Circulating profile of the appetite-regulating hormone ghrelin during moult-fast and chick provisioning in southern rockhopper penguins (Eudyptes chrysocome chrysocome).

A multitude of animal species undergo prolonged fasting events at regularly occurring life history stages. During such periods of food deprivation, individuals need to suppress their appetite. The satiety signalling gut hormone ghrelin has received much attention in this context in studies looking at mammalian systems. In wild birds, however, knowledge on the ghrelin system and its role during extended fasts is still scarce. In this study, we collected plasma samples for measurements of circulating ghrelin concentrations from adult southern rockhopper penguins (Eudyptes chrysocome chrysocome) during the three to four week-long moult-fast that they repeat annually to replace their feathers. We further sampled chicks before and after feeding bouts and non-moulting adults. Circulating ghrelin levels did not differ significantly between fed and unfed chicks but chicks had significantly lower plasma ghrelin levels compared to adults. Furthermore, penguins in late moult (i.e. individuals at the end of the prolonged fasting bout) had higher ghrelin levels compared to non-moulting adults. Our results show elevated levels of circulating ghrelin during moult and generally lower levels of ghrelin in chicks than in adults regardless of feeding state. Given the scarcity or absence of knowledge on the function of ghrelin in seabirds and in fasting birds in general, our results add greatly to our understanding of the avian ghrelin system.

Developing δ15N and δ13C isoscapes using whole blood from Magellanic penguins, Spheniscus magellanicus.

RATIONALE: Understanding the migration of marine animals is hindered by the limitations of traditional tracking methods. It is therefore crucial to develop alternative methods. Stable isotope-based tracking has proven useful for this task, although it requires detailed isoscapes in the focal area. Here, we present predator-based isoscapes of the coastal zone of the Patagonian Shelf Large Marine Ecosystem (PSLME), which offers a novel tool for geolocation. METHODS: Whole-blood samples from breeding Magellanic penguins nesting at 11 colonies were used to create δ15N and δ13C isoscapes. Isotopic values were assigned to random positions inside their corresponding foraging area. Spatial analysis and data interpolation resulted in δ15N and δ13C isoscapes for the coastal zone of the PSLME, which were validated through cross-validation. RESULTS: The isoscapes mean standard error ranged from 0.05 to 0.41 for δ15N and from 0.07 to 0.3 for δ13C, similar to the error range of the mass spectrometer used for measuring isotope ratios. Predictive surfaces reflected the latitudinal trends, with δ13C and δ15N values increasing northwards. δ13C values showed a strong latitudinal gradient, while δ15N values had two distinct domains, with higher values in the north. The error surface indicated the highest certainty within 130 km from the shore and within the reported Magellanic penguin foraging areas. CONCLUSIONS: Both isoscapes revealed strong spatial variation. The δ13C isoscape showed a latitudinal gradient, consistent with patterns in other oceans. The δ15N isoscape clearly separated northern and southern colonies, likely influenced by nitrogen sources. The error obtained fell within the measurement error ranges, adding credibility to the models.

Evolved increases in hemoglobin-oxygen affinity and the Bohr effect coincided with the aquatic specialization of penguins.

Dive capacities of air-breathing vertebrates are dictated by onboard O2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O2 transport. It has been hypothesized that increased hemoglobin (Hb)-O2 affinity improves pulmonary O2 extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O2 affinity at low pH). Although an increased Hb-O2 affinity reduces the gradient for O2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O2 affinity in combination with the augmented Bohr effect maximizes both O2 extraction from the lungs and O2 unloading from the blood, allowing penguins to fully utilize their onboard O2 stores and maximize underwater foraging time.

Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (Aptenodytes patagonicus).

