No description available.

No description available.

Two distinct nonapeptide systems, consisting of the vasotocin- and oxytocin-related peptides have evolved in vertebrates, and their role in male reproduction is well-described in mammals. In contrast, their comparative role in reproduction in basal vertebrate species, and teleost fishes in particular, has not been investigated in great detail. Using two cyprinid species, the zebrafish (D. rerio) and the goldfish (C. auratus), I address the hypothesis that the teleost nonapeptides vasotocin and isotocin stimulate male cyprinid reproductive physiology by affecting central neuronal and/or peripheral endocrine pathways. To test this hypothesis in zebrafish, an indeterminate breeder, I conducted pharmacological inhibition experiments employing vasotocin and isotocin-specific antagonists in males, a treatment predicted to inhibit reproductive success in mating trials. Because nonapeptides can act both as central peptide neuromodulators and as secreted hormone, I further quantified indices of male courtship behavior (nudging, circling and chasing) and major androgens (testosterone and 11-keto-testosterone) as key endocrine indices of the male reproductive axis. Together, these experiments revealed a dose-dependent, differential inhibition of spawning success, with significant reductions (-65%) in egg fertilization rate observed in pairs in which males had been i.p. injected with 5 ng/g vasotocin and significant reductions (-79%) observed at 500 ng/g i.p injected isotocin. In either case, these partial inhibitions of reproductive success were correlated with significant decreases in specific indices of male courtship behavior, but not endocrine indices, suggesting that individual nonapeptides mediate their effects via central modulation of behavioural neurocircuits. Interestingly, a co-administration of vasotocin and isotocin antagonists completely abolished reproductive success, however this effect was neither correlated with decreases in male courtship behavior, nor endocrine indices, suggesting a separate mode of action, possibly at the level of male pheromone release. To further probe the role of nonapeptides in male zebrafish reproduction, I subsequently tested the hypothesis that nonapeptide systems are acutely activated by key reproductive cues, specifically the releaser pheromone PGF2α, which serves as a chemoattractant and acutely stimulates male reproductive behavior in male cyprinids. Using a chemoattractant choice assay in conjunction with immunohistochemistry and gene expression approaches, I determined whether male zebrafish are attracted to pheromonal cues and acutely activate isotocinergic neurons in the short term and/or regulate nonapeptide gene expression in the longer term. My results show that individual male zebrafish are attracted to PGF2α in an acute choice test. Furthermore, an increase in p-ERK immunoreactivity, a marker of neuronal activation, was observed in the olfactory bulb 10 min following exposure, suggesting a specific response to the pheromone compared to EtOH vehicle. However, no co-localization of p-ERK and IT-positive perikarya was observed in the preoptic area (POA), refuting the hypothesis that PGF2α exposure acutely activates isotocinergic neurons in zebrafish. Analysis of whole brain relative mRNA transcript abundance revealed that PGF2α exposure time-dependently regulates whole brain isotocin, but not vasotocin transcript abundance, suggesting secondary longer-term effects of PGF2α exposure on the isotocinergic system. Using an analogous experimental approach, I further tested the hypothesis that nonapeptides stimulate male reproductive physiology in goldfish, a determinate breeder. Sexually mature male goldfish pretreated with saline or vasotocin or isotocin antagonists were exposed to saline or PGF2α-injected stimulus females and male courtship behavior (chasing, circling), endocrine indices (circulating testosterone) and milt release were quantified. Both nonapeptide antagonists reduced strippable male milt quantity in response to PGF2α-injected females, suggesting a neuronal or hormonal action of both nonapeptides on goldfish milt release. Together, I show that nonapeptides contribute to male reproductive physiology in two species of cyprinids with different reproductive tactics. However, the mode of action may differ from one species to another, with evidence suggesting that nonapeptides play a role in the regulation of reproductive behavior and, possibly, male pheromone, release in zebrafish, while effects on male goldfish seem to be exclusively related to the release of milt. Future studies should compare other teleost species with specific reproductive biology and focus on the gonadal roles of nonapeptides in sperm maturation and/or release.

