MATERIALS AND METHODS

Ethical approval

This study was conducted with the authorization and institutional support of Universidad San Francisco de Quito (USFQ) and the Galapagos National Park Directorate (GNPD) under research permits PC-19-23, PC-12-24, and MAAE-DNB-CM-2021-0178-M-0001. All procedures complied with Ecuadorian national regulations governing wildlife research and followed animal care and handling guidelines approved by both institutions. Only free-ranging GSL pups were included in the study, and no animals were maintained in captivity or under human management. Field procedures were designed to minimize handling time, stress, and disturbance to breeding rookeries. Individuals were manually restrained to avoid the risks associated with chemical immobilization. All procedures were performed by USFQ-authorized veterinarians, assisted by trained GNPD park rangers experienced in pinniped handling. Following sample collection, pups were briefly monitored and released at the site of capture.

Study period and location

The study was conducted during March–April 2024 at multiple rookeries distributed throughout the Galapagos archipelago. A total of 40 GSL pups, representing 10.49% of the pups counted across the sampled rookeries, were examined (Table 1, Figure 1). Environmental conditions during the study coincided with the 2024 El Niño event.

Sampling date | Island | Rookery | Pups sampled | Pups counted
31 Mar 2024 | Pinta | Cabo Ibetson | 2 | 26
01 Apr 2024 | Marchena | Punta Mejía | 2 | 2
02 Apr 2024 | Genovesa | Salvaje de Corazón | 2 | 37
03 Apr 2024 | Rábida | Beagles | 5 | 19
03 Apr 2024 | Santiago | Sombrero Chino | 2 | 4
04 Apr 2024 | Seymour | Mosquera | 5 | 31
04 Apr 2024 | Seymour | Seymour Norte | 5 | 67
05 Apr 2024 | Santa Cruz | Plazas Sur | 5 | 61
05 Apr 2024 | Santa Fe | Barrington | 4 | 25
06 Apr 2024 | Floreana | Champion | 2 | 38
06 Apr 2024 | Floreana | Post Office | 2 | 38
07 Apr 2024 | Española | Bahía Gardner | 2 | 32
07 Apr 2024 | Española | Punta Suárez | 2 | 2
Total | 10 | 13 | 40 | 381

Figure 1: Archipelago-wide spatial distribution of chlorophyll-a (mg m⁻³) in the Galapagos Islands during 2023 (non-El Niño–Southern Oscillation [ENSO] year, left panel) and 2024 (ENSO year, right panel). Oceanic Niño Index values for each year are indicated on the maps. Rookery locations of Galapagos sea lions (yellow triangles) and Galapagos fur seals (red circles) are shown, highlighting site-specific patterns of marine productivity across the archipelago.

Study design

This cross-sectional field study evaluated morphometric, hematological, and biochemical characteristics of free-ranging GSL pups. Pups were identified based on external morphology, particularly the presence of natal fur before molting, which enabled clear differentiation from older juveniles. However, precise chronological age could not be determined. Each pup was captured, handled, and sampled only once. To prevent resampling, individuals were marked with a haircut on the dorsal surface of the hind flippers.

Pups were captured using a hoop net whenever possible during periods of maternal absence to minimize disruption to mother-pup interactions. Capture and manual restraint were performed by trained park rangers, while two wildlife veterinarians simultaneously conducted the procedures to reduce total handling time. To minimize inter-observer variability, the same veterinarian performed all morphometric measurements and blood collections throughout the study. Each pup underwent a brief physical examination and was classified as apparently healthy based on adequate body condition, normal posture and behavior, appropriate responsiveness to handling, and the absence of visible wounds or clinical signs of disease. Sex was determined through external genital examination.

Morphometric measurements

Morphometric variables included body weight, standard length, and axillary girth, following the same field procedures used in the comparative GFS dataset [23].

Body weight (kg) was measured using a digital scale and net sling (precision ± 0.1 kg), ensuring full support of the animal during weighing. Standard length was measured from the tip of the nose to the end of the tail using a flexible measuring tape to the nearest 0.5 cm. Axillary girth was measured immediately caudal to the fore flippers. Body condition was calculated as body weight (kg) divided by standard length (cm).

