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.

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.

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 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).

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.

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.

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.

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.

No significant sex-related differences were detected in GSL pups with respect to body weight, standard length, axillary girth, or body condition (Table 2). Welch’s t-tests showed that all comparisons were non-significant (all p ≥ 0.89). Mean differences between males and females were minimal, effect sizes were negligible (Hedges’ g ranging from −0.03 to 0.05), and the 95% confidence intervals for all mean differences included zero. Overall, these findings indicate similar morphometric profiles between female and male GSL pups during the sampled postnatal period.

Body condition was significantly higher in GSL pups than in sympatric GFS pups in both sexes (Table 3). In females, mean body condition was higher in GSL than in GFS, and this difference was significant in both Welch’s t-test and Mann–Whitney U test (both p < 0.001), with a large effect size. A similar pattern was observed in males, with significantly higher body condition in GSL than in GFS (both p < 0.001) and a large effect size. These findings indicate consistently higher relative body condition in GSL pups than in GFS pups during the study period.

Sex-stratified serum chemistry values for GSL and GFS pups are summarized in Table 4. In females, significant interspecific differences were observed for ALB, GLOB, TP, and BUN. ALB concentrations were higher in GFS females than in GSL females, whereas GLOB and TP concentrations were higher in GSL females than in GFS females. BUN concentrations were also higher in GFS females. No significant interspecific differences were detected for ALT, ALP, TB, CRE, PHO, GLU, or Na⁺.

In males, GLOB and TP concentrations were higher in GSL than in GFS, whereas GLU concentrations were higher in GFS than in GSL. No other analytes differed significantly between species in males. Within GSL, females exhibited significantly higher GLOB and TP concentrations than males. No significant sex-related differences were observed within GFS for any serum chemistry analyte.

Hematological values are summarized in Table 5. In females, hematocrit, neutrophils, lymphocytes, monocytes, and eosinophils did not differ significantly between GSL and GFS, whereas basophil percentages were significantly lower in GSL females. In males, hematocrit values were significantly higher in GSL than in GFS, whereas monocyte and eosinophil percentages were significantly lower in GSL males. Neutrophil, lymphocyte, and basophil percentages did not differ significantly between species in males.

Published serum chemistry ranges for GSL and GFS pups broadly overlapped with those reported for other otariid species for most analytes. The most notable differences were observed for ALP, TB, and Na⁺. Both Galapagos species exhibited lower ALP and higher TB ranges than Antarctic fur seals, South American fur seals, and Steller sea lions, whereas Na⁺ concentrations were generally higher in Galapagos otariids. In contrast, ALT, ALB, TP, BUN, CRE, PHO, and GLU values generally fell within the broad ranges reported for other otariid pups (Supplementary Table S1).

Hematological profiles of GSL and GFS pups also broadly overlapped with published values reported for other otariids. Hematocrit, neutrophil, lymphocyte, monocyte, and eosinophil values were generally comparable across species, whereas basophil values were consistently low or were not reported in the available literature (Supplementary Table S2).

The 2024 sampling period coincided with a weak-to-moderate El Niño event (ONI: +0.8 to +1.3°C), whereas 2023 represented an ENSO-neutral baseline period (ONI < +0.5°C). Rookery-level chlorophyll-a concentrations were significantly lower in 2024 than in 2023 (Wilcoxon signed-rank test, V = 37, p = 0.005). Across rookeries, the median paired change indicated an overall decline in chlorophyll-a concentrations, although the magnitude of change varied among sites (Supplementary Table S3).

Despite this general reduction in marine productivity, some local areas retained relatively high chlorophyll-a concentrations, indicating spatial heterogeneity in ENSO-related productivity changes across the archipelago (Figures 1 and 2).

The present study provides an integrated clinical assessment of GSL pups during the 2024 El Niño event and compares these findings with those of sympatric GFS pups using morphometric, biochemical, and hematological parameters. Sampling coincided with a weak-to-moderate ENSO phase and reduced rookery-level chlorophyll-a concentrations relative to 2023, suggesting lower marine productivity during the study period. This environmental context is relevant because Galapagos pinnipeds are known to be sensitive to El Niño-driven oceanographic variability, although the magnitude of ecological effects differs among events [21].

No significant sex-related differences were detected in body weight, standard length, axillary girth, or body condition in GSL pups. These findings indicate morphometric similarity between females and males during early postnatal development and are consistent with previous studies showing that sexual size dimorphism in otariids generally becomes more apparent later in life [28, 29].

