Establishing a reference interval for acute phase proteins, cytokines, antioxidants and commonly measured biochemical and hematologic parameters in the northern fur seal (Callorhinus ursinus).

Over the past several decades there has been a precipitous decline of northern fur seals (Callorhinus ursinus; NFS) at their breeding grounds on the Pribilof Islands in the Bering Sea. The cause of this decline is likely multifactorial and could include changes in environmental parameters, prey abundance and distribution as well as exposure to pathogens and pollutants. Evaluation of inflammatory markers and antioxidant levels of the current population of fur seals in addition to hematologic and biochemical profiles could provide important information regarding health and subclinical or clinical disease in this population. Serum and plasma samples were obtained from clinically healthy adult female NFS and references intervals were determined for multiple parameters that can be altered in response to the presence of disease and environmental stressors. We established a reference interval for cytokines involved in acute inflammation and infection (TNFa, IL1, IL6, IL8, KC, IL10, C-reactive Protein) by utilizing commercially available canine cross-reactive antibodies. Reference intervals were also established for reactive oxygen species (hydrogen peroxide and malondialdehyde), as well as antioxidant levels (vitamin E and selenium) and acute phase proteins evaluated by serum electrophoresis. To improve the ability to compare and interpret indicators of health and disease in this species, we developed reference intervals for commonly utilized hematologic and biochemical tests in addition to the aforementioned markers of oxidative stress and inflammatory biomarkers. There were several animals identified as outliers indicating that they may have had subclinical illness or inflammation. Further investigation utilizing these tests in clinically ill animals and comparison to animals that exhibit normal behavior and no overt signs of illness could increase our understanding of the utility of measuring these parameters in this species.

Isotopic variation in blood components based on their biochemistry and physiology: A comparison between sharks and fur seals.

Research using stable isotopes analysis (SIA) of carbon (δ13 C) and nitrogen (δ15 N) in blood components is lacking, because of the challenge of sample collection, processing, and storage in remote areas. There also is a paucity of information regarding the effect of tissue biochemical composition on isotopic ratios with few comparisons among taxa. We collected blood samples from shortfin mako sharks (n = 70; 2016) and Guadalupe fur seals (n = 25; 2017). All samples were centrifuged to obtain plasma from sharks and serum from the Guadalupe fur seals, and all the samples were prepared for SIA and analyzed using a Costech 4010 elemental analyzer interfaced with a Delta V Plus isotope ratio mass spectrometer. We found significant differences between plasma δ13 C values of shortfin mako sharks (-17.6 ± 0.9‰) and serum of Guadalupe fur seals (-20.3 ± 1.2‰), but we did not find any differences for δ15 N values between the two species. The differences in δ13 C values between species are probably due to the specific blood composition and to the different biochemical characteristics and different adaptations within taxa. These findings highlight the importance of further research on the influence of biochemistry features on isotopic results, in this way a more accurate assessment will be possible for this factor, separating it from the dietary influences on stable isotopic values.

Normal hematology and serum chemistry of northern fur seals (Callorhinus ursinus) in captivity.

Northern fur seals (Callorhinus ursinus) are endemic to the North Pacific Ocean. They were hunted for their fur and became endangered in the late 1800s, but their populations recovered following the introduction of protection laws. Recently, populations have been decreasing again, although the reasons are unclear. For individuals that are bred and reared in captivity as part of ex situ conservation projects, details of blood characteristics are essential to ensure good health. However, the normal ranges of hematology and serum chemistry of captive northern fur seals have not been defined. This study determined the normal ranges of hematology and serum chemistry of captive fur seals. Blood samples were collected every month for 2 years from four captive northern fur seals in Japan (three born in an aquarium and one kept in the same aquarium following rescue). Fifteen blood characteristics and 29 serum chemistry properties were compared with those previously reported for wild northern fur seals in the USA. Several parameters were not within the normal ranges reported previously in wild northern fur seals. In particular, levels of alkaline phosphatase was outside of the normal ranges previously reported. The hematological and serum chemistry ranges in this study can help provide a guideline for understanding the health of northern fur seals in captivity.

