endocrin

Brown, J.L., Kersey, D.C., Freeman, E.W., Wagener, T., 2009. Assessment of diurnal urinary cortisol excretion in Asian and African elephants using different endocrine methods. Zoo. Biol.
Abstract: Longitudinal urine samples were collected from Asian and African elephants to assess sample processing and immunoassay techniques for monitoring adrenal activity. Temporal profiles of urinary cortisol measured by RIA and EIA, with and without dichloromethane extraction, were similar; all correlation coefficients were >0.90. However, based on regression analyses, cortisol immunoactivity in extracted samples was only 72-81% of that of unextracted values. Within assay technique, RIA values were only 74-81% of EIA values. Collection of 24-hr urine samples demonstrated a clear diurnal pattern of glucocorticoid excretion, with the lowest concentrations observed just before midnight and peak concentrations occurring around 0600-0800 hr. These results indicate that elephants fit the pattern of a diurnal species, and that glucocorticoid production is affected by a sleep-wake cycle similar to that described for other terrestrial mammals. Cortisol can be measured in both extracted and unextracted urine using RIA and EIA methodologies. However, unexplained differences in quantitative results suggest there may be sample matrix effects and that data generated using different techniques may not be directly comparable or interchangeable. Zoo Biol 28:1-10, 2009. (c) 2009 Wiley-Liss, Inc

Mason, G.J., Veasey, J.S., 2009. How should the psychological well-being of zoo elephants be objectively investigated?
47. Zoo. Biol.
Abstract: Animal welfare (sometimes termed "well-being") is about feelings - states such as "suffering" or "contentment" that we can infer but cannot measure directly. Welfare indices have been developed from two main sources: studies of suffering humans, and of research animals deliberately subjected to challenges known to affect emotional state. We briefly review the resulting indices here, and discuss how well they are understood for elephants, since objective welfare assessment should play a central role in evidence-based elephant management. We cover behavioral and cognitive responses (approach/avoidance; intention, redirected and displacement activities; vigilance/startle; warning signals; cognitive biases, apathy and depression-like changes; stereotypic behavior); physiological responses (sympathetic responses; corticosteroid output - often assayed non-invasively via urine, feces or even hair; other aspects of HPA function, e.g. adrenal hypertrophy); and the potential negative effects of prolonged stress on reproduction (e.g. reduced gametogenesis; low libido; elevated still-birth rates; poor maternal care) and health (e.g. poor wound-healing; enhanced disease rates; shortened lifespans). The best validated, most used welfare indices for elephants are corticosteroid outputs and stereotypic behavior. Indices suggested as valid, partially validated, and/or validated but not yet applied within zoos include: measures of preference/avoidance; displacement movements; vocal/postural signals of affective (emotional) state; startle/vigilance; apathy; salivary and urinary epinephrine; female acyclity; infant mortality rates; skin/foot infections; cardio-vascular disease; and premature adult death. Potentially useful indices that have not yet attracted any validation work in elephants include: operant responding and place preference tests; intention and vacuum movements; fear/stress pheromone release; cognitive biases; heart rate, pupil dilation and blood pressure; corticosteroid assay from hair, especially tail-hairs (to access endocrine events up to a year ago); adrenal hypertrophy; male infertility; prolactinemia; and immunological changes. Zoo Biol 28:1-19, 2009. (c) 2009 Wiley-Liss, Inc

Steinetz, B., Lasano, S., de Haas van, D.F., Glickman, S., Bergfelt, D., Santymire, R., Songsassen, N., Swanson, W., 2009. Relaxin concentrations in serum and urine of endangered and crazy mixed-up species
66. Ann. N. Y. Acad. Sci. 1160, 179-185.
Abstract: The human population explosion has pushed many mammalian wildlife species to the brink of extinction. Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. We previously reported that serum immunoactive relaxin provided a reliable means of distinguishing between true and pseudopregnancy in domestic dogs, and this method has since been found to be a reliable indicator of true pregnancy in endangered Asian and African elephants and Sumatran rhinoceroses. Our canine relaxin radioimmunoassay (RIA) has now been adapted and validated to measure relaxin in the serum and urine of felids, including domestic and wild species. Moreover, a commercially available canine serum relaxin kit (Witness) Relaxin Kit; Synbiotics, San Diego, CA), has been adapted for reliable detection of relaxin in urine of some felid species. Our porcine relaxin RIA has also been utilized to investigate the role of relaxin in reproductive processes of the spotted hyena, a species in which the female fetuses are severely masculinized in utero. Indeed, this species might well now be extinct were it not for the timely secretion of relaxin to enable copulation and birth of young through the clitoris. Additional studies have suggested relaxin may be a useful marker of pregnancy in the northern fur seal and the maned wolf (the former species has been designated as "depleted" and the latter as "near threatened"). Given appropriate immunoassay reagents, relaxin determination in body fluids thus provides a powerful tool for conservationists and biologists investigating reproduction in a wide variety of endangered and exotic species

Thitaram, C., Pongsopawijit, P., Chansitthiwet, S., Brown, J.L., Nimtragul, K., Boonprasert, K., Homkong, P., Mahasawangkul, S., Rojanasthien, S., Colenbrander, B., van der Weijden, G.C., van Eerdenburg, F.J., 2009. Induction of the ovulatory LH surge in Asian elephants (Elephas maximus): a novel aid in captive breeding management of an endangered species
49. Reprod. Fertil. Dev. 21, 672-678.
Abstract: A unique feature of the reproductive physiology of Asian elephants (Elephas maximus) is the occurrence of two LH surges before ovulation, instead of one. An anovulatory LH (anLH) surge, the function of which is unknown, occurs consistently 3 weeks before the ovulatory LH (ovLH) surge that induces ovulation. Thus, the ability to induce an ovLH surge would be useful for scheduling natural mating or artificial insemination. The present study tested the efficacy of a gonadotrophin-releasing hormone agonist (GnRH-Ag) to induce LH surges during the follicular phase of the oestrous cycle, which resulted in varied LH responses, but generally none were as high as previously documented natural surges. Thus, for the ovulation-induction trials, nine females were administered 80 microg GnRH-Ag intravenously at three time periods during the oestrous cycle, namely the anovulatory follicular phase, the ovulatory follicular phase and the luteal phase. During the late anovulatory follicular phase, nine of 10 females (90%) responded with an immediate LH surge followed 15-22 days later by an ovLH surge or a post-ovulatory increase in progestagens. In contrast, despite responding to the GnRH-Ag with an immediate increase in LH, none of the females treated during other periods of the oestrous cycle exhibited subsequent ovLH surges. One cow got pregnant from natural mating following the induced ovLH surge. In conclusion, ovLH induction is possible using a GnRH-Ag, but only during a specific time of the anovulatory follicular phase

Tripp, K.M., Dubois, M., Delahaut, P., Verstegen, J.P., 2009. Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS
54. Theriogenology 72, 365-371.
Abstract: Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee

Gobush, K.S., Mutayoba, B.M., Wasser, S.K., 2008. Long-term impacts of poaching on relatedness, stress physiology, and reproductive output of adult female african elephants. Conserv. Biol. 22, 1590-1599.
Abstract: Widespread poaching prior to the 1989 ivory ban greatly altered the demographic structure of matrilineal African elephant (Loxodonta africana) family groups in many populations by decreasing the number of old, adult females. We assessed the long-term impacts of poaching by investigating genetic, physiological, and reproductive correlates of a disturbed social structure resulting from heavy poaching of an African elephant population in Mikumi National Park, Tanzania, prior to 1989. We examined fecal glucocorticoid levels and reproductive output among 218 adult female elephants from 109 groups differing in size, age structure, and average genetic relatedness over 25 months from 2003 to 2005. The distribution in group size has changed little since 1989, but the number of families with tusked old matriarchs has increased by 14.2%. Females from groups that lacked an old matriarch, first-order adult relatives, and strong social bonds had significantly higher fecal glucocorticoid values than those from groups with these features (all females R(2)= 0.31; females in multiadult groups R(2)= 0.46). Females that frequented isolated areas with historically high poaching risk had higher fecal glucocorticoid values than those in low poaching risk areas. Females with weak bonds and low group relatedness had significantly lower reproductive output (R(2)[U]=0.21). Females from disrupted groups, defined as having observed average group relatedness 1 SD below the expected mean for a simulated unpoached family, had significantly lower reproductive output than females from intact groups, despite many being in their reproductive prime. These results suggest that long-term negative impacts from poaching of old, related matriarchs have persisted among adult female elephants 1.5 decades after the 1989 ivory ban was implemented

Rasmssen, H.B., Ganswindt, A., Douglas-Hamilton, I., Vollrath, F., 2008. Endocrine and behavioral changes in male African elephants: Linking hormone changes to sexual state and reproductive tactics. Hormones and Behavior.
Abstract: Endocrine and behavioral changes in male African elephants: Linking hormone changes to sexual state and reproductive tactics.Henrik B Rasmussen, Andre Ganswindt, Iain Douglas-Hamilton, and Fritz VollrathHormones and Behavior, May 22, 2008
Hormones play a crucial role in mediating genetic and environmental effects into morphological and behavioral phenotypes. In systems with alternative reproductive tactics (ART) shifts between tactics are hypothesized to be under proximate hormonal control. Most studies of the underlying endocrine changes behind ART have focused on fish and amphibians rather than mammals and few have investigated the potential interaction between different endocrine axes in regulating shifts between conditional dependent tactics. Using a combination of endocrine and behavioral data from male African elephants we expand on our previously published analysis and show that the initial increase in androgens predates the behavioral shifts associated with reproductively active periods, supporting the role of androgens in activating sexually active periods in males. A strong interactive effect between androgens and glucocorticoids was found to determine the presence or absence of temporal gland secretion and urine dribbling, signals associated with the competitive reproductive tactic of musth, with elevated glucocorticoids levels suppressing the occurrence of musth signals. In addition external environmental conditions affected hormone levels. The presence of receptive females resulted in elevated androgens in dominant musth males but increased glucocorticoids in subordinate non-musth males. The presented data on hormones, behavior and reproductive tactics strongly support an underlying endocrine mechanism for mediating the translation of intrinsic as well as extrinsic local conditions into the conditional dependent reproductive tactics in male elephants via interactions between the hypothalamic-pituitary-gonadal and -adrenal axes.

Rasmussen, H.B., Ganswindt, A., Douglas-Hamilton, I., Vollrath, F., 2008. Endocrine and behavioral changes in male African elephants: linking hormone changes to sexual state and reproductive tactics. Horm. Behav. 54, 539-548.
Abstract: Hormones play a crucial role in mediating genetic and environmental effects into morphological and behavioral phenotypes. In systems with alternative reproductive tactics (ART) shifts between tactics are hypothesized to be under proximate hormonal control. Most studies of the underlying endocrine changes behind ART have focused on fish and amphibians rather than mammals and few have investigated the potential interaction between different endocrine axes in regulating shifts between conditional dependent tactics. Using a combination of endocrine and behavioral data from male African elephants we expand on our previously published analysis and show that the initial increase in androgens predates the behavioral shifts associated with reproductively active periods, supporting the role of androgens in activating sexually active periods in males. A strong interactive effect between androgens and glucocorticoids was found to determine the presence or absence of temporal gland secretion and urine dribbling, signals associated with the competitive reproductive tactic of musth, with elevated glucocorticoids levels suppressing the occurrence of musth signals. In addition external environmental conditions affected hormone levels. The presence of receptive females resulted in elevated androgens in dominant musth males but increased glucocorticoids in subordinate non-musth males. The presented data on hormones, behavior and reproductive tactics strongly support an underlying endocrine mechanism for mediating the translation of intrinsic as well as extrinsic local conditions into the conditional dependent reproductive tactics in male elephants via interactions between the hypothalamic-pituitary-gonadal and -adrenal axes

Thongtip, N., Saikhun, J., Mahasawangkul, S., Kornkaewrat, K., Pongsopavijitr, P., Songsasen, N., Pinyopummin, A., 2008. Potential factors affecting semen quality in the Asian elephant (Elephas maximus). Reprod. Biol. Endocrinol. 6, 9.
Abstract: BACKGROUND: One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant METHODS: Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. RESULTS: The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05). CONCLUSION: This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species

van der Kolk, J.H., van Leeuwen, J.P., van den Belt, A.J., van Schaik, R.H., Schaftenaar, W., 2008. Subclinical hypocalcaemia in captive Asian elephants (Elephas maximus). Veterinary Record 162, 475-479.
Abstract: The hypothesis that hypocalcaemia may play a role in dystocia in captive Asian elephants (Elephas maximus) was investigated. The objectives of the study were to measure the total calcium concentration in elephant plasma; assess the changes in parameters of calcium metabolism during a feeding trial; investigate a possible relationship between calcium metabolism and dystocia; and assess bone mineralisation in captive Asian elephants in vivo. The following parameters were measured: total and ionised calcium, inorganic phosphorous and magnesium, the fractional excretions of these minerals, intact parathyroid hormone, 25-OH-D(3) and 1,25-OH-D(3). Radiographs were taken from tail vertebrae for assessment of bone mineralisation. The mean (sd) heparinised plasma total calcium concentration was 2.7 (0.33) mmol/l (n=43) ranging from 0.84 to 3.08 mmol/l in 11 Asian elephants. There was no significant correlation between plasma total calcium concentration and age. Following feeding of a calcium rich ration to four captive Asian elephant cows, plasma total and ionised calcium peaked at 3.6 (0.24) mmol/l (range 3.4 to 3.9 mmol/l) and 1.25 (0.07) mmol/l (range 1.17 to 1.32 mmol/l), respectively. Plasma ionised calcium concentrations around parturition in four Asian elephant cows ranged from 0.37 to 1.1 mmol/l only. The present study indicates that captive Asian elephants might be hypocalcaemic, and that, in captive Asian elephants, the normal plasma concentration of total calcium should actually be around 3.6 mmol/l and normal plasma concentration of ionised calcium around 1.25 mmol/l. Given the fact that elephants absorb dietary calcium mainly from the intestine, it could be concluded that elephants should be fed calcium-rich diets at all times, and particularly around parturition. In addition, normal values for ionised calcium in captive Asian elephants should be reassessed

Wallis, M., 2008. Mammalian genome projects reveal new growth hormone (GH) sequences. Characterization of the GH-encoding genes of armadillo (Dasypus novemcinctus), hedgehog (Erinaceus europaeus), bat (Myotis lucifugus), hyrax (Procavia capensis), shrew (Sorex araneus), ground squirrel (Spermophilus tridecemlineatus), elephant (Loxodonta africana), cat (Felis catus) and opossum (Monodelphis domestica). Gen. Comp Endocrinol. 155, 271-279.
Abstract: Mammalian growth hormone (GH) sequences have been shown previously to display episodic evolution: the sequence is generally strongly conserved but on at least two occasions during mammalian evolution (on lineages leading to higher primates and ruminants) bursts of rapid evolution occurred. However, the number of mammalian orders studied previously has been relatively limited, and the availability of sequence data via mammalian genome projects provides the potential for extending the range of GH gene sequences examined. Complete or nearly complete GH gene sequences for six mammalian species for which no data were previously available have been extracted from the genome databases-Dasypus novemcinctus (nine-banded armadillo), Erinaceus europaeus (western European hedgehog), Myotis lucifugus (little brown bat), Procavia capensis (cape rock hyrax), Sorex araneus (European shrew), Spermophilus tridecemlineatus (13-lined ground squirrel). In addition incomplete data for several other species have been extended. Examination of the data in detail and comparison with previously available sequences has allowed assessment of the reliability of deduced sequences. Several of the new sequences differ substantially from the consensus sequence previously determined for eutherian GHs, indicating greater variability than previously recognised, and confirming the episodic pattern of evolution. The episodic pattern is not seen for signal sequences, 5' upstream sequence or synonymous substitutions-it is specific to the mature protein sequence, suggesting that it relates to the hormonal function. The substitutions accumulated during the course of GH evolution have occurred mainly on the side of the hormone facing away from the receptor, in a non-random fashion, and it is suggested that this may reflect interaction of the receptor-bound hormone with other proteins or small ligands

Woolley, L.A., Millspaugh, J.J., Woods, R.J., van Rensburg, S.J., Mackey, R.L., Page, B., Slotow, R., 2008. Population and individual elephant response to a catastrophic fire in Pilanesberg National Park. PLoS. One. 3, e3233.
Abstract: In predator-free large herbivore populations, where density-dependent feedbacks occur at the limit where forage resources can no longer support the population, environmental catastrophes may play a significant role in population regulation. The potential role of fire as a stochastic mass-mortality event limiting these populations is poorly understood, so too the behavioural and physiological responses of the affected animals to this type of large disturbance event. During September 2005, a wildfire resulted in mortality of 29 (18% population mortality) and injury to 18, African elephants in Pilanesberg National Park, South Africa. We examined movement and herd association patterns of six GPS-collared breeding herds, and evaluated population physiological response through faecal glucocorticoid metabolite (stress) levels. We investigated population size, structure and projected growth rates using a simulation model. After an initial flight response post-fire, severely injured breeding herds reduced daily displacement with increased daily variability, reduced home range size, spent more time in non-tourist areas and associated less with other herds. Uninjured, or less severely injured, breeding herds also shifted into non-tourist areas post-fire, but in contrast, increased displacement rate (both mean and variability), did not adjust home range size and formed larger herds post-fire. Adult cow stress hormone levels increased significantly post-fire, whereas juvenile and adult bull stress levels did not change significantly. Most mortality occurred to the juvenile age class causing a change in post-fire population age structure. Projected population growth rate remained unchanged at 6.5% p.a., and at current fecundity levels, the population would reach its previous level three to four years post-fire. The natural mortality patterns seen in elephant populations during stochastic events, such as droughts, follows that of the classic mortality pattern seen in predator-free large ungulate populations, i.e. mainly involving juveniles. Fire therefore functions in a similar manner to other environmental catastrophes and may be a natural mechanism contributing to population limitation. Welfare concerns of arson fires, burning during "hot-fire" conditions and the conservation implications of fire suppression (i.e. removal of a potential contributing factor to natural population regulation) should be integrated into fire management strategies for conservation areas

Brown, J.L., Somerville, M., Riddle, H.S., Keele, M., Duer, C.K., Freeman, E.W., 2007. Comparative endocrinology of testicular, adrenal and thyroid function in captive Asian and African elephant bulls. Gen. Comp Endocrinol. 151, 153-162.
Abstract: Concentrations of serum testosterone, cortisol, thyroxine (free and total T4), triiodothyronine (free and total T3) and thyroid stimulating hormone (TSH) were measured to assess adrenal and thyroid function as they relate to testicular activity and musth in captive elephants. Blood samples were collected approximately weekly from Asian (n=8) and African (n=12) bulls at seven facilities for periods of 4 months to 9.5 years. Age ranges at study onset were 8-50 years for Asian and 10-21 years for African elephants. Based on keeper logs, seven Asian and three African bulls exhibited behavioral and/or physical (temporal gland secretion, TGS, or urine dribbling, UD) signs of musth, which lasted 2.8+/-2.5 months in duration. Serum testosterone was elevated during musth, with concentrations often exceeding 100 ng/ml. Patterns of testosterone secretion and musth varied among bulls with no evidence of seasonality (P>0.05). Only three bulls at one facility exhibited classic, well-defined yearly musth cycles. Others exhibited more irregular cycles, with musth symptoms often occurring more than once a year. A number of bulls (1 Asian, 9 African) had consistently low testosterone (<10 ng/ml) and never exhibited significant TGS or UD. At facilities with multiple bulls (n=3), testosterone concentrations were highest in the oldest, most dominant male. There were positive correlations between testosterone and cortisol for six of seven Asian and all three African males that exhibited musth (range, r=0.23-0.52; P<0.05), but no significant correlations for bulls that did not (P>0.05). For the three bulls that exhibited yearly musth cycles, TSH was positively correlated (range, r=0.22-0.28; P<0.05) and thyroid hormones (T3, T4) were negatively correlated (range, r=-0.25 to -0.47; P<0.05) to testosterone secretion. In the remaining bulls, there were no clear relationships between thyroid activity and musth status. Overall mean testosterone and cortisol concentrations increased with age for all bulls combined, whereas thyroid activity declined. In summary, a number of bulls did not exhibit musth despite being of adequate physical maturity. Cortisol and testosterone were correlated in most bulls exhibiting musth, indicating a possible role for the adrenal gland in modulating or facilitating downstream responses. Data were generally inconclusive as to a role for thyroid hormones in male reproduction, but the finding of discrete patterns in bulls showing clear testosterone cycles suggests they may facilitate expression or control of musth in some individuals

Brown, J.L., Somerville, M., Riddle, H.S., Keele, M., Duer, C.K., Freeman, E.W., 2007. Comparative endocrinology of testicular, adrenal and thyroid function in captive Asian and African elephant bulls. General and Comparative Endocrinology 151, 153-162.
Abstract: Concentrations of serum testosterone, cortisol, thyroxine (free and total T4), triiodothyronine (free and total T3) and thyroid stimulating hormone (TSH) were measured to assess adrenal and thyroid function as they relate to testicular activity and musth in captive elephants. Blood samples were collected approximately weekly from Asian (n = 8) and African (n = 12) bulls at seven facilities for periods of 4 months to 9.5 years. Age ranges at study onset were 8-50 years for Asian and 10-21 years for African elephants. Based on keeper logs, seven Asian and three African bulls exhibited behavioral and/or physical (temporal gland secretion, TGS, or urine dribbling, UD) signs of musth, which lasted 2.8 +/- 2.5 months in duration. Serum testosterone was elevated during musth, with concentrations often exceeding 100 ng/ml. Patterns of testosterone secretion and musth varied among bulls with no evidence of seasonality (P > 0.05). Only three bulls at one facility exhibited classic, well-defined yearly musth cycles. Others exhibited more irregular cycles, with musth symptoms often occurring more than once a year. A number of bulls (I Asian, 9 African) had consistently low testosterone (< 10 ng/ml) and never exhibited significant TGS or UD. At facilities with multiple bulls (n = 3), testosterone concentrations were highest in the oldest, most dominant male. There were positive correlations between testosterone and cortisol for six of seven Asian and all three African males that exhibited musth (range, r = 0.23-0.52; P < 0.05), but no significant correlations for bulls that did not (P > 0.05). For the three bulls that exhibited yearly musth cycles, TSH was positively correlated (range, r = 0.22-0.28; P < 0.05) and thyroid hormones (T3, T4) were negatively correlated (range, r = -0.25 to -0.47; P < 0.05) to testosterone secretion. In the remaining bulls, there were no clear relationships between thyroid activity and musth status. Overall mean testosterone and cortisol concentrations increased with age for all bulls combined, whereas thyroid activity declined. In summary, a number of bulls did not exhibit musth despite being of adequate physical maturity. Cortisol and testosterone were correlated in most bulls exhibiting musth, indicating a possible role for the adrenal gland in modulating or facilitating downstream responses. Data were generally inconclusive as to a role for thyroid hormones in male reproduction, but the finding of discrete patterns in bulls showing clear testosterone cycles suggests they may facilitate expression or control of musth in some individuals.

Dehnhard, M., 2007. Characterisation of the sympathetic nervous system of Asian (Elephas maximus) and African (Loxodonta africana) elephants based on urinary catecholamine analyses. Gen. Comp Endocrinol. 151, 274-284.
Abstract: Assessing the welfare status of captive animals using non-invasive measurements of hormones is of growing interest because this can serve as an effective tool to facilitate the optimization of environmental and husbandry conditions. Both the African elephant (Loxodonta africana) and the Asian elephant (Elephas maximus) exhibit extremely low breeding success in captivity, and because elevated levels of stress may negatively influence reproductive functions, this study sought to establish a method for assessing sympathoadrenal activity in captive female elephants. We found a circadian variation in urinary noradrenaline (norepinephrine, NE), adrenaline (epinephrine, Epi) and dopamine (DA) under short day length. Peak activity of noradrenaline and dopamine was noted at 3 a.m. Adrenaline showed a biphasic pattern with a minor peak recorded at 3 a.m. and a major peak 9 a.m. Under long-day photoperiodic conditions, simultaneous peaks of noradrenaline and adrenaline were again noted at 3 a.m. whereas dopamine does not appear to have a distinct circadian pattern under long-day length. A transfer of two elephant cows resulted in a marked increase in urinary adrenaline and noradrenaline levels, confirming that the transfer represented a stressful event. During the peripartal period, noradrenaline concentrations increased and maximum concentrations were obtained at delivery. Daily measurements of urinary dopamine throughout the follicular phase revealed an increase in dopamine secretion close to ovulation. This increase might indicate a role of dopamine in the ovulatory mechanisms. These results suggest that changes in urinary catecholamine excretion reflect fluctuations in sympathoadrenal activity and may be a useful indicator of stress

Wittemyer, G., Ganswindt, A., Hodges, K., 2007. The impact of ecological variability on the reproductive endocrinology of wild female African elephants. Hormones and Behavior 51, 346-354.
Abstract: Non-invasive endocrine methods enable investigation of the relationship between ecological variation and ovarian activity and how this impacts on demographic processes. The underlying physiological factors driving high variation in inter-calving intervals among multi-parous African elephants offer an interesting system for such an investigation. This study investigates the relationship between Normalized Differential Vegetation Index (NDVI), an ecosystem surrogate measure of primary productivity, and fecal progestin concentrations among wild female elephants. Matched fecal samples and behavioral data on reproductive activity were collected from 37 focal individuals during the two-year study. Linear mixed models were used to explore the relationship between fecal 5 alpha-pregnane-3-ol-20-one concentrations and the independent variables of NDVI, calf sex, female age, gestation day, and time since last parturition. Among both non-pregnant and pregnant females, fecal 5 alpha-pregnane-3-ol-20-one concentrations were significantly correlated with time-specific NDVI indicating a strong relationship between ecological conditions and endocrine activity regulating reproduction. In addition, the age of a female and time since her last parturition impacted hormone concentrations. These results indicate that the identification of an individual's reproductive status from a single hormone sample is possible, but difficult to achieve in practice since numerous independent factors, particularly season, impact fecal hormone concentrations. Regardless of season, however, fecal 5 alpha-pregnane-3-ol-20-one concentrations below 1 mu g/g were exclusively collected from non-pregnant females, which could be used as a threshold value to identify non-pregnant individuals. Collectively the information generated contributes to a better understanding of environmental regulation of reproductive endocrinology in wild elephant populations, information salient to the management and manipulation of population dynamics in this species.

