Biochemistry and hematology parameters of the San Cristóbal Galápagos tortoise (Chelonoidis chathamensis)

The San Cristóbal Galapagos tortoise, Chelonoidis chathamensis, is native to San Cristóbal, the most eastern and oldest island of the Galapagos Archipelago. Although numerous studies on the ecology, behavior and genetics of Galapagos tortoises have been published, little is known about the health status of these animals. Here we report blood values from clinically healthy tortoises.


Introduction
The San Cristóbal Galapagos tortoise, Chelonoidis chathamensis, is a species of chelonian native to San Cristóbal, the most eastern and oldest island of the Galapagos Archipelago, and the second settled by humans (Parent et al., 2008). While the taxonomy of Galapagos tortoises has undergone a number of major shifts and revisions over the past several decades (Loveridge and Williams, 1957;Pritchard, 1996;Caccone et al., 1999Caccone et al., , 2002Caccone et al., , 2004Paquette et al., 2012), it is currently widely accepted that 15 species are extant, and most of these are endemic to a specific island (Poulakakis et al., 2015).
Although numerous studies on the ecology, behavior and genetics of Galapagos tortoises have been published, little is known about the health status of these animals. Peripheral blood chemistry and hematology parameters are useful diagnostic tools in health assessment and management in the  reptiles (Geffre et al., 2009;Zhang et al., 2011;Gibbons et al., 2013;Lewbart et al., 2014;Muñoz-Pérez et al., 2017) and many other taxa. To detect and monitor changes in the health of individuals and populations, it is paramount to determine species-specific normal values for blood parameters of interest. Reference intervals for many tortoise species exist (Marks and Citino, 1990;Gottdenker and Jacobson, 1995;Hart et al., 1991;López-Olvera et al., 2003;Zaias et al., 2006;Scope et al., 2013;Eshar et al., 2014;Andreani et al., 2014;López et al., 2017), in addition to recommended handling methods of blood films in the field (Sheldon et al., 2016): characteristics that allow a comparative margin when generating new information.
In this study, we evaluated selected blood gas, biochemical, and hematology parameters along with vital signs and standard measurements from 32 semi-free-ranging Galapagos tortoises (C. chathamensis) at a captive breeding facility on the island of San Cristóbal. While this is an important component of a pre-introduction health assessment it should be noted that infectious disease screening might be warranted in some cases and can be a valuable part of such an effort.

Ethics statement
This study was performed as part of a population health assessment authorized by the Galapagos National Park Service (GNP) through agreement and request from the GNP resident veterinarian Andrea Loyola and Diego Páez-Rosas; Juan Pablo Muñoz-Pérez from the Galápagos Science Center (GSC). Approved by the GSC ethics and animal handling protocol. All handling and sampling procedures were consistent with standard vertebrate protocols and veterinary practices.

Study area
The Galapageura de Cerro Colorado is a captive giant tortoise breeding facility and sanctuary located on the northeast side of the San Cristóbal island,~20 km from the town of Puerto Baquerizo Moreno. This~12 hectares facility is home to~140 free-ranging tortoises that are part of the breeding program or are progeny of the adults.

Sampling
Captures and sampling were performed in March 2017 with the assistance and support of Galapagos National Park (PNG) personnel. Tortoises were located, positively identified by microchip, and selected for examination. This group was part of a program introducing tortoises to small farms (fincas) in the highlands of the San Cristóbal Island. This program, a collaborative effort between the PNG and local farmers, is designed to engage the farmers in conservation of a native species with the potential to damage crops. The hope is that tourism and the good will generated will result in mutual sustainability.
After a blood sample was obtained from each tortoise (see below), a thorough physical examination was performed including body temperature, morphometric measurements and fecal sample obtained (see below).

Blood sample collection and handling
Blood samples were collected within an average of 5 min of capture. Blood samples of~2.5 ml were obtained from either the brachial sinus or dorsal coccygeal vein using a heparinized (Heparin Sodium USP, 1000 units/ml; APP Pharmaceuticals, LLC, Schaumburg, IL, USA) 1.0 or 1.5 inch 22-gauge needle attached to a 3.0 ml syringe. The blood was then immediately divided into sub-samples which were used to make blood films on clean glass microscope slides, stored on ice in Eppendorf vials for laboratory analyses of manual hematocrit and total solids, and loaded into iSTAT cartridges or onto lactate strips within 10 min of sample collection.