Interspecific comparisons revealed clearer differences. Both female and male GSL pups exhibited higher body condition values than GFS pups, indicating better relative body condition during the study period. Body condition indices are widely used as size-adjusted indicators of nutritional status and maternal investment in marine mammals [30, 31]. These interspecific differences may be related, at least in part, to contrasting foraging ecologies. GSLs typically forage in more coastal environments and consume mainly small epipelagic fish, whereas GFSs exploit more pelagic habitats and show a greater contribution of cephalopods to their diet [32]. In addition, GSLs have been reported to exhibit trophic flexibility during ENSO conditions, which may facilitate adjustments in prey use when marine productivity declines [33].

El Niño-associated environmental variability can reduce prey availability and increase maternal foraging effort, with potential downstream effects on pup growth and nursing frequency [20, 34, 35]. However, despite these constraints, the morphometric indicators observed in GSL pups remained relatively stable. This pattern may reflect the moderate intensity of the 2024 ENSO event or short-term physiological resilience, although sampling bias toward apparently healthy individuals should also be considered.

The serum chemistry results revealed a partially similar pattern. GSL pups had higher GLOB and TP concentrations than GFS pups in both sexes, paralleling the interspecific differences observed in body condition. Serum protein levels in marine mammals may vary with nutritional status, hydration, immune activity, and inflammatory processes [10, 11, 36]. Therefore, the higher GLOB and TP concentrations observed in GSL pups may reflect broader interspecific differences in physiological condition, immune stimulation, or both. Nevertheless, because these markers are influenced by multiple biological processes, their interpretation should remain cautious.

In contrast, differences in ALB, BUN, and GLU were restricted to a single sex and should therefore be interpreted conservatively. ALB and BUN concentrations were higher in female GFS pups, whereas GLU concentrations were higher in male GFS pups. Because these analytes can be influenced by recent feeding, fasting duration, hydration, and short-term metabolic variation, such differences may reflect transient physiological states rather than stable species-level patterns [37].

A similarly cautious interpretation applies to the hematological findings. Most leukocyte differential values overlapped between species, indicating broadly comparable hematological profiles. The few significant differences observed, including higher hematocrit values in GSL males and higher monocyte and eosinophil values in GFS males, may reflect variation in immune maturation, inflammatory status, or parasite exposure rather than consistent interspecific divergence. Previous studies in otariid pups have shown that parasitism and immune development can influence hematological parameters during early life [38, 39].

Comparisons with published data from other otariid pups further support the overall interpretation of the present findings. Most biochemical and hematological analytes fell within ranges previously reported for other otariid species, suggesting that the clinical profiles of both Galapagos pinnipeds remained broadly within expected physiological limits. Nevertheless, ALP and TB showed some divergence from certain published datasets, with both species exhibiting relatively lower ALP and higher TB values. These differences should be interpreted with caution. Variation among studies may reflect differences in age class, nutritional status, health status, sampling context, environmental conditions, or analytical methodology rather than clear biological separation. Although lower ALP and higher TB values could be compatible with differences in metabolic or nutritional status, the present data do not allow a specific causal interpretation [11, 40–43].

Several limitations should be considered when interpreting this study. First, this was a cross-sectional assessment conducted during a single breeding season, which limits inference regarding temporal variability in physiological parameters. Hematological and biochemical values in marine mammals may vary across seasons, years, and life-history stages according to differences in diet, development, and metabolic demands.

Second, sample size limitations are inherent to studies of free-ranging marine mammals, particularly in remote and protected environments such as the Galapagos archipelago. Logistical constraints, animal welfare considerations, and restricted access to rookeries can limit the number of animals sampled and reduce statistical power.

Third, the study focused on apparently healthy pups accessible within breeding rookeries. Consequently, the reported values likely represent relatively healthy individuals and may underestimate the full range of health variability within the population.

Fourth, interpretation of clinical pathology in wildlife is constrained by the limited availability of species-specific reference intervals and methodological differences among studies. Variability in analytical platforms, laboratory procedures, and sample handling may influence reported values and complicate direct interstudy comparisons.

Fifth, capture and restraint may have influenced some blood analytes, particularly GLU concentrations and leukocyte-related variables, despite efforts to minimize handling time and disturbance.

Despite these limitations, this study provides the first direct comparison of GSL and GFS pup health during an active ENSO event. GSL pups exhibited higher relative body condition and higher GLOB and TP concentrations than GFS pups, whereas most hematological variables were similar between species. These findings suggest species-specific differences in physiological responses to reduced marine productivity, while also indicating broadly comparable clinical status across most measured parameters.

The baseline data generated in this study provide a useful foundation for long-term monitoring, early detection of nutritional stress, and conservation-oriented health assessment of Galapagos pinnipeds under increasingly variable oceanographic conditions.

The present study evaluated morphometric and standard clinical pathology parameters but did not assess pathogen exposure, endocrine responses, or chronic stress biomarkers. Future studies integrating parasitological, immunological, and endocrine markers, together with longitudinal monitoring during both ENSO and non-ENSO periods, would provide a more comprehensive understanding of pinniped health under changing oceanographic conditions.