This study described the PCV and several plasma chemistry analytes of green sea turtles, upon admission to a rehabilitation centre, during treatment and upon recovery, and suggests RIs for PCV, plasma glucose and potassium concentrations, as well as AST and ALP activity. The results of this study for recovered turtles from the EMS were compared with published data for a wild Mediterranean loggerhead population and two wild Atlantic green populations. We examined serum biochemical and hematological profile trends associated with body size and health status. PCV values were found to be indicative of recovery success rate after the initial treatment and during the rehabilitation phase.
This study encountered several challenges from, among other things, small sample sizes, a scarcity of published data, and differences in methodologies and reporting formats used in previous studies. The results published herein should, therefore, be regarded as of preliminary nature, and they comprise the initial parts of an ongoing research, expected to further elaborate and enhance these products.
Correlations between analytes
The PCV constitutes the relative red blood cell (RVC) volume of whole blood volume and is therefore highly corelated with whole blood hemoglobin concentrations (Emery 1986), as also demonstrated herein.
Cholesterol is a lipid that plays an essential role in energy transport, cell membrane formation, and steroid hormone synthesis (Gallagher et al. 2017). Well-fed, healthy animals, such as the turtles who accomplished full recovery at ISTRC, are likely to display adequate levels of serum cholesterol and PCV, as demonstrated by the positive correlation found between these analytes in this study.
Hyperglycemia can be induced by stress (Aguirre et al. 1995). Increased plasma leakage enzyme activity (i.e., CK) result from cellular injury (i.e., skeletal muscle tissue) (Bain 2011). As stress and tissue damage go hand in hand, we indeed observed a positive correlation between serum glucose concentrations and CK activity.
Exertional myopathy reduces sodium-potassium transport across cell membranes and causes cell disruption, leading to elevated extracellular potassium concentrations. In green sea turtles, hyperkalemia has been linked to forced submergence and capture stress (Phillips et al. 2015). Traumatic injuries also cause muscle cell damage and elevated serum potassium concentrations. Thus, the positive correlation observed between glucose and potassium levels can be assigned to concurrent stress-related effects and cellular damage.
Although ALP is found in reptile kidneys, its plasma levels do not rise following renal damage due to its release in urine rather than to blood. It is also widely dispersed throughout reptilian tissues and the source of its plasma activity is not organ-specific. Information regarding the clinical interpretation of elevated plasma ALP activity in reptiles is scarce, although it is commonly associated with bone formation during growth (Campbell 2006).Gregory and Schmid (2001) reported that size affected stress-induced increase of corticosterone, with higher corticosterone levels observed in smaller turtles after capture. Elevated stress hormones levels may obstruct salt gland function, which in sea turtles is responsible for most of the body’s salt excretion. Sea turtle salt gland function is also inhibited during cold stunning. The inhibition of salt gland activity can result in elevated blood concentrations of different ions, including potassium (Milton and Lutz 2003). Sea turtles are poikilotherms, and small turtles have higher surface area to volume ratios, therefore more prone to cold stunning-related salt gland inhibition. This common effect of age and body size on ALP and stress- or cold-related inhibition of potassium mediation by the salt glands may account for the positive correlation observed between these analytes in this study.
Lower total protein concentrations were previously documented in imperilled loggerhead sea turtles compared to healthy individuals, hypothesized as being associated with decreased food intake and nutritional status, or reduced protein assimilation (Alberghina et al. 2015). Imperilled turtles admitted to the rescue centre are often emaciated and malnourished, and presumably suffering physical trauma as well. Such trauma is considered the main reason for them to become beached and a cause of high CK activity, a skeletal muscle lesion marker. However, the rehabilitation process should reduce CK activity, while elevating total protein concentrations.
