DETERMINATION OF PLASMA BIOCHEMISTRIES, IONIZED CALCIUM, VITAMIN 03, AND HEMATOCRIT VALUES IN CAPTIVE GREEN IGUANAS (Iguana iguana) FROM EI SALVADOR

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1 DETERMINATION OF PLASMA BIOCHEMISTRIES, IONIZED CALCIUM, VITAMIN 03, AND HEMATOCRIT VALUES IN CAPTIVE GREEN IGUANAS (Iguana iguana) FROM EI SALVADOR Javier G. Nevarez 1, DVM, Mark A. MitcheI1 1 *, DVM, MS, PhD, Casey Le Blanc 1, DVM Peter Graham BVMS, PhD, CertVR, MRCVS 2 1Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA 70803, USA; 2Veferinary Diagnostic Endocrinology, Animal Health Diagnostic Laboratory, Michigan State University, Lansing, MI 48909, USA Abstract: The green iguana is a reptile that is routinely presented to veterinary hospitals for diagnostic evaluation. Hematology is an important diagnostic tool used by veterinarians to assess the health status of their patients. The purpose of this study was to evaluate and characterize the hematological parameters of captive adult male and female green iguanas in EI Salvador and establish appropriate reference ranges. Key Words: Iguana, Iguana iguana, iguana plasma chemistry, reptile Introduction As non-traditional species medicine continues to expand, so does the need for better diagnostic tests and accurate interpretation of the tests currently available. One major factor affecting test reliability has been the limited amount of information concerning reference intervals for blood parameters from nontraditional species. The complete blood count (CSC) and plasma/serum biochemical 'assay are frequently used diagnostic tools when evaluating the health status of reptiles. However, interpretation of the diagnostic test results is often dependent on the veterinarian's personal experience or a limited number of reference sources. Unfortunately, for the average veterinary practitioner, the paucity of scientifically derived reference values undermines their confidence in interpretation of test results. Therefore, it is only reasonable that we continue to investigate the importance of hematologic data for reptiles and other nontraditional species. The purpose of this study was to determine the hematological and biochemical reference values from captive, adult green iguanas, Iguana iguana, from EI Salvador. Materials and Methods Sixty-seven adult (two to four years old) green iguanas were randomly sampled from a commercial iguana farm in Costa del Sol, EI Salvador in March Of the sixty-seven iguanas, 29 were males, 17 were non-gravid females, and 21 were gravid females. The iguanas were kept in open outdoor pens on a dirt substrate, with almond trees and bamboo shelters for shade. Iguanas were housed in groups of up to 20,000, however, each of the three groups (males, non-gravid females, and gravid females) were housed separately. Water stations 2002 Proceedings Association of Reptilian and Amphibian Veterina~ians 81 ~

2 within the pens were refilled as needed. The iguanas were fed locally grown forage and a commercial iguana chow four times a week. The environmental temperature in the direct sunlight ranged between C ( F), while the temperature recorded under the bamboo shelters ranged between C( F) during the same time period. The environmental humidity ranged from 24%-36% and 280/0-37% respectively. A single night temperature 25 C (78.3 F) and humidity (56%) were also recorded. Sample collections were performed between 9:00 am and 3:00 pm daily and the samples were collected over a 72 hour period. The iguanas were captured by hand and physically restrained for venipuncture. A 6cc syringe with a 22gauge needle was used to obtain 3ml of blood from either the jugular vein or the ventral coccygeal vein. Blood samples were equally divided into a lithium heparin tube and a serum clot tube for analysis. Following blood collection, samples were taken to an on-site field laboratory for initial testing. Blood smears were made immediately after mixing five drops of blood with one drop of 220/0 bovine albumin (gamma Biologicals Inc., Houston TX 77092) to help preserve integrity of the cells. After being air-dried, the blood smeared slides were transported to the Louisiana State University School of Veterinary Medicine (LSU-SVM) where they were stained using a modified Wright's stain (Hema Tek Stain Pak, Bayer Corporation, Elkhart, Indiana, USA). Packed cell volume was measured using micro-centrifugation. Blood biochemical analyses were performed on site using an j-stat portable analyzer (i-stat Corporation, 104 Windsor Ce r Dr., East Windsor, NJ, USA). An EG 7+ cartridge (Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO USA) was used tp measure ionized calcium. Plasma samples were transported to the LSU-SVM on frozen gel packs and the samples were processed using an Olympus AU 600 chemistry machine (Olympus, Melville, NY, USA) within hours after collection. The following values were measured: glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total protein (TP), albumin, globulin, creatinine, calcium, phosphorus, sodium, potassium, chloride, TC02, anion gap, and uric acid. Serum samples were transported on frozen gel packs to the Michigan State University Animal Health Diagnostic Laboratory for 25-hydroxy-vitamin 0 analysis. Statistical analyses The mean, median, standard deviation and range were determined for each parameter. Any value recorded as a percentage was log transformed. A Shapiro Wilk test was used to determine if the data was normally distributed. A 950/0 confidence interval was calculated for those parameters that had a normal distribution. A 950/0 frequency distribution was calculated for data that was not normally distributed. An ANOVA test was used to assess between group differences for normally distributed data. Any between group differences were further evaluated using a Tukey's test to determine within group differences. For Proceedings Association of Reptilian and Amphibian Veterinarians

