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Humboldt penguins (Spheniscus humboldti) from the Barcelona zoo (n=9) were followed to assess their physiological stress status using conservative protocols. Corticosterone levels were measured in feathers and plasma as indicative of chronic and acute physiological stress, respectively. Other markers: B-esterases, potentially indicative of xenobiotic exposure were measured in plasma of these same individuals and reported for the first time in this species. The sensitivity to chemicals of environmental concern, employed as plastic additives, was assessed in vitro with plasma of this species using the inhibition of carboxylesterase (CE) and acetylcholinesterase enzymatic measurements. Among the tested additives, the organophosphorus flame retardants displayed the highest in vitro inhibitory potential on basal CE activity, suggesting their potential utility as biomarkers of this particular chemical class. Additionally, enzymatic measurements in plasma are determined for the first time in Humboldt penguin and can be regarded as baseline values for a potential field monitoring application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Only limited data on concentrations of trace elements in the blood of avian species have been published. This information can play an important role in the conservation of endangered species and their protection from environmental pollutants and can also be clinically relevant in managed individuals. Some elements are essential for the health of the animals in human care, but little is known about expected concentrations for some of these elements. Therefore, 21 elements (silver, arsenic, gold, barium, cadmium, cobalt, chromium, copper, iron, iodine, mercury, magnesium, manganese, molybdenum, nickel, platinum, antimony, selenium, thallium, vanadium, and zinc) were measured by inductively coupled plasma mass spectrometry in lithium-heparinized plasma samples from Humboldt penguins ( Spheniscus humboldti ) from a single zoological collection in Germany ( n = 39). The plasma concentrations of cadmium, nickel, platinum, antimony, and thallium were under the limit of detection in most of the analyzed samples. The results also showed that the females had significantly ( P ≤ 0.05) higher concentrations of silver, barium, cobalt, chromium, iron, mercury, magnesium, vanadium, and zinc in their plasma in comparison with males. A correlation between the element plasma concentration and the age of the individuals could not be found. The differences in plasma concentrations of individual element between males and females could be due to hormonal influences on the metabolism. Further studies in different seasons and other age groups are needed in the future to investigate environmental and host effects in more detail and to understand physiological mechanisms responsible for observed differences.

Antarctica is one of the most remote regions on the planet, where many species of penguins inhabit. One of these species is the Adélie penguin (Pygoscelis adeliae), which occupies a high trophic level. Blood is an important indicator of short-term exposure to mercury (Hg) in avian species, however there are few publications investigating methylmercury (MeHg) in Antarctic penguin blood. During the austral summer of 2016, thirty adult specimens of Adélie penguins were captured at three locations of the King George Island and the north-west Antarctic Peninsula. This study determined by atomic fluorescence spectroscopy, Hg levels in the blood of adult Adélie penguins. Concentrations of MeHg ranged from 22.0 to 323.3 (ng/g dw), while concentrations of total Hg (THg) ranged from 31.9 to 410.1 (ng/g dw). To our knowledge, this is the first study to report MeHg levels in the blood of adult Adélie penguins in these locations. The results showed that the highest Hg levels were found where human activities are more prevalent. Future studies are needed to determine the actual impact of these activities on local Hg contamination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 δ 15 N and δ 13 C isoscapes. Isotopic values were assigned to random positions inside their corresponding foraging area. Spatial analysis and data interpolation resulted in δ 15 N and δ 13 C 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 δ 15 N and from 0.07 to 0.3 for δ 13 C, similar to the error range of the mass spectrometer used for measuring isotope ratios. Predictive surfaces reflected the latitudinal trends, with δ 13 C and δ 15 N values increasing northwards. δ 13 C values showed a strong latitudinal gradient, while δ 15 N 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 δ 13 C isoscape showed a latitudinal gradient, consistent with patterns in other oceans. The δ 15 N 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., (© 2024 John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (T eye ) increased and minimum beak temperature (T beak ) 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 T eye and T beak ) 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 T eye and T beak 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., Competing Interests: Declaration of competing interest Antoine Stier was an Associate Editor of J Thermal Biol at the time of submitting the manuscript but took no part in the peer review and decision-making processes for this research article., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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, β-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 β-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.

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.

Dive capacities of air-breathing vertebrates are dictated by onboard O 2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O 2 transport. It has been hypothesized that increased hemoglobin (Hb)-O 2 affinity improves pulmonary O 2 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-O 2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O 2 affinity at low pH). Although an increased Hb-O 2 affinity reduces the gradient for O 2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O 2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O 2 affinity in combination with the augmented Bohr effect maximizes both O 2 extraction from the lungs and O 2 unloading from the blood, allowing penguins to fully utilize their onboard O 2 stores and maximize underwater foraging time., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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 (E 2 ) to support vitellogenesis and egg production, but E 2 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 E 2 -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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

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 β-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., (© 2020 Wiley Periodicals, Inc.)

