Circulatory water concentration in suckling and fasting northern elephant seal pups

Summary This study examined circulatory water concentrations in the neonatal northern elephant seal (Mirounga angustirostris) in order to determine how suckling and fasting would alter the percentage of water in the whole blood, plasma, and red blood cells (RBC). Plasma water concentration dropped by about 3% during suckling and recovered about 1% during the fast (92.38±0.48% <1 week old, 90.15±0.36% weaning, 91.02±0.68% end of fast). RBC water values during this time were more variable than plasma values: RBC water increased about 1% during the first 2 weeks of suckling (from 67.80±0.28% to 68.68±0.51%) but dropped to slightly below original neonatal values by weaning 67.15±0.63%. The first several weeks of fasting were marked by wide variability in RBC water, but by the end of the fast RBC water was comparable to that at weaning. These results indicate: 1) Northern elephant seal pups do not exhibit circulatory dehydration during 10 weeks of fasting; 2) Measurements of plasma or RBC metabolites (such as plasma glucose or RBC hemoglobin) may show variations or trends due not to metabolic regulation but rather to changes in circulatory water concentration.Abbreviations Hb Hemoglobin - Hct Hematocrit - MCHC Mean corpuscular - RBC Red blood cell - WB Whole blood     

Renal function in suckling and fasting pups of the northern elephant seal.

Elephant seals fast for prolonged periods without access to water. This is made possible, in part, by reductions in urine production. However, the mechanisms involved in reducing urine production are not understood. In this study, glomerular filtration rate (GFR) was measured in five northern elephant seal pups (Mirounga angustirostris) via the inulin clearance technique. Measurements were made during day 9 and day 18-22 of nursing and the second and eighth week of the postweaning fast. Plasma aldosterone and cortisol concentrations, quantified by radioimmunoassay, were measured in eight other weanlings during the second and eighth week of the fast. Mean GFR was 79.3+/-29.3 ml/min during the early suckling period and 78.2+/-17.1, 89.8+/-52.7, and 80.4+/-12.2 ml/min during the late suckling, early fasting and late fasting periods, respectively. Differences between nursing and fasting were insignificant, possibly because reduced protein oxidation during suckling and rapid recruitment of protein for tissue synthesis obviated the need for postprandial hyperfiltration. Alternatively, maintenance of GFR during fasting may facilitate urea concentration by compensating for reductions in the fractional excretion of urea. It is further hypothesized that aldosterone is primarily responsible for mediating renal water reabsorption in this system.     

Glucose metabolism during lactation in a fasting animal, the northern elephant seal

Fasting is associated with a series of physiological responses that protect body tissues from degradation by efficiently using expendable energy reserves while sparing protein. Lactation requires the mobilization of maternal nutrients for milk synthesis. The rare life history trait of fasting simultaneous with lactation results in the conflicting demands of provisioning offspring while meeting maternal metabolic costs and preserving maternal tissues for her own survival and future reproduction. Certain tissues continue to require glucose for operation during fasting and might constrain tissue mobilization for lactogenesis due to a need for gluconeogenic substrates. This study investigated glucose flux, glucose cycle activity, and the influence of regulatory hormones in fasting lactating northern elephant seals. Measurements were taken early (5 days) and late (21 days) during the lactation period and, as a nonlactating comparison, after the completion of molting. Glucose cycle activity was highly variable in all study groups and did not change over lactation (P > 0.3), whereas endogenous glucose production decreased during lactation (t = -3.41, P = 0.008). Insulin and insulin-to-glucagon molar ratio decreased across lactation (t = 6.48, 4.28; P = 0.0001, 0.002), while plasma cortisol level increased (t = 4.15, P = 0.002). There were no relationships between glucose production and hormone levels. The glucose production values measured exceeded that predicted from available gluconeogenic substrate, indicating substantial glucose recycling in this species.     

