Genetic assessment of captive breeding program of Indian Pangolin: implications for conservation and management.

BACKGROUND: Captive breeding programs play a vital role in conservation of threatened species, necessitating an understanding of genetic diversity among captive individuals to ensure long-term genetic viability, appropriate mate selection, and successful reintroduction to native habitats. METHODS AND RESULTS: We did not observe any recent genetic bottleneck, and population showed moderate genetic diversity. The estimated effective population size, representing individuals capable of contributing genetically to future generations, was estimated as 18.6 individuals (11.4-35.1 at 95% CI). Based on the genetic make-up and allelic diversity, we found seventeen pangolins (11 females and 6 males) were genetically unrelated and relatively more potent than others. CONCLUSION: In this study, we evaluated the captive breeding program of the Indian pangolin population at the Pangolin Conservation Breeding Centre in Nandankanan Zoological Park, Bhubaneswar, Odisha. We highlight the significance of genetic monitoring within the captive population of Indian pangolin for preserving genetic diversity and ensuring the long-term survival of the species. We established the genetic profiles of all 29 pangolins and identified 17 pangolins to be prioritized for enhanced breeding and future zoo exchange programs. We appreciate the zoo authorities for promoting genetic assessment of pangolin for better and more effective monitoring of the captive breeding of the endangered Indian pangolin.

Embryonic development of the world's smallest puffer fish, Carinotetraodon travancoricus - a threatened freshwater fish of the Western Ghats Biodiversity Hotspot.

The Malabar dwarf puffer, Carinotetraodon travancoricus is the smallest known pufferfish (family Tetraodontidae) and one of the smallest freshwater fishes of the Indian subcontinent. Due to their miniature size, wacky behaviour and appearance, they are much preferred in the international aquarium fish trade, although little is known regarding their breeding activity in captivity and their embryonic development. The purpose of this study was to fill these knowledge gaps. Wild-caught Malabar dwarf puffers were acclimatised to conditions, and pairs were introduced to breeding tanks. Adult fishes were fed with live and frozen diets including Artemia nauplii, moina and bloodworm. During spawning seasons, adult fish displayed elaborate courtship behaviour around sunset. Carinotetraodon travancoricus is a batch spawner releasing 1 to 5 eggs per diem. The eggs were spherical, and non-sticky, with a diameter of 1.48 ± 0.1 mm, and hatching took place after 108 to 116 h post-incubation. The newly hatched larvae were 3.5 ± 0.2 mm in length, and weighed 2.9 ± 0.4 mg. The early larvae have substantial yolk and oil globules as an energy reserve. Histological studies on mature females suggested the batch spawning nature of the species and low fecundity. Given its unique reproductive behaviour and characters, in situ protected habitats are required to ensure their continued survival in the wild, apart from encouraging captive breeding to augment the demand in the international aquarium fish trade.

First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni.

The Louisiana pinesnake (Pituophis ruthveni) is considered one of the rarest snakes in North America and was federally listed under the Endangered Species Act in 2018. Captive breeding and reintroduction of zoo-bred hatchlings has been successful, however, limited founders in the captive population and the inability to bring new, wild genes into the captive colony presents a major concern for the conservation of this species. The use of artificial insemination (AI) was first applied to snakes in the 1980s but further development of the technique has since received little attention. Our goal was to develop a method of AI for use in breeding Louisiana pinesnakes to facilitate gene flow from wild to captive populations. We inseminated two captive Louisiana pinesnakes with semen collected from one donor male, novel to both females. Timing of AI occurred following the emergence of females from brumation, and when large, distinct follicles were detected using digital palpation. Females were inseminated four and five times over a period of 14 and 19 days, respectively, using fresh and 2-day refrigerator stored semen. One female laid seven eggs, which resulted in four fertile eggs and two viable hatchlings, while the second female produced three fertile of seven eggs laid but no viable hatchlings. Genetic analyses confirmed the donor male was the sire of hatchlings. This is the first successful AI of an endangered snake species and provides a framework for the use and optimization of assisted reproductive technologies for use in conservation breeding programs.

Gut bacterial composition shows sex-specific shifts during breeding season in ex situ managed black-footed ferrets.

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus - all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets.

