Serum estradiol and progesterone profiles during estrus, pseudopregnancy, and active gestation in Steller sea lions.

While the proximate driver behind the decline of the Western stock of Steller sea lions (Eumetopias jubatus, >80% since 1970s) is likely multifactorial, the population reduction may have been powered by a decrease in fecundity. A harvest of Steller sea lions in the 1970s and 80s revealed a 30% reduction in the proportion of pregnant females from early (October-November) to late gestation (April-May). Identification and quantification of these reproductive failures are difficult when we lack species-specific data on endocrinology associated with discrete stages of the reproductive cycle (i.e., estrus, implantation, and gestation). We tracked changes in serum estradiol and progesterone in three adult female Steller sea lions from 2011 to 2015. In all years and most females, a discrete increase in estradiol was observed during the breeding season (June-August), indicative of estrus. Estradiol concentrations from October to May in a pregnant female compared to her corresponding values when non-pregnant did not consistently differ through gestation. An elevation in progesterone was observed in all females and all years beginning approximately in June and lasting through November. This likely results from progesterone production by the corpus luteum in both pregnant and pseudopregnant females. Serum progesterone shows promise as a diagnostic tool to identify pregnancy during months 3-5 (December-February) of the 8-month active gestation following embryonic implantation. This study provides ranges of key hormones during estrus, embryonic diapause/pseudopregnancy, and gestation in pregnant and non-pregnant females for studying reproduction in Steller sea lions.

Updating a gas dynamics model using estimates for California sea lions (Zalophus californianus).

Theoretical models are used to predict how breath-hold diving vertebrates manage O2, CO2, and N2 while underwater. One recent gas dynamics model used available lung and tracheal compliance data from various species. As variation in respiratory compliance significantly affects alveolar compression and pulmonary shunt, the current study objective was to evaluate changes in model output when using species-specific parameters from California sea lions (Zalophus californianus). We explored the effects of lung and dead space compliance on the uptake of N2, O2, and CO2 in various tissues during a series of hypothetical dives. The updated parameters allowed for increased compliance of the lungs and an increased stiffness in the trachea. When comparing updated model output with a model using previous compliance values, there was a large decrease in N2 uptake but little change in O2 and CO2 levels. Therefore, previous models may overestimate N2 tensions and the risk of gas-related disease, such as decompression sickness (DCS), in marine mammals.

Disentangling the cause of a catastrophic population decline in a large marine mammal.

Considerable uncertainties often surround the causes of long-term changes in population abundance. One striking example is the precipitous decline of southern sea lions (SSL; Otariaflavescens) at the Falkland Islands, from 80 555 pups in the mid 1930s to just 5506 pups in 1965. Despite an increase in SSL abundance over the past two decades, the population has not recovered, with the number of pups born in 2014 (minimum 4443 pups) less than 6% of the 1930s estimate. The order-of-magnitude decline is primarily attributed to commercial sealing in Argentina. Here, we test this established paradigm and alternative hypotheses by assessing (1) commercial sealing at the Falkland Islands, (2) winter migration of SSL from the Falkland Islands to Argentina, (3) whether the number of SSL in Argentina could have sustained the reported level of exploitation, and (4) environmental change. The most parsimonious hypothesis explaining the SSL population decline was environmental change. Specifically, analysis of 160 years of winter sea surface temperatures revealed marked changes, including a period of warming between 1930 and 1950 that was consistent with the period of SSL decline. Sea surface temperature changes likely influenced the distribution or availability of SSL prey and impacted its population dynamics. We suggest that historical harvesting may not always be the "smoking gun" as is often purported. Rather, our conclusions support the growing evidence for bottom-up forcing on the abundance of species at lower trophic levels (e.g., plankton and fish) and resulting impacts on higher trophic levels across a broad range of ecosystems.

Domoic acid-induced seizures in California sea lions (Zalophus californianus) are associated with neuroinflammatory brain injury.