Reproductive success of endangered Humboldt penguin (Spheniscus humboldti) colonies in Peru has been associated with nesting habitat type, presumably due to differences in environmental exposure and activity patterns that may affect energy demands and metabolism. Gas chromatography and mass spectrometry were used to determine serum concentrations of 19 saccharides from 30 Humboldt penguins nesting at Punta San Juan, Peru in order to evaluate differences in metabolic state between penguins nesting in a sheltered burrow or crevice (n = 17) and those in exposed surface nests (n = 13). Univariate and multivariate statistical analyses identified serum saccharides (arabinose, maltose, glucose-6-phosphate, and levoglucosenone in particular) that were nest-dimorphic with substantial differences between surface- and sheltered-nesting penguins. Four sugars (arabinose, xylose, fructose-6-phosphate, and sucrose) had ≥ 2-fold difference in concentration between nest types. Seven saccharides were in the top five subsets generated by discriminant analysis; four of these are simple sugars (D-glucopyranose, α ⇄ β; D-glucose; D-maltose; and D-mannose) and three are derivatives (glucose 6-phosphate, levoglucosenone, and N-acetylglucosamine). D-ribose had the highest information values (generated from weight-of-evidence values) followed by glucose 6-phosphate, levoglucosenone, and D-galactose. Sex was not a significant predictor of saccharide concentration. Levoglucosenone, which is a metabolite of the environmental contaminant levoglucosan, was significantly higher in surface-nesting penguins, reflecting a higher rate of exposure in non-sheltered penguins. Differences in the saccharide profiles of surface- and sheltered-nesting Humboldt penguins likely reflect increased metabolic requirements of surface-nesters at Punta San Juan. Conservation of appropriate sheltered-nesting habitat for penguins is essential for sustained reproductive success and colony health., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Analysis of the stable isotope ratios of carbon and nitrogen (δ 13 C and δ 15 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 (δ 13 C and δ 15 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 δ 13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ 15 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 δ 13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ 15 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., (© 2019 John Wiley & Sons, Ltd.)

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.

Vitamin A comprises vitamin A 1 and vitamin A 2 ; vitamin A 1 is retinol and its fatty-acid esters and vitamin A 2 is 3,4-didehydroretinol and its fatty-acid esters. Although vitamin A 1 is generally recognized as the major vitamin A, vitamin A 2 is found in some birds and mammals that eat fish containing vitamin A 2 . Plasma concentration of retinyl esters, but not retinol, is known to increase postprandially in humans. The objectives of this study were to confirm the presence of vitamin A 2 in fish fed to penguins, and in penguin plasma, and the postprandial changes in vitamin A concentration in penguin plasma. Blood was collected from six male African penguins (Spheniscus demersus) before and after feeding on jack mackerels (Trachurus japonicus) along with a vitamin premix containing vitamin A 1 . Vitamin A 1 concentration in fish was much higher than the requirement, and was 5-fold higher than the vitamin A 2 concentration. Vitamin A 2 was present in plasma but its concentration was at least 100-fold below that of plasma retinol, suggesting that vitamin A 2 is much less bioavailable than vitamin A 1 in penguins. Plasma retinol and retinyl palmitate concentrations were found to be stable after the meal. Plasma retinol concentration is suggested to be homeostatically controlled in penguins against the rapid flow of vitamin A 1 after meal. The absorbed vitamin A 1 is thought to be transported to the liver via the portal vein for storage in penguins, resulting in stable retinyl palmitate concentration in plasma after meal.

We monitored the post-release survival of little penguins (Eudyptula minor) rehabilitated after the 2011 C/V Rena oil spill in New Zealand to assess the effectiveness of the rehabilitation process. Surveys were conducted over a 23-month period after the spill to assess whether survival differed between rehabilitated and control penguins. Survival probabilities from mark-recapture analyses were lower for both oiled and control penguins in the first six months of the study (monthly probability 0.92) but increased and remained high thereafter (monthly probabilities 0.97-1.0). Importantly, survival did not differ significantly between oiled and control birds throughout the study. Post-release survival of rehabilitated birds was not influenced by the degree of oiling, body mass (at admission or release), blood parameters (admission packed cell volume, total protein or blood glucose) or the duration of captivity. Rehabilitation therefore appeared to successfully reverse the negative effects of oiling on the post-release survival of treated penguins., (Copyright © 2019. Published by Elsevier Ltd.)