Genetic assessment improves conservation efforts for the critically endangered oceanic island endemic Hibiscus liliiflorus.

Hibiscus liliiflorus, endemic to the Indian Ocean island of Rodrigues, is one of the rarest plant species in the world; only 2 wild individuals remain. Previously, when 4 wild individuals remained, the Mauritian Wildlife Foundation (MWF) in Rodrigues propagated cuttings of them in their nursery, then planted seedlings produced in the nursery into 3 outplanted populations on the island. Our goals were to: 1) assess whether all 4 original wild genotypes are represented in the MWF nursery; 2) determine whether ex situ living collections at international botanical gardens maintain unique genotypes of H. liliiflorus; 3) assess whether nursery individuals have crossed or self-fertilized to produce seed and quantify their relative contributions to outplanted populations; and 4) provide recommendations for future conservation actions. We used a 2b-RADseq approach to produce 2,711 genome-wide single nucleotide polymorphisms (SNPs) from 98 samples. Genotype identity analysis, principal component analysis, and model-based clustering in STRUCTURE found 4 genotypes extant in Rodrigues but no unique genotypes in ex situ botanic garden collections. Only 3 genotypes are represented in the MWF nursery; the one remaining genotype is represented by an extant wild individual. Parentage analysis showed that seeds produced in the MWF nursery resulted from both self-fertilization and crossing between genotypes, a result supported by internal relatedness and hybrid index calculations. Each outplanted population is dominated by a subset of parental genotypes, and we propose actions to balance the parental contributions to outplanted populations. Our study highlights how genetic assessments of ex situ conservation projects help conserve critically endangered species.

Cryogenic storage increases the longevity of butternut (Juglans cinerea, L.) seed embryogenic axes.

Butternut (Juglans cinerea L.), a species listed as endangered, is currently undergoing rapid decline due to habitat loss and the introduction of Ophiognomonia clavigignenti-juglandacearum, a non-native pathogen causing butternut canker. The decline of butternut has led to the development of cryobiological methods for the ex-situ conservation of the species since viability is not maintained using conventional methods. In this study, we assess the survivability and growth of butternut embryogenic axes into plantlets after 7 years of cryopreservation. Results show that cryopreservation is a highly effective method for the long-term storage of embryogenic axes since both survival and subsequent acclimatization into plantlets was successful with an average of 76% survival and 71% acclimatization, respectively. These results surpass the actual duration viability for non-cryogenic storage of butternut seed and we hope that future testing will result in longer term success of this cryobiological method and provide much needed germplasm for future restoration.

Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas.

The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.

Backcrossing as a species restoration technique.

An investigation was conducted on the phenotypic results of mouse hybridization and seven generations of backcrossing, observing reciprocal F1 hybrids and backcrosses of Mus spretus and a laboratory strain of Mus domesticus C57BL/6J. F1 hybrids, backcrosses, and pure control specimens were measured for 6 body characteristics, 4 pelage coloration characteristics, 14 behaviors, and reproduction as reflected in litter size. Backcrossing was pursued for seven generations to FBC7 (i.e., "Backcross 7" or seven generations from commencement of backcrossing from an F1 hybrid female) where species restoration is mathematically calculated to be at 99.7%. Except for a minority of FBC7 M. spretus specimens failing to conform completely to one pelage characteristic, FBC7 specimens were indistinguishable from controls both subjectively and in all areas of measurement. The M. spretus backcross line was followed generation by generation and was largely conforming to controls by FBC4 at latest. The same effect was observed in the reciprocal M. domesticus backcross line. Fertility was negatively affected in F1 hybrids but restored or improved in backcross generations. Discussion is offered on hybridization and backcrossing as it occurs in nature and how it has been used or could be used as an additional ex situ tool in wildlife conservation efforts. It is concluded that conservation-oriented backcrossing is a practical species/subspecies restoration technique and has the potential to make genetic rescue feasible with minimal gene flow at the binomial level. Backcrossing is most applicable in closely monitored ex situ settings (1) where only one sex remains of a given taxon; and (2) where inbreeding depression seriously threatens a remnant taxon's ability to recover, and the only gene flow option is from another distinct species.

Tracking population genetic signatures of local extinction with herbarium specimens.