Blood gas and biochemistry parameters
An iSTAT portable clinical analyzer (Abbot Point of Care Inc., Princeton, NJ, USA) was used to obtain biochemistry, blood gas, and electrolyte results utilizing Chem8 cartridges (Abbot Point of Care Inc., Princeton, NJ, USA). The following parameters were measured and recorded: sodium (Na), potassium (K), chloride (Cl), ionized calcium (iCa), Total CO 2 , glucose (GLU), blood urea nitrogen (BUN), hematocrit (HCT), hemoglobin (Hb), anion gap and lactate. Blood lactate was determined using a portable Lactate Plus TM analyzer (Nova Biomedical, Waltham, MA, USA).

Hematology
Manual hematocrit (PCV%) was determined using highspeed centrifugation of blood-filled micro-hematocrit tubes and total solids (TS) via plasma analysis with a refractometer. Differential white blood differential counts were conducted by examining 100 white blood cells on a peripheral smear stained with Wright-Giemsa stain. The estimated total white blood cell counts were determined by averaging the number of white cells in 10 high-powered fields and multiplying by 2000 (Durbin et al., 2009).

Morphometric measures, body temperature and fecal sample collection
For each tortoise captured morphometric measurements were taken using a flexible 100 cm measuring tape. These included curved carapace length (CCL), curved carapace width (CCW), plastron length and shell depth. In addition, each tortoise was weighed with a precision of ±0.5 kg. Sex was determined in mature animals by plastron concavity and tail length. An EBRO ® Compact J/K/T/E thermocouple thermometer was used to obtain all temperature readings (model EW-91219-40; Cole-Parmer, Vernon Hills, IL, USA 60061). Core body temperatures were recorded from the cloaca using the probe EBRO ® T PVC epoxy tip 24GA × 1 m in length, which was inserted~10 cm into the vent.
Respiratory rates per minute were measured by visualization and the heart rate with a Doppler ultrasound probe (Parks Medical Electronics, Inc., Aloha, OR, USA) in the area of the carotid or femoral artery.
Twenty-five of the tortoises defecated during handling. Fecal samples from these tortoises were collected, placed in Eppendorf tubes, and brought back to the laboratory for direct microscopic examination. A small fecal sample was placed on a slide with a coverslip and examined with a compound microscope using a 10× and 40× objective.

Statistical analysis
We used ANOVA to test for differences in all morphometric, blood gas and hematology parameters between males, females and juveniles. We then ran post-hoc pairwise Wilcoxon rank sum tests when appropriate. A standard alpha of P = 0.05 was used for all statistical tests. We compared manually and iSTAT-derived hematrocrit values with a Passing-Bablok regression. All analyses were performed in R 3.4.1.
All of the tortoises were bright, alert and responsive. Thirty-one of the 32 tortoises were deemed healthy enough for release to the fincas with the recommendation that one tortoise with an ocular discharge be reexamined by a veterinarian prior to release. Blood samples were drawn from a total of 32 animals but complete results were not obtained for every individual due to sample or analysis errors.

Blood biochemical and hematology analysis
Tables 2 and 3 report the biochemistry, blood gas and hematology results. There were no statistically significant differences between males, females and juveniles in most of the blood chemistry and hematology parameters, including Similarly, males and females did not differ in any hematology parameters, so we lumped them into a single 'adult' category for these traits (Table 3). However, we did find significant differences between some of the age/sex groups in Na Post-hoc analyses showed that juveniles had lower total solids than adults (W = 103, P = 0.004), males had lower Na levels than juveniles (W = 14.5, P = 0.004), males had lower Cl levels than females (W = 28, P = 0.041) or juveniles (W = 15, P = 0.004), and juveniles had lower BUN levels than males (W = 93.5, P = 0.031). Manually determined hematocrit values were not systematically and proportionally different from iSTAT hematocrit values, with manually determined values being higher.

Fecal analysis
No helminth ova or other parasites were observed on direct microscopic examination of feces from 25 tortoises.

