Near infrared spectroscopy (NIRS) has been successfully used for non-invasive diagnosis of diseases and abnormalities where water spectral patterns are found to play an important role. The present study investigates water absorbance patterns indicative of estrus in the female giant panda. NIR spectra of urine samples were acquired from the same animal on a daily basis over three consecutive putative estrus periods. Characteristic water absorbance patterns based on 12 specific water absorbance bands were discovered, which displayed high urine spectral variation, suggesting that hydrogen-bonded water structures increase with estrus. Regression analysis of urine spectra and spectra of estrone-3-glucuronide standard concentrations at these water bands showed high correlation with estrogen levels. Cluster analysis of urine spectra grouped together estrus samples from different years. These results open a new avenue for using water structure as a molecular mirror for fast estrus detection.

Near infrared (NIR) spectroscopy has been used to answer a wide variety of questions in wildlife and biodiversity research. Whereas agricultural systems and manufacturing seek to limit variation in production systems, wildlife and biodiversity research must embrace it. Variation amongst individuals is the material on which natural selection operates and NIR spectroscopy provides a means to catalogue this variation and to use it in broader ecological and evolutionary analyses and for practical conservation outcomes. In this review we describe how NIR spectroscopy has been applied in wildlife and biodiversity research to obtain data that we could not obtain otherwise. Here we describe a range of applications for which NIR spectroscopy has been applied to questions in taxonomy, physiology, habitat evaluation and population monitoring and highlight new approaches that will allow NIR spectroscopy to be used more widely in wildlife and ecological studies.

Highlights d During the breeding season, females are attracted by male wrist glandular odor d Three C12 and C14 aldehydes are seasonally secreted by the male antebrachial gland d The amounts of the identified aldehydes increase in a testosterone-dependent manner d Females are interested in cotton pads soaked in the identified aldehydes

Graphical abstractSummary of the experimental process. The items colored blue affected the results of the hair cortisol analysis in the present study. The stippled items were found to affect the results obtained in our previous study [1].

Age is an important ecological tool in wildlife conservation. However, it is difficult to estimate in most animals, including felines—most of whom are endangered. Here, we developed the first DNA methylation-based age-estimation technique—as an alternative to current age-estimation methods—for two feline species that share a relatively long genetic distance with each other: domestic cat (Felis catus; 79 blood samples) and an endangered Panthera, the snow leopard (Panthera uncia; 11 blood samples). We measured the methylation rates of two gene regions using methylation-sensitive high-resolution melting (MS-HRM). Domestic cat age was estimated with a mean absolute deviation (MAD) of 3.83 years. Health conditions influenced accuracy of the model. Specifically, the models built on cats with chronic kidney disease (CKD) had lower accuracy than those built on healthy cats. The snow leopard-specific model (i.e. the model that resets the model settings for snow leopards) had a better accuracy (MAD = 2.10 years) than that obtained on using the domestic cat model directly. This implies that our markers could be utilised across species, although changing the model settings when targeting different species could lead to better estimation accuracy. The snow leopard-specific model also successfully distinguished between sexually immature and mature individuals.

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

The ability of animals to survive dramatic climates depends on their physiology, morphology and behaviour, but is often influenced by the configuration of their habitat. Along with autonomic responses, thermoregulatory behaviours, including postural adjustments, social aggregation, and use of trees for shelter, help individuals maintain homeostasis across climate variations. Japanese macaques (Macaca fuscata) are the world's most northerly species of nonhuman primates and have adapted to extremely cold environments. Given that thermoregulatory stress can increase glucocorticoid concentrations in primates, we hypothesized that by using an available hot spring, Japanese macaques could gain protection against weather-induced cold stress during winter. We studied 12 adult female Japanese macaques living in Jigokudani Monkey Park, Japan, during the spring birth season (April to June) and winter mating season (October to December). We collected faecal samples for determination of faecal glucocorticoid (fGC) metabolite concentrations by enzyme immunoassay, as well as behavioural data to determine time spent in the hot springs, dominance rank, aggression rates, and affiliative behaviours. We used nonparametric statistics to examine seasonal changes in hot spring bathing, and the relationship between rank and air temperature on hot spring bathing. We used general linear mixed-effect models to examine factors impacting hormone concentrations. We found that Japanese macaques use hot spring bathing for thermoregulation during the winter. In the studied troop, the single hot spring is a restricted resource favoured by dominant females. High social rank had both costs and benefits: dominant females sustained high fGC levels, which were associated with high aggression rates in winter, but benefited by priority of access to the hot spring, which was associated with low fGC concentrations and therefore might help reduce energy expenditure and subsequent body heat loss. This unique habit of hot spring bathing by Japanese macaques illustrates how behavioural flexibility can help counter cold climate stress, with likely implications for reproduction and survival.

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