Wittemyer, G., Ganswindt, A., Hodges, K., 2007. The impact of ecological variability on the reproductive endocrinology of wild female African elephants. Horm. Behav. 51, 346-354.
Abstract: Non-invasive endocrine methods enable investigation of the relationship between ecological variation and ovarian activity and how this impacts on demographic processes. The underlying physiological factors driving high variation in inter-calving intervals among multi-parous African elephants offer an interesting system for such an investigation. This study investigates the relationship between Normalized Differential Vegetation Index (NDVI), an ecosystem surrogate measure of primary productivity, and fecal progestin concentrations among wild female elephants. Matched fecal samples and behavioral data on reproductive activity were collected from 37 focal individuals during the two-year study. Linear mixed models were used to explore the relationship between fecal 5alpha-pregnane-3-ol-20-one concentrations and the independent variables of NDVI, calf sex, female age, gestation day, and time since last parturition. Among both non-pregnant and pregnant females, fecal 5alpha-pregnane-3-ol-20-one concentrations were significantly correlated with time-specific NDVI indicating a strong relationship between ecological conditions and endocrine activity regulating reproduction. In addition, the age of a female and time since her last parturition impacted hormone concentrations. These results indicate that the identification of an individual's reproductive status from a single hormone sample is possible, but difficult to achieve in practice since numerous independent factors, particularly season, impact fecal hormone concentrations. Regardless of season, however, fecal 5alpha-pregnane-3-ol-20-one concentrations below 1 microg/g were exclusively collected from non-pregnant females, which could be used as a threshold value to identify non-pregnant individuals. Collectively the information generated contributes to a better understanding of environmental regulation of reproductive endocrinology in wild elephant populations, information salient to the management and manipulation of population dynamics in this species

Yon, L., Kanchanapangka, S., Chaiyabutr, N., Stanczyk, F., Meepan, S., Lasley, B., 2007. ACTH stimulation in four Asian bull elephants (Elephas maximus): an investigation of androgen sources in bull elephants. Gen. Comp Endocrinol. 151, 246-251.
Abstract: The phenomenon of musth is a very stressful event, both behaviorally and physiologically. An ACTH stimulation test was conducted in four adult Asian bull elephants to investigate the possibility that the classical hypothalamic-pituitary-adrenal (HPA) axis is active during musth, resulting in an increase in adrenally produced steroids. Serum cortisol, testosterone (T), androstenedione (A4), androstenediol (A5), and dehydroepiandrosterone (DHEA) were measured. Cortisol increased 3-10 times above baseline in response to ACTH stimulation, and DHEA doubled. A4 and A5 were erratic, while testosterone decreased significantly in all bulls. The pattern of results suggests that the adrenal steroid increase which occurs during musth results from some mechanism other than the classical HPA axis

Yon, L., Chen, J., Moran, P., Lasley, B., 2007. An analysis of the androgens of musth in the Asian bull elephant (Elephas maximus). Gen Comp Endocrinol. Mar 24; [Epub ahead of print].
Abstract: During musth in bull elephants, the androgens testosterone (T), dihydrotestosterone (DHT), and androstenedione all increase significantly. Given the unusual endocrine physiology that has been discovered in female elephants, it is also possible that bull elephants produce some unusual androgens. A cell-based androgen receptor assay was used to explore this possibility using two different methods. The first method compared the level of T measured by radioimmunoassay (RIA) with the level of androgen receptor (AR) activity measured in the serum of eight bull elephants during musth and non-musth periods. A ratio was calculated for T/AR activity for non-musth and musth, to determine if there was a change in the ratio between these two states. The second method used HPLC to separate two pooled serum samples (one non-musth and one musth) into fractions using a protocol which separates known androgens into specific, previously identified fractions. Each fraction was then tested with the AR assay to determine the androgenicity of any compounds present. This was done to determine if there were any fractions which had androgenic activity but did not contain any previously identified androgens. Results from the first analysis indicated no change in the T/AR ratio between non-musth and musth states. Clearly whatever active androgens are present during musth, they increase proportionately with T. Findings from the second analysis suggested that the only bioactive androgen present in the serum of non-musth Asian bulls is a low level of T. During musth, the only bioactive androgens detected were T and DHT; of these, T was by far the predominant active androgen present. Taken together, these two analyses suggest that T is by far the predominant active androgen present during musth in Asian bull elephants, and that no previously unidentified bioactive androgen is present.

Yon, L., Kanchanapangka, S., Chaiyabutr, N., Meepan, S., Stanczyk, F.Z., Dahl, N., Lasley, B., 2007. A longitudinal study of LH, gonadal and adrenal steroids in four intact Asian bull elephants (Elephas maximus) and one castrate African bull (Loxodonta africana) during musth and non-musth periods. Gen. Comp Endocrinol. 151, 241-245.
Abstract: During their annual musth cycle, adult African and Asian bull elephants have increased gonadal androgens (testosterone [T], dihydrotestosterone [DHT], androstenedione [A4]). Because musth is a physiologically and psychologically stressful time, this study was conducted to investigate whether the adrenal glands (stimulated by stress) increase production of both glucocorticoids and androgens during musth. Weekly serum samples were taken for 11-15 months from four intact adult Asian bull elephants, and from a castrate African bull elephant who exhibits musth. Testosterone, androstenediol (A5), A4, luteinizing hormone (LH), cortisol, and dehydroepiandrosterone (DHEA) were measured in each sample. In three of the four intact bulls, all hormones measured increased during musth. Adrenal androgens were strongly correlated with LH and testicular androgens, though not to cortisol. None of the hormones measured in the castrate bull increased during his musth cycles. While the significance of adrenal activity in the elephant during musth has yet to be determined, this study provides evidence that the adrenal gland actively produces both glucocorticoids and androgens during musth in the Asian elephant

Ball, R., Fad, O. Serum cortisols in captive Asian elephants (Elephas maximus) in different management systems at Busch Gardens Tampa Bay. 2006 Proceedings American Association of Zoo Veterinarians. 177-180. 2006.
Ref Type: Conference Proceeding
Abstract: Introduction:Cortisol is a widely accepted measure of stress in wild and captive animals. In the past, captive elephant management systems have been criticized as potential stress inducers. The analysis of fecal cortisols is non-invasive and has been used to give long term evalutions of social and ecologic pressures in elephants and other species. Salivary cortisols have also been used as a minimally invasive technique to measure social stress in captive elephants. The herd of Asian elephants at Busch Gardens Tampa Bay (BGT) changed from a traditional contact managemnt (free ccontact, FC) to a protected contact (PC) system utilizing positive-reinforcement based operant conditioning in 2004. Serum cortisols were measured after the change and evaluated along wth banked samples from before. Long term sampling will be utilized to measure this transition but evaluating a single process will hopefully reflect the overall changes that can be expected with this change in management. While the individual variations are notable and other issues potentially confound the issue, it appears that this transition has lowered the serum cortisols in this herd. In addition to serum cortisol measurements, the actual process of collecting the samples appears to be less stressful behaviorally. Pathologic processes should not be discounted when considering cortisol levels in evalauting stress in captive elephants.

Methods and Materials: Six female Asian elephants (Studbook numbers 30, 32, 304, 34, 35, 3) had been managed in a free contact system for many years. Studbook number 304 was captive born and the others were wild born. Serum was collected intermittently during this management system to bank and for reproductive hormone analysis. The elephants were placed in lateral recumbency by the handlers and blood collected from the ear vein on the caudal aspect of the down ear. Reproductively sound animals were bled more frequently than the others. Serum was frozen at -80°C until analyzed. In August 2004, the first group of three animals was moved to the new barn and started the new positive-reinforcement, PC management system. Within 5 wk, all animals had been moved over. All animals had been trunkwashed and were culture negative for Mycobacterium tuberculosis and negative on the newly developed MultiAntigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test) developed to detected antigen to M. tuberculosis. As the caudal aspect of the ear was used for sampling, each elephant was asked to station in a static chute designed to allow training of voluntary ear-presentation for manipulation and blood collection. Handler safety and creating an effective learning environment for the elephants required training each to proceed to the chute solo and station there calmly. General desensitization techniques were applied as session durations were increased. Within the chute,individual elephants had significant room to maneuver. Since no physical restraint or sedation was utilized,animals were trained to cooperate fully and voluntarily allowing for blood sampling and other husbandry procedures. By May 2005, training for voluntary bloods draws was firmly established on all six animals. The first approximately 20 samples collected under this new system were matched against the samples collected in the previous system. Samples were selected against if the animal had an active problem or was on therapy for any reason. Several animals had undergone a drug trial and these samples were selected against as well. Serum was again stored in -80°C freezer until analyzed at Conservation and Research Center (CRC) Endocrine Research Laboratory, Smithsonian Institution, National Zoological Park, Front Royal, VA. T-tests were utilized to discern any statistically significant results in the mean serum cortisols collected from animals before and after the implementation of the new husbandry systems. Results were considered significant at alpha levels <0.05.

Results: The results and simple means of serum cortisols are listed in Table 1. Elephant No. 34 had essentially the same level of cortisol in both systems. Elephant No. 32 had a reduction in the mean cortisol level of approximately 32% (20.84 versus 14.28 ng/ml) from the FC to the PC system. Elephant No. 304 had a similar reduction of 37% in the mean cortisol (22.59 versus 14.29 ng/ml). Statistical analyses results are reported here (means, standard deviations, t-test results).

Discussion: Serum was chosen over salivary and fecal sampling as a means to measure cortisol for several reasons. While fecal and salivary cortisol changes can reflect stresses within a reasonable period after the stressor (approximately 24 hr), serum cortisols is more likely to be reflective of the stressors closer to the moment of sampling. The methodology is straightforward and less subject to the hazards for sample storage. Timeliness of the sample result is also a benefit to serum sampling. Blood sampling is a required husbandry practice in all elephant holding facilities belonging to the American Zoo and Aquarium Association (AZA). While fecal cortisol samples may be useful to look at over a long term period to evaluate the transition from FC to PC, we choose to additionally look at how one specific task, blood collection, was affected by making this transition. Fecal cortisols have been used to measure stress in transportation and environmental stress in some species, but are not thought to be reflective of the stress in a diagnostic procedure itself. For this evaluation, the lag time period between the potential stressor (blood collection) and the means to measure the stressor are same. Elephants No. 304 and 32 both had significant reductions in the mean serum cortisol levels. Both are in good health and had no apparent inflammatory problems. The logical deduction here is that the sampling process itself is less stressful in the PC management than the FC management. Elephant 34 and 30 had essentially the same level of serum cortisol as measured by the mean in the different management systems. Elephant 34 has developed significant uterine leiomyomas during the time period measured. Elephant 30 has recently had clinical bouts of anterior enteritis and is suspected of having a dietary hypersensitivity to wheat. Even with these two pathologic processes, the serum cortisol did not rise. Elevations in cortisol are quite often explained as resulting from social, behavioral, or environmental causes and little attention is paid to inflammatory causes. Associations between infections and elevated cortisols have been noted in wild animals. It is reasonable to assume that if these two processes did not exist, these levels would indeed be lower. Based on the other two elephants, a reduction of approximately 30% could be expected. Overall it appears that collecting blood from the elephants at BGT in the PC system is less stressful that the FC system. As this is an example of how the routine husbandry and medical husbandry is now conducted, it can be expected that the overall net effect is going to be lowered stress in the elephants at BGT. ……………………………………………………………………………

Ball, R.L., Fad, O. Serum cortisols in captive Asian elephants (Elephas maximus) in different management systems at Busch Gardens Tampa Bay. Proceedings International Elephant Conservation & Research Symposium. 244-247. 2006. 2006.
Ref Type: Conference Proceeding

Ball, R.L., Brown, J. Preliminary results of a cabergoline trial in captive elephant with hyperprolactinemia. 2006 Proceedings American Association of Zoo Veterinarians. 174-176. 2006.
Ref Type: Conference Proceeding
Abstract: Introduction: An Asian elephant (Elephas maximus) at Busch Gardens Tampa Bay (BGT) was diagnosed with hyperprolactinemia, with a persistently elevated serum prolactin concentration greater than 15 ng/ml, by the Conservation & Research Center (CRC) laboratory in January 1996. She also had a number of other problems, including uterine disorders that resulted in consistently elevated progesterone. In March 2002, she was given cabergoline orally at a dose of 1 mg twice weekly p.o. for 6 mo. Cabergoline is a long-acting dopamine receptor agonist with a high affinity for D2 receptors. It exerts a direct inhibitory effect on the secretion of prolactin. Cabergoline (Dostinex®, Pfizer Inc. Kalamazoo, Michigan 49007 USA) was purchased from a local pharmacy. Serum prolactin concentrations declined almost immediately after treatment initiation, followed about 1 mo later by a drop in progesterone to baseline. Progesterone secretion remained low until November 2002 when she resumed cycling based on the observation of a normal luteal phase based on serum progesterone profile. From November 2002 through January 2004 she exhibited four normal estrous cycles. Prolactin secretion also remained within the normal range for elephants, over 1 yr after treatment withdrawal. This female suffered no adverse effects due to the cabergoline treatment. There were no behavioral changes noted or changes in appetite. Given the need to increase reproductive rates of African elephants (Loxodonta africana) to prevent captive extinction, it might be efficacious to treat genetically valuable females with cabergoline in the hope it will reinitiate reproductive cyclicity. Nearly 1/3 of African elephants with hormone data are not cycling normally, and in an earlier study 1/3 of these (11 of 30) were found to have increased serum prolactin levels.

Methods and Materials:
A clinical trial was undertaken with six captive African elephant females that were identified as good candidates for a cabergoline treatment study (i.e., they are acylic and had mean prolactin concentrations of >15 ng/ml). The treatment consisted of 1 mg cabergoline given twice weekly p.o. for 6 mo. Serum was banked and then analyzed at the CRC for progesterone and prolactin. All elephants were thought to be otherwise healthy. Because prolactin is known to be an inflammatory marker, all candidates were required to have a negative lateral flow immunochromatograpy (Rapid Test) and multiple antigen immunoassay (MAPIA) for Mycobacteria tuberculosis.

Results:
A summary of the results is given in Table 1. The treatment period is complete for three elephants, all of which showed a decrease in prolactin levels. Elephant 1 showed a good response while on treatment, but did not cycle and serum prolactin has subsequently risen to pretreatment levels. Increasing the dose in Elephant 2 and 3 reduced prolactin to baseline levels, but again did not result in a return to ovarian cyclicity. Elephant 4 was taken off the study after only a few doses due to increased aggressive behaviors. This is believed to be due to changes in the group social dynamics and not related to the cabergoline, as this behavior has continued after withdrawal of the drug. Based on these findings, the two newest candidates, Elephant 5 and 6, with very high prolactin concentrations have been placed on 2 mg/twice weekly for 1 yr pending continuation of this project.

Discussion:
Normalization of prolactin levels facilitated the return of normal cycles in an Asian elephant, but none of the African elephants have resumed cycling so far. Thus, while the use of cabergoline shows promise in reducing elevated prolactin levels in both Asian and African elephants, other factors may need to be considered or a longer course at higher doses may be required for treatment to be successful in reinitiating ovarian activity. The latter suggestion is supported by two of the animals (Elephants 2 and 3) in this limited trial, in which a decline in prolactin occurred after the dose was increased. Understanding the etiology of hyperprolactinemia in elephants may also help in returning females to normal cycling.Relapse of hyperprolactinemia is more common in humans with micro- or macroprolactinomas. Chronic estrogen stimulation is also known to increase prolactin levels. A proposed pathophysiology is that elevated estrogen levels from persistent cycling will lead to elevated prolactin levels and acyclicity. A difference between the two species in the causes of and potential treatment options for hyperprolactinemia should also be evaluated more closely.

1Busch Gardens Tampa Bay, 3605 Bougainvillea Drive, Tampa, FL 33674 USA;2Smithsonian Institution, National Zoological Park, Conservation & Research Center, Front Royal, VA 22630 USA
ACKNOWLEDGMENTS
We would like to thank the participating zoos for their cooperation and patience during this trial.
LITERATURE CITED
1Brown, J.L., S.L. Walker and T. Moeller. 2004. Comparative endocrinology of cycling and noncycling Asian (Elephas maximus) and African (Loxodonta africana) elephants. Gen. Comp. Endocrinol. 136:360-370.
2 Colao, A., A. Di Sarno, P. Cappabianca, C. Di Somma, R. Pivonello, and G. Lombardi. 2003. Withdrawal of long-term cabergoline therapy for tumoral and non-tumoral hyperprolactinemia. New Engl. J. Med. 349:20232033.
3 Ismail, M.S., G.I. Serour, U. Torsten, H. Weitzel, and H.P. Berlien. 1998. Elevated serum prolactin level with high-dose estrogen contraceptive pills. .Eur. J. Contracept. Reprod. Health Care. 3(1):45-50.
4 Montero, A.M., O.A. Bottasso, M.R.Luraghi, A.G. Giovannoni, and L. Sen. 2001. Association between high serum prolactin and concomitant infections in HIV-infected patients. Human Immunol.62: 191-196.
5 Lyashchenko, K., M. Miller, and W.R. Waters. 2005. Application of multiple antigen print immunoassay and rapid lateral flow technology for tuberculosis testing of elephants. . Proc. Am. Assoc. Zoo Vet. Annu. Meet. Pp. 64-65

Bertschinger, H., Delsink, A., Kirkpatrick, J.F., Human, A., Grobler, D., van Altena, J.J. Management of elephant populations in private South African game reserves with porcine zona pellucida vaccine. 2006 Proceedings American Association of Zoo Veterinarians. 283-285. 2006.
Ref Type: Conference Proceeding
Abstract: Control of African elephant populations has become an absolute necessity in a number of game reserves in southern Africa. The two main methods used to control populations so far are culling and translocation. Culling, besides being regarded as inhumane and unacceptable in many quarters, is not suitable for smaller populations. It requires that whole family units are culled simultaneously which could mean that in reserves with 10 to 50 elephants a considerable portion, if not the entire population, is killed. As far as translocation is concerned, limited new space is available for elephants. The only alternative to the two above options is to control the rate of reproduction. The porcine zona pellucida (pZP) vaccine has been used to successfully contracept wild horses and other wildlife species. Work on the contraception of African elephants was initiated in the Kruger National Park in 1995 when the potential for using the porcine zona pellucida (pZP) was investigated. Subsequently the first field trials on wild elephants were carried out in Kruger and the results clearly showed that elephants could be contracepted with the pZP vaccine, although the efficacy achieved was 80%. During these field trials safety and reversibility werecould be demonstrated. In 2000 an elephant contraceptive program was initiated at Makalali Private Game Reserve, RSA, which has become the flagship model for immunocontrol in African elephants. The preliminary findings have been reported in three publications.During the first year, all 18 cows that were individually identified and older than 12 yr of age were treated. During the next 4 yr the number of cows contracepted increased to 23 as young animals were added to the program. The standard vaccination procedure during the first year consisted of a primary vaccination (600 μg or 400 μg pZP with 0.5 ml Freund's modified complete adjuvant) followed by boosters (200 μg pZP with 0.5 ml Freund's incomplete adjuvant) at 3 to 6-wk intervals. Annual boosters to maintain antibody titers and contraceptive effect followed. To date, the success rate on cows that have passed reserve-specific intercalving period of 56 mo has been 100%. The population stabilized within 3 yr by which time when all cows that had been pregnant at the time of first vaccination in 2000 had calved. Once again safety during pregnancy (14 cows pregnant at 2-21 mo gestation when first treated gave birth to normal healthy calves) as well as side effects that were limited to occasional lumps at the site of vaccination could be shown. Following ground darting, behavioral patterns returned to pre-darting status within 2 days. During 2003 and 2004 most boosters were administered from a helicopter; whereas, previously they had been done from a vehicle or on foot. In all cases, drop-out darts were used. Time taken for vaccination from helicopter take-off to landing was about 30 min (1.5 min per cow; 30 min for total time). This required prior knowledge of the locations of family units or that an individual in each unit is radio-collared. Herds settled down much more quickly (1-2 days) than if darted from the ground. Since then we have vaccinated another 107 elephant cows in eight game reserves. The cow populations have ranged from 4 to 43. In one of the reserves, Mabula, RSA, two of the four cows vaccinated have passed the mean intercalving intervals of the reserve with neither of them producing a calf. Treatment at the remaining reserves was initiated in 2004 or 2005 and it is too early to evaluate results. The most difficult reserve in terms of the vaccination process was Welgevonden, RSA, (35 000 ha) with 43 cows. The reserve is mountainous and heavily wooded. None of the elephants were collared and individuals could not be easily identified on the day of primary vaccination. The total flying time during which individuals were identified and vaccinated was 4.5 hr. Administration of the first booster took about 2 hr to locate and vaccinate each cow. Between the first and second booster the first rains occurred, followed by the spring flush of the vegetation. By the time the second booster was attempted late in November, the trees all had foliage. Only half the cows were located and darted because the elephants were very difficult to spot under the tree canopies. The valuable lessons we learned from this were: 1) that helicopter vaccinations should be performed when most trees are bare, and 2) when larger populations are vaccinated repeatedly during the first year, one cow in each family unit should be radio-collared. This makes rapid location of each unit possible and cuts down on the major cost factor that is flying time. Elephant behavior is being monitored in all eight reserves where contraception is being applied. Because most of them have been contracepted recently, only the data from Makalali is available. The elephants at Makalali have been monitored intensively almost on a daily basis. To date, no anomalies in terms of aggressive or indifferent behavior with regards to nursing time, nursing behavior and calf proximity have been noted. No change in the cows' social hierarchy has been noted. Since January 2003, a total of 15 heats were observed in 10 cows (nine in 2003 and six in 2004) with four mating episodes. For the same period, 38 musth occasions were seen in five bulls (26 in 2003 and 12 in 2004). These occasions include musth displayed in the same bull during consecutive days or within the same musth cycle. The greatest occurrence of musth was recorded in the largest, dominant bull. Bulls were not observed harassing or separating cows off from their herds or calves as a result of increased estrous frequency. Thus, the Makalali program demonstrates that pZP does not cause herd fragmentation, harassment by bulls, change in rank and other negative behaviors normally associated with hormonal contraceptives. In conclusion we feel that it is important to emphasize the following points: The pZP vaccine can be used successfully to contracept African elephants The vaccine is safe during pregnancy and has no negative effect on birth or calf raising It has no side effects other than occasional swelling at the site of vaccination It is reversible Other than an increased incidence of heat no behavioral side effects were seen.

Brown, J.L., Somerville, M., Riddle, H.S., Keele, M., Duer, C. Comparative endocrinology of testicular and thyroid function in captive Asian and African elephant bulls. Proceedings International Elephant Conservation & Research Symposium. 58-75. 2006. 2006.
Ref Type: Conference Proceeding

Keay, J.M., Singh, J., Gaunt, M.C., Kaur, T., 2006. Fecal glucocorticoids and their metabolites as indicators of stress in various mammalian species: a literature review. Journal of Zoo and Wildlife Medicine 37, 234-244.

Bonar, C.J., Lewandowski, A.H., Arafah, B., Capen, C.C., 2005. Pheochromocytoma in an aged female African elephant (Loxodonta africana). J. Zoo. Wildl. Med. 36, 719-723.
Abstract: A 43 yr-old female African elephant (Loxodonta africana) collapsed acutely and died. Necropsy revealed an enlarged right adrenal medulla. Histologic appearance was typical of pheochromocytoma. Special stains and electron microscopy demonstrated chromaffin granules, suggesting that the tumor was derived from catecholamine secreting cells of the adrenal medulla, and may have been functionally secretory. Serum levels of both norepinephrine and epinephrine were elevated at time of death, supporting the functional nature of the tumor. Histologic findings of arteriolar sclerosis and smooth muscle hyperplasia suggested that the animal may have suffered from chronic systemic hypertension. Pheochromocytoma should be considered as a differential diagnosis in cases of suspected hypertension and acute death in elephants

Delves, P.J., Roitt, I.M., 2005. Vaccines for the control of reproduction--status in mammals, and aspects of comparative interest
592. Dev. Biol. (Basel) 121, 265-273.
Abstract: The objective of producing vaccines which target elements of the reproductive system to control fertility has been pursued for many years. Of the many targets for such vaccines, several sperm-associated antigens have been proposed for antibody-mediated intervention before fertilization but the very abundance of antigen to be neutralized has been a barrier. Zona pellucida antigens associated with the surface of the oocyte have also been targeted and used successfully for control of 'wild' elephant populations but worries concerning immunopathologically-mediated tissue damage have been mooted. Vaccines using human chorionic gonadotropin (hCG) which is required for the implantation and maintenance of the fertilized egg, although of interest for the development of fertility control in human populations, has no relevance in the context of the present conference because external fertilization of fish eggs is independent. The pathways by which gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus promote release of luteinizing (LH) and follicle-stimulating hormone (FSH) which govern the physiological maturation and maintenance of the reproductive organs, provide many targets for immunological intervention. Most consistent success has been reported using GnRH-based vaccines which are immunosterilizing in a variety of mammalian species such as pigs, rodents and white-tailed deer. The fact that the structure of the decapeptide, GnRH, has been maintained over so many years of evolution and been conserved across so many animal species, encourages the view that a strategy for control of sexual maturation in fish based upon stimulation of GnRH antibodies may well prove to be a practical proposition, provided the formulation of an appropriate highly immunogenic vaccine can be achieved

Ganswindt, A., Heistermann, M., Hodges, K., 2005. Physical, physiological, and behavioral correlates of musth in captive African elephants (Loxodonta africana)
594. Physiol Biochem. Zool. 78, 505-514.
Abstract: Although musth in male African elephants (Loxodonta africana) is known to be associated with increased aggressiveness, urine dribbling (UD), temporal gland secretion (TGS), and elevated androgens, the temporal relationship between these changes has not been examined. Here, we describe the pattern of musth-related characteristics in 14 captive elephant bulls by combining long-term observations of physical and behavioral changes with physiological data on testicular and adrenal function. The length of musth periods was highly variable but according to our data set not related to age. Our data also confirm that musth is associated with elevated androgens and, in this respect, show that TGS and UD are downstream effects of this elevation, with TGS responding earlier and to lower androgen levels than UD. Because the majority of musth periods were associated with a decrease in glucocorticoid levels, our data also indicate that musth does not represent a physiological stress mediated by the hypothalamic-pituitary-adrenal axis. Furthermore, we demonstrate that the occurrence of musth is associated with increased aggression and that this is presumably androgen mediated because aggressive males had higher androgen levels. Collectively, the information generated contributes to a better understanding of what characterizes and initiates musth in captive African elephants and provides a basis for further studies designed to examine in more detail the factors regulating the intensity and duration of musth

Ganswindt, A., Rasmssen, H.B., Heistermann, M., Hodges, J.K., 2005. The sexually active states of free-ranging male African elephants (Loxodonta africana): defining musth and non-musth using endocrinology, physical signals, and behavior. Horm Behav 47, 83-91.
Abstract: Musth in male African elephants, Loxodonta africana, is associated with increased aggressive behavior, continuous discharge of urine, copious secretions from the swollen temporal glands, and elevated androgen levels. During musth, bulls actively seek out and are preferred by estrous females although sexual activity is not restricted to the musth condition. The present study combines recently established methods of fecal hormone analysis with long-term observations on male-female associations as well as the presence and intensity of physical signals to provide a more detailed picture about the physical, physiological, and behavioral characteristics of different states of sexual activity in free-ranging African elephants. Based on quantitative shifts in individual bull association patterns, the presence of different physical signals, and significant differences in androgen levels, a total of three potential sub-categories for sexually active bulls could be established. The results demonstrate that elevations in androgen levels are only observed in sexually active animals showing temporal gland secretion and/or urine dribbling, but are not related to the age of the individual. Further, none of the sexually active states showed elevated glucocorticoid output indicating that musth does not represent an HPA-mediated stress condition. On the basis of these results, we suggest that the term "musth" should be exclusively used for the competitive state in sexually active male elephants and that the presence of urine dribbling should be the physical signal used for defining this state.

Ganswindt, A., Rasmussen, H.B., Heistermann, M., Hodges, J.K., 2005. The sexually active states of free-ranging male African elephants (Loxodonta africana): defining musth and non-musth using endocrinology, physical signals, and behavior
652. Horm. Behav. 47, 83-91.
Abstract: Musth in male African elephants, Loxodonta africana, is associated with increased aggressive behavior, continuous discharge of urine, copious secretions from the swollen temporal glands, and elevated androgen levels. During musth, bulls actively seek out and are preferred by estrous females although sexual activity is not restricted to the musth condition. The present study combines recently established methods of fecal hormone analysis with long-term observations on male-female associations as well as the presence and intensity of physical signals to provide a more detailed picture about the physical, physiological, and behavioral characteristics of different states of sexual activity in free-ranging African elephants. Based on quantitative shifts in individual bull association patterns, the presence of different physical signals, and significant differences in androgen levels, a total of three potential sub-categories for sexually active bulls could be established. The results demonstrate that elevations in androgen levels are only observed in sexually active animals showing temporal gland secretion and/or urine dribbling, but are not related to the age of the individual. Further, none of the sexually active states showed elevated glucocorticoid output indicating that musth does not represent an HPA-mediated stress condition. On the basis of these results, we suggest that the term "musth" should be exclusively used for the competitive state in sexually active male elephants and that the presence of urine dribbling should be the physical signal used for defining this state

Glickman, S.E., Short, R.V., Renfree, M.B., 2005. Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies
566. Horm. Behav. 48, 403-417.
Abstract: The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion

Glickman, S.E., Short, R.V., Renfree, M.B., 2005. Sexual differentiation in three unconventional mammals: Spotted hyenas, elephants and tammar wallabies. Hormones and Behaviour 48, 403-417.
Abstract: The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the ''specific'' requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the ''expanded'' version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5α androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion.

