Hematology and Biochemistry Reference Intervals for American Alligator (Alligator mississippiensis) in South Florida, USA

The American alligator (Alligator mississippiensis) is a well-studied crocodilian species, yet a comprehensive set of hematologic and biochemistry reference intervals (RI) does not exist for wild populations. Most studies reporting on alligator blood parameters present summaries of values as some combination of means, SDs, and ranges rather than true reference intervals (Barnett et al. 1997; Lance and Elsey 1999; Schoeb et al. 2002; Teare 2010 in Carpenter 2013; Hamilton et al. 2016; DiGeronimo et al. 2017; Faulkner et al. 2019). We used the American Society of Veterinary Clinical Pathology guidelines (Friedrichs et al. 2012) to establish RI for 48 hematological and blood biochemistry parameters for alligators in South Florida, US. Blood parameters included a reptile panel augmented with parameters that could be related to nutrition, dehydration, and stress. We compare our results to other studies reporting alligator blood parameter values.

Our reference population of subadult and adult alligators (≥125 cm total length) includes animals of both sexes captured in fall and spring from freshwater wetlands in South Florida: Arthur R. Marshall Loxahatchee National Wildlife Refuge, Florida Panther National Wildlife Refuge, Water Conservation Area 3, and Everglades National Park. We captured alligators at night from October 2017 through April 2019 using standard protocols (Mazzotti et al. 2010). Our target was >30 animals in each season to reach >120 animals in total. Alligators were captured in natural habitats at least 1 km from any canal to minimize the influence of human-made habitat. We recorded capture location and time using a handheld GPS unit (GPSMap 64s or 60CSx Garmin Ltd., Olathe, Kansas, USA), along with the alligator's snout-vent-length, total length, tail girth, mass, sex, and time (after capture) of blood draw (see Jennings et al. 2020). All alligators were released at their capture site following sampling and data collection.

Alligators were excluded from analysis if they had a major deformity that would affect or compromise their fitness in the wild or their health and ability to function normally. This included major injuries such as loss of their tail after single scute 8, or a combination of two or more of the following: broken jaw, missing eye, missing more than two limbs. Alligators deemed unhealthy with a body condition index (calculated by Fulton's K) below 1.60 were excluded. This cutoff was determined by reviewing >4,000 records of alligator captures by University of Florida, in which alligators with a K value below 1.60 were found to be emaciated and otherwise in extremely poor condition.

We collected blood samples soon after capture to minimize effects of capture stress on analytes informative of stress. Samples were withdrawn from the ventral coccygeal vein below the transverse process of tail vertebrate using a 20 G × 1.5-in Luer-Lok tip with 6 mL syringe (BD Medical Company, Franklin Lakes, New Jersey, USA). Depending on volume collected, samples were either transferred into a 3 mL Lithium Heparin Vacutainer (BD Medical Company) or microvette 500 µL Lithium Heparin vials (Starsted Inc., Nümbrecht, Germany) and inverted 7–8 times to ensure even mixing. Due to humid and rainy field conditions, blood smears were not performed at time of blood draw. Finger et al. (2015) found that alligator blood samples can remain stable for hematologic analysis within 24 hr if properly stored; we stored vials in a cooler with an insulating layer to avoid direct ice-sample contact, minimizing risk of freezing and lysing blood cells.