Whole blood, serum, and feather samples from 29 Humboldt Penguins ( Spheniscus humboldti) at the Punta San Juan Marine Protected Area, Peru, were analyzed for 55 toxic and essential elements by using inductively coupled plasma mass spectrometry. Mercury (Hg) was analyzed by cold vapor atomic fluorescence. Maximum Hg concentrations in serum (0.0056 mg/g), whole blood (0.297 mg/kg), and feathers (1.8 mg/kg dry weight) were at levels generally not considered to cause health impairment. Of the elements analyzed, only eight (aluminum, calcium, iron, Hg, potassium, magnesium, sodium, and zinc) were detected in serum. These elements, plus selenium and titanium, were also quantifiable in whole blood. Feather analysis detected quantifiable values for the elements found in serum, plus arsenic, boron, barium, copper, manganese, and titanium. Results indicate this important breeding population of endangered penguins did not appear to be exposed to environmental elemental contaminants at levels detrimental to health and reproductive success. However, identification of measurable concentrations of toxic elements at low levels underscores the need for continued environmental monitoring, particularly in the face of expanding regional human populations and industrial growth. These results provide important reference data for temporospatial monitoring of this and other penguin populations.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (© 2018 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.)

The main motivation for this study was to determine the occurrence of Toxoplasma gondii, a cosmopolitan widespread zoonotic parasite distribution that can infect a wide variety of mammals and birds, in Magellanic penguins (Spheniscus magellanicus) in Brazil. In recent decades there has been a significant increase in the number of penguins originating from Argentinian and Chilean Patagonia, where these birds are born, that arrive on the Brazilian coast, where many of them are stranded and rescued. Tissue samples were collected from 330 individuals surveyed from 2012-2015 at the Institute for Marine Animal Research and Rehabilitation (IPRAM) located in Cariacica, state of Espirito Santo, Brazil. Serum were collected from 145 animals surveyed in 2015 for the detection of anti-T. gondii antibodies using the Modified Agglutination Test (MAT ≥20) and 18 birds were positive, with titers of 20 (7 birds), 40 (9 birds) and 80 (2 birds). Mouse bioassay for the isolation of T. gondii was performed using tissues from 54 penguins that were also surveyed in 2015, but no isolates were obtained. DNA from tissue samples of 330 individuals was PCR amplified and sequenced to detect tissue cyst forming coccidians by using pan sarcocystids-directed primers (based on 18S rDNA). These samples were from animals surveyed in 2015 and from frozen stocked tissues from animals surveyed in the years 2012 and 2013. The positives were PCR amplified and sequenced with genus Sarcocystis-specific primers (based on internal transcribed spacer 1, RNA polymerase beta subunit coding gene, and cytochrome B coding gene) and with Sarcocystis falcatula/Sarcocystis neurona- specific primers (based on surface antigens SAG2, SAG3 and SAG4). Sixteen (3.0%) of pectoral muscle samples were positive by all the seven molecular markers and all the samples were identical to each other. Organisms close related to Sarcocystis falcatula were confirmed in all cases. This is the first report on molecular detection of infection by S. falcatula-related organisms and the first report of seropositivity for T. gondii in free-living Magellanic penguins in Brazil. Felids and didephid opossums are definitive hosts of T. gondii and S. falcatula, respectively. Where the penguins acquire the infective forms of the parasites shed by the terrestrial mammals remains to be elucidated., Competing Interests: The authors have declared that no competing interests exist.

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 (β-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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2018 Blackwell Verlag GmbH.)

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 β-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, β-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.

In a practical feeding trial at Ouwehand Zoo, plasma concentrations of vitamin A 1 , calcidiol (D 3 ), α-tocopherol (E), and B 1 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 A 1 , D 3 , tocopheryl acetate, and B 1 supplementation placed in their fish immediately prior to feeding and nine control penguins received no supplementation. Concentrations of vitamins A 1 , D 3 , α-tocopherol, and B 1 were also measured in the thawed ready-to-feed fish. Concentrations of vitamins B 1 and α-tocopherol were below the Association of Zoos and Aquariums (AZA) recommendations for penguin diets, while concentrations of vitamins A 1 and D 3 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 B 1 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 D 3 and α-tocopherol did not show a significant change. Vitamin A 1 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 B 1 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.

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 (Ca 2+ ) values. As for age-gender interaction, significant positive correlation was shown in MCHC and K + , and the reverse was true in H/L ratio., (© 2018 Wiley Periodicals, Inc.)