Naz, R.K., Gupta, S.K., Gupta, J.C., Vyas, H.K., Talwar, A.G., 2005. Recent advances in contraceptive vaccine development: a mini-review
577. Hum. Reprod. 20, 3271-3283.
Abstract: Contraceptive vaccines (CV) may provide viable and valuable alternatives to the presently available methods of contraception. The molecules that are being explored for CV development either target gamete production [luteinizing hormone-releasing hormone (LHRH)/GnRH, FSH], gamete function [sperm antigens and oocyte zona pellucida (ZP)], and gamete outcome (HCG). CV targeting gamete production have shown varied degrees of efficacy; however, they either affect sex steroids causing impotency and/or show only a partial rather than a complete effect in inhibiting gametogenesis. However, vaccines based on LHRH/GnRH are being developed by several pharmaceutical companies as substitutes for castration of domestic pets, farm and wild animals, and for therapeutic anticancer purposes such as in prostatic hypertrophy and carcinoma. These vaccines may also find applications in clinical situations that require the inhibition of increased secretions of sex steroids, such as in uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty. CV targeting molecules involved in gamete function such as sperm antigens and ZP proteins are exciting choices. Sperm constitute the most promising and exciting target for CV. Several sperm-specific antigens have been delineated in several laboratories and are being actively explored for CV development. Studies are focused on delineating appropriate sperm-specific epitopes, and increasing the immunogenicity (specifically in the local genital tract) and efficacy on the vaccines. Anti-sperm antibody (ASA)-mediated immunoinfertility provides a naturally occurring model to indicate how a vaccine might work in humans. Vaccines based on ZP proteins are quite efficacious in producing contraceptive effects, but may induce oophoritis, affecting sex steroids. They are being successfully tested to control feral populations of dogs, deer, horses and elephants, and populations of several species of zoo animals. The current research for human applicability is focused on delineating infertility-related epitopes (B-cell epitopes) from oophoritis-inducing epitopes (T-cell epitopes). Vaccines targeting gamete outcome primarily focus on the HCG molecule. The HCG vaccine is the first vaccine to undergo Phase I and II clinical trials in humans. Both efficacy and lack of immunopathology have been reasonably well demonstrated for this vaccine. At the present time, studies are focused on increasing the immunogenicity and efficacy of the birth control vaccine, and examining its clinical applications in various HCG-producing cancers. The present article will focus on the current status of the anti-sperm, anti-ZP, anti-LHRH/GnRH and anti-HCG vaccines

Rasmussen, L.E.L., Krishamurthy, V., Sakumar, R., 2005. Behavioural and chemical confirmation of the preovulatory pheromone, (Z)-7-dodecenyl acetate, in wild Asian elephants: its relationship to musth. Behaviour 142, 351-396.
Abstract: Mammalian breeding strategies vary depending on particular social contexts and sensory systems emphasized in various species. Among sexually dimorphic non-territorial Asian elephants, Elephas maximus, a multiplex olfactory chemical signaling system has been implicated in ensuring effective reproduction. This study explores how, using chemosensory mechanisms, widely roaming, wild male elephants locate periovulatory females in matriarchal-led female family units and precisely assess their ovulatory status. In this species, the dual obstacles of separately living sexes and infrequent oestrus are overcome by lengthy female cycles. During an extended preovulatory period captive females release increasing concentrations of the urinary pheromone (Z)-7-dodecenyl acetate, timed to reach a maximum just before ovulation. The current field studies combined chemical identification and quantification of female urinary (Z)-7-dodecenyl acetate with behavioural observations, monitoring the frequencies of chemosensory responses and premating behaviours by various categories of males. The results suggest the temporal extension of the preovulatory period effectively provides a synchrony between sexes for successful reproduction. Male elephants undergo a two-decade-long maturation process that involves physical, sexual, social, and physiological maturation. Males older than 30 years are generally large, sexually active, socially adept and capable of sustaining long periods of musth, during which they release secretions distinctive of adult musth. These older adult males in musth demonstrated significantly more chemosensory responses and premating behaviours than their younger or nonmusth counterparts; they apparently are more skilled at detecting the precise ovulatory status of females. Male-male interactions are affected by size, age, and musth; the winners gain greater access to females, as indicated by the high incidence of mate guarding. The Asian elephant shares some breeding tactics common to other mammals including some primates (e.g. orangutans) and whales, while the musth parameter adds a unique feature. Fusion-fission events are influenced by elephant reproductive strategies, as roving males join female groups while tracking preovulatory pheromone concentrations.

Steinetz, B.G., Brown, J.L., Roth, T.L., Czekala, N., 2005. Relaxin concentrations in serum and urine of endangered species: correlations with physiologic events and use as a marker of pregnancy
596. Ann. N. Y. Acad. Sci. 1041, 367-378.
Abstract: Many mammalian species are facing extinction due to problems created by human encroachment, agriculture, pollution, and willful slaughter. Among those at risk are the Asian and African elephant, Sumatran rhinoceros, and giant panda. Conservation groups try to save species in the wild by preserving habitat and limiting animal-human conflicts, often with limited success. Another alternative is to preserve the extant gene pool through captive breeding as a hedge against extinction. Measurement of circulating reproductive hormones is impractical for most wildlife species; determination of urinary or fecal hormone metabolites provides a more viable approach. To aid breeding management, one important tool is the ability to diagnose and monitor pregnancy, especially in species with long gestations (e.g., rhinos over 15 mo and elephants over 20 mo). Unfortunately, measuring progestins often is not useful diagnostically, because concentrations are similar during at least part of the pregnancy and the nonpregnant luteal phase in some species (e.g., elephants, rhinoceroses, and giant pandas). As serum relaxin reliably distinguishes between pregnancy and pseudopregnancy in bitches, relaxin measurement might also provide a method for detecting a successful pregnancy in endangered species. Appropriate immunoassay reagents have enabled the estimation of relaxin concentrations in the serum of elephants and rhinos and the determination of pregnancy establishment and the outcome. Relaxin was also detected in panda serum and urine. However, the extreme variability of the time between observed mating and parturition and the confounding factors of delayed implantation, pseudopregnancy, and frequent fetal resorptions made it impossible to use the panda relaxin data as a specific marker of pregnancy

Brown, J.L., Walker, S.L., Moeller, T., 2004. Comparative endocrinology of cycling and non-cycling Asian (Elephas maximus) and African (Loxodonta africana) elephants
729. Gen. Comp Endocrinol. 136, 360-370.
Abstract: Up to 14% of Asian and 29% of African elephants in captivity are not cycling normally or exhibit irregular cycles based on progestin profiles. To determine if ovarian acyclicity is related to other disruptions in endocrine activity, serum pituitary, thyroid, adrenal, and ovarian hormones in weekly samples collected for 6-25 months were compared between normal cycling (n=22 each species) and non-cycling (n=6 Asian; n=30 African) elephants. A subset of cycling females (n=4 Asian, 7 African) also were blood sampled daily during the follicular phase to characterize the peri-ovulatory period. In normal cycling females, two leutinizing hormone (LH) surges were observed 3 weeks apart during a normal follicular phase, with the second inducing ovulation (ovLH). Serum FSH concentrations were highest at the beginning of the non-luteal phase, declining to nadir concentrations within 4 days of the ovLH surge. FSH remained low until after the ovLH surge and then increased during the luteal phase. A species difference was noted in prolactin secretion. In the African elephant, prolactin was increased during the follicular phase, but in Asian elephants concentrations remained stable throughout the cycle. Patterns of thyroid hormones (thyroid-stimulating hormone, TSH; free and total thyroxine, T4; free and total triiodothyronine, T3) and cortisol secretion were not affected by estrous cycle stage or season in cycling elephants. In non-cycling elephants, there were no fluctuating patterns of LH, FSH, or prolactin secretion. Overall mean concentrations of all hormones were similar to those in cycling animals, with the exception of FSH, prolactin, and estradiol. Mean serum FSH concentrations were lower due to females not exhibiting normal cyclic increases, whereas serum estradiol was higher overall in most acyclic females. Prolactin concentrations were significantly increased in 11 of 30 non-cycling females, all of which were African elephants. In sum, while there were no consistent endocrine anomalies associated with ovarian acyclicity, hyperprolactinemia may be one cause of ovarian dysfunction. The finding of elevated estrogens in some acyclic females also deserves further investigation, especially determining how it relates to reproductive tract pathologies

Buchanan, K.L., Goldsmith, A.R., 2004. Noninvasive endocrine data for behavioural studies: The importance of validation. Animal Behaviour 67, 183-185.
Abstract: There has been a substantial growth recently in the use of noninvasive methods to quantify hormone production, through the measurement of excreted hormones or hormone levels from saliva, sweat or hair (e.g.Wasser et al. 2000; Cook 2002; Pfeffer et al. 2002). These measures can quantify either current (e.g. Berg & Wynne-Edwards 2002; Maso et al. 2002) or past (e.g. Wasser et al. 2000; Ostner et al. 2002) levels of hormone production and the data can be used to determine the relations between a range of hormone levels and animal behaviour across taxa (Wasser et al. 2000). Such techniques have been used extensively to examine social stress (Goymann et al. 2001), the effects of environmental stress (Creel et al. 2002), reproductive cycles (Curtis et al. 2000) and social dominance (von Engelhardt et al. 2000; Langmore et al. 2002). They may have important applications in conservation science (Ishii 1999). There are several reasons why noninvasive methods of sampling are highly desirable. Importantly, animal suffering can potentially be reduced. In practical terms there are also several advantages: noninvasive methods allow samples to be obtained retrospectively, which represent average hormone production over a certain time frame, and the time spent handling the animal does not affect the levels obtained, which is advantageous for highly pulsatile hormones such as corticosteroids. In addition, the licensing constraints for noninvasive methods of sampling are less restrictive. However, such techniques also have disadvantages. In particular, faecal, hair or feather samples can indicate only average hormone levels over a considerable, and possibly unknown, period. Compared with plasma levels, noninvasive measures may result in a loss of sensitivity in any further analyses examining the relations between hormone levels and other variables (Shirtcliff et al. 2002). Furthermore, faecal samples in particular may not be available from known individuals a known amount of time after excretion, preventing reliable determination of individual hormone levels. It is also worth considering that while noninvasive sampling will not cause large increases in pulsatile 'stress' hormones as caused by capture and restraint, some increase may occur merely as a result of the presence of the sampler. In addition, there are a number of validation issues concerning the quantification of steroids from noninvasive samples which we outline below. Koren et al. (2002) documented a protocol for the extraction of testosterone and cortisol from hair obtained from the rock hyrax, Procavia capensis. They used this technique to quantify the levels of hormones contained in plucked hair samples, allowing hormone levels during the period of hair production to be determined, noninvasively. They found that the levels of testosterone extracted correlated positively with the dominance rank of male hyraxes. Although such methods are highly desirable, it is important to emphasize that all new methods of measuring levels of hormone production using hormone extracted from organic substrates should be appropriately validated, such that the limitations of the technique can be defined. This requires: (1) that the assay is validated for each new species and substrate and (2) that the extraction efficiency is determined for the target hormone in the species and substrate of interest. Although ready-made endocrine kits are provided with some data on the assay validation, the validation is relevant only for the species and substrate tested by the commercial supplier, generally in a limited range of biological media. It is essential to extend these validations for the species and substrate to which the kit is being applied. For example, a methanol extract of hair may contain substances that interfere with the assay procedure and thus would give misleading results.

Carter, A.M., Enders, A.C., 2004. Comparative aspects of trophoblast development and placentation. Reproductive Biology and Endocrinology 2.
Abstract: Based on the number of tissues separating maternal from fetal blood, placentas are classified as epitheliochorial, endotheliochorial or hemochorial. We review the occurrence of these placental types in the various orders of eutherian mammals within the framework of the four superorders identified by the techniques of molecular phylogenetics. The superorder Afrotheria diversified in ancient Africa and its living representatives include elephants, sea cows, hyraxes, aardvark, elephant shrews and tenrecs. Xenarthra, comprising armadillos, anteaters and sloths, diversified in South America. All placentas examined from members of these two oldest superorders are either endotheliochorial or hemochorial. The superorder Euarchontoglires includes two sister groups, Glires and Euarchonta. The former comprises rodents and lagomorphs, which typically have hemochorial placentas. The most primitive members of Euarchonta, the tree shrews, have endotheliochorial placentation. Flying lemurs and all higher primates have hemochorial placentas. However, the lemurs and lorises are exceptional among primates in having epitheliochorial placentation. Laurasiatheria, the last superorder to arise, includes several orders with epitheliochorial placentation. These comprise whales, camels, pigs, ruminants, horses and pangolins. In contrast, nearly all carnivores have endotheliochorial placentation, whilst bats have endotheliochorial or hemochorial placentas. Also included in Laurasiatheria are a number of insectivores that have many conserved morphological characters; none of these has epitheliochorial placentation. Consideration of placental type in relation to the findings of molecular phylogenetics suggests that the likely path of evolution in Afrotheria was from endotheliochorial to hemochorial placentation. This is also a likely scenario for Xenarthra and the bats. We argue that a definitive epitheliochorial placenta is a secondary specialization and that it evolved twice, once in the Laurasiatheria and once in the lemurs and lorises.

de Oliveira, C.A., West, G.D., Houck, R., Leblanc, M., 2004. Control of musth in an Asian elephant bull (Elephas maximus) using leuprolide acetate
716. J. Zoo. Wildl. Med. 35, 70-76.
Abstract: The results of long-term administration of leuprolide acetate (LA) depot in a 52-yr-old Asian elephant bull (Elephas maximus) for control of musth are presented. Twelve injections were administered for 6 yr during our interpretation of early musth or "premusth." Intervals between musth periods during the study varied from 2 to 34 mo. Blood samples, drawn weekly, were assayed for serum testosterone concentrations; mean levels were 11.78 +/- 1.97 nmol/L throughout the first 26 mo of the study, 7.28 +/- 1.28 nmol/L during the following 21 mo, and 0.45 +/- 0.035 nmol/L in the last 34 mo of this study. Early musth signs ceased within 3 days of drug administration after 10 of 12 injections. The mean serum testosterone concentrations were significantly decreased by the last 34 mo of the study. The results suggest leuprolide is a suitable alternative for controlling or preventing (or both) musth in captive Asian elephants, although permanent reproductive effects may occur. Zoos and wildlife conservation institutions could benefit from the use of LA in Asian elephants to increase the male availability in captivity, consequently ensuring genetic diversity and the perpetuation of the species

Hermes, R., Hildebrandt, T.B., Goritz, F., 2004. Reproductive problems directly attributable to long-term captivity-asymmetric reproductive aging. Animal Reproduction Science 82-83, 49-60.
Abstract: Problems attributable to long-term captivity have been identified and are responsible for the difficulties in establishing successful reproduction in captive populations of wildlife, specifically, elephants and rhinoceroses. Historically, non-reproductive periods of 10-15 years in nulliparous female rhinoceroses and elephants have not been considered problematic. New evidence suggests that prolonged exposure to endogenous sex steroids and that long stretches of non-reproductive periods induce asymmetric reproductive aging in captive animals. The consequences are reduced fertility, shortened reproductive life-span and, eventually, irreversible acyclicity. Although age-related reproductive lesions have also been documented in male rhinoceroses, they continue to maintain a longer reproductive life-span than females. Since human and domestic animal models have already indicated that early pregnancy provides natural protective mechanism against asymmetric reproductive aging processes and premature senescence, it is imperative that appropriate counter measures such as assisted reproductive technologies (ART) be utilized to ensure early pregnancy in captive animals for their preservation and to ensure increased genetic diversity of the captive populations.

Meyer, J.M., Walker, S.L., Freeman, E.W., Steinetz, B.G., Brown, J.L., 2004. Species and fetal gender effects on the endocrinology of pregnancy in elephants
682. Gen. Comp Endocrinol. 138, 263-270.
Abstract: Quantitative and temporal progestin profiles vary during gestation in the elephant, sometimes making it difficult to determine if a pregnancy is progressing normally. The aim of the present study was to determine if circulating progestin variability was related to species or fetal gender effects. A similar comparison also was conducted for secretory profiles of prolactin, relaxin, and cortisol. Overall mean progestin concentrations during gestation in Asian (n = 19) and African (n = 8) elephants were similar; however, the temporal profiles differed (P < 0.001). Concentrations were higher in African elephants during the first half of pregnancy, but then declined to levels below those observed in Asian elephants (P < 0.05). There also was a fetal gender effect in Asian, but not African elephants. Progestin concentrations were higher in Asian cows carrying male calves (n = 9) as compared to those carrying females (n = 10) (P < 0.001). Overall prolactin concentrations were higher in Asian than in African elephants between 8 and 15 months of gestation ( P< 0.001). There were no species differences in the secretory patterns of relaxin. Cortisol was relatively stable until the end of gestation when significant surges were observed, mainly between 8 and 11 days before parturition, and again on the day of birth. In sum, a comparison of progestin patterns between Asian and African elephants identified notable differences related to species and fetal gender. A role for cortisol in the initiation of parturition also was inferred from these data. From a practical standpoint, understanding the factors affecting gestational hormone characteristics and recognizing what the species differences are will help ensure that data used in diagnosing and monitoring elephant pregnancies are properly interpreted

Wilson, M.L., Bloomsmith, M.A., Maple, T.L., 2004. Stereotypic swaying and serum cortisol concentrations in three captive African elephants (Loxodonta africana). Animal-Welfare 13, 39-43.
Abstract: The behaviour and serum cortisol concentrations of three captive female African elephants (Loxodonta africana) were studied to determine whether their stereotypic swaying was more prevalent before regularly scheduled events in the elephants' routine, and whether the elephants that exhibited more stereotyped swaying had lower mean serum cortisol concentrations. Behavioural data were collected during hour-long observations balanced across three periods, and during 15-min observations prior to the elephants being moved to different portions of their enclosure. Observational data were collected using instantaneous focal sampling of behaviours every 30 s. Serum cortisol measures were obtained through weekly blood withdrawal from the elephants' ears. Of the three elephants, two exhibited stereotyped swaying, which accounted for a mean of 0.4% of the scans during the hour-long observations and a mean of 18% of the scans prior to the elephants being moved between different parts of the enclosure. Swaying was highly variable among the individual elephants during both categories of observations. Additionally, both elephants swayed more prior to moving in the afternoon than prior to moving in the morning. Analyses of serum cortisol concentrations indicated that each elephant had a different mean cortisol level, which did not clearly correspond with the expression of swaying. The findings indicate that a rigidly scheduled management event may elicit stereotyped swaying in the studied elephants. Future research should document the behavioural and physiological effects of an altered management routine to improve captive elephant welfare.

Dehnhard, M., Hatt, J.M., Eulenberger, K., Ochs, A., Strauss, G., 2003. Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for the determination of 5alpha-androst-2-en-17-one and -17beta-ol in the female Asian elephant: application for reproductive monitoring and prediction of parturition. J Steroid Biochem Mol Biol. 84, 383-391.
Abstract: Asian elephants are not self-sustaining in captivity. The main reasons for this phenomenon are a low birth rate, an aging population, and poor calf-rearing. Therefore, it is essential that reproductive rates had to be improved and there is need for rapid quantitative measures to monitor reproductive functions focussing on estrous detection and the prediction of the period of parturition. The objective of this study was to develop a method which combines headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for analyses of 5alpha-androst-2-en-17beta-ol and -17-one to prognose estrous and to predict the period of parturition. SPME was carried out with a CTC Combi Pal system.The course of the luteal phase-specific substance 5alpha-androst-2-en-17beta-ol and -17-one followed a cyclic pattern in which the follicular and luteal phases could be clearly distinguished (mean estrous cycle length, 15+/-1.4 weeks). Based on daily urine samples, estrous prognosis might be possibly based on the initial 5alpha-androst-2-en-17beta-o1 increase at the end of the follicular phase. Parturition prognosis was performed in three elephant cows based on the 5alpha-androst-2-en-17beta-o1 drop to baseline levels 5-4 days prior parturition. Experiments revealed that 5alpha-androst-3alpha-ol-17-one and probably 5alpha-androst-3alpha-ol-17beta-ol are generated from sulfate conjugates by a thermal process. Institute for Zoo Biology and Wildlife Research, PF 601103, D-10252 Berlin, Germany. dehnhard@izw-berlin.de

Ganswindt, A., Palme, R., Heistermann, M., Borragan, S., Hodges, J.K., 2003. Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth. Gen Comp Endocrinol 134, 156-166.
Abstract: German Primate Centre, Department of Reproductive Biology, Kellnerweg 4, 37077 Gottingen, Germany. ganswindt@www.dpz.gdwg.de

Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.

Hunt, K.E., Wasser, S.K., 2003. Effect of long-term preservation methods on fecal glucocorticoid concentrations of grizzly bear and african elephant. Physiol Biochem Zool. 76, 918-928.
Abstract: Center for Conservation Biology, Department of Biology, University of Washington, Seattle, WA, 98195-1800, USA.

Rajaram, A., Krishnamurthy, V., 2003. Elephant temporal gland ultrastructure and androgen secretion during musth. Current Science 85, 1467-1471.
Abstract: We have investigated the ultrastructure of the temporal gland of the Asian elephant (Elephas maximus) in the musth condition. We find that the organelles are highly evolved for the production of the androgen, testosterone which is reported to be very high in the Asian male elephant in full musth. The mitochondria bear cristae which are profuse and tubular, and occur along with many Golgi bodies. There is hypertrophy of smooth endoplasmic reticulum. All the structures involved in the production of androgen, as in the Leydig cell or the cells of the adrenal cortex, are thus found in abundance. Cellular structures also seem singularly evolved for the secretion of androgen and its degradation products.

Rasmussen, L.E., Greenwood, D.R., 2003. Frontalin: a chemical message of musth in Asian elephants (Elephas maximus). Chemical Senses 28, 433-446.
Abstract: Musth is an important male phenomenon affecting many aspects of elephant society including reproduction. During musth, the temporal gland secretions (as well as the urine and breath) of adult male Asian elephants (Elephas maximus) discharge a variety of malodorous compounds together with the bicyclic ketal, frontalin. In contrast, teenage male elephants in musth release a sweet-smelling exudate from their facial temporal gland. We recently demonstrated that the concentration of frontalin becomes increasingly evident as male elephants mature. In the present study, we demonstrate that behaviors exhibited towards frontalin are consistent and dependent on the sex, developmental stage and physiological status of the responding conspecific individual. To examine whether frontalin functions as a chemical signal, perhaps even a pheromone, we bioassayed older and younger adult males, and luteal- and follicular-phase and pregnant females for their chemosensory and behavioral responses to frontalin. Adult males were mostly indifferent to frontalin, whereas subadult males were highly reactive, often exhibiting repulsion or avoidance. Female chemosensory responses to frontalin varied with hormonal state. Females in the luteal phase demonstrated low frequencies of responses, whereas pregnant females responded significantly more frequently, with varied types of responses including those to the palatal pits. Females in the follicular phase were the most responsive and often demonstrated mating-related behaviors subsequent to high chemosensory responses to frontalin. Our evidence strongly suggests that frontalin, a well-studied pheromone in insects, also functions as a pheromone in the Asian elephant: it exhibits all of the determinants that define a pheromone and evidently conveys some of the messages underlying the phenomenon of musth. Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, OR 97006-8921, USA. betsr@bmb.ogi.edu

Wilson, J.D., Leihy, M.W., Shaw, G., Renfree, M.B., 2003. Androgen physiology: unsolved problems at the millennium. Molecular and Cellular Endocrinology 198, 1-5.
Abstract: Androgen physiology differs from that of other steroid hormones in two major regards. First, testosterone, the predominant circulating testicular androgen, is both an active hormone and a prohormone for the formation of a more active androgen, the 5alpha-reduced steroid dihydrotestosterone. Genetic evidence indicates that testosterone and dihydrotestosterone work via a common intracellular receptor, and studies involving in vitro reporter gene assays and intact mice in which both steroid 5alpha-reductase isoenzymes have been disrupted by homologous recombination indicate that dihydrotestosterone acts during embryonic life to amplify hormonal signals that can be mediated by testosterone at higher concentrations. However, in post-embryonic life dihydrotestosterone plays unique roles that have not been elucidated. Studies of other 5alpha-reduced steroids, including the plant hormone brassinolide, the hog pheromones androstanol and androstenol, and 5alpha-dihydroprogesterone (in horses and elephants) indicate that this reaction serves different functions in different systems. Second, during embryonic life androgen causes the formation of the male urogenital tract and hence is responsible for development of the tissues that serve as the major sites of androgen action in postnatal life. It has been generally assumed that androgens virilize the male fetus by the same mechanisms as in the adult, namely by the conversion of circulating testosterone to dihydrotestosterone in target tissues. However, in marsupial mammals there is no sexual dimorphism in the levels of testosterone or dihydrotestosterone at the time the male phenotype forms, and in the pouch young of one marsupial, the tammar wallaby, the testes secrete another 5alpha-reduced steroid, 5alpha-androstane-3alpha, 17beta-diol (5alpha-adiol), into plasma. The administration of 5alpha-adiol to female pouch young causes profound virilization of the urogenital sinus and external genitalia, but within target tissues 5alpha-adiol appears to work after oxidation to dihydrotestosterone. Thus, two separate mechanisms evolved for the formation of dihydrotestosterone in target tissues. 5alpha-adiol is the predominant androgen in neonatal testes in several placental mammals, but it is unclear whether it plays a similar role in other mammalian species.

Alex, P.C., 2002. The Musth, the vicious and the rogue elephants - a review. Journal of Indian Veterinary Association Kerala 7, 26-27.

Allen, W.R., Mathias, S.S., Wooding, F.B., Skidmore, J.A., van Aarde, R.J., 2002. Placentation in the African elephant, Loxodonta africana. I. Endocrinological aspects. Reprod Suppl 60, 105-116.
Abstract: Placental and fetal tissues were recovered from the uteri of 59 pregnant elephant that ranged in estimated age from day 18 to month 21 of gestation. Incubation of placenta and fetal gonad, alone or in combination, with tritium-labelled cholesterol, pregnenolone and androstenedione failed to yield any labelled progestagens or oestrogens from placenta, but did produce small amounts of labelled progesterone and 5alpha-dihydroprogesterone from fetal gonad. Immunochemical staining of tissues with four antisera specific for enzymes involved in the steroidogenic pathway revealed no staining in sections of placenta but positive labelling for P450 side chain cleavage enzyme (SCC450) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the interstitial cells that comprise the bulk of the enlarged fetal gonads during the second half of gestation. Saline extracts of placental tissue showed no activity in three different gonadotrophin assays. In view of this endocrinological inactivity in the zonary elephant placenta and the probable reliance on maternal luteal sources of progestagens for maintenance of the pregnant state, the argument is advanced that uncomplicated abortion would probably follow a single administration of a PGF analogue given at any stage of pregnancy. If so, the treatment might constitute an efficacious method for controlling population increases in elephants maintained in enclosed game parks in Africa.

Cheeran, J.V., 2002. Elephant facts. Journal of Indian Veterinary Association Kerala 7, 12-14.

Cheeran, J.V., Radhakrishnan, K., Chandrasekharan, K., 2002. Musth. Journal of Indian Veterinary Association Kerala 7, 28-30.

Forsyth, I.A., Wallis, M., 2002. Growth hormone and prolactin--molecular and functional evolution. J Mammary Gland Biol Neoplasia 7, 291-312.
Abstract: Growth hormone, prolactin, the fish hormone, somatolactin, and related mammalian placental hormones, including placental lactogen, form a family of polypeptide hormones that share a common tertiary structure. They produce their biological effects by interacting with and dimerizing specific single transmembrane-domain receptors. The receptors belong to a superfamily of cytokine receptors with no intrinsic tyrosine kinase, which use the Jak-Stat cascade as a major signalling pathway. Hormones and receptors are thought to have arisen as a result of gene duplication and subsequent divergence early in vertebrate evolution. Mammalian growth hormone and prolactin show a slow basal evolutionary rate of change, but with episodes of accelerated evolution. These occurred for growth hormone during the evolution of the primates and artiodactyls and for prolactin in lineages leading to rodents, elephants, ruminants, and man. Placental lactogen has probably evolved independently on three occasions, from prolactin in rodents and ruminants and from growth hormone in man. Receptor sequences also show variable rates of evolution, corresponding partly, but not completely, with changes in the ligand. A principal biological role of growth hormone, the control of postnatal growth, has remained quite consistent throughout vertebrate evolution and is largely mediated by insulin-like growth factors. Prolactin has many and diverse roles. In relation to lactation, the relative roles of growth hormone and prolactin vary between species. Correlation between the molecular and functional evolution of these hormones is very incomplete, and it is likely that many important functional adaptations involved changes in regulatory elements, for example, altering tissue of origin or posttranscriptional processing, rather than change of the structures of the proteins themselves. The Babraham Institute, Babraham, Cambridge, United Kingdom. isabel.forsyth@bbsrc.ac.uk

Goodwin, T.E., Brown, F.D., Counts, R.W., Dowdy, N.C., Fraley, P.L., Hughes, R.A., Liu, D.Z., Mashburn, C.D., Rankin, J.D., Roberson, R.S., Rasmussen, E.L., Riddle, S.W., Riddle, H.S., Schulz, S.J., 2002. African Elephant Sesquiterpenes. II. Identification and Synthesis of New Derivatives of 2,3-Dihydrofarnesol. Natural Products 65, 1319-1322.
Abstract: A search for potential semiochemicals revealed nerolidol (6), albicanol (7), and the new 2,3-dihydrofarnesol derivatives 8-10 in the temporal gland secretions of African elephants. A novel synthesis from (E,E)-farnesol (1) provided compounds 8-10 for GC-MS comparison to the natural products. This study confirms the farnesol family as frequently occurring secondary metabolites in African elephant temporal gland secretions.