3 the data that did not have a normal distribution, a Kruskal-Wallis one-way ANOVA and Dunn's test were used to assess differences between and within groups respectively. The observed power was calculated for those parameters where no statistical difference was found. Values of P<O.05 were considered statistically different. Statistical analysis was performed using SPSS 8.0 (SPSS Inc., Chicago, IL). Results No statistical differences were detected between the three groups (males, females, gravid females) for ALP, total protein, globulin, calcium, phosphorus, and hematocrit. Significant differences (p<0.05) were observed between males and gravid females for glucose, AST, ALT, CK, albumin, sodium, potassium, chloride, uric acid, and vitamin 03. Also a significant difference (p<o.05) was observed between females and gravid females for AST, ALT, CK, albumin, sodium, potassium, TC02, uric acid, and ionized calcium. Significant differences (p<o.05) were detected between males and females for TC02 and chloride. Reference ranges were calculated for those values with a normal distribution within the groups (Table 1-3). iscussion/conclusion Glucose, AST, ALT, CK, albumin, sodium, potassium, TC02, uric acid and vitamin 0 were higher in gravid females than non-gravid females and males. These findings are different from those previously reported (Harr at ai., 2001). This difference may be associated with the time in the breeding cycle that the samples were collected. Female iguanas in this popu'lation generally oviposit in January or February. The population of gravid females used in this study was considered to be late for oviposition. These animals may have either had underlying health problems or a delayed reproductive cycle, which the farm manager reported occurs on an annual basis with a small population of iguanas on the farm. The biochemical parameters may have been different if the gravid females were sampled earlier in the season. An increase in plasma calcium, phosphorus and total protein is considered a normal consequence of folliculogenesis (Harr, et ai, 2001; Campbell, 1996). However, there was no statistical difference between males, females, and gravid females for any of these parameters. Because the gravid females were in late term and the eggs were shelled, the transient increase of the aforementioned values had likely already occurred. Further research should be performed to evaluate these parameters before, during and after folliculogenesis to increase our understanding of the reproductive physiology of the female green iguana. Of additional importance was the measurement of ionized calcium, the biologically active form of calcium in the plasma. Levels of ionized calcium can decrease with alkalosis and increase with acidosis (Dennis, et ai, 2001.) Therefore, various changes in metabolism can alter the fraction of ionized calcium available during the reproductive cycle. In this study, there was a 2002 Proceedings Association of Reptilian and Amphibian Veterinarians 89

4 significant difference in the levels of ionized calcium between females (1.30 mmol/l) and gravid females (1.21 mmol/l). This differs from the previously published report by Dennis eta al. (2001). Because gravid females mobilize large quantities of protein (e.g., albumin) bound to calcium during the reproductive cycle, it might be expected that gravid females have lower ionized calcium than non-gravid females. However, because the iguanas tested in this study had already produced fully developed eggs, the expectation that they were still mobilizing large quantities of protein might not be as great. A future study that evaluated the ionized calcium throughout the reproductive cycle of the green iguana may provide more insight to the interpretation of these results. The metabolic status of gravid and non-gravid females must also be taken into consideration. Vitamin 0 3 is an essential hormone that plays a key role in the absorption and utilization of calcium in reptiles. In this study, vitamin D31evels were normally distributed and there was a significant difference between males and gravid females. The increased levels of vitamin D3 in females is likely associated with the importance of the hormone in the reproductive cycle. Lower values of vitamin D3 in males may be explained by the lack of a high demand for calcium during folliculogenesis and egg preparation. It is important to take these values in the context of which they were collected. These values are applicable to iguanas exposed to natural sunlight in EI Salvador or similar locations. Variations in these values may be observed at different geographical locations. A significant difference between and within groups was noted in creatine kinase (CK) values. Males ( Ull) and gravid females ( U/L) had higher levels of CK than non-gravid females (845.6 U/L). The values reported in the gravid females could result from muscle necrosis, which is generally associated with the cachexia/anorexia many gravid female iguanas experience during gestation. Other factors contributing to the high CK levels reported in both males and gravid females may include venipuncture and stress during capture. The mean AST values for gravid females (80.2 U/L) were significantly higher than those of males (39.6 U/L) and non-gravid females (24.4 U/L). These higher values in AST may also be associated with muscle necrosis or changes in the hepatocytes associated with the mobilization of albumin. There was no significant difference between groups for total protein or globulin. A significant difference for albumin was observed between gravid females and both males and non-gravid females. Although the difference between the groups was statistically significant, the biological relevance of these differences is probably minimal (males=2.4, females=2.5, gravid females=2.7 G/dL). Similar findings were observed for sodium and chloride, which had significant differences between and within groups. Higher mean values of sodium and chloride might be explained by dehydration. However, there was no significant difference for hematocrit or total protein values between the groups to support Proceedings Association of Reptilian and Amphibian Veterinarians