Persistent organic pollutants were assessed in Humboldt Penguins ( Spheniscus humboldti) from the Punta San Juan Marine Protected Area, Peru, in the austral winter of 2009. Plasma samples from 29 penguins were evaluated for 31 polychlorinated biphenyl (PCB) congeners and 11 organochlorine pesticides (OCPs) by using gas chromatography coupled to an ion trap mass spectrometer and for 15 polybrominated diphenyl ether (PBDE) congeners by using gas chromatography coupled with high-resolution mass spectrometry. The detection rate for PCBs in the samples was 69%, with congeners 105, 118, 180, and 153 most commonly detected. The maximum ΣPCB concentration was 25 ng/g. The detection rate for DDT, DDD, and/or DDE was higher than for other OCP residues (90%; maximum concentration=10 ng/g). The detection rate for PBDEs was 86%, but most concentrations were low (maximum ΣPBDE concentration=3.81 ng/g). This crucial breeding population of S. humboldti was not exposed to contaminants at levels detrimental to health and reproductive success; however, the identified concentrations of legacy and recently emerged toxicants underscore the need for temporal monitoring and diligence to protect this endangered species in the face of regional human population and industrial growth. These results also provide key reference values for spatial comparisons throughout the range of this species.

Although many studies have investigated organohalogenated contaminants (OHCs) in yolk, little is known about the mechanisms and timing of transfer of OHCs from the female to the egg. Vitellogenin, a yolk precursor, has been suggested to play a role in this transport. We here report for the first time the temporal changes in OHC and an index of vitellogenin concentrations in female plasma from the pre-laying period to clutch completion in free-living birds: the southern rockhopper penguin (Eudyptes chrysocome chrysocome) breeding in the Falkland/Malvinas Islands. In addition, OHC concentrations in the corresponding clutches were analysed. OHC concentrations in female plasma and in the yolk of both the first (A-) and the second (B-)eggs followed a similar pattern, with hexachlorobenzene (HCB) > Σpolychlorinated biphenyls (PCBs) > Σdichlorodiphenyltrichloroethanes (DDTs) > Σmethoxylated polybrominated diphenyl ethers (MeO-PBDEs) > Σchlordanes (CHLs) > Σpolybrominated diphenyl ethers (PBDEs) ≈ Σhexachlorocyclohexanes (HCHs). The higher concentrations of MeO-PBDEs compared to PBDEs indicate a diet containing naturally-produced MeO-PBDEs. All OHC compounds except for PBDEs increased from the pre-laying period to A-egg laying and subsequently declined from A-egg laying to B-egg laying, and female plasma vitellogenin showed the same pattern. For ΣPCBs and ΣMeO-PBDEs, we found positive correlations between female plasma during A-egg laying and both eggs, and for HCB between female plasma and A-eggs only. During pre-laying, only ΣMeO-PBDEs correlated between both eggs and female plasma, and no correlations between OHC concentrations in eggs and female plasma were found during B-egg laying, highlighting that maternal transfer of OHCs is time- and compound-specific. Finally, female vitellogenin concentrations did not significantly correlate with any OHC compounds in either female plasma or eggs, and our results therefore did not confirm the suggested role of vitellogenin in the maternal transfer of OHC molecules into their eggs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 (AUC 0-24 ) 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.

Piscivorous species like the Little Penguin (Eudyptula minor) are particularly at risk of being negatively impacted by pollution due to their heightened exposure through aquatic food chains. Therefore, determining the concentration of heavy metals in the fish prey of seabirds is an essential component of assessing such risk. In this study, we report on arsenic, cadmium, mercury, lead and selenium concentrations in three fish species, which are known to comprise a substantial part of the diet of Little Penguins at the urban colony of St Kilda, Melbourne, Australia. Metal concentrations in the fish sampled were generally within the expected limits, however, arsenic and mercury were higher than reported elsewhere. Anchovy (Engraulis australis) and sandy sprat (Hyperlophus vittatus) contained higher Hg concentrations than pilchard (Sardinops sagax), while sandy sprat and pilchard contained more selenium. We present these findings together with metal concentrations in Little Penguin blood and faeces, sampled within weeks of the fish collection. Mercury concentrations were highest in the blood, while faeces and fish prey species contained similar concentrations of arsenic and lead, suggesting faeces as a primary route of detoxification for these elements. We also investigated paired blood - faecal samples and found a correlation for selenium only. Preliminary data from stable isotope ratios in penguin blood indicate that changes in penguin blood mercury concentrations cannot be explained by trophic changes in their diet alone, suggesting a variation of bioavailable Hg within this semi-enclosed bay., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

While safety of fasting therapy is debated in humans, extended fasting occurs routinely and safely in wild animals. To do so, food deprived animals like breeding penguins anticipate the critical limit of fasting by resuming feeding. To date, however, no molecular indices of the physiological state that links spontaneous refeeding behaviour with fasting limits had been identified. Blood proteomics and physiological data reveal here that fasting-induced body protein depletion is not unsafe "per se". Indeed, incubating penguins only abandon their chick/egg to refeed when this state is associated with metabolic defects in glucose homeostasis/fatty acid utilization, insulin production and action, and possible renal dysfunctions. Our data illustrate how the field investigation of "exotic" models can be a unique source of information, with possible biomedical interest.