California sea lions (CSLs) exposed to the marine biotoxin domoic acid (DA) develop an acute or chronic toxicosis marked by seizures and act as sentinels of the disease. Experimental evidence suggests that oxidative stress and neuroinflammation are important mechanisms underlying the seizurogenic potential of environmental toxicants but these pathways are relatively unstudied in CSLs. In the current study, we investigated the role of glutamate-glutamine changes and gliosis in DA-exposed CSLs to better understand the neurotoxic mechanisms occurring during DA toxicity. Sections from archived hippocampi from control and CSLs diagnosed with DA toxicosis were immunofluorescently stained for markers of gliosis, oxidative/nitrative stress and changes in glutamine synthetase (GS). Quantitative assessment revealed increasing loss of microtubule associated protein-2 positive neurons with elevations in 4-hydroxynonenal correlating with chronicity of exposure, whereas the pattern of activated glia expressing nitric oxide synthase 2 and tumor necrosis factor followed pathological severity. There was no significant change in the amount of GS positive cells but there was increased 3-nitrotyrosine in GS expressing cells and in neurons, particularly in animals with chronic DA toxicosis. These changes were consistently seen in the dentate gyrus and in the cornu ammonis (CA) sectors CA3, CA4, and CA1. The results of this study indicate that gliosis and resultant changes in GS are likely important mechanisms in DA-induced seizure that need to be further explored as potential therapies in treating exposed wildlife.

Trihalomethanes in marine mammal aquaria: occurrences, sources, and health risks.

Disinfecting water containing the high levels of dissolved organic carbon (DOC) commonly generated during pinniped husbandry may cause the formation of carcinogenic disinfection byproducts (DBPs). Little information is available on DBP levels, sources, and health risks in marine mammal aquaria. Using the commonly observed trihalomethanes (THMs) as a DBP indicator, we monitored concentrations for seven months at The Marine Mammal Center in Sausalito, California, one of the largest pinniped rehabilitation facilities in the world. Concentrations of THMs ranged 1.1-144.2 µg/L in pool waters and generally increased with number of animals housed (P < 0.05). To identify the sources of THM precursors in marine mammal aquaria, we intensively monitored the mass flows of potential THM precursors (i.e. food and wastes) in an isolated system with nine individual California sea lions to evaluate the sources and reactivity of dissolved organic carbon (DOC) for 2-5 weeks. The common frozen foods used in feeding pinnipeds, including herring, sardine, and squid, produced an average of 22-34 mg-DOC/g-food in water and 836-1066 µg-THM/g-food after chlorination, whereas the fecal materials, including fresh scat, decomposed scat, and urine, produced 2-16 mg-DOC/g-waste and 116-768 µg-THM/g-waste. Food not eaten by animals could cause a sharp increase of DOC and DBP production and therefore should be removed rapidly from pools. Marine mammal husbandry staff and trainers are at risk (5.16 × 10(-4) to 1.30 × 10(-3)) through exposure of THMs, exceeding the negligible risk level (10(-6)) defined by the US Environmental Protection Agency.

Seasonal variation in blood and muscle oxygen stores attributed to diving behavior, environmental temperature and pregnancy in a marine predator, the California sea lion.

Survival depends on an animal's ability to find and acquire prey. In diving vertebrates, this ability is directly related to their physiological capability (e.g. oxygen stores). We studied the seasonal variation in oxygen stores, body temperature and body condition in California sea lions (Zalophus californianus) (CSL) as a function of seasonal variation in temperature, primary productivity, diving behavior and reproductive stage. During summer, blood oxygen stores were significantly greater and muscle oxygen stores were significantly lower than in winter. Total oxygen stores, body condition and body temperature did not change between seasons but variations in body temperature were greater during summer. Changes in oxygen stores are partly attributed to diving behavior, temperature and pregnancy that could increase oxygen consumption. Blood and muscle oxygen stores appear to be influenced by reproductive state. Blood oxygen stores are more likely influenced by diving behavior and temperature than muscle oxygen stores.

PBDE flame retardants and PCBs in migrating Steller sea lions (Eumetopias jubatus) in the Strait of Georgia, British Columbia, Canada.

Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) were measured in blubber biopsy samples from 22 live-captured Steller sea lions (Eumetopias jubatus) that had just entered the Strait of Georgia, British Columbia, Canada, for their overwintering feeding season. ∑PBDE ranged from 50µgkg(-1) (lipid weight) in adult females to 3780µgkg(-1) in subadult individuals. ∑PCBs ranged from 272µgkg(-1) in adult females to 14280µgkg(-1) in subadult individuals. While most PBDE and PCB congeners were transferred through milk to pups, PCBs with logK(OW)>7.0 (PCBs 206, 207, 208 and 209) appeared constrained, resulting in a lighter mixture in pups compared to adult females. The ratio of individual PCB congeners by metabolic group (Groups I, II, III, IV and V) to PCB-153 regressed against length of males suggested poor biotransformation of these compounds (slopes did not differ from zero, p>0.05). PBDE congeners 49, 99, 153 and 183 appeared bioaccumulative (slopes of ratio BDE/PCB 153 versus length were higher than zero, p<0.05), but the dominance of the single congener, BDE-47 (64% of total PBDEs), likely due in part to debromination pathways, reduced our ability to explore congener-specific dynamics of PBDEs in these pinnipeds. With 80% of our Steller sea lions exceeding a recent toxicity reference value for PCBs, the fasting-associated mobilization of these contaminants raises concerns about a heightened vulnerability to adverse effects during annual migrations.