Protein catabolism in suckling and fasting northern elephant seal pups (Mirounga angustirostris)

Nursing elephant seal pups are hypothesized to be preadapted to the postweaning fast, yet no comparison of lipid or protein use for meeting metabolic costs has been made between these contrasting nutritional periods. To address this, protein catabolism was estimated in five elephant seal pups from measurements of urea turnover made twice during nursing and twice during the postweaning fast. Changes in body composition were measured in ten separate weaned pups via tritiated water dilution and matched to fasting urea turnover measurements in order to assess errors in protein catabolism derived from urea turnover rates. Estimates of lean mass loss based upon urea turnover and tritiated water dilution were in general agreement, supporting estimates of protein catabolism derived from urea turnover measurements. Protein catabolism was estimated to contribute less than 4% to the average metabolic rate of suckling and fasting pups implying strict protein conservation during both periods and supporting the shypothesis that suckling pups are pre-adapted to fasting. It is proposed that strict protein conservation across suckling and fasting compensates for relative reductions in maternal investment associated with the abbreviated lactation period of the elephant seal.     

Fatty acid metabolism in fasting elephant seal pups

Summary The turnover of two plasma free fatty acids (FFA) were measured in 5 northern elephant seal pups (Mirounga angustirostris) after approximately 2 months of post-weaning fasting. Turnover was determined using simultaneous bolus injections of¹⁴C-palmitate,³H-oleate and Evans blue (EB) administered via an indwelling extradural intravertebral catheter. At this time in their natural fast, the seals exhibited plasma FFA levels and turnover values higher than reported for other marine mammals and most terrestrial carnivores. There was no consistent difference between plasma FFA turnover measured by palmitate or oleate tracers. The results imply that FFA metabolism is the primary source of energy during fasting. This is interesting in light of previous observations of minimal ketoacid accumulation and low levels of glucose and protein energy production during fasting in these juvenile seals.     

Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups

Northern elephant seal (Mirounga angustirostris) pups rely on the oxidation of fat stores as their primary source of energy during their 8- to 12-wk postweaning fast; however, potential endocrine mechanisms involved with this increased fat metabolism have yet to be examined. Therefore, 15 pups were serially blood sampled in the field during the first 7 wk of their postweaning fast to examine the changes in plasma concentrations of cortisol and thyroid hormones (TH), which are involved in fat metabolism in other mammals. Cortisol increased, indicating that it contributed to an increase in lipolysis. Increased total triiodothyronine (tT(3)) and thyroxine (tT(4)) may not reflect increased thyroid gland activity, but rather alterations in hormone metabolism. tT(3)-to-tT(4) ratio decreased, suggesting a decrease in thyroxine (T(4)) deiodination, whereas the negative correlation between total proteins and free T(4) suggests that the increase in free hormone is attributed to a decrease in binding globulins. Changes in TH are most similar to those observed during hibernation than starvation in mammals, suggesting that the metabolic adaptations to natural fasting are more similar to hibernation despite the fact these animals remain active throughout the fasting period.     

Glut4 is upregulated despite decreased insulin signaling during prolonged fasting in northern elephant seal pups

Postprandial cellular glucose uptake is dependent on an insulin-signaling cascade in muscle and adipose tissue, resulting in the translocation of the insulin-dependent glucose transporter 4 (Glut4) into the plasma membrane. Additionally, extended food deprivation is characterized by suppressed insulin signaling and decreased Glut4 expression. Northern elephant seals are adapted to prolonged fasts characterized by high levels of plasma glucose. To address the hypothesis that the fasting-induced decrease in insulin is associated with reduced insulin signaling in prolonged fasted seals, we compared the adipose protein levels of the cellular insulin-signaling pathway, Glut4 and plasma glucose, insulin, cortisol, and adiponectin concentrations between Early (n = 9; 2-3 wks postweaning) and Late (n = 8; 6-8 wks postweaning) fasted seals. Plasma adiponectin (230 ± 13 vs. 177 ± 11 ng/ml), insulin (2.7 ± 0.4 vs. 1.0 ± 0.1 μU/ml), and glucose (9.8 ± 0.5 vs. 8.0 ± 0.3 mM) decreased, while cortisol (124 ± 6 vs. 257 ± 30 nM) doubled with fasting. Glut4 increased (31%) with fasting despite the significant decreases in the cellular content of phosphatidylinositol 3-kinase as well as phosphorylated insulin receptor, insulin receptor substrate-1, and Akt2. Increased Glut4 may have contributed to the decrease in plasma glucose, but the decrease in insulin and insulin signaling suggests that Glut4 is not insulin-dependent in adipose tissue during prolonged fasting in elephant seals. The reduction of plasma glucose independent of insulin may make these animals an ideal model for the study of insulin resistance.     