5 differences in hydration status. Because the animals were all fed a similar diet, the likelihood that the difference in the electrolytes was associated with dietary intake should also be considered limited. There was a significant difference in the uric acid levels between gravid females (9.4 mg/dl) and both males (5.3 mg/dl) and non-gravid females (4.6 mg/dl). This elevation in uric acid does not correlate with changes in the electrolytes as non-gravid females had a higher mean value of chloride (118 mmol/l) than gravid females (116.3 mmoi/l). Because these animals may be utilizing muscle tissue for energy (CK), and uric acid is the end product of nitrogen break down in reptiles, the elevated uric acid levels in these gravid females would be expected. Similarities between our results and those previously published must be interpreted within the context of the sample size and conditions of the population analyzed. Future studies that focus on specific physiologic behaviors (e.g., the complete reproductive cycle of the green iguana) are needed to increase our understanding of these popular pet reptiles. References Harr KE, Alleman AR, Dennis PM, et sl Morphologic and cytochemical characteristics of blood cells and hematological and plasma biochemical reference ranges in green iguanas. J Am Vet Med Assoc, 218(6): Campbell TW Clinical pathology. In Mader DR (ed) Reptile Medicine and Surgery. WB Saunders Co., Philadelphia, PA Dennis PM, Bennett RA, Harr KE, Lock SA Plasma concentrations of ionized calcium in healthy iguanas. J Am Vet Mad Assoc, 219(3): Proceedings Association of Reptilian and Amphibian Veterinarians 91

6 Table 1-Reference ranges ofplasma biochemical values Males (n = 29) Females (n =17) Gravid Females (n =21) Variable Mean (+/- SD) Range Mean (+/- SD) Range Mean (+/- SD) Range Glucose (mg/dl) (+/- 78.2) ALT (U/L) 7.1 (+/- 4.0) ALP (U/L) 49.3 (+/- 38.8) Tot. Prot. (G/dL) 5.8 (+/-.98) (+/- 1.24) Albumin (G/dL) (+/- 0.4) (+/- 0.4) Globulin (G/dL) 3.4 (+/- 0.8) (+/- 1.0) (+/- 1.0) Calcium (mg/dl) 11.1 (+/- 1.4) Phosphorus (mg/dl) 5.4 (+/- 2.6) Sodium (mmol/l) (+/- 6.6) (+/- 7.0) Potassium Chloride (mmoill) (+/- 7.8) (+/- 4.4) TC02 (mmoill) (+/- 8.2) (+/- 11.2) (+/- 17.0) Uric Acid (mg/dl) 5.3 (+/- 2.0) (+/- 2.4) SO = two standard deviations 1- Statistical difference for females and gravid females 2- Statistical difference for males and females 3- Statistical difference for males and females; females and gravid females Proceedings Association of Reptilian and Amphibian Veterinarians

7 Table 2 - Reference ranges ofvitamin D3 (25 hydroxy-vitamin D) Males (n =18) Females (n =17) Gravid Females (n =15) Variable Mean (+/- SD) Range Mean (+1- SO) Range Mean (+1- SO) Range Vitamin 0 3 (nmoi/l)* 188.9( ) (+1-159) ( ) so =two standard deviations * Statistical difference for males and gravid females Table 3 - Reference ranges ofionized calcium Variable i-calcium (mmoi/l) Mean (+1- SO) Range Mean (+1- SO) Range 1.32 ( ) (+1-0.2) SD = two standard deviations 2002 Proceedings Association of Reptilian and Amphibian Veterinarians 93