BACKGROUND AND AIMS: Habitat degradation and landscape fragmentation dramatically lower population sizes of rare plant species. Decreasing population sizes may, in turn, negatively affect genetic diversity and reproductive fitness, which can ultimately lead to local extinction of populations. Although such extinction vortex dynamics have been postulated in theory and modelling for decades, empirical evidence from local extinctions of plant populations is scarce. In particular, comparisons between current vs. historical genetic diversity and differentiation are lacking despite their potential to guide conservation management. METHODS: We studied the population genetic signatures of the local extinction of Biscutella laevigata subsp. gracilis populations in Central Germany. We used microsatellites to genotype individuals from 15 current populations, one ex situ population, and 81 herbarium samples from five extant and 22 extinct populations. In the current populations, we recorded population size and fitness proxies, collected seeds for a germination trial and conducted a vegetation survey. The latter served as a surrogate for habitat conditions to study how habitat dissimilarity affects functional connectivity among the current populations. KEY RESULTS: Bayesian clustering revealed similar gene pool distribution in current and historical samples but also indicated that a distinct genetic cluster was significantly associated with extinction probability. Gene flow was affected by both the spatial distance and floristic composition of population sites, highlighting the potential of floristic composition as a powerful predictor of functional connectivity which may promote decision-making for reintroduction measures. For an extinct population, we found a negative relationship between sampling year and heterozygosity. Inbreeding negatively affected germination. CONCLUSIONS: Our study illustrates the usefulness of historical DNA to study extinction vortices in threatened species. Our novel combination of classical population genetics together with data from herbarium specimens, an ex situ population and a germination trial underlines the need for genetic rescue measures to prevent extinction of B. laevigata in Central Germany.

DNA matchmaking in captive facilities: a case study with tigers.

BACKGROUND: Genetics driven interventions if adopted in conservation breeding projects may enhance the overall success by prioritizing breeding among genetically most competent individuals and delaying or completely diminishing the ill effects of inbreeding. METHODS AND RESULTS: In the present study, we investigated genetic make-up of 15 tigers housed at five different captive facilities of West Bengal in India and report the moderate level of genetic variation. We identified five tigers based on individual genetic attributes that may be prioritized for future breeding or animal exchange programmes. The occurrence of first and second order related individuals in captivity require management attention and they should be paired considering their immediate genetic background. CONCLUSION: Considering tiger as a case study, we highlight the use of genetic assessment and necessity to validate the studbook records in formulating adaptive management strategies for long-term conservation and management of species of interest.

Mortality analysis of captive red panda cubs within Chengdu, China.

BACKGROUND: The red panda has been classified as an endangered species due to the decreased number in the world and disease is considered as a great threat to the health and survival of the cubs in captivity. RESULTS: This study analyzed 32 red panda cub mortalities (15 females and 17 males, age less than two months) through gross necropsy, microbiological examination, and histopathological observation at the Chengdu Research Base of Giant Panda Breeding, China, during 2014-2020. The results showed that screenings for canine distemper virus, canine parvovirus, rotavirus and parasite infection were all negative, however bacteria such as Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, Enterococcus faecalis, Pseudomonas were isolated from the tissue samples of some cubs. The major causes of death were respiratory (43.75%) and digestive system disease (28.13%), followed by cardiovascular disease (12.5%) and neonatal stillbirths (9.38%). Renal system diseases and trauma were also detected, at lower incidence (one case for each). The mortality rate within 15 days of birth was 68.75% and gradually decreased with age, there was no significant difference in gender. CONCLUSION: This study can provide a scientific basis for the analysis of the cause of death among red panda cubs in captivity, so as to improve the survival rate, help build the captive population and further the ex-situ conservation management of this endangered species. Additionally, our research may also provide insights into the in-situ conservation of wild red pandas by identifying emerging disease threats within the wild population and potential treatment for rescued individuals.

Effects of copper accumulation on growth and development of Scopelophila cataractae grown in vitro.