Assessing the physiological stress responses of wild animals opens a window for understanding how organisms cope with environmental challenges. Since stress response is associated with changes in body temperature, the use of body surface temperature through thermal imaging could help to measure acute and chronic stress responses non-invasively. We used thermal imaging, acute handling-stress protocol and an experimental manipulation of corticosterone (the main glucocorticoid hormone in birds) levels in breeding king penguins (Aptenodytes patagonicus), to assess: 1. The potential contribution of the Hypothalamo-Pituitary-Adrenal (HPA) axis in mediating chronic and acute stress-induced changes in adult surface temperature, 2. The influence of HPA axis manipulation on parental investment through thermal imaging of eggs and brooded chicks, and 3. The impact of parental treatment on offspring thermal's response to acute handling. Maximum eye temperature (Teye) increased and minimum beak temperature (Tbeak) decreased in response to handling stress in adults, but neither basal nor stress-induced surface temperatures were significantly affected by corticosterone implant. While egg temperature was not significantly influenced by parental treatment, we found a surprising pattern for chicks: chicks brooded by the (non-implanted) partner of corticosterone-implanted individuals exhibited higher surface temperature (both Teye and Tbeak) than those brooded by glucocorticoid-implanted or control parents. Chick's response to handling in terms of surface temperature was characterized by a drop in both Teye and Tbeak independently of parental treatment. We conclude that the HPA axis seems unlikely to play a major role in determining chronic or acute changes in surface temperature in king penguins. Changes in surface temperature may primarily be mediated by the Sympathetic-Adrenal-Medullary (SAM) axis in response to stressful situations. Our experiment did not reveal a direct impact of parental HPA axis manipulation on parental investment (egg or chick temperature), but a potential influence on the partner's brooding behaviour.

Capillary Zone Electrophoresis in Humboldt Penguins (Spheniscus humboldti).

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, ß-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of ß-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

PHARMACOKINETICS OF PRIMAQUINE PHOSPHATE AFTER A SINGLE ORAL ADMINISTRATION TO AFRICAN PENGUINS (SPHENISCUS DEMERSUS).

Primaquine is an 8-aminoquinolone drug commonly used for the chemoprophylaxis and treatment of avian malarial infections in managed penguin populations worldwide. Little is known about its pharmacokinetic properties in avian species. The objective of this study was to describe the disposition of primaquine phosphate after a single oral dose in 15 healthy African penguins (Spheniscus demersus). A single tablet containing 26.3 mg of primaquine phosphate (equivalent to 15 mg primaquine base) was administered orally to each bird in a herring fish. Blood samples were collected prior to drug administration and at predetermined timepoints through 144 hr postadministration. Plasma was analyzed for drug concentration by high-performance liquid chromatography with ultraviolet detection. Mean maximum plasma concentration of primaquine phosphate was 277 ± 96 ng/ml at approximately 3.1 hr following oral administration. The mean disappearance half-life was 3.6 ± 1.6 hr. Plasma concentrations were below detectable limits in all but one penguin by 36 hr. A single oral administration of 26.3 mg of primaquine phosphate in African penguins resulted in a pharmacokinetic profile comparable to those attained in human studies. These results suggest that a dosing interval similar to human regimens may be of potential use in the prevention and treatment of avian malaria in penguins. Additional clinical studies are needed to determine the efficacy and safety of this regimen.

Diet-tissue discrimination factors (δ15 N and δ13 C values) for blood components in Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins.

RATIONALE: Analysis of the stable isotope ratios of carbon and nitrogen (δ13 C and δ15 N values) is increasingly being used to gain insight into predator trophic ecology, which requires accurate diet-tissue discrimination factors (DTDFs), or the isotopic difference between prey and predator. Accurate DTDFs must be calculated from predators consuming an isotopically constant diet over time in controlled feeding experiments, but these studies have received little attention to date, especially among seabird species. METHODS: In this study, aquarium-housed Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins were fed a single-prey source diet (capelin Mallotus villosus) for eight weeks. Stable isotope ratios (δ13 C and δ15 N values) of penguin blood (cellular component and plasma) and capelin were measured using mass spectrometry and then used to calculate DTDFs for both components of penguin blood by comparison with prey values. RESULTS: The DTDFs for plasma were -0.63 ± 0.49 (mean ± SD) and -0.27 ± 0.22 for δ13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ15 N values for Magellanic and southern rockhopper penguins, respectively, while the DTDFs for the cellular component were 1.22 ± 0.03 and 1.26 ± 0.03 for δ13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ15 N values. CONCLUSIONS: We compare our DTDFs with published values from blood components of penguins and discuss the effects that lipid extraction, sample storage, and diet have on the DTDFs of penguin blood components. This study provides accurate DTDFs of blood components for two seabird species of conservation concern, and is one of the first to provide plasma DTDFs for penguins, which are underrepresented in the seabird literature.