Blood collection

Approximately 5 mL of blood was collected from the caudal gluteal vein using 21-gauge needles. Blood was immediately transferred into ethylenediaminetetraacetic acid (EDTA) tubes (Jiangsu Kangjian Medical Apparatus Co., Ltd., Jiangsu, China) and plain serum tubes (Suzhou Medmay Biotechnology Co., Ltd., Suzhou, China) for hematological and biochemical analyses. Samples were kept in a cooler containing ice packs, transported to a shipboard laboratory, and processed within 4 h of collection. Total handling and sampling time was approximately 10 min per pup to minimize stress and potential handling-related alterations in blood parameters.

Laboratory analyses

Blood smears were prepared from EDTA-anticoagulated whole blood, air-dried, and stained using a Diff-Quik stain kit (Química Clínica Aplicada S.A., Tarragona, Spain) according to standard hematological procedures [24]. Hematocrit and serum biochemical analyses were performed shortly after sample collection. For serum chemistry, whole blood was centrifuged at 3,500 × g for 10 min to separate serum.

Serum analytes were measured using a portable VetScan VS2 analyzer (Abaxis, Zoetis, Parsippany, NJ, USA) with the VS2 Mammal Profile Plus rotor. The analytes evaluated were alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), globulin (GLOB), total protein (TP), total bilirubin (TB), blood urea nitrogen (BUN), creatinine (CRE), phosphate (PHO), glucose (GLU), and sodium (Na⁺).

Hematocrit was determined using the microhematocrit method by centrifugation of EDTA-anticoagulated blood in capillary tubes at 12,000 × g for 5 min, followed by direct reading with a microhematocrit reader. The remaining serum and cellular fractions were stored at −20°C and transported to the USFQ laboratory for potential future analyses.

Differential white blood cell counts were performed 4–6 weeks later at the USFQ laboratory. Stained blood smears were examined under a light microscope (Eclipse Ci-L, Nikon Corporation, Tokyo, Japan) at 1000× magnification using oil immersion. The relative proportions of neutrophils, lymphocytes, monocytes, eosinophils, and basophils were determined by counting 100 leukocytes per smear. All leukocyte differentials were performed by a single trained observer to minimize inter-observer variability.

Environmental data collection

ENSO conditions during the study period were characterized using the Oceanic Niño Index (ONI) values for seasons overlapping the sampling period. According to the National Oceanic and Atmospheric Administration, the ONI represents the 3-month running mean of sea surface temperature anomalies in the Niño 3.4 region (5°N–5°S, 120°–170°W) [15]. El Niño conditions were classified as ONI > +0.5°C, with weak, moderate, and strong events corresponding to +0.5–0.9°C, +1.0–1.4°C, and ≥ +1.5°C, respectively.

Satellite-derived chlorophyll-a data for the Galapagos archipelago were obtained from the Copernicus Marine Environment Monitoring Service [25] using the “Global Ocean Color Bio-Geo-Chemical, Level-4 monthly and interpolated” product as a proxy for marine primary productivity. Chlorophyll-a concentrations at a spatial resolution of 4 km were extracted for March–April 2023 and March–April 2024. Raster datasets were processed using ArcGIS Pro 3.2 (Esri, Redlands, CA, USA), converted into point features, and mean chlorophyll-a values were calculated within 50-km circular buffers surrounding each rookery. This buffer size was selected to characterize near-rookery marine productivity potentially relevant to local otariid foraging ecology [26, 27]. Each rookery contributed one mean chlorophyll-a value per year.

Statistical analysis

All analyses were conducted in R version 4.5.1 (R Core Team, Vienna, Austria; https://www.r-project.org/). Descriptive statistics, including mean, standard deviation, and range, were calculated for biochemical, hematological, and morphometric parameters. Normality of each variable was assessed using the Shapiro–Wilk test. Sex-related differences were analyzed using parametric methods for normally distributed data and non-parametric methods for non-normally distributed data. Published reference ranges from related otariid species were used to contextualize the health status of Galapagos pinnipeds.

Descriptive analyses were performed in R version 4.5.1, whereas inferential analyses were conducted using Python 3.11 (Python Software Foundation, Wilmington, USA; https://www.python.org/) with NumPy (NumPy Developers; https://numpy.org/), pandas (The pandas development team; https://pandas.pydata.org/), and SciPy (SciPy Developers; https://scipy.org/) libraries. Statistical significance was set at p < 0.05. Rookery-level mean chlorophyll-a values were evaluated for normality using the Shapiro–Wilk test and for homogeneity of variance using the median-centered Levene's test. Interannual paired differences were analyzed using the Wilcoxon signed-rank test because the data deviated from normality, with each rookery contributing one paired value.