Hemoparasite Analysis and Hematological Parameters of Four Freshwater Turtle Species (Testudines: Podocnemididae) from the Brazilian Amazon.

METHODS: Sampling was performed at three sites in the Tapajós River mouth, Pará State, Brazil, and an aliquot of blood was collected from each turtle by post-occipital dorsal vein puncture. The aliquots were used for the determination of hematological parameters and for the study of hemoparasites in the blood samples. RESULTS: A total of 37 turtles were sampled: 21 Podocnemis expansa, three P. sextuberculata, nine P. unifilis, and four P. erythrocephala. Parasitological analyses showed inclusions in erythrocytes compatible with Haemogregarina sp., representing an overall prevalence of parasitized animals of 64.8% with an average of 94,789 parasites/ml of blood. In this study, the evolutionary forms of Haemogregarina spp. from trophozoites, pre-meronts, meronts, and mature and immature gamonts were observed in three species of freshwater turtle species, and one morphotype of the parasite with high similarity to Haemogregarina podocnemis has been observed. CONCLUSION: Despite recent research on freshwater turtles, this study provides new information on the intensity of blood parasite infection and hematological parameters of free-living animals, highlighting the need for studies on infected and uninfected animals from the same population.

An 18-µm microaggregate blood filter does not cause hemolysis during in vitro whole blood transfusions in sea turtles.

OBJECTIVE: Determine the hemolytic effect of an 18-µm microaggregate blood filter during in vitro sea turtle whole blood transfusions as well as describe the average diameter of leatherback (Dermochelys coriacea) and Kemp's ridley sea turtle (Lepidochelys kempii) RBCs. ANIMALS: 5 green (Chelonia mydas), 5 loggerhead (Caretta caretta), and 5 Kemp's ridley sea turtles (total n = 15). METHODS: Heparinized sea turtle blood was infused at 60 mL/h through a microbore extension set without and then with a postsyringe, inline 18-µm microaggregate blood filter. Pre- and postfiltration PCV, Hct, total solids, sodium, chloride, potassium, glucose, and free plasma hemoglobin concentrations were measured. With the use of light microscopy and archived blood smears, the maximum and minimum diameter of 20 RBCs from each of the 5 leatherback and 5 Kemp's ridley sea turtles were measured with a calibrated ocular micrometer using 400X magnification. RESULTS: There were no significant differences between pre- and postfiltration samples for Hct, total solids, sodium, chloride, potassium, glucose, and free plasma hemoglobin concentrations; however, there was a significant median postfiltration decrease in PCV of approximately 4%, representing a 13% decrease of the total RBCs transfused. Average maximum diameters for leatherback and Kemp's ridley sea turtle RBCs were 19.7 and 16.1 µm, respectively. CLINICAL RELEVANCE: Although the 18-µm microaggregate blood filter does not hemolyze transfused sea turtle RBCs and is likely safe for in vivo blood transfusions, the filter's pores may retain a small proportion of infused RBCs given their diameter.

Subcutaneous administration of ceftazidime at 20 and 40 mg/kg produces theoretically therapeutic plasma concentrations for at least 120 hours in red-eared sliders (Trachemys scripta elegans).

OBJECTIVE: To evaluate and compare the pharmacokinetic parameters of SC ceftazidime administered at 20 and 40 mg/kg to red-eared sliders. ANIMALS: 8 adult red-eared sliders (Trachemys scripta elegans). METHODS: In a sequential, 2-period study with a 3-week washout period between treatments, ceftazidime was administered SC to turtles at 20 and 40 mg/kg. Blood samples were collected from the subcarapacial sinus at 0, 24, 48, 72, 96, and 120 hours after ceftazidime administration. Plasma ceftazidime concentrations were quantified using reversed-phase HPLC. RESULTS: Mean plasma half-life after 20- and 40-mg/kg dosing was 39.75 ± 8.0 hours and 33.03 ± 6.56 hours, respectively. Mean maximum plasma concentration after 20- and 40-mg/kg dosing was 71.0 ± 15.93 µg/mL and 120.0 ± 30.62 µg/mL, respectively. Mean plasma ceftazidime concentrations remained ≥ 8 µg/mL, the theoretical MIC for various reptile pathogens for all time points. CLINICAL RELEVANCE: Results indicate that ceftazidime dosed at either 20 or 40 mg/kg produces plasma concentrations exceeding the theoretical MIC of various reptile pathogens for at least 120 hours. An ideal dosing interval could not be determined, as all plasma concentrations remained above the threshold of interest for all time points. Follow-up studies should focus on establishing a dosing interval and more rigorous monitoring for potential adverse effects.