Trade-off between aerobic performance and egg production in migratory macaroni penguins.

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E2) to support vitellogenesis and egg production, but E2 secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E2-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 µg Zn ml-1, and 9.70 ± 0.19 mmol l-1, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

Circulating nutrient concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Punta San Juan, Peru.

Free-ranging Humboldt penguin (HP, Spheniscus humboldti) populations are under pressure from resource competition with industrial fisheries, habitat loss, and El Niño Southern Oscillation events. Foraging patterns for this top marine predator change during periods of aberrant oceanographic conditions and scarce fish stock numbers. These radical dietary fluctuations can lead to poor fertility, early embryonic death, poor hatchability, suppressed immune function, high chick mortality, and illness. To understand the variability of nutrient status in reproductive seasons, we measured select circulating nutrient concentrations (fat-soluble vitamins A, D, E, and carotenoids, fatty acids, amino acids, minerals, and electrolytes) of 105 HP at Punta San Juan, Peru during the first reproductive seasons of 2007 and 2008. We determined significant differences in nutrient status between sexes, years of sampling, and reproductive stages. Males (4.5 ± 0.38 kg) weighed more than females (4.0 ± 0.29 kg) and exhibited higher concentrations of vitamin A (0.71 ± 0.11 vs. 0.61 ± 0.12 µg/ml) and docosahexaenoic acid (6.70 ± 1.61 vs. 5.65 ± 1.59%). Males also displayed lower concentrations of ß-carotene (0.01 ± 0.01 vs. 0.012 ± 0.001 µg/ml) and phosphorus (3.43 ± 0.83 vs. 4.40 ± 1.66 mg/dl). Comparison between the 2 years showed most circulating amino acid concentrations were higher in 2007. Significant differences in circulating amino acids and vitamins were also noted between different reproductive stages. These results demonstrate concentrations of nutrients can vary due to the physiological state of the animal, as well as the overall dynamics of their marine ecosystem habitat.

HEMATOLOGY AND PLASMA CHEMISTRY VALUES IN BEACHED MAGELLANIC PENGUIN (SPHENISCUS MAGELLANICUS) IN NORTHERN ARGENTINA DURING THE NONBREEDING SEASON.

Seabirds have been widely used for monitoring the health of the oceans in diverse marine regions. Among low-cost survey strategies, systematic surveys of seabirds beached on coasts have been developed since the 20th century. However, these studies do not always address blood aspects. The assessment of the health status of birds based on the analysis of hematological and plasma chemistry is crucial to evaluate the overall health status profile of live organisms. Here, the authors study the variability of blood parameters by sex, age class, and year of beached Magellanic Penguin during the nonreproductive period in northern Argentina. Of 44 penguins, 77% were categorized as younger juveniles and the rest as older juveniles, and were captured and studied in coastal areas of Buenos Aires Province during the summers of 2017 and 2018. The mean body weight of beached penguins was affected by the age class of the individuals; most of the younger juveniles showed poor condition in terms of body mass (1,761 ± 235 g). No significant differences were observed in body weight between years and sex. Still, there were significant differences between years for alkaline phosphatase (ALP) and creatine phosphokinase (CPK) values. Twelve of the 20 blood parameters analyzed differ significantly with the age class of the beached penguins; younger juveniles were in a state of inanition. Our results may serve as a necessary first step in improving the conservation status of the Magellanic Penguin in nonbreeding grounds of Argentina, and call for a better knowledge of the health status of the species along its annual cycle.

Effect of molting on the concentration of plasma 25-hydroxyvitamin D in captive African penguins (Spheniscus demersus).