OBJECTIVE: To examine the likelihood of self-reporting a diagnosis for PCOS with the presentation of menstrual irregularity in a diverse, multiethnic population, based on data collected between August 9th, 2017 and October 24th, 2017 for the pilot of the Ovulation and Menstruation (OM) Study at Boston University School of Medicine. BACKGROUND: Polycystic ovary syndrome, or PCOS, is the most common endocrine disorder among reproductive age women. It is typically diagnosed by variable combinations of menstrual irregularity, clinical or biochemical hyperandrogenism, and polycystic ovaries on ultrasound. An alternative is its diagnosis as one of exclusion due to similarities in presentation to other endocrine disorders. As a result, PCOS may often be misdiagnosed and mismanaged in the course of a patient’s care, further exacerbated by a poor understanding of the syndrome, a lack of easily available resources, and patient frustration with clinician interactions. The early identification of key hallmarks of the disorder, such as menstrual irregularity, and awareness of its linkage to PCOS, could lead to early diagnosis and intervention. METHODS: 248 participants enrolled and participated in the Ovulation and Menstruation (OM) Health Study’s as members of its pilot cohort. Inclusion criteria were women ages 18-45 currently experiencing menstrual periods without a history of chemotherapy, radiation, or surgical menopause. Participants completed the relevant sections of the OM Study survey related to demographics, menstrual cycle patterns, and history of PCOS. Demographic questions pertained to the age, race/ethnicity, country of birth, and education levels of the participants. The menstrual cycle questions provided information regarding the age of menarche, length and pattern of menses and the menstrual cycle overall. The questions regarding history of PCOS ascertained the presence of an official or self-diagnosis for PCOS for the participant, and the age at which this was determined. The descriptive measures were presented for comparison before determining the concurrence of the presence of menstrual irregularity and the diagnosis of PCOS across demographic categories and calculating an associated prevalence ratio. RESULTS: Among women reporting a history of menstrual irregularity for 3 months or greater, PCOS was the second-highest self-reported cause for menstrual irregularity, with 20.7% of participants endorsing it as the cause for their irregularity. The presence of menstrual irregularity for 3 or more months was also more likely to be present in concurrence with a clinician diagnosis, or to a lesser extent, a self-diagnosis, for PCOS. Participants were also far more likely to have a clinician diagnosis for PCOS if they were White, US-born, young, or educated. The same applied for the likelihood of a self-diagnosis with the exception of age. CONCLUSIONS: The association between menstrual cycle irregularities and likelihood of being diagnosed with PCOS is supported by the data and appears to be influenced by demographic factors such as race/ethnicity, age, and education.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20964号 / 理博第4416号 / 新制||理||1634(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 Michael Alan Huffman, 准教授 足立 幾磨, 教授 友永 雅己 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

<p>Sex steroids are critical for the post-natal development of the female reproductive system, and are involved in ovulatory cycling and pregnancy. In mice, <em>Mus musculus</em>, female development, cycling, and pregnancy can be affected by the urine of conspecifics, which is known to contain active steroids. Specifically, puberty can be accelerated (the Vandenbergh effect), estrous cycling can be prolonged (the Lee-Boot effect) or synchronized (the Whitten effect), and blastocyst implantation can be disrupted (the Bruce effect). Since steroids alone can affect females in ways that are indistinguishable from these social reproductive effects, I hypothesized that urinary steroids of conspecifics may be absorbed by females, arrive in the reproductive system, and thereby affect females through known mechanisms. First I showed that tritium-labelled 17β-estradiol (³H-E₂) injected into males is excreted in their urine, and that application of urine from these males to the nose of an inseminated female results in detectable levels in her uterus. When I paired inseminated females with non-sire males injected with ³H-E₂, radioactivity was detected in the brain and reproductive tissues of the females. This was the first demonstration of steroids from one animal directly entering the body of another. Similar results were found when I exposed juvenile females to adult males injected with ³H-E₂, and when I exposed nulliparous adult females to same-strain ³H‑E₂- or ³H-progesterone- (³H-P₄) treated adult males or females. Taken with the existing literature, these results suggest that steroid transfer may underlie various social reproductive phenomena in mice, with potential implications for many other species.</p> / Doctor of Philosophy (PhD)