Reference intervals of blood analytes in recovered turtles and comparison with non-Mediterranean Sea conspecifics and other Mediterranean Sea turtle species
Suggested RIs with 90% CI for their upper and lower limits were computed for five serum chemistry analytes, while medians and ranges are provided for recovered green turtles for the remaining nine analytes. With relatively low numbers of green turtles present in the EMS and admitted in the ISTRC, the cohort size was limited. Nevertheless, as rehabilitation and research efforts are ongoing, and the database is continuously expanding, and we expect to compute additional RIs and validate the presently proposed ones. However, it is important to keep in mind the effects of captivity and feeding on analyte concentrations and activity in the process of RIs determination for turtles that went through rehabilitation.
Since vertebrate blood indices are affected by species, geographic area, ecological habitat, genetically distinct populations, sex, life stage, and more (Jensen et al. 1994; Maceda-Veiga et al. 2015; Page-Karjian et al. 2015), it is not a common practice to compare measurements from a specific population with values of different species or populations from other geographical areas. However, due to the scarcity of available data on green turtles in the Mediterranean Sea, such comparisons were the only ones applicable. It is important to note that environmental and nutritional conditions directly affect sea turtle serum chemistry. Additionally, differences in laboratory equipment, reagents and methodology might also account for plasma chemistry variations between studies (Casal et al. 2009).
AST activity can be found in many soft tissues and is non-organ-specific (Bain 2011), although is routinely employed as a biomarker of hepatic stress in turtles. In loggerhead turtles rehabilitated in Croatia, AST levels were higher just prior to turtle release than upon arrival to the rescue centre (Jakšić et al. 2022), in contrast to that observed herein, which may be due individual turtle physiological status and condition (Jakšić et al. 2022).
The differences in plasma triglycerides and cholesterol concentrations between the current results in recovered green turtles and previous ones of wild Mediterranean Sea loggerhead turtles (Gelli et al. 2009) are likely species-related, also influenced by interspecific dietary differences between omnivorous loggerheads and herbivorous green turtles, and likely not attributed to feeding differences between wild and captive turtles, as triglyceride concentrations of rehabilitated Canary Islands loggerhead turtles were similar to those of the Mediterranean Sea (Bolten and Bjorndal 1992; Casal et al. 2009; Page-Karjian et al. 2015), while the current triglyceride concentrations were similar to those reported in wild green turtles from the Bahamas (Bolten and Bjorndal 1992).
Plasma total protein concentrations in this study are somewhat higher compared to those reported in Mediterranean Sea loggerhead turtles and Atlantic green turtles, both wild and rehabilitated (Bolten and Bjorndal 1992; Casal et al. 2009; Gelli et al. 2009; Osborne et al. 2010). The reasons for this are unclear, but species related differences (regarding loggerhead turtles), and dietary and environmental variations, as well as differences in laboratory methods likely played roles in these findings.
Comparison between life stages and correlations with body size
We observed higher plasma AST and ALP activity in juvenile vs. subadult recovered turtles, and a significant negative correlation between CCL and these analytes. CCL also correlated negatively with potassium concentrations, and positively with PCV and cholesterol concentrations.
It is hard to interpret variations in AST activity without CK measurements, as AST activity is prevalent in many tissues, including muscle and liver, while CK is muscle-specific and most of its serum activity originates from muscle cells. Thus, if AST activity is increased, with no increase concurrently of CK activity, the former is very likely not of muscle origin, and may suggest liver damage (Bain 2011). Our results indicated higher AST activity in smaller turtles, also reported in green turtles from Taiwan (Fong et al. 2010) and Kemp’s ridley turtles (Perrault et al. 2020). It has been suggested by Perrault et al. (2020) that AST activity correlates negatively with CCL due to faster tissue growth at small turtle sizes, with a gradual decrease in growth rate as turtles become larger.
This trend of growth rate also applies to the process of bone formation, and accordingly, ALP activity is higher in juvenile turtles and decreases with an increase in CCL. These findings were observed by several other researchers in green and other sea turtle species (Bolten and Bjorndal 1992; Kakizoe et al. 2007; Stacy et al. 2018).