Feed intake and body weight are drastically altered in penguins during peri-molting period, and molting is known to affect the nutritional status of vitamin A and E. Although vitamin D status is not known in penguins during peri-molting period, vitamin D intake is supposed to be remarkably altered. The objective of the present study was to clarify the alterations in plasma 25-hydroxyvitamin D (25(OH)D) concentration, the most reliable biomarker for assessing vitamin D status, and vitamin D intake during peri-molting period. Blood samples were collected from seven adult male African penguins (Spheniscus demersus) in the control period, pre-molting period, early-molting period, and late-molting period. The dietary content of vitamin D and calcium (Ca) were higher than that of the estimated requirements. Feed intake increased in the pre-molting period and drastically decreased during the molting periods. Body weight increased in the pre-molting period, followed by the loss of weight towards the end of the experiment. Although vitamin D and Ca intakes decreased during the molting periods, plasma 25(OH)D concentration increased during the molting periods and the increase in plasma Ca concentration was also observed in the late-molting period. These results suggest that the reduction in body fat induced by reducing feed intake stimulated the release of vitamin D from body fat, which increased plasma 25(OH)D and Ca concentrations in molting penguins. Penguins are unlikely to suffer from typical hypervitaminosis D even during molting and vitamin D toxicity is not a realistic problem in penguins because of the short duration of molting.

Application of different pharmacokinetic models to describe and predict pharmacokinetics of voriconazole in magellanic penguins following oral administration.

Aspergillosis is a condition causing serious morbidity and mortality in captive penguins and other bird species. It can be treated with antifungal drugs, such as voriconazole. However, the pharmacokinetics of voriconazole are variable between different animal and bird species. Therefore, the pharmacokinetics of voriconazole were investigated in this study in Magellanic penguins. Pharmacokinetic models were constructed and applied to predict the pharmacokinetics of voriconazole during long-term treatment in Magellanic penguins, since the voriconazole treatment duration in chronic aspergillosis cases can last up to several months. Plasma voriconazole concentration-time data from adult Magellanic penguins (Spheniscus magellanicus; n = 15) following a single oral (PO) dose of either 2.5 mg/kg or 5 mg/kg in a herring in three separate study periods 7-12 months apart were collected. Mean plasma voriconazole concentrations were above the targeted MIC for Aspergillus fumigatus for 2 hr following a single 2.5 mg/kg voriconazole dose while the plasma concentrations exceeded the MIC for least 24 hr following a 5 mg/kg dose. Nonlinear mixed-effects modeling was used to fit two pharmacokinetic models, one with first-order and another with saturable elimination, to the single-dose data. Fits were good for both, as long as dose was included as a covariate for the first-order model so that clearance was lower and the half-life longer for animals receiving the 5 mg/kg dose. Although the single-dose data suggested saturated elimination at higher concentrations, the model with saturable elimination did not predict plasma voriconazole concentrations well for a clinical aspergillosis case receiving long-term treatment, possibly because of induction of metabolizing enzymes with chronic exposure. Pharmacokinetic models should accurately predict plasma drug concentrations for different dosage regimens in order to be applicable in the field. Future studies should focus on determining clearance at steady-state to be able to refine the pharmacokinetic models presented here and improve model performance for long-term oral voriconazole administration in Magellanic penguins.

An integrative appraisal of the hormonal and metabolic changes induced by acute stress using king penguins as a model.

A large number of studies have focused on the reactivity of the hypothalamic-pituitaryadrenal (HPA) axis and the consequences of glucocorticoids (GC) in mediating life-history trade-offs. Although short-term increases in GCs are viewed as adaptive, mobilizing energy substrates allowing animals to deal with impending threats (e.g. stimulating hepatic gluconeogenesis, stimulating lipolysis, mobilizing amino acids), few studies have actually measured the exact time-course of substrate mobilisation in response to acute stress in natural conditions. We evaluated the hormonal and metabolic components of the stress response to acute stress in 32 free-living king penguins (Aptenodytes patagonicus). We monitored changes in blood GCs (corticosterone, CORT), glucose, lactate, ketone bodies (ß-hydroxybutyrate), non-esterified fatty acids, and uric acid in response to a standardized capture-restraint protocol lasting for up to 90min. Furthermore, we tested whether the vigilance status of the animal (alert or asleep) affected its perception of the capture, thereby modulating the hormonal and metabolic stress responses. The time course of energy mobilisation followed the characteristic pattern expected from laboratory and theoretical models, with a rapid depletion of those energy stores linked to rapid adrenergic responses (i.e. glucose and ketone bodies), followed by a mobilisation of energy stores associated with the sustained longer-term GC response (i.e. fats and protein stores). HPA reactivity was generally slower than reported in other birds, and there was high inter-individual variability. Sleeping birds had higher GC and glucose responses to acute stress, suggesting a more rapid mobilization of energy stores. Our results highlight the importance of considering HPA and metabolic responses to acute stress against species-specific life history and ecological relevant backgrounds.