Analysis of risk factors associated with gas embolism and evaluation of predictors of mortality in 482 loggerhead sea turtles.

Sea turtles that are entrapped in static and towed nets may develop gas embolism which can lead to severe organ injury and death. Trawling characteristics, physical and physiologic factors associated with gas-embolism and predictors of mortality were analysed from 482 bycaught loggerheads. We found 204 turtles affected by gas-embolism and significant positive correlations between the presence of gas-embolism and duration, depth, ascent rate of trawl, turtle size and temperature, and between mortality and ascent time, neurological deficits, significant acidosis and involvement of > 12 cardiovascular sites and the left atrium and sinus venosus-right atrium. About 90% turtles with GE alive upon arrival at Sea Turtle Clinic recovered from the disease without any supportive drug therapy. Results of this study may be useful in clinical evaluation, prognostication, and management for turtles affected by gas-embolism, but bycatch reduction must become a priority for major international organizations. According to the results of the present study the measures to be considered to reduce the catches or mortality of sea turtles for trawling are to be found in the modification of fishing nets or fishing operations and in greater awareness and education of fishermen.

Plasma electrophoresis profiles of Blanding's turtles (Emydoidea blandingii) and influences of month, age, sex, health status, and location.

Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.

Evaluation of a dexmedetomidine-midazolam-ketamine combination administered intramuscularly in captive ornate box turtles (Terrapene ornata ornata).

OBJECTIVE: To characterize the effects of a combination protocol of dexmedetomidine-midazolam-ketamine (DMK) administered intramuscularly (IM) in ornate box turtles (Terrapene ornata ornata). STUDY DESIGN: Prospective experimental trial. ANIMALS: A total of 16 apparently clinically healthy adult ornate box turtles (eight male, eight female). METHODS: Each turtle was treated with dexmedetomidine (0.1 mg kg-1), midazolam (1 mg kg-1) and ketamine (10 mg kg-1) administered IM. Time to first response, time to maximal effect, the plateau phase and time to recovery from reversal administration were recorded. Physiologic variables, muscle tone, reflexes and the ability to perform endotracheal intubation were recorded at 5 minute intervals. Movement in response to an IM injection of 0.1 mL sterile 0.9% NaCl administered in the left pelvic limb, using a 25 gauge needle to a depth of just past the bevel of the needle, was assessed every 15 minutes. Atipamezole (0.5 mg kg-1) IM and flumazenil (0.05 mg kg-1) SC were administered 60 minutes after the initial DMK injections. RESULTS: The mean time to first response, time to maximal effect, the plateau phase and time to recovery were 2.1, 14.9, 38.7 and 7.8 minutes, respectively. A respiratory rate was not observed in most turtles. The body temperature significantly increased over time. The palpebral reflex was persistent in 43% of turtles and the tail pinch reflex remained intact in 13% of turtles. All turtles recovered with no observed adverse effects. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, this DMK protocol administered to ornate box turtles resulted in a rapid-onset, light anesthesia lasting approximately 40 minutes and a smooth recovery with no adverse effects noted.

Circulating miRNome of Trachemys scripta after elective gonadectomy under general anesthesia.