Whether oiled wildlife should be rehabilitated during an oil spill is internationally debated. Research on little penguins (LP, Eudyptula minor) rehabilitated and released back into a cleaned environment after the New Zealand C/V Rena grounding oil spill in 2011 found the rehabilitation process was effective at treating and reversing the negative effects of oil-contamination on penguin post-release survival, productivity and diving behaviour. Here we investigated the acute corticosterone stress response of LPs to determine if responses of rehabilitated birds differed from those of "control" birds. Corticosterone responses of LPs two years after an oil spill did not differ between rehabilitated and non-rehabilitated penguins. These results show that the rehabilitation process for LP did not affect their long-term physiological responses to humans. This indicates that wildlife can be rehabilitated and returned to the wild with similar human tolerance levels to non-rehabilitated birds and an absence of habituation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Seabirds are ideal model organisms to track mercury (Hg) through marine food webs as they are long-lived, broadly distributed, and are susceptible to biomagnification due to foraging at relatively high trophic levels. However, using these species as biomonitors requires a solid understanding of the degree of species, sexual and age-specific variation in foraging behaviors which act to mediate their dietary exposure to Hg. We combined stomach content analysis along with Hg and stable isotope analyses of blood, feathers and common prey items to help explain inter and intra-specific patterns of dietary Hg exposure across three sympatric Pygoscelis penguin species commonly used as biomonitors of Hg availability in the Antarctic marine ecosystem. We found that penguin tissue Hg concentrations differed across species, between adults and juveniles, but not between sexes. While all three penguins species diets were dominated by Antarctic krill (Euphausia superba) and to a lesser extent fish, stable isotope based proxies of relative trophic level and krill consumption could not by itself sufficiently explain the observed patterns of inter and intra-specific variation in Hg. However, integrating isotopic approaches with stomach content analysis allowed us to identify the relatively higher risk of Hg exposure for penguins foraging on mesopelagic prey relative to congeners targeting epipelagic or benthic prey species. When possible, future seabird biomonitoring studies should seek to combine isotopic approaches with other, independent measures of foraging behavior to better account for the confounding effects of inter and intra-specific variation on dietary Hg exposure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2016. Published by The Company of Biologists Ltd.)

Little Penguins (Eudyptula minor) are high-trophic coastal feeders and are effective indicators of bioavailable pollutants in their foraging zones. Here, we present concentrations of metals and metalloids in blood of 157 Little Penguins, collected over three years and during three distinct seasons (breeding, moulting and non-breeding) at two locations: the urban St Kilda colony and the semi-rural colony at Phillip Island, Victoria, Australia. Penguin metal concentrations were foremostly influenced by location (St Kilda>Phillip Island for non-essential elements) and differed among years and seasons at both locations, reflecting differences in seasonal metal bioaccumulation or seasonal exposure through prey. Mean blood mercury concentrations showed an increasing annual trend and a negative correlation with flipper length at St Kilda. Notably, this study is the first to report on blood metal concentrations during the different stages of moult, showing the mechanism of non-essential metal mobilisation and detoxification., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rationale: The use of stable isotopes for ecological studies has increased exponentially in recent years. Isotopic trophic studies are based on the assumption that animals are what they eat plus a discrimination factor. The discrimination factor is affected by many variables and can be determined empirically. The Magellanic penguin is a highly abundant marine bird that plays a key role in the southern oceans. This study provides the first estimation of the Magellanic penguin blood discrimination factor for (13) C and (15) N., Methods: A two and a half month feeding experiment was performed, in which ten captive penguins were fed their main natural prey (anchovy Engraulis anchoita). The discrimination factors were estimated by comparing anchovy δ(13) C and δ(15) N values (obtained with isotope ratio mass spectrometry using lipid-extracted and bulk anchovy muscle) with penguin blood δ(13) C and δ(15) N values., Results: Penguin blood was shown to be enriched, compared with anchovies, for (13) C and (15) N. No changes were observed in the stable isotope ratios of anchovies and discrimination factors during the experiment. The overall discrimination factors were 0.93 ± 0.12 (bulk) and 0.41 ± 0.12 (lipid-free) for (13) C; and 2.81 ± 0.17 (bulk) and 2.31 ± 0.17 (lipid-free) for (15) N., Conclusions: Having an accurate discrimination factor for the studied species is key in any trophic or food web isotopic study. Comparisons of estimated diet-to-blood discrimination factors with published values of aquatic piscivore birds showed that the (13) C discrimination factor is particularly variable, and therefore ecologists should be cautious when using a surrogate value from other species. In this study, the Magellanic penguin discrimination factor of a tissue that does not require euthanasia was obtained, a fundamental input for trophic isotopic modeling of the species. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Research was performed to increase our understanding of male Magellanic penguin (Spheniscus magellanicus) reproductive biology and to develop artificial insemination (AI) technology to assist with maintaining the species' genetic diversity. Seminal traits were characterized from seven males with noncontaminated ejaculates (n = 123) displaying high in vitro motion parameters, membrane integrity, and morphology. Seven females were maintained in nest sites that permitted visual, auditory, and tactile contact with their paired male but not copulation for 18.3 ± 2.4 days before egg lay. After cloacal AI (2.6 ± 0.4 inseminations/female) with semen chilled for up to 20.5 hr at 5°C, all females produced one to two fertile eggs, with the first oviposition occurring within 7 days of plasma progesterone concentrations exceeding 0.8 ng/ml. Overall fertility was 91.7%, hatchability was 63.6%, and genetic analyses confirmed that all embryos and hatchlings were sired by AI males. The heterospermic AI design demonstrated that eggs were fertilized by spermatozoa chilled for 1.5-19.8 hr before AI and were laid 4.5-11.5 days post AI. These results contribute new data on Magellanic penguin sperm biology and demonstrate that high fertility rates after AI of chilled semen can be achieved with females remaining in proximity to their paired mate.