Estradiol and testosterone concentrations increase with fasting in weaned pups of the northern elephant seal (Mirounga angustirostris)

Although neonatal development is generally associated with increased levels of circulating testosterone (T) and estradiol (E2), food deprivation may inhibit steroidogenesis. Therefore, these potentially conflicting stimuli were examined in fasting weaned northern elephant seal (Mirounga angustirostris) pups by measuring serum concentrations of T, E2, progesterone (P4), and luteinizing hormone (LH) by either radioimmunoassay (P4, LH) or enzymeimmunoassay (T, E2). Blood samples were obtained from 20 male and 20 female pups at both early (<1 wk postweaning) and late (6-8 wk postweaning) periods during their natural postweaning fast. T in males (early: 2.9 +/- 0.4 ng/mL; late: 16 +/- 2 ng/mL; P < 0.0001) and E2 in females (early: 42 +/- 6 pg/mL; late: 67 +/- 5 pg/mL; P < 0.01) increased between the two measurement periods, while P4 (early: 2.5 +/- 0.3 ng/mL; late: 2.1 +/- 0.3 ng/mL; P > 0.05) did not. LH increased (early: 46 +/- 4 pg/mL; late: 65 +/- 6 pg/mL; P < 0.05) in males but not in females (early: 69 +/- 9 pg/mL; late: 65 +/- 6 pg/mL; P > 0.05). Increases in LH and T suggest that LH may stimulate T secretion. Alternatively, relatively low concentrations of LH in both males and females may reflect negative feedback inhibition imposed by elevated T and E2 concentrations. Despite the inherent postweaning fast, concentrations of T and E2 increased, suggesting that they may be critical for the continued development of pups. Therefore, compensatory mechanisms may exist that alleviate the fasting-induced inhibition of gonadal steroidogenesis during neonatal development in elephant seal pups.     

Individual differences in threat calls of northern elephant seal bulls

This study describes the range and reliability of differences between individual elephant seal bulls, Mirounga angustirostris, on several parameters of the threat call: call type, spacing of pulses within calls, addition of snorts to calls, pulse rate of calls, and number of pulses in calls. In addition, some preliminary analyses of the social function of these individual differences are made. Two previously suggested hypotheses, that specific features of the call communicate dominance status and that differences in pulse rate represent dialects, are investigated.     

Body water handling in response to hypertonic-saline induced diuresis in fasting northern elephant seal pups (Mirounga angustirostris)

During natural fasting conditions in postweaned northern elephant seal (NES) (Mirounga angustirostris) pups, urinary water loss is minimized and percent total body water (TBW) is maintained constant. However, following infusion of hypertonic saline, glomerular filtration rate (GFR) and urine output increased in fasting pups. Therefore, we quantified the magnitude of the hypernatremia-induced diuresis relative to the animal's total body water (TBW) pool and the percentage of filtered water reabsorbed. Following a 24 h control period, naturally fasting NES pups (n=7) were infused (4 ml min(-1)) with hypertonic saline (16.7%) at a dose of 3 mmol NaCl kg(-1) body mass. Total body water was estimated prior to infusion by tritium dilution, GFR was estimated by standard creatinine clearance, and urine output (V) was measured for 24 h during the control and post infusion periods. Percentage of filtered water reabsorbed was calculated as (1-(V/GFR))x100. Twenty-four hours following the infusion, GFR (control: 69+/-12 ml min(-1) and post-infusion: 118+/-19 ml min(-1); mean+/-S.E.) increased 77+/-28% above control and the percentage of filtered water reabsorbed was decreased 0.4+/-0.1%. The increase in urine output (control: 218+/-47 ml d(-1) and post-infusion: 883+/-92 ml d(-1)) accounted for 1.7+/-0.2% of the pups' TBW. The hypernatremia-induced diuresis was accompanied by the loss of body water indicating the lack of water retention. Although the 77% increase in GFR was only associated with a 0.4% decrease in the percentage of filtered water reabsorbed, this decrease was significant enough to result in a 4-fold increase in urine output. Despite the observed diuresis, fasting NES pups appear to possess an efficient water recycling mechanism requiring only a small percentage of body water to excrete an excess salt load. This water recycling mechanism may allow pups to avoid negative perturbations in body water as they initiate feeding in a marine environment following the fast.     

