Satoshi Kusuda

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.

Relationship between gonadal steroid hormones and vulvar bleeding in southern tamandua, Tamandua tetradactyla.

This study aimed at demonstrating the profiles of circulating gonadal steroid hormones during the estrous cycle and pregnancy in a southern tamandua (Tamandua tetradactyla). Additionally, this study clarified the relationship between vulvar bleeding and hormonal changes. The concentrations of serum progesterone (P(4)) and estradiol-17β (E(2)) were determined by enzyme immunoassays. Serum P(4) and E(2) concentrations changed cyclically and the estrous cycle length (± SD) based on the E(2) cycles was 44.3 ± 4.5 days. Vulvar bleeding started to be seen at the decreasing of P(4). The cycle length for vulvar bleeding was 43.3 ± 4.2 days. Interval from the first day of bleeding to the peak of E(2) concentration was 23.1 ± 3.1 days. Serum P(4) during pregnancy remained high and E(2) increased 8 weeks after conception and remained high until parturition. The female delivered normally after a 165 day-pregnancy period and reared the offspring well. Approximately 3 weeks after parturition, serum E(2) and P(4) cycles resumed. Visual bleeding may be useful as a real-time indicator for understanding the ovarian cycle of southern tamanduas, and estrus could be expected approximately 3 weeks after the first bleeding.

Noninvasive Monitoring of Reproductive Activity Based on Fecal Progestagen Profiles and Sexual Behavior in Koalas, Phascolarctos cinereus

Studies on the reproductive endocrinology of koalas have been performed mainly by using blood samples; however, in practice it is difficult to collect blood periodically because koalas are easily stressed. The purposes of the present study were to establish a noninvasive endocrine monitoring technique and to investigate the reproductive physiology of female koalas. Feces were collected from female northern and southern koalas, and progestagen was extracted from lyophilized fecal samples and determined by enzyme immunoassay. In nonpregnant northern and southern koalas, fecal progestagen markedly increased after copulation and remained high for 36.3 ± 2.5 days and 38.9 ± 1.4 days (luteal phase, mean ± SEM), respectively. Mean (±SEM) progestagen levels (6.34 ± 0.49 μg/g) during the luteal phase in northern koalas were significantly higher than in southern koalas (4.19 ± 0.24 μg/g). Fecal progestagen in parturient northern koalas remained high for 36.2 ± 1.9 days (gestation period, 34.1 ± 0.3 days). In northern koalas, the mean levels and profiles of progestagen during pregnancy (6.44 ± 0.37 μg/g) were consistent with those during nonpregnancy after copulation (6.34 ± 0.49 μg/g). The duration of behavioral estrus in northern koalas was 13.5 ± 0.9 days without copulation. In contrast, when estrous females mated, the estrous sign disappeared just after copulation. The mean (±SEM) length of the estrous cycle in northern koalas, as determined by behavioral estrus intervals, was 33.5 ± 2.2 days without the luteal phase and 69.2 ± 7.6 days with the luteal phase. Fecal progestagen analysis is a helpful and noninvasive tool to monitor ovulatory activity in northern and southern koalas and could help us to understand the reproductive activity of koalas by the combination approach with behavioral estrus.

Ovarian cycle approach by rectal temperature and fecal progesterone in a female killer whale, Orcinus orca†

This study aimed to validate the measurements of body temperature and fecal progesterone concentrations as minimally invasive techniques for assessing ovarian cycle in a single sexually mature female killer whale. Rectal temperature data, fecal and blood samples were collected in the dorsal position using routine husbandry training on a voluntary basis. The correlations between rectal temperature and plasma progesterone concentration and between fecal and plasma progesterone concentrations were investigated. Fecal progesterone metabolites were identified by a combination of high-performance liquid chromatography and enzyme immunoassay. Plasma progesterone concentrations (range: 0.2-18.6 ng/ml) and rectal temperature (range: 35.3-35.9°C) changed cyclically, and cycle lengths were an average (±SD) of 44.9±4.0 days (nine cycles) and 44.6±5.9 days (nine cycles), respectively. Rectal temperature positively correlated with the plasma progesterone concentrations (r=0.641, P<0.01). There was a visual trend for fecal progesterone profiles to be similar to circulating plasma progesterone profiles. Fecal immunoreactive progestagen analysis resulted in a marked immunoreactive peak of progesterone. The data from the single killer whale indicate that the measurement of rectal temperature is suitable for minimally invasive assessment of the estrous cycle and monitoring the fecal progesterone concentration is useful to assess ovarian luteal activity.

Diversity in the Matrix Structure of Eggshells in the Testudines (Reptilia)

The eggshells of 56 chelonians were examined by electron microscopy and X-ray diffractometry. They were classified into six types in terms of the matrix structure of their calcareous layer; type I was composed of a thin calcareous layer with minerals in an amorphous structure; type II with shell units composed of mammillary cores calcified with aragonite crystals; type III with shell units composed of mammillary cores, plus a single palisade layer also calcified with aragonite crystals, and with each shell unit separated; type IV with shell units the same as type III, but tightly packed together; type V with shell units composed of mammillary cores plus two palisade layers; and type VI with a cuticle layer calcified with calcite crystals over the same structure as that of type V. X-ray diffraction analyses at the outer surface of eggshells showed a gradual change in crystal disposition from the random disposition of type II to the single direction-oriented disposition of type V. The shell height was approximately parallel to the development of the palisade-layer matrix. The limiting membrane of all eggshell types was perforated with canals and that of type I was partially missing. Type I had a parchment shell, types II and III had a pliable shell (some were rigid) and types IV to VI had rigid shells. The present study showed that the hardness of eggshells can be determined by the composition of the shell matrices, as shell matrices are the framework for mineralization.