Kumar, G.A., Ghosh, K.N.A., Sreekumaran, T., Chandrasekharan, K., 2002. Reproduction in elephants. Journal of Indian Veterinary Association Kerala 7, 38-40, 48-48.

Nayar, K.N.M., Chandrasekharan, K., Radhakrishnan, K., 2002. Management of surgical affections in captive elephants. Journal of Indian Veterinary Association Kerala 7, 55-59.

Rasmussen, L.E., Riddle, H.S., Krishnamurthy, V., 2002. Mellifluous matures to malodorous in musth. Nature 415, 975-976.
Abstract: Male Asian elephants in musth--an annual period of heightened sexual activity and intensified aggression--broadcast odoriferous, behaviourally influential messages from secretions of the temporal gland. From our observations in the wild, together with instantaneous chemical sampling and captive-elephant playback experiments, we have discovered that young, socially immature males in musth signal their naivety by releasing honey-like odors to avoid conflict with adult males, whereas older musth males broadcast malodorous combinations to deter young males, facilitating the smooth functioning of male society. As elephant--human conflicts can upset this equilibrium, chemically modulating male behaviour may be one way to help the conservation of wild elephants.

Rasmussen, L.E.L., Riddle, H.S., Krishnamurthy, V., 2002. Mellifluous matures to malodorous in musth; Mood-altering secretions by excited male elephants smooth out social interactions. Nature 415, 975-976.

Welsch, U., Unterberger, P., Hofter, E., Cuttitta, F., Martinez, A., 2002. Adrenomedullin in mammalian and human skin glands including the mammary gland. Acta Histochem 104, 65-72.
Abstract: Adrenomedullin is a peptide that has been ascribed numerous functions. In the present paper, adrenomedullin has been localized immunhistochemically in a variety of skin glands of humans, elephants and impalas: apocrine scent glands, eccrine sweat glands, holocrine glands and mammary glands. In the apocrine glands expression of adrenomedullin varied with respect to staining intensity and intracellular localization. In general, glands which appeared to be actively secreting were more strongly stained than quiescent glands. However, within a single glandular tubule, individual cells differed considerably in the staining intensity of adrenomedullin. Adrenomedullin was present in both non-lactating and lactating mammary secretory epithelia, both ducts and alveoli reacted positively. In human mammary glands displaying apocrine metaplasia, the apical protrusions were strongly positive. Furthermore, positive immunostaining was found in endothelium and often in smooth muscle cells of small arteries and veins and in mast cells as well. Many of the adrenomedullin-positive epithelial cells were most strongly stained in the area of the Golgi apparatus, the cellular apex and particularly close to the basal side of the cell membrane.This pattern suggests packaging of adrenomedullin into secretory granules and secretion both at the apex of cells and at their basis. The first form of secretion suggests exocrine secretion, the latter form endocrine secretion of adrenomedullin. A possible hormonal function is in line with basally located electron dense small secretory granules, which have been found by electron microscopy in the glandular epithelia studied.

Wielebnowski, N.C., Fletchall, N., 2002. Noninvasive assessment of adrenal activity associated with husbandry and behavioral factors in the North American clouded leopard population. Zoo Biology 21, 77-98.

Dehnhard, M., Hildebrand, T., Rohleder, M., Strauss, G., Meyer, H.H.D., Goritz, F., 2001. Application of an enzyme-immunoassay (EIA) for rapid screening of 5alpha-pregnane-3,20-dione (DHP) in blood plasma of the Asian elephant, Elephas maximus. Berliner und Munchener Tierarztliche Wochenschrift. 114, 161-165.
Abstract: Populations of African (Loxodonta africana) and Asian elephants (Elephas maximus) in zoos and safari parks are at risk due to their low reproductive success. To extend the limited knowledge of their reproductive physiology, easy and practical methods for the analysis of relevant reproductive hormones must be developed to support assisted reproduction, for instance. Blood samples from 2 nonpregnant and 2 pregnant Asian elephants were used in the following study. For the measurement of 5alpha-pregnane-3,20-dione (DHP), the predominant ovarian gestagen in both species, an enzyme-immunoassay (EIA) based on commercial reagents was applied. Advantages of this EIA are the small volume of plasma needed for evaluation (5 µl) and the possibility of direct processing without an extraction stage. The lower limit of detection was 0.16 ng/ml, mean recovery was 101% and the mean coefficients of variation were 7.3 (intra-assay) and 9.9% (inter-assay). In the Asian elephants, DHP levels reached 15 ng/ml during the luteal phase and 21 ng/ml during pregnancy. Oestrous cycle lengths based on the lowest DHP concentrations varied from 12 to 20 weeks (mean of 15.4±2.3). In two Asian elephant cows, a calf was stillborn. Thereafter, ovarian activity in the animals resumed after approximately 8 and 13 weeks, respectively. In one animal, estradiol implants for hormonal contraception caused a down regulation of ovarian function as demonstrated by an irregular pattern of DHP secretion over a period of 48 weeks. It is proposed that the direct DHP-EIA is a suitable method for reproductive monitoring in elephants, as it can be easily established in laboratories.

Dehnhard, M., Hesitermann, M., Goritz, F., Hermes, R., Hildebrand, T., Haber, H., 2001. Demonstration of 2-unsaturated C19-steroids in the urine of female Asian elephants, Elephas maximus, and their dependence on ovarian activity. Reproduction-Cambridge 121, 475-484.
Abstract: An oestrous-related pheromone of the female Asian elephant (Elephas maximus) is known to induce behavioural responses in elephant bulls. Additional data revealed that timing of oestrus in females with close social relationships tends to be synchronized. Therefore, urine from female Asian elephants might be expected to contain luteal phase-dependent volatile substances, which may function as additional chemical signals in this species. The aim of the present study was to identify such compounds and to investigate their pattern of excretion throughout the ovarian cycle. Urine samples were collected 3 times a week during the follicular phase and 1 to 3 times a week during the luteal phase from 5 adult female Asian elephants from a total of 13 non-conception cycles and one conception cycle, including the first 72 weeks of pregnancy. A simple headspace solid-phase microextraction method has been developed for quantification of urinary volatile substances and analysis was performed by gas chromatography. The comparison of urine collected during the follicular and the luteal phase indicated the presence of two luteal phase-dependent substances. Mass spectrometry was used to identify one substance as 5alpha-androst-2-en-17-one and a second substance as the corresponding alcoholic compound 5alpha-androst-2-en-17beta-ol. The 5alpha-androst-2-en-17beta-ol and -17-one profiles reflected cyclic ovarian activity with clear (10-20-fold) luteal phase increases. Furthermore, measurements of both compounds were correlated positively with the concentration of urinary pregnanetriol and indicated cycle duration (15.1±1.2 weeks) similar to that obtained from pregnanetriol measurements (15.2±1.6 weeks). The results demonstrated the presence of 2 luteal phase-specific steroidal volatile compounds in elephant urine. One of the substances, 5alpha-androst-2-en-17-one, has been demonstrated in human axillary bacterial isolates. The measurement of both volatile substances in elephant urine can be used for rapid detection of the stage of the ovarian cycle, as the analysis can be completed within 2 h.

Ganswindt, A., Heistermann, M., Hodges, J.K. Faecal Glucocorticoid and Androgen Metabolite Excretion in Male African Elephants (Loxodonta africana). A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001. 258. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Ganswindt, A., Heistermann, M., Borragan, S., Hodges, J.K., 2001. Assessment of testicular endocrine function in captive African elephants by measurement of urinary and fecal androgens. Zoo Biology 21, 27-36.

Lamps, L.W., Smoller, B.R., Rasmussen, L.E., Slade, B.E., Fritsch, G., Goodwin, T.E., 2001. Characterization of interdigital glands in the Asian elephant (Elephas maximus). Research in Veterinary Science 71, 197-200.
Abstract: In the Asian elephant, wetness akin to perspiration is commonly observed on the cuticles and interdigital areas of the feet; this observation has lead to speculation regarding the existence of an interdigital gland. Our goal was to search for interdigital glands and characterize them morphologically, histochemically, and immunohistochemically. Necropsy samples of interdigital areas from two Asian elephants were obtained. Multiple sections were fixed and processed routinely, then stained with hematoxylin/eosin and differential mucin stains. Immunohistochemistry was also performed for cytokeratins 8 and 10. Interdigital glands resembling human eccrine glands were detected deep within the reticular dermis. Histochemical staining indicated neutral mucopolysaccharides and nonsulphated acid mucopolysaccharides in glandular secretions, and the glandular epithelium also showed immunoreactivity to cytokeratins 8 and 10. Both the histochemical and immunohistochemical staining patterns are analogous to human eccrine structures. This study shows with certainty that Asian elephants possess sweat glands as they are defined histologically.

Pucher, H.E., Stremme, C., Tu, N.C., Ly, C.T., Holzmann, A., Schwarzenberger, F. Endocrine and Spermatological Evaluations of Semi-Wild Ranging Male Asian Elephants (Elephas maximus) in Vietnam - Preliminary Results. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001. 283. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Rasmussen, L.E.L., 2001. Source and cyclic release pattern of (Z)-7-dodecenyl acetate, the pre-ovulatory pheromone of the female Asian elephant. Chemical Senses 26, 611-623.
Abstract: Female Asian elephants (Elephas maximus) release a pre-ovulatory urinary pheromone, (Z)-7-dodecenyl acetate (Z7-12:Ac), to signal males of their readiness to mate. Z7-12:Ac is quantitatively elevated during the follicular stage of oestrus, reaching maximum concentrations just prior to ovulation, as demonstrated by two complementary headspace techniques: (1) evacuated canister capture followed by cryogenic trapping and (2) solid phase microextraction (SPME) used prior to gas chromatography/mass spectrometry (GC/MS). These patterns were coincident with observed male behaviours and were consistent with biochemical and binding properties of the active ligand, including optimal binding pH. To release maximum amounts of Z7-12:Ac for quantitation, serum and urine samples from three mature female Asian elephants in their luteal and follicular stages of several oestrous cycles were subjected to heat and pH changes and were then treated with protease prior to SPME-GC/MS analyses. When the post-luteal serum progesterone concentrations declined to baseline levels, Z7-12:Ac became detectable in the female urine. Throughout the follicular stage, pheromone concentrations increased linearly with no apparent relationship to the two serum luteinizing hormone peaks. Pre-ovulatory urine also contained related compounds, including (Z)-7-12-dodecenol. The relative amount of this alcohol increased relative to acetate during long-term storage, with a proportional reduction in bioactivity. Z7-12:Ac was not detected in mucus samples from the urogenital tract. A potential precursor of Z7-12:Ac was identified in liver homogenates from female elephants in the follicular stage. Erratum in: Chem Senses 2001 Sep;26(7):935

Rasmussen, L.E.L., Krishnamurthy, V., 2001. Urinary, temporal gland and breath odors from Asian elephants of Mudumalai National Park. Gajah 20, 1-7.

Suedmeyer, W.K. Serum hydrocortisone levels in a manually restrained African elephant (Loxodonta africana) pre- and post- semen collection. Kirk Baer, C. and Wilmette, M. W. Proceedings American Association of Zoo Veterinarians, American Association of Wildlife Veterinarians, Association of Reptilian and Amphibian Veterinarians and the National Association of Zoo and Wildlife Veterinarians Joint Conference. 388-389. 2001. American Association of Zoo Veterinarians. 9-18-2001.
Ref Type: Conference Proceeding

Wilson, M.L., Bloomsmith, M.A., Crane, M., Maple, T.L. Behavior and serum cortisol concentrations of three captive African elephants ( Loxodonta africana): preliminary results. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001. 147-149. 2001. Vienna, Austria, Schuling Verlag.
Ref Type: Conference Proceeding

Wingate, L., Lasley, B. Is Musth a Reproductive Event: An Examination of Arguments For and Against this View. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001. 150-156. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Wyatt, J. Elephant breeding soundness examination forms and database. Kirk Baer, C. and Wilmette, M. W. Proceedings American Association of Zoo Veterinarians, American Association of Wildlife Veterinarians, Association of Reptilian and Amphibian Veterinarians, and the National Association of Zoo and Wildlife Veterinarians Joint Conference 2001. 396-400. 2001. USA, AAZV. 1.
Ref Type: Conference Proceeding
Abstract: Routine use of standardized soundness examination forms for male and female elephants provides data for an inter-relational database. Through queries we may answer population-based reproductive questions essential for promoting self-sustaining populations. This poster presentation demonstrates breeding soundness examination forms and accompanying database used in a pilot project to evaluate 25 elephants.

Brown, J.L., 2000. Reproductive endocrine monitoring of elephants: an essential tool for assisting captive management. Zoo Biology 19, 347-367.
Abstract: Considerable information now is available about the basic reproductive biology of elephants, especially females. However, as important as this knowledge is, it no longer is enough to simply compile it into a database. The potential exists for using endocrine monitoring techniques to solve real problems. This review summarizes our current knowledge of elephant endocrinology and offers suggestions on how to use the technology to maximize reproductive potential. The oestrous cycle can be monitored through the analysis of serum progestogens, primarily 5alpha-reduced compounds, and consists of an 8- to 12-week luteal phase and a 4- to 6-week inter-luteal period. Proof of ovarian cyclicity currently is mandatory before Species Survival Plan breeding recommendations are approved. However, because many adult females are not cycling normally, the reproductive monitoring of all cows throughout their life span is now encouraged. Complete endocrine evaluations in conjunction with ultrasound examinations and behavioral assessments are needed to identify causes of reproductive failure and develop mitigating treatments. Progestogen analyses also are effective for monitoring pregnancy, but only if longitudinal samples are collected. Alternatively, pregnancy can be diagnosed in occasional samples using serum prolactin or possibly relaxin measurements after 20 weeks of gestation. Parturition can be predicted on the basis of the rapid decrease in progestogens that occurs about 2-5 days before birth. An updated model of ovarian dynamics during the oestrous cycle suggests that two waves of follicular development occur 3 weeks apart during the non-luteal phase, possibly under the control of follicle-stimulating hormone. Each follicular wave culminates in a luteinizing hormone (LH) surge, with the second surge inducing ovulation and corpus luteum formation. The functional significance of the first, anovulatory LH surge is under investigation, but from a practical perspective it can be used to schedule breeding (by artificial insemination or natural mating) to coincide with the ovulatory LH surge. Less is known about the reproductive biology of bulls, aside from the fact that musth is associated with dramatic changes in androgen secretion. Studies are needed to determine whether poor libido and inadequate semen quality observed in some mature elephants are due to testicular steroidogenic dysfunction. When blood samples cannot be collected for routine hormone analysis, gonadal activity can be monitored non-invasively through the measurement of excreted steroid metabolites (males: androgens; females: estrogens, progestogens) in urine and faeces. Lastly, suggestions for future research priorities are provided.

Brown, J.L. Zoo Biology. Special Issue on elephant biology 19[5], 1-184. 2000.
Ref Type: Journal (Full)
Abstract: This issue focuses on elephant biology and includes the following topics: ultrasonography of the urogenital tract in elephants Loxodonta africana and Elephas maximus (an important tool for assessing female and male reproductive function); reproductive endocrine monitoring of elephants (an essential tool for assisting captive management); ultrasonography of the oestrous cycle in female African elephants; review of a newly recognized disease of elephants caused by endotheliotropic herpesviruses; tuberculosis in elephants in North America; how chemical signals integrate Asian elephant society; elephant communication; social structure and helping behaviour in captive elephants; a postcryogenic comparison of membrane fatty acids of elephant spermatozoa; and first disclosure and preliminary investigation of a liquid released from the ears of African elephants.

Doi, O., Komatsumoto, M., Terazono, M., Wada, S., 2000. Exfoliative cytology in vaginal vestibule of female Asian elephants: relation to circulating progesterone concentrations. Zoological Science 17, 1303-1309.
Abstract: The oestrous cycle of the Asian elephant (Elephas maximus) was monitored by analysis of exfoliative cytology in the vaginal vestibule and serum progesterone concentrations. Appearance frequency of each 5 exfoliative cells; parabasal, intermediate, superficial anuclear and nuclear cells and leukocytes; on the smear collected from two elephants was calculated, and serum progesterone concentrations were measured by radioimmunoassay. Serum progesterone concentrations changed regularly with the cycle between 14 and 17 weeks. Using spectrum analysis (Yule-Walker method) to appearance frequency of exfoliative cells, it was found that the time when a superficial cell markedly appeared in vaginal vestibule corresponded to the time when serum progesterone concentration was almost negligible. It is suggested that the time when numbers of two kinds of superficial (anuclear and nuclear) cells and parabasal and intermediate cells increase to the smear of the elephant, means the period from pro-oestrus to oestrus and from metoestrus to dioestrus, respectively.

Goodwin, T.E. The Secrets in Secretions: Unraveling Elephant Mysteries via Chemical Methodologies. Elephants: Cultural, Behavioral, and Ecological Perspectives; Program and Abstracts of the Workshop. 11-12. 2000. Davis, CA. 2000.
Ref Type: Conference Proceeding

Hermes, R., Olson, D., Goritz, F., Brown, J.L., Schmitt, D.L., Hagan, D., Peterson, J.S., Fritsch, G., Hildebrandt, T.B., 2000. Ultrasonography of the estrous cycle in female African elephants (Loxodonta africana). Zoo Biology 19, 369-382.
Abstract: The endocrinology of the elephant oestrous cycle has been well characterized, but little emphasis has been placed on evaluating corresponding changes in the reproductive tract. Ultrasound was used to document changes in reproductive tract morphology throughout the oestrous cycle in four cycling female African elephants. During a 7-month period, frequent ultrasound examinations (n=190) during the luteal and non-luteal phase were compared with serum progesterone and luteinizing hormone (LH) concentrations during a 7-month period. Ultrasonographic images documented vaginal and cervical oedema and changes in mucus consistency during the non-luteal phase. The cross-sectional diameter of the endometrium showed a dramatic increase during the non-luteal phase and followed cyclic changes. A different pattern of follicular development on the ovary was associated with the two LH surges. Follicle growth associated with the first, anovulatory LH surge was characterized by the formation of multiple small follicles, in contrast to the maturation of a single large follicle at the second, ovulatory LH (ovLH) surge. Ovulation and the subsequent formation of a corpus luteum (CL) were observed only after the ovLH surge. Ultrasound data in combination with endocrine assessments suggest that the African elephant is non-ovulatory, although multiple non-ovulatory luteal structures developed during the late non-luteal phase of each cycle. Both ovulatory CL and non-ovulatory luteal structures were present only through one cycle and regressed at the end of the luteal phase in conjunction with the drop in serum progesterone. We conclude that periodic reproductive-tract ultrasound assessments in association with continued endocrine monitoring of the oestrous cycle should be incorporated into the routine reproductive health assessment of elephants. This information is necessary for determining reproductive fitness before making breeding recommendations. It also has proven to be an invaluable tool for use with assisted reproductive techniques and has enormous potential for evaluating the efficiency of hormonal therapies used to treat reproductive dysfunction.

Rasmussen, L.E.L. Wild and Non-wild Elephants: How Two Modes of Olfaction and a Multitude of Chemical Signals and Pheromones Influence Elephant Behavior. Elephants: Cultural, Behavioral, and Ecological Perspectives; Program and Abstracts of the Workshop. 19-20. 2000. Davis, CA. 2000.
Ref Type: Conference Proceeding

Rasmussen, L.E.L., Krishnamurthy, V., 2000. How chemical signals integrate Asian elephant society: the known and the unknown. Zoo Biology 19, 405-423.
Abstract: The importance of chemical senses to elephants was recognized in anecdotal observations by ancient humans. Modern scientific tools, such as molecular biological techniques, highly sensitive gas chromatographic/mass spectrometric instrumentation, and statistically valid ethological methods, have allowed the study of real events of chemical communication between elephants. Such communication encompasses long- and short-range navigation, relationship recognition, and inter- and intra-sexual exchange of reproductive condition, metabolic state, and social status. Asian elephants emit large amounts of complex chemical mixtures in breath and urine, and in secretions from the temporal gland, inter-digital glands, and ears. Some emitted chemicals originate in blood and may be metabolic products; others are secretory products, at times apparently under hormonal control. The wide variety of emitted compounds includes hormones, proteins, and volatile compounds; selected volatile ketones and an acetate ((Z)-7-dodecenyl acetate) function as chemical signals and a pheromone, respectively. Some of these specific chemicals identified in emissions from Asian elephants dwelling in the United States have been found to be present in the exudates from elephants in India. This similarity is demonstrable for three metabolic conditions: pregnancy in females and pre- and post-musth males. Future chemical communication studies on male elephants should focus on musth and its relevance to reproduction and male social structures. Such investigations should include hormones, metabolites, brain chemistry, and possible primer pheromones. For females, the factors influencing possible estrous synchrony, what role primer pheromones play in female reproduction, how chemical signals influence social behavior, and whether luteinizing hormone influences pheromone production are among remaining fundamental questions.

Riddle, H.S., Riddle, S.W., Rasmussen, L.E.L., Goodwin, T.E., 2000. First disclosure and preliminary investigation of a liquid released from the ears of African elephants. Zoo Biology 19, 475-480.
Abstract: This report is the first documentation, both behaviorally and chemically, of a phenomenon observed among African elephants (Loxodonta africana) whereby a sudden, often stream-like discharge of liquid is seen from the auricular orifice. During this initial investigation, multiple samples of the fluid have been collected for analysis of physical properties and components. Trace organic chemicals which are apparently of elephant origin have been identified in the ear liquid, and the aqueous nature of the liquid has been demonstrated. The continuing objectives of this work and related studies are to determine the specific source of the liquid with particular focus on a search for auricular glands, to further characterize potential conspecific chemical signals, and to document more precisely particular social situations when this phenomenon occurs.

Schulte, B.A., Feldman, E., Lambert, R., Oliver, R., Oliver, R., 2000. Temporary ovarian inactivity in elephants: relationship to status and time outside. Physiol Behav 71, 123-131.
Abstract: The captive elephant population in North America is in reproductive decline and,without importation from the wild, may cease to be viable within the next several decades. The estrous cycle of three captive, reproductive-age African elephants was monitored for 3 years by measuring serum progesterone concentrations. Each elephant experienced one or more episodes of extended low progesterone (>12 weeks), analogous to supposed terminal cessation of estrous cyclicity or 'flatlining' that has been described in some captive Asian and
African elephants. Other studies have reported lengthy non-luteal (follicular)phases that indicate extended episodes of ovarian inactivity; however, this phenomenon has not been examined in detail. In this study, total duration of temporary ovarian inactivity or acyclicity followed a social rank pattern, with the most subordinate female having the longest and the dominant female the shortest duration. During periods of acyclicity, the number of hours the elephants spent outside was significantly less than during non-luteal or luteal phases of the cycle. Except in one instance, behavioral data recorded by elephant keepers during their interactions with the elephants showed no change in handling during periods of ovarian inactivity. Further study is necessary to distinguish the causative agent for temporary cessation of estrous cyclicity. Understanding this phenomenon is imperative for the future reproductive viability of captive elephant populations.

Stead, S.K., Meltzer, D.G.A., Palme, R., 2000. The measurement of glucocorticoid concentrations in the serum and faeces of captive African elephants (Loxodonta africana) after ACTH stimulation. Journal of the South African Veterinary Medical Association 71, 192-196.
Abstract: Recently established enzyme immunoassays that measure faecal glucocorticoid metabolites in elephants were evaluated, and a preliminary investigation into the biological relevance of this non-invasive method for use in assessing the degree of stress in this species was performed. Four juvenile African elephants were injected i.m. with 2.15 mg of synthetic adrenocorticotrophic hormone. Blood and faecal samples were collected over 4 h and 7 days, respectively. Concentrations of serum cortisol and faecal cortisol metabolites were determined using immunoassay. Variability of basal and peak values in blood and faeces was observed among the elephants. After ACTH injection, serum cortisol concentrations increased by 400-700%. An 11-oxoaetiocholanolone enzyme immunoassay (EIA) proved best suited to measure cortisol metabolites (11, 17-dioxoandrostanes) when compared to a cortisol and corticosterone EIA in faecal samples. Concentrations of faecal 11,17-dioxoandrostanes increased by 570-1070%, reaching peak levels after 20.0-25.5 h. Greater levels of glucocorticoid metabolites were measured in faecal samples from elephants kept in small enclosures compared with levels in the faeces of animals ranging over a larger area. The results of this preliminary study suggest that non-invasive faecal monitoring of glucocorticoid metabolites is useful in investigating adrenal activity in African elephants.

Wallis, M., 2000. Episodic evolution of protein hormones: molecular evolution of pituitary prolactin. J Mol Evol 50, 465-473.
Abstract: Previous studies have shown that pituitary growth hormone displays an episodic pattern of evolution, with a slow underlying evolutionary rate and occasional sustained bursts of rapid change. The present study establishes that pituitary prolactin shows a similar pattern. During much of tetrapod evolution the sequence of prolactin has been strongly conserved, showing a slow basal rate of change (approx 0.27x10(9) substitutions/amino acid site/year). This rate has increased substantially ( approximately 12- to 38-fold) on at least four occasions during eutherian evolution, during the evolution of primates, artiodactyles, rodents, and elephants. That these increases are real and not a consequence of inadvertent comparison of paralogous genes is shown (for at least the first three groups) by the fact that they are confined to mature protein coding sequence and not apparent in sequences coding for signal peptides or when synonymous substitutions are examined. Sequences of teleost prolactins differ markedly from those of tetrapods and lungfish, but during the course of teleost evolution the rate of change of prolactin has been less variable than that of growth hormone. It is concluded that the evolutionary pattern seen for prolactin shows long periods of near-stasis interrupted by occasional bursts of rapid change, resembling the pattern seen for growth hormone in general but not in detail. The most likely basis for these bursts appears to be adaptive evolution though the biological changes involved are relatively small.

Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, C.M., Bechert, U., Millspaugh, J.J., Larson, S., Monfort, S.L., 2000. A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. Gen Comp Endocrinol 120, 260-275.
Abstract: Noninvasive fecal glucocorticoid analysis has tremendous potential as a means of assessing stress associated with environmental disturbance in wildlife. However, interspecific variation in excreted glucocorticoid metabolites requires careful selection of the antibody used in their quantification. We compared four antibodies for detecting the major fecal cortisol metabolites in yellow baboons following (3)H cortisol administration, ACTH challenge, and HPLC separation of fecal glucocorticoid metabolites. The most effective antibody (ICN corticosterone RIA; Cat. No. 07-120102) demonstrated relatively high cross-reactivities to the major cortisol metabolites present in feces during peak excretion, following both radiolabel infusion and ACTH challenge. This same antibody also detected increased fecal glucocorticoid metabolites after ACTH administration in the African elephant, black rhinoceros, Roosevelt elk, gerenuk, scimitar-horned oryx, Alaskan sea otter, Malayan sun bear, cheetah, clouded leopard, longtailed macaque, and northern spotted owl. Results suggest that (1) fecal glucocorticoid assays reliably detect endogenous changes in
adrenal activity of a diverse array of species and (2) where comparisons were made, the ICN corticosterone antibody generally was superior to other antibodies for measuring glucocorticoid metabolites in feces.

Weissengruber, G.E., Kubber-Heiss, A., Forstenpointner, G., Riccaboni, P., 2000. On the morphology of the temporal gland (Glandula temporalis) in the African elephant (Loxodonta africana). Wiener Tierarztliche Monatsschrift 87, 303-308.