Scopelophila cataractae was cultured in vitro for 16 weeks to assess the contrasting effects of Cu on growth and reproduction, as well as gametophore stage. To induce buds and gametophores of S. cataractae, ten treatments (tr 1 to tr 10) of culture media were prepared using a combination of mineral salts, sugar, vitamin B complex, CuSO4, and exogenous hormones. Highest numbers of gametophores and buds were formed in media containing 500 µM CuSO4 in co-application with auxin and cytokinin, as shown in the modest Cu treatments (tr 6 and tr 7, 26 per cushion and 255 per 25 mm2, respectively). A 5000 µM CuSO4 concentration inhibited development of protonema, possibly due to Cu toxicity, resulting in chloronema forming contorted filaments or short cells containing lipid bodies, and brood body diaspores but no gametophore or bud formation. In this study, S. cataractae Cu accumulation in tissue was substantial (up to 2843.1 mg kg-1; tr 6) with no or minimal adverse effects, reflecting its potential for phytoremediation of Cu in terrestrial and aquatic ecosystems. The highest atomic percentages of Cu and Zn were detected in the stem surfaces of gametophores treated with 500 µM CuSO4 (11% atomic Cu and 7% atomic Zn), which served as a primary heavy metal storage site, ultimately protecting cells from metal toxicity. The success of this in vitro study on S. cataractae should also aid ex situ conservation efforts for a variety of rare moss taxa in the wild.

Cryobanking European Mink (Mustela lutreola) Mesenchymal Stem Cells and Oocytes.

The European mink (Mustela lutreola) is one of Europe's most endangered species, and it is on the brink of extinction in the Iberian Peninsula. The species' precarious situation requires the application of new ex situ conservation methodologies that complement the existing ex situ and in situ conservation measures. Here, we report for the first time the establishment of a biobank for European mink mesenchymal stem cells (emMSC) and oocytes from specimens found dead in the Iberian Peninsula, either free or in captivity. New emMSC lines were isolated from different tissues: bone marrow (emBM-MSC), oral mucosa (emOM-MSc), dermal skin (emDS-MSC), oviduct (emO-MSc), endometrium (emE-MSC), testicular (emT-MSC), and adipose tissue from two different adipose depots: subcutaneous (emSCA-MSC) and ovarian (emOA-MSC). All eight emMSC lines showed plastic adhesion, a detectable expression of characteristic markers of MSCs, and, when cultured under osteogenic and adipogenic conditions, differentiation capacity to these lineages. Additionally, we were able to keep 227 Cumulus-oocyte complexes (COCs) in the biobank, 97 of which are grade I or II. The European mink MSC and oocyte biobank will allow for the conservation of the species' genetic variability, the application of assisted reproduction techniques, and the development of in vitro models for studying the molecular mechanisms of infectious diseases that threaten the species' precarious situation.

Living with a giant, flowering parasite: metabolic differences between Tetrastigma loheri Gagnep. (Vitaceae) shoots uninfected and infected with Rafflesia (Rafflesiaceae) and potential applications for propagation.

MAIN CONCLUSION: Metabolites in Rafflesia-infected and non-infected Tetrastigma were compared which may have applications in Rafflesia propagation. Benzylisoquinoline alkaloids, here reported for the first time in Vitaceae, were abundant in non-infected shoots and may be a form of defense. In Rafflesia-infected shoots, oxylipins, which mediate immune response, were elevated. Endemic to the forests of Southeast Asia, Rafflesia (Rafflesiaceae) is a genus of holoparasitic plants producing the largest flowers in the world, yet completely dependent on its host, the tropical grape vine, Tetrastigma. Rafflesia species are threatened with extinction, making them an iconic symbol of plant conservation. Thus far, propagation has proved challenging, greatly decreasing efficacy of conservation efforts. This study compared the metabolites in the shoots of Rafflesia-infected and non-infected Tetrastigma loheri to examine how Rafflesia infection affects host metabolomics and elucidate the Rafflesia infection process. Results from LC-MS-based untargeted metabolomics analysis showed benzylisoquinoline alkaloids were naturally more abundant in non-infected shoots and are here reported for the first time in the genus Tetrastigma, and in the grape family, Vitaceae. These metabolites have been implicated in plant defense mechanisms and may prevent a Rafflesia infection. In Rafflesia-infected shoots, oxygenated fatty acids, or oxylipins, and a flavonoid, previously shown involved in plant immune response, were significantly elevated. This study provides a preliminary assessment of metabolites that differ between Rafflesia-infected and non-infected Tetrastigma hosts and may have applications in Rafflesia propagation to meet conservation goals.

