What Type Of Animal Is Reptile Mammal Amphibian Radiated Tortoises Radiated Tortoises

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Hematology and biochemistry of critically endangered radiated tortoises (Astrochelys radiata): Reference intervals in previously confiscated subadults and variability based on common techniques

• Maris Brenn-White,
• Bonnie L. Raphael,
• Ny Aina Tiana Rakotoarisoa,
• Sharon L. Deem

x

• Published: March 14, 2022
• https://doi.org/ten.1371/periodical.pone.0264111

Figures

Abstract

Madagascar's radiated tortoises (Astrochelys radiata) are critically endangered, threatened by illegal collection, and confiscated in alarming numbers in recent years. Robust population- and technique-specific hematology and biochemistry reference intervals are valuable still heretofore missing tools for triage, rehabilitation, and reintroduction of confiscated radiated tortoises. Nosotros determined reference intervals in 120 previously confiscated, clinically healthy subadult radiated tortoises living under homo care inside their native habitat at the Tortoise Conservation Middle (TCC). Specific analytes measured were manual packed cell volume, total solids, white blood cell (WBC) count and differentials, and biochemistry analytes using a point of care system. To evaluate the effects of unlike normally used techniques on these analytes, we compared results between ii venipuncture sites (subcarapacial sinus and brachial vein) and iii unlike WBC quantification methods (Natt and Herrick, Leukopet, and slide estimate). Reference intervals were narrower for most analytes, and sodium and potassium were qualitatively college in the TCC population compared to previously published values from radiated tortoises housed in Due north American institutions. Creatine kinase, aspartate aminotransferase, glucose and inorganic phosphorus were all significantly greater in brachial samples than in subcarapacial samples. There was poor agreement and evidence of constant and/or proportional bias between all WBC quantification methods. Differences based on fourth dimension of sample collection were incidentally found in some analytes. These results highlight the need for considering technique, demographic, and environmental factors in creating and applying reference intervals, and contribute foundational knowledge for improving care of radiated tortoises throughout the confiscation-to-release pathway.

Citation: Brenn-White 1000, Raphael BL, Rakotoarisoa NAT, Deem SL (2022) Hematology and biochemistry of critically endangered radiated tortoises (Astrochelys radiata): Reference intervals in previously confiscated subadults and variability based on common techniques. PLoS 1 17(iii): e0264111. https://doi.org/x.1371/journal.pone.0264111

Editor: Jorge Ramón López-Olvera, Universitat Autònoma de Barcelona, Kingdom of spain

Received: March xix, 2021; Accepted: Feb 3, 2022; Published: March 14, 2022

Copyright: © 2022 Brenn-White et al. This is an open access article distributed nether the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in whatever medium, provided the original author and source are credited.

Data Availability: All data are included in a single file (a.radiate_bloodwork) available from Zenodo at http://doi.org/10.5281/zenodo.4614664 (DOI 10.5281/zenodo.4614664). Data and Read Me file including metadata take been uploaded.

Funding: Chief funding was provided by grant #2019.07 from the Saint Louis Zoo WildCare Institute Field Inquiry for Conservation grant (Principal investigator: MBW, Co-investigators: SLD and NATR). Bacon for MBW was provided by an anonymous donor to the Saint Louis Zoo. Support for additional project members was provided past the Saint Louis Zoo, Turtle Survival Alliance, and Wildlife Conservation Guild Bronx Zoo. The funders had no role in written report design, data collection and analysis, determination to publish, or preparation of the manuscript.

Competing interests: All authors have declared that no competing interests exist.

Introduction

Radiated tortoises (Astrochelys radiata) are critically endangered chelonians endemic to the spiny forests of southern Madagascar [one]. Habitat loss and poaching for trade and food are considered the primary threats to conservation of the species [1–5]. The scale of poaching is reflected in the massive confiscations of radiated tortoises that take taken identify in contempo years with over 10,000 tortoises recovered in a unmarried 2018 event [half-dozen,7]. Over 24,000 confiscated radiated tortoises are currently in rehabilitation facilities in Madagascar, all managed by the Turtle Survival Alliance (TSA), the primary organization responsible for the care of rescued tortoises. Forth with measures to minimize illegal drove, successful treatment, rehabilitation, and reintroduction of these and futurity confiscated radiated tortoises are critical to reversing the steep decline of this species in the wild.

Baseline biomedical information is essential to ensuring effective rehabilitation and rubber reintroduction of wildlife, including chelonian species [8–10]. Hematology and biochemistry reference intervals based on clinically healthy tortoises are necessary for improving treatment of confiscated animals, selecting healthy candidates for release, and making evidence-based decisions about triage in confiscation events if and when resource allow [eleven–15]. Species and population-specific reference intervals also provide foundational data for subsequent enquiry evaluating the efficacy of rehabilitation protocols and how these analytes may vary under different environmental conditions.