There are few publications on the clinical haematology and biochemistry of African penguins (Spheniscus demersus) and these are based on captive populations. Baseline haematology and serum biochemistry parameters were analysed from 108 blood samples from wild, adult African penguins. Samples were collected from the breeding range of the African penguin in South Africa and the results were compared between breeding region and sex. The haematological parameters that were measured were: haematocrit, haemoglobin, red cell count and white cell count. The biochemical parameters that were measured were: sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, cholesterol, serum glucose, uric acid, bile acid, total serum protein, albumin, aspartate transaminase and creatine kinase. All samples were serologically negative for selected avian diseases and no blood parasites were detected. No haemolysis was present in any of the analysed samples. Male African penguins were larger and heavier than females, with higher haematocrit, haemoglobin and red cell count values, but lower calcium and phosphate values. African penguins in the Eastern Cape were heavier than those in the Western Cape, with lower white cell count and globulin values and a higher albumin/globulin ratio, possibly indicating that birds are in a poorer condition in the Western Cape. Results were also compared between multiple penguin species and with African penguins in captivity. These values for healthy, wild, adult penguins can be used for future health and disease assessments.

Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens.

A morphologic and molecular epidemiologic investigation was conducted on a captive African black-footed penguin (Spheniscus demersus) colony with a history of Plasmodium infections at La Palmyre Zoo (France). Each penguin received 12.5 mg of pyrimethamine twice a week as a prophylaxis every year from April to November. Although Plasmodium parasites were not detected in blood smears and tissues collected from the penguins, various blood parasites were recorded in blood smears from wild Eurasian magpies (Pica pica) and carrion crows (Corvus corone) sampled at the same time in the study area. These parasites consisted of several Plasmodium spp. (P. lenoblei, P. dorsti, P bioccai, P. relictum, P. dherteae, P. beaucournui, P. maior, P. tranieri, and P. snounoui), Parahaemoproteus spp., Trypanosoma spp., and Leucocytozoon spp. On the other hand, nested polymerase chain reaction enabled detection of Plasmodium DNA in 28/44 (64%) penguins, 15/25 (60%) magpies, and 4/9 (44%) crows. Sequencing and phylogenetic analyses indicated that the parasite DNA amplified from the penguins, magpies, and crows were similar. Magpies and crows could therefore act as a reservoir for penguin Plasmodium infections, which may be more prevalent than previously thought. Morphologic characterization of the Plasmodium spp. detected in the penguins, as well as further biological and epidemiologic studies, are needed to fully understand the transmission of Plasmodium parasites to captive penguins.

Analgesia is an important part of veterinary medicine, but until recently there have been limited studies on analgesic drugs in avian species. Tramadol represents an orally administered opioid drug that has shown analgesic potential in numerous species, including mammals, birds, and reptiles. The objective of this study was to determine the pharmacokinetic parameters of tramadol and its primary metabolite, O-desmethyltramadol (M1), after oral administration of tramadol hydrochloride (HCl) in African penguins (Spheniscus demersus). A dose of 10 mg/kg of tramadol HCl was administered orally to 15 birds, and blood was collected at various time points from 0 to 36 hr. Tramadol and M1 concentrations were determined and were consistent with therapeutic concentrations in humans through 12 hr in 9/15 birds for tramadol and 36 hr in 14/15 birds for M1. Based on these findings and a comparison with other avian studies, an oral dose of 10 mg/kg of tramadol once daily appears to be a promising analgesic option for African penguins.

