Longitudinal patterns in progesterone metabolites in pregnant and non-pregnant Steller sea lions (Eumetopias jubatus).

Similar to the several pinniped and a few terrestrial carnivore species, the Steller sea lion has a seasonal synchronized mating scheme enabled by a female reproductive cycle that includes embryonic diapause, delayed implantation, and pseudopregnancy (a state in which the corpus luteum produces progesterone for approximately as long as in pregnant females). Due to this, circulating systemic progesterone concentrations cannot be used to differentiate pregnant and nonpregnant females during early gestation. With the use of advanced measurement technologies such as liquid chromatography tandem mass spectrometry (LC-MS/MS) additional steroid hormones are measurable which can provide additional information on the endocrine pathways throughout gestation. Our objectives were to further characterize endocrine patterns in female Steller sea lion pregnancy by 1) quantifying longitudinal profiles of hormone metabolites in pregnant and non-pregnant female sera, and 2) evaluating hormone profiles to identify pregnant animals within the early stage of gestation. Three gestation stages were delineated based on what is believed to be the period of implantation (September-October): EARLY (August- November), MID (December-February), and LATE (March to May). Five steroids, Progesterone (P4), 5α-dihydroprogesterone (DHP), 17αOH-progesterone (17OHP), 20αOH-progesterone (20OHP), and androstenedione (A4), were detected in both pregnant and non-pregnant animals. A significant difference in P4 concentrations was measured between EARLY and MID gestation (p ≤ 0.01) in both pregnant and non-pregnant animals. During MID gestation there was a significant difference (p ≤ 0.05) between pregnant and non-pregnant animals in all pregnanes measured. Significant patterns of correlation between P4 and 17OHP and between P4 and DHP were detected during EARLY and MID gestation in non-pregnant animals. While those significant correlations also exist in EARLY pregnant animals, this pattern was lost by MID gestation. This loss of correlation suggests a potential shift in progesterone metabolism from ovarian to alternative tissue (e.g. fetal gonads or adrenal glands) by MID gestation in Steller sea lions. We were unable to identifying a steroid hormone biomarker capable of differentiating pseudopregnancy from pregnant animals and conclude that such a biomarker likely falls outside of the traditional progesterone metabolic pathway.

Convergent evolution of forelimb-propelled swimming in seals.

Modern pinnipeds (true and eared seals) employ two radically different swimming styles, with true seals (phocids) propelling themselves primarily with their hindlimbs, whereas eared seals (otariids) rely on their wing-like foreflippers.1,2 Current explanations of this functional dichotomy invoke either pinniped diphyly3-5 or independent colonizations of the ocean by related but still largely terrestrial ancestors.6-8 Here, we show that pinniped swimming styles form an anatomical, functional, and behavioral continuum, within which adaptations for forelimb swimming can arise directly from a hindlimb-propelled bauplan. Within phocids, southern seals (monachines) show a convergent trend toward wing-like, hydrodynamically efficient forelimbs used for propulsion during slow swimming, turning, bursts of speed, or when initiating movement. This condition is most evident in leopard seals, which have well-integrated foreflippers with little digit mobility, reduced claws, and hydrodynamic characteristics comparable to those of forelimb-propelled otariids. Using monachines as a model, we suggest that the last common ancestor of modern seals may have been hindlimb-propelled and aquatically adapted, thus resolving the apparent contradiction at the root of pinniped evolution.

Characterizing estrus by trans-abdominal ultrasounds, fecal estrone-3-glucuronide, and vaginal cytology in the Steller sea lion (Eumetopias jubatus).