The negative size-correlation with potassium concentrations observed in this study was also reported by (Stacy et al. 2018), who proposed this may suggest limited osmoregulatory capacity (i.e., decreased potassium excretion) of smaller turtles due to underdeveloped or small salt glands, or alternatively indicate changes in bone metabolism.
Positive size-correlation with PCV, as observed herein, is widely documented in captive and wild sea turtles of various species, including green turtles (Frair 1977; Wood and Ebanks 1984; Stamper et al. 2005; Kakizoe et al. 2007; Casal et al. 2009; Basile et al. 2012; Rousselet et al. 2013; Kelly et al. 2015; Stacy et al. 2018; Perrault et al. 2020). An increase in age and body size is associated with a concomitant increase in red blood cell (RBC) volumes and decrease in RBC counts (Frair 1977). Sea turtle diving activity increases with size, making higher PCV advantageous for meeting the increasing oxygen demands in larger turtles (Stacy et al. 2018).
Cholesterol concentrations were shown to increase with increasing body size in green, loggerhead, and Kemp’s ridley sea turtles, possibly in relation to dietary shifts and changes in hormone levels and reproductive state (Kakizoe et al. 2007; Arthur et al. 2008; Perrault et al. 2020), similar to the current findings.
Comparison between treatment stages and correlations with percent of rehabilitation time
In this study, plasma total protein, potassium, and triglycerides concentrations increased from rehabilitation up to recovery, while plasma AST and CK activities decreased significantly, consistent with previous results in Atlantic green turtles rehabilitated in GA, USA (Bloodgood et al. 2019). This change in plasma total protein and potassium concentrations likely reflects malnutrition or digestive disfunctions, cachexia and a negative energy balance upon admission, with gradual improvement in body condition and energy balance over the rehabilitation with a protein-rich and high-fat diet and administration of medical care. These findings are in agreement with previous reports of stranded loggerhead turtles sustaining malnutrition and restricted food intake exhibiting significantly lower total protein, potassium, and triglyceride concentrations than healthy foraging turtles (Deem et al. 2009; Casale and Margaritoulis 2010; Osborne et al. 2010; Alberghina et al. 2015)
AST and CK are intracellular leakage enzymes found in myocytes. Their concomitant high activities in turtles upon admission, decreasing later during rehabilitation, towards recovery, are consistent with muscle damage occurring due to trauma or myopathy prior to admission, which gradually improved during treatment. Increased CK activity was also reported in stranded loggerhead turtles compared to healthy turtles (Deem et al. 2009; Flower et al. 2015), similar to AST activity (Casal et al. 2009).
Potential predictors of rehabilitation success
In samples obtained during the rehabilitation phase, PCV was significantly higher in turtles which ultimately recovered than in non-survivors. The ROC curve AUC of PCV during 10–65% percent of the total rehabilitation time as a predictor of the overall survival (0.84), indicates that PCV is a good prognostic marker (Ekelund 2012).
Pagano et al. (2019) examined and compared hematological and chemistry analytes of survivor and non-survivor loggerhead turtles stranded and rehabilitated in Sicily. In that study, the PCV was not measured, but the RBC count and hemoglobin concentration were recorded. Severe anemia was noted in all turtles upon admission, and both analytes were significantly lower in non-survivor turtles than in survivors.
No hematological or biochemical differences were detected to indicate a prognosis in the data of admitted turtles, likely due to all turtles suffering from poor health and possible anaemia. However, later during rehabilitation, turtles with lower PCV displayed lower chances of surviving, which is useful in alerting turtle caregivers of individuals requiring more intensive care and monitoring, as PCV measurements are simple, cost-effective, and widely available.
It should be noted, however, that the non-survivor sample size (N = 7) was three times smaller than the survivor sample size (N = 21), indicating the need for further assessments in this regard for the Mediterranean population.