UV-light and dietary vitamin D and their effects on ionized calcium and 25-OH-D plasma concentrations in captive gentoo penguins (Pygoscelis papua).

In this study, the effect of ultraviolet (UV) light and dietary vitamin D on calcium metabolism in permanently indoor-housed gentoo penguins (Pygoscelis papua) was investigated. The study consisted of three periods, each completed with blood samples to analyse plasma concentrations of 25-OH-D, 1,25-(OH)2 -D, ionized (iCa) and total calcium (tCa). During the first study period (D), animals were housed under routine conditions without UV-light and fed a diet of different fish species, supplemented with 1,000 IU vitamin D per animal and day. The following study period (Baseline) of 28-day duration consisted of the same diet without any vitamin D supplementation and without UV-light. During the study period (UVB) artificial UV-light was added for 3 weeks. The vitamin D content of fish was measured by high-performance liquid chromatography. It varied between fish species and between facilities, ranging from no measurable content in capelin (Mallotus villosus) to 7,340 IU vitamin D/kg original matter (OM) in herring (Clupea spp). The average dietary vitamin D content was 311 IU/kg OM at facility 1 and 6,325 IU/kg OM at facility 2, resulting in a vitamin D intake per animal and day without supplementation of 130 IU (25.5 IU/kg body weight BW) and 2,454 IU (438.2 IU/kg BW) respectively. The supplementation of vitamin D elevated significantly the plasma concentrations of 25-OH-D by an intraindividual difference of 15 (range -2 to 59) nmol/L and tCa by 0.1 (0.0-0.3) mmol/L only at facility 2. The exposure to UV-light raised the blood concentrations of tCa at facility 2 by 0.15 (0.1-0.2) mmol/L, and of iCa and tCa for females at facility 1 by 0.23 (0.13-0.41) mmol/L and 1.8 (1.1-2.5) mmol/L respectively. No significant influence of the study periods (D) and (UVB) was found for the concentrations of 1,25-(OH)2 -D at both facilities.

Influence of biological factors on hematological and serum biochemistry values in captive Adélie, Chinstrap, Gentoo, and Macaroni penguins in Pingtung, Taiwan.

Hematology and serum biochemistry reference values are essential for health evaluation and disease diagnosis in penguins. However, there are currently no published physiological values for captive Adélie (Pygoscelis adeliae) and Chinstrap penguins (P. antarcticus), nor for wild or captive Macaroni penguins (Eudyptes chrysolophus). The present study is the first investigation regarding hematology and serum biochemistry reference values for captive Adélie, Gentoo (P. papua), Chinstrap, and Macaroni penguins in Asia. Fixed effect models for repeated measure were applied to determine the influence of penguin species, age, gender, and age-gender interaction on each blood parameter. Hematology and serum biochemical data from 122 apparently healthy penguins (24 Adélie, 38 Chinstrap, 46 Gentoo, and 14 Macaroni) were collected between 2009 and 2014. The effects of penguin species were observed for most blood parameters, except total bilirubin, creatine kinase (CK), creatinine, and potassium ion (K+ ). Values of mean corpuscular volume, mean corpuscular hemoglobin (MCH), heterophil, ratio of heterophils to lymphocytes (H/L), alanine aminotransferase (ALT), and chloride ion (Cl- ) had significant positive correlation with age, while significant negative correlation with age was observed in total red blood cells (RBCs), lymphocytes, thrombocytes, alkaline phosphatase (ALP), CK, lactate dehydrogenase (LDH), and plasma iron. Compared to male penguins, females had lower mean corpuscular hemoglobin concentration (MCHC) and blood urea nitrogen (BUN) but higher calcium ion (Ca2+ ) values. As for age-gender interaction, significant positive correlation was shown in MCHC and K+ , and the reverse was true in H/L ratio.

