Health Status of Galápagos Sea Lions (Zalophus wollebaeki) on San Cristóbal Island Rookeries Determined by Hematology, Biochemistry, Blood Gases, and Physical Examination

The Galápagos sea lion (GSL; Zalophus wollebaeki) is an endemic marine mammal of the Galápagos Islands, with the majority of the population residing in the eastern and southern islands of the archipelago (San Cristóbal and Floreana) (Alava and Salazar 2006; Páez-Rosas 2011). The species was listed as endangered in the Red List of Threatened Species by the International Union for Conservation of Nature because of a large population reduction over the past 30 yr through exposure to new diseases through human and animal contact (i.e., cats [Felis catus], dogs [Canis lupus familiaris], rats [Rattus norvegicus]) (Aurioles and Trillmich 2008; Denkinger et al. 2015).

Negative consequences of anthropogenic impacts may be amplified by acting synergistically with environmental variability such as the El Niño Southern Oscillation (Trillmich et al. 1991). These oceanographic events lead to a lack of food resources in marine ecosystems and may cause nutritional stress, thus increasing mortality of GSLs (Páez-Rosas et al. 2012; Villegas-Amtmann et al. 2013).

Although isolated in the Pacific, the Galápagos Islands are not exempt from the dangers of introduced diseases (Aurioles and Trillmich 2008; Denkinger et al. 2015). As the numbers of visitors, residents, and domestic animals that arrive on the archipelago increase each year, so does the threat of disease to resident wildlife (Kilpatrick et al. 2006; Lorden et al. 2012). Under this premise, health assessments become important for understanding the response mechanisms of species that are endangered, as evidenced by the time trajectories of their population abundance (Brock et al. 2013).

Reference intervals for certain pinniped species have been reported (Castellini et al. 1996; Horning and Trillmich 1997; Rea et al. 1998; Trillmich et al. 2008). Additionally, the hematology of Galápagos pinnipeds has been studied but not widely reported (Trillmich et al. 2008; Actis 2012) and data regarding the assessment of clinically normal animals is sparse.

We collected data in two rookeries of a large colony at Puerto Baquerizo Moreno (0°54′8″S, 89°36′44″W) on San Cristóbal Island (Fig. 1). This colony represents the largest breeding site in the Galápagos Archipelago and has an average of 630 animals (Páez-Rosas and Guevara 2012).

In September 2014, we sampled 30 juvenile sea lions 2–4 yr old; age classes were determined according to Jeglinski et al. (2010). Before sampling the animals, we evaluated body condition using weight/length relationship and external physical condition to avoid sampling sick animals (Trillmich 1986; Hall et al. 2002). Sea lions resting ashore were captured with hoop nets and briefly restrained in a prone position without chemical immobilization. To minimize potential effects of handling on blood parameters, samples were obtained within approximately 10 min of capture. An EBRO^® compact J/K/T/E thermocouple thermometer (model EW-91219-40; Cole-Parmer, Vernon Hills, Illinois, USA) was applied to obtain internal body temperature from the rectum (5–7 cm insertion) using the probe T polyvinyl chloride epoxy tip 24GA (some body temperatures were obtained with a simple mammalian digital rectal thermometer). The animals were then weighed in the hoop-net using a digital scale (precision: 0.1 kg) hanging from a large tripod and standard body length (SL), curved body length, and axillary girth were measured to the nearest 0.5 cm. To approximate nutritional status a body condition index was calculated as weight/SL (Hall et al. 2002). Heart rate was measured using a Doppler flow detector (Parks Medical Electronics Inc., Aloha, Oregon, USA) and sex was determined on the basis of external sexual dimorphism.

We captured, examined, and sampled 20 sea lions at Punta Carola (0°54′3″S, 89°34′46″W) and 10 animals at the naval base (0°54′6″S, 89°36′55″W) (Fig. 1). To avoid capturing the same animal more than once, a small line of fur was trimmed from the back, close to the posterior flippers, after a blood sample had been obtained and before the animal was released. All captured animals were clinically healthy on the basis of appearance, behavior, and response to handling.