The ability to monitor the estrus cycle in wild and captive marine species is important for identifying reproductive failures, ensuring a successful breeding program, and monitoring animal welfare. Minimally invasive sampling methods to monitor estrus in captive populations have been developed, but results suggest these tools can be species-specific in their precision and accuracy. Therefore, the minimally invasive sampling methods of trans-abdominal ultrasounds, a fecal steroid analysis (estrone-3-glucuronide, E1G), and vaginal cytology, were evaluated for their efficacy to characterize and monitor estrus in a captive breeding population of Steller sea lions (Eumetopias jubatus). Three adult females were sampled over five breeding seasons, resulting in six estrus profiles characterized by trans-abdominal ultrasounds, five by fecal E1G, and four by vaginal cytology. Animals were trained to allow trans-abdominal ultrasounds, fecal samples, and vaginal swabs to be collected approximately daily. Of the 76 trans-abdominal ultrasound sessions attempted, 8 successfully visualized both ovaries. From these scans, the chronology of ovarian changes during proestrus and estrus was estimated. The time from the detection of developing follicles to the identification of a dominate follicle occurred in 2-5 days and a corpus hemorrhagicum formed approximately 4 days later. However, because visualization of the ovaries was prevented by the gastrointestinal system in 88% of scans, this tool was overall unreliable for monitoring changes associated with estrus. To detect fine scale physiological changes associated with estrus, we analyzed changes in fecal E1G (n = 62) and vaginal cytology (n = 157) 15 days before and after each female's single copulation event (Day = 0). Changes in fecal E1G had the highest accuracy at detecting Day = 0. Fecal E1G increased leading up to estrus, peaked at Day = 0, and then declined. Although we did observe the characteristic increase in superficial cells associated with impending estrus, the type of cell which peaked closest to Day = 0 was intermediate. The uncertainty around the peak in intermediate cells, indicating estrus, was greater than the uncertainty associated with detecting estrus from fecal E1G. Collectively, these results suggest that changes in fecal E1G and vaginal cytology are viable tools to detect estrus in Steller sea lions, but require daily sampling to detect gradual changes, limiting their applicability to studies of wild populations.

Clawed forelimbs allow northern seals to eat like their ancient ancestors.

Streamlined flippers are often considered the defining feature of seals and sea lions, whose very name 'pinniped' comes from the Latin pinna and pedis, meaning 'fin-footed'. Yet not all pinniped limbs are alike. Whereas otariids (fur seals and sea lions) possess stiff streamlined forelimb flippers, phocine seals (northern true seals) have retained a webbed yet mobile paw bearing sharp claws. Here, we show that captive and wild phocines routinely use these claws to secure prey during processing, enabling seals to tear large fish by stretching them between their teeth and forelimbs. 'Hold and tear' processing relies on the primitive forelimb anatomy displayed by phocines, which is also found in the early fossil pinniped Enaliarctos. Phocine forelimb anatomy and behaviour therefore provide a glimpse into how the earliest seals likely fed, and indicate what behaviours may have assisted pinnipeds along their journey from terrestrial to aquatic feeding.

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.

Decline of heterozygosity in a large but isolated population: a 45-year examination of moose genetic diversity on Isle Royale.

Wildlife conservation and management approaches typically focus on demographic measurements to assess population viability over both short and long periods. However, genetic diversity is an important predictor of long term population vitality. We investigated the pattern of change in genetic diversity in a large and likely isolated moose (Alces alces) population on Isle Royale (Lake Superior) from 1960-2005. We characterized samples, partitioned into five different 5-year periods, using nine microsatellite loci and a portion of the mtDNA control region. We also simulated the moose population to generate a theoretical backdrop of genetic diversity change. In the empirical data, we found that the number of alleles was consistently low and that observed heterozygosity notably declined from 1960 to 2005 (p = 0.08, R2 = 0.70). Furthermore, inbreeding coefficients approximately doubled from 0.08 in 1960-65 to 0.16 in 2000-05. Finally, we found that the empirical rate of observed heterozygosity decline was faster than the rate of observed heterozygosity loss in our simulations. Combined, these data suggest that genetic drift and inbreeding occurred in the Isle Royale moose populations over the study period, leading to significant losses in heterozygosity. Although inbreeding can be mitigated by migration, we found no evidence to support the occurrence of recent migrants into the population using analysis of our mtDNA haplotypes nor microsatellite data. Therefore, the Isle Royale moose population illustrates that even large populations are subjected to inbreeding in the absence of migration.