APPLICATION OF 3-HYDROXYBUTYRATE MEASUREMENT AND PLASMA PROTEIN ELECTROPHORESIS IN THE DIAGNOSIS OF ASPERGILLOSIS IN AFRICAN PENGUINS ( SPHENISCUS DEMERSUS).

New alternative laboratory means are needed to improve the options for antemortem diagnosis of avian aspergillosis. In this study, 3-hydroxybutyrate was measured in plasma samples collected from a cohort of African penguins ( Spheniscus demersus) maintained under human care. Results were interpreted in combination with those of protein electrophoresis and compared with anti- Aspergillus antibody and galactomannan antigen detection. Overall, 3-hydroxybutyrate levels were found significantly increased in Aspergillus-diseased cases versus the control penguin group ( P = 0.002). Mean absolute concentration of ß-globulins was increased >20% in samples from infected birds, and α2-globublins were also found to be significantly increased versus clinically normal controls ( P < 0.001 and P = 0.001 respectively). Of note, the α2-globulins were also significantly increased versus penguins with inflammatory (non-aspergillosis) diseases ( P = 0.001). The specificity of 3-hydroxybutyrate, ß-globulins, and α2-globulins for aspergillosis was 78.6%, 79.6%, and 92.2%, respectively. Using these measures in tandem resulted in high specificity (>90%) and negative predictive value (≥80%). In contrast, anti- Aspergillus antibody and galactomannan antigen did not distinguish between infected cases and controls ( P > 0.05). This study demonstrates that basic testing in tandem with the new biomarker 3-hydroxybutyrate may provide reliable evidence for the diagnosis of aspergillosis in penguins.

PLASMA CONCENTRATIONS OF VITAMIN A1, B1, D3, AND E IN HUMBOLDT PENGUINS ( SPHENISCUS HUMBOLDTI) BEFORE AND AFTER DIETARY VITAMIN SUPPLEMENTATION OF THEIR FISH DIET.

In a practical feeding trial at Ouwehand Zoo, plasma concentrations of vitamin A1, calcidiol (D3), α-tocopherol (E), and B1 in 17 Humboldt penguins ( Spheniscus humboldti) were measured before and after supplementation to gain insight into the effect of supplementing these vitamins in animals being fed thawed frozen-fish diets. None of the penguins received vitamin supplements for at least 6 mo before the supplementation trial, which was conducted prior to their normal nesting and molting period. During the trial period, eight penguins received daily vitamin A1, D3, tocopheryl acetate, and B1 supplementation placed in their fish immediately prior to feeding and nine control penguins received no supplementation. Concentrations of vitamins A1, D3, α-tocopherol, and B1 were also measured in the thawed ready-to-feed fish. Concentrations of vitamins B1 and α-tocopherol were below the Association of Zoos and Aquariums (AZA) recommendations for penguin diets, while concentrations of vitamins A1 and D3 were far above AZA recommendations. At the start of the study and after 70 days of supplementation, plasma concentrations were determined for these vitamins. Vitamin B1 concentrations in plasma increased significantly ( P < 0.05) between Day 0 (mean 39.9 µg/L) and day 70 (mean 160.5 µg/L) in the supplemented group. Plasma vitamin D3 and α-tocopherol did not show a significant change. Vitamin A1 levels in the supplemented group decreased significantly from 1.65 mg/L on day 0 to 1.4 mg/L on day 70. In the control group no significant changes were observed. The results of the study support the necessity of supplementing vitamin B1 in penguins fed thawed frozen fish. Depletion of vitamin A and E concentrations in frozen food fish over time support recommendations to regularly measure vitamin concentrations in different batches of frozen fish.

PHARMACOKINETICS OF ORALLY ADMINISTERED VORICONAZOLE IN AFRICAN PENGUINS (SPHENISCUS DEMERSUS) AFTER SINGLE AND MULTIPLE DOSES.