Moult energetics of the northern elephant seal (Mirounga angustirostris)

Northern elephant seals, Mirounga angustirostris , undergo an annual moult during which they shed all of their pelage and underlying epidermis. Moulting takes place on land and lasts a mean of 32.0±6.6 days. During this time the mean mass loss of adult females was 24.7±6.1%. Mean body composition at arrival (25.6±4.8%fat) did not differ significantly from that at departure (24.9±3.2%fat). Fat catabolism accounted for 93.6%of derived energy and 41%of mass lost. Approximately 3.5%of total mass loss was associated with the shedding of the pelage and epidermis. Moulting female northern elephant seals express an average daily metabolic rate of 2.0±0.6 times that predicted for adult terrestrial mammals. This energy demand was met by losing 3.0 kg d^-1 of total body mass. These energy expenditures suggest that, similar to data for harbour seals, the moult period is a time of relatively low energy expenditure.     

Three-dimensional movements and swimming activity of a northern elephant seal

We attached a video system and data recorder to a northern elephant seal to track its three-dimensional movements and observe propulsive strokes of the hind flippers. During 6 h of recording, the seal made 20 dives and spent 90% of the time submerged. Average dive duration, maximum depth and swimming speed were 14.9 min+/-6.1 S.D., 289 m+/-117 S.D. and 1.1 m s(-1)+/-0.12 S.D., respectively. The distance swum during a dive averaged 925 m+/-339 S.D., and the average descent and ascent angles were 41 degrees +/-18 S.D. and 50 degrees +/-21 S.D., respectively. Dive paths were remarkably straight suggesting that the seal was navigating while submerged. We identified three modes of swimming based on the interval between propulsive strokes: continuous stroking; stroke-and-glide swimming; and prolonged gliding. The seal used continuous stroking from the surface to a mean depth of 20 m followed by stroke-and-glide swimming. Prolonged gliding started at a mean depth of 60 m and continued to the bottom of dives. For dives to depths of 300 m or more, 75% of the descent time was spent in prolonged gliding and 10% in stroke-and-glide swimming, amounting to 5.9-9.6 min of passive descent per dive. Average swimming speed varied little with swimming mode and was not a good indicator of propulsive effort. It appears that the seal can use prolonged gliding to reduce the cost of transport and increase dive duration. Energetically efficient locomotion may help explain the long and deep dives that routinely exceed the theoretical aerobic dive limit in this species.     

Biliary adenocarcinoma in a stranded northern elephant seal (Mirounga angustirostris)

A stranded adult female northern elephant seal (Mirounga angustirostris) was admitted to a rehabilitation hospital grossly underweight and lethargic in April 2001. The animal was icteric, had severe bilirubinemia, and elevated serum gamma glutamyl transferase concentrations. Laparoscopy under anesthesia revealed multiple masses up to 3 cm diameter throughout the liver and spleen and the animal was euthanized. Abnormal gross postmortem findings included green serous fluid in the abdominal cavity and 0.5 to 3 cm diameter yellow nodules scattered throughout the liver, spleen, and lymph nodes. Histopathology revealed a biliary adenocarcinoma with metastases to the liver, spleen, lymph nodes, adrenal gland, and pancreas. This is believed to be the first reported case of neoplasia in a northern elephant seal.     