Post-surgical management is an important issue in veterinary medicine, requiring biomarkers with high sensitivity and specificity for timely and effective treatment. Emerging evidence suggests that miRNAs are promising stress- and pain-related markers. The aims were to profile the circulating miRNA signature in plasma of turtles (Trachemys scripta) and point out potential candidate biomarkers to assess the status of the animal. The plasma of female turtles underwent surgical gonadectomy were collected 24 h pre-surgery, and 2.5 h and 36 h post-surgery. The expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves. The results showed that 14 miRNAs were differentially expressed over time. RT-qPCR validation highlighted that 2-miR-499-3p and miR-203-5p-out of 8 miRNAs tested were effectively modulated. The Area Under the Curve (AUC) of miR-203-5p was fair (AUC 0.7934) in discriminating pre- and 36 h post-surgery samples and poor for other time points; the AUC of miR-499-3p was excellent (AUC 0.944) in discriminating pre-surgery and 2.5 h post-surgery samples, and fair in discriminating pre-surgery and 36 h post-surgery (AUC 0.7292) and 2.5 h and 36 h post-surgery (AUC 0.7569) samples. In conclusion, we demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers to assess animals' status. Further studies are necessary to confirm their diagnostic value and to investigate functional and mechanistic networks to improve our understanding of the biological processes.

TERBINAFINE PHARMACOKINETICS FOLLOWING SINGLE-DOSE ORAL ADMINISTRATION IN RED-EARED SLIDER TURTLES (TRACHEMYS SCRIPTA ELEGANS): A PILOT STUDY.

In this pilot study, the pharmacokinetics of terbinafine were determined in six apparently healthy red-eared slider turtles (Trachemys scripta elegans) after a single PO administration. Terbinafine suspension (15 mg/kg, once) was administered via gavage tube to all turtles. Blood samples were collected immediately before (time 0) and at 1, 2, 4, 8, 24, and 48 h after drug administration. Plasma terbinafine concentrations were quantified by ultra-performance liquid chromatography-mass spectrometry, and noncompartmental pharmacokinetic analysis was performed. None of the animals showed any adverse responses following terbinafine administration. Mean area under the curve from time 0 to 24 h was 1,213 h × ng/ml (range 319-7,309), mean peak plasma concentration was 201.5 ng/ml (range 45.8-585.3), mean time to maximum plasma concentration was 1.26 h (range 1-4), mean residence time was 7.71 h (range 3.85-14.8), and mean terminal half-life was 5.35 h (range 2.67-9.83). The administration of terbinafine (15 mg/kg, PO) may be appropriate for treatment of select fungal organisms with low minimum inhibitory concentrations in red-eared slider turtles but may require q12h administration even for organisms with low minimum inhibitory concentrations. Multiple-dose studies as well as clinical studies are needed to determine ideal dosages and efficacy.

PLASMA VORICONAZOLE CONCENTRATIONS FOLLOWING SINGLE- AND MULTIPLE-DOSE SUBCUTANEOUS INJECTIONS IN WESTERN POND TURTLES (ACTINEMYS MARMORATA).

A recently characterized fungal pathogen, Emydomyces testavorans, has been associated with ulcerative shell disease and significant morbidity in Western pond turtles. Voriconazole is a second-generation triazole antifungal medication that prevents fungal growth through disruption of ergosterol synthesis, causing abnormalities in the fungal cell membrane. Preliminary reports of minimum inhibitory concentrations (MIC) indicate that voriconazole is effective in vitro against E. testavorans. Ultraperformance liquid chromatography was used to measure voriconazole plasma concentrations in blood samples from healthy Western pond turtles after administration of a single SC injection of 10 mg/kg and after multiple doses (10 mg/kg SC q48h for seven doses). The data were analyzed using a naïve pooled approach. Mean (SE) observed time to maximum concentration was 2 (0.18) h for a single dose and 50 (2.2) h for multiple doses; the multiple-dose trial observed mean (SE) maximum concentration was 12.4 (2.2) µg/ml, and observed mean (SE) trough concentration was 1.7 (0.7) µg/ml. Multifocal skin sloughing following the single-dose trial was observed in one turtle and there was a significant increase in polychromatophilic cells amongst the study turtles after the multiple-dose voriconazole trial. No other adverse effects were observed. When voriconazole was administered at 10 mg/kg SC q48h, observed trough plasma concentrations were consistently higher than reported E. testavorans MIC concentrations. Further study is needed to determine the clinical safety and in vivo efficacy of this dose in Western pond turtles.