Devido as atuais mudanças globais é esperado que um grande número de espécies necessitem de uma integração de ações dentro e fora do seu ambiente natural para a conservação. O bioma cerrado é um dos ecossistemas mais ricos em biodiversidade, mas devido a ação antrópica, resta menos de 20% de sua cobertura vegetal original. A Raposinha do Campo (Lycalopex vetulus) é um canídeo de pequeno porte (2,5-4kg) endêmico do cerrado do Brasil central e está presente na lista dos animais ameaçados de extinção do Estado de São Paulo. Não existe em literatura uma descrição detalhada do ciclo estral desta espécie. O presente estudo teve como objetivo caracterizar a ciclicidade ovariana de Lycalopex vetulus com o uso de método não-invasivo: Extração e mensuração de metabólitos fecais de progesterona e estradiol, com o uso da técnica de enzimaimunoensaio, verificando também a possível existência de diferenças mensais entre as médias das concentrações dos referidos metabólitos em um período de 12 meses. Foram utilizadas 8 fêmeas, adultas, presentes em cinco instituições no estado de São Paulo, onde foram coletadas fezes 3 vezes por semana de cada indivíduo, durante 12 meses, para a extração e mensuração dos metabólitos de progesterona, estradiol e corticosterona. Os perfis das excreções dos metabólitos fecais dos hormônios sexuais de 6 das 8 fêmeas, foram muito semelhantes, sendo período de maior atividade reprodutiva entre os messes de julho, agosto e setembro, ocorrendo mais de um ciclo ovulatório dentro desse período. Nos outros meses do ano, apesar de algumas variações acima da linha basal, não foram encontrados indícios de atividade reprodutiva. De acordo com essas características, semelhante entre a maioria das fêmeas do estudo, podemos sugerir que a raposa-do-campo seja poliestrica sazonal, com atividade reprodutiva ocorrendo nos meses de julho, agosto e setembro. Esses achados são muito próximos do visualizado em populações de vida livre, onde acasalamentos são observados entre junho e julho. O perfil de excreção dos metabólitos fecais de glicocorticóides segue o mesmo padrão observado para os metabólitos de progesterona e estradiol, com um aumento mais significativo da excreção nos meses de julho, agosto e setembro. Esses resultados indicam que além da grande quantidade de estressores que podem alterar a excreção de glicocorticóides, também deve-se considerar a flutuação sazonal e o status reprodutivo do indivíduo ao avaliar as concentrações de metabólitos de glicocorticóides. Duas fêmeas, que dividem o mesmo recinto, não apresentaram um padrão de ciclicidade reprodutiva. Os resultados obtidos nesse estudo indicam que a dosagem de metabólitos fecais de progesterona e estradiol podem ser usadas para diferenciar o período reprodutivo do período não reprodutivo em fêmeas de Lycalopex vetulus, fornecendo informações importantes sobre a biologia reprodutiva da espécie, o que pode contribuir no desenvolvimento de estratégias para a conservação desta espécie, como por exemplo aumentar o sucesso reprodutivo ex situ. / Due to the current global changes is expected that a large number of species require an integration of actions inside and outside their natural environment for them conservation. The cerrado is one of the richest ecosystems in biodiversity, but due to human action, there remains less than 20% of its original vegetation cover. The Hoary fox (Lycalopex vetulus) is a small canid (2,5-4kg) endemic of the cerrado from the central Brazil and is present in the list of endangered species in the state of São Paulo. It was not found in literature any detailed description of the estrous cycle of this species. The present study aimed to characterize ovarian cyclicity in Lycalopex vetulus using a noninvasive method: Extraction and measurement of fecal metabolites of estradiol and progesterone, using the technique of enzymeimmunoassay, It was also verified the possible differences between the monthly mean concentrations of these metabolites in a period of 12 months. 