Current research on seabirds suggests a key role of hormones in the trade-off between self-maintenance and parental investment through their influence on foraging decisions during the breeding period. Although prolactin is known to have major effects on parental care, its role in foraging behavior has rarely been investigated in seabirds to date. The aim of this study was to assess the influence of an experimental decrease in prolactin levels on foraging decisions and its consequences on breeding success in free-living seabirds. To achieve this, we implanted bromocriptine (an inhibitor of prolactin secretion) in male Adélie penguins (Pygoscelis adeliae), monitored their foraging behavior using time-depth recorders over several trips, and recorded their reproductive output. On average 8±0.5days after implantation, we showed that bromocriptine administration led to an efficient decrease in prolactin levels. However, no differences were seen in foraging trip durations between bromocriptine-implanted birds and controls. Moreover, the time spent diving and the number of dives performed per trip were similar in both groups. By contrast, all diving parameters (including diving efficiency) were negatively affected by the treatment during the first at-sea trip following the treatment. Finally, the treatment did not affect adult body condition or chick growth and survival. Our study highlights the short-term negative effect of low prolactin levels on diving effort, but indicates that a short-term and/or low-magnitude decrease in prolactin levels alone is not sufficient to modify consistently the body maintenance or the parental investment of Adélie penguins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

During the cold austral winter, king penguin chicks are infrequently fed by their parents and thus experience severe nutritional deprivation under harsh environmental conditions. These energetic constraints lead to a range of energy sparing mechanisms balanced by the maintenance of efficient thermogenic processes. The present work investigated whether the high thermogenic capacities exhibited by winter-acclimatized king penguin chicks could be related to an increase in lipid substrate supply and oxidation in skeletal muscle, the main site of thermogenesis in birds. To test this hypothesis, we examined i) the effect of an experimental rise in plasma triglyceride on the whole metabolic rate in winter-acclimatized (WA) and de-acclimatized king penguin chicks kept at thermoneutrality (TN), and ii) investigated the fuel preference of muscle mitochondria. In vivo, a perfusion of a lipid emulsion induced a small 10% increase of metabolic rate in WA chicks but not in TN group. In vitro, the oxidation rate of muscle mitochondria respiring on lipid-derived substrate was +40% higher in WA chicks than in TN, while no differences were found between groups when mitochondria oxidized carbohydrate-derived substrate or succinate. Despite an enhanced fuel selection towards lipid oxidation in skeletal muscle, a rise of circulating lipids per se was not sufficient to fully unravel the thermogenic capacity of winter-acclimatized king penguin chicks., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pharmacokinetics of a single dose of enrofloxacin administered orally, both pilled and in fish, and i.v. to African penguins (Spheniscus demersus) at 15 mg/kg were determined. Plasma concentrations of enrofloxacin and its metabolite ciprofloxacin were measured via high-pressure liquid chromatography with mass spectrometry. An i.v. administration of enrofloxacin resulted in an extrapolated mean plasma concentration of 7.86 microg/ml at time zero. Plasma volume of distribution for i.v. administration was 3.00 L/kg, with a mean elimination half-life of 13.67 hr and a mean total body clearance rate of 3.03 ml/min/kg. Oral administration of enrofloxacin achieved a mean maximum plasma concentration of4.38 microg/ml at 4.8 hr after administration when pilled, whereas mean maximum plasma concentration was 4.77 microg/ml at 1.59 hr after administration when given in fish. Mean terminal elimination half-life was 13.79 hr pilled and 11.93 hr when given in fish. Low concentrations of ciprofloxacin were detected after both oral and i.v. enrofloxacin administration. Enrofloxacin administered to African penguins at 15 mg/kg p.o.q. 24 hr, whether in fish or pilled, is expected to achieve the surrogate markers of efficacy for bacteria with a minimum inhibitory concentration of 0.5 microg/ml or less; however, clinical studies are needed to determine efficacy.

Ecological niche theory predicts segregation mechanisms that mitigate potential competition between closely related organisms. However, little is known outside the breeding season, when central-place foraging animals may move on larger scales. This study tested for segregation mechanisms within the same 2007 inter-breeding period on three neighboring populations of avian predators from the southern Indian Ocean: Eastern Rockhopper Penguins Eudyptes filholi from Crozet and Kerguelen and Northern Rockhopper Penguins E. moseleyi from Amsterdam. Using state-of-the-art geolocation tracking and stable isotope analysis techniques, we quantified and compared the ecological niches in time, space, and diet. The three populations showed large-scale movements over deep oceanic waters near the Subantarctic Front, with generally little individual variation. The two neighboring populations of Eastern Rockhopper Penguins showed strikingly distinct distribution in space, while foraging in similar habitats and at the same trophic level (crustacean-eaters). In contrast, Northern Rockhoppers showed marked spatial overlap with birds of the sibling Eastern species, but their temporal delay of two months enabled them to effectively avoid significant overlap. Our results highlight parsimonious mechanisms of resource partitioning operating at the population level that may explain how animals from neighboring localities can coexist during the nonbreeding period.