Robust hematology and biochemistry reference intervals for radiated tortoises have not previously been determined. Blood samples from more than 40 healthy individuals from a comparable reference population are recommended to plant de novo reference intervals with an absolute minimum of 20 individuals and 120 or more than considered ideal [16–18]. To date, hematology and biochemistry research published for this species has been descriptive and based on sample sizes below 19 [19,twenty]. Additionally, prior published values and unpublished reference intervals accept been based entirely on mixed age groups of individuals bred and/or managed within Northward American institutions [19–21]. As hematology and biochemistry values of reptile species change beyond demographic and environmental factors such as ambience temperature, humidity, rainfall patterns, food availability, and diet, there may exist important differences betwixt previously bachelor hematology and biochemistry data and values in previously-confiscated tortoises being rehabilitated and conditioned for release in their native habitat [22–24].

Hematology and biochemistry values can vary not merely past population, simply besides by sample collection and analytical methods. Choice of venipuncture site solitary or in combination with lymph contamination has been shown to change hematologic and biochemical analytes in a number of chelonian species [25–31]. Similarly, groovy variability in leukocyte counts using different quantification methods has been demonstrated in Galápagos tortoises (Chelonoidis spp.), eastern box turtles (Terrapene carolina carolina), and marine turtle species [32–36]. Veterinary and para-veterinarian practitioners working with radiated tortoises may need to choose from multiple venipuncture sites and analytic methods based on patient size and temperament, practitioner training, toll and availability of supplies, and field conditions. Agreement how hematology and biochemistry analytes vary by technique is required to accurately apply population-specific reference intervals to estimation of private results.

To address these knowledge gaps in radiated tortoise health, our study had iii objectives: 1) establish population-specific hematology and biochemistry reference intervals for previously confiscated, clinically healthy subadult radiated tortoises living nether human care within their natural range; 2) evaluate differences in hematology and biochemistry values in blood from subcarapacial and brachial venipuncture sites within this population (between-animal comparison); and iii) evaluate agreement betwixt iii common leukocyte quantification methods within this population (within-beast comparison).

Materials and methods

Written report site & population

In Feb of 2020, we examined and sampled radiated tortoises housed at the TCC, a TSA rehabilitation and convict care facility located in the Ala Mahavelo spiny forest in southern Madagascar within the radiated tortoise's electric current range [5]. For security reasons, GPS coordinates of the TCC cannot be provided.

Nosotros selected study subjects from a population of 1,000 juvenile and subadult radiated tortoises (carapacial length 15–26 cm) [37] undergoing pre-release health screening. All tortoises were illegally collected from their native habitat, confiscated past law enforcement, and placed under TSA management for rehabilitation and conditioning for release. Fourth dimension at the TCC prior to sampling was between half-dozen months and 2 years for all individuals. Only subadults were included in this study as this is the age grouping targeted for this and subsequent releases. Sex activity of these tortoises is unknown as sexual dimorphism is not reliably apparent in this age group. The tortoises are housed year-round in 1000m² open pens filled with native vegetation and natural substrate. Diets are supplemented with locally cultivated plants and native vegetation foraged past neighboring communities.

Study subjects, design, & sample collection

To allow time for same-twenty-four hours laboratory analyses, we screened the kickoff viii to 18 tortoises examined betwixt six:30 AM and eleven:00 AM each day for inclusion in this written report. Occasional afternoon screening and sampling of tortoises occurred between 4:00 PM and five:xxx PM when time permitted. Screening in the field consisted of a concrete exam by a veterinarian. This included routine visual inspection and palpation, oral and cloacal examination, and coelomic palpation as size and temperament immune. We nerveless oral-cloacal swabs for mail service-hoc screening for Mycoplasma spp., herpesviruses, and the intranuclear coccidian parasite of Testudines via polymerase chain reaction conducted by the Wildlife Conservation Society Molecular Lab as previously described [10,38–41]. Only tortoises that were apparently healthy on physical examination and negative for all pathogens via polymerase chain reaction were included in the study. We sampled 136 tortoises, with 120 samples of sufficient volume and gross quality for assay. All treatment and sample collection was performed according to Saint Louis Zoo'southward Institutional Creature Care and Employ Committee application 19–09 and Direction de la Gestion des ressources naturelles renouvelables et des ecosystemes inquiry permit 026/20/MEDD/SG/DGEF/DGRNE.

