Kodzue Kinoshita

Spectral pattern of urinary water as a biomarker of estrus in the giant panda

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.

Long-Term Monitoring of Fecal Steroid Hormones in Female Snow Leopards (Panthera uncia) during Pregnancy or Pseudopregnancy

Knowledge of the basic reproductive physiology of snow leopards is required urgently in order to develop a suitable management conditions under captivity. In this study, the long-term monitoring of concentrations of three steroid hormones in fecal matter of three female snow leopards was performed using enzyme immunoassays: (1) estradiol-17β, (2) progesterone and (3) cortisol metabolite. Two of the female animals were housed with a male during the winter breeding season, and copulated around the day the estradiol-17β metabolite peaked subsequently becoming pregnant. The other female was treated in two different ways: (1) first housed with a male in all year round and then (2) in the winter season only. She did not mate with him on the first occasion, but did so latter around when estradiol-17β metabolite peaked, and became pseudopregnant. During pregnancy, progesterone metabolite concentrations increased for 92 or 94 days, with this period being approximately twice as long as in the pseudopregnant case (31, 42, 49 and 53 days). The levels of cortisol metabolite in the pseudopregnant female (1.35 µg/g) were significantly higher than in the pregnant females (0.33 and 0.24 µg/g) (P<0.05). Similarly, during the breeding season, the levels of estradiol-17β metabolite in the pseudopregnant female (2.18 µg/g) were significantly higher than those in the pregnant females (0.81 and 0.85 µg/g) (P<0.05). Unlike cortisol the average levels of estradiol-17β during the breeding season were independent of reproductive success. The hormone levels may also be related to housing conditions and the resulting reproductive success in female leopards. The female housed with a male during the non-breeding season had high levels of cortisol metabolites and low levels of estradiol-17β in the breeding season, and failed to become pregnant. This indicates that housing conditions in snow leopards may be an important factor for normal endocrine secretion and resulting breeding success.

Near infrared spectroscopy of urine proves useful for estimating ovulation in giant panda (Ailuropoda melanoleuca)

The usefulness of near infrared spectroscopy (NIRS) to monitor urine estrogen concentrations was studied in order to determine optimal timing for breeding captive female giant pandas. NIR spectra of daily urine samples from a female giant panda (Ailuropoda melanoleuca) were acquired in the period between March 1st and 25th, 2007 (n = 53). Estrone-3-glucuronide (E1G) concentrations in the samples were also measured by enzyme immunoassay (EIA). Transmittance spectra of all urine samples were obtained in the wavelength range from 1100 to 2432 nm (excluding the range from 1884 to 2012 nm) with sample thickness of 1 mm. Partial least square regression was applied to the spectra and good correlation was obtained between E1G concentration measured by EIA and predicted values by NIR (R2 = 0.94, SECV = 10.04 ng ml−1). The results of both soft-independent modeling of class analogy (SIMCA) and moving principal component analysis (MPCA) could detect the time changes in E1G concentration as measured by EIA (the Pearsons correlation coefficients between E1G concentration and the interclass distances of SIMCA or the index of MPCA were r = 0.64 and r = 0.81 respectively, P < 0.01). As for MPCA index, the index sharply dropped on March 24th corresponding to the decrease of the E1G concentration indicating ovulation. Finally, artificial insemination was performed for 3 consecutive days including the peak day, March 24th, and the female became pregnant. These results indicated that NIRS and the following MPCA analysis of the respective urine spectral data could detect the changes of urinary hormones during estrous cycle at a nanogram level. The NIRS can find the optimal timing for breeding quicker and easier than EIA, so this technique can be useful for captive breeding of this threatened species.

Daily fecal sex steroid hormonal changes and mating success in captive female cheetahs (Acinonyx jubatus) in Japan

Daily fecal estrogen and progestin concentrations were measured by enzyme immunoassay in five female cheetahs (Acinonyx jubatus) for 4-6 months. The animals were housed under different conditions: (1) a female always housed in a group including one or more males; (2) two females isolated individually for short or long periods; (3) the other two females housed together. These females were separately housed with males for mating around the time of the estrogen peaks. The hormone profiles were similar in all five females regardless of the housing conditions. However, only the female that had been isolated from other cheetahs for over a year mated and reproduce cubs successfully, whereas the remaining four did not (one was isolated for only 6 weeks, another was always housed with males and the other two were housed together). In all females, the estrogen peaks were obtained at regular intervals of approximately 8-15 days. Unlike estrogen, the progestin concentrations were always low in all females except during pregnancy and they did not increase following the estrogen surges. These results showed that female cheetahs are typically reflex ovulators and female receptiveness may not be reflected to her hormonal states. It was also suspected that individual housing and long-term separation are advantageous for breeding this wild cat in captivity, mimicking the ecological/behavioral patterns in the wild, though housing condition might have no effect on the estrous cycle.

Analysis of hair cortisol levels in captive chimpanzees: Effect of various methods on cortisol stability and variability

Graphical abstract Summary 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].

Review: Near infrared spectroscopy in wildlife and biodiversity

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.

Detection of urinary estrogen conjugates and creatinine using near infrared spectroscopy in Bornean orangutans (Pongo Pygmaeus).