Samples from 120 alligators (40 male, 80 female) ranging in size from 136.2 cm to 279.7 cm in length were determined suitable for RI calculation. Samples were transferred to University of Miami's Comparative Pathology laboratory (Miami, Florida, USA) within 12 hr of collection, for blood smear preparation and analysis. Samples were rejected if they did not meet University of Miami's quality check. Blood smears were prepared on full slides. Plasma was obtained by spinning blood samples for 5 min at 9,391 × G in an Eppendorf 5254 R centrifuge (Eppendorf AG, Hamburg, Germany). We performed complete blood counts with differential using the Natt and Herrick (1952) method with Wright Giemsa stain at 1,000×. We used Natt and Herrick's stain (Vetlab, Miami, Florida, USA) and a hemacytometer with improved Neubauer counting chamber (VWR International, Radnor, Pennsylvania, USA) for absolute white and red blood cell counts. Hematocrit (packed cell volume) was determined using a Haematokrit 200 centrifuge (Andreas Hettich GimbH & Co. KG, Tuttingen, Germany) at 10,000 rpm (9,903 × G) for 5 min. Serum biochemistry was performed using Vitros 250 (Ortho, Rochester, New York, USA), bile acids and hydroxybutyrate reagents (Randox, Kearneysville, West Virginia, USA) with RX Daytona (Randox). Total protein, albumin/globulin ratio, albumin, alpha-1 globulins, alpha-2 globulins, beta globulins, and gamma globulins were measured via electrophoresis, using Helena reagents and SPIFE 300 analyzer (Helena, Beaumont, Texas, USA). Plasma was banked at –80 C for later corticosterone analysis in batches at the end of the sampling season using MP Biomedical reagents (Solon, Ohio, USA) and Genesys counter (Laboratory Technologies, Inc., Elburn, Illinois, USA). Biochemistry values outside any instrument's linear range were excluded from analysis.

We performed statistical analyses using MedCalc Statistical Software for Windows, version 1 (MedCalc Software Limited, Ostend, Belgium) and calculated 95% RIs and associated 90% confidence intervals (Tables 1, 2). We used the robust method for determining RI based on Friedrichs et al. (2012) for sample sizes between 40 and 120. Data were assessed for normality using Shapiro-Wilk test. If normality was rejected, we transformed data using logarithmic or box-cox transformations, evaluated histograms, and reassessed for normality. Outliers were identified using Tukey's interquartile range approach and excluded from the data set.

We found seven publications that reported blood parameter values for alligators as means or medians with SDs or SEs and, in some cases, ranges. Four used wild-caught animals; two of these involved adult alligators. The remaining studies were of juveniles and captive animals (Barnett et al. 1997; Lance and Elsey 1999; Schoeb et al. 2002; Teare 2010 in Carpenter 2013; Hamilton et al. 2016; DiGeronimo et al. 2017; Faulkner et al. 2019). The majority of reported blood parameter values were similar to ours and fell within our RI (Tables 1, 2). Basophils, eosinophils, aspartate aminotransferase, albumin/globulin ratio, and albumin showed the most deviation from our reference intervals.

Comparing our RI to blood parameter values measured in other wild-caught alligators could provide insight into what factors (e.g., environmental stressors, diet, habitat, food availability, geographic location) might contribute to variation in these values. Furthermore, comparisons to other wild populations will allow us to determine if RI calculated for South Florida alligators are more broadly representative.

Data collected for this study were conducted under University of Florida IACUC study 201509072, Arthur R. Marshall Loxahatchee National Wildlife Refuge Research and Monitoring Special Use Permit 6B16-003, Florida Panther National Wildlife Refuge Research and Monitoring Special Use Permit 41545-201705R, Florida Fish and Wildlife Conservation Commission Scientific Collecting Permit SPGS-13-58 and SPGS-17-62, and Department of the Interior National Park Service Scientific Research and Collection Permit EVER-2016-SCI-0014 under study EVER-00093. Funding for this project was provided by US Fish and Wildlife Service through a Cooperative Agreement (F17AC01071) through the South Florida Caribbean Cooperative Ecosystems Studies Unit. Blood analyses were conducted by the Comparative Pathology laboratory at the University of Miami Miller School of Medicine in Miami, FL. The following people helped capture alligators and collect blood: M. Barazowski, M. S. Cherkiss, S. Cooke, J. Dalaba, M. Danaher, M. Denton, S. Farris, A. Godahewa, S. Godfrey, B. Mason, E. Metzger, and other University of Florida Croc Docs staff. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the US Fish and Wildlife Service. Any use of trade, firm, or product names is for descriptive purposes and does not imply endorsement by the US Government.