Hematology, Serum Chemistry, and Early Hematologic Changes in Free-Ranging South American Fur Seals ( Arctocephalus australis ) at Guafo Island, Chilean Patagonia.

The establishment of clinical pathology baseline data is critical to evaluate temporal and spatial changes in marine mammal groups. Despite increased availability of studies on hematology and biochemistry of marine mammals, reference ranges are lacking for many populations, especially among fur seal species. During the austral summers of 2014 and 2015, we evaluated basic hematologic and biochemical parameters in clinically healthy, physically restrained South American fur seal ( Arctocephalus australis ) lactating females and 2-mo-old pups. We also assessed the temporal variation of hematology parameters on the pups during their first 2 mo of life. Reference ranges of lactating females were similar to those previously reported in other fur seal species. In the case of pups, reference ranges are similar to values previously reported in sea lion species. As expected, most biochemical and hematologic values differ significantly between adult females and pups. As in other otariids, South American fur seals pups are born with higher values of total red blood cells, hemoglobin, and packed cell volume, and lower numbers of total leukocytes, neutrophils, lymphocytes, and eosinophils. To the best of our knowledge, data on hematology reference values for South American fur seals has not been previously reported and is useful for continued health monitoring of this species, as well as for comparisons with other otariid groups.

Stable Isotope Models Predict Foraging Habitat of Northern Fur Seals (Callorhinus ursinus) in Alaska.

We developed models to predict foraging habitat of adult female northern fur seals (Callorhinus ursinus) using stable carbon (δ13C) and nitrogen (δ15N) isotope values from plasma and red blood cells. Binomial generalized linear mixed models were developed using blood isotope samples collected from 35 adult female fur seals on three breeding colonies in Alaska during July-October 2006. Satellite location and dive data were used to define habitat use in terms of the proportion of time spent or dives made in different oceanographic/bathymetric domains. For both plasma and red blood cells, the models accurately predicted habitat use for animals that foraged exclusively off or on the continental shelf. The models did not perform as well in predicting habitat use for animals that foraged in both on- and off-shelf habitat; however, sample sizes for these animals were small. Concurrently collected scat, fatty acid, and dive data confirmed that the foraging differences predicted by isotopes were associated with diet differences. Stable isotope samples, dive data, and GPS location data collected from an additional 15 females during August-October 2008 validated the effective use of the models across years. Little within year variation in habitat use was indicated from the comparison between stable isotope values from plasma (representing 1-2 weeks) and red blood cells (representing the prior few months). Constructing predictive models using stable isotopes provides an effective means to assess habitat use at the population level, is inexpensive, and can be applied to other marine predators.

Serum chemistry and antibodies against pathogens in antarctic fur seals, Weddell seals, crabeater seals, and Ross seals.

Information on health parameters, such as antibody prevalences and serum chemistry that can reveal exposure to pathogens, disease, and abnormal physiologic conditions, is scarce for Antarctic seal species. Serum samples from Antarctic fur seals (Arctocephalus gazella, n=88) from Bouvetøya (2000-2001 and 2001-2002), and from Weddell seals (Leptonychotes weddellii, n=20), Ross seals (Ommatophoca rossii, n=20), and crabeater seals (Lobodon carcinophagus, n=9) from the pack-ice off Queen Maud Land, Antarctica (2001) were analyzed for enzyme activity, and concentrations of protein, metabolites, minerals, and cortisol. Adult Antarctic fur seal males had elevated levels of total protein (range 64-99 g/l) compared to adult females and pups (range 52-79 g/l). Antarctic fur seals had higher enzyme activities of creatine kinase, lactate dehydrogenase, and amylase, compared to Weddell, Ross, and crabeater seals. Antibodies against Brucella spp. were detected in Weddell seals (37%), Ross seals (5%), and crabeater seals (11%), but not in Antarctic fur seals. Antibodies against phocine herpesvirus 1 were detected in all species examined (Antarctic fur seals, 58%; Weddell seals, 100%; Ross seals, 15%; and crabeater seals, 44%). No antibodies against Trichinella spp., Toxoplasma, or phocine distemper virus (PDV) were detected (Antarctic fur seals were not tested for PDV antibodies). Antarctic seals are challenged by reduced sea ice and increasing temperatures due to climate change, and increased anthropogenic activity can introduce new pathogens to these vulnerable ecosystems and represent a threat for these animals. Our data provide a baseline for future monitoring of health parameters of these Antarctic seal species, for tracking the impact of environmental, climatic, and anthropogenic changes in Antarctica over time.