Reversing extinction trends: new uses of (old) herbarium specimens to accelerate conservation action on threatened species.

Although often not collected specifically for the purposes of conservation, herbarium specimens offer sufficient information to reconstruct parameters that are needed to designate a species as 'at-risk' of extinction. While such designations should prompt quick and efficient legal action towards species recovery, such action often lags far behind and is mired in bureaucratic procedure. The increase in online digitization of natural history collections has now led to a surge in the number new studies on the uses of machine learning. These repositories of species occurrences are now equipped with advances that allow for the identification of rare species. The increase in attention devoted to estimating the scope and severity of the threats that lead to the decline of such species will increase our ability to mitigate these threats and reverse the declines, overcoming a current barrier to the recovery of many threatened plant species. Thus far, collected specimens have been used to fill gaps in systematics, range extent, and past genetic diversity. We find that they also offer material with which it is possible to foster species recovery, ecosystem restoration, and de-extinction, and these elements should be used in conjunction with machine learning and citizen science initiatives to mobilize as large a force as possible to counter current extinction trends.

Contrasting gut microbiota in captive Eurasian otters (Lutra lutra) by age.

Understanding the gut microbiota characteristics of endangered species such as the Eurasian otter (Lutra lutra), especially in their early stages of life, could be essential for improving their management and ex situ conservation strategies. Here, we analyzed the gut microbiota diversity, composition, and function of captive Eurasian otters at different ages using high-throughput 16S rRNA gene sequencing. We found that: (1) Clostridiaceae was abundant in all age stages; (2) Lactococcus in cubs is thought to predominate for digesting milk; (3) bacteria associated with amino acid metabolism increase with age, while bacteria associated with carbohydrate metabolism decrease with age, which is likely due to decrease in dietary carbohydrate content (e.g., milk) and increase in dietary protein contents (e.g., fishes) with age; and (4) fish-related bacteria were detected in feces of healthy adults and juveniles. Overall, the gut microbiota of captive Eurasian otters was taxonomically and functionally different by age, which is thought to be attributed to the difference in the diet in their life stages. This study provided baseline information regarding the gut microbiota of Eurasian otters for the first time and contributes to improvement in their management in captivity.

Seed longevity of maize conserved under germplasm bank conditions for up to 60 years.

BACKGROUND AND AIMS: The long-term conservation of seeds of plant genetic resources is of key importance for food security and preservation of agrobiodiversity. Nevertheless, there is scarce information available about seed longevity of many crops under germplasm bank conditions. METHODS: Through germination experiments as well as the analysis of historical monitoring data, we studied the decline in viability manifested by 1000 maize (Zea mays subsp. mays) seed accessions conserved for an average of 48 years at the CIMMYT germplasm bank, the largest maize seedbank in the world, under two cold storage conditions: an active (-3 °C; intended for seed distribution) and a base conservation chamber (-15 °C; for long-term conservation). KEY RESULTS: Seed lots stored in the active chamber had a significantly lower and more variable seed germination, averaging 81.4 %, as compared with the seed lots conserved in the base chamber, averaging 92.1 %. The average seed viability detected in this study was higher in comparison with that found in other seed longevity studies on maize conserved under similar conditions. A significant difference was detected in seed germination and longevity estimates (e.g. p85 and p50) among accessions. Correlating seed longevity with seed traits and passport data, grain type showed the strongest correlation, with flint varieties being longer lived than floury and dent types. CONCLUSIONS: The more rapid loss of seed viability detected in the active chamber suggests that the seed conservation approach, based on the storage of the same seed accessions in two chambers with different temperatures, might be counterproductive for overall long-term conservation and that base conditions should be applied in both. The significant differences detected in seed longevity among accessions underscores that different viability monitoring and regeneration intervals should be applied to groups of accessions showing different longevity profiles.

Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation.