Wingate, L., Lasley, B. The Significance of Musth in Bull Elephants: Is It a Reproductive Event? Elephants: Cultural, Behavioral, and Ecological Perspectives; Program and Abstracts of the Workshop. 24. 2000. Davis, CA. 2000.
Ref Type: Conference Proceeding

Bechert, U.S., Swanson, L., Wasser, S.K., Hess, D.L., Stormshak, F., 1999. Serum prolactin concentrations in the captive female African elephant (Loxodonta africana): potential effects of season and steroid hormone interactions. Gen Comp Endocrinol 114, 269-278.
Abstract: Research was conducted to determine whether seasonal changes in prolactin secretion occur in nonpregnant female African elephants and to examine potential functional interrelationships between secretion of prolactin, cortisol, and progesterone. Weekly blood samples were taken for 18 months from four female African elephants and the sera were analyzed by RIA for progesterone, cortisol, and prolactin concentrations. There was no significant effect of season on serum concentrations of prolactin. Estrous cycles averaged 14 weeks in length and were composed of a 9-week luteal phase and a 5-week follicular phase (based on progesterone concentrations consistently >200 and <200 pg/ml, respectively). Estrous cycle synchronicity was evident between pairs of elephants. Serum concentrations of prolactin (3.91 +/- 0.69 ng/ml; range: 0.84-15.8 ng/ml) were significantly lower during the luteal, compared with the follicular, phase (P < 0.0001; t test) and were positively correlated with serum concentrations of cortisol (r = 0.14; P < 0.05). Mean (+/-SE) serum concentration of cortisol was 5.7 +/- 1.3 ng/ml (range: 1.4-19.3 ng/ml), and concentrations of this adrenal steroid were negatively correlated with progesterone concentrations (r = -0.15; P < 0.01). Increased serum concentrations of prolactin detected during the follicular phase suggest that this hormone may be regulated by ovarian estrogens and may play a role in modulating ovarian function in the elephant.

Brown, J.L., Schmitt, D.L., Bellem, A., Graham, L.H., Lehnhardt, J., 1999. Hormone secretion in the Asian elephant (Elephas maximus): characterization of ovulatory and anovulatory luteinizing hormone surges. Biology of Reproduction 61, 1294-1299.
Abstract: In the elephant, 2 distinct LH surges occur 3 weeks apart during the non-luteal phase of the oestrous cycle: the first is an anovulatory surge (anLH) while the second surge (ovLH) induces ovulation. To understand how the timing of these 2 surges is regulated, serum concentrations of LH, FSH, progesterone, inhibin, oestradiol and prolactin were measured throughout the oestrous cycle. Long-term dynamics of hormone secretion were examined from weekly blood samples taken from 3 Asian elephants for up to 3 years. To determine whether differences exist in the secretory patterns between the anLH and ovLH surges, daily blood samples were analysed from 21 non-luteal-phase periods from 7 Asian elephants. During the non-luteal phase, serum LH was elevated for 1-2 days during the anLH and ovLH surges; there was, however, no difference in peak concentration between the 2 surges. The anLH surge occurred 19.9±1.2 days after the end of the luteal phase and was followed by the ovLH surge 20.8±0.5 days later. Serum FSH concentrations were highest at the beginning of the non-luteal phase and gradually declined to nadir concentrations within 4 days of the ovLH surge. FSH remained low until after the ovLH surge and then increased during the luteal phase. Inhibin concentration was negatively correlated with FSH during the non-luteal phase (-0.53). Oestradiol and prolactin concentrations fluctuated throughout the oestrous cycle, without a discernible pattern. The results did not identify clear differences in hormone patterns between the anLH and ovLH surges. It is suggested that elevated FSH at the beginning of the non-luteal phase may be important for follicle recruitment, and that the anLH surge acts so as to complete the follicle selection process before ovulation.

Brown, J.L., 1999. Difficulties Associated with Diagnosis and Treatment of Ovarian Dysfunction in Elephants - The Flatliner Problem. Journal of the Elephant Managers Association 10, 55-61.

Brown, J.L., Hildebrandt, T.B., Theison, W., Neiffer, D.L., 1999. Endocrine and ultrasound evaluation of a non-cycling African elephant: identification of an ovarian follicular cyst. Zoo Biology 18, 223-232.
Abstract: The reproductive rate of captive African elephants is low because of logistical difficulties associated with transporting animals for breeding, the danger of maintaining bulls and medical or physiological problems. There also is growing evidence that a significant number of mature female elephants are not experiencing normal estrous cycles. The case described in this report involves the diagnosis and attempted treatment of an ovarian follicular cyst in an African elephant at the Pittsburgh Zoo. On the basis of serum progesterone analysis, the female exhibited regular ovarian cycles from July 1993 through March 1994, but from November 1994 to the present has not shown any evidence of reproductive cyclicity. In April 1996, a large follicular structure was identified on the right ovary using transrectal ultrasound. In an attempt to luteinize the cyst, 500 ug gonadotropin-releasing hormone (GnRH) was administered intravenously in October 1996, which stimulated a modest increase in serum luteinizing hormone (LH) (approximately twofold over baseline), but no resumption of ovarian activity. The elephant was treated again 5 months later with a higher dose of GnRH (5 mg, i.v.) with the same results. An ultrasound evaluation in July 1997 indicated the structure was still present. In October 1997, the female was given human chorionic gonadotropin (hCG; 10,000 IU, i.m.), which induced estrus and breeding, but no ovulation or luteinizing of the cyst, and she remains acyclic. These results suggest that conventional methods developed in other species for treating ovarian follicular cysts may not necessarily be effective in the elephant. It is also important that reproductive age females be monitored via continuous progesterone analysis and occasional reproductive tract ultrasound evaluations to understand better the etiology of ovarian dysfunction so that effective treatments can be developed to induce consistent ovarian activity.

Fies, M., Heistermann, M., Hodges, J.K., 1999. Patterns of urinary and fecal steroid excretion during the ovarian cycle and pregnancy in the African elephant (Loxodonta africana). Gen Comp Endocrinol 115, 76-89.
Abstract: The aims of the present study were to (I) determine the relative abundance of the 5alpha-reduced progestins 5alpha-pregnane-3-ol-20-one (5alpha-P-3OH) and 5alpha-dihydroprogesterone (5alpha-DHP) and progesterone (P4) in African elephant feces and to establish improved fecal progestin assays for monitoring ovarian function; and (ii) describe longitudinal profiles of urinary and fecal progestin and estrogen metabolites during pregnancy. Matched urine and fecal samples were collected weekly from six adult females throughout 18 nonfertile cycles and two complete pregnancies (89 and 93 weeks duration). Fecal samples were lyophilized and extracted with 80% methanol in water and immunoreactive 5alpha-P-3OH, 5alpha-DHP, and P4 and (for pregnant females only) estrone (E1) and estradiol (E2) determined by enzyme immunoassay. Urine samples were hydrolyzed, ether-extracted, and assayed for 5alpha-P-3OH, E1, and E2. HPLC cochromatography of fecal extracts with various radioactive progestin tracers confirmed the presence of large amounts of both 5-reduced progestins (5alpha-P-3OH > 5alpha-DHP) but not of P4. 5-Reduced progestins (but not P4) were excreted in a cyclic pattern and levels were significantly correlated with urinary 5alpha-P-3OH. Fecal 5alpha-P-3OH showed the more pronounced and consistent luteal-phase elevation and a better correspondence to urine with respect to timing of the luteal-phase rise. Fecal and urinary 5-reduced progestins increased gradually during early pregnancy to maximum values around week 40-45. Levels gradually declined during the second half of pregnancy, reaching baseline values 2 days before parturition. Urinary estrogens did not show any cyclic pattern during the preconception period and levels remained low during the first 30 weeks of gestation. Thereafter, there was a rapid 10- to 20-fold increase to maximum values at mid-pregnancy, followed by a gradual decline to birth. There was no mid-pregnancy elevation in fecal estrogens, but there was a modest increase in E1 during the second half of gestation.

Goodwin, T.E., Rasmussen, E.L., Guinn, A.C., McKelvey, S.S., Gunawardena, R., Riddle, S.W., Riddle, H.S., 1999. African elephant sesquiterpenes. J Nat Prod 62, 1570-1572.
Abstract: GC-MS analysis of extracts from temporal gland secretions of an African elephant has revealed the presence of several farnesol-related sesquiterpenes. Among these are (E)-2, 3-dihydrofarnesol (3), a bumblebee pheromone not seen before in mammals, and a rare component of a Greek tobacco, drimane-8alpha, 11-diol (4), never observed before in an animal.

Göritz, F., Hildebrandt, T.B., Hermes, R., Quandt, S., Grobler, D., Jewgenow, K., Rohleder, M., Meyer, H.H.D., Hofmann, R.R. Results of hormonal contraception program in free-ranging African elephants. Verh ber Erkrg Zootiere. 39-40. 1999.
Ref Type: Conference Proceeding

Hodges, J.K., Fieb, M., Heistermann, M. Non-Invasive Reproductive Assessment in the African Elephant: Applications to Studies in Captivity and in the Wild. Roth, T. L., Swanson, W. F., and Blattman, L. K. Seventh World Conference on Breeding Endangered Species: Linking Zoo and Field Research to Advance Conservation. 1999. Cincinnati, OH. 1999.
Ref Type: Conference Proceeding

Poole, J.H., 1999. Signals and assessment in African elephants: evidence from playback experiments. Animal Behaviour 58, 185-193.
Abstract: A series of playback experiments using two elephant vocalizations, the 'musth rumble' and the 'oestrous call', was carried out in Amboseli National Park to examine signaling and assessment in African elephants, Loxodonta africana. In response to the musth rumble of a high-ranking male other musth males approached the speaker aggressively, whereas nonmusth males walked away from the stimulus. The call of an oestrous female, too, attracted musth males who approached the speaker rapidly, while nonmusth males listened and then walked away. Females listened and often showed considerable interest in the musth rumbles of males, approaching the speaker and sometimes responding by vocalizing and or secreting from the temporal glands. The experiments bear out earlier observational data and game theory predictions which suggest that by being in or out of musth a male may be conveying information about the relative value he places on contesting his dominance rank and his access to oestrous females. When not visibly in musth, a male may be indicating his intention not to contest access to oestrous females.

Rasmussen, L.E.L., Perrin, T.E., 1999. Physiological correlates of musth: lipid metabolites and chemical composition of exudates. Physiology and Behavior 67, 539-549.
Abstract: Physiological changes related to lipid metabolism, behaviour and chemicals released in body exudates were studied during musth in the Asian elephant (Elephas maximus) as a case study. During musth, changes in serum testosterone and triglyceride concentrations followed similar patterns, with the former increasing sooner than the latter. Deviant behaviour increased during changing androgen levels. The observed high concentrations of testosterone were positively and significantly correlated with increased triglycerides. Lipase activity elevated significantly immediately before and after musth. Blood pH increased significantly in alkalinity. Urine and temporal gland secretions released variable amounts of compounds, some of which may be chemical signals. During musth, temporal gland and urinary exudates demonstrated increased acetone and other ketones indicative of lipid metabolic alterations. Large quantities of nonmethane hydrocarbons, especially 2-butanone, were released from the seemingly dry orifice of the temporal gland before the start of over musth and before maximum blood elevations were observed; isoprene release was similar. However, maximal acetone levels occurred simultaneously in blood, temporal gland secretions, and urine. Metabolically, musth is a series of interwoven, changing stages of increasing and decreasing hormones and lipid-related constituents. Released chemicals can be quantitatively related to these internal physiological events; some observed behaviours appear to result from altered chemical signals.

Rasmussen, L.E.L., 1999. Evolution of chemical signals in the Asian elephant, Elephas maximus: behavioural and ecological influences. Journal of Biosciences 24, 241-251.
Abstract: In antiquity, the Asian elephant, Elephas maximus, gradually spread southward and eastward to become a successfully surviving, ecologically dominant megaherbivore in the tropical environment of south-east Asia. The changing physical environment forced dynamic fluxes in its social structure and altered its metabolism. Such events shaped the production and ultimately the stability of certain chemicals released by body effluvia. Some of these chemicals took on significance as chemical signals and/or pheromones. This article demonstrates by experimental and observational evidence, and hypothesizes based on speculative reasoning, how and why specific chemical signals evolved in the modern Asian elephant. Evidence, including the functional criteria required by elephant social structure and ecology, is presented for the hypothesis that the recently identified female-emitted, male-received sex pheromone, (Z)-7-dodecenyl acetate evolved first as a chemical signal. Subsequently, the cohesiveness and harmony of small, matriarchally-led female groups were strengthened by a female-to-female chemical signal, recently defined behaviourally. The looser societal structure of freer, roaming males also became bounded by chemical signals; for the males, breath and temporal gland emissions, as well as urinary ones function in chemical signaling. Basic knowledge about elephant chemical signals is now linking chemical information to behaviour and beginning to demonstrate how these signals affect elephant social structure and enable the species to cope with environmental changes.

Rasmussen, L.E.L., Schulte, B.A., 1999. Ecological and biochemical constraints on pheromonal signaling systems in Asian elephants and their evolutionary implications. In: Johnston, R.E., Muller-Schwarze, D., Sorenson, P.W. (Eds.), Advances in Chemical Communication in Vertebrates 8. Kluwer/Academic/ Plenum Press, pp. 49-62.
Abstract: The Asian elephant is an unusual example of how intraspecies chemical communication helps maintain societal cohesiveness within familial and herd units. The amount of multi-directional chemical communication is surprising, because long-lived elephants have a highly organized society, are capable of trans-generational passage of information, possess a sophisticated vocalization system, and are capable of complex learning and tool use. This paper discusses the ecological, behavioral, and biochemical aspects of chemical signals in elephants from an evolutionary perspective. Diverse bodily emissions are utilized as intraspecies chemical signals (including pheromones), often with imposed biochemical constraints. In this chapter, chemosignals released from the temporal gland secretions and breath of male Asian elephants in musth and a urinary female-to-male preovulatory pheromone are utilized as examples of these concepts. Furthermore, specific behavioral and biochemical studies with (Z)-7-dodecenyl acetate (a female-to-male urinary sex pheromone) demonstrate that social context significantly influences responsivity (demonstrated by field studies in Myanmar) and that additional biochemical requirements, perhaps lipocalin-like proteins, may be required for full bioactivity. The remarkable convergent evolution of (Z)-7-dodecenyl acetate, both structurally and functionally, in elephants and Lepidoptera, allows the use in elephant studies of effective biochemical tools developed for insect investigations. This convergence of chemical signaling systems of elephants and insects has several interesting implications.

Schulte, B.A., Rasmussen, L.E.L., 1999. Musth, sexual selection, testosterone and metabolites. In: Johnston, R.E., Muller-Schwarze, D., Sorenson, P.W. (Eds.), Advances in Chemical Communication in Vertebrates 8. Kluwer Academic/Plenum Press, pp. 383-397.
Abstract: Musth is an annual, yet asynchronous, rut-like condition that is experienced by many adult African and Asian male elephants. Behaviorally, musth is characterized by heightened aggression, decreased feeding, urine dribbling, temporal gland secretion and enhanced sexual activity. Musth improves the access of a male to reproductively active females through increased mobility and a higher dominance ranking (intrasexual competition). Whether females prefer males in musth as mates is as yet uncertain (intersexual choice). Females can distinguish among the odors of males in musth and nonmusth. Although behavioral musth has been associated with greatly elevated plasma testosterone levels, a recent study in Sri Lanka shows that intensified aggressiveness follows maximal testosterone secretion and proposes that behavioral musth is a consequence of declining androgen levels. Our data from an Asian male elephant in North America suggest that either declining or rising serum testosterone may be related to "musth behaviors." Our report demonstrates that certain aspects of body physiology are greatly altered during musth. Rather than a single state, our data suggest that musth is an ever-changing condition with some typical stages. Specific chemical compounds released at different stages of musth may serve individually or in combination as honest signals of male condition.

Wimalaratne, O., Kodikara, D.S., 1999. First reported case of elephant rabies in Sri Lanka. Veterinary Record 144, 98.
Abstract: An 84-year-old female domesticated elephant presented with a 4-day history of lethargy. Appetite and water intake was normal but the following day she was unsteady, aggressive and restless. There were secretions from both temporal glands. On the sixth day she was completely anorectic, had developed paralysis of the trunk and was unable to stand, falling each time she tried to stand up, and she was noticed to be blind. She died on the ninth day after the first symptoms were observed. PM examination showed the brain to be more vascular than normal and a brain smear was positive for rabies antigen. A serum sample went to the WHO Collaborating Center for Rabies in Bangkok, Thailand, which determined a rabies virus neutralizing antibody titre of 0.68 IU/ml by the rapid fluorescent focus inhibition test. Antigenic typing and genetic sequencing showed the virus to be similar, but not identical, to the common Sri Lankan dog rabies variant, although there was no history of an animal bite to the elephant.

Carden, M., Schmitt, D., Tomasi, T., Bradford, J., Moll, D., Brown, J., 1998. Utility of serum progesterone and prolactin analysis for assessing reproductive status in the Asian elephant (Elephas maximus). Animal Reproduction Science 53, 133-142.
Abstract: Concentrations of serum progesterone and prolactin were measured in blood samples collected during the perioestrous period and throughout gestation in captive Asian elephants. In >95% of perioestrous periods (n = 35), behavioural oestrus (as determined by bull interest, mounting and/or mating) coincided with the onset of increased serum progesterone concentrations at the beginning of the luteal phase and continued through day 7 (day 1 = first significant serum progesterone rise). A transient decrease (P<0.05) in serum progesterone, lasting 1-2 days, occurred between days 2 and 9. No sexual behaviour was observed in any female after this transient fall in progesterone. Prolactin concentrations fluctuated randomly throughout the perioestrous period, with no clear pattern. During the study, 4 females conceived (1 conceived twice), and delivered 3 viable offspring. Serum progesterone concentration was above baseline throughout gestation, and declined rapidly 2-3 days before parturition. Serum prolactin concentrations were higher than baseline values (P<0.05) after 5-6 months of gestation and remained high until after parturition. It is suggested that progesterone and prolactin concentrations could be used to detect oestrus, pregnancy and impending parturition.

Greyling, M.D., Ford, M., Potgieter, H.C., van Aarde, R.J., 1998. Influence of gestation on uterine endometrial steroid receptor concentrations in the African elephant, Loxodonta africana. Biol Reprod 58, 60-64.
Abstract: The modulatory effects of gestational age and circulating concentrations of progesterone, 5alpha-pregnane-3,20-dione, and estradiol-17beta on the uterine sex steroid hormone receptor levels of the African elephant were investigated. Uterine tissue biopsies and blood samples were obtained from animals culled in the Kruger National Park. Estrogen and progesterone receptor concentrations were determined in uterine biopsies from subadult, lactating, early-, mid-, and late-pregnant elephants, by equilibrium binding assays. Circulating estradiol-17beta and progesterone concentrations were measured by means of RIAs, while plasma concentrations of 5alpha-pregnane-3,20-dione were determined with an amplified ELISA. Significant inverse correlations of the concentrations of estrogen and progesterone receptors with the gestational stage of the elephants were observed. Pregnant uterine horns of individual animals contained lower levels of estrogen and progesterone receptors than the nonpregnant horns of the same animals. A strong positive correlation existed between uterine estrogen and progesterone receptors levels. Circulating concentrations of 5alpha-pregnane-3,20-dione and progesterone decreased with an increase in the concentrations of progesterone receptors as well as with fetal age. We conclude that the progesterone receptor concentrations are down-regulated with progressing gestation in the African elephant. This down-regulation appears to be linked to an increase in circulatory 5alpha-pregnane-3,20-dione concentration in the plasma of pregnant animals.

Hodges, J.K., 1998. Endocrinology of the ovarian cycle and pregnancy in the Asian (Elephas maximus) and African (Loxodonta africana) elephant. Animal Reproduction Science 53, 3-18.
Abstract: In reviewing the literature, this paper assesses the current level of understanding of the hormonal control of the ovarian cycle and pregnancy in the
elephant. Data are compiled from two genera, Elephas maximus and Loxodonta africana, recognizing differences where known to exist. Measurements of various steroid and peptide hormones, but most notably immunoreactive progesterone (iP), indicate an overall cycle length of 15-16 weeks, comprising an 8-11-week luteal phase and a shorter inter-luteal (follicular) period of 4-6 weeks. Oestrogen related phenomena suggest the occurrence of two (possibly more) waves of follicular development with a 3-week periodicity, although oestrogen levels are low and fluctuate without clear pattern. The inter-luteal period is characterized by two discrete LH peaks also 3 weeks apart, but only the second is associated with an iP rise. Ovulation rate is variable and additional luteal structures lacking ovulation points are probably formed each cycle. The post-ovulatory period is associated with elevated progestogen levels, mainly 5alpha-reduced compounds, while progesterone itself is a minor secretory product. A revised model for the elephant ovarian cycle is presented. Detailed information on the endocrinology of pregnancy is lacking. Elevated progestins beyond the normal luteal phase indicate that conception extends the functional lifespan of the CL, with maximum steroidogenic activity between 3-15 months. The nature of the luteotrophic support is not known and placental gonadotrophins have not been conclusively demonstrated. Progestins fluctuate at or slightly above luteal phase values throughout pregnancy. There is a marked increase in prolactin levels after 16-24 weeks and a relationship with oestrogen secretion may exist since circulating and urinary levels show a progressive increase from 6-8 months. A placental contribution to progestin secretion is likely, although direct evidence is lacking. Considerable enlargement of foetal gonads during the second half of pregnancy in African elephants suggests a possible steroidogenic function, but none has been defined. The endocrine events leading to parturition are unknown. In the Asian elephant, a fall in iP precedes parturition; oestrogen levels decline and cortisol increases at the time of birth. The paper concludes with a brief selection of priorities for future research.

Niemuller, C.A., Gray, C., Cummings, E., Liptrap, R.M., 1998. Plasma concentrations of immunoreactive relaxin activity and progesterone in the pregnant Asian elephant (Elephas maximus). Animal Reproduction Science 53, 119-131.
Abstract: Immunoreactive relaxin activity and progesterone concentrations in weekly plasma samples were measured throughout 6 pregnancies in 5 Asian elephants maintained at the African Lion Safari in Ontario. One animal aborted, one delivered a stillborn calf at term and the remaining pregnancies produced living calves. Duration of pregnancy ranged from 89 to 95 weeks. Immunoreactive relaxin activity increased by week 20 of pregnancy to reach a mean peak concentration of >6 ng/ml in the 2nd trimester. This was followed by a slow decline beginning approximately 30 weeks before parturition. A smaller, secondary relaxin peak was observed during the final 8 weeks before parturition and circulating concentrations remained above the prepregnancy basal value for 72 h after birth. Progesterone concentration increased immediately after mating and rose to a maximum of >2 ng/ml by midgestation. As with relaxin, progesterone concentrations began to decline gradually approximately 30 weeks before birth. More frequently collected samples before, during and after parturition revealed a decline to the prepregnancy level between 2 and 5 days before parturition, in contrast to an elevated relaxin level (1 ng/ml) during this time. The aborted pregnancy at week 35 was accompanied by a sudden decline in immunoreactive relaxin and progesterone concentrations 1 week before the visible termination of the pregnancy. Delivery of the stillborn calf occurred 5 weeks after immunoreactive relaxin and progesterone concentrations had declined to the prepregnancy value.

Rasmussen, L.E.L., Schulte, B.A., 1998. Chemical signals in the reproduction of Asian (Elephas maximus) and African (Loxodonta africana) elephants. Animal Reproduction Science 53, 19-34.
Abstract: Asian (Elephas maximus) and African (Loxodonta africana) elephants exhibit polygynous mating that involves female choice of mates and male-male competition for access to females. Chemical signals mediate intersexual and intrasexual interactions associated with reproduction. The need for reliable and honest signals is accentuated by the markedly different social structure of adult males and females. Adult female elephants live in matriarchal herds consisting of a dominant female and several generations of offspring. Adult males are solitary or travel with other males except during breeding periods. Because females have a long 16-week oestrous cycle with a brief 1-week receptive period and a 4-5 year interval between births, a sexually active female is a limited resource. Asian elephant females advertise a forthcoming ovulation by releasing (Z)-7-dodecen-1-yl acetate in their urine during the preovulatory period. African elephants probably produce a sex pheromone as well. Females regularly contact the ano-genital region of other females and show heightened chemosensory responsiveness to urine during the follicular phase. The physiological impacts of this ability to detect reproductive condition (e.g. possible synchronizing or suppressing of oestrus) are uncertain. Males experience an annual period of heightened aggressiveness and highly elevated testosterone concentrations known as musth. Males secrete fluid copiously from their temporal gland and dribble strongly odoriferous urine during musth. Females appear to prefer musth males as mates, and captive Asian females exhibit greater chemosensory responses to urine from males in musth than not. Males in musth are competitively dominant to all other males, even those larger than themselves. Nonmusth males avoid males in musth, and captive Asian bulls show greater interest in musth than nonmusth urine. In captivity subordinate Asian females back away from musth secretions, and females with calves sometimes display protective behaviour. Clearly, chemical signals play an important role in communication by elephants between and within the sexes. Further work is needed to identify more of these chemical messengers and to understand their complete function in mediating reproductive interactions in the elephant social system.

Rasmussen, L.E.L., 1998. Chemical communication: An integral part of functional Asian elephant (Elephas maximus) society. Ecoscience 5, 410-426.
Abstract: The matriarchally organized Asian elephant society is characterized by long-term stability and continuity. Flux within this society results from changing ecological conditions and the dynamics of its population. Its structure is influenced by age composition and physiological states within the female herd and by impinging influences of the peripheral males, especially during reproductive times. Recent behavioral studies of captive populations have substantiated older field studies and have demonstrated that chemical signals play a significant role in elephant society. Chemical investigations, based on previously substantiated behavioral interactions, have identified specific compounds or combinations of compounds in elephant emissions (especially urine, temporal gland secretions and breath) that retain bioactivity throughout chemical extractions and playback experiments, based on behavioral and/or chemosensory responses. Chemosensory neuroreceptive systems in Asian elephants are reviewed, as well as behavioral and chemosensory effects of whole exudate chemical signals on lifestyles, especially related to mating. Several discrete and composite chemical signals have been deciphered in elephants, one of which is a preovulatory female-to-male pheromone, (Z)-7-dodecen-1-yl acetate. This pheromone and other recently described or potential chemical signals are compared to compounds in insect pheromone blends. Such knowledge of the chemical ecology of the Asian elephant has potentially important implications for conservation.

Brown, J.L., Lehnhardt, J., 1997. Secretory patterns of serum prolactin in Asian (Elephas maximus) and African (Loxodonta africana) elephants during different reproductive states: comparison with concentrations in a noncycling African elephant. Zoo Biology 16, 149-159.
Abstract: Serum prolactin was quantified in adult female Asian (Elephas maximus) and African (Loxodonta africana) elephants during various reproductive states and the profiles compared to that in a noncycling African elephant. In reproductively normal elephants, there was no effect of season, estrous cycle stage, or lactational status on quantitative or qualitative prolactin secretion (P > 0.05), nor were there any differences (P > 0.05) in overall prolactin concentrations between species. In pregnant elephants, prolactin concentrations remained at baseline for the first 4-6 months of gestation. Thereafter, concentrations during early pregnancy averaged ~four-fold higher than those during the estrous cycling, increasing to ~100-fold over baseline during mid- to late gestation in both species. In contrast to cycling elephants, prolactin concentrations in an African elephant exhibiting chronic anovulation (on the basis of an acyclic serum progesterone profile) and mild galactorrhea were consistently about five-fold higher (P < 0.05) suggesting she is hyperprolatinemic. Other endocrinological assessments confirmed the hypogonadal state of this female. Serum estradiol concentrations were consistently at or below dectectable levels. Additionally, no preovulatory leutinizing hormone (LH) surges occured in daily serum samples analyzed over a 12-month period. the pituitary was not totally, refractory, however, and responded with a several-fold increase in serum LH concentration (peak, 3.07 ng/ml) over baseline (0.75 ng/ml) after i.v. injection of gonadotropin-releasing hormone. this study describes normal baseline serum prolactin values for Asian and African elephants and is the first to identify hyperprolactinemia as a possible cause of reproductive acyclicity and galactorrhea in and African elephant.

