBC count, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume between the cohorts from the 2 vendors is likely due to variation in environmental factors such as diet, and water quality, along with divergent genetic backgrounds. Notably, compared with those of other species, the RBC count, hemoglobin, hematocrit, and mean corpuscular hemoglobin values reported here for laboratory Xenopus are more similar to those reported for a semiterrestrial amphibian species (the common toad, Bufo bufo),⁵ the American bullfrog (R. catesbeiana), and the Peru coast toad (B. s. limensis) rather than finfish.^3,5 Not surprisingly, the X. laevis hematogram reference range intervals (Figure 2) are most similar those of to another fully aquatic amphibian, the Eastern hellbender (C. alleganiensis), with most intervals overlapping.¹⁹

In comparing serum electrolyte values for X. laevis with those obtained for other frogs, the values are comparable to those obtained for both the American bullfrog, B. bufo, and the boreal toad, Anaxyrus (Bufo) boreas boreas.^3,15 Not unexpectedly, the electrolyte values for fully aquatic Xenopus were not significantly different from those of the fully aquatic Eastern hellbender, C. alleganiensis. In addition, serum calcium and sodium values were statistically higher in female than male frogs in the current study. More male frogs need to be sampled to confirm the finding, but this trend has also been observed with larger samples in both hellbenders and bullfrogs.^3,19

The dearth of published clinical hematologic reference intervals for frogs and amphibians in general precluded extensive comparison of clinical biochemistry of all analytes. However, rigorously calculated values for some analytes are available for the Eastern hellbender, C. alleganesis,¹⁹ and the American bullfrog, R. catesbeiana.³ Blood protein values—albumin, globulin, and total protein—are similar between hellbenders, bullfrogs, and X. laevis, and values were not significantly different for blood urea nitrogen and uric acid.^3,19 The terrestrial bullfrog has higher uric acid and creatinine values, but its BUN level was not statistically different from that for Xenopus or Cryptobranchus.^3,19 In general, the laboratory values we report here are in line with those described for other amphibian species and are most like those reported for the only other fully aquatic species studied in detail to date, C. alleganiensis (Figure 2).

The recent completion of the X. tropicalis genome¹¹ (the smaller, genetically close, South African clawed frog species) revealed striking similarities to the chicken genome.¹¹ To determine whether the recently identified genetic similarity with chickens translated to hematologic or biochemical similarities with reference ranges for chickens or with the well-established references ranges for more genetically distant species (fish, mice, dogs), we compared the calculated Xenopus laevis reference ranges with those of the domestic chicken (Gallus gallus),^4,6,14 the finfish tilapia (Oreochromis hybrid),¹³ mice, ^16,18 and dogs¹⁷. As illustrated through the findings we report here, the clinical hematologic and serum biochemical laboratory values for X. laevis, a fully aquatic and ectothermic amphibian species, differ from those of mammals, birds, and fish. The reference range values we describe for X. laevis likely will be useful to clinicians and researchers who may not have access to a veterinary diagnostic laboratory that maintains range intervals for laboratory X. laevis.