The purpose of this study was to examine the validity of albumin determinations in penguin plasma by the bromcresol green (BCG) method and the gold standard of protein electrophoresis (EPH). Plasma from 96 clinically normal and abnormal penguins (Spheniscus species) was analyzed. The 2 methods did not yield equivalent results. The BCG method underestimated the albumin level in samples from normal patients (indicated by a normal albumin:globulin ratio) and overestimated the albumin level in samples from clinically abnormal penguins (indicated by a decreased albumin:globulin ratio). After EPH of plasma samples from clinically abnormal penguins samples was performed to separate albumin and globulin fractions, the globulins exhibited marked binding to the BCG dye. There were no significant differences between the variable reaction of paired serum and plasma samples when using the BCG method. These results demonstrated marked differences in the determination of albumin levels when using the BCG method and protein EPH. They further demonstrated that the BCG method can provide erroneous results, which have the potential to significantly impact clinical diagnosis and treatment. This study confirmed findings from previous studies in other avian species that the BCG method yields unreliable results in avian species. It is our conclusion that the BCG method, commonly found on automated analyzers in commercial laboratories and on point-of-care analyzers, should not be used to determine albumin concentration in avian samples.

The immune system is essential for health and survival of vertebrates, yet still little is known about the ontogeny of the immune system in wild birds. The southern rockhopper penguin (Eudyptes chrysocome chrysocome) is a semi-altricial seabird with a long developmental period and reversed hatching asynchrony, favouring the survival of B-chicks. We compared leucocyte counts and baseline corticosterone levels of southern rockhopper penguin chicks under different preconditions such as sex and origin from an A- or B-egg from 4 to 51 days of age. We conducted an experiment to compare leucocyte profiles and baseline corticosterone levels in A- and B-chicks in single-egg clutches as well as in B-chicks from normal two egg-clutches (one A- and one B-egg). None of these treatments influenced leucocyte counts or corticosterone levels, indicating a similar investment in the immune system. Our main finding was an increase of leucocytes/10,000 erythrocytes with age, which was mainly caused by an increase in lymphocyte numbers. This suggests differential investment into acquired immunity at this stage of development. As the granulocyte/lymphocyte (G/L) ratio did not change with age or body condition, G/L ratios seem not to reflect stress caused by poor provisioning of penguin chicks. This was also reinforced by the decrease of plasma corticosterone levels with age. Body condition was negatively correlated with monocyte numbers, suggesting a poorer health status of penguin chicks in poorer body condition. Yet, there was no link between body condition and other leucocyte parameters, indicating that chicks in a good body condition did not additionally invest into their immune system.

According to life-history theory, long-lived birds should favor their survival over the current reproductive attempt, when breeding becomes too costly. In seabirds, incubation is often associated with spontaneous long-term fasting. Below a threshold in body reserves, hormonal and metabolic shift characteristics of a switch from lipid to protein utilization (phase III, PIII) occur. These metabolic changes are paralleled by nest abandonment and stimulation of refeeding behavior. Parental behavior is then under control of two hormones with opposite effects: corticosterone (CORT) and prolactin which stimulate foraging and incubation behavior, respectively. The aim of this study was to determine the respective role of these two hormones in nest abandonment by Adélie penguins. To this end, plasma hormone levels were measured before egg-laying and at departure from the colony (i.e. when birds were relieved by their partner or abandoned their nest), and related to nutritional state and incubation success. We found that males abandoning their nest in PIII presented high CORT levels and low prolactin levels. Interestingly, males which presented high plasma levels of prolactin in PIII did not abandon. We show that although CORT is the first hormone to be affected by prolonged energy constraints, the combined effects of high CORT and low prolactin levels are necessary for parents to favor self-maintenance and abandon the nest. We provide insights into time-course changes of the endocrine profile as PIII proceeds and report that reaching proteolytic late fasting is not sufficient to induce nest abandonment in a long-lived bird., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Unsuccessful attempts to identify the leptin gene in birds are well documented, despite the characterization of its receptor (LEPR). Since leptin and LEPR have poor sequence conservation among vertebrates, we speculated that a functional assay should represent the best way to detect leptin in birds. Using a leptin bioassay that is based on activation of the chicken LEPR in cultured cells, blood samples from wild birds with extreme seasonal variation in voluntary food intake and fat deposition (Adélie penguins and bar-tailed godwits) were tested for leptin activity. In these experiments, blood samples collected during the pre-incubation and the chick-rearing periods of Adélie penguins, and during the migratory flight and refueling stages of bar-tailed godwits, were found to contain no detectable leptin activity, while the sensitivity of the assay to activation by human blood samples from donor subjects representing a variety of body mass indices and fat contents was clearly demonstrated. These results suggest that in birds, an alternative control mechanism to that of mammals operates in the communication between the body fat tissues and the central control on energy homeostasis.