Caudal gluteal venipuncture was performed using a heparinized 20-gauge needle attached to a 5.0-mL syringe to collect up to 3.5 mL of blood per sea lion. The blood was immediately divided into subsamples, which were either used for instant analysis with portable blood chemistry and lactate analyzers, or stored on ice in sterile plastic vials within 10 min of sample collection for hematology and future analyses.

Blood gas, electrolyte, and biochemistry results were obtained using an iSTAT Portable Clinical Analyzer (Abbott Point of Care Inc., Princeton, New Jersey, USA) with CG8+ cartridges. The iSTAT is a portable, handheld, battery-operated electronic device that measures a variety of blood gas, chemistry, and basic hematology parameters with only a few drops (0.095 mL) of whole, noncoagulated blood. We measured and recorded: pH, partial pressure of oxygen (pO₂₎, partial pressure of carbon dioxide (pCO₂₎, bicarbonate (HCO₃⁻), hematocrit (Hct), hemoglobin (Hb), Na, K, ionized Ca (iCa), and glucose. The device analyzed the blood at 37 C and then corrected pH, pO₂, pCO₂, iCa, and HCO₃⁻ for body temperature once this information was entered. Blood lactate was determined using a portable Lactate Plus^TM analyzer (Nova Biomedical, Waltham, Massachusetts, USA). Remaining samples were immediately stored on ice during fieldwork and centrifuged within 3 h of collection. Hematocrit was determined using high-speed centrifugation of blood-filled microhematocrit tubes at 7,155 × G for 5 min.

The iSTAT results for Hct were compared with the results of manually determined Hct using a paired t-test. We grouped values for each blood analyte by sex and compared the groups using Mann–Whitney U-test. Spearman’s rank correlations were used to examine the relationship between the sea lions’ body condition (Hall et al. 2002) and the measured blood parameters. An alpha of P=0.05 was used for all statistical tests using R statistical software 3.0.2 (R Development Core Team 2014).

Table 1 summarizes morphologic and physiologic measurements and the time to obtain blood samples. There was no statistical difference between sexes for any biochemistry, blood gas, or hematology results (Table 2). The mean difference between the manually determined Hct values and the automatic iSTAT results was −4.95 (t=−7.7432, df=29, P<0.001) (Table 2). Of all blood parameters only Hb was significantly correlated (r=0.39, P=0.04) with sea lion body condition. We compared blood chemistry and hematology results with values reported by Roletto (1993) for juvenile California sea lions (CSLs; Zalophus californianus), the closest related species (Wolf et al. 2007) (Table 3).

Most measured blood chemistry and hematology values are comparable with other pinniped species and reflect parameters for a healthy population of juvenile GSLs (Needham et al. 1980; McConnell and Vaughan 1983; Roletto 1993; Trumble and Castellini 2002; Lander et al. 2013; Witte et al. 2014).

The largest differences in blood parameters between GSLs and CSLs were found in Hct, Hb, calcium, and potassium (Table 3). Discrepancies in calcium and potassium might result from dietary salt supplementation of CSLs in captivity (Roletto 1993). Healthy CSLs and GSLs in this study had similar protein and glucose levels, indicating fair nutritional status (Trites and Donnelly 2003; Mellish et al. 2006). The robust GSL Hct is also an indicator of good nutritional status. Increased Hct and Hb levels have been found in free-living versus captive harbor seals (Phoca vitulina), which might explain the difference in both blood parameters due to increased physical activity and deep diving in free-ranging GSLs (McConnell and Vaughan 1983).

Other investigators found an increased immune activity in GSL pups at the same study site, due to exposure to human disturbances. Nevertheless, the authors did not find that the total protein or Hct values of disturbed GSL pups were altered relative to undisturbed controls (Brock et al. 2012). We therefore gauge the validity of our data set as a reference for juvenile GSLs in areas with high human use, but emphasize the importance of extending blood chemistry and hematology data to include sampling of nonaffected rookeries, as well as rookeries under nutritional stress from environmental variability and anthropogenic impacts.

We thank Carlos Mena, Philip Page, Kent Passingham, Stephen Walsh, the Galápagos National Park Service, and especially the Heska Corporation for support and assistance.