8 captive adult females, were studied in five different institutions in the state of São Paulo, where feces were collected three times per week for each individual for 12 months for extraction and measurement of metabolites of progesterone, estradiol and glucocorticoids. The profile of the concentration of fecal metabolites of sexual hormones from 6 of 8 females was very similar, being the period of major reproductive activity between the months of July, August and September, with the occurrence of more than one ovulatory cycle within that period. In the others months of the year, despite some variations above the baseline, no evidence of reproductive activity were found. According to these characteristics, similar in most females of the study, we can suggest that the hoary fox is seasonal polyestrous with reproductive activity occurring in the months of July, August and September. These findings are very close to the observed in wild populations, where mating is observed between June and July. The profile of excretion of fecal glucocorticoid metabolites follows the same pattern observed for the metabolites of progesterone and estradiol, with a more significant increase in excretion in the months of July, August and September. These results indicate that besides the large amount of stressors that can alter the excretion of glucocorticoids also it must be considered the seasonal fluctuation and the reproductive status of the individual to evaluate the levels of glucocorticoids. Two females, who share the same captivity, did not show a pattern of reproductive cyclicity. The results of this study indicate that the dosage of fecal metabolites of estradiol and progesterone can be used to differentiate the reproductive period of non-reproductive period in females of Lycalopex vetulus, providing important information about the reproductive biology, which may contribute to the development of the species conservation strategies, such as increasing the reproductive ex situ success.

Devido as atuais mudanças globais é esperado que um grande número de espécies necessitem de uma integração de ações dentro e fora do seu ambiente natural para a conservação. O bioma cerrado é um dos ecossistemas mais ricos em biodiversidade, mas devido a ação antrópica, resta menos de 20% de sua cobertura vegetal original. A Raposinha do Campo (Lycalopex vetulus) é um canídeo de pequeno porte (2,5-4kg) endêmico do cerrado do Brasil central e está presente na lista dos animais ameaçados de extinção do Estado de São Paulo. Não existe em literatura uma descrição detalhada do ciclo estral desta espécie. O presente estudo teve como objetivo caracterizar a ciclicidade ovariana de Lycalopex vetulus com o uso de método não-invasivo: Extração e mensuração de metabólitos fecais de progesterona e estradiol, com o uso da técnica de enzimaimunoensaio, verificando também a possível existência de diferenças mensais entre as médias das concentrações dos referidos metabólitos em um período de 12 meses. Foram utilizadas 8 fêmeas, adultas, presentes em cinco instituições no estado de São Paulo, onde foram coletadas fezes 3 vezes por semana de cada indivíduo, durante 12 meses, para a extração e mensuração dos metabólitos de progesterona, estradiol e corticosterona. Os perfis das excreções dos metabólitos fecais dos hormônios sexuais de 6 das 8 fêmeas, foram muito semelhantes, sendo período de maior atividade reprodutiva entre os messes de julho, agosto e setembro, ocorrendo mais de um ciclo ovulatório dentro desse período. Nos outros meses do ano, apesar de algumas variações acima da linha basal, não foram encontrados indícios de atividade reprodutiva. De acordo com essas características, semelhante entre a maioria das fêmeas do estudo, podemos sugerir que a raposa-do-campo seja poliestrica sazonal, com atividade reprodutiva ocorrendo nos meses de julho, agosto e setembro. Esses achados são muito próximos do visualizado em populações de vida livre, onde acasalamentos são observados entre junho e julho. O perfil de excreção dos metabólitos fecais de glicocorticóides segue o mesmo padrão observado para os metabólitos de progesterona e estradiol, com um aumento mais significativo da excreção nos meses de julho, agosto e setembro. Esses resultados indicam que além da grande quantidade de estressores que podem alterar a excreção de glicocorticóides, também deve-se considerar a flutuação sazonal e o status reprodutivo do indivíduo ao avaliar as concentrações de metabólitos de glicocorticóides. Duas fêmeas, que dividem o mesmo recinto, não apresentaram um padrão de ciclicidade reprodutiva. Os resultados obtidos nesse estudo indicam que a dosagem de metabólitos fecais de progesterona e estradiol podem ser usadas para diferenciar o período reprodutivo do período não reprodutivo em fêmeas de Lycalopex vetulus, fornecendo informações importantes sobre a biologia reprodutiva da espécie, o que pode contribuir no desenvolvimento