Lipolysis and glycerol gluconeogenesis in simultaneously fasting and lactating northern elephant seals

Adult female elephant seals (Mirounga angustirostris) combine long-term fasting with lactation and molting. Glycerol gluconeogenesis has been hypothesized as potentially meeting all of the glucose requirements of the seals during these fasts. To test this hypothesis, a primed constant infusion of [2-(14)C]glycerol was administered to 10 ten adult female elephant seals at 5 and 21-22 days postpartum and to 10 additional adult females immediately after the molt. Glycerol kinetics, rates of lipolysis, and the contribution of glycerol to glucose production were determined for each period. Plasma metabolite levels as well as insulin, glucagon, and cortisol were also measured. Glycerol rate of appearance was not significantly correlated with mass (P = 0.14, r2 = 0.33) but was significantly related to the percentage of glucose derived from glycerol (P < 0.01, r2 = 0.81) during late lactation. The contribution of glycerol to glucose production was <3% during each fasting period, suggesting a lower contribution to gluconeogenesis than is observed in other long-term fasting mammals. Because of a high rate of endogenous glucose production in fasting elephant seals, it is likely that glycerol gluconeogenesis still makes a substantial contribution to the substrate needs of glucose-dependent tissues. The lack of a relationship between glucoregulatory hormones and glycerol kinetics, glycerol gluconeogenesis, and metabolites supports the proposition that fasting elephant seals do not conform to the traditional insulin-glucagon model of substrate metabolism.     

An empirical genetic assessment of the severity of the northern elephant seal population bottleneck

A bottleneck in population size of a species is often correlated with a sharp reduction in genetic variation. The northern elephant seal (Mirounga angustirostris) has undergone at least one extreme bottleneck, having rebounded from 20-100 individuals a century ago to over 175,000 individuals today. The relative lack of molecular-genetic variation in contemporary populations has been documented, but the extent of variation before the late 19th century remains unknown. We have determined the nucleotide sequence of a 179 base-pair segment of the mitochondrial DNA (mtDNA) control region from seals that lived before, during and after a bottleneck low in 1892. A 'primerless' PCR was used to improve the recovery of information from older samples. Only two mtDNA genotypes were present in all 150+ seals from the 1892 bottleneck on, but we discovered four genotypes in five pre-bottleneck seals. This suggests a much greater amount of mtDNA genotypic variation before this bottleneck, and that the persistence of two genotypes today is a consequence of random lineage sampling. We cannot correlate the loss of mtDNA genotypes with a lowered mean fitness of individuals in the species today. However, we show that the species historically possessed additional genotypes to those present now, and that sampling of ancient DNA could elucidate the genetic consequences of severe reductions in population size.     

Major histocompatibility complex variation at three class II loci in the northern elephant seal

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.     

Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast

Urine production and N output were monitored in northern elephant seal (Mirounga angustirostris) pups progressing through 10 weeks of a natural postweaning fast. Urine output declind by 84% (to 69±12 ml·day^–1) at 10 weeks (P<0.05). Glomerular filtration rate at 10 weeks was 51% of the 67±3 ml serum·min^–1 observed during week 1 (P<0.05). Urine N excretion fell by 69% to 1.2±0.17 g·day^–1, while urinary concentration increased (P<0.05). Serum urea declined from an initial 11 mmol·1^–1 to 5–7 mmol·1^–1 by 5 weeks. The fall in urinary N loss (and thus amino acid oxidation) was concomitant with depressed metabolic rate. Therefore, protein contributed little toward meeting energy demands (i.e., <4% of average metabolic rate) throughout fasting. These data indicate that fasting pups improve water conservation and minimize protein catabolism during prolonged natural fasts without an exogenous source of water.Abbreviations AA amino acid(s) - AMR average metabolic rate - ANOVA one-way analysis of variance - BMR basal metabolic rate - BUN blood urea nitrogen - EP end product - EWL evaporative water loss - [Gr]_s serum creatinine concentration - GFR glomerular filtration rate - LBM lean body mass - LML Long Marine Laboratory - MR metabolic rate - NEFA non-esterified fatty acids - RMR resting metabolic rate - TCA tricarboxylic acid - U:C ulinary urea: creatinine concentration ratio