Steven Osborn

This investigation compared the fatty acid (FA) levels found in erythrocyte (RBC) membranes, plasma, whole blood (WB), and blubber from wild Alaskan (Bristol Bay) belugas (Delphinapterus leucas) (BBB, n = 9) with oceanaria-based belugas (OBB, n = 14) fed a controlled diet consisting of primarily herring (Clupea harengus) and capelin (Mallotus villosus). FA patterns in RBCs, WB, and plasma varied considerably between BBB and OBB animals. Focusing on RBC FA levels of known dietary origin, the OBBs had markedly higher levels of 20:1n9,11 and 22:1n9,11. RBC levels of these fatty acids were 1% and 0.2% in the BBBs, but 8.2% and 4.5%, respectively, in the OBBs (p < 0.05 both). These long-chain mono-unsaturated FAs (LC-MUFAs) are rich in herring and capelin but not in the prey species (i.e., salmonids, smelt, cod, and shrimp) generally available to BBBs. As for the marine omega-3 poly-unsaturated fatty acids [PUFAs; 20:5n3 (eicosapentaenoic acid) and 22:6n3 (docosahexaenoic acid)], the ...

ABSTRACTAccurate identification of individual ages within wild bottlenose dolphins (Tursiops truncatus) is critical for determining population health and the development of population management strategies. As such, we analyzed DNA methylation patterns by applying a custom methylation array (HorvathMammalMethyl40) to both blood (n = 140) and skin samples (n = 87) from known age or approximate age (0 to 57 years) bottlenose dolphins. We present three bottlenose dolphin specific age estimation clocks using combined blood and skin (48 CpGs, R = 0.93, median absolute error = 2.13 years), blood only (64 CpGs, R = 0.97, error= 1.46 years) and skin only (39 CpGs, R = 0.95, error= 2.53). Our sex estimator based on 71 CpGs predicts the sex of any odontocete species with 99.5% accuracy. We characterize individual cytosines that correlate with sex and age in dolphins.The presented epigenetic clocks are expected to be useful for conservation efforts and for studying anthropogenic events.

The development of a precise blood or skin tissue DNA Epigenetic Aging Clock for Odontocete (OEAC) would solve current age estimation inaccuracies for wild odontocetes. Therefore, we determined genome-wide DNA methylation profiles using a custom array (HorvathMammalMethyl40) across skin and blood samples (n = 446) from known age animals representing nine odontocete species within 4 phylogenetic families to identify age associated CG dinucleotides (CpGs). The top CpGs were used to create a cross-validated OEAC clock which was highly correlated for individuals (r = 0.94) and for unique species (median r = 0.93). Finally, we applied the OEAC for estimating the age and sex of 22 wild Norwegian killer whales. DNA methylation patterns of age associated CpGs are highly conserved across odontocetes. These similarities allowed us to develop an odontocete epigenetic aging clock (OEAC) which can be used for species conservation efforts by provide a mechanism for estimating the age of free rang...

Accurate identification of individual ages within wild bottlenose dolphins (Tursiops truncatus) is critical for determining population health and the development of population management strategies. As such, we analyzed DNA methylation (DNAm) patterns by applying a custom methylation array (HorvathMammalMethyl40) to both blood (n= 140) and skin samples (n= 87) from known age or approximate age (0–57 years) bottlenose dolphins. We present three bottlenose dolphin specific age estimation clocks using combined blood and skin [48 CpGs,R= 0.93, median absolute error (MAE) = 2.13 years], blood only (64 CpGs, R = 0.97, error = 1.46 years) and skin only (39 CpGs,R= 0.95, error = 2.53). We characterized individual cytosines that correlate with sex and age in dolphins and developed a sex estimator based on 71 CpGs that predicts the sex of any odontocete species with 99.5% accuracy. The presented epigenetic clocks are expected to be useful for conservation efforts and for determining if anthro...

The reproductive physiology of the Pacific white-sided dolphin,Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%,n=37) and conceptions (83%,n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm2), and sperm concentration (144.3×107 sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were ...

Persistent organic pollutants (POPs) pose a health risk for southern resident killer whales (SRKWs). Data on maternal contaminant transfer to calves are needed to inform models that estimate future contaminant loads as well as assess risk to newborn killer whale calves exposed to POPs. We conducted a study on trained female killer whales and their calves to fill these data gaps. POPs and lipid content were quantified in blood serum (POP levels in serum and blubber are highly correlated) collected during gestation and in milk and serum collected post-partum from female killer whales. Serum samples were also collected from calves during the lactation period. POP concentrations in milk collected over 15 months post-partum from the primiparous female decreased by 47-65%, depending on the contaminant class. The highest influx of contaminants to calves tended to occur soon after birth. Greater contaminant transfer rates during early lactation were also reflected in maternal serum POP levels. POP levels in maternal serum decreased significantly during the first 144-158 days post-partum, depending on contaminant class, and then leveled off through the remaining lactation period. This resulted in 67-81% reductions in POP levels in maternal serum over 15 months. By 15 months post-partum, serum POP levels from the primiparous female had dropped to levels measured in the multiparous female during pregnancy. Dissimilar to the multiparous female, body mass and blubber thickness in the primiparous female also declined significantly during the first 3-5 months post-partum, demonstrating linkages between lipid and POP transfer from blubber stores to milk in early lactation. By the end of lactation, lipid-corrected POP concentrations in serum from the first-born calf were 5-8 times greater than the corresponding POP levels from her primiparous mother. These results demonstrate that very young neonatal SRKW calves, particularly first-born calves, are at high risk from contaminant exposure

