Various ecological and human-induced disturbances play an important role in defining the health of an animal. To cope up with such threats or challenges to its homeostasis, an animal responds by secreting stress hormones (glucocorticoids) to mobilize the energy. In general, glucocorticoids help animals to cope up with the ‘stressors’. Prolonged exposure to any ‘stressors’ may lead to an elevation in the levels of glucocorticoids, thereby leading to reproductive inhibition, immune system suppression, neuronal death and impaired cognitive function and hence, threatening the survival and fitness of an animal. However, the long-term effects of environmental or anthropogenic disturbances on the fitness and survival are difficult to analyze in a slow reproducing, long-lived species such as elephants. Spread over 3% of India’s geographical area, India shelters around 28,000 to 30,000 Asian elephants (Elephas maximus; Bist, 2002; Santiapillai and Sukumar, 2006). Protected areas in India are estimated to form 22% of elephant habitat; while the rest of the elephant habitats extend outside the protected areas (Lenin and Sukumar, 2011) making them vulnerable to human-induced threats. Consequently, there are increased incidences of human-elephant conflicts leading to crop and property depredation and human-elephant mortality. Most of the studies, in context of such conflicts, highlight the temporal, spatial scales of conflicts majorly focusing on mitigation measures. None of the studies so far have elucidated how escalating human-elephant conflicts would influence the physiological health of Asian elephants. This study, hence, focused on addressing the proximate causation of the stress-response in free-ranging Asian elephants of the Bandipur National Park, the Nagarahole National Park and Hassan district of Karnataka, using a non-invasive technique for measuring faecal glucocorticoid metabolites (fGCM). There are several factors which could act as possible stressors to the physiological state of an elephant, but depending upon the feasibility and time limitations of conducting the study, we selected those factors which we thought are essential to address the proximate causation of stress-response in Asian elephants. Being the first detailed study to explore the stress-response in free-ranging Asian elephants, this study attempted to understand the influence of some of the fundamental ecological (seasonality, group size, and body condition) and human-induced stressors. The main body of the dissertation is divided into six chapters. The first and the last chapters discuss the general introduction and conclusion while the rest four chapters highlight the main four objectives of the study. The main objectives of the thesis were: 1. The first objective of this thesis was to validate and standardize some of the crucial parts of protocols to avoid any technique-based bias while interpreting the levels of faecal glucocorticoid metabolites. Under the first objective, the influences of some of the fundamental intrinsic and extrinsic factors on the fGCM levels were also assessed. 2. The second objective was to assess the association between body condition, seasonality and stress response. 3. Having assessed the relationship between body condition and stress-status, the third objective focused on the influence of socio-ecological correlates such as group size, lactational status and the presence of adult females in a herd on the stress status of female adult elephants. 4. With above three objectives majorly highlighting the influence of ecological factors, the fourth objective was to assess the influence of anthropogenic factors on stress-response of free-ranging elephants under which we compared the stress-response between crop-raiding in human-dominated and nonraiding elephants in forested habitats. The study was conducted during dry season (February to May) and wet season (August to December) of 2013 and 2015. The dissertation is organized in the following way: Chapter 1: General Introduction The first chapter provides a general introduction about stress response and its physiological pathway. It also discusses the importance of non-invasive technique used in our study. It highlights the significance of this study based on the available literature on both African and Asian elephants. Chapter 2: General validation and standardization of field techniques and assays; influence of intrinsic and extrinsic factors on faecal glucocorticoid metabolites This chapter is divided into two parts. The first part deals with general validation and standardization of field techniques and the second part assesses the influence of fundamental intrinsic and extrinsic factors. Being the first study on stress-response of free-ranging Asian elephants, influence of various field-based techniques and variations were studied to facilitate the reliable interpretation of fGCM levels. We first conducted the experiments to standardize the field protocol and the laboratory protocol. Under the field protocol, we found that there was no within sample variation (as observed in many other species) in fGCM levels from thoroughly mixed faecal samples and samples from the center of the same dung bolus. The time of collection of samples had a significant influence, suggesting that the concentration of fGCM remains stable until 6-9 hours. Influence of storage of lyophilized samples suggested that the concentrations of fGCM levels decline if exposed to moisture. Under the laboratory protocol, A group specific 11-oxoetiocholanolone EIA, used for analyzing fGCM levels (μg/g), was modified to enhance the repeatability, accuracy and precision. EIA was analytically validated in every hormonal analysis by calculating the inter- and intra-assay coefficients of variation, specificity and parallelism. In the second part, we assessed the influence of age, sex and season on fGCM levels and found there was no age-wise variation and sex-wise variation. Season-wise variation was significant only in female elephants. We discuss the potential physiological reasons behind such variation. These findings suggest that aspects such as time of collection, proper storage of samples and the influence of extrinsic stresses (seasonality) should be considered for reliable and unbiased interpretation of fGCM levels. Chapter 3: Assessment of season-dependent body condition scores in relation to faecal glucocorticoid metabolites in free-ranging Asian elephants. (Published in Conservation Physiology; Pokharel et al., 2017; doi:10.1093/conphys/cox039) We studied seasonal and annual changes in visual body condition scores (BCS), and assessed how these scores were related to levels of fGCM levels in free-ranging Asian elephants in the seasonally dry tropical forests of the Mysore and Nilgiri Elephant Reserves in southern India. We assessed the animals’ BCS visually on a scale of 1 to 5; where 1 represents a very thin and 5 represents a very fat elephant. To understand the influence of seasonality on BCS, we sampled the population during dry and wet seasons of 2013 and 2015 while, for annual changes in BCS, we sampled nine free-ranging adult females from different family groups that had been repeatedly sighted over seven years. To evaluate the influence of body condition on fGCM, we measured the fGCM levels from fresh fecal samples collected from the body-condition scored animals. Effect of age and season on BCS in relation to fGCM was also studied. We found that the BCS was related with season, i.e. individuals with low BCS were more frequent during the dry season as compared to the wet season and fGCM levels were negatively associated with BCS. To be precise, concentrations of fGCM were highest in individuals with the lowest BCS (BCS-1) and then significantly declined till BCS-3. fGCM levels were almost comparable for BCS 3, 4 and 5. This pattern was more conspicuous in female than in male elephants. We discussed the possible reasons behind such relationship between fGCM and BCS. The findings from this chapter suggest that season-dependent BCS, hence, reflect the stress status as measured by fGCM, especially in female Asian elephants and could be used as an important non-invasive approach to monitor the physiological health of free-ranging elephant populations. This is the first study to compare the body condition scores and stress-response in free-ranging elephants. Chapter 4: Influence of ecological and physiological correlates on stress physiology of free-ranging female Asian elephants. We investigated the influence of herd size (HS), lactational status and number of adult females present in a herd on adrenocortical function in free-ranging adult female Asian elephants by measuring their fecal glucocorticoid metabolites (fGCM) levels. We found that there was a negative association between herd size and fGCM levels when only HS and fGCM were compared. Lactating females showed higher fGCM than that observed in non-lactating females, which might reflect the nutritional stress on mother and anti-predatory challenges against the calves. Herds with only one adult female had higher fGCM levels than herds with more than one adult female which could be because of social challenges. The poor association between herd size and fGCM when all predictor variables were combined elucidate that group size may not necessarily influence the adreno-cortical function. However, the numbers of adult females present in a herd and their lactational status has a significant role in defining the stress-response in free-ranging adult female Asian elephants. This study is the first study to document the influence of lactational status with the stress-response in free-ranging elephants. Chapter 5: How physiologically costlier it is to be a crop-raider in a human-dominated landscape? Diet quality as a possible ‘pacifier’ against stress. We studied the stress-response in crop-raiding elephants in a human-dominated landscape and non-raiding elephants in the protected forested areas. While raiding agricultural crops, elephants face various associated threats such as retaliation by humans, human-induced disturbances and stress of raiding which could enhance the energetic costs, ultimately elevating their stress levels. We hypothesized that crop-raiders (in human-dominated landscape; Hassan district of Karnataka, India) will exhibit higher faecal glucocorticoid metabolite (a proxy of stress-response; fGCM) levels as compared to the nonraiders (in forested areas; Bandipur and Nagarahole National Parks). Contrary to our hypothesis, fGCM levels were found to be higher in both nonraiding female and male elephants than female and male crop-raiders. To assess the influence of benefits obtained from crop-raiding as one of the possible factors for the lower fGCM in crop-raiders, the difference in vegetation between human-dominated landscape and forested areas were further analyzed by using a remotely sensed NDVI (Normalized Differential Vegetation Index) and was ground-truthed by measuring the quality of diet (C:N ratio through fecal samples). Interestingly, the NDVI values were higher at the human-dominated and the C:N ratio was lower (higher Nitrogen content) in faecal samples of crop-raiding elephants (both in females and in males) than the nonraiders in the forested areas. Positive correlation was observed between C:N ratio and fGCM levels. These findings suggest that crop-raiding comes with the benefits of easy access to good quality of diet which may help in reducing the stress-response in elephants while being in the human-dominated landscapes, provided there is a low intensity of human disturbance. This is the first study to document and compare the stress-status of crop-raiding Asian elephants with elephants in their natural habitats using NDVI and C:N ratio. Chapter 6: Conclusion In conclusion, the work presented in this dissertation provides an overview of how ecological and anthropogenic factors could influence the physiological health of free-ranging Asian elephants. This dissertation focused on providing detailed insights about the stress-response in free-ranging Asian elephants. The objectives have been achieved by assessing the relationship between ecological stressors such as seasonality, body condition, herd size, lactational status, a presence of individuals in a herd and anthropogenic stressors such as human-induced threats or crop-raiding using well-validated and standardized laboratory and field protocols. This study provides valuable insights into the physiological health or stress-response being synergistically influenced by various ecological, social and anthropogenic factors in free-ranging Asian elephants. Findings obtained from this study could help in addressing the issues related to the management of free-ranging elephant populations.

Earlier studies investigated the genetic structure of fragmented or isolated elephant populations by comparing the genetic characteristics of pre-defined populations. This study aimed to determine if there was genetic evidence for spatial structuring in a continuous elephant population in the Kavango-Zambezi Transfrontier Conservation Area (KAZA-TFCA). I sequenced one mtDNA gene region for 88 individuals and genotyped 100 individuals for 10 nuclear microsatellite loci. Bayesian Clustering Algorithms incorporated in the program Geneland were used to identify groups of genetically similar individuals. An Analysis of Molecular Variance (AMOVA) determined if these groups (henceforth referred to as subpopulations) were significantly differentiated. I used a Geographic Information System (GIS) landscape genetic toolbox to identify areas in the landscape with high genetic divergence between individual samples to determine if there were identifiable genetic barriers in the landscape. There were three significantly differentiated mtDNA sub-populations (Fst = 0.787), and two nDNA sub-populations that were not significantly differentiated (Fst = -0.02; Rst = -0.045), implying obstructed mtDNA, but high nDNA gene flow across the study region. Also, gene flow was apparent between Chobe and Kafue National Parks, where telemetry data has as of yet not recorded inter-population movements between these parks. The three mtDNA sub-populations were geographically differentiated and followed political boundaries as apparent sub-populations in Botswana, Zambia and Zimbabwe. The differences between mtDNA and nDNA genetic structuring may be explained by i) historical events that shaped the current genetic structure (e.g. through founder-effects and persistent poaching hotspots) and ii) intrinsic variables that influence genetic structure at a local scale (e.g. through resource dependencies and social behaviour). The KAZA elephant population has a genetic diversity (mtDNA diversity as the pairwise number of differences (π) = 2.59; nDNA diversity as the mean alleles/locus and He = 7.5, 0.71) higher than other southern African populations, and inter-population movements may be responsible for maintaining this genetic diversity. I recommend continued support for conservation initiatives that aim at maintaining and restoring connectivity between populations through landscape linkages, which in so doing may ensure inter-population gene flow and uphold the current genetic state of the KAZA-TFCA elephant population. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Zoology and Entomology / unrestricted

ŠIMKOVÁ, D. Reproduction of an African elephant (Loxodonta africana) in a Zoo Zlín – Lešná. Mendel University in Brno, 2019. Diploma thesis. The thesis analyzes the issue of reproduction of critically endangered species of African elephant. The work is focused on monitoring of animal weight and blood test results during the reproductive cycle. The first part describes the general characteristics of the reproduction cycle of the African elephant, the issue of pregnancy and childbirth, and the female reproductive system. In the second part, there are given data from three particular elephants from the Zoo in Zlín. The data are processed into graphs and these are then commented on and compared with the conclusions of already conducted studies and scientific papers. At the end, all the knowledge and forecast of the African elephant reproduction in the Zoo - Lešná in the future are evaluated. All the results show that females are fully capable of reproduction and there is no need to make any changes in the breeding of these animals.

A research report submitted to the Faculty of Science, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Masters of Science (Environmental Sciences). September 2015. / African elephants (Loxodonta africana) are still being poached and hunted and this could potentially affect their movement. As a keystone species, crucial to maintaining ecosystem function and therefore food webs, it is important to understand the effects hunting may have on their movements. This study was undertaken in the Associated Private Nature Reserves adjacent to the Kruger National Park, where elephants are hunted. I looked at changes in daily displacement distance and distance from hunting and natural mortality events to determine if elephants were affected by hunting events, up to what distance, and to determine whether elephants reacted in the same way to hunting and natural mortality events. I found that elephants are only affected by hunting events when within 1 km of the event, which is consistent with the literature. When considering natural mortality events, they actually diverted movements towards the event location, which is also consistent with literature. These results, however, are based on a very small sample size. In addition, the time of day of the events was not recorded by the wardens of the reserve, and this provided a further limitation for the data analysis and interpretation.

Philosophiae Doctor - PhD / The survival of free-ranging (in situ) African elephant and Southern white rhinoceros populations are currently being challenged on a daily basis in Africa. Reproductive health is considered a vital component of species conservation. Conservation of the last mega land mammals may ultimately require intervention by breeding management or combined with assisted reproductive technologies (ART). There is a strong case for gathering baseline information, both physiological and biological, of any species, as opportunities arise. During this study a total number of 21 ejaculates collected over two seasons from 12 free-ranging African elephant bulls were characterised, as well as 10 ejaculates collected from 10 free-ranging Southern white rhinoceros bulls from two populations. Ejaculates were collected from adult bulls by means of electroejaculation under anaesthesia. Routine semen analysis was combined with Computer-aided sperm analysis (CASA), Computer-aided sperm morphology analysis (CASMA), Transmission electron microscopy (TEM) and Genomics as diagnostic tools. Additionally, sperm functionality within different media was investigated and sperm subpopulation classification according to the motion pattern displayed. The results presented is based on the evaluation and classification of ≈ 45 000 individual African elephant spermatozoa and ≈ 18 000 individual Southern white rhinoceros spermatozoa. The average elephant ejaculate contained a total number of 47 x 10⁹ spermatozoa (volume of 56 ± 38mL x concentration of 818 ± 750 x 10⁶/mL) that recorded a total motility of 81 ± 29% of which 62 ± 26% were progressively motile. CASA recorded velocities for curvilinear velocity (VCL 241 ± 58μm/s), straight-line velocity (VSL 173 ± 181μm/s) and average path velocity (VAP 201 ± 54μm/s), and kinematics at straightness of track (STR 86 ± 85%), linearity of track (LIN 67 ± 16%), amplitude of lateral head displacement (ALH 4 ± 0.75μm) and beat cross frequency (BCF 21 ± 3Hz). Structural analysis revealed 68 ± 11% of the spermatozoa were viable (intact plasma membrane) and 77 ± 11% maintained acrosome integrity. Ejaculates contained 55 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 6.83 ± 0.26μm and width 3.32 ± 0.18μm (total head area of 20.17 ± 1.96μm²) of which 38.95 ± 0.92% is covered by an acrosomal cap. The average rhinoceros ejaculate contained a total number of 1.1 x 10⁹ spermatozoa (volume of 24 ± 24mL x concentration of 83 ± 96 x 10⁶/mL) that recorded a total motility at 82 ± 8% of which 28 ± 23% were progressively motile. CASA recorded velocities for VCL (85 ± 29μm/s), VSL (44 ± 25μm/s) and VAP (69 ± 30μm/s, and kinematics at STR (63 ± 14%), LIN (51 ± 16%), ALH (2 ± 0.16μm) and BCF (16 ± 6Hz). Structural analysis revealed 73 ± 10% of the spermatozoa were viable (intact plasma membrane) and 76 ± 4% maintained acrosome integrity. Ejaculates contained 62 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 5.5 ± 0.17μm and width 2.9 ± 0.19μm (total head area of 14.8 ± 1.43μm²) of which 36.3 ± 0.59% is covered by an acrosomal cap. Based on a Boolean argument and CASA data exploration it was possible to derive elephant and rhinoceros CASA cut-off criteria to sort between activated and hyperactivated motile spermatozoa. For the genomic component of this study, the CatSper1 (Loxodonta africana) gene was identified,sequenced and verified in a free-ranging (natural) African elephant population. Multivariate analysis(MVA) was applied to examine the associations between the semen and sperm parameters and the traits they accounted for in this study. Our understanding of wildlife reproductive sciences can substantially progress as the analytical techniques applied and the combination thereof is expanded. This investigation presents a new set of comprehensive semen and sperm threshold values for future investigations.

The aim of the study was to determine the consequences of elephant (Loxodonta africana) impact on selected nesting sites of avian fauna and other species in the Associated Private Nature Reserves. The study also aimed at answering key questions on how the architecture of trees influence nest site suitability and what landscape features affect nest site location. Furthermore the type of impact that elephants have on specific nesting sites was determined and how this would affect the short term persistence of these trees. The facillitatory role of elephants was examined by looking at the type of impact that produces gum exudants as well as what gum is selected for by primates and whether primary branch breaking may lead to the creation of nesting sites for species such as the southern ground hornbill (Bucorvus leadbeateri). / Environmental Sciences / M. Sc. (Environmental Management)

Compromised welfare and wellbeing of elephants (Loxodonta africana and Elephas maximus) in captive facilities are significant and global problems. The period between birth and two years old is crucial for calf survival and social and environmental learning. Behaviour and developmental processes among captive elephant calves in these first years were compared with those seen in wild calves. Wild elephants calves develop within a complex, varied social context and provide one reference for normal patterns of development. Such comparisons enable insights into welfare at captive facilities. Eleven captive elephant calves born at three UK facilities were studied from birth to 18 months (AsianN=6; AfricanN=5). Older calves (AsianN=2; AfricanN=2) were also sampled up to 3.5 years; making a total of 15 calves studied from 2009 to 2014. Due to the small sample size, the 11 younger calves were also discussed as individual case studies. By 2017, only two of these case study calves were both alive and not orphaned. Three additional calves (AsianN=1; AfricanN=2) died on their day of birth and were not sampled. This small sample highlights the ongoing lack of self-sustaining populations of captive elephants. This thesis collated systematic behavioural observations on captive calves across 373 days (483.5hrs). Calf maintenance activities (feeding, resting, moving), associations with mother and others, interactions and calf play were compared with behavioural observations of wild AsianN=101 (74hrs, Uda Walawe, Sri Lanka) and wild AfricanN=130 (252hrs, Amboseli, Kenya) calves from ~birth to five yrs. Mothers’ (captive: AsianN=4; AfricanN=4; wild: AsianN=90; AfricanN=105) activities were also recorded to explore synchrony with calves. Captive calves raised by their mothers had similar activity budgets to those seen in the wild. Expected age-related declines in suckling were found in captivity. However, captive calves were more independent than wild calves for their age in distance from mother and spent significantly more time in play. A Decision Tree for whether to breed elephants in captivity was developed; benefits that a calf potentially brings to companions, e.g. multi-generational matrilineal groups, enabling social bonding and reducing abnormal behaviours, were considered against space required for families to grow and divide naturally over time, as well as ensuring that captive-bred males are socially sustained. It was recommended that facilities invest in future enclosure/housing designs which permit: free-access to other elephants; 24hr trickle feeding; juvenile males allowed to stay with their maternal group for longer, encouraging learning opportunities and further retaining age-structure/composition. Conversely, facilities unwilling to house a male or provide appropriate group size/composition are recommended to cease breeding.

The aim of the study was to determine the consequences of elephant (Loxodonta africana) impact on selected nesting sites of avian fauna and other species in the Associated Private Nature Reserves. The study also aimed at answering key questions on how the architecture of trees influence nest site suitability and what landscape features affect nest site location. Furthermore the type of impact that elephants have on specific nesting sites was determined and how this would affect the short term persistence of these trees. The facillitatory role of elephants was examined by looking at the type of impact that produces gum exudants as well as what gum is selected for by primates and whether primary branch breaking may lead to the creation of nesting sites for species such as the southern ground hornbill (Bucorvus leadbeateri). / Environmental Sciences / M. Sc. (Environmental Management)