Hormonal responses to extreme fasting in subantarctic fur seal (Arctocephalus tropicalis) pups.

Surviving prolonged fasting implies closely regulated alterations in fuel provisioning to meet metabolic requirements, while preserving homeostasis. Little is known, however, of the endocrine regulations governing such metabolic adaptations in naturally fasting free-ranging animals. The hormonal responses to natural prolonged fasting and how they correlate to the metabolic adaptations observed, were investigated in subantarctic fur seal (Arctocephalus tropicalis) pups, which, because of the intermittent pattern of maternal attendance, repeatedly endure exceptionally long fasting episodes throughout their development (1-3 mo). Phase I fasting was characterized by a dramatic decrease in plasma insulin, glucagon, leptin, and total l-thyroxine (T(4)) associated with reductions in mass-specific resting metabolic rate (RMR), plasma triglycerides, glycerol, and urea-to-creatine ratio, while nonesterified fatty acids (NEFA) and ß-OHB increased. In contrast, the metabolic steady-state of phase II fasting reached within 6 days was associated with minimal concentrations of insulin, glucagon, and leptin; unchanged cortisol and triiodothyronine (T(3)); and moderately increased T(4). The early fall in insulin and leptin may mediate the shift to the strategy of energy conservation, protein sparing, and primary reliance on body lipids observed in response to the cessation of feeding. In contrast to the typical mammalian starvation response, nonelevated cortisol and minimal glucagon levels may contribute to body protein preservation and downregulation of catabolic pathways, in general. Furthermore, thyroid hormones may be involved in a process of energy conservation, independent of pups' nutritional state. These original hormonal settings might reflect an adaptation to the otariid repeated fasting pattern and emphasize the crucial importance of a tight physiological control over metabolism to survive extreme energetic constraints.

Plasma cortisol and thyroid hormone concentrations in pre-weaning Australian fur seal pups.

The hormonal factors that influence development from birth to weaning in otariid seals is still largely unknown. In the present study, a suite of thyroid hormones and cortisol were measured in Australian fur seal pups in order to determine baseline concentrations as well as to describe their endocrinology over this critical developmental period. A cross-section of newborn pups from a breeding colony located on Kanowna Island, Australia were sampled at six different times over the course of the 10 month lactation period. Sample times were designed to correspond to periods of heightened physiological change during pre-weaning development: post-natal, pre-molt, the initiation of molt, mid-molt, period of peak milk intake and weaning. Results indicate that the greatest hormonal changes were associated with the post-natal stage and molt, with molt showing the greatest changes, as has been reported for several species of pinnipeds. Two forms of thyroid hormones analyzed (Total T(4), and Free T(3)), increased with the initiation of the molt, and Free T(3) exhibited a second increase that was associated with the period of peak milk intake. The T(3):T(4) ratio was significantly lower during the initiation of molt than either pre- or mid-molt. The study was able to describe physiological change during the first year of life in Australian fur seals as well as document basal concentrations of thyroid hormones and cortisol in pups of this species.

Hematology of free-ranging, lactating northern fur seals, Callorhinus ursinus.

Thirteen standard hematology values were determined for a healthy and growing population of free-ranging, lactating northern fur seals (Callorhinus ursinus) from Lovushki Island in the Kuril Islands of far-east Russia. Results are presented from 24 females sampled between June and August during the 3-yr period of 2006-08. Hematologic values have been made available for future comparisons with the declining population of northern fur seals on the Pribilof Islands, Alaska, and are compared with published values for other otariid species.