BLOOD FATTY ACID PROFILES OF NERITIC JUVENILE WILD GREEN TURTLES (CHELONIA MYDAS) AND KEMP'S RIDLEYS (LEPIDOCHELYS KEMPII).

Blood fatty acid profiles can indicate an animal's wild-type diet composition and fatty acid status, but have not been reported in sea turtles. Newer technologies allow for fatty acid profiles from very small (less than three drops) samples of whole blood. This study examined whole blood fatty acid profiles of presumably healthy, neritic, juvenile, wild green (Chelonia mydas) (n = 9; 6 males, 3 females) and Kemp's ridley (Lepidochelys kempii) (n = 8; 6 males, 2 females) turtles from North Carolina, USA. Saturated fatty acids, which can be synthesized de novo, consisted primarily of 16:0, although green turtle blood had a higher proportion of 18:0 (P < 0.001) than Kemp's ridleys, while Kemp's ridley blood had higher proportions of 17:0 (P = 0.007), 20:0 (P = 0.03), 22:0 (P= 0.002), and 24:0 (P < 0.001) as compared with green turtles. Total monounsaturated w7 fatty acids, which can be synthesized de novo or may be diet derived, were higher in Kemp's ridleys and predominantly in the form of 16:1 and 18:1w7 fatty acids. Kemp's ridley blood had more than double the relative proportion of 16:1w7 as compared with green turtles (P= 0.03). Green turtles had higher levels of 18:2w6 than Kemp's ridleys (P= 0.02). In both turtle species, 20:4w6 was detected, despite predicted low dietary proportions, suggesting bioconversion from precursors. Finally, green turtles had higher levels of 18:3w3 compared with Kemp's ridleys, while Kemp's ridleys had higher proportions of 20:5w3 compared with green turtles (P < 0.001, = 0.007, respectively). Whole blood fatty acid profiles generally correlate to previous work with lipid depots, supporting the use of this less invasive methodology to advance the understanding of fatty acid nutrition of sea turtles. These data can be used to assess and guide nutrition and health programs for sea turtles under human care.

PARTIAL HEMATOLOGY, BIOCHEMISTRY, AND PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR CAPTIVE SPOTTED TURTLES (CLEMMYS GUTTATA).

Spotted turtles (Clemmys guttata) are an endangered species and are commonly encountered in the pet trade and in many zoological collections across the United States, yet peer-reviewed published reference intervals (RI) for common clinicopathologic tests are unavailable for this species. The objectives of this study were to calculate partial RI for routine hematology, biochemistry, and electrophoretic analyses, as well as to perform an initial comparison of capillary zone electrophoresis (CZE) and agarose gel electrophoresis (AGE) in this species. A single blood sample was obtained from a single collection of 32 apparently healthy captive spotted turtles weighing at least 100 g and was submitted for standard hematologic and biochemistry analyses, as well as electrophoresis via CZE and AGE methods. Partial RI were calculated for corresponding analytes for each type of testing. While CZE and AGE protein fractions were found to have good correlation, some significant differences were observed, reinforcing that RI should be reported with the specific method used for their determination. The spotted turtle electrophoretograms were distinctly different from those previously reported from turtles in the same taxonomic family, including differences in the number and relative prominence of protein fractions.

Hematologic variables of the Arrau turtle (Podocnemis expansa) under the effects of different anticoagulants and cytologic stains.

BACKGROUND: The hematology of turtles of the Amazon, such as the Arrau turtle (Podocnemis expansa), has not been well described in the literature. Referencing how to collect and analyze blood samples is fundamental for the medical practice of these exotic animals. OBJECTIVES: The aim of this study was to evaluate the hematologic parameters of Arrau turtles of the Amazon to determine the best anticoagulant (EDTA, citrate, or sodium heparin) and cytologic stain (Giemsa, Quick Panoptic, or Rosenfeld) for use with this species. METHODS: Blood samples from eight turtles were collected. Three blood smears were made using blood without anticoagulant, and the rest of the sample was distributed in three tubes, containing EDTA, citrate, and sodium heparin, for erythrogram and leukogram analyses. RESULTS: All blood samples anticoagulated with EDTA showed marked hemolysis. Blood collected with citrate showed minimal hemolysis, and blood collected with sodium heparin did not have any hemolysis. Some analyses were not performed on blood containing EDTA because of the intense hemolysis. The erythrocyte variables were obtained and showed better results for blood samples in heparin. Blood smears without anticoagulant were appropriate for differential leukocyte counts with the three stain types, while blood with any of the anticoagulants made it difficult to differentiate leukocytes. CONCLUSIONS: Sodium heparin showed the best results and should be the anticoagulant of choice for hematologic studies in P expansa. All cytologic stains were efficient, allowing adequate identification of distinct cell groups. Leukocyte differentiation was assured and had better contrast using blood smears made from blood without an anticoagulant.

Assessment of captive rearing conditions on loggerhead hatchlings: Effect of handling frequency and stocking density.

Frequently, stranded sea turtles require rehabilitation under controlled conditions. Currently, few publications have described the conditions under which rehabilitation is to take place, particularly with respect to the hatchling life stage. To address this paucity of data, we conducted some experiments to assist rehabilitating facilities assess their handling of hatchlings. While in captivity, hatchlings are routinely handled, for example, for data collection and cleaning. Standardization of handling and housing protocols is necessary to define the most adequate rearing conditions to maintain hatchling welfare. Accordingly, the aim of this study was to assess plasma circulating corticosterone (Cort) concentration and growth, as a biomarker for the stress of hatchling loggerhead sea turtles (Caretta caretta) under controlled conditions. We performed two experiments to analyze handling frequency and stocking density. In both, Cort was measured and correlated with variations in animal weight and length. In handling experiments, Cort exhibited no significant increase when hatchlings were handled once a week, whereas Cort was significantly elevated when hatchlings were handled once every 2 weeks, suggesting that hatchlings have the ability to acclimate to frequent handling. However, hatchlings exhibited similar growth and mortality, regardless of handling regime. In stocking density experiments, hatchling isolation induced a significant elevation of Cort, in comparison with hatchlings placed with conspecifics at increasing densities. Growth increased in singly housed hatchlings, while mortality increased in tanks with three or more hatchlings. The results obtained suggest that Cort, growth, and mortality should be measured to assess hatchling welfare when kept under controlled conditions.

SAFETY OF MULTIPLE-DOSE INTRAMUSCULAR KETOPROFEN TREATMENT IN LOGGERHEAD TURTLES (CARETTA CARETTA).

Sea turtles are frequently presented for rehabilitation with injuries for which analgesic treatment is warranted. Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) widely used in clinical veterinary medicine for musculoskeletal pain relief. Pharmacokinetics of 2 mg/kg IM have been studied in loggerhead sea turtles (Caretta caretta) as a single and a repeated dose q24hr for 3 days. Safety of longer term administration has not been performed, however, and NSAID use carries a risk of potential complications, including gastrointestinal ulceration, kidney damage, and bleeding. The objective of the current study was to determine the effects of a 5-day course of ketoprofen on thromboelastography (TEG) and hematological (including thrombocytes) and plasma biochemical analytes in loggerheads. A secondary objective was to determine 24-hr trough concentrations of ketoprofen after 5 days of treatment. Eight loggerheads were treated with ketoprofen 2 mg/kg IM q24hr for 5 days, and TEG, hematology, and plasma biochemistry panels were performed before and at the conclusion of treatment. Eight controls were treated with an equivalent volume of saline intramuscularly. Virtually no changes were detected before and after treatment or between treatment and control groups in any of the 24 endpoints evaluated, and marginal differences were not considered clinically relevant. Plasma ketoprofen concentrations after 5 days of treatment indicated no accumulation over that duration. Ketoprofen at 2 mg/kg IM q24hr for up to 5 days in loggerheads appears safe with respect to blood clotting and blood data, although other potential effects were not evaluated.

BLOOD LACTATE CONCENTRATIONS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) FOLLOWING CAPTURE BY A CANINE SEARCH TEAM.

Studies to assess wildlife health commonly evaluate clinical pathology changes, immune responses, pathogen presence, and contaminant exposure, but novel modalities are needed to characterize the unique physiologic responses of reptiles. Lactate is an indicator of hypoperfusion and/or anaerobic respiration and can be quickly and easily measured using a point-of-care analyzer. This study evaluated baseline blood lactate concentrations in free-living eastern box turtles (Terrapene carolina carolina, n = 116) using a point of care analyzer and then determined the effect of handling time, physical examination (PE) abnormalities, and quantitative polymerase chain reaction pathogen detection (Terrapene herpesvirus 1, Mycoplasma sp., Terrapene adenovirus) on lactate concentrations. Blood lactate concentrations were higher in turtles with Terrapene herpesvirus 1 (n = 11), quiet mentation, and increased packed cell volume (P < 0.05). Lactate concentrations increased between initial capture and PE, with peak values reaching 129 min after capture. Lactate at PE was positively associated with baseline lactate concentrations. Turtles with Terrapene herpesvirus 1 may have alterations in blood flow, oxygen delivery, or activity patterns, driving increases in baseline lactate. Increased handling time likely leads to more escape behaviors and/or breath holding, causing turtles to undergo anaerobic metabolism and raising lactate concentrations. Overall, lactate measured by a point of care analyzer shows variability caused by capture and health factors in eastern box turtles and may be a useful adjunctive diagnostic test in this species after full methodologic validation.

PHARMACOKINETIC BEHAVIOR OF MELOXICAM IN LOGGERHEAD (CARETTA CARETTA), KEMP'S RIDLEY (LEPIDOCHELYS KEMPII), AND GREEN (CHELONIA MYDAS) SEA TURTLES AFTER SUBCUTANEOUS ADMINISTRATION.

The objective of this study was to determine the pharmacokinetics of a single dose of meloxicam administered subcutaneously (SQ) to three species of sea turtles: loggerheads (Caretta caretta), Kemp's ridley (Lepidochelys kempii), and greens (Chelonia mydas). A dose of 1 mg/kg was given to the Kemp's ridleys and greens, whereas the loggerheads received 2 mg/kg. After SQ administration, the half-life (t1/2) of meloxicam administered at 1 mg/kg in the Kemp's ridleys was 5.51 hr but could not be determined in the greens. The half-life of meloxicam administered at 2 mg/kg in the loggerheads was 2.99 hr. The maximum concentration (Cmax) for meloxicam after SQ administration at 1 mg/kg in the Kemp's ridleys was 6.76 µg/ml and in the greens was 9.35 µg/ml. The Cmax in loggerheads for meloxicam after SQ administration at 2 mg/kg was 3.63 µg/mL. Meloxicam administered SQ at a dose of 1 mg/kg to the Kemp's ridley and greens provided measurable plasma concentrations of meloxicam for 48 and 120 hr, respectively, with no adverse side effects. In loggerheads, meloxicam administered SQ at a dose of 2 mg/kg provided measurable plasma levels of meloxicam for only 24 hr. Plasma levels of meloxicam of greater than 0.5 µg/ml are considered to be therapeutic in humans. Results suggested that administration of meloxicam SQ at 1 mg/kg in Kemp's ridleys and greens would result in plasma concentrations greater than 0.5 µg/ml for 12 and 120 hr, respectively. The administration of 2 mg/kg meloxicam to loggerhead turtles resulted in plasma concentrations greater than 0.5 µg/ml for only 4 hr.

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species.

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.

Comparison of 2 glucose analytical methodologies in immature Kemp's ridley sea turtles: dry chemistry of plasma versus point-of-care glucometer analysis of whole blood.

Blood glucose measurements provide important diagnostic information regarding stress, disease, and nutritional status. Glucose analytical methodologies include dry chemistry analysis (DCA) of plasma and point-of-care (POC) glucometer analysis of whole blood; however, these 2 methods differ in cost, required sample volume, and processing time. Because POC glucometers use built-in equations based on features of mammalian blood to convert whole blood measurements to plasma equivalent units, obtained glucose data must be compared and validated using gold-standard chemistry analytical methodology in reptiles. For in-water, trawl-captured, immature Kemp's ridley sea turtles (Lepidochelys kempii) from Georgia, USA, we observed significant, positive agreement between the 2 glucose determination methods; however, the glucometer overestimated glucose concentrations by 1.4 mmol/L on average in comparison to DCA and produced a wider range of results. The discordance of these results suggests that POC glucometer glucose data should be interpreted in the context of methodology- and brand-specific reference intervals along with concurrent packed cell volume data.