de estratégias para a conservação desta espécie, como por exemplo aumentar o sucesso reprodutivo ex situ. / Due to the current global changes is expected that a large number of species require an integration of actions inside and outside their natural environment for them conservation. The cerrado is one of the richest ecosystems in biodiversity, but due to human action, there remains less than 20% of its original vegetation cover. The Hoary fox (Lycalopex vetulus) is a small canid (2,5-4kg) endemic of the cerrado from the central Brazil and is present in the list of endangered species in the state of São Paulo. It was not found in literature any detailed description of the estrous cycle of this species. The present study aimed to characterize ovarian cyclicity in Lycalopex vetulus using a noninvasive method: Extraction and measurement of fecal metabolites of estradiol and progesterone, using the technique of enzymeimmunoassay, It was also verified the possible differences between the monthly mean concentrations of these metabolites in a period of 12 months. 8 captive adult females, were studied in five different institutions in the state of São Paulo, where feces were collected three times per week for each individual for 12 months for extraction and measurement of metabolites of progesterone, estradiol and glucocorticoids. The profile of the concentration of fecal metabolites of sexual hormones from 6 of 8 females was very similar, being the period of major reproductive activity between the months of July, August and September, with the occurrence of more than one ovulatory cycle within that period. In the others months of the year, despite some variations above the baseline, no evidence of reproductive activity were found. According to these characteristics, similar in most females of the study, we can suggest that the hoary fox is seasonal polyestrous with reproductive activity occurring in the months of July, August and September. These findings are very close to the observed in wild populations, where mating is observed between June and July. The profile of excretion of fecal glucocorticoid metabolites follows the same pattern observed for the metabolites of progesterone and estradiol, with a more significant increase in excretion in the months of July, August and September. These results indicate that besides the large amount of stressors that can alter the excretion of glucocorticoids also it must be considered the seasonal fluctuation and the reproductive status of the individual to evaluate the levels of glucocorticoids. Two females, who share the same captivity, did not show a pattern of reproductive cyclicity. The results of this study indicate that the dosage of fecal metabolites of estradiol and progesterone can be used to differentiate the reproductive period of non-reproductive period in females of Lycalopex vetulus, providing important information about the reproductive biology, which may contribute to the development of the species conservation strategies, such as increasing the reproductive ex situ success.

The Asian elephant (Elephas maximus), a charismatic ‘flagship species’, is threatened by extinction in the wild, and the development of self-sustainable captive populations is a key conservation challenge. A third of the Asian elephant population is presently in captivity and information on the reproductive status, especially in females, is still lacking to a large extent. The onset of estrus in female Asian elephants is not associated with any visible physical signs, thus making the assessment of the reproductive status rather difficult. One approach to understanding reproductive cyclicity of animals is through generating profiles of reproductive hormones in blood (Wiseman et al. 1983; Brown et al. 1999, 2004). Profiles of reproductive hormones such as progesterone (P4), estrogen, luteinizing hormone and follicle stimulating hormone have already being demonstrated in Asian elephants (Brown et al. 1999, 2004; Brown 2000). In these studies, the reproductive status of females, maintained in zoos/captivity, was characterized based on circulating levels of hormones in blood samples. This is difficult to implement in the case of semi-captive or wild populations of elephants due to practical, legal and ethical considerations. In order to overcome this problem and to better understand the estrous status of female elephants, it is important to develop and validate non-invasive methods to monitor the reproductive status of female Asian elephants. An alternative approach to evaluating the reproductive status of females is to consider behavioral responses shown by males towards chemical signals produced