Effects of hot-iron branding on heart rate, breathing rate and behaviour of anaesthetised Steller sea lions.

This study assessed the heart rate, breathing rate and behavioural responses of 12 juvenile Steller sea lions during hot-iron branding under isoflurane anaesthesia. Physiological and behavioural measures were recorded in four periods: baseline (five minutes), sham branding (one minute), branding (approximately 2.7 minutes) and postbranding (five minutes). No difference in heart rate was noted from baseline to sham branding, but heart rate increased from mean (sem) 78.3 (2.4) bpm in the baseline period to 85.6 (2.5) bpm in the branding period. Heart rate remained elevated in the postbranding period, averaging 84.7 (2.5) bpm. Breathing rate averaged 2.5 (1.0) breaths/minute in the baseline and sham branding periods increased to 8.9 (1.0) breaths/minute during branding, but returned to baseline by the postbranding period. Behaviourally, half of the sea lions exhibited trembling and head and shoulder movements during branding.

The effects of two analgesic regimes on behavior after abdominal surgery in Steller sea lions.

This study examined the effects of two non-steroidal anti-inflammatory drug (NSAID) treatment protocols on the behavioral responses of juvenile Steller sea lions after abdominal surgery. Sea lions were randomly assigned to one of two treatments designed to control post-operative pain. The flunixin group (n=6) received flunixin meglumine (1mg/kg) administered as a single intramuscular (IM) injection before extubation from surgery. The carprofen group (n=5) received carprofen (4.4 mg/kg) as an IM injection before extubation, then orally at 24, 48 and 72 h after surgery. Seven behaviors related to post-operative pain were monitored by observers, blinded to treatment, for a total of 10 days (3 days pre-, day of surgery, and 6 days post-surgery). All seven behaviors changed after surgery regardless of NSAID treatment, two of which returned to baseline within 6 days of surgery. Only one behavior was mildly affected by analgesic treatment: sea lions in the carprofen group tended to spend less time lying down in Days 1-3 following surgery (i.e., the days which they received oral carprofen). These results suggested that neither treatment, at the dose administered, was effective in controlling pain in the days following this surgery.

The normal genital tract of the female California sea lion (Zalophus californianus): cyclic changes in histomorphology and hormone receptor distribution.

Changes in reproductive tract histomorphology, and estrogen (ERalpha) and progesterone receptor (PR) expression throughout the breeding cycle were evaluated in free-ranging stranded female California sea lions (Zalophus californianus). Hormone receptor expression in the ovaries, uterus, cervix, and vagina was evaluated using an immunohistochemical technique with monoclonal antibodies. During a large portion of the cycle, ovaries contained both a corpora lutea (CL) and follicles in varying stages of development. In the periods of pupping and estrus during June and July, and in the spring morphologic features of the endometrium suggested estrogen influence. There were areas of squamous differentiation in the pseudostratified columnar epithelium of the cervix and vagina in some animals during estrus and in the spring. Estrogen receptor immunohistochemical scores were highest during pupping and estrus and in the spring and lowest during embryonic diapause. Cyclic changes in uterine PR expression throughout the cycle were minimal. Both ERalpha and PR were expressed in epithelial and stromal cells throughout the cervix and vagina, however, receptor expression was typically higher in the stroma. Stromal cell hormone receptors may play an important role in epithelial responses to circulating sex hormones. The results of this investigation add to the general knowledge of California sea lion reproduction and establish baseline information on reproductive tract hormone receptors that will aid in determining the factors involved in urogenital cancer development in sea lions.

Normal morphology and hormone receptor expression in the male California sea lion (Zalophus californianus) genital tract.

Histomorphology and estrogen alpha (ER alpha), and progesterone receptor (PR) expression were evaluated in free-ranging stranded male California sea lions (Zalophus californianus). Hormone receptor expression was evaluated using an immunohistochemical technique with monoclonal antibodies. Estrogen and PRs were identified in the efferent ductules, prostate gland, corpus cavernosa, corpus spongiosium, penile urethra, and in the epithelium and stroma of both the penis and prepuce. In some tissues, ER alpha expression was more intense in the stroma, emphasizing the importance of the stroma in hormone-mediated growth and differentiation of reproductive organs. To our knowledge, this is the first study to localize ER alpha and PR to the epithelium of the glans penis. The results of this investigation add to the general knowledge of male California sea lion reproduction and suggest that estrogens could have a role in the function of the male reproductive tract.

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to Steller sea lion lung cells.

Hexavalent chromium is an environmental contaminant. Within the environment, marine waters are a common site for hexavalent chromium deposition. We have recently reported significantly high levels of chromium in skin tissue from North Atlantic right whales. These findings demonstrate that marine species are being exposed to chromium. It is possible that such exposures may be playing a role in population declines evident among certain marine mammals, such as the Steller sea lion. We developed a Steller sea lion lung cell line from Steller sea lion lung tissue. Hexavalent chromium was cytotoxic to these primary lung fibroblasts as 1, 2.5, 5, 10 and 25microM sodium chromate induced 104, 99, 92, 58 and 11% relative survival, respectively. It was also genotoxic as 0, 1, 2.5, 5 and 10microM sodium chromate damaged chromosomes in 6, 11, 21, 36, and 39% of metaphases and damaged 6, 12, 27, 49 and 57 total aberrations in 100 metaphases, respectively. We also considered the toxicity of particulate hexavalent chromium, as it is the more potent carcinogen in humans. We found that 0.1, 0.5, 1, 5 and 10microg/cm(2) particulate chromate induced 95, 88, 91, 70, and 52% relative cell survival, respectively. These concentrations were genotoxic and damaged chromosomes in 9, 13, 18, and 23% of metaphases and induced 9, 15, 20 and 30 total aberrations per 100 metaphases, respectively. These data indicate that if sufficiently exposed, chromium may adversely affect the struggling Steller sea lion population. It would be prudent to investigate the effects chromium has in other Steller sea lion organs in order to derive a better understanding of how chromium in the marine environment may be affecting the declining Steller sea lion population.

Monitoring glucocorticoid response to rehabilitation and research procedures in California and Steller sea lions.

We used serum and fecal corticosteroid analysis to study the physiological response to a range of invasive and non-invasive procedures in sea lions. Four experimental groups of California sea lions (Zalophus californianus; Group A: restraint only [n=9], Group B: gas anesthesia without surgery [n=10], Group C: minimally invasive surgery [n=10], and Group D: invasive surgery [n=5]) were monitored for adrenal response. Feces were collected opportunistically from 72 hr before 72 hr post procedure for corticosterone analysis. All experimental groups showed substantial individual variation and no significant change in corticosterone levels after the procedures. Additional fecal and serum corticoid samples were collected from six free-ranging Steller sea lions (Eumetopias jubatus) in temporary captivity undergoing abdominal implantation of long-term telemetry devices. Only one sea lion exhibited a significant fecal corticosterone increase in response to the surgery. Capture and restraint appear to elicit a greater glucocorticoid response than invasive or non-invasive procedures.

Seasonal variability in otariid energetics: implications for the effects of predators on localized prey resources.

Otariids, like other wild mammals, contend with a wide variety of energetic demands across seasons. However, due to the cryptic behaviors of this marine group, few studies have been able to examine longitudinal energetic costs or the potential impact of these costs on seasonal or annual prey requirements. Here we evaluated the changes in energy demand and intake of female California sea lions (Zalophus californianus) during reproductive (n=2 sea lions) and nonreproductive (n=3) periods. Monthly measurements included resting metabolic rate, blood hormone levels, body condition (blubber thickness and body mass), and caloric intake for adult sea lions throughout molting, late pregnancy, lactation, and postweaning. We found that maintenance energy demands decreased from 32.0 to 23.1 MJ d(-1) before pupping, remaining stable at 19.4+/-0.6 MJ d(-1) during lactation and postweaning. Energy intake rates to meet these demands showed marked changes with activity level and the reproductive cycle, reaching a peak intake of 3.6 times baseline levels during lactation. Translating this into prey demands, we find that 20,000 reproductively active females on San Nicolas Island rookeries would maximally require 4,950 metric tons of Pacific whiting during a month of the breeding season. This localized impact is reduced significantly with postbreeding dispersal and demonstrates the importance of considering spatial and temporal factors driving the energetic requirements of predators when designing marine protected areas.

Isoflurane anaesthesia in four sea lions (Otaria byronia and Zalophus californianus).

OBJECTIVE: To (1) evaluate a purpose-built chamber for inducing isoflurane anaesthesia in sea lions and (2) assess isoflurane as an anaesthetic for dental surgery in these species. STUDY DESIGN: Prospective case study. ANIMALS: Four sea lions, aged between 5 and 12 years and weighing 74-110 kg, with dental disease. METHODS: Sea lions were restrained in a custom-built acrylic chamber into which 5% isoflurane (vapourizer setting) was delivered in O(2) (30 L minute(-1)) from three anaesthetic machines. When the animals were recumbent, the chamber was opened and induction completed using a face mask. Anaesthesia was maintained with 1.5-3.0% isoflurane (vapourizer setting) for 10-15 minutes and after tracheal intubation, was maintained with 1.0-3.0% isoflurane (end-tidal) for 41-255 minutes, using a partial rebreathing system with CO(2) absorption. RESULTS: During induction, the sea lions attempted to support their weight on their thoracic limbs before slipping into sternal or lateral recumbency. Sea lions underwent either root canal surgery, 'crowning' or canine tooth extraction. The animals were completely unresponsive during these operations and apnoea was never encountered at any point during anaesthesia. After surgery, the sea lions were placed into transport cages and their tracheae extubated once they began to move their heads. Thereafter, recovery from anaesthesia was smooth and no signs of distress were observed. Animals were able to raise their forequarters using their flippers within 1 hour of the operation and were returned to the aquarium about 2 hours after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: The purpose-built induction chamber is safe for inducing isoflurane anaesthesia in spontaneously breathing sea lions and isoflurane is an effective anaesthetic in this species.

Reversible anesthesia of captive California sea lions (Zalophus californianus) with medetomidine, midazolam, butorphanol, and isoflurane.

Two adult California sea lions (Zalophus californianus) were effectively anesthetized 13 times with medetomidine (0.010-0.013 mg/kg), midazolam (0.2-0.26 mg/kg), and butorphanol (0.2-0.4 mg/kg) by i.m. hand or pole syringe injection. For each anesthetic event, atropine (0.02 mg/kg, i.m.) was administered 6-20 min after initial injections, and oxygen administration via face mask or nasal insufflation began at the same time. Light anesthesia was induced in 8-22 min and lasted 13-78 min. During eight of the procedures, isoflurane (0.5-2.0%) was administered via face mask or endotracheal tube for an additional 30-120 min to facilitate longer procedures or surgery. Anesthesia was antagonized with atipamezole (0.05-0.06 mg/kg) and naltrexone (0.1 mg/kg) in seven events, with the addition of flumazenil (0.0002-0.002 mg/kg) in six events. The antagonists were administered by i.m. injection 42-149 min after administration of the induction agents. All sea lions recovered to mild sedation within 4-17 min after administration of the antagonists.

Isoflurane anesthesia in free ranging sea lion pups.

California sea lion pups, Zalophus californianus, (n = 115), were captured and anesthetized for (mean +/- SD) 17.6 +/- 7.8 min on San Miguel Island, California (USA) in November of 1992. Mask isoflurane anesthesia allowed intubation in 7.1 +/- 2.74 min. Pups recovered and walked in 7.32 +/- 4.8 min. Mask anesthesia in pups resulted in relaxation in 45 +/- 14 sec. Safe, brief anesthesia was delivered in support of weighing, medical evaluation, and short surgical procedures. Recovery character of all pups was sufficient to permit release to the free ranging state immediately after surgery, saving labor, supervision personnel, and postoperative time.

Evidence for a prolonged postimplantation period in the Australian sea lion (Neophoca cinerea).

Concentrations of circulating progesterone and oestradiol were measured in 96 free-ranging, female Australian sea lions Neophoca cinerea from Kangaroo Island, South Australia. There was a marked increase in the concentrations of both hormones (progesterone from approximately 12 ng ml-1 to approximately 24 ng ml-1; oestradiol from approximately 1.5 pg ml-1 to approximately 14 pg ml-1) about 3.5 months after the probable date of mating, reaching peak values in the 5 months after parturition. Progesterone concentrations remained at peak concentrations for about 2 months, decreasing at approximately 8 months to concentrations approximating those of the first 3 months after parturition. Oestradiol concentrations decreased, after reaching a peak, to 3-4 pg ml-1 at about 8 months after parturition. The timing of the increase in the concentrations of circulating progesterone and oestradiol provides evidence that the blastocyst reactivates and implants between 3.5 and 5 months of pregnancy in Australian sea lions, indicating an embryonic diapause of similar duration to that of other pinnipeds. This would suggest a prolonged postimplantation period of up to 14 months (to fit with the gestation period of 18 months reported for this species) the longest postimplantation period recorded for pregnancy in any pinniped.