Dickerman, R.D., Zachariah, N.Y., Fouraker, M., McConathy, W.J., 1997. Neuroendocrine-associated behavioral patterns in the male Asian elephant (Elephas maximus). Physiology and Behavior 61, 771-773.
Abstract: Steroid-responsive behaviors have been reported in various species; however, the reports thus far on the male Asian elephant (bull) during musth are few in number and most have been conducted on single captive animals for short time periods. The purpose of this investigation was to perform a longitudinal study on steroid-responsive behavior in 3 male Asian elephants from a captive herd of 11 male Asian elephants in Nepal. Male Asian elephants were 18, 25, and 43 years old. The animals had serum collected for 11 months and were observed on a daily basis for aggressive behavior according to the Species Survival Plan (SSP) collection protocol on SSP data sheets. Testosterone (T) and dihydrotestosterone (DHT) were measured in each animal by radioimmunoassay. Testosterone levels rose during musth 26-fold compared to nonmusth, and DHT was elevated 12-fold in musth. Maximal aggressive behavior episodes occurred during peak elevations of T and DHT, with correlation coefficients of 0.82 and 0.89, respectively. Therefore, we suggest that the aggressive episodes are dependent on elevated circulating androgens acting on androgen-responsive neural tissues.

Goodwin, T.E., 1997. Notes from a Fledgling Elephant Researcher. Journal of the Elephant Managers Association 8, 42.

Heisterman, M., Trohorsch, B., Hodges, J.K., 1997. Assessment of ovarian function in the African elephant (Loxodonta africana) by measurement of 5a-reduced progesterone metabolites in serum and urine. Zoo Biology 16, 273-284.
Abstract: We have previously shown that 5a-pregnane-3,20-dione (5a-DHP) and 5a-pregnane-3-ol-20-one (5a-OH) are the major luteal and circulating progestins in the African elephant. Therefore, the aim of the present study was to determine 1) circulating levels and patterns of secretion of 5a-DHP in relation to progesterone (P4) throughout the ovarian cycle, 2) the presence and relative abundance of 5a-reduced progestins in urine and 3) whether their measurement in urine would provide a non-invasive method for monitoring luteal function. Urine samples were collected weekly throughout a total of 13 ovarian cycles from 5 females. In addition, matched blood samples were collected during 6 cycles from 2 of the 5 animals. All hormone measurements were carried out by enzymeimmunoassay following extraction. Urine was hydrolized prior to extraction. Profiles of P4 and 5a-DHP in serum followed a similar cyclic pattern and both measurements were significantly correlated (r = 0.78-0.98, mean 0.89, P < 0.001). Concentrations of 5a-DHP were, however 10-20-fold higher than those if P4. In addition, 5a-DHP measurements showed a more pronounced luteal phase increase than that of immunoreactive P4. HPLC co-chromatography confirmed the presence of large amounts of 5a-P-3-OH in urine as a single immunoreactive peak, whereas 5a-DHP was present in very low levels and measurable only as one of several immunoreactive substances. Measurements of urinary 5a-P-3-OH were significantly correlated to serum 5a -DHP measurements in each of the 6 cycles (r = 0.72-0.93, mean 0.81, P < 0.001), whereas correlation coefficients between urinary and serum 5a-DHP values were generally lower (r = 0.34-0.83, mean 0.69) and significant in only 4 of 6 cycles. Accordingly, only urinary excretion of 5a-P-3-OH, but not of 5 a-DHP, exhibited a clear cyclic pattern, with consistently low levels of 0.15-.020 mg/mg Cr in the follicular phase and 10-fold elevated levels (1.8-2.2 mg/mg Cr) in the luteal phase. Based on the intervals between successive luteal phase increases in urinary 5a-P-3-OH, a mean cycle length of 14.1+/- 1.8 weeks, comprising a follicular phase of 5.0 +/- 0.9 weeks and a luteal phase of 9.10+/-01.4 weeks was determined for the 13 cycles studied. The results indicate that measurements of 5a-P-3-OH in urine provide a reliable non-invasive method for monitoring luteal function in the African elephant.

Hodges, J.K., Heisterman, M., Beard, A., van Aarde, R.J., 1997. Concentrations of progesterone and the 5a-reduced progestins, 5a-pregnane-3,20-dione, and 3-a-hydroxy-5a-pregnan-20-one, in luteal tissue and circulating blood and their relationship to luteal function in the African elephant (Loxodonta africana). Biology of Reproduction 56, 640-646.
Abstract: The 5 alpha-reduced metabolites 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha-P-3 alpha-OH) are the principal progestins biosynthesized by the African elephant corpus luteum. The aim of the present study was to determine luteal and circulating concentrations of these 5 alpha-reduced progestins in relation to progesterone (P4) and to examine whether their measurement reflects corpus luteum function. Ovarian (luteal) tissue (30 corpora lutea and 3 corpora rubra from 8 animals) and plasma samples (30 animals) were collected from pregnant and nonpregnant adult elephants shot in the Kruger National Park. Specific immunological measurement for both 5 alpha-reduced progestins and P4 was achieved by enzymeimmunoassay of tissue and plasma extracts following purification by HPLC. Mean (+/- SEM) luteal concentrations of 5 alpha-DHP and 5 alpha-P-3 alpha-OH were 79.5 +/- 9.4 micrograms/g and 196.5 +/- 24.8 micrograms/g, respectively, approximately 2-3 orders of magnitude higher than those of P4 (mean +/- SEM, 0.16 +/- 0.01 microgram/g). Whereas 5 alpha-reduced progestin concentrations tended to be lower in corpora lutea from late pregnancy compared with earlier stages and were lowest in corpora rubra, P4 levels were similar in all tissues/stages examined. The 5 alpha-reduced progestins also predominated over P4 in plasma (mean 5 alpha-DHP:P4 and 5 alpha-P-3 alpha-OH:P4 ratios 20.3 and 13.4, respectively). Similar to results for luteal tissue, plasma concentrations of 5 alpha-reduced progestins, but not of P4, were lower in late pregnancy than in earlier gestation stages and in nonpregnant animals. Moreover, plasma levels of both 5 alpha-reduced metabolites were negatively correlated with gestation age, whereas those of P4 were not. Levels of 5 alpha-reduced metabolites (without chromatography) were also measured in weekly blood samples throughout two complete ovarian cycles in one captive female. Both measurements showed a cyclic profile (similar to that of P4) with a luteal-phase elevation of 10- to 15-fold. The results indicate that 5 alpha-reduced compounds are the predominant progestins contained within and secreted by the corpus luteum of the African elephant, both during the ovarian cycle and throughout pregnancy. They also provide preliminary evidence to suggest that measurements of 5 alpha-reduced metabolites may reflect corpus luteum function more closely than those of P4.

Meyer, H.H.D., Jewgenow, K., Hodges, J.K., 1997. Binding activity of 5a-reduced gestagens to the progestin receptor from African elephant (Loxodonta africana). General and Comparative Endocrinology 105, 164-167.
Abstract: Recent findings in the African elephant (Loxodonta africana) indicate that the major progestins contained within and biosynthesized by corpora lutea are 5alpha-reduced metabolites and that progesterone is quantitatively of minor importance. The specific gestagenic action within the reproductive tract of elephants was determined by measurement of relative binding affinity of the respective progestins to the gestagen receptor extracted from elephant endometrium. The cytosol was incubated with 40 nmol/liter [3H]ORG-2058 and increasing concentrations of the tested progestin. Progesterone (P4), 5alpha-pregnane-3,20-dione (DHP), and 5alpha-pregnane-3alpha-ol-20-one (5alpha-P-3OH) were used. The competition for binding sites on the progestin receptor was shown by decreasing counts measured after extraction with scintillation fluid. The progestin concentration which induced a 50% reduction of measured counts was estimated (C50) and relative binding affinity of progesterone to other progestins was calculated (RBA = C50progestin/C50p4). The relative binding affinity of DHP to P4 at the gestagen receptor of elephant endometrium was equivalent. The other 5alpha-reduced progestin (5alpha-P-3OH) showed no competition to the [3H]ORG-2058 receptor binding. We conclude that the biological significance of P4 and DHP at the receptor level is very similar. The higher quantitative levels of DHP in corpus luteum and serum support the hypothesis that this progestin is the major gestagen in the elephants, whereas 5alpha-P-3OH is an inactive metabolite.

Niemuller, C., Shaw, H.J., Hodges, J.K., 1997. Pregnancy determination in the Asian elephant (Elephas maximus): A change in the plasma progesterone to 17a hydroxyprogesterone ratio. Zoo Biology 16, 415-426.
Abstract: The measurement of circulating progesterone (P) is widely used to confirm and monitor pregnancy in the captive Asian elephant. Consistently elevated progesterone (P) concentrations for a minimum of 12 weeks can be used as a positive indication of pregnancy, although two instances of prolonged P secretion during the cycle have been recorded [Rubel, 1987; Olsen et al, 1994]. Previously, we demonstrated that pregnanetriol was the major urinary gestagen metabolite enabling the noninvasive monitoring of the reproductive cycle of the Asian elephant [Niemuller et al., 1993] as well as pregnancy [unpublished data]. the importance of this unusual urinary metabolite triggered an investigation into the secretion of circulating concentrations of 17a hydroxyprogesterone (17a OHP) during pregnancy and reproductive cycles, as this steroid is the only steroid precursor of pregnanetriol. Comparison of the profiles between 17a OHP and P during early pregnancy (n = 5) and nonconceptive cycles (n = 15) demonstrated a decline in 17a OHP , but not P, as early as week 3 postmating (designated as week 1) and lasting up to week 13. Otherwise, secretions of 17a OHP mimicked P concentrations throughout pregnancy and in nonconceptive cycles. Examination of the mean ratio values of 17a OHP to P demonstrated a significant drop in the ratio during weeks 2-7 of early pregnancy from >/= 0.7 to < 0.7 compared with the same time period in a nonreceptive cycles (p < 0.05m N = 5), A 2x2 table analysis of the 17a OHP:P ratio during weeks 2-7 indicated that the possibility of a false positive or false negative result was 3.4 and 6.5%, respectively, based on the sensitivity and specificity of the test. Overall, gestation lengths of the pregnancies completed during this study (N = 4) ranged from 91 to 98 weeks, with a mean of 93+/-2.9 (SEM) weeks. A birth-to-conception interval of 47 weeks was noted in one animal. The results described in this report provide additional data on the reproductive endocrinology of the pregnant Asian elephant and also present the earliest means to date of determining pregnancy by analysis of the 17a OHP:P ratio.

Schwarzenberger, F., Strauss, G., Hoppen, H.-O., Schaftenaar, W., Dieleman, S.J., Zenker, W., Pagan, O., 1997. Evaluation of progesterone and 20-oxo-progestins in the plasma of Asian (Elephas maximus) and African (Loxodonta africana) elephants. Zoo Biology 16, 403-413.
Abstract: The corpus luteum of African elephants produces high amounts of 5a-reduced progesterone metabolites (5a-pregnane-3,20-dione and 5-a-pregnane-3a-ol-20-one), whereas progesterone itself is quantitatively less important, and plasma levels of progesterone during the estrous cycle in elephants are considerably lower than those of other mammals. The objective of this study was to compare the concentration of progesterone in plasma of Asian and African elephants as determined by specific progesterone assays with those of total immunoreactive progestagens containing a 20-oxo-group (20-oxo-P). These metabolites were determined by an enzyme immunoassay using an antibody against 5-a-pregnane-3b-ol-20-one, 3HS:BSA. Plasma of non-pregnant Asian (n = 4) and African (n = 4) elephants was collected at weekly intervals for periods of 8-15 months and at random intervals during pregnancy in one Asian elephant. High-performance liquid chromatography separation of plama samples of both species demonstrated that in the 20-oxo-P assay, 5a-pregnane-3,20-dione makes up ~60% of the total immunoreactive material. The progesterone and 20-oxo-P values during the estrous cycle showed a parallel pattern and were significantly correlated (P < 0.001; Asian: r = 0.80; y = 3.76 x -0.10; African: r = 0.75; y = 2.66 x -0.08. Progesterone and 20-oxo-P values in Asian and African elephants were < 15 ng/mL during the follicular phase (weeks -4 to 0) of the estrous cycle; progesterone values during the luteal phase (weeks 2-9) were 0.60+/-0.03 and 053+/-0.03 ng/mL, and the 20-oxo-P values were 2.19+/-0.16 and 1.48+/-0.12 ng/mL, respectively. The 20-oxo-P values of the pregnant animal, although slightly higher, were comparable to those of non-pregnant elephants during the luteal phase. Total immunoreactive 20-oxo-P values are about three times higher than those of progesterone during the luteal phase, and 5a-pregnane-3,20-dione is the major immunoreactive 20-oxo-P in the plasma of Asian and African elephants.

Kapustin, N., Critser, J.K., Olson, D., Malven, P.V., 1996. Nonluteal estrous cycles of 3-week duration are initiated by anovulatory lutienizing hormone peaks in African elephants. Biology of Reproduction 55, 1147-1154.
Abstract: Previous attempts to characterize the estrous cycle of elephants have yielded conflicting estimates of cycle length and LH profiles. In order to establish artificial breeding programs in this species, resolution of these issues is needed. Therefore, four female African elephants housed at the Indianapolis Zoo were studied for approximately 6 mo beginning in December 1994. Blood was collected weekly, and the serum was immediately analyzed for progesterone (P4). Whenever the weekly concentration of P4 was found to be low, blood was collected one or four times per day. All serum samples were assayed for LH, and the daily samples were assayed for P4 and estradiol. Transient increases of serum LH (designated as peaks) were observed four times in each of the four females. Of these 16 LH peaks, 8 were classified as ovulatory LH (ovLH) peaks and 8 were classified as anovulatory LH (anLH) peaks. Peaks designated ovLH averaged 3.60 +/- 0.67 ng/ml (mean +/- SEM); serum P4 measured during these peaks began to increase 2-3 days before each ovLH peak and continued to increase for several weeks thereafter, reaching a peak of 675 +/- 35 pg/ml. The eight other LH peaks, designated anLH peaks, were of similar (p > 0.05) magnitude averaging 3.07 +/-0.72 ng/ml, but the serum concentration of P4 remained very low (< 80 pg/ml) during and for several weeks after these peaks. Six peaks designated anLH occurred an average of 12.2 +/- 1.4 days after serum P4 had declined below 80 pg/ml. In each elephant, there was a regular sequence in which each ovLH peak was followed by a luteal-active period lasting about 60 days and then about 12 days later by one anLH peak. Each anLH peak was followed 19-22 days later by one ovLH peak, but serum P4 remained at nonluteal levels throughout this interval between peaks. The authors propose to designate this interval after the anLH peak and before the next ovLH peak as a nonluteal (i.e., low P4) estrous cycle of only 3-wk duration. Following each short nonluteal estrous cycle, there was a single ovLH peak that initiated one luteal-active estrous cycle lasting 10-11 wk until terminated by the next anLH peak. The present results demonstrate that nonpregnant African elephants, housed in the absence of males, alternate between short nonluteal estrous cycles and long luteal-active estrous cycles. Daily measurements of serum P4 can be used to distinguish between the two types of estrous cycles and thereby provide a clinical prediction about the optimum time for artificial insemination.

Lincoln, G.A., Ratnasooriya, W.D., 1996. Testosterone secretion, musth behaviour and social dominance in captive male Asian elephants living near the equator. Journal of Reproduction and Fertility 108, 107-113.
Abstract: Blood samples collected weekly over a 5-year period from 6 adult (19- to 40-year old) male Asian elephants (Elephas maximus maximus) living in captivity in Sri Lanka (7°N). Testosterone profiles were very variable within and between animals. Long-term phasic changes in blood concentrations of testosterone, associated with periods of musth (temporal gland secretion, drip urination and aggressive behaviour), occurred in 3 of the 6 elephants, the most pronounced cyclicity occurring in the oldest animal. Musth occurred annually after periods of high androgen secretion and the duration of musth was positively correlated with the mean concentration of testosterone during the previous 2 months. The time of musth, while consistent for an individual, varied between animals. In 4 bulls living in 1 social group, social rank was positively correlated with the mean concentration of testosterone over the 5-year period, and only the dominant animal showed periodic musth. Short-term changes in testosterone concentration occurred in blood samples collected every 15 min for 7 h, and after the injection of 20 µg GnRH, consistent with regulation through the pulsatile secretion of LH. The results support the view that fully mature male Asian elephants living near the equator express an asynchronous, cyclical, circannual pattern of gonadal activity. The periodic increase in testosterone secretion during the gonadal cycle induces the development of musth; however, androgen withdrawal following a period of hypersecretion may be the cause of some aspects of musth behaviour (aggression, unpredictability, disobedience) which make bull elephants very difficult to manage in captivity.

Perrin, T.E., Rasmussen, L.E.L., Gunawardena, R., Rasmussen, R.A., 1996. A method for collection, long-term storage, and bioassay of labile volatile chemosignals. J. Chemical Ecology 21, 207-221.
Abstract: A procedure for headspace sampling and long-term storage of organic volatiles coupled with gas chromatographic-mass spectrometric (GC-MS) analysis was used to study the volatile chemosignals in a biological secretion prior to bioassay. The approach involved collecting the volatiles in evacuated canisters from an apparatus in which 1 ml of secretion was dispersed for headspace sampling. These canisters, stainless steel, 850 ml, and 100% internally eletropolished, have been demonstrated to store volatile compounds, in chemically stable form, for several weeks. The GC-MS analyses provided the quantitation and identification of compounds from C₃ through C₁₄at concentrations as low as 0.10 parts per billion volume. The approach was used to study chemosignals of musth temporal gland secretions (TGS) from a male Asian elephant (Elephas maximus). Fresh TGS material loses its biological activity within 1 hr. TGS material stored at -20ºC usually loses its activity within 30 days. The usefulness of this method for long-term storage of the volatile chemosignals was demonstrated by the retention of biologically active TGS headspace compounds, as determined through bioassays, stored in these canisters for one year.

Rasmussen, L.E.L., Hall-Martin, A.J., Hess, D.L., 1996. Chemical profiles of African bull elephants, Loxodonta africana: physiological and ecological implications. Journal of Mammalogy 77, 422-439.
Abstract: This study reports concentrations of testosterone and dihydrotestosterone in both serum and temporal-gland secretion of male African elephant (Loxodonta africana), including radiocollared elephants, and identifies a spectrum of volatile components in the temporal-gland secretions. Androgens in the serum (testosterone and dihydrotestosterone) were measured in 111 adult male African elephants, ages 21-40 years, from two national parks in South Africa during several years and seasons. About one-fifth (18.6%) of these mature, male, African elephants exhibited dramatically increased concentrations of testosterone in serum characteristic of male Asian elephants during musth. In Krueger National Park, six radiocollared male African elephants, ages 25-35 years, were tracked and serially samples for both serum and temporal-gland secretions during a 5-year period. Concentrations of testosterone in serum and temporal gland secretions were elevated cyclically at times when typical musth behaviors, including aggression, were observed. This study reports the first chemical characterization of the volatile compounds of the temporal gland secretions from male African elephants in musth. It reveals many similarities between the chemical constituents of the temporal-gland secretions of these male African elephants and the compounds identified in male Asian elephants. In addition, several compounds, not previously identified in temporal-gland secretions of African elephants, are described. Such chemical data support the behavioral observations by ourselves and other researchers that male African elephants experience musth. Especially convincing are the concurrent hormonal and chemical data from the radiocollared males during episodic periods of behavioral musth. Implications of the incidence of musth in the past and present ecology of African elephants are discussed in view of the increasing compression within national parks.

Schwarzenberger, F., Mostl, E., Palme, R., Bamburg, E., 1996. Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild, and zoo animals. Animal Reproduction Science 42, 515-526.

Wasser, S.K., Papageorge, S., Foley, C., Brown, J.L., 1996. Excretory fate of estradiol and progesterone in the African elephant (Loxodonta africana) and patterns of fecal steroid concentrations. General and Comparative Endocrinology 102, 255-262.
Abstract: We developed and validated a noninvasive method to quantify fecal estrogens and progestins as a tool for monitoring long-term ovarian activity in free-ranging African elephants. The lag times between iv injection of [(3)H]estradiol and [(14)C]progesterone and peak excretion of radioactivity in urine and feces were approximately 4 hr and 48 hr, respectively. The majority of progesterone metabolites recovered was excreted in feces (55%) versus urine (45%), whereas comparatively little of the recovered estradiol metabolites were excreted in feces (5%) compared to urine (95%). Intrasample variation in fecal hormone concentrations was extremely high but could be substantially reduced by extracting well-mixed fecal powder from freeze-dried samples, taken from the central or premixed portion of the wet sample. This method resulted in a close correspondence between matched serum and fecal progestins (mean correlation =0.81, range 0.61-0.94) collected from five nonpregnant adult females over a 7-month period. Fecal estrogen profiles were more ambiguous, tending to overlap with those of fecal progestins. We conclude that analyses of fecal progestins can provide an effective, noninvasive means of characterizing ovarian activity in free-ranging African elephants.

Brown, J.L., Lehnhardt, J., 1995. Serum and urinary hormones during pregnancy and the peri- and postpartum period in an Asian elephant (Elephas maximus). Zoo Biology 14, 555-564.

Brown, J.L., Wemmer, C.M., Lehnhardt, J., 1995. Urinary Cortisol Analysis for Monitoring Adrenal Activity in Elephants. Zoo Biology 14 , 533-542.
Abstract: Cortisol was measured in dichloromethane-extracted elephant urine using an ¹²⁵I solid-phase radioimmunoassay (RIA). The cortisol RIA was validated by demonstrating 1) parallelism between dilutions of pooled urinary extracts and the standard curve, 2) significant recovery of exogenous cortisol added to elephant urine, and 3) a relationship between changes in the peripheral and urinary cortisol after an adrenocorticotropin hormone (ACTH) challenge. One African (Loxodonta africana) and one Asian (Elephas maximus) elephant were given three injections of ACTH (1.25 mg) at 2 h intervals. Serum cortisol increased four- to eightfold within 30 min after the first injection and peaked (nine- to twelvefold increase) after the second injection. Serum concentrations began to decline 2-3 h after the last injection but were still approximately fourfold higher than baseline at the end of the collection period (hour 8). In the urine, cortisol concentrations were increased in the first sample postinjection (1.5 - 4 h) and peaked twenty- to fortyfold by ~6 h. Urinary cortisol remained elevated at 8 h, but returned to baseline by the following morning. Analysis of high performance liquid chromatography fractions of extracted urine revealed that immunoactivity was associated with free cortisol (~90% of total immunoactivity) and a more polar, unidentified metabolite. A method for preserving urine was developed to allow storing unfrozen samples. One pool of urine from each of one African and two Asian elephants was divided into aliquots, placed in tubes containing absolute ethanol (10%), sodium azide (0.1%) or distilled water (control), and frozen after 0, 1 , 2, 3, 4, 6, 8, 10, 12, and 24 weeks of storage at ~25^ºC. In unpreserved samples, cortisol concentrations were reduced 46% by 2 weeks and 95% by 24 weeks. In contrast, ethanol- and sodium azide-preserved samples retained 100 and 95% of cortisol immunoactivity through 8 weeks and 93 and 85% of activity through 12 weeks, respectively. We infer from these data that changes in urinary cortisol excretion in the elephant reflect fluctuations in adrenal activity and may be a useful indicator of stress. Additionally, urine samples can be collected and stored unfrozen for at least 2 months before any appreciable loss in cortisol immunoactivity occurs, a finding potentially useful to field application of this technique.

Magunna, C. Oestrous cycle and pregnancy in Asian elephants (Elephas maximus). Zyklus und Trachtigkeit beim asiatischen Elefanten (Elephas maximus). 1-117. 1995. Tierarztliche Hochschule Hannover.
Ref Type: Thesis/Dissertation
Abstract: Blood samples were collected at weekly intervals over a period of 18 months from 10 females at a zoo in Germany. Increases in blood progesterone to >0.4 ng/ml indicated the onset of the oestrous cycle; the concentration averaged 0.72±0.14 and 0.2±0.07 ng/ml in the luteal and follicular phases respectively. The average duration of 45 oestrous cycles of 8 cycling females was 13.93±0.59 weeks, the luteal phase averaging 8.11 weeks and the follicular phase 5.86 weeks. Blood LH concentration increased at the end of the follicular phase. Of 6 females mated on the basis of their blood progesterone curve, 5 conceived. Data on the cycles of individual females are tabulated.

Papageorge, S., Wasser, S.K., Foley, C., Brown, J. Fecal steroid analysis: validation of extraction and radioimmunoassay for estradiol and progestagens in African elephants (Loxodonta africana) and analysis of fecal samples utilizing a validated method. Joint Conf AAZV/WDA/AAWV. 447. 1995.
Ref Type: Conference Proceeding

1994. Veterinary Laboratory Medicine. Iowa State University Press, Ames, IA.

Huang, H.B., Wellner, D., Naude, R., Oelofsen, W., Oosthuizen, M.M., Breslow, E., 1994. Amino acid sequence and properties of vasopressin-associated elephant neurophysin. Int J Pept Protein Res 44, 270-277.
Abstract: The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(abstract truncated at 250 words).

Olsen, J.H., Chen, C.L., Boules, M.M., Morris, L.S., Coville, B.R., 1994. Determination of reproductive cyclicity and pregnancy in Asian elephants (Elephas maximus) by rapid radioimmunoassay of serum progesterone. Journal of Zoo and Wildlife Medicine 25, 349-354.
Abstract: The stages of the reproductive cycle and pregnancy of 15 Asian elephants at Busch Gardens in Tampa, Florida were determined by measuring serum progesterone concentrations using a commercial radioimmunoassay (RIA) kit with a sensitivity of 40 pg/ml of serum. Blood samples were collected and analyzed weekly for a 45-mo period. Serum progesterone concentrations ranged from nondetectable (<40 pg/ml) in nonpregnant elephants to 2110 pg/ml in pregnant elephants. The highest concentration of progesterone during the oestrous cycle was 1490 pg/ml. The length of oestrous cycle ranged from 10 to 23 wk, with an average of 15.1±0.3 wk for 103 oestrous cycles. The duration of the nonluteal phase was 4.6±0.2 wk, and the duration of the luteal phase was 10.5±0.2 wk. The serum progesterone concentration during the oestrous cycle was 214.0±5.2 pg/ml. The individual elephant average cycle length was used to estimate the time of oestrus and ovulation. During the nonluteal phase, elephant cows were placed with a bull for mating. Two weeks after oestrus began (based on rising progesterone at the end of the nonluteal phase), the cow was separated from the bull. Ovulation was predicated to occur during the first week after oestrus began. Eight elephants became pregnant, with a serum progesterone concentration of 554.6±16.4 pg/ml and a range of <40-2110 pg/ml. These results confirm that weekly measurement of serum progesterone by RIA can be used to characterize the luteal and nonluteal phases of the Asian elephant oestrous cycle. Using this information, cows can be managed and placed with a bull at the appropriate time for breeding. Serum concentrations of progesterone can also be used to confirm pregnancy and to monitor and evaluate potential problems during pregnancy. The approximate date of parturition can then be predicted from average gestation length data, thus allowing staff to prepare facilities and be ready to assist with delivery.

Perrin, T.E., Rasmussen, L.E.L., 1994. Chemosensory responses of female Asian elephants (Elephas maximus) to cyclohexanone. Journal of Chemical Ecology 20, 2577-2586.
Abstract: Cyclohexanone, a naturally occurring component of male Asian elephant temporal gland secretion, was tested as a candidate elicitor of bioresponses from female Asian elephants (Elephas maximus). Four female Asian elephants were presented with synthetic cyclohexanone samples during a standardized bioassay. Four types of bioresponses, some or all of which may be important in intersexual communication, were monitored: flehmen, palatal pit area contact, scrub, and check responses. Cyclohexanone evoked persistent responses of all bioresponse types by two females, moderate response by a third female, and very few responses by a relatively unresponsive female. The results suggest that cyclohexanone may provide chemical information to females about male elephants, particularly regarding their state of musth.

Plouzeau, E., daCunha, S., Shaw, H.J., 1994. The ovarian cycle in Asian and African elephants (Elephas maximus and Loxodonta africana). Techniques for the monitoring of female fertility in captivity. Revue de Medecine Veterinaire 145, 905-911.
Abstract: A discussion. During the oestrous cycle, circulating blood progesterone in elephants alternates between high and low concentrations over a cycle of 16±2 weeks. Some of the data have suggested a 3-week cycle and other data a 15- to 16-week cycle. Captive females show no signs of oestrus, although males show a Flehmen-like response to female urine, which is inversely related to plasma progesterone concentration. Pregnancy is associated with a sustained increase in circulating progesterone concentration and with an increase in total oestrogen, prolactin and oestrogen concentrations during the second half of pregnancy. The most reliable method of monitoring the oestrous cycle and pregnancy is by weekly analysis of plasma progesterone in both species, or of its urinary metabolite (pregnanetriol) in Asian elephants.

Rasmussen, L.E.L., Perrin, T.E., Rasmussen, R.A., Gunawardena, R., 1994. Isolation of potential musth-alerting signals from temporal gland secretions of male Asian elephants (Elephas maximus). Chemical Senses 19, 540.

Van-Aswegen, G., Schoeman, J.H., De-Vos, V., Van-Noorden, S., 1994. The oesophagus and stomach of the African elephant: a histological, immunocytochemical and immunofluorescence study. Onderstepoort Journal of Veterinary Research 61, 223-229.
Abstract: Histological, immunocytochemical and immunofluorescence methods were employed to study the oesophagus and stomach of the elephant. The histological findings were similar to those in monogastric species like pigs and humans. In the mucosa of the stomach, endocrine cells were immunoreactive to gastrin, somatostatin, chromogranin A and serotonin. Nerve cells immunoreactive to somatostatin, bombesin, VIP, PHI and CGRP were detected in the submucosal and myenteric plexus of the stomach. In the stomach, the absence of glucagon cells and the presence of endocrine cells immunoreactive to PYY, are in contrast to the situation in other mammals and need further investigation. Small gastric ulcers were observed in some of the specimens.

Brown, J.L., Bush, M., Wildt, D.E., Raath, J.P., de Vos, V., Howard, J.G., 1993. Effects of GnRH analogues on pituitary-testicular function in free-ranging African elephants (Loxodonta africana). Journal of Reproduction and Fertility 99, 626-634.
Abstract: In the first of 2 experiments, 6 free-living adult male elephants were given 4 or 12 mg GnRH antagonist (Detirelix) or saline intramuscularly on day 0. Animals were then recaptured about 48 h later and given 300 µg GnRH intravenously to assess the ability of the antagonist to block pituitary activity. Detirelix reduced (P<0.05) basal concentrations of serum LH and testosterone on day 2 compared with day 0, with no effect of dose. Similarly, LH and testosterone release induced by GnRH were also reduced (P<0.05) in the Detirelix-treated bulls (50-70% reduction in peak concentration). In the 2nd experiment, elephants were given 30 mg of a structurally similar GnRH antagonist (103-201-40; n = 6), 22.5 mg of a long-acting GnRH agonist (Lupron Depot; n = 4) or D-mannitol carrier (n = 4) intramuscularly on day 0. All bulls were recaptured and given GnRH on day 2 (103-201-40 treatment group) or on days 2 and 20 (Lupron Depot group) after the initial injection. In contrast to Detirelix, the antagonist 103-201-40 did not inhibit basal or GnRH-induced LH or testosterone secretion. Pituitary-testicular responses to Lupron Depot were initially stimulatory, as evidence by increased (P<0.05) LH and testosterone secretion on days 0 and 2. By day 20, basal LH concentrations had returned to baseline values and the response to GnRH was markedly reduced (P<0.05), indicating that the pituitary was at least partially desensitized. Basal testosterone concentrations had also returned to baseline values by day 20 after Lupron Depot treatment. However, despite the attenuated LH response to GnRH, subsequent testosterone secretion was increased (P<0.05) compared with controls, suggesting that the testes of agonist-treated bulls had become hyper-responsive to small increases in LH secretion. It is suggested that GnRH analogues can suppress the pituitary-gonadal axis in African elephants. However, longer treatment periods, more frequent injection intervals or higher doses are probably needed to completely inhibit testosterone secretion and, thus, musth.

Niemuller, C.A., Shaw, H.J., Hodges, J.K., 1993. Non-invasive monitoring of ovarian function in Asian elephants (Elephas maximus) by measurement of urinary 5beta-pregnanetriol. Journal of Reproduction and Fertility 99, 617-625.
Abstract: An enzymeimmunoassay for 5beta-pregnanetriol is described. Immunoreactive pregnanetriol concentrations were significantly correlated with the concentrations of progesterone (0.98, n = 269, P<0.01) and 17alpha-hydroxyprogesterone (0.95, n = 205, P<0.01), the precursor of pregnanetriol. The duration of cycles as determined by measurements of plasma progesterone, plasma 17alpha-hydroxyprogesterone and urinary pregnanetriol averaged 15.54±1.5 weeks (23 cycles), 15.21±1.7 weeks (15 cycles) and 15.45±0.94 weeks (20 cycles) respectively. The results demonstrated that it is possible to monitor ovarian function in Asian elephants by measuring urinary pregnanetriol concentration.

Schmidt, M.J., 1993. Breeding elephants in captivity. In: Fowler, M.E. (Ed.), Zoo and wild animal medicine. W.B. Saunders Company, Philadelphia, PA, USA, pp. 445-448.

Taya, K., 1993. The reproductive physiology of the elephant. Journal of Reproduction and Development 39, 77-91.
Abstract: A discussion. In the females the vagina opens ventrally, ovarian cycles average 15-17 weeks in length, the gestation period is 22 months, and the ovary has a number of corpora lutea, although elephants are monovular. In males, there are permanent intra-abdominal testes, there is no distinct epididymis, but instead there is an extremely tortuous and convoluted duct which connects the testes to the openings of the seminal vesicles, the accessory organs are extremely well developed, especially the seminal vesicles and the bulbo-urethral glands. During musth, adult bulls become disobedient, aggressive and extremely dangerous, often attempting to kill their mahouts, or anybody else who comes within range. Bulls in musth have high testosterone levels and show urine dribbling and swollen temporal glands.

Turczynski, C.J. The endocrinology of musth in the male Asiatic elephant (Elephas maximus): Serum estradiol, serum LH and serum, fecal and urinary testosterone. 1993. College Station, TX. USA, Texas A&M University.
Ref Type: Thesis/Dissertation

Czekala, N.M., Roocroft, A., Bates, M., Allen, J., Lasley, B.L., 1992. Estrogen metabolism in the Asian elephant (Elephas maximus). Zoo Biology 11, 75-80.
Abstract: Estradiol-17B metabolism was studied in two female Asian elephants (Elephas maximus). In an initial study, 500 microCi of tritiated estradiol-17B was injected iv into a single animal, and 0, 30, and 60 min serum samples were collected as well as all excreted urine and feces for 24hr. In a second study, 1.5 mg unlabeled estradiol-17B was injected iv into a second animal and 0, 5, 15, 30 and 60 min serum samples and a 30 min urine sample were collected postinjection. Analyses of samples from both studies demonstrated a rapid conversion of free estradiol to conjugated forms in the serum. The first (5 min) serum sample following the injection of unlabelled estradiol contained unconjugated estradiol:conjugated estradiol: conjugated estrone at a ratio of 60:29:10, respectively, and at 30 min a ratio of 33:43:24. The urinary estrogen metabolites were in the conjugated form with an estradiol:estrone ratio of 60:40. No radiolabelled estrogen was found in the fecal samples during the 24 hr following administration of the radiolabelled estradiol. These data indicate a rapid clearance of circulating free estradiol in the elephant, with a major metabolite in the serum and urine being estradiol conjugate.

Dathe, H.H., Kuckelkorn, B., Minnemann, D., 1992. Salivary cortisol assessment for stress detection in the Asian elephant (Elephas maximus): A pilot study. Zoo Biology 11, 285-289.
Abstract: Effects of introducing an unfamiliar female into an Asian elephant herd at Tierpark Berlin were monitored by means of salivary cortisol assessment. Saliva samples were obtained from a second female for comparative purposes. The period of familiarization was characterized by an enhanced cortisol level in both animals, with a maximum on the second day after joining. Cortisol returned to normal on the following day. Manipulations of the keepers caused a transitory increase on two other days. Possibilities for the use of this noninvasive method of stress monitoring in various management situations are indicated.

Harshan, K.R., Chungath, J.J., Paily, L., Ommer, P.A., 1992. Histology of the temporal gland of the Indian elephant (Elephas maximus). In: Silas, E.G., Nair, M.K., Nirmalan, G. (Eds.), The Asian Elephant: Ecology, Biology, Diseases, Conservation and Management (Proceedings of the National Symposium on the Asian Elephant held at the Kerala Agricultural University, Trichur, India, January 1989). Kerala Agricultural University, Trichur, India, pp. 46-48.

Mar, K.U., 1992. Development of artificial insemination in Myanmar elephants. Captive Breeding Specialist Group News 3, 32.
Abstract: So far, no elephants have been reproduced successfully by artificial insemination (AI). Researchers have determined the estrous cycle in cow elephants, which come into heat regularly about every 13-16 weeks. After tracing the hormonal pattern of progesterone by radioimmunoassay, zoo veterinarians were able to identify when the cows would ovulate in order to attempt AI. Four main problems need to be solved: 1) understanding the estrous cycle of female elephants in order to correctly time AI; 2) developing a safe and reliable method of collecting semen from bulls; 3) developing a way to preserve elephant semen for storage and transportation and ; 4) devising a method for insemination through the cow's twisting 3-4 foot long urogenital canal. Timber production, which is the second largest source of Myanmar's export earnings (about $100 million U.S. per year), is still dependent to a large extend on elephant logging. The ultimate goal of AI for elephants is not only to maintain the numbers of domestic working elephants but also to establish a reserve stock of new-generation elephants by preventing or reducing inbreeding.

Ommer, P.A., Harshan, K.R., Chungath, J.J., Paily, L., 1992. Histology of the adrenal gland of the Indian elephant (Elephas maximus). In: Silas, E.G., Nair, M.K., Nirmalan, G. (Eds.), The Asian Elephant: Ecology, Biology, Diseases, Conservation and Management (Proceedings of the National Symposium on the Asian Elephant held at the Kerala Agricultural University, Trichur, India, January 1989). Kerala Agricultural University, Trichur, India, pp. 49-50.

Ratnasooriya, W.D., Fernando, S.B.U., Manatunga, A.N.V.R., 1992. Serum testosterone levels of Sri Lankan female elephants (Elephas maximus maximus). Med. Sci. Res. 20, 79-80.

Brown, J.L., Citino, S.B., Bush, M., Lehnhardt, J., Phillips, L.G., 1991. Cyclic patterns of luteinizing hormone, follicle-stimulating hormone, inhibin and progesterone secretion in the Asian elephant (Elephas maximus). Journal of Zoo and Wildlife Medicine 22, 49-57.
Abstract: Serum samples were collected one to three times weekly from four unanesthetized Asian elephants (Elephas maximus) for 6-18 consecutive months. Based on circulating progesterone profiles, 14 complete ovarian cycles were observed. The estrous cycle averaged 13.2 + 0.7 wk in length, with an active luteal phase of 9.8 + 0.7 wk. Increases in serum luteinizing hormone (LH) were observed immediately before or during the progesterone rise in 11 of 14 cycles. In eight cycles, a second LH surge was detected 11-19 days later. Radioimmunoassays for follicle-stimulating hormone (FSH) and inhibin were validated for elephant serum. Fluctuations in FSH and inhibin secretion were observed at 12-14 wk intervals, although their temporal profiles differed from each other and from that of progesterone. Follicle-stimulating hormone concentrations were lowest during the late follicular and early luteal phases and then increased to peak levels during the later part of the luteal phase. In contrast, serum inhibin concentrations were inversely related to FSH levels throughout the estrous cycle (r = -0.78, P < 0.01). In summary, progesterone analyses confirm that the luteal phase in the Asian elephant is approximately 10 weeks long. Furthermore, the 12-14-wk oscillations in serum FSH and inhibin secretion provide additional evidence that the ovarian cycle of this species is several months in duration. The inverse relationship between serum FSH and inhibin suggests that inhibit may regulate FSH secretion, as is described for other species. Elevated FSH secretion throughout the mid-and late luteal phase may stimulate waves of follicular growth that are responsible for the short "follicular cycles" described in earlier reports.

Cheng, H.C., Yamashiro, D., 1991. Synthesis and receptor binding activity of elephant beta- endorphin, a beta-endorphin homolog with highly potent analgesic activity. International Journal of Peptide and Protein Research 38, 66-69.
Abstract: Elephant beta-endorphin and its analog, elephant beta- endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant beta-endorphin was five to six times more potent than human beta-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16, 33-37), tail flick test for intracerebroventricularly administered beta-endorphin showed that the antinociceptive potency of elephant beta-endorphin was seven to eight times higher than that of human beta-endorphin in mice. Results from both studies suggest that elephant beta-endorphin was a much more potent antinociceptive agent than human beta- endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.

Niemuller, C., Liptrap, R.M., 1991. Altered androstenedione to testosterone ratios and LH concentrations during musth in the captive male Asian elephant (Elephas maximus). Journal of Reproduction and Fertility 91, 139-146.
Abstract: Greater concentrations of androstenedione than testosterone were usually present during periods of non-musth in plasma collected weekly for periods up to 2 years in 8 male Asian elephants (4-35 years of age). For the 6 males that exhibited musth the androstenedione/testosterone ratio shifted greatly in favour of testosterone. The severity of musth was assessed weekly using a scale of 1 to 5 for each of 8 behavioural traits including urine dribbling, temporal gland secretion and aggression. Brief shifts in the ratio of the two androgens when testosterone predominated (n=106) were seen during the non-musth period in 3 of the males studied continuously for 2 years. In 82% of these instances, stimuli of a sexual or aggressive nature had occurred in the preceding 48 h (x², p < 0.01). A heterologous bovine assay was used to measure LH values in plasma collected every 15 minutes for 12h. Increases in testosterone concentrations followed pulsatile increases in plasma LH concentrations during 7 non-musth periods in 4 animals. Apart from pulse frequency, increases in the variables describing pulsatile LH secretion were seen in 2 strong musth and 2 mild musth episodes compared to non-musth values. A strong musth, however, was characterized by a much greater increase in pulsatile testosterone secretion than was a mild musth and which may be a function of the duration of musth.

Taya, K., Komura, H., Kondoh, M., Ogawa, Y., Nakada, K., Watanabe, G., Sasamoto, S., Tanabe, K., Saito, K., Tajima, H., Narushima, E., 1991. Concentrations of progesterone, testosterone and estradiol-17B in the serum during the estrous cycle of Asian elephants (Elephas maximus). Zoo Biology 10, 299-307.

Cooper, K.A., Harder, J.D., Clawson, D.H., Fredrick, D.L., Lodge, G.A., Peachey, H.C., Spellmire, T.J., Winstel, D.P., 1990. Serum testosterone and musth in captive male African and Asian elephants. Zoo Biology 9, 297-306.
Abstract: Testosterone concentrations in serum samples collected weekly over a 5-year period from a young adult male Asian elephant (Elephas maximus) and a young adult male African forest elephant (Loxodonta africana cyclotis) were measured by radioimmunoassay. Testosterone profiles during this maturational period were compared between the two species and related to the occurrence of musth, a recurring physiological and behavioral condition exhibited by most mature Asian, and some African, bull elephants. Musth is characterized by secretion from the bull's temporal glands, dribbling urine, and increased aggression. Serum testosterone concentrations in the Asian bull were elevated substantially between April and September each year, coincident with the presence of temporal gland secretion, urine dribbling, and aggressive behavior. Testosterone levels from April through September averaged (± SEM) 41.2 ± 2.8 ng/ml, compared to 7.6 ± 1.0 ng/ml during the rest of the year. In contrast, the testosterone profile of the African bull showed greater variability and lower levels overall, the only pattern being a tendency for levels to be lowest from November to February (avg. 6.8 ± 1.5 vs. 10.3 ± 0.8 ng/ml during the rest of the year). Temporal gland secretion and other signs of musth were first observed in this bull in 1988, at age 17. While his testosterone values did not show a pattern comparable to that in the Asian bull, average testosterone values were significantly greater in 1988 compared to previous years. The Asian bull showed sexual attention to preovulatory (estrous) cows whether in musth or not, and exposure to estrous cows did not appear to alter the highly consistent, annual pattern of musth as evidenced by temporal gland flow.

Diaz-Samayoa-de-Aguirre, L. Sex hormones in blood plasma, urine and faeces from female and male Indian elephants. Vergleichende Hormonuntersuchungen in Blutplasma, Harn und Kot beim weiblichen und mannlichen asiatischen Elefanten (Elephas maximus). 1-145. 1990. Hannover, Germany, Tierarztliche Hochschule.
Ref Type: Thesis/Dissertation

Mainka, S.A., Lothrop, C.D., Jr., 1990. Reproductive and hormonal changes during the estrous cycle and pregnancy in Asian elephants. Zoo Biology 9, 411-419.
Abstract: Serum progesterone and urinary total estrogen concentrations were determined weekly to bi-weekly in 2 female Asian elephants for 96 weeks. The mean estrous interval was approximately 16 weeks in the nonpregnant animal. A total of 5 cycles were observed in the 96 weeks study period. The serum progesterone concentration ranged from 150 pg/ml to greater than 350 pg/ml during the luteal phase of the estrous cycle. The serum progesterone was elevated for 8-12 weeks of the 16 week estrous cycle. The urinary total estrogen concentration ranged from less than 10 to greater than 300 pg/microgram creatinine. The second animal was pregnant at the beginning of the study period. The serum progesterone concentration was elevated (> 100 pg/ml) in the pregnant animal until parturition. The urinary total estrogens increased from approximately 50 pg/microgram creatinine to greater than 400 pg/microgram creatinine during the first year of pregnancy and remained elevated until parturition. Estrous cycling had not resumed by 3 months post partum.

McFarlane, J.R., Cabrera, C.M., Oosthuizen, M.M.J., Papkoff, H., 1990. Elephant pituitary gonadotropins. General and Comparative Endocrinology 79, 193-200.
Abstract: We describe for the first time the purification and some properties of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) isolated from anterior pituitary tissue of the African Elephant (Loxodonta africana). Methodology previously applied to equine and donkey pituitaries was used to obtain purified preparations of elephant LH and FSH in yields of 8.8 and 0.48 mg, respectively, per 10 g pituitary powder. The preparations were characterized by HPLC gel filtration and amino acid analysis, both of which showed the elephant LH and FSH to be very similar to ovine LH and FSH. The preparations were also characterized by radioimmunoassays and bioassays for LH and FSH and a radioreceptor assay for FSH. Results showed virtually no cross-contamination of hormonal activities in the elephant LH and FSH preparations. Elephant LH potencies ranged from 50 to 66% of highly purified ovine LH and elephant FSH potencies ranged from 21 to 52% of highly purified ovine FSH in the various assays employed. No evidence was found for any demonstrable intrinsic FSH activity in elephant LH. The assays employed suggest possible usage for making physiological measurements of gonadotropins in elephants.

Niemuller, C., Gentry, P.A., Liptrap, R.M., 1990. Longitudinal study of haematological and biochemical constituents in blood of the Asian elephant (Elephas maximus). Comparative Biochemistry and Physiology [A] 96, 131-134.
Abstract: 1. Haematological parameters and biochemical analyses were determined in four elephants over a period of one year. 2. The haematological profile remained constant over time and was similar between animals. 3. Values for biochemical analyses were stable except for alkaline phosphatase, gamma glutamyl transferase and creatinine which rose during musth in male elephants. 4. The association of elevated enzyme levels and increased testosterone concentration is discussed.

Rasmussen, L.E.L., Hess, D.L., Haight, J.D., 1990. Chemical analysis of temporal gland secretions collected from an Asian bull elephant during a four-month musth episode. Journal of Chemical Ecology 16, 2167-2181.
Abstract: The temporal glands, modified facial apocrine sweat glands unique to elephants, release collectable secretions during an unusual physiological state termed "musth" in the Asian bull elephant (Elephas maximus). Recently we began the characterization of the chemical components of musth, especially in the temporal gland secretions (TGS), and the examination of the role of such secretions as agents for chemical communication among elephants. The presents study focuses on possible correlations between testosterone levels and the serum and temporal gland secretions. We were especially interested in possible qualitative and/or quantitative changes in volatile compounds as the testosterone levels varied during a discrete musth period. Ouantitative changes in TGS and serum testosterone were determined by radioimmunoassay. Qualitative and semiquantitative changes occurring in volatile composition were studied by high-resolution gas chromatography (fused silica capillary column, on column injection). Compound identification was by nuclear magnetic resonance, gas chromatography-mass spectrometr, and gas chromatography internal standards. Twenty-three major compounds and a number of minor components were identified. Androgen concentrations were correlated with TGS-specific volatiles including benzoic acid, 2-nonanone, 5-nonanol, tetradecanoic acid and decanoic acid. The latter two compounds and (E)-farnesol, a major component of African TGS, demonstrated an inverse relationship to T levels.

Rasmussen, L.E.L., Hess, D.L., Hall-Martin, A. Chemical profiles of temporal gland secretions from captive Asian bull elephants during musth and from African bull elephants living in wild but crowded conditions. Chemical Senses 15, 628. 1990.
Ref Type: Abstract
Abstract: Full-text: This study compares the volatile components of the temporal gland secretions of captive Asian bull elephants in musth and a distinctive group of wild African bull elephants, confined to a national park. The captive Asian population has been well studied (Rasmussen et al., 1984; Rasmussen, 1988). Serum testosterone was elevated at specific times; aggressive behaviors occurred concomitantly with temporal gland secretions, although aggression and elevated serum testosterone were not always related. Selected volatiles among the 23 compounds identified demonstrated concentration changes during the progression of musth, at times simultaneously with alterations in testosterone levels (Rasmussen et al., in press). The African bull elephants have been monitored, behaviorally and physiologically, by radiocontrolled tracking and monthly sampling during the past 5 years. Aggressive behaviors similar to those of Asian bull elephants have been documented; serum and temporal gland testosterone were elevated concomitantly in a cyclical fashion similar to musth in Asian elephants. Chemical characterization of the volatiles of the temporal gland secretions from these bulls revealed several similarities to the compounds described in Asian bulls, including several compounds not previously described in African temporal gland secretions. It is suggested that these chemicals, or other, more ephemeral compounds, may chemically inform other bulls and cows of the musth-like state of these bulls.

Roach, R., Briggs, M., Fithian, C. Determining the estrous cycle in a group of African elephants by evaluating serum progesterone levels. AAZPA Reg.Conf.Proc. 185-188. 1990.
Ref Type: Conference Proceeding

Wong, C.L., Wai, M.-K., Cheng, H.-C., Chung, D., Yamashiro, D., 1990. Preliminary study on the antinociceptive effect of elephant beta-endorphin. Clinical and Experimental Pharmacology and Physiology 17, 33-37.
Abstract: 1. Intraventricular administration of human beta-endorphin and elephant beta-endorphin significantly prolonged the tail flick response tested 30 min later. However, elephant beta-endorphin was about 7-8 times more potent than human beta-endorphin in the tail flick test. 2. beta-Endorphin antagonized the antinociceptive effect of both human beta-endorphin and elephant beta-endorphin by the same extent. Naloxone also antagonized the antinociceptive effects of the beta-endorphins but it was less effective than beta-endorphin. 3. Human beta-endorphin and elephant beta-endorphin were of equal potency in inhibiting the abdominal constriction response induced by intraperitoneal (i.p.) acetic acid. Both beta-endorphin and naloxone antagonized these effects of the beta-endorphins with naloxone being more effective. 4. The present study showed that different opioid receptor subtypes may be involved in the tail flick test and the abdominal constriction test. Furthermore, elephant beta-endorphin was a better antinociceptive agent than human beta-endorphin in the tail flick test.

Brannian, J.D., Terranova, P.F., Griffin, F. Long and short estrous cycles in the African elephant: an endocrine profile. AAZPA Reg.Conf.Proc. 16-18. 1989.
Ref Type: Conference Proceeding

Brannian, J.D., Griffin, F., Terranova, P.F., 1989. Urinary androstenedione and luteinizing hormone concentrations during musth in a mature African elephant. Zoo Biology 8, 165-170.
Abstract: Musth has not been well documented in captive African elephants. A 37-year-old African bull elephant in the Kansas City Zoological Park was observed during periods of behavioral musth and non-musth. Androstenedione and luteinizing hormone (LH) concentrations in urine were measured by radioimmunoassay. Urinary androstenedione and LH levels were significantly higher in musth urine than in non-musth samples. A positive correlation (P> 0.001) existed between urinary LH and androstenedione concentrations. These results indicate that musth can occur in a zoo-maintained African elephant and that urinary androgen levels are elevated during musth, possibly as a result of LH stimulation of testicular steroidogenesis.

Cooper, K.A. Reproductive endocrinology of male and female Asian and African elephants at the Columbus Zoo. 1989. Columbus, Ohio, USA, Ohio State Unversity.
Ref Type: Thesis/Dissertation

Czekala, N.M., Roocroft, A., Bates, M. Estrogen metabolism in the Asian elephant (Elephas maximus). Biology of Reproduction 40[suppl.1], 119. 1989.
Ref Type: Abstract
Abstract: Understanding follicular development in elephants has been confounded by an inability to detect serum estradiol (E₂) in cyclic patterns. Serum levels remain < 20 pg/ml throughout the cycle. To understand E₂ dynamics in the elephant, two metabolic studies were initiated. First 500 microCi tritiated E₂ was injected (iv). All urine and feces voided during the following 24 hrs. and two blood samples (30 min and 2 hrs) were collected. No radiolabel appeared in feces during the 24 hrs. Urinary radiolabel analyzed by HPLC appeared 100% conjugated, 52% E₂-3-conjugate (E₂C) and 48% estrone conjugate (E₁C). Peak levels appeared in urine 30 min post-injection, decreased rapidly during the next 2 hrs, then gradually to 16.5 hrs when levels stabilized. Serum radiolabel at 30 min appeared as conjugated and free (66:34). In the second study, unlabelled E₂ (1.5 mg) was injected (iv). Blood was taken at 0, 5, 15, 30, and 60 min and urine at 30 min. In serum, peak estrogen levels appeared at 5 min (E₂:E₂C:E₁C, 38:39:22 or 61:38 conjugated:free). Half-life of E₂ is ca.10 min and 60 min for E₂C. E₁C peaked at 15 min and declined by 20% in 45 min. Urine yielded similar results as the label study (E₂C:E₁C, 60:40). Daily urine E₂C and weekly serum E₂C and progesterone (P) were measured by RIA in five mature female elephants. Preliminary results indicated elevated levels of E₂C during the luteal phase similar to serum P profile. The follicular phase contains a biphasic profile of E₂C. These results suggest that E₂ is rapidly converted to E₂C and excreted in urine.

Dahl, K.D., Czekala, N.M., Hsueh, A.J.W. Measurement of urinary bioactive follicle-stimulating hormone (FSH) levels during reproductive cycles in diverse mammalian species. Biology of Reproduction 40[suppl.1], 119. 1989.
Ref Type: Abstract
Abstract: Recently an in vitro granulosa cell aromatase bioassay (GAB) was used to measure FSH in serum and urine samples (JCEM, March, 1987). We now adapted the GAB assay to measure urinary bio-FSH levels in conjunction with the determination of urinary immunoreactive pregnanediol-3-glucoronide (PdG) and/or estrone conjugates (EC). Daily urine samples were collected from 2 monkey species, lion-tailed macaque (Macaca silenus) and golden monkey (Rhinopithecus roxellana), and 3 ungulates, giraffe (Giraffa camelopardalis), okapi (Okapi johnstoni) and Asian elephant (Elephas maximus). The monkeys displayed a follicular phase rise in EC followed by a midcycle bio-FSH surge which declined as PdG concentrations increased during the luteal phase. Although both monkey species had similar cycle lengths (30-35 days), the golden monkey's follicular phase was much longer and displayed 2 bio-FSH peaks compared to 1 peak for the macaque. Although EC was not detectable in the ungulates, a midcycle FSH surge was followed by a luteal phase increase in PdG. The closely related giraffe and okapi had similar cycle lengths (16-17 days), follicular phase lengths (8-9 days), and only 1 follicular phase FSH peak. Conversely, the elephants had cycles lasting 16-19 weeks, and multiple FSH peaks were observed during the 5-6 week follicular phase. Thus, 1) in contrast to the single follicular phase increase and preovulatory surge of bio-FSH seen in the macaque, giraffe and okapi, the finding of multiple follicular phase peaks in the golden monkey and elephants suggests the involvement of complex regulatory mechanisms; 2) the GAB assay provides a noninvasive and practical method for monitoring reproductive cycles in endangered species for future breeding programs.

de Villiers, D.J., Skinner, J.D., Hall-Martin, A.J., 1989. Circulating progesterone concentrations and ovarian functional anatomy in the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 86, 195-201.
Abstract: Mean plasma progesterone concentrations measured in pregnant and non-pregnant elephants did not differ significantly from each other because of considerable variation, particularly for stage of pregnancy. Maximum progesterone values were recorded during early pregnancy (5-8 months) and declined towards term (22 months). The numbers of corpora lutea or total luteal tissue volume were not critical in maintaining progesterone secretion. An increase in plasma progesterone concentrations with the luteal phase of the ovarian cycle was evident. A possible role of the placenta in the second half of gestation is indicated by an increase in fetal progesterone concentrations towards term.

Li, C.H., Oosthuizen, M.M.J., Chung, D., 1989. Primary structure of elephant pituitary prolactin. International Journal of Peptide and Protein Research 33, 67-69.
Abstract: Tryptic digests of elephant pituitary prolactin (ele PRL) were separated by reverse-phase HPLC and paper electrophoresis. From the amino acid composition, the amino acid sequencing of selected peptides and from their alignment with expected tryptic peptides from ovine prolactin (based on the assumption that protein hormones with similar origins and bioactivities show significant degrees of homogenity), the primary sequence of ele PRL was proposed.

Richter, P. Comparative morphology of the thyroid gland of mammals: shape, size, position, blood supply, innervation and histology. Vergleichende, morphologische Studie an der Glandula thyreoidea der Mammalia unter Berucksichtigung von Form, Grosse, Lage, Gefassversorgung, Innervation und histologischem Aufbau. 1-274. 1989. Giessen, Fachbereich Veterinarmedizin, Justus-Liebig-Universitat .
Ref Type: Thesis/Dissertation
Abstract: Literature concerning the thyroid gland in primates, rodents, insectivores, carnivores, artiodactyles, perissodactyles, elephants, lagomorphs, bats and other classes of mammals is reviewed.

Brannian, J.D., Griffin, F., Papkoff, H., Terranova, P.F., 1988. Short and long phases of progesterone secretion during the oestrous cycle of the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 84, 357-365.
Abstract: Serum samples were collected from 3 mature female African elephants once each week for 15-18 months. Circulating concentrations of progesterone, oestradiol and LH were determined by radioimmunoassay (RIA). The LH RIA was validated by demonstrating parallel cross-reaction with partly purified elephant LH pituitary fractions. Changing serum progesterone concentrations indicated an oestrous cycle length of 13.3 ± 1.3 weeks (n=11). The presumed luteal phase, characterized by elevated serum progesterone values, was 9.1 ± 1.1 weeks (n=11). Two abbreviated phases of progesterone in serum lasting 2-3 weeks were observed in two elephants, indicating short luteal phases. Oestradiol concentrations in serum were variable, with no clear pattern of secretion. More frequent blood samples were collected during periovulatory periods and 9 distinct LH peaks were detected; all followed by rises in serum progesterone concentrations. Periovulatory changes in progesterone and LH in sera correlated with external signs of oestrus and mating.

Hromadka, J. Observations made on an Asian elephant (Elephas maximus) during gestation. Proc.Ann.Elephant Workshop 9. 93-105. 1988.
Ref Type: Conference Proceeding

Koch, E., Elsaesser, F., Boer, M., Dittrich, L., Ellendorff, F., 1988. Pregnancy diagnosis in Asian and African elephants. Deutsche Tierarztliche Wochenschrift 95, 111-114.
Abstract: Serum progesterone concentrations were regularly measured by radioimmunoassay in non-pregnant African and Asiatic elephants after collection of blood from an ear vein. This revealed a cyclic pattern which did not occur in a pregnant Asiatic cow elephant. Mid-stream, morning urine was collected from spontaneously urinating animals. After cooling, both serum and urine were stored at -25°C and analyzed within 2 months. The urine of the pregnant elephant, as well as that of 3 other females which had previously been mated, was examined with human chorionic gonadotropin (HCG) cross-reacting substances, with the help of a commercial haemagglutination-inhibition test. The first female's pregnancy followed mating in May, 1983. A dead calf was born at term in February, 1985. In mid-November, 1983 the progesterone reading was 512 pg/ml and the HCG test was positive. At approximately monthly intervals for the following 3 months, the progesterone/HCG readings were, respectively 875 pg/ml positive, 265 pg/ml positive, and 439 pg/ml negative. In the 3 other females which had previously been mated, immunological cross-reactions with HCG were also detected. These results suggest that both methods, individually or in combination, might be used for pregnancy diagnosis in elephants.

Li, C.H., Oosthuizen, M.M., Chung, D., 1988. Isolation and primary structures of elephant adrenocorticotropin and beta-lipotropin. International Journal of Peptide and Protein Research 32, 573-578.
Abstract: Adrenocorticotropin and beta-lipotropin have been isolated and purified from elephant pituitary glands. The primary structures of these two hormones were determined by amino acid and sequence analyses of enzymatically cleaved peptides from the hormones. Peptide purification involved the use of gel filtration, reverse phase high performance liquid chromatography, and paper electrophoresis.

Niemuller-Hare, C., Gray, C., Liptrap, R. A preliminary report on musth in male Asian elephants. Proc.Ann.Elephant Workshop 9. 106-111. 1988.
Ref Type: Conference Proceeding

Plotka, E.D., Seal, U.S., Zarembka, F.R., Simmons, L.G., Teare, A., Phillips, L.G., Hinshaw, K.C., Wood, D.G., 1988. Ovarian function in the elephant: luteinizing hormone and progesterone cycles in African and Asian elephants. Biology of Reproduction 38, 309-314.
Abstract: Serum samples were collected weekly for 3 yr from two female African elephants, for 18 mo from two other female African elephants, and for 2 yr from two female Asian elephants. Animals were not sedated at the time of blood collection. Ovarian cycles, characterized by changes in progesterone and immunoreactive luteinizing hormone (ILH) concentrations, averaged 15.9 + 0.6 wk (n=25) for African females and 14.7 + 0.5 wk for Asian females (n=10). The length of the active luteal phase averaged 10.0 + 0.3 wk for African elephants (range 8-14 wk) and 10.6 + 0.6 wk for Asian females (range 9-13 wk). One African female (Maliaca) had two extended interluteal phases, both occurring between the months of February and May. Excluding these two periods, there were no differences in the length of the luteal phase between species of elephant. Serum progesterone in both species ranged from less than 50 pg/ml to 933 pg/ml. Average progesterone concentrations during the luteal phase were significantly lower in African elephants compared with Asian elephants (328 + 13, n = 30 cycles vs. 456 + 23, n = 14 cycles; p<0.001). ILH ranged from nondetectable to 11.6 ng/ml. These data suggest that the length of the ovarian cycle in the African elephant is about 16 wk and confirm that the length of the ovarian cycle in the Asian elephant is about 15 wk.

Rasmussen, L.E.L., 1988. Chemosensory responses in two species of elephants to constituents of temporal gland secretion and musth urine. Journal of Chemical Ecology 14, 1687-1711.
Abstract: This report discusses three areas of investigation: (1) The chemical components in the temporal gland secretion (TGS) of Asian (Elephas maximus) and African (Loxodonta africana) elephants were characterized by radioimmunoassay (RIA) for testosterone (T) and dihydrotestosterone(DHT) levels and by on-column capillary column gas chromatographic analysis of volatiles. An inverse relationship between TGS testosterone levels and (E)-farnesol levels was observed. (2) African elephants responded preferentially toward a particular constituent of African elephant (TGS). (3) Urine from Asian bull elephants in musth was partially fractionated by high-performance liquid chromatography. Specific chromatographic regions elicited dramatic avoidance responses from female African elephants. These results support the suggestion that the TGS plays multiple chemocommunicative roles.

Teubner, V., Wells, S. Serum progesterone and cortisol levels in female Asian elephants (Elephas maximus). 1988.
Ref Type: Unpublished Work
Abstract: Poster presented at Symposium on Vertebrate Models in Endocrinology. National Institutes of Health, Bethesda, MD. 1988. Abstract. In the past zoological gardens and parks have not been at the forefront of endocrine research. However, zoos and wild animal parks represent a relatively untapped source of vertebrates for use as endocrine models. The collaboration of academic institutions with zoo personnel on various projects will not only enhance the management of exotic species but will also add to the data base in endocrine research. This is of critical importance to the propagation of endanged species. The developement of radioimmunoassay techniques had made it possible to detect minute amounts of hormones in the blood or excreta of various species. This serves as a valuable tool in assessing the reproductive status of a given animal. In species that do not exhibit the classic behavioral signs of estrous, measurement of hormonal activity can be the only link to evaluating fertility. The female Asian elephant (Elephas maximus) has a covert estrous cycle that only an adult bull elephant can detect. Due to the problems associated with the maintenance of bull elephants in captivity the measurement of the hormonal activity of the female is a feasible alternative. The goal of our study is to measure sex hormones (i.e. progesterone,and estradiol) and cortisol levels in two adult, female Asian elephants housed at the Audubon Zoo and to determine their estrous cycles. Blood is sampled weekly from the ear vein of the each elephant. Whole blood is then centrifuged and serum is stored at -70C for hormone measurement via radioimmunoassay. Attempts to measure estradiol were unsuccessful as Asian elephants secrete a combination of estrone and estradiol. Therefore, we chose to limit our analysis to progesterone, as the indicator of ovulation and to the glucocorticoid cortisol. We determined that the elephants' estrous cycle has a duration of approximately 12 weeks with a 3.2 week follicular phase followed by a luteal phase of approximately 9 weeks. Elevation of serum progesterone of 50 pg/ml above baseline and remaining elevated for 2 weeks was used as an indicator of luteal activity. Cortisol levels (ug/dl) were also measured and expressed as a percentage. Serum cortisol was elevated either prior to a cycle or during a cycle. This suggests that cortisol may also be a useful indicator of fertility. The hormonal data from the Asian elephant indicate that this species has a predictable estrous cycle that can be readily and accurately assessed using radioimmunoassay. THe methodology used for this species may serve as a valuable research model to aid in captive breeding programs and to further our understanding of the endocrine systems of exotic species.

Easa, P.S., 1987. Chemical composition of the temporal gland secretion of an Asian elephant (Elephas maximus). Elephant 2(3), 67-68.
Abstract: The non-volatile chemical constituents of a temporal gland secretion of a male Asian elephant are reported for the first time, and they seem to be different, in part, from those of African elephant.

Hall-Martin, A.J., 1987. The role of musth in the reproductive strategy of the African elephant (Loxodonta africana). South African Journal of Science 83, 616-620.
Abstract: Behavioral and endocrinological data on African elephants ( Loxodonta africana) are integrated to provide a hypothesis of the adaptations reflected in the phenomenon of musth in bulls. Occupation of home ranges, movements, male dominance hierarchies and intra-specific agonistic behavior are reviewed. Bulls in musth leave their home range, travel far and fast, imitate more contacts with distant breeding herds, show aggression which overrides normal social male hierarchies, probably mate more frequently than non-musth bulls and then return to their home range. This behaviour is associated with elevated levels of serum testosterone and dihydrotestosterone. Elephants normally show a high degree of fidelity to sexually segregated adjoining home ranges, which results in regular contact between the same bulls and cows. This breeding strategy is applicable to older, dominant bulls within the locally resident hierarchy. The musth adaptation is a second strategy, whereby younger, lower ranking bulls (25-35 years) can ensure more contacts with cows and maximize their chances of breeding. Because musth bulls mate far from their normal ranges the strategy promotes gene flow and ensures outbreeding. In English with Afrikaans summary.

Hodges, J.K., Mcneilly, A.S., Hess, D.L., 1987. Circulating hormones during pregnancy in the Asian and African elephants, Elephas maximus and Loxodonta africana: a diagnostic test based on the measurement of prolactin. International Zoo Yearbook 26, 285-289.

Li, C.H., Bewley, T.A., Chung, D., Oosthuizen, M.M.J., 1987. Elephant growth hormone: Isolation and characterization. International Journal of Peptide and Protein Research 29, 62-67.
Abstract: Growth hormone has been purified to homogeneity from elephant pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 15% when compared with bovine hormone in the radioreceptor binding assay. From circular dichroism spectra alpha-helical content of elephant growth hormone is estimated to be 50%. Difference absorption spectra of the hormone suggest the presence of a hydrogen bond between the single Trp and a carboxylate ion.

Li, C.H., Chung, D., Bewley, T.A., Cabrera, C.M., 1987. Elephant prolactin: Isolation and characterization. International Journal of Peptide and Protein Research 29, 472-477.
Abstract: Prolactin was isolated from anterior pituitary lobes of elephant pituitary glands. It consisted of 199 amino acids with three disulfide bridges and two tryptophan residues as found in prolactin from other species. The sequence of the NH₂ terminal 28 amino acids was determined and shown homologous with the ovine hormone. In comparison with ovine prolactin, a marked difference was seen in the methionine content; the elephant hormone possessed only 18-34% lactogenic potency. The conformation of elephant prolactin was examined by zero order, second order and circular dichroism spectroscopy. The alpha helical content was estimated to be about 60%. In comparison with prolactins from other species, the second order spectra of elephant prolactin suggest [sic] that the local microenvironment for one or both tryptophan residues is somewhat different.

Matteri, R.L., Roser, J.F., Baldwin, D.M., Lipovetsky, V., Papkoff, H., 1987. Characterization of a monoclonial antibody which detects luteinizing hormone from diverse mammalian species. Domestic Animal Endocrinology 4, 157-165.
Abstract: The present study describes the development and characterization of a monoclonal antibody (518B7) generated against bovine LH (bLH). Although 518B7 was extremely specific for LH, very low species specificity was observed. A RIA using this antibody and radioiodinated equine LH (eLH) showed good sensitivity for all mammalian LH preparations tested, with the exception of human LH (15% relative to the eLH reference standard). Activities of most mammalian LH's ranged between approximately 50-200%. Much less activity was dectected with reptilian LH (<1.5%). Amphibian and avian LH fractions were essentially inactive. The reactivities of LH alpha and beta subunits from a variety of mammals clearly showed that the antibody reacts with the beta subunit. Sensitive RIAs were also developed utilizing 125I-bovine and 125I-rat LH. Interestingly, all hormone preparations which showed sufficient reactivity for statistical analysis within the dose ranges used in the present study (0.01-1000 ng/tube) produced a displacement curve parallel to the reference standard. We have also validated the use of 518B7 in detecting LH in serum. Parallel dilution curves relative to purified LH reference standards were observed with equine and bovine serum samples and equine pituitary extract. High (average 94%) recoveries were also seen with bovine serum with known amounts of exogenously added bLH and 518B7 and a previously described polyclonal antibody-based RIA in bovine serum samples during estrus. Thus, a monoclonal antibody for LH has been produced which can be used to develop sensitive and specific RIAs in many different mammalian species. This antibody can be readily produced in amounts sufficient to provide a stable source of a high quality LH antibody, which may find wide applications in endocrinological research dealing with both domestic and laboratory animals.

Rubel, A. Physiological and pathological conditions associated with reproduction of female Asian elephants at the Zurich zoo. Proc.1st.Intl.Conf.Zool.Avian Med. 379. 1987.
Ref Type: Conference Proceeding

Smith, D.A., Nadaraja, R., Beck, B., Honhold, N., Hale, D., Knottenbelt, D.C., Hill, F.W.G., 1987. Serum testosterone levels in male African elephants, Loxodonta africana, in Hwange National Park, Zimbabwe. Zimbabwe Veterinary Journal 18, 58-63.
Abstract: Age and serum testosterone levels were determined for 44 male African elephants (Loxodonta africana) from Hwange National Park. Testicular weight was measured in 26 animals. Age and testicular weight were found to be highly correlated (r=0.94), while serum testosterone levels were correlated to both age (r=0.54) and testicular weight (r=0.43). Although the range of serum testosterone levels and maximum value increased with age, the minimum value did not.

Gorman, M.L., 1986. The secretion of the temporal gland of the African elephant, Loxodonta africana as an elephant repellant. Journal of Tropical Ecology 2, 187-190.

Kahl, V.A.L. The study of the reproductive cycle of the African elephant (Loxodonta africana) through correlation of behavior and cytology with serum and urinary hormonal patterns. 1986. Nebraska, Omaha, USA, University of Nebraska.
Ref Type: Thesis/Dissertation

Mainka, S.A. Monitoring reproductive cycles in two Sri Lankan elephants. Proc.Ann.Elephant Workshop. 7, 4-9. 1986.
Ref Type: Conference Proceeding

Baranga, J., 1984. The adrenal gland weights of the African elephant, Loxodonta africana. Zeitschrift fur Saugetierkunde 49, 341-348.

Hadley, M.E., 1984. Endocrinology. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.

Hall-Martin, A.J., van der Walt, L.A., 1984. Plasma testosterone levels in relation to musth in the male African elephant. Koedoe 27, 147-149.

Hattingh, J., Wright, P.G., de Vos, V., McNairn, I.S., Ganhao, M.F., Silove, M., Wolverson, G., Cornelius, S.T., 1984. Blood composition in culled elephants and buffaloes. Journal of the South African Veterinary Medical Association 55, 157-164.
Abstract: Blood composition of succinyldicholine culled elephants and buffaloes was compared with that of undisturbed animals shot in the brain. The results show statistically significant differences in a number of variables including plasma ACTH and cortisol concentrations. The observed changes are attributed to stress induced by a combination of herding and darting with succinyldicholine and asphyxia. Extrapolation from blood oxygen tensions suggests that this stress may be perceived for an undetermined period which is probably longer in elephants than buffaloes.

Howard, J., Bush, M., de Vos, V., Wildt, D.E., 1984. Electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging African elephants (Loxodonta africana). Journal of Reproduction and Fertility 72, 187-195.
Abstract: A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93.3 ml, 2408.6 x 10(6) spermatozoa/ml, 70%, 3.9 and 7.4, respectively. A high percentage (mean 77.5%) of spermatozoa whin each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic defect. When sperm viability was tested in vitro at 37 C, sperm motility rating declined by at least half of the initial assessment within 3.5 h of semem collection. Generally, spermatozoa maintained motility in vitro for < 6 h. Serum testosterone ranged from 1.4 to 8.2 ng/ml in 4 males evaluated in the morning (07:30 - 08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00 - 18:00 h), testosterone levels were < 0.9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25.6 ng/ml) and a preputial discharge potentially indicative of "musth." The present study demonstrates that high quality semen call be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.

Poole, J.H., Kasman, L.H., Ramsay, E.C., Lasley, B.L., 1984. Musth and urinary testosterone concentrations in the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 70, 255-260.
Abstract: Urine samples were obtained from free-ranging African elephants that were considered to be in and out of musth. Testosterone concentrations, measured by radioimmunoassay were significantly greater in males that were in or around the time of behavioral musth. This study supports a correlation between the observed behavioral characteristics of musth and urinary testosterone levels.

Rasmussen, L.E., Buss, I.O., Hess, D.L., Schmidt, M.J., 1984. Testosterone and dihydrotestosterone concentrations in elephant serum and temporal gland secretions. Biology of Reproduction 30, 352-362.
Abstract: Serum and termporal gland secretions (TGS) were obtained from mature wild African (Loxodonta africana) and captive Asian (Elephas maximus). Samples were obtained from five cows and eight bulls culled for management purposes in Kruger National Park, South Africa, and from four females and two males residing at the Washington Park Zoo, Portland, Oregon. Our purpose was to describe the levels of androgens, testosterone, and dihydrotestosterone, and to correlate these observations with sex, species, and behavioral status. Male-female differences in serum T were pronounced in the Asian species, whereas male and female concentrations overlapped in the African elephant serum. Serum T concentrations in African females were > than in Asian females. Serum DHT reflected T levels, except that the striking elevation of testosterone in Asian bulls during musth was not paralleled by = increases in DHT. A species difference observed among males was higher serum T levels in nonmusth Asian bulls (1.84-5.35ng/ml) compared to levels in African bulls (0.38-0.68ng/ml), except for one dominant African bull (6.64ng/ml). This single African value was still considerably lower than the serum T values of the Asian males during musth. These musth values were the highest serum androgen concentrations: T was between 19 and 40ng/ml (average 26.1 ng/ml). The TSG values of T and DHT were much higher than serum levels except in the Asian female. T/DHT ratios in TGS were more similar than in serum. One dominant African bull had a T TGS value of 78ng/ml, which was much higher than the rest of the African males or females, but considerably lower than an Asian bull in musth (547ng/ml). It seems apparent that a change in androgen status as reflected in serum and TGS levels of T and DHT precedes or is concomitant with overt alteration in behavior in the Asian male. The temporal gland appears to actively concentrate androgens in both African males and females, but in the Asian male the gland secretes only during musth when the greatest concentration of both T and DHT were observed. The apparent difference in the degree of temporal gland secretory activity between the 2 species suggests a more specific communicative function within the Asian male.

Siegel, R.K., 1984. LSD-induced effects in elephants: comparisons with musth behavior. Bulletin of the Psychonomic Society 22, 53-56.
Abstract: Musth is a condition observed in male Asiatic elephants and is characterized by aggression and temporal gland secretion. A classic and controversial 1962 study attempted to induce a musth syndrome in an elephant via treatment with LSD. Two elephants in the present study survived dosages of LSD (.003 -.10 mg/kg) and exhibited changes in the frequency or duration of several behaviors as scored according to a quantitative observational system. LSD increased aggression and inappropriate behaviors such as ataxia. Results are discussed in terms of musth and drug-induced perceptual-motor dysfunction.

Hodges, J.K., Henderson, C., Mcneilly, A.S., 1983. Circulating oestrogen concentrations during pregnancy in the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 67, 121-127.
Abstract: Oestrone, Oestradiol-17B and oestriol were measured in plasma samples from non-pregnant and pregnant African elephants shot in the wild. Enzymatic hydrolysis of plasma showed that approximately 90 and 96% of the total (i.e. conjugated plus unconjugated) concentrations of oestrone and oestradiol-17B, respectively were represented by conjugated hormones. Unconjugated oestrogens remained low (<50pg/ml) in all samples, with no distinction between non-pregnant and pregnant animals. Levels of total oestrone during pregnancy varied between 160 and 594 pg/ml but were not significantly different from non-pregnant values. Total oestradiol-17B concentrations were significantly elevated during pregnancy (P < 0.01) and, despite considerable individual variation (193-1428pg/ml), were consistently higher than non-pregnant values after 6 months gestation. The elevated levels of oestradiol-17B resulted in a reversal of the total oestradiol-17B: oestrone concentration ratio at about 6 months of pregnancy. Concentrations of total oestriol did not exceed 103 pg/ml. An indirect method of measurement indicated that oestradiol-17B sulfate was probably the most abundant circulating oestrogen during pregnancy in the African elephant.

Mcneilly, A.S., Martin, R.D., Hodges, J.K., Smuts, G.L., 1983. Blood concentrations of gonadotropins, prolactin and gonadal steroids in males and in non-pregnant and pregnant female African elephants (Loxodonta africana). Journal of Reproduction and Fertility 67, 113-120.
Abstract: No seasonal variation in any of the hormones measured was apparent in males or females. Testosterone levels in males increased around puberty (10-11 years) and remained significantly higher in adults than prepubertal males. This was not accompanied by any significant change in levels of LH, FSH or prolactin. In non-pregnant females there was no apparent difference in levels of LH, FSH or prolactin with age.There was a significant increase in progesterone around puberty (12 years) but there was considerable overlap in values between prepubertal and adult females. During pregnancy, progesterone levels were significantly higher than in non-pregnant females with maximum levels occurring at mid-pregnancy (9-12 months). However, there was considerable overlap in values between non-pregnancy and pregnancy. Concentrations of LH and FSH decreased significantly during mid-pregnancy while prolactin levels increased dramatically during pregnancy; after 7 months gestation until term levels were always at least 8ng/ml greater than in any non-pregnant female. It is suggested that this consistent increase in plasma/serum levels of prolactin can be used to diagnose pregnancy in the elephant.

Pichaicharnarong, A., Loypetjra, P., Chaiyabutr, N., Komonmena, V., Usanakornkul, S., Boonnamsiri, V., Devakul, C., 1983. The thyroid activity of Asiatic elephants in Thailand. Acta Veterinaria (Beograd) 33, 73-80.
Abstract: Values of serum T3 and T4 were determined by radioimmunoassay in 58 normal Asiatic elephants (Elephas maximus, Linnaeus 1758) with ages ranging from 1 to 80 years. The mean concentrations of T4 and T3 were 113.6 ± 27.0 umol/l and 1.8 ± 0.7 umol/l respectively. The RT3U values (resin triiodothyronine125 uptake) were assessed in 65 elephants. The mean was 30.1 ± 3.8%. Both serum T4 and T3 decreased with age. The RT3U of 1-10 year old elephants was higher than that of older elephants, but there was not statistical difference between any age range. Details of the effects of sex and age of the Asiatic elephant on the values of T3 (RIA), T4 (RIA) and RT3U are discussed.

Wheeler, J.W., Rasmussen, L.E., Ayorinde, F., Buss, I.O., Smuts, G.L., 1982. Chemical constituents of temporal gland secretion of the African elephant, Loxodonta africana. Journal of Chemical Ecology 8, 821-835.
Abstract: Temporal gland secretion (TGS), obtained from 15 different mature African elephants in Kruger National Park was analyzed for volatile constituents. Only five volatile components were present. p-Cresol was present in all samples, but phenol was found as an appreciable component of only one sample and as trace amounts in six others. Three sesquiterpenes were identified, the latter two being new natural products: E-farnesol, farnesol hydrate (3,7,11-trimethyl-2,10-dodecadien-1,7 diol), and farnesol dihydrate (3,7,11-trimethyl-2-dodecen-1,7,11-triol). These sesquiterpenes represent the first isolated from mammals. Ten samples of TGS, serum, and saliva were assayed for cholesterol, urea, and proteins including several enzymes.

Poole, J.H., Moss, C.J., 1981. Musth in the African elephant, Loxodonta africana. Nature 292, 830-831.
Abstract: The phenomenon of musth in male Asian elephants, Elephas maximus, has long been recognized. Musth, which has been likened to rutting behaviour in ungulates, refers to a set of physical and behavioural characteristics displayed periodically by adult male elephants. The most obvious manifestations are a sharp rise in aggressive behaviour, copious secretions from and enlargement of the temporal glands, and a continuous discharge of urine. It has been speculated that a similar phenomenon occurs in males of the African genus, Loxodonta africana, but most workers have concluded that it does not exist. Here we show that musth does occur in the African elephant and that its manifestations are similar to those in the Asian elephant.

Ramsay, E.C., Lasley, B.L., Stabenfeldt, G.H., 1981. Monitoring the estrous cycle of the Asian elephant (Elephas maximus), using urinary estrogens. American Journal of Veterinary Research 42, 256-260.
Abstract: The estrous cycle of the Asian elephant (Elephas maximus) was monitored by analysis of urinary estrogens. Daily morning urine samples were analyzed for estrone (E₁), estradiol (E₂), and total immunoreactive estrogen (E_T). The E_T values were shown to correlate poorly with E₁ and E₂ and failed to reveal any patterns of reproductive cycling. Daily E₁ and E₂ values, indexed by creatinine concentrations, demonstrated cyclic profiles in those samples of sufficient concentrations. The technique offered a simple, noninvasive method for determining ovarian function in the elephant.

Schmidt, M.J., Hess, D.L., Schmidt, A.M., Henneous, R.L., Groves, D.A., Haight, J.D. The estrous cycle of the Asian elephant. Proc.Am.Assoc.Zoo Vet. 91-95. 1981.
Ref Type: Conference Proceeding

Norris, D.O., 1980. Vertebrate endocrinology. Lea & Febiger, Philadelphia.

Chappel, S.C., Schmidt, M.J., 1979. Cyclic release of luteinizing hormone and the effects of luteinizing hormone-releasing hormone injection in Asiatic elephants. American Journal of Veterinary Research 40, 451-453.
Abstract: Cyclic changes in serum concentrations of luteinizing hormone (LH) were observed throughout the estrous cycle of Asiatic elephants (Elephas maximus). The increase in serum LH was correlated with a slight increase in serum estradiol concentration and the onset of behavioral heat (willingness to mate). In a second series of studies, injection of luteinizing hormone-releasing hormone after 3 days of estrone administration induced an increase in serum LH. These studies indicate that the Asiatic elephant exhibits a cyclic LH release that can be experimentally induced by estrone and luteinizing hormone-releasing hormone administration.

Adams, J., Garcia III, A., Foote, C.S., 1978. Some chemical constituents of the secretion from the temporal gland of the African elephant (Loxodonