Introduction

Sea turtles are exposed to a wide range of environmental changes in each of their life stages, so assessing their health is of paramount importance (^{Aguirre and Lutz 2004}). These ectothermic organisms depend on ambient temperature to carry out their main activities, so changes in water temperature can affect the function of their immune system and represent different threats (^{Foley et al. 2007}, ^{Hunt et al. 2019}). Although sea turtles have thermoregulatory adaptations that allow them to save energy and maintain stable body temperatures, such as torpor (^{Southwood et al. 2003}), when temperatures drop below 10 ºC, they become lethargic and are unable to swim or dive, entering a status of hypothermia (^{Schwartz 1978}, ^{Innis et al. 2007}) that disrupts metabolic pathways leading to imbalances in their different systems (^{Innis et al. 2007}). Changes in temperature may also increase the risk of infectious diseases such as fibropapillomatosis (^{Arthur et al. 2008}, ^{Van Houtan et al. 2014}). Monitoring vital signs is therefore important for physiological and medical assessments and it becomes a basic tool for the conservation of these species. In laboratory studies with captive turtles, physical factors such as temperature can be controlled and metabolic and physiological responses can be closely monitored (^{Southwood et al. 2003}); however, in free-ranging populations handling sea turtles is often more complicated, so improving some methods and techniques used in their health assessment can facilitate vital-sign monitoring and even sample collection (^{Reséndiz et al. 2019}). The aims of this study were to (1) assess body temperature and heart rate in black turtles (Chelonia mydas) in the Ojo de Liebre lagoon complex, Baja California Sur, Mexico; (2) validate a novel, non-invasive temperature measurement technique; and (3) compare the body temperature and heart rate data obtained in this study with previous reports.

Materials and methods

Ojo de Liebre Lagoon (OLL) and Guerrero Negro Lagoon (GNL) in the Ojo de Liebre lagoon complex are part of El Vizcaíno Biosphere Reserve, on the northern Pacific coast of Baja California Sur (Mexico). These coastal lagoons are located between latitude 27.5833 and 28.25º N and longitude 113.8333 and 114.3333º W, and they are characterized by having extensive shallow, hypersaline areas, no contributions of fresh water, high evaporation, and mean sea surface temperatures of 21.29 ºC (OLL) and 20.53 ºC (GNL) (^{ESSA 2018}). Both lagoons are inhabited by black turtles (^{Reséndiz et al. 2018}), which were considered a single group of organisms because of the geographical proximity of both lagoons and the similarity in habitat characteristics.

A total of 20 field trips were carried out from May 2017 to December 2019 to capture black turtles in OLL and GNL. All captures were made using monofilament nets (100 m long by 5 m deep, 60-cm mesh width) during daylight hours. Nets were periodically checked every hour to avoid excess stress on the turtles and to minimize lesion risk.

To assess vital signs, the turtles were systematically examined in detail in prone and supine positions on the boat (^{Reséndiz et al. 2018}). The carapace, plastron, and left inguinal area skin temperatures were taken with a digital infrared thermometer gun (Steren HER-425); the gun was held at a distance of 10 cm from the surface of each area, and temperatures were taken immediately (1-30 seconds) after the turtles were captured. For each anatomical region, temperature was measured 3 times and the mean was calculated (^{Reséndiz et al. 2018}). Cloacal temperature was measured with an electronic thermometer (Suretemp Plus 690, Welch Allyn). During the physical examination and after resting for 10 min alone in a free space, the turtles were auscultated with a stethoscope (IUMED model 400), which was placed on the skin between the neck and the proximal front flipper. The curved carapace length (CCL, centimeters) and turtle weight (kilograms) were then recorded (^{Bolten 1999}); immediately after, the turtles were aseptically tagged with metallic Inconel 681 tags (National Band & Tag) in the skin of the distal hind limbs (^{Balazs 1999}) and released. Black turtles under 77.5 cm CCL were considered juveniles and those 77.5 cm CCL or over were classified as adults according to ^{Márquez (1990)}.

For this study, juvenile and adult turtles were considered a single group of organisms since there were no significant differences in temperature and heart rate between age classes (P < 0.05). The Kolmogorov-Smirnov statistic, skewness, and kurtosis were used to test data for normality. The mean and standard deviation were determined for each variable. Student’s t-tests between cloacal (control) temperature and carapace, plastron, and inguinal area temperatures were performed to assess the effectiveness of the temperature measurements. A linear regression between cloacal and inguinal area temperatures was made for black turtles examined in the present study. Comparisons of cloacal temperature and heart rate between black turtles examined in the present study, black turtles in OLL and San Ignacio Lagoon (SIL) (^{Reséndiz et al. 2018}), and loggerhead turtles (Caretta caretta) in the Gulf of Ulloa (GU, ^{Reséndiz et al. 2019}) were made using a one-way analysis of variance, and specific between-group differences were evaluated using a Tukey’s test. Values of P < 0.05 were considered statistically significant. Statistical analyses were performed using R v.3.6.2 (Integrated Development for R. RStudio; Boston, MA).