For promoting in situ conservation, it is important to estimate the density distribution of fertile individuals, and there is a need for developing an easy monitoring method to discriminate between physiological states. To date, physiological state has generally been determined by measuring hormone concentration using radioimmunoassay or enzyme immunoassay (EIA) methods. However, these methods have rarely been applied in situ because of the requirements for a large amount of reagent, instruments, and a radioactive isotope. In addition, the proper storage of the sample (including urine and feces) on site until analysis is difficult. On the other hand, near infrared (NIR) spectroscopy requires no reagent and enables rapid measurement. In the present study, we attempted urinary NIR spectroscopy to determine the estrogen levels of orangutans in Japanese zoos and in the Danum Valley Conservation Area, Sabah, Malaysia. Reflectance NIR spectra were obtained from urine stored using a filter paper. Filter paper is easy to use to store dried urine, even in the wild. Urinary estrogen and creatinine concentrations measured by EIA were used as the reference data of partial least square (PLS) regression of urinary NIR spectra. High accuracies (R2xa0>xa00.68) were obtained in both estrogen and creatinine regression models. In addition, the PLS regressions in both standards showed higher accuracies (R2xa0>xa00.70). Therefore, the present study demonstrates that urinary NIR spectra have the potential to estimate the estrogen and creatinine concentrations.

Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)

The number of sea turtles in the wild is decreasing because of human activities, such as fishery bycatch (Peckham et al. 2007), oil spills, and marine pollution (Witherington 2001). Furthermore, illegal hunting for their oil, meat, and shells are still continuing (Koch et al. 2013). Hence, protection measures are being implemented in various countries to conserve the endangered species of sea turtles. In the past few decades, a major concern regarding sea turtles in the wild has been hybridization. Interspecific hybridization has been reported in several areas of the world (Karl et al. 1995; Barber et al. 2003; Lara-Ruiz et al. 2006). The original sea turtle species, such as hawksbill, loggerhead, and olive ridley, had established as independent species for more than millions of years despite overlaps among their habitats (Bowen et al. 1993). However, recent reports on intensive hybridization among these species clearly indicate that these unique original species are at a high risk of extinction in the near future. Acquiring information about the basic genetic background of these species, such as karyotype, is necessary to understand the cause of this intensive hybridization. In this study, we established a primary culture from olive ridley sea turtles and determined the karyotype of the primary cells. Since 1976, the San Diego Zoo started the preservation of biological specimens derived from critically endangered species in order to facilitate the use of these specimens as research materials for the following generations; this project was named “Frozen Zoo.” The significance of this project was internationally recognized, and similar projects, such as the Frozen Ark project in the UK, are underway. The establishment of cell cultures from critically endangered animals might contribute to these cryopreservation projects by increasing the stability of cryopreserved materials and facilitating efficient expansion of cultured cells. Since cultured cells have intact genetic information of the critically endangered animals, the cells have a potential to be used for the genetic analysis of future generations. The olive ridley sea turtles were maintained at the Port of Nagoya Public Aquarium. Small (3×3 mm) dermal tissue biopsy specimens were obtained from the flipper-like fin of two olive ridley sea turtles. The tissue biopsy specimens were immediately immersed in the cell culture medium. The biopsy process was supervised by a veterinary doctor of the Port of Nagoya Public Aquarium. For the primary culture, a six-well cell culture dish was coated with type I collagen. The detailed method for collagen coating has been described in our previous study (Fukuda et al. 2012). The cell culture was maintained at 26°C under 5% CO2 in a humidified chamber T. Fukuda (*) :M. Katayama :K. Donai : T. Uchida : E. Isogai Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi Aoba-ku, Sendai 981-8555, Japan e-mail: tomofukuda@bios.tohoku.ac.jp

Beneficial effect of hot spring bathing on stress levels in Japanese macaques

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.

Effect of castration on social behavior and hormones in male Japanese macaques (Macaca fuscata)

Population control is essential for animal welfare and human safety in free-ranging or captive settings, especially when resources are limited. As an alternative to lethal control, contraceptive methods such as castration in males can be a practical solution, because the testicles are a visual cue to determine which males have been sterilized. However, careful analyses should be carried out to ensure no disruption in the social structure of the population. Japanese macaques (Macaca fuscata) live in a society centered around dominance rank and matrilineal kin relationships. Testosterone and glucocorticoids have been correlated with dominance rank in humans and other species, but previous studies in Japanese macaques were inconclusive. This study aimed to investigate behavioral and physiological differences between castrated and intact male Japanese macaques, and to examine the effect of season and behavior in hormonal concentrations in intact males. Our subjects were six intact males from Jigokudani Monkey Park (Japan) and 13 castrated males from Born Free Primate Sanctuary (USA). We collected behavioral data using both focal and ad libitum sampling, and fecal samples for determination of testosterone (fT) and glucocorticoids (fGC) by enzyme immunoassay. We found that castrated males exhibited a social hierarchy, but not a linear hierarchy, as was the case in intact males. Castrated males were less aggressive than intact males, probably because fT concentrations were lower in the castrated males. Age was positively correlated with fGC levels, while fT concentrations were lower in old males than younger adult males. Fecal T levels correlated with both rank and atmospheric temperature. In intact males, both fGC and fT levels were elevated during the mating season. We found a negative correlation between fGC levels and the amount of grooming received. Our findings indicate that castration had a minimal impact on sociality, with season, temperature, and rank all influencing male sex steroid levels in intact males. Our study indicates that castration can be adopted as a population control mechanism without drastically altering the social relationships of males.