Discussion
Although many studies have reported baseline biochemical, blood gas and hematology values for other chelonian species (Gottdenker and Jacobson, 1995;Andreani et al., 2014;Eshar et al., 2014Eshar et al., , 2016 nothing comprehensive has yet been published on Galápagos tortoises. Knafo et al. (2011) and Rivera et al. (2011) conducted studies based on sterilization of Galapagos tortoises and included PCV and total solids values on females and males. In both studies the PCV % and TS values are higher than those of our study. One difference is the mean TS of the females was 7.8 g/dl in Knafo et al. (2011) and that of our females was 4.8 g/dl. Blake et al. (2015) measured PCV and TS values for 37 wild adult female tortoises (Chelonoidis porteri) on Santa Cruz in two locations (Cerro Fatal and La Reserva) as part of a feeding ecology study. While the both PCV and TS were positively correlated with elevation, the differences were not significant. The average mean PCV and TS for the two groups was 20.9% and 7.9 mg/dl, respectively. This PCV value is just slightly higher than the tortoises in our study but the TS is much higher. This could be related to the reproductive status of the females. 1974b; Rostal et al., 1998). While a definitive explanation for this discrepancy is unknown, another possibility includes intrageneric differences between C. chathamensis, C. porteri and C. nigra. However, Rivera et al. (2011) report that males of C. nigra have similar TS values in relation to the males monitored in our study (5.6 and 5.2 g/dl, respectively), so the interspecific difference between males is much smaller than between females.
We judged the tortoises we examined to be clinically healthy and the hematology (total white cell/differential counts) and blood chemistry values were generally consistent with those reported for other healthy tortoise species (Marks and Citino, 1990;Gottdenker and Jacobson, 1995;López-Olvera et al., 2003;Zaias et al., 2006;Scope et al., 2013;Eshar et al., 2014;Andreani et al., 2014;López et al., 2017). One exception was the PCV%, which on average was lower in the Galapagos tortoises compared to desert tortoises, Gopherus agassizii, (Christopher et al., 1999), Hermann's tortoises, Testudo hermanni (Andreani et al., 2014), ploughshare tortoises, Astrochelys yniphora (López et al., 2017), radiated tortoises, Geochelone radiata (Zaias et al., 2006), and African spurred tortoises, Geochelone sulcata (Eshar et al., 2016). This could be related to species size, as the next lowest mean PCV% of this group was the sulcata, second largest of the included species. A finding relevant to future methodologies is that the iSTAT proved accurate in determining PCV in Galapagos tortoises. In some animals iSTAT hematocrit values are lower than the manually obtained PCV%. These species include loggerhead sea turtles (Wolf et al., 2008), rainbow trout (Harter et al., 2014), marine iguanas (Lewbart et al., 2015) and Quaker parrots (Rettenmund et al., 2014). Thus, for Galápagos tortoises, the Chem 8 cartridge used with an iSTAT reader provided an accurate estimation of the PCV%.
The mean total white blood cell counts (WBC) of the Galapagos tortoises in our study were lower than those reported in 42 wild Santa Cruz Galapagos tortoises (Sheldon et al., 2016). This robust study examined several methods of determining the WBC, including an estimated count using multiple fields with a 40× objective (Sheldon et al., 2016). While there was overlap with the ranges, their mean total count was 8.85 × 10 3 cells/μl, compared to our mean of 3.79 × 10 3 cells/μl. The San Cristóbal Galápagos tortoise WBC values were just slightly lower than those reported for other tortoise  The asterisks indicate significant differences between males and juveniles.  (Christopher et al., 1999;Zaias et al., 2006;López et al., 2017). With so many variables between the species, like age, size, and time of year, making direct comparisons with differential counts is challenging. One noteworthy finding was that the San Cristóbal Galapagos tortoises had higher numbers of monocytes than the Santa Cruz tortoises and other species in the family Testudinidae. This fact might be worth pursuing with a larger and more comprehensive hematology study.
The small sample size of our study precluded the calculation of formal reference intervals, a process that would require a minimum of 120 individuals (Campbell, 1995;Geffre et al., 2009;Friedrichs et al., 2012). In addition, all of the samples were obtained on one day; thus, the possibility exists that sampling at different times of the year would yield different results (Campbell, 2006;Zaias et al., 2006;Scope et al., 2013). This was the case with a population of radiated tortoises (G. radiata) on St. Catherine's Island, GA, USA (Zaias et al., 2006) that was sampled in both January and August of the same year.
Although direct fecal examination of 25 animals did not reveal any helminth parasites or ova, this does not represent a comprehensive parasite screening, as there are far more sensitive tests that were not undertaken for logistical and cost reasons.
In summary, data reported in this study represent an important step toward determining a baseline range of values against which future blood gas and biochemistry results in Galapagos tortoises can be compared. Future research should work toward establishing reference values in the other Chelonoidis species, as well as expanding sampling to facilitate comparisons of blood values across age groups and seasons.