Results

A total of 600 black turtles measuring 72.38 ± 7.54 cm CCL and weighing 42.24 ± 20.80 kg were captured. OLL and GNL turtles were classified as juveniles and adults and determined clinically healthy according to physical examination. Data on morphometrics and vital signs (body temperatures and heart rate) are shown in Table 1. There was no significant difference between cloacal temperature and inguinal area temperature for black turtles from the present study (P = 0.13), and both variables showed a positive linear relationship (R ² = 0.44, Fig. 1). There was significant variation in cloacal temperatures between the 4 groups of marine turtles (F = 4.33, d.f. = 3, P < 0.05) (Fig. 2a). A Tukey’s post hoc test indicated that cloacal temperatures for black turtles in the present study (20.74 ± 2.82 ºC) and those for black turtles in OLL (19.85 ± 2.25 ºC) (^{Reséndiz et al. 2018}) were significantly lower than cloacal temperatures for black turtles in SIL (22.42 ± 3.13 ºC) (^{Reséndiz et al. 2018}). There was no significant variation in mean heart rates between the 4 groups (F = 0.53, d.f. = 3, P = 0.66) (Fig. 2b).

Figure 1 Linear regression between cloacal and inguinal area temperatures for black turtles (Chelonia mydas) in the present study. 

Figure 2 Cloacal temperature (a) and heart rate (b) for black turtles (Chelonia mydas) in Ojo de Liebre Lagoon (OLL) and Guerrero Negro Lagoon (GNL) (present study), black turtles in OLL and San Ignacio Lagoon (SIL) (^{Reséndiz et al. 2018}), and loggerhead turtles (Caretta caretta) in the Gulf of Ulloa (GU) (Reséndiz et al. 2019). 

Discussion

In sea turtles cloacal temperature can be representative of ambient temperature (^{Southwood et al. 2003}, ^{Foley et al. 2007}). By using data from healthy organisms as reference intervals, wildlife rehabilitators can more rapidly respond to events of cold-stunned turtles, increasing their chances of recovery (^{Wyneken et al. 2006}). However, the technique for taking cloacal temperature is moderately invasive and may be painful for organisms (^{Manire et al. 2017}). The left inguinal area temperature values reported in the present study coincided with the body temperature value previously reported for healthy turtles (Southwood et al. 2003, ^{Hunt et al. 2019}), and the present study confirmed that the use of the digital infrared thermometer in the left inguinal area is an easier, effective, and less invasive measurement technique. Although both areas showed a positive statistical relationship, the value of R ² is medium-low and must be interpreted carefully. We recommend using this type of thermometer (properly calibrated) and recording body temperature when the turtle has just been captured in order to obtain the most representative values and avoid bias due to stress or external factors such as ambient temperature. The comparison of temperature between groups showed that cloacal temperatures were significantly higher in SIL turtles than in OLL and GNL turtles, which can be attributed to the conditions of the water masses they inhabit. OLL and GNL are interconnected and together occupy an area of 381 km² (^{Contreras 1985}). Both lagoons flow into the Sebastián Vizcaíno Bay, a biological active center that is located within the California Current region, where northwesterly winds create one of the main coastal upwelling regions (^{Lluch-Belda 2000}). SIL is located further south and occupies an area of 175 km² (^{Contreras 1985}). This lagoon flows into the GU, which is also considered a biological active center with coastal upwelling (^{Lluch-Belda 2000}). The surface, geographic location, and oceanographic conditions of the different sites can explain the differences in water temperature and hence the organisms.

Cardiac auscultation in sea turtles allows us to identify basic physiological alterations or certain pathological processes (^{Butler et al. 1984}, ^{Hunt et al. 2019}), which are often initially described as tachycardia and bradycardia. Under controlled conditions (sea turtle hospitals and rehabilitation centers) heart rate is recorded with a Doppler probe or ultrasound, and the use of a stethoscope is unconventional (^{Norton 2005}). However, our results showed that the use of the stethoscope is a novel and effective technique for assessing heart rate in free-ranging sea turtle populations. The heart rates for black turtles in the present study were similar to those reported by ^{Southwood et al. (1999)}, ^{Norton (2005)}, and ^{Reséndiz et al. (2018}, ²⁰¹⁹⁾ for healthy sea turtles at a temperature of 24 ºC, which ranged from 30 to 60 beats per minute. There were no significant differences between black turtle heart rates in this study and previously reported data for black turtles in OLL and SIL, and loggerheads in GU, indicating that these data are an accurate representation for the species. This basic diagnostic orientation tool allows us to rule out health abnormalities such as cold-stunning, signs of respiratory problems, lung lesions, and other pathological changes related to the respiratory system (^{Reséndiz et al. 2019}).

In summary, body temperature and heart rate data for OLL and GNL black turtles were generated and compared with previous reports. This information provides further insight into the variation of vital signs between different species of sea turtles in nearby areas. The use of the digital infrared thermometer in the left inguinal area proved to be an easier, effective, and less invasive measurement technique than taking the cloacal temperature. The minimally invasive diagnostic approach using vital signs described here allows us to evaluate and infer core physiological functions, including general cardiopulmonary status, and rule out health threats such as cold-stunning, resulting in a useful preliminary systemic assessment of free-ranging sea turtles.