Aspergillosis is a common respiratory fungal disease in African penguins ( Spheniscus demersus ) under managed care, and treatment failures with itraconazole due to drug resistance are increasingly common, leading to recent use of voriconazole. Empirical dosing with voriconazole based on other avian studies has resulted in adverse clinical drug effects in penguins. The objective of this study was to determine oral voriconazole pharmacokinetics (PK) in African penguins (n = 18). Single and once daily multiple oral doses of 5 mg/kg voriconazole were evaluated with a 4-mo washout period between trials. Plasma voriconazole concentrations were determined via high-performance liquid chromatography. Data was modeled using 3-compartamental population methodologies that supported first-order elimination. Observed mean peak concentration (1.89 µg/ml) after single dosing PK analysis was determined within the first hour following voriconazole administration. In the multiple-dose trial average plasma voriconazole concentrations were significantly higher on days 4 and 7 as compared with day 2. The mean estimates for volume of distribution (V/F) and clearance (Cl/F) for the multiple-dose study were 3.34 L and 0.18 L/hr, respectively. Monte Carlo simulations determined the median area under the curve (AUC0-24) at 84 hr was 37.7 µg·h/ml. As this assessment was comparable with the average AUC in humans receiving the recommended human oral dosage 200 mg b.i.d., it suggests that 5 mg/kg p.o. s.i.d. could be a safe and effective regimen in African penguins for treatment of aspergillosis. However, due to potential drug accumulation and subsequent toxicity, therapeutic drug monitoring with dosage adjustments is recommended to individualize dosing.

THE PASSAGE AND DURATION OF ANTIBODIES TO WEST NILE VIRUS IN HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI).

West Nile virus (genus Flavivirus) outbreaks and mortality events have been documented in both wild and captive avian species, including penguins. Serologic response to vaccination in avian species has varied and appears to be largely species dependent; however, Humboldt penguins ( Spheniscus humboldti ) previously showed excellent rates of seroconversion. The goal of this study was to determine virus neutralization titers of 17 Humboldt penguin hens and their subsequent eggs, chicks, or both following vaccination with a killed West Nile vaccine. Chicks were also vaccinated at 56, 70, and 84 days old. Titers were measured from 10-346 days prior to lay as well as serially in seven chicks. Data collected showed positive rank correlation between maternal titers and yolk titers (ρ = 0.90, P < 0.0001, n = 14) but no association between booster vaccination and yolk titers. All seven chicks had detectable antibody on days 14 and 28, and antibody levels had increased (relative to day 56) in 3 of 6 chicks (50%; 95% confidence interval 14-86%) by day 112. Further information is provided on a suggested vaccination schedule for Humboldt penguin chicks based on a time-dependent decline in maternal antibody titers. Cell-mediated immunity and experimental challenge following vaccination have not yet been investigated in this species.

The oxidative debt of fasting: evidence for short- to medium-term costs of advanced fasting in adult king penguins.

In response to prolonged periods of fasting, animals have evolved metabolic adaptations helping to mobilize body reserves and/or reduce metabolic rate to ensure a longer usage of reserves. However, those metabolic changes can be associated with higher exposure to oxidative stress, raising the question of how species that naturally fast during their life cycle avoid an accumulation of oxidative damage over time. King penguins repeatedly cope with fasting periods of up to several weeks. Here, we investigated how adult male penguins deal with oxidative stress after an experimentally induced moderate fasting period (PII) or an advanced fasting period (PIII). After fasting in captivity, birds were released to forage at sea. We measured plasmatic oxidative stress on the same individuals at the start and end of the fasting period and when they returned from foraging at sea. We found an increase in activity of the antioxidant enzyme superoxide dismutase along with fasting. However, PIII individuals showed higher oxidative damage at the end of the fast compared with PII individuals. When they returned from re-feeding at sea, all birds had recovered their initial body mass and exhibited low levels of oxidative damage. Notably, levels of oxidative damage after the foraging trip were correlated to the rate of mass gain at sea in PIII individuals but not in PII individuals. Altogether, our results suggest that fasting induces a transitory exposure to oxidative stress and that effort to recover in body mass after an advanced fasting period may be a neglected carryover cost of fasting.