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Maury, M., Murphy, K., Kumar, S., Mauerer, A., & Lee, G. (2005). Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G. Eur. J. Pharm. Biopharm., 59(2), 251–261.
Abstract: An immunoglobulin G (IgG) was spray-dried on a Büchi 190 laboratory spray-dryer at inlet and outlet air temperatures of 130 and 190°C, respectively. The IgG solution contains initially 115mg/ml IgG plus 50mg/ml sorbitol. After dialysis, at least 80% of low molecular weight component was removed. After spray-drying the dialyzed IgG and immediate redissolution of the powder, an increase in aggregates from 1 to 17% occurred. A major shift towards increase β-sheet structure was detected in the spray-dried solid, which, however, reverted to native structure on redissolution of the powder. A correlation between aggregation determined by size exclusion chromatography and alterations in secondary structure determined by Fourier transformation infra-red spectroscopy could not therefore be established. On spray-drying a non-dialyzed, sorbitol-containing IgG only some 0.7% aggregates were formed. The sorbitol is therefore evidently able to stabilize partially the IgG during the process of spray-drying. Addition of trehalose to the liquid feed produced quantitatively the same stabilizing action on the IgG during spray-drying as did the sorbitol. This finding again points towards a water replacement stabilization mechanism. The IgG spray-dried powder prepared from the dialyzed liquid feed showed continued substantial aggregation on dry storage at 25°C. This was substantially less in the non-dialyzed, sorbitol-containing spray-dried powder. Addition of trehalose to both dialyzed and non-dialyzed system produced substantial improvement in storage stability and reduction in aggregate formation in storage. The quantitative stabilizing effect of the trehalose was only slightly higher than that of the sorbitol. Taken together, these results indicate that both the sorbitol and trehalose stabilize the IgG primarily by a water replacement mechanism rather than by glassy immobilization. The relevance of this work is its questioning of the importance of the usually considered dominance of glassy stabilization of protein in dried systems of high glass transition temperature, such as trehalose. The low glass transition temperature sorbitol produces almost equal process and storage stability in this case.
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Palme, R. (2019). Non-invasive measurement of glucocorticoids: Advances and problems. Physiol. Behav., 199, 229–243.
Abstract: Glucocorticoids (GCs; i.e. cortisol/corticosterone) are a central component of the stress response and thus their measurement is frequently used to evaluate the impact of stressful situations. Their metabolites from faeces of various animal species are more and more taken as a non-invasive aid to assess GC release and thus adrenocortical activity. The current literature review includes an extensive collection (1327 papers) and evaluation (see also Supplementary Tables) of the literature on faecal cortisol/corticosterone metabolite (FCM) analysis published to date. It aims at giving reference for researchers interested in implementing FCM analysis into their study or seeking to improve such methods by providing background knowledge on GC metabolism and excretion, conveying insights into methodological issues and stating caveats of FCM analysis and by highlighting prerequisites for and some examples of a successful application of such methods. Collecting faecal samples and analysing FCMs may appear simple and straightforward, but researchers have to select and apply methods correctly. They also need to be aware of the many pitfalls and potentially confounding factors and, last but not least, have to carefully interpret results. Applied properly, measurement of FCMs is a powerful non-invasive tool in a variety of research areas, such as (stress) biology, ethology, ecology, animal conservation and welfare, but also biomedicine.
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Griebenow, K., & Klibanov, A. M. (1995). Lyophilization-induced reversible changes in the secondary structure of proteins. Proc Natl Acad Sci USA, 92(24), 10969–10976.
Abstract: Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.
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Palme, R., Touma, C., Arias, N., Dominchin, M.N., & Lepschy, M. (2013). Steroid extraction: Get the best out of faecal samples. Wien Tierärztl Monat – Vet Med Austria, 100, 238–246.
Abstract: Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. The extraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artifi- cial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids. We recommend extracting faecal steroids by simply suspending the faeces in a high percentage of a primary alcohol (for glucocorticoid metabolites 80% aqueous methanol proved best suited for virtually all mammalian species tested so far). Not only does the procedure significantly increase the total amount of recovered radioactivity, it also increases the percentage of unconjugated metabolites, which are more likely to be recognized by the antibodies used in various immunoassays. The advantages of this extraction procedure are clear: it is very easy to use (no evaporation step is needed), it yields high recoveries and variation based on the extraction procedure is reduced to a minimum.
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Gholib, G., Heistermann, M., Agil, M., Supriatna, I., Purwantara, B., Nugraha, T. P., et al. (2018). Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques. Primates, 59(3), 281–292.
Abstract: Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.
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Fisher, J., & Hinde, R. A. (1994). The opening of milk bottles by birds. British Birds, (42), 347–357.
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Kuczaj, S. A., Makecha, R., Trone, M., Paulos, R. D., & Ramos, J. A. (2006). Role of Peers in Cultural Innovation and Cultural Transmission: Evidence from the Play of Dolphin Calves. Int. J. Comp. Psychol, 19(2), 223–240.
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Lonsdorf, E. V. (2005). Sex differences in the development of termite-fishing skills in the wild chimpanzees, Pan troglodytes schweinfurthii, of Gombe National Park, Tanzania. Anim. Behav., 70(3), 673–683.
Abstract: By the age of 5.5 years, all of the young chimpanzees of Gombe National Park have acquired a skill known as 'termite fishing'. Termite fishing involves inserting a flexible tool made from vegetation into a termite mound and extracting the termites that attack and cling to the tool. Although tool use is a well-known phenomenon in chimpanzees, little is known about how such skills develop in the wild. Prior studies have found adult sex differences in frequency, duration and efficiency of tool-using tasks, with females scoring higher on all measures. To investigate whether these sex differences occurred in youngsters, I performed a 4-year longitudinal field study during which I observed and videotaped young chimpanzees' development of the termite-fishing behaviour. Critical elements of the skill included identifying a hole, making a tool, inserting a tool into a hole and extracting termites. These elements appeared in the same order during the development of all subjects, but females typically peaked at least a year earlier than males in their performance of the skills that precede termite fishing. In addition, young females successfully termite-fished an average of 27 months earlier than young males and were more proficient at the skill after acquisition had occurred. Furthermore, the techniques of female offspring closely resembled those of their mothers whereas the techniques of male offspring did not, suggesting that the process by which termite fishing is learned differs for male and female chimpanzees.
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Liker, A., & Bókony, V. (2009). Larger groups are more successful in innovative problem solving in house sparrows. Proc Natl Acad Sci USA, 106(19), 7893–7898.
Abstract: Group living offers well-known benefits to animals, such as better predator avoidance and increased foraging success. An important additional, but so far neglected, advantage is that groups may cope more effectively with unfamiliar situations through faster innovations of new solutions by some group members. We tested this hypothesis experimentally by presenting a new foraging task of opening a familiar feeder in an unfamiliar way to house sparrows in small and large groups (2 versus 6 birds). Group size had strong effects on problem solving: sparrows performed 4 times more and 11 times faster openings in large than in small groups, and all members of large groups profited by getting food sooner (7 times on average). Independently from group size, urban groups were more successful than rural groups. The disproportionately higher success in large groups was not a mere consequence of higher number of attempts, but was also related to a higher effectiveness of problem solving (3 times higher proportion of successful birds). The analyses of the birds' behavior suggest that the latter was not explained by either reduced investment in antipredator vigilance or reduced neophobia in large groups. Instead, larger groups may contain more diverse individuals with different skills and experiences, which may increase the chance of solving the task by some group members. Increased success in problem solving may promote group living in animals and may help them to adapt quickly to new situations in rapidly-changing environments.
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Morand-Ferron, J., & Quinn, J. L. (2011). Larger groups of passerines are more efficient problem solvers in the wild. Proc Natl Acad Sci USA, 108(38), 15898–15903.
Abstract: Group living commonly helps organisms face challenging environmental conditions. Although a known phenomenon in humans, recent findings suggest that a benefit of group living in animals generally might be increased innovative problem-solving efficiency. This benefit has never been demonstrated in a natural context, however, and the mechanisms underlying improved efficiency are largely unknown. We examined the problem-solving performance of great and blue tits at automated devices and found that efficiency increased with flock size. This relationship held when restricting the analysis to naive individuals, demonstrating that larger groups increased innovation efficiency. In addition to this effect of naive flock size, the presence of at least one experienced bird increased the frequency of solving, and larger flocks were more likely to contain experienced birds. These findings provide empirical evidence for the “pool of competence” hypothesis in nonhuman animals. The probability of success also differed consistently between individuals, a necessary condition for the pool of competence hypothesis. Solvers had a higher probability of success when foraging with a larger number of companions and when using devices located near rather than further from protective tree cover, suggesting a role for reduced predation risk on problem-solving efficiency. In contrast to traditional group living theory, individuals joining larger flocks benefited from a higher seed intake, suggesting that group living facilitated exploitation of a novel food source through improved problem-solving efficiency. Together our results suggest that both ecological and social factors, through reduced predation risk and increased pool of competence, mediate innovation in natural populations.
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