Validation of a homologous canine relaxin radioimmunoassay and application with pregnant and non-pregnant Northern fur seals (Callorhinus ursinus).

The primary objectives of this study were to validate a canine relaxin RIA for use in otariids and phocids and consider practical applications. For 6 captive Northern fur seal females, serum samples were grouped and examined according to pregnancy (n=13), post-partum (n=8) and non-pregnancy (n=6), and, for 2 captive Northern fur seal males, serum samples were grouped and examined together regardless of age (2 mo-15 yrs, n=6). Placental tissue was available for examination from one Northern fur seal, Steller sea lion and harbor seal. The validation process involved several steps using an acid-acetone extraction process to isolate a relaxin-containing fraction in pools of serum from each group of fur seals and placental tissue from each seal species. A relaxin-like substance was detected in extracts of pregnant, non-pregnant and male serum and placental tissue in a dose-responsive manner as increasing volumes of respective extracts or amounts of canine relaxin were introduced into the assay. In raw serum samples, mean immuno-reactive relaxin concentrations were higher (P<0.05) during pregnancy than post-partum and non-pregnancy, and lower (P<0.05) in male than female fur seals. During pregnancy, mean serum concentrations of relaxin progressively increased (P<0.05) over Months 4-10 and, in serial samples collected from the same fur seals before and after parturition, mean concentrations were higher (P<0.06) pre-partum than post-partum. In conclusion, validation of a homologous canine relaxin RIA for use in otariids and phocids resulted in the discovery of a relaxin-like substance in extracted and raw serum and placental tissue from Northern fur seals, a Steller sea lion and harbor seal. Distinctly higher immuno-reactive concentrations during pregnancy indicated the potential for relaxin to serve as a hormonal marker to differentiate between pregnant and non-pregnant or pseudopregnant pinnipeds.

Serum chemistry values of free-ranging, lactating northern fur seals (Callorhinus ursinus).

Reference range clinical serum chemistry values were established for free-ranging lactating northern fur seals (Callorhinus ursinus). Fur seals sampled for this study were part of a healthy and growing population in the Kuril Islands of far-east Russia. Blood was collected from 45 females between June and August 2005 to 2007. Fresh serum was assayed for 16 components. Packed-cell volume was determined from fresh whole blood. Results are made available for future comparisons with the declining population of northern fur seals on the Pribilof Islands and are compared with published values for other otariid species.

Sex steroid concentrations and localization of steroidogenic enzyme expression in free-ranging female northern fur seals (Callorhinus ursinus).

Recent precipitous population declines in northern fur seal (Callorhinus ursinus) and other Alaskan pinniped populations are due in part to reduced fecundity and have emphasized deficits in basic reproductive knowledge of these species. Following estrus and mating, fertilized female pinnipeds experience an obligatory delayed implantation lasting several months and non-pregnant (pseudopregnant) females are indistinguishable by sex steroid levels during this time. The current study examined circulating steroid concentrations and ovarian expression of key steroid-synthesizing enzymes in northern fur seals to identify changes associated with embryonic implantation, data necessary for estimating early pregnancy rates of the population. Blood samples were collected from 84 female fur seals captured on an Alaskan rookery from October 15 to November 30, a period spanning the end of the delay and subsequent uterine implantation of the fertilized blastocyst in this species. Concentrations of progesterone, estradiol, and estrone measured in blood collected during boreal fall were contrasted with samples collected during late summer from post-ovulatory females and males. Serum sex steroids were also related to ovarian expression of key enzymes responsible for androgen (17alpha-hydroxylase/17,20-lyase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase) and estrogen (aromatase cytochrome P450) synthesis. Enzymes necessary for androgen synthesis were highly expressed in ovaries, and accordingly, endocrine profiles were expanded to include DHEA, androstenediol, androstenedione, and testosterone. Estradiol concentrations were universally low and free and conjugated estrone were the primary circulating estrogens in fur seal sera. No implantation-associated peak was identified for estrogens or progesterone and mean values of progesterone and estrone were actually greater in female serum samples collected during summer than fall. However, there was a significant positive relationship between fall sampling date and testosterone concentration. Additionally, DHEA concentrations in females were lower in samples collected during the implantation period than during the summer embryonic delay. These data suggest androgens may play a substantial regulatory role in the embryonic delay of northern fur seals.