PREMISE: Many cultivated coffee varieties descend from Coffea canephora, commonly known as Robusta coffee. The Congo Basin has a century-long history of Robusta coffee cultivation and breeding, and is hypothesized to be the region of origin of many of the cultivated Robusta varieties. Since little is known about the genetic composition of C. canephora in this region, we assessed the genetic diversity of wild and cultivated C. canephora shrubs in the Democratic Republic of the Congo. METHODS: Using 18 microsatellite markers, we studied the genetic composition of wild and backyard-grown C. canephora shrubs in the Tshopo and Ituri provinces and multiple accessions from the INERA Yangambi Coffee Collection. We assessed genetic clustering patterns, genetic diversity, and genetic differentiation between populations. RESULTS: Genetic differentiation was relatively strong between wild and cultivated C. canephora shrubs, and both gene pools harbored multiple unique alleles. Strong genetic differentiation was also observed between wild populations. The level of genetic diversity in wild populations was similar to that of the INERA Yangambi Coffee Collection, but local wild genotypes were mostly missing from that collection. Shrubs grown in the backyards were genetically similar to the breeding material from INERA Yangambi. CONCLUSIONS: Most C. canephora that is grown in local backyards originated from INERA breeding programs, while a few shrubs were obtained directly from surrounding forests. The INERA Yangambi Coffee Collection could benefit from an enrichment with local wild genotypes to increase the genetic resources available for breeding purposes and to support ex situ conservation.

Stress behaviors in captive robust capuchins: Effects of humidity, visitors, management and sex.

Wild robust capuchins (Sapajus spp.) are omnivorous neotropical primates that live in relatively large groups in extensive home and daily ranges with activity budgets dominated by traveling, foraging, and object manipulation, meaning that enclosed spaces can result in significant deprivation. Space restriction, manipulation by caretakers, and the chronic presence of visitors, can disrupt the animals' welfare, altering their normal activities and inducing stress behaviors. We aimed to study the behavioral repertoire, activity budget, and frequency of stress behaviors (stereotypes and self-directed behaviors) between two captive groups of robust capuchins in a public zoo in Foz do Iguaçu, Brazil, to understand how much their behavioral homeostasis has been affected. More specifically, we assessed the effect of environmental variables (temperature, relative air humidity, number of visitors, and food management) and sex on the frequency of stress behaviors. Capuchins showed a high frequency of stress behaviors, which represented around 10% of their activity budget (though the frequencies were unevenly distributed among the individuals), and traveling was positively correlated with stereotypes. We found that high relative air humidity appears to induce more stereotypes, high numbers of visitors appear to increase self-directed and vigilance behaviors and reduce stereotypes, food management can increase both kinds of stress behaviors, and females demonstrated more frequent stress behaviors than males, but individual variation may play a role. Capuchins in the group with a greater space restriction showed more stereotypes, while those in the group with more individuals showed more self-directed behaviors. Our study shows that the stress behaviors performed by the capuchins are complex and it is difficult to determine a single cause, because many traits could be involved. Despite that, this study enlightens us to direct some approaches to help these animals to meet their ecological and social needs, mitigating their stress.

Lipid Remodeling Confers Osmotic Stress Tolerance to Embryogenic Cells during Cryopreservation.

Plant species conservation through cryopreservation using plant vitrification solutions (PVS) is based in empiricism and the mechanisms that confer cell integrity are not well understood. Using ESI-MS/MS analysis and quantification, we generated 12 comparative lipidomics datasets for membranes of embryogenic cells (ECs) of Magnolia officinalis during cryogenic treatments. Each step of the complex PVS-based cryoprotocol had a profoundly different impact on membrane lipid composition. Loading treatment (osmoprotection) remodeled the cell membrane by lipid turnover, between increased phosphatidic acid (PA) and phosphatidylglycerol (PG) and decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PA increase likely serves as an intermediate for adjustments in lipid metabolism to desiccation stress. Following PVS treatment, lipid levels increased, including PC and PE, and this effectively counteracted the potential for massive loss of lipid species when cryopreservation was implemented in the absence of cryoprotection. The present detailed cryobiotechnology findings suggest that the remodeling of membrane lipids and attenuation of lipid degradation are critical for the successful use of PVS. As lipid metabolism and composition varies with species, these new insights provide a framework for technology development for the preservation of other species at increasing risk of extinction.