Population statistics and biological traits of endangered kiso horse.

The objective of this study was to clarify the current status of endangered Kiso horse, population statistics and biological traits, in order to take a step for the conservation by scientific approach. We surveyed 125 Kiso horses (86.2% of the whole breed), analyzed the construction of the population, and calculated the coefficient of inbreeding and effective population size. Moreover, we confirmed coat color variations and the traditional traits of the Kiso horse, and measured their height at the withers and chest circumference to clarify their physical characteristics. The population pyramid of the horses was stationary or contractive, suggesting a reduction of the population in the near future. The effective population size of the horse (47.9) suggested that the diversity was much less than their census size, and the high coefficient of inbreeding, 0.11 ± 0.07 on average, suggested that the horses were surely inbred. The horses had only 4 coat colors; bay, dark bay, buckskin dun, and chestnut, and 116 horses (92.8%) were bayish color, suggesting the fixation in their coat color. Moreover, the majority of them had dorsal stripe (83 horses; 66.4%), and the average heights at withers(131.9 ± 4.4 cm) and chest circumference (167.1 ± 10.1 cm) were not significantly different between males and females.

Reproductive characteristics of female lesser mouse deers (Tragulus javanicus) based on fecal progestagens and breeding records

Information about breeding and the reproductive biology of mouse deer is limited in the wild and captivity. No reports on reproductive endocrinology are available. The objective of the present study was to observe the reproductive biology based on breeding records, to validate the utility of the non-invasive endocrine monitoring technique using feces of the female lesser mouse deer (Tragulus javanicus), and thus to clarify the reproductive physiology. Breeding records from 2 females were investigated and the fecal progestagen profile was monitored in captivity. Fecal progestagens were extracted using methanol and measured by enzyme immunoassay. From the breeding records, many births occurred in May (spring) and November-December (winter); however, fecal progestagen profile showed cyclical changes throughout the year in a female mouse deer. Most mounting and mating behaviors were observed 2-3 days after the peak of progestagen concentration during luteal phase. The ovarian cycle length based on the fecal progestagen profile averaged 14.5±0.3 days. The fecal progestagen concentration remained high during pregnancy. Fecal progestagen monitoring is useful for evaluating ovarian activity and pregnancy in the lesser mouse deer.

Intrauterine insemination with fresh semen in Amur leopard cat (Pionailurus bengalensis eutilura) during non-breeding season

Equine and human chorionic gonadotropins were administered to two female Amur leopard cats to induce estrus and ovulation during non-breeding season. Fresh semen collected from male cats was surgically inseminated into the uterine horn of the females. In one animal, two fetal sacs without heartbeats were observed on abdominal ultrasonography 31 days after insemination, which indicated that embryo death had occurred. In the other animal, fetal heartbeats were detected in two fetal sacs 29 days after insemination, which confirmed as pregnancy. This animal delivered two newborns 68 days after insemination; the one of the kittens was assumed to be stillbirth, and the other grew normally. In this study, we successfully obtained a kitten from an Amur leopard cat by artificial breeding for the first time in Japan.

Induced estrus in female small Asian mongooses (Herpestes javanicus) for the purpose of controlling invasive alien species in Okinawa Island

The small Asian mongoose (Herpestes javanicus) belongs to the family Herpestidae, order Carnivora, and originally inhabited Iraq through northern India to extreme southern China and Indonesia, the Malay Peninsula, Hainan, Java (Nowak 2005). Mongooses, including small Asian mongooses, have been widely introduced by people to exterminate rats for pest control (Yamada and Sugimura 2004). In Japan, small Asian mongooses were introduced to eliminate yellow-spotted pit vipers (Habu snakes; Protobothrops flavoviridis) and sugarcane rats on Okinawa Island in 1910 and Amami-Oshima Island, Kagoshima Prefecture in 1979 (Yamada 2002). Naturalized and overbred mongooses, which prey on insects, reptiles, small mammals, wild birds and domestic fowl, have impacted the poultry industry, and threaten the survival of rare endemic species, such as the Amami rabbit (Pentalagus furnessi), Okinawa rail (Galirallus okinawae) and so on (Yamada 2002). A mongoose eradication project led by the Ministry of the Environment, Government of Japan, has been conducted on Amami-Oshima Island since 1996 and northern Okinawa Island since 2000. Moreover, mongooses were designated as invasive alien species by the Invasive Alien Species Act in Japan (Ministry of the Environment, 2004); however, the eradication project has not yet succeeded. The distribution of mongooses has expanded on Okinawa and Amami-Oshima Islands and, in addition, mongooses were found in Kagoshima city (Kyusyu Island) in 2007. Currently, cage-type traps and cylindrical trapping with bait, chicken meat, have been mainly used for capturing mongooses on Okinawa and Amami-Oshima Islands. Mongoose-drawing techniques require a more effective for increase of the capture number. For effective capture, studies searching for and identifying attractant substances for species-specific capture have been performed in small Asian mongooses, and the previous study attempted to identify volatile fatty acids from anal sac secretions in this species (Ogura et al. 2000b). In the present study, to establish a trapping method with an estrous female or its excreta, we tried to create a protocol to artificially induce estrus in female small Asian mongooses using exogenous hormonal treatment. Estrus was determined based on the profile of urinary estrogen concentrations.

Immunohistochemical analysis of the development of olfactory organs in two species of turtles Pelodiscus sinensis and Mauremys reevesii

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeves turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeves turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeves turtle.