by females to advertise their reproductive status. In order to understand the reproductive status of the individual belonging to the opposite sex, studies have shown that elephants rely on a variety of chemical signals produced in biological fluids such as urine, temporal gland secretion, inter-digital gland secretion, etc. (Krishnan 1972; Rasmussen & Schulte 1998). Chemical signaling is one of the prominent modes of communication in elephants, especially with respect to locating potential mates (Sukumar 2003). Thus, in most cases, elephants usually employ specific behavioral responses, for example sniff, check and place behaviors of trunk, for investigating the reproductive status of the conspecific individual, belonging either to the same or the opposite sex (Rasmussen et al. 1996; Schulte & Rasmussen 1999; Bagley et al. 2006). The objectives of the thesis are two-fold. First, to develop a non-invasive method of reproductive monitoring from fecal hormonal metabolites and also to understand the possible role of feces as an inter-sexual signal. The main body of thesis is divided into four chapters. 1) Development and validation of a non-invasive method to estimate progesterone metabolite in feces, to monitor the reproductive cyclicity of female elephants (Chapter 2). 2)Generation and characterization of progesterone and its metabolite, 5α-P-3-OH, profiles of semi-captive females using the developed non-invasive method to measure fecal metabolites (Chapter 3). 3) Validation of developed methodology and assay systems to a wild-population of female elephants (Chapter 4). 4) Feces as a potential source for inter-sexual chemical signaling in Asian elephants (Chapter 5). The above studies were carried out on semi-captive male and female elephants maintained in the forest camps of Mudumalai Wildlife Sanctuary (MWLS), Tamil Nadu and Bandipur National Park (BNP), Karnataka, India (Chapters 2, 3 and 5). For Chapter-4, free-ranging females of the MWLS were examined. 1. Development and validation of a non-invasive method to estimate progesterone metabolite in feces, to monitor the reproductive cyclicity of female elephants Niemuller et al. (1993) generated a profile of the progesterone metabolite, 5βpregnanetriol, to assess the estrous phase of Asian elephants based on non-invasive urine sampling. However, the collection of urine is difficult and to some extent impossible in the case of semi-captive and as well as that of wild elephants. Thus, the method of choice in this study was the development and validation of a non-invasive approach to measure fecal progesterone metabolites to assess reproductive status of females. Sampling was carried out at monthly intervals on three female elephants at the MWLS forest camp, while three other females maintained at the BNP forest camp were sampled fortnightly. An enzyme linked immuno-sorbent assay was developed to measure the concentration of the progesterone metabolite, 5α-P-3OH in the fecal samples of the semi-captive females. Using varying concentrations of the hapten (5α-P3OH), from low (0.1 mM) to high (1 mM), a standard curve was first generated, which had a linear range between 0.25 mM to 62.5 mM, with an EC50 of 1.37 mM. The linear range was then used to detect the concentrations of 5α-P-3OH in the fecal samples of females examined. The non-invasive method was further validated as there existed a positive correlation (p<0.1) between the levels of fecal 5α-P-3OH and that of concentration of circulating P4, measured in blood samples. This is the first valid documentation of a non-invasive method based on fecal progesterone metabolite pattern in order to assess the reproductive status of the female Asian elephants. 2. Generation and characterization of reproductive hormone profiles of semi-captive females using the developed non-invasive method to measure fecal metabolites With the establishment of a non-invasive method to understand occurrence of estrus in female elephants (described in Chapter 2), attempts were made to generate hormonal profiles over a longer time interval through more frequent sampling. Based on sampling at weekly intervals, the concentrations of both fecal 5α-P-3OH and that of native P4 hormone in the blood were determined, and the females were then identified as belonging to different reproductive states of hormonal cycling (follicular and luteal phases), non-cycling and pregnant categories. Of the 7 females sampled at both MWLS and BNP, three distinct categories of hormone-metabolite profiles emerged. The first category included four females that showed regular cyclicity throughout the sampling period, as monitored through the measurement of fecal 5α-P-3OH and serum P4 levels. The estrous cycle of all the four females was divided into two phases (follicular and luteal), based on the patterns of fecal 5α-P-3OH and serum P4 concentrations. The follicular phase of the estrous cycle was assigned when the values of both fecal 5α-P-3OH and serum P4 remained below 0.3 µg/gm and 0.3 ng/ml, respectively, for a considerable time period (viz. >2 wk). However, the luteal phase was characterized, when the levels of fecal 5α-P-3OH and serum P4 remained at or above 0.3 µg/gm and 0.3 ng/ml respectively, over a period of more than 2 - 3 wk. The second category had two females showing a ‘flat-lining pattern’ for the levels of fecal 5α-P-3OH and serum P4 concentrations, without any peak or dip in their concentrations. Since the reproductive pattern for both these females was flat-lined throughout their sampling period (51 wk), maintaining the levels of fecal 5α-P-3OH and serum P4 below 0.3 µg/gm and 0.3 ng/ml respectively, the females were considered to be non-cycling or anestrus. The third category had one female in which the levels of both fecal 5α-P-3OH and serum P4 measured were consistently high throughout the entire sampling period (26 wk). The levels of both fecal -P-3OH and serum P4 were above 0.3 µg/gm and 0.3 ng/ml, respectively. At the end of the sampling period, this female delivered a male calf; thus, the measured concentrations of fecal 5α-P-3OH and serum P4 signified the levels maintained during the gestational phase of this female. Sampling and hormonal analyses were also carried out for a male in the MWLS forest camps to investigate the baseline concentrations of fecal 5α-P-3OH and serum P4. The male showed consistently low concentrations of both fecal 5α-P-3OH and serum P4 throughout the sampling period (16 wk), below the margin of 0.3 µg/gm and 0.3 ng/ml, respectively. In this part of the study, it was confirmed that the reproductive status of a female elephant can be correctly assessed on the basis of measurements of fecal 5α-P-3OH alone, with repeated sampling of the female over a longer time scale. It was also shown that the strength of the positive correlation between the concentrations of the fecal 5α-P3OH and the serum P4 increased (p<0.01) for a larger sample size than that obtained for a comparatively smaller sample (described in Chapter 2). 3. Validation of developed methodology and assay systems to wild-population of female elephants So far, the non-invasive method to estimate fecal progesterone metabolite in order to predict occurrence of estrus of elephants was largely applied to semi-captive females (described in Chapters 2, 3). However, the necessity of such a method is being recognized for assessing the reproductive status of free-ranging females. Several findings have described differences in the rate and type of steroid metabolite excretion among individuals maintained under different diet regimes (Wasser et al. 1993; Smith et al. 2006). For instance, female elephants in the forest camps are provided with supplementary diet consisting of sugarcane, rice and millets. This diet is strikingly different from the feeding materials consumed by wild/free-ranging elephants (Sukumar 2003). Therefore, differences in dietary components can potentially influence the fecal steroid metabolites’ excretory patterns shown by semi-captive vis-a-vis wild females, which can affect the validity of measuring fecal 5α-P-3OH to predict females’ reproductive status. In order to examine this problem, the non-invasive method was applied to the population of wild elephants in the forests of MWLS, through random one time sampling of 30 individual female elephants. The steroid extraction efficiency (73 ± 11.0%, mean ± S.D., n =30) determined for the fecal samples collected from the wild females was not significantly different from the coefficient calculated in the case of the semi-captive females (80 ± 4.3%, mean ± S.D., n = 38). This indicated that dietary differences between wild and captive elephants did not influence levels of fecal hormonal metabolites in feces, unlike earlier observations on baboons (Wasser et al. 1993), old world primates (Wasser et al. 1988) and sheep (Smith et al. 2006). The values of the fecal allopregnanolone determined in the case of the wild females, ranged from as low as 0.06 µg/gm to as high as 23µg/gm of the sample, thus showing the heterogeneity of the samples, indicating that the females may be belonging to different reproductive phases. However, since sampling was carried out randomly, with an adult female being sampled just once, at this stage, it is not possible to identify or elaborate on the reproductive phase of the females. This is the first study reporting the values of the fecal progesterone metabolite in female Asian elephants in the wild. Further studies may be required to carry out long term monitoring of the wild females, through repeated collection of fecal samples over time from particular female. 4. Feces as a potential source for inter-sexual chemical signaling in Asian elephants In this Chapter-5, behavioral trials were conducted on male elephants to understand the role of fecal matter in conveying inter-sexual chemical signals. This was demonstrated by analyses of specific chemosensory behavioral responses shown by males towards the fecal samples of females that were strangers and belonging to different reproductive stages. Males showed four prominent behavioral responses namely ‘distant sniff’, ‘close sniff’, ‘check’ and ‘place’ towards the fecal samples of females. The sum of frequencies of these four responses (distant sniff, close sniff, check and place) was much higher for samples of the follicular (pre-ovulatory) phase females as compared to those of the luteal (post-ovulatory) phase females (p<0.005). Thus, for the first time, it was shown that male elephants are able to discriminate the different reproductive phases of females based on their specific behavioral response towards the fecal samples of the opposite sex. In conclusion, the thesis has focused on understanding and providing new insights regarding the reproductive biology of the female Asian elephants. This has been achieved through the development of the non-invasive method based on measuring the concentrations of the fecal progesterone metabolite and through the analyses of the chemosensory responses performed by the males towards the fecal samples of strange females. These methods can potentially be applied to the populations of both wild and captive/semi-captive female elephants in order to evaluate their reproductive status, through non-invasive measures. The information derived from the application of such methods will help in understanding the reproductive potential of the wild elephants under various environmental and ecological conditions. Further, the non-invasive measurement of reproductive hormones will help in monitoring the reproductive state of the individuals and thus aid in planning strategies for the welfare and management of the elephants maintained in captive or semi-captive conditions.

Treatment success in IVF-ICSI is mainly limited by female age, but differences in ovarian reserve (OR; the remaining pool of oocytes and their quality) between individuals modify treatment prerequisites among women of similar age. OR may be assessed by OR tests (ORTs). The main aims of this work were to study menstrual cycle length (MCL), basal levels of circulating gonadotrophins, antral follicle count (AFC) and serum Anti-Müllerian hormone (AMH) levels and their associations with and prognostic capacities regarding IVF-ICSI outcome in large cohorts of unselected women. Age-adjusted MCL was positively and linearly associated with pregnancy rates (PRs), live-birth rates (LBRs) and ovarian response to controlled ovarian hyperstimulation. An MCL of &gt;34 days almost doubled the LBR compared with an MCL of &lt;26 days. The grouped variable ‘combined FSH and LH levels’ was superior to both individual gonadotrophin levels and the LH:FSH ratio. The highest mean PR was seen in connection with a combination of FSH &lt;6.7 U/l with LH &gt;4.9 U/l; PRs were lowest when FSH-LH levels were opposite to this (high-low) and intermediate when FSH-LH levels were low-low or high-high. Associations with LBR and ovarian response were similar as those for PR. AFCs and serum AMH levels were positively and log-linearly associated with PR, LBR and ovarian response. Success rates levelled out above AFC 30 or AMH 5 ng/ml. Treatment outcome was superior among women with polycystic ovaries. Among the studied ORTs, logAFC and logAMH concentration correlated most strongly. After multivariate testing, entering all studied ORTs, AMH and female age remained independently associated with LBR. AMH + AFC + age predicted both poor and excessive ovarian responses with high accuracy. Adjusting for age and oocyte yield, all ORTs remained significant for LBR, implying that ORTs also capture information on oocyte quality. In conclusion, measures of OR are strongly associated with PR, LBR and ovarian response in a log-linear fashion, and partly reflect oocyte quality. The OR spectrum is continuous, from small ‘oligofollicular’ ovaries (the low extreme) to polycystic ovaries (the high extreme). Among the studied ORTs, AMH together with age provide the most powerful basal estimate for IVF/ICSI outcome.