Steller sea lion pup plasma metabolites

Geographic differences in population growth trends are well-documented in Steller sea lions (Eumetopias jubatus), a species of North Pacific pinniped listed under the U.S. Endangered Species Act in 1990 following a marked decline in population abundance that began during the 1970s. As population growth is intrinsically linked to pup production and survival, examining factors related to pup physiological condition provides useful information to management authorities regarding potential drivers of regional differences.  During dam foraging trips, pups predictably transition among three fasting phases, distinguished by the changes in the predominant metabolic byproduct. We used standardized ranges of two plasma metabolites (blood urea nitrogen and β–hydroxybutyrate) to assign pups to fasting categories (n=1528, 1990–2016, 12 subpopulations): Recently Fed–Phase I (digestion/assimilation–expected hepatic/muscle glycogen usage), Phase II (expected lipid utilization), transitioning between Phases II–III (expected lipid utilization with increased protein reliance), or Phase III (expected protein catabolism). As anticipated, the majority of pups were classified as Recently Fed–Phase I (overall mean proportion=0.72) and a few pups as Phase III (overall mean proportion=0.04). By further comparing pups in Short (Recently Fed–Phase II) and Long (all other pups) duration fasts, we identified three subpopulations with significantly (p<0.03) greater proportions of pups dependent upon endogenous sources of energy for extended periods, during a life stage of somatic growth and development: the 1) central (0.27 ± 0.09) and 2) western (0.36 ± 0.13) Aleutian Island (declining population trend) and 3) southern Southeast Alaska (0.32 ± 0.06; increasing population trend) subpopulations had greater Long fast proportions than the eastern Aleutian Islands (0.10 ± 0.05; stabilized population).  Due to contrasting population growth trends among these highlighted subpopulations over the past 50+ years, both density-independent and density-dependent factors likely influence the dam foraging trip duration, contributing to longer fasting durations for pups at some rookeries.

Methods

Steller sea lion pups were captured live on their natal rookeries. They were restrained physically or chemically. Blood samples were drawn from the vein of the caudal gluteal plexus into blood tubes with anticoagulants (ethylenediaminetetraacetic acid or sodium heparin). During field physical examinations, morphometrics (mass, standard length, axillary girth) and sex were recorded for most pups, as well as any observed external abnormalities. Blood samples were kept chilled in the field until centrifuging (3000–3500 rpm for ten minutes), typically within four hours of collection. Plasma aliquots were stored at -20°C during the remainder of the field research trips (3–10 days) and at -80°C thereafter (months to years, maximal interval of 8 years) following return to the laboratory.
Plasma-derived [BUN] and [β-HBA] were measured via spectrophotometer (SpectraMax 340PC384, Molecular Devices, San Jose, CA) using commercially-available endpoint assay kits: [BUN] via StanBio Kit #2050 and Sigma Aldrich Kit #66-20 and #MAK006; [β-HBA] via StanBio Kit #2440 and Sigma Aldrich Kits #310 and #MAK001 (StanBio, EKF Diagnostics USA, Boerne, TX; Sigma Aldrich, now Millipore Sigma, St. Louis, MO). Plasma samples with moderate to severe hemolysis were not included.  Technical replicates were ≤10% coefficient of variation. Calculations of metabolite concentrations were made using Softmax Pro (v. 4.8) software or the open-access interface MyAssays.com, applying a four-parameter logistic curve. A point-of-care ketometer (Precision Xtra™, Abbott Laboratories, Abbott Park, IL, ketometer precision=0.1 mmol/L [β-HBA]) was also used. Samples measuring between 0.2–0.4 mmol/L via ketometer were further analyzed via the biochemical assay to improve precision around that threshold important to fasting category assignment (threshold=0.3mmol/L; assay precision≈0.01 mmol/L [β-HBA]). The means of the technical replicates are reported in mmol/L for each metabolite.