Reproductive performance is associated with seasonal plasma reproductive hormone levels, steroidogenic enzymes and sex hormone receptor expression levels in cultured Asian yellow pond turtles (Mauremys mutica).

In order to understand the endocrine mechanism associated with fecundity of seasonally breeding animals, we investigated the plasma reproductive hormones levels and detected the differences in steroidogenic enzymes and sex hormone receptor mRNA levels in female Mauremys mutica. These turtles were divided into higher fecundity (HF) group than those in lower fecundity (LF) group based on paternity identification in our previous research. The plasma estrogen (E2), testosterone (T) and progesterone (P4) levels were significantly higher in pre-breeding season (PBS) than those in non-breeding season (NBS) and were markedly higher in the HF group than those in LF group. In the hypothalamus, there was significantly higher mRNA abundance of P450-cholesterol side-chain cleavage enzyme (P450Scc) encoded by Cyp11α1, aromatase (Cyp19α1) and 5-reductase (5α-R), but significantly lower mRNA levels of follicular stimulating hormone receptor (FSHR) and progesterone receptor (PR) detected in PBS than those in NBS. The pituitary steroidogenic acute regulatory protein (StAR), cytochrome P450-17alpha-hydroxylase (Cyp17α1), 3-hydroxy-steroid dehydrogenase (3ßHSD), 17-hydroxy-steroid dehydrogenase 3 (17ßHSD3), Cyp19α1, 5α-R, FSHR, estrogen receptor 1 (ESR1), androgen receptor (AR) and PR transcriptional levels in HF group were up-regulated significantly compared with the LF group. In the ovary, Cyp17α1 and 17ßHSD3 transcriptional levels were markedly higher in PBS than those in NBS. We detected significantly increased expression levels of all steroidogenic enzymes, but notably lower mRNA levels of FSHR and PR in uterus during the PBS, and the HF group has significantly higher expression levels of StAR, Cyp17α1, 5α-R and AR than LF group. Our work reveals seasonal variations in hormone regulation as well as gene regulation in turtles, providing reliable information to understand the mechanisms underlying the different reproductive capacity of reptiles.

PLASMA CAPILLARY ZONE ELECTROPHORESIS AND PLASMA CHEMISTRY ANALYTES IN TORTOISES (TESTUDO HERMANNI, TESTUDO GRAECA) AND TURTLES (TRACHEMYS SCRIPTA ELEGANS, GRAPTEMYS SPP.) IN FALL.

Clinical health checks including blood testing before brumation in fall are an important tool in reptile medicine and help to reduce the risk of complications during brumation. Electrophoresis may be useful for the evaluation of liver cell function and the detection of antigenic stimulation. The goal of this study was to compare species-specific reference intervals for plasma chemistry analytes and capillary zone electrophoresis (CZE) for a variety of pet chelonian species in fall. Lithium heparinized samples were collected from 68 clinically healthy spur-thighed tortoises (Testudo graeca), 79 red-eared sliders (Trachemys scripta elegans), and 30 map turtles (Graptemys spp.) from September to November 2016 and 2017. During the same period, 128 equivalent samples were collected from Hermann's tortoises (Testudo hermanni) and the obtained data was used for comparison. Chemistry analytes were measured from plasma using an autoanalyzer and electrophoresis was carried out by CZE. Significant variations (P < 0.0001) between the species were found for several analytes including total protein, percent albumin, and albumin: globulin ratio, α-, ß-, and γ-globulin, alkaline phosphatase, glutamate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bile acids, creatine kinase, urea, uric acid, inorganic phosphorus, total calcium, and potassium. The variations in albumin (g/l) measured by CZE were also significant (P = 0.0064). No significant variations were detected for sodium levels. The results demonstrate the importance of species-specific reference intervals and provide reference intervals for the plasma chemistry and capillary zone electrophoresis in three chelonian species.