Variation in plasma leptin levels in response to fasting in Antarctic fur seals (Arctocephalus gazella).

Plasma leptin levels were determined in 8 lactating female and 20 pup Antarctic fur seals (Arctocephalus gazella) during fasting periods of normal duration. Plasma leptin levels ranged from 1.35 3.19 ng x ml(-1) in lactating females and 1.79-4.80 ng x ml(-1) in pups and were not positively correlated with body mass or condition. A negative trend, however, was observed between plasma leptin levels and body condition in lactating females upon their arrival at the colony following a foraging trip (beginning of fast). In accordance with findings in other species, plasma leptin levels dropped significantly (P < 0.02) in response to the 17-19% drop in body mass experienced by pups during fasting. In contrast, plasma leptin levels in lactating females increased during the first 24 h of fasting before decreasing throughout the remaining 48 h of the fast. This unexpected result could be due to the high level of energy expenditure by seals as they swim back to the colony (i.e. post-exercise response) or may be influenced by the intense suckling activity experienced by females during the onshore fasting periods. The results of this study support recent findings in other carnivore species which suggest the primary physiological role of leptin in these species may not necessarily be as a signal of the magnitude of body energy reserves.

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella).

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.

Factors affecting organochlorine contaminant concentrations in milk and blood of northern fur seal (Callorhinus ursinus) dams and pups from St. George Island, Alaska.

Levels of organochlorine contaminants in blood of northern fur seal (Callorhinus ursinus) pups and the blood and milk of their dams early in the lactation period are reported here. The contaminants included 15 selected individual polychlorinated biphenyl (PCB) congeners and DDT metabolites identified through high-performance liquid chromatography. Congeners CB-77, -81, -126, -169 and -189 were below the limits of detection in milk and blood samples analyzed. Congener-specific concentrations of PCBs in the blood of pups were compared based on the age of their dam (< or = 5 years or > 7 years). Pups of young (presumably primiparous) dams had significantly elevated levels of CBs-101, -118, -128, -138, -153/87, -170/194, and -180 than pups of older (multiparous) dams. Congeners CB-128 and -170/194 were detected in the blood of pups of young dams but not in the blood of pups of older dams nor in any of the dams blood. Additionally, pups had higher blood levels in seven of 10 detected PCB congeners as compared to the levels measured in milk when adjusted for lipid content. Levels of DDT metabolites and toxic equivalency quotients of dioxin-like congeners followed similar trends. Lipid-normalized concentrations of CB-101 and total PCBs were significantly higher in the blood of dams than in their milk. CB-128, -156, -157, -170/194, p,p'-DDT, o,p'-DDT, p,p'-DDD and o,p'-DDD were not detected in dam blood samples, but were detected in milk samples. Calculation of 'biomagnification factors' from milk to pup blood indicated a biomagnification of CB-101, -105, -118, -138, -153/87, and -180. Significant mean accumulation factors ranged from 1.5 to 7.5. Inter-annual differences in exposure levels and specific congener concentrations in both milk and blood were apparent. Northern fur seal pups, especially first-born, have a substantial exposure to organochlorine contaminants at a critical developmental stage.

The complete primary structure of the marine Carnivora, galapagoes fur seal (Arctocephalus galapagoensis, Otariidae) hemoglobins.

The complete primary structure of the two hemoglobin components of the fur seal (Arctocephalus galapagoensis) is presented. The two components (HbI and HbII) occur in nearly equal amounts and have identical beta-chains; whereas the two alpha-chains (alpha I/alpha II) differ by six exchanges Ile/Val, Met/Thr, Ser/Ala, Pro/His, Lys/Gly, and Thr/Ala at positions 10, 34, 35, 50, 78, and 131, respectively. The components were isolated by DEAE-Sephacel chromatography and were separated into the globin chains by RP-HPLC on a column of Nucleocil-C4. The sequences have been determined by Edman degradation in liquid- and gas-phase sequencer, using the native chains and tryptic peptides. The sequences compared with those of other Carnivora species and an adult human globin chains. An identical beta-chain is found in fur seal and walrus, whereas larger differences were found between alpha I and alpha II compared to beta-chains.

Changes in plasma progesterone and prolactin concentrations during the annual cycle and the role of prolactin in the maintenance of lactation and luteal development in the Antarctic fur seal (Arctocephalus gazella).

Progesterone in Antarctic fur seals was undetectable from 1-2 days before parturition to 4-6 days after parturition. There was a rapid increase in progesterone to 20 ng/ml between 6 and 10 days post partum and this increase coincided with peak concentrations of oestradiol-17 beta at the time normally associated with oestrus and mating in this species. Newly formed corpora lutea were present in the ovaries by Day 9 post partum even though the seals had been isolated in an enclosure and not mated. Thereafter, progesterone remained detectable, but at a low concentration (5 ng/ml) throughout embryonic diapause. A similar pattern was observed in unmated females which suggests they enter a period of pseudopregnancy. Progesterone increased to 35 ng/ml between late February and mid-March, indicating activation of the corpus luteum at the end of diapause, and then declined slowly through the remainder of gestation. Plasma prolactin, measured against a human prolactin standard, was elevated from 1-2 days before parturition and peaked at 0-3 days post partum. It then declined slowly throughout the post-partum period and remained at a low level throughout embryonic diapause. Prolactin concentration declined to undetectable at the end of diapause and before the end of lactation. Reduction of prolactin secretion by injections of bromocriptine from Days 3 to 5 post-partum terminated lactation. Mothers, which normally leave their pups to feed at sea on about Day 7 post partum, did not continue to lactate beyond Day 7 although this did not appear to be associated with reduced prolactin secretion. Bromocriptine treatment appeared to prevent the post-ovulatory surge of progesterone although there was no long-term effect of bromocriptine on progesterone secretion during the early stages of embryonic diapause/pseudopregnancy. This study has shown that prolactin is an important hormone for maintaining early lactation in the fur seal and it probably also has a role in the control of ovulation and luteal development. Prolactin does not appear to be implicated in the control of lactation cycles in fur seals. Changes in plasma progesterone during the annual cycle show that the pattern in fur seals resembles that of some carnivores with embryonic diapause.

Electrophoretic transferrin variation in fur seals (Arctocephalus spp.) at Marion Island.

1. Variation in transferrin types were investigated in sympatric populations of two fur seal species which are undergoing limited hybridization at Marion Island. 2. Vertical polyacrylamide gel electrophoresis of serum was performed to demonstrate the transferrin types. 3. The two species appear fixed for alternative alleles: A. tropicalis being homozygous for the fast allele, and A. gazella (with one exception) being homozygous for the slow allele, indicating low gene flow between these two species. The single hybrid tested was homozygous for the slow allele. 4. The use of electrophoretically determined transferrin variation holds promise for the investigation of these and other sympatric fur seal populations.

Clinical blood values of the northern fur seal, Callorhinus ursinus. III. Comparison of eye fluid and serum values.

The levels of 13 components in the serum and eye fluids of the northern fur seal, Callorhinus ursinus, are compared. The wide variations observed would appear to limit the usefulness of eye fluid values as a substitute for serum values.

Clinical blood values of the northern fur seal, Callorhinus ursinus. II. Comparison of fresh versus stored frozen serum.

Analysis of 14 blood components in fresh and stored (107-166 days) frozen serum of the northern fur seal (Callorhinus ursinus) revealed significant changes in the values of all but total protein, globulins, phosphorus and creatinine. While most values decreased during storage, cholesterol and bilirubin showed small but significant increases.