Abstract: Brucella ceti, associated with neurobrucellosis, has been isolated from cerebrospinal fluid (CSF) of postmortem cetaceans. A 106-kg, stranded female bottlenose dolphin (Tursiops truncatus) presented with serum antibodies to Brucella spp. via competitive enzyme-linked immunosorbent assay and fluorescence polarization assay. Polymerase chain reaction (PCR) and culture of whole blood, bronchoalveolar fluid, and rectal, nasal, and genital swabs for Brucella spp. were consistently negative. Serial computed tomography revealed mild focal dilatation of brain ventricles. CSF sampling was warranted to exclude neurobrucellosis. Sedation was achieved with 30 mg diazepam (0.28 mg/kg) orally 2.5 hours prior to arrival in hospital, followed by 5.3 mg midazolam (0.05 mg/kg) intramuscularly, and anesthetic induction with 2.5 mg midazolam (0.02 mg/kg) and 200 mg propofol (2 mg/kg) administered slowly intravenously, followed by intubation and maintenance on sevoflurane using controlled mech...

Abstract: Neutrophils are one of the initial cell lines of protection against pathogens, and when their concentrations in the blood are low, animals are highly susceptible to infections. Neutropenia has been reported in cetaceans secondary to administration of systemic sulfa antibiotics or antifungal medications and severe, overwhelming infection. Filgrastim was administered to treat neutropenia over a 15-y period in 11 cetaceans comprising four species—beluga (Delphinapterus leucas, n = 1), bottlenose dolphin (Tursiops truncatus, n = 4), killer whale (Orcinus orca, n = 5), and short-finned pilot whale (Globicephala macrorhynchus, n = 1)] ranging in age from 1 wk to >24 y. Seven study animals received multiple doses (2–6). All animals responded to at least one dose (1–7 µg/kg) of parenteral filgrastim characterized by an increase in peripheral immature (band) neutrophils, segmented neutrophils, or both. In most cases (9/11), neutrophil counts increased within 48 h of a single dose. Duration of response varied but was at least 2 wk in eight of the 11 animals and 5–9 d in the remaining animals. No adverse reactions were observed in any cases.

Steroid hormone analysis in blow (respiratory vapor) may provide a minimally invasive way to assess the reproductive status of wild cetaceans. Biological validation of the method is needed to allow for the interpretation of hormone measurements in blow samples. Utilizing samples collected from trained belugas (Delphinapterus leucas, n=20), enzyme immunoassays for testosterone and progesterone were validated for use with beluga blow samples. Testosterone concentrations in 40 matched blood and blow samples collected from 4 male belugas demonstrated a positive correlation (R(2)=0.52, p<0.0001). Progesterone concentrations in 64 matching blood and blow samples from 11 females were also positively correlated (R(2)=0.60, p<0.0001). Testosterone concentrations (mean±SD) in blow samples collected from adult males (119.3±14.2pg/ml) were higher (p<0.01) than that of a juvenile male (<8years) (59.4±6.5pg/ml) or female belugas (54.1±25.7pg/ml). Among adult males, testosterone concen...

For wild belugas (Delphinapterus leucas), gestation length estimates based on fetal size have produced extreme ranges. Ex situ populations thereby provide unique opportunities to define this important life history event. Accordingly, research with ultrasound was conducted on six beluga whales over 11 gestations with known conception dates to serially measure fetal changes in biparietal diameter (BP), thoracic diameter (TD), thoracic circumference (TC), and total length (TL). Incremental polynomial regression analyses were performed on each fetal measurement to develop predictive models for determining age based on fetal size and days from parturition. Gestation length (n = 11) was a mean 467 ± 5.4 d with male calves (478 ± 8.6 d) experiencing a longer gestation (P = 0.04) than females (457 ± 3.9 d). Age at TL was best described using a 2nd order polynomial model, while linear relationships existed for BPD, TD, and TC. Accuracy was improved for predicting age (P = 0.001) or days prior to parturition (P = 0.038) using data from the first vs. the second half of gestation. The results provide accurate models for aging beluga fetuses based on size in both in situ and ex situ populations.

A 22-yr-old multiparous beluga, Delphinapterus leucas, with consistently elevated serum progesterone concentrations post-artificial insemination was diagnosed with viable twins at 149 days postconception. Twins were of similar size at least until day 264, the last point when ultrasound measurements of both twins were made. However, ultrasound was used to determine the viability of both fetuses on days 365, 393, and 400. After 90% of normal gestation, or 434 days, steroids were administered to encourage fetal lung maturation. Seven days later a 40.9-kg live female calf was delivered headfirst. A second 22.7-kg stillborn calf was delivered in fluke-first presentation 8 hr later. Immediately after birth, the live calf was administered surfactant intratracheally. The next day, it was given beluga immunoglobulin intramuscularly, and started on oral antibiotics and provided nutritional support via an oral gastric tube. The calf started nursing voluntarily on day 3. Antibiotic and nutritional support was discontinued on day 10. Bimonthly weight and length measurements demonstrated that after an initial increased growth rate, the calf has grown within normal parameters after birth. This calf represents the first known successful surviving twin of any cetacean species and sets an important precedent for treatment modalities that may be available to assist the premature cetacean neonate.

This article discusses the utility and limitations of urethroscopy and cystoscopy in both male and female cats. The special equipment and techniques necessary to adequately visualize the urethra and bladder of female cats are described in some detail. The emerging ability to visualize the male urethra is introduced. Photographs taken through the cystoscope are used to illustrate both normal anatomy and lesions likely to be encountered with cats with lower urinary tract disease. Cystoscopy is an important diagnostic tool for an inclusionary and exclusionary diagnosis of interstitial cystitis (sterile idiopathic inflammation).