We manually restrained tortoises [42] for claret collection from either the subcarapacial sinus (SC) or brachial vein using pre-heparinized 1 ml or iii ml syringes with 0.64 mm (23 chiliad) or 0.51 mm (25 one thousand) needles of 25.4 mm (i") or 38.i mm (1.5") in length equally appropriate for patient size and temperament [43–45]. Nosotros collected no more than 3 ml of blood per tortoise, which was equivalent to less than 0.5% of body weight [45], and discarded samples with evidence of lymph contagion or clotting on gross examination. Using fresh blood directly from the syringe, nosotros fabricated two blood smears on drinking glass slides and fixed them for more than one minute in methanol after air-drying. We removed the needle from the syringe and immediately transferred the remaining blood to lithium heparin claret collection tubes (Becton, Dickinson and Visitor, Franklin Lakes, New Jersey 07417, U.s.), which were filled > 2/3 of capacity and kept in coolers until analysis.

Hematology and biochemistry analyses

Nosotros performed all sample processing and analyses at an on-site field laboratory inside 0.5 to 11 hours of sample collection with the exception of review of blood smears, which were stained the aforementioned day with a modified Wright-Giemsa stain (Dip Quick Stain, Jorgensen Laboratories, Loveland, Colorado 80538, Us) and evaluated within xiv days. We measured packed cell book (PCV) using not-heparinized microhematocrit tubes centrifuged for six minutes at 1534 G (three,500 rpm) using hematocrit-specific adaptors (Crit Carrier, LW Scientific, Lawrenceville, GA 30045, USA) in a portable angled centrifuge (E8, LW Scientific, Lawrenceville, GA 30045, USA). Nosotros measured total solids (TS) in plasma from the microhematocrit tube using a manually calibrated, handheld clinical refractometer (J-351, Jorgensen Laboratories, Loveland, Colorado 80538, USA). We performed biochemistry analyses on whole blood using a VetScan® VS2 chemistry analyzer (Abaxis Inc., Union Metropolis, California 94584, USA) with VetScan Avian Reptilian Profile Plus rotors (Abaxis Inc., Matrimony City, California 94584, USA). The biochemical console included: albumin (Alb), bile acids (BA), aspartate aminotransferase (AST), total calcium (Ca), creatinine kinase (CK), glucose (GLU), potassium (One thousand), sodium (Na), inorganic phosphorus (P), globulin (Glob), full protein (TP), and uric acid (UA).

Nosotros performed full white blood cell (WBC) quantification or estimates on each sample using three techniques. The Natt and Herrick'southward method (NH) was performed using Natt-Herricks-TIC® 1:200 staining kits (Bioanalytic GmbH, D79224, Umkirch, Germany) per manufacturer instructions, charged into a Neubauer hemocytometer and calculated equally: WBC 10 10^nine/L = number of WBC counted in 9 squares x 0.2/ nine. The Leukopet method (LP) was performed using Avian Leukopet^TM Kits (Vetlab Supply, Palmetto Bay, FL) per manufacturer instruction, charged into a Neubauer hemocytometer and calculated equally: WBC x x^ix/L = (number of eosinophilic staining cells [heterophils and eosinophils] counted in 18 squares 10 ane.1 x 16 x 0.1)/(% heterophils + % eosinophils determined from differential counts). For NH and LP, samples were incubated at room temperature with stain for 5 and ten minutes, respectively, and all were allowed to settle in the hemocytometer in a humidity chamber for three minutes prior to reading. Total WBC estimates (EST) were performed on the highest quality blood film from each tortoise by counting all leukocytes in ten fields of the monolayer using a 40x objective lens and calculated as: WBC x ten⁹/Fifty = (boilerplate number of leukocytes per field) x (objective power)ⁱⁱ x 0.001 [46]. We performed 100-prison cell leukocyte differentials on the same claret moving picture using a 100x objective lens under oil immersion. We calculated method-specific absolute values for each WBC type past multiplying the per centum of a given cell blazon from the differential by the total leukocyte count from each WBC quantification method. All NH, EST, and differentials were performed by i observer. All LP were performed past a second observer later on verifying that LP results performed by both observers were identical on a subset of samples.

Statistical analyses

All statistical analyses were performed in R v.four.0.3 [47] using the RStudio five. 1.3.1093 interface [48] unless specified otherwise. For all statistical tests, p < 0.05 was considered meaning with the exception of the Anderson-Darling test for Gaussian distribution for reference interval calculation, where p < 0.3 was used per recommendations for samples of this size [49].

Pre-assay information preparation.

Xx samples were excluded from all analyses for gross lymph contamination (SC: due north = 14/94 [14.8%]; brachial: due north = 6/54 [11.i%]) and viii for clotting (SC: northward = 0/94 [0.0%]; brachial: due north = viii/54 [14.8%]). Slide quality issues (northward = 9) and cell clumping on LP (n = 8) precluded additional samples from applicable WBC analyses. For values that vicious outside of the dynamic range of the VetScan VS2 analyzer, we substituted the reported qualitative values with the quantitative value closest to the analyzer threshold to reduce the bias that would be introduced by removing these high and low values from the dataset [50]. Substitutions were made as follows: Ca– 4.01 for > 4.00 mmol/L; Yard– 8.vi for > viii.5 mmol/L; TP– 19 for < 20 one thousand/L; and UA– 17 for < xviii μmol/L.

We evaluated the distribution of each hematologic and biochemical analyte past inspection of histograms, the Anderson-Darling test for Gaussian distribution [51], and a distribution-costless exam for symmetry [52]. For data that had a non-Gaussian distribution, nosotros applied a Box-Cox transformation [53] and repeated evaluation of the distribution every bit to a higher place. We used Tukey'southward interquartile range to identify statistical outliers on untransformed Gaussian data or on Box-Cox transformed data for non-Gaussian data. We removed only those outliers that could be attributed to sample, treatment, or analytical error. Afterwards removing an outlier, we repeated the higher up procedures until no further outliers warranting removal were identified. Outliers removed from applicable analyses were: NH– xviii.87 WBC ten x⁹/Fifty, LP– 28.86 WBC x 10⁹/L, EST– 29.lx WBC 10 ten⁹/L, and proportion of eosinophils– 0.15. Values contradistinct specifically past hemolysis or extended time from sample collection to assay were identified and handled as described beneath.

Effects of venipuncture site, sample artifacts, and fourth dimension of sample drove (between-brute).

To identify potential confounds and presence of sample artifacts, we tested the associations betwixt venipuncture site (SC, brachial), hemolysis (0, one+, ii+, 3+), fourth dimension of sample drove (AM, PM), and time from sample collection to biochemistry analysis using Kruskal-Wallis, Wilcoxon rank sum, and chi-square tests. As significant relationships existed between hemolysis and both venipuncture site and time to assay also as between time of sample collection and time to analysis, we used univariate generalized linear models (GLM) fit past the glm function [47] to simultaneously evaluate the effects of these four variables on our variables of interest. We fit split models with each hematological analyte as the response and venipuncture site, hemolysis, and time of sample collection equally predictors. Nosotros evaluated furnishings on biochemical analytes in the same mode with time to analysis included every bit an boosted predictor. Model assumptions were checked using residual vs. fitted, normal Q-Q, scale-location, and residual vs. leverage plots. When model fit was poor, Box-Cox transformation was used. If this failed to provide sufficient fit, we performed stratified not-parametric tests with Hommel corrected p-values for each predictor-response pair. We conditionally averaged the best fit models divers every bit ΔAICc < 2. Nosotros determined the threshold at which hemolysis and/or time to analysis no longer exhibited a statistically pregnant issue on a given hematological or biochemical analyte by iteratively removing samples with the greatest hemolysis and/or time to analysis from the dataset and rerunning the above models or tests. We additionally evaluated differences in sample quality (hemolysis, gross clotting, and gross lymph contamination) based on venipuncture site using chi-square and Fisher exact tests.

Reference interval & descriptive statistics.

For a given analyte, we excluded samples determined to be affected by hemolysis or time to analysis as described above from reference interval calculation. For all analytes, samples collected in both the AM and PM were included in reference interval calculation as we considered variation based on time of sample collection to stand for physiologic variation rather than pathology or handling artifact. Additionally, the number of samples collected in the PM (n = thirteen) was below the threshold recommended by American Society of Veterinary Clinical Pathology (ASVCP) guidelines for partitioning of reference intervals [16]. Nosotros used Reference Value Counselor v. 2.1 [54] to summate reference intervals, xc% confidence intervals (CI) of the lower and upper reference interval limits, and the descriptive statistics mean, standard deviation, and median for each hematological and biochemical analyte. We calculated these values separately for each WBC quantification method and for each venipuncture site when venipuncture site was found to have an effect on an analyte. Nosotros calculated reference intervals and CIs according to the ASVCP guidelines [16] equally follows: non-parametric methods for reference intervals and CIs when n = 120; robust method for reference intervals and bootstrap method for CIs when 40 ≤ n < 120 and distribution was symmetric; non-parametric method for reference intervals and bootstrap method for CIs when 40 ≤ n < 120 and distribution was not symmetric; and robust method for reference intervals and bootstrap method for CIs when xx ≤ due north < twoscore and distribution was symmetric. When n < 120, we used Box-Cox transformed information for reference intervals and CI calculations of non-Gaussian distributed analytes when transformation improved distribution symmetry. Reference intervals were not calculated when due north < 20 or when 20 ≤ n < 40 and analyte distribution was neither Gaussian nor symmetric.

Agreement between WBC quantification method (within-animal).

We evaluated agreement in full WBC count from the NH, LP, and EST methods using Passing-Bablok (Pb) regression and Bland Altman plots. Passing-Bablok assumes a high positive correlation between the diagnostic methods existence evaluated [55,56], which we tested using Kendall's tau. We considered the methods in statistical agreement per PB when the 95% CI for the slope, the indicator of constant bias, contained ane and the 95% CI for the y-intercept, the indicator of proportional bias, independent zero. We used Bland-Altman plots, estimates of bias (defined as the hateful difference betwixt methods), and limits of agreement (defined every bit the bias ± 1.96 times the standard deviation of the differences) to further evaluate agreement, systematic bias, and proportional bias [57]. As it has been proposed that LP results vary according to lymphocyte and heterophil proportions [24], we used Kendall'southward tau to test for correlation between proportions of these cell types and the deviation in total WBC count for each method pair.

Results

Reference intervals & descriptive statistics

Reference intervals calculated for hematology and biochemistry analytes of tortoises are reported in Tables 1 and 2 respectively. Run into S1 Tabular array for biochemistry reference intervals and descriptive statistics presented in United States conventional units.

Table 1. Subadult radiated tortoise hematology reference intervals and descriptive statistics based on clinically good for you individuals living in a rehabilitation setting within their natural range.

https://doi.org/10.1371/journal.pone.0264111.t001

Tabular array ii. Subadult radiated tortoise biochemistry reference intervals and descriptive statistics based on clinically salubrious individuals living in a rehabilitation setting inside their natural range.

https://doi.org/10.1371/journal.pone.0264111.t002

Reference intervals could not exist calculated for iii biochemistry analytes due to large proportions of the results falling outside of the dynamic range of the VetScan VS2 analyzer. All bile acid results (n = 120) were reported as < 35 μmol/L. Of 120 albumin results, 107 were reported as < ten one thousand/Fifty with the remaining 13 results yielding a mean and standard departure of 12 +/- ii 1000/L, median of 11 yard/L, and range of 10–17 thou/L. As globulin concentration is calculated from total protein and albumin, only thirteen values were reported for globulin with a mean and standard deviation of 24 +/- iii g/L, median of 23 thou/L, and range of 17–28 g/Fifty.

Furnishings of venipuncture site (betwixt-animal)

No significant furnishings on hematology analytes were found. All biochemistry analytes on which venipuncture site had a significant effect were greater in brachial samples than in subcarapacial samples. These included: AST (GLM: β = 19.ix [iii.35–36.5] U/L, p = 0.018), CK (GLM: β = 893 [564–1222] U/L, p < 0.001), Glu (GLM: β = 0.630 [0.851–0.410] mmol/L, p < 0.001), and Phos (GLM: β = 0.107 [0.191–0.0222] mmol/L, p = 0.013). Conditionally averaged best-fit GLMs evaluating the furnishings of venipuncture site along with time of sample collection, hemolysis, and time from sample collection to biochemistry assay are reported in Table 3. Effects of venipuncture site on uric acid, proportion of eosinophils, and presence of immature lymphocytes were evaluated with non-parametric tests due to disability to obtain appropriate GLM fit and no pregnant effects were identified. Gross clotting and hemolysis levels greater than 1+ were more frequent in brachial samples than SC samples (chi-foursquare p < 0.001 and Fisher verbal p < 0.001, respectively). There was no meaning departure in gross lymph contagion (chi-square p = 0.517) between venipuncture sites.

Tabular array 3. Effects of venipuncture site, hemolysis, and time from sample drove to biochemistry analysis on hematology and biochemistry analytes in clinically healthy subadult radiated tortoises living in a rehabilitation setting within their natural range.

https://doi.org/10.1371/journal.pone.0264111.t003

Effects of time of sample collection (between-fauna)

Time of sample collection did not have a significant upshot on full WBC counts by any method, but the proportion of individual WBC types was significantly affected with PM samples being associated with increased heterophils (GLM: β = 0.0769 [0.0153–0.139], p = 0.016), decreased lymphocytes (GLM: β = -0.111 (-0.191–-0.0315), p = 0.006), and increased heterophil:lymphocyte ratios (GLM: β = 0.580 [0.161–1.000], p = 0.007) compared to AM samples. Of the biochemistry analytes, but Ca (GLM: β = -0.226 (-0.406–-0.0471) mmol/L, p = 0.013) and K (GLM: β = 1.06 [0.431–ane.68] mmol/50, p < 0.001) were significantly affected.

Agreement between WBC quantification method (within-animal)

There was poor statistical agreement between all method pairs based on Lead regression and Banal Altman plots (Fig 1, Table 4) with limits of understanding wider than 12.00 WBC 10 10⁹/L.

Fig ane. Agreement between WBC quantification methods in clinically healthy, subadult radiated tortoises.

(a, c, due east) Bland-Altman–solid line is the line of perfect agreement, dashed lines are hateful divergence between the methods (center), upper limit of understanding (upper), and lower limit of understanding (lower) with shading indicating associated 95% confidence intervals. (b, d) Passing-Bablok regression–dashed line is the line of perfect understanding, solid line is the regression line with shading indicating associated 95% confidence intervals. WBC = white blood cell.

https://doi.org/10.1371/periodical.pone.0264111.g001

Banal Altman plots revealed systematic bias between each pair with the EST method yielding higher values on average than the LP method (bias = ii.57 [i.49–3.66] WBC x ten⁹/L), which in plough yielded college values than the NH method (bias = 1.26 [0.21–ii.30] WBC x 10⁹/L). Passing-Bablok revealed constant bias between LP and EST values (intercept = v.37 [3.08–seven.forty] WBC x 10⁹/L) and proportional bias betwixt EST and NH values (slope = ane.44 [1.03–1.83]), though the 95% CI approaches one. Results of the LP and NH methods failed the test for positive correlation prohibiting Atomic number 82 regression analysis. On Bland Altman plots, all pairs showed a tendency toward larger differences as mean WBC counts increased. The departure betwixt LP and NH values was positively correlated with lymphocyte proportion (τ = 0.332, p < 0.001) and negatively correlated with heterophil proportion (τ = -0.399, p < 0.001), as was the difference between LP and EST values (τ = 0.348, p < 0.001 and τ = -0.427, p < 0.001 respectively). The departure betwixt NH and EST values was not significantly correlated with either lymphocyte proportion (τ = 0.0152, p = 0.814) or heterophil proportion (τ = -0.0313, p = 0.632).

Discussion

Robust population and technique-specific hematology and biochemistry reference intervals are valuable however previously missing tools throughout the confiscation-to-release pathway that is important to reversing radiated tortoise population declines. Low cost, low technology analytes such as PCV and TS have proven useful equally triage criteria with avian species in big scale oil spills [58–sixty]. They could be similarly informative in directing resource during tortoise confiscation events, though measuring even these 2 analytes may be challenging during big scale confiscations in low resource settings. Afterwards triage and when resource let, hematology and biochemistry reference intervals can be used to assistance guide clinical care of confiscated tortoises just every bit they are used for a wide multifariousness of taxa in rehabilitation settings [thirteen,61–64]. These reference intervals also contribute to evaluating the health status of tortoises prior to release, as was the context of this study, with the goal of identifying and removing tortoises with evidence of illness from the release population [10,65]. Lastly, baseline values in the pre-release population provide a foundation for hereafter research aimed at evaluating and improving management at all stages of care from confiscation until release [10,14,66–68].

This report provides statistically robust population- and technique-specific reference intervals for clinically healthy radiated tortoises in the priority age group for reintroduction living in a managed setting within native habitat. While reference intervals reported here are largely comparable to previously reported ranges for radiated tortoises in North American institutions [19–21], some of import differences are apparent. Potassium and Na are notably higher in this population, which is nearly likely attributable to differences in water residual and diet betwixt these settings. Drought-adapted chelonians tolerate wide variation in circulating K, Na, and Cl levels [69–71], all of which accept been institute to exist higher in free-living desert tortoises (Gopherus agassizii) during seasons with relatively low rainfall and where diets are high in Thou [23,71]. This study was performed at the terminate of an extended dry out flavor and thus under conditions of decreased water availability and contradistinct forage, which likely too contributed to the relative elevations in K and Na in this population. In full general, reference intervals for this population are substantially narrower than reference intervals calculated by ZIMS, which combine values from multiple age groups, venipunctures sites, biochemistry analyzers, and seasons. When based on adequate sample sizes, narrower intervals provide increased sensitivity in detecting abnormalities and highlight the value of determining and applying reference intervals on a population- and technique-specific basis.

We additionally quantified the effects of venipuncture site on hematology and biochemistry analytes in radiated tortoises and, to the authors' knowledge, provide the kickoff straight comparing between SC and brachial venipuncture sites in whatever species. While jugular blood is generally considered the gold standard sample in chelonians [72], SC and brachial venipuncture are the two most accessible techniques in unsedated radiated tortoises beyond the age groups in this written report. Statistically meaning differences were observed in AST, CK, Glu, and Phos, all of which were greater in brachial samples than in SC samples. These differences were nowadays contained of level of hemolysis, time from sample collection to biochemistry analysis, and fourth dimension of venipuncture. Of these, the magnitude of differences in CK, Glu, and Phos exceeded the thresholds adopted by the ASCVP for creation of separate reference intervals for clinical use [73,74]. Venipuncture site-based differences in CK (GLM: β = 893 [564–1222] U/L) and Glu (GLM: β = 0.630 [0.851–0.410] mmol/L) are particularly relevant for evaluating the physiologic effects of field capture and handling techniques and accurately identifying hypoglycemia in emergency or triage scenarios.

Relatively increased AST and CK are most probable attributable to restraint technique and relatively increased, although minor, iatrogenic tissue trauma during brachial venipuncture. This is supported by greater proportions of brachial samples exhibiting gross clotting (north = 8/54 [14.8%], Fisher exact p < 0.001) and hemolysis levels greater than 1+ (n = 15/xl [37.5%], chi-foursquare p < 0.001) as compared to SC samples (due north = 0/94 [0.0%] and n = 6/80 [7.5%], respectively). The etiology of the relatively decreased Glu and Phos in SC samples is less articulate. This finding is consistent with presumed or observed lymph dilution during SC venipuncture in other chelonian species [25,26,28]. While gross lymph contamination was noted in a slightly greater proportion of SC (due north = 14/94 [14.8%]) compared to brachial (n = 6/54 [eleven.one%]) samples in this study, this difference was far from statistically significant (chi-square p = 0.517). Further, lymph contagion is a form of hemodilution that most commonly reduces PCV, WBC count, and TP [25,26,28–xxx], none of which were observed here. Alternatively, an undetected difference betwixt tortoises in the SC and brachial sample groups may be responsible, though the overall study population is homogenous in terms of age, size, credible health condition, husbandry, and ecology atmospheric condition.

As with venipuncture site, option of leukocyte quantification method has been shown to take marked impacts on results in all chelonian species in which comparison of methods has been performed [31–36]. Leukocyte results in this study demonstrated poor clinical understanding between all methods with LOAs ranging from 12.64–21.58 WBC x 10⁹/L and markedly different method-specific reference intervals. Width of the LOAs is due in part to random fault inherent in transmission leukocyte quantification methods even when performed by a unmarried highly-trained observer. While imprecision has rarely been quantified, recent studies reported coefficients of variation of 8.two% using the LP method in eastern box turtles (Terrapene carolina carolina) [36] and 12% using the NH method in multiple avian species [75]. Despite the furnishings of random fault, systematic and/or proportional biases are evident betwixt all method pairs. On average, EST overestimated both LP (bias = 2.57 [i.49–3.66] ten 10⁹/L) and NH (bias = iii.51 [2.89–iv.xiii] WBC x ten⁹/L). This is consistent with comparisons between EST and NH in Galapagos tortoises (Chelonoidis spp.) [35], simply contrary to studies comparing EST and LP in other chelonian species, all of which take found higher WBC counts using the aforementioned or similar phloxine-based stains compared to EST [33–36]. The magnitude of disagreement between all methods increased every bit hateful WBC count increased on corresponding Banal Altman plots, though this proportional bias was only statistically supported by Passing-Bablok regression for EST vs. NH (slope = i.44 [1.03–1.83]). When the proportional bias between EST and NH is adjusted for via Passing-Bablok regression, the constant bias between the methods decreases substantially (intercept = -0.160 [-three.02–3.12] WBC x 10⁹/L). Given this and the relatively narrow LOA (12.64 [ten.50–fourteen.78] WBC 10 10^nine/L), EST and NH showed the greatest agreement of all method pairs. In contrast, LP and NH exhibited the poorest agreement with the widest LOA (21.58 [18.00–25.16] WBC ten 10^ix/L) and a lack of even basic positive correlation. Additionally, the departure in total WBC count by LP and both other methods was positively correlated with the proportion of lymphocytes and negatively correlated with the proportion of heterophils. This supports the supposition that LP overestimates total WBC count in high lymphocyte/low heterophil samples [24,35] and raises questions about its accuracy and utility in lymphocyte dominant species such as the radiated tortoise. Without an accepted gilded standard or method-specific measures of precision, it is non possible to make up one's mind which method is the most accurate or precise. Yet, our findings support the apply of NH or EST over LP in radiated tortoises and emphasize the inadvisability of comparing results from different methods in individual patients or across populations.

The result of time of sample collection on some hematological and biochemical analytes was an incidental finding in this study that warrants further investigation. Statistically significant differences in PM samples relative to AM samples included higher Yard, heterophil proportion, and heterophil:lymphocyte ratio and lower Ca and lymphocyte proportion. These differences may be due to cyclic rhythm, temperature, or activity level, all of which have been testify to affect certain hematology and biochemistry analytes in other reptile species [xiii,22,76–79]. For case, the changes in heterophil and lymphocyte proportions are consistent with shifts seen in some reptile species as glucocorticoids increase [fourscore], which in turn has been shown to vary with temperature [81,82] and diurnally [83]. Similarly, Yard and Ca may be secondarily afflicted by the demonstrated effects of temperature and activeness-level on blood gas and acid-base status [77,79,84]. As this report was not designed to evaluate the furnishings of fourth dimension of twenty-four hours, reasons for observed differences are speculative. Future experiments that measure hematological and biochemical analytes in a time series with concurrent ambient and tortoise temperature, an alphabetize of activity level, and blood corticosterone would elucidate underlying etiologies and clinical implications of differences along these axes.

Certain limitations of this study should exist considered when interpreting and applying our results. Reference intervals must be based on salubrious individuals and while tortoises included in this study were apparently healthy based on concrete examination and pathogen screening, subtler indicators of poor wellness such equally changes in activity level and appetite may have been overlooked in this high book rehabilitation setting. Additionally, biochemistry results were restricted by the dynamic range of the VetScan VS2 for certain analytes, imposing artificial limits on reference intervals and hampering the ability to find effects of venipuncture site, sample artifacts, and time of day. By substituting out of range values with the value closest to the analyzer threshold rather than removing out of range values from analyses, these effects were reduced but non eliminated. Bile acids, UA, Alb, and Glob (which is calculated from Alb and TP) were well-nigh afflicted with results for these analytes below the reportable range for several or all tortoises. Other researchers have encountered similar constraints using the VetScan in alligator snapping turtles (Macrochelys temminckii) [85] and loggerhead turtles (Caretta caretta) [86,87]. As liver and renal disease are associated with elevated BA and UA, the VetScan and values reported here are likely useful for detecting clinical disease, simply lack precision for ecophysiological enquiry applications. In contrast, the current VetScan arrangement cannot be recommended for measurement of Alb or Glob in this species for either clinical or research purposes with poly peptide electrophoresis being the preferred method where resource allow [88,89]. For other biochemistry analytes, the VetScan remains a valuable tool for clinical care and enquiry in chelonian populations where access to reference laboratories is limited, though species-specific validation is strongly recommended.

By determining population- and technique-specific hematology and biochemistry reference intervals and quantifying how these analytes vary betwixt commonly used techniques, this study provides a valuable tool for improving care along the confiscation-to-release pathway critical to radiated tortoise conservation. These results also contribute foundational cognition for time to come clinical research for this critically endangered species. We focused our chief efforts on the target population for release, subadults at the TCC, at a single time betoken. Further research is needed to characterize variation in hematology and biochemistry reference intervals across age groups and seasons within the rehabilitation population and to evaluate associations between these analytes, clinical outcomes, and mail-release survival.

Supporting information

S1 Table. Subadult radiated tortoise biochemistry reference intervals and descriptive statistics in The states conventional units based on clinically healthy individuals living in a rehabilitation setting within their natural range.

https://doi.org/x.1371/journal.pone.0264111.s001

(DOCX)

Acknowledgments

We give thanks Jane Merkel, Dalia Ferguson, Christel Griffioen, Riana Rakotondrainy, Tony Ralivaniaina, Paul Calle, Vontsoa, Mena, and all TCC staff for their many contributions and invaluable back up in the field. We are also grateful to Rick Hudson, Herilala Randriamahazo, and all Turtle Survival Alliance Madagascar staff for their organizational back up and the ongoing care of they provide to these and thousands of other radiated tortoises. Thank you to Jamie Palmer, Ainoa Nieto-Claudín, and all Saint Louis Zoo Found for Conservation Medicine staff for assistance with report logistics, design, and analysis. Lastly, nosotros give thanks the Ecole Supérieure des Sciences Agronomiques (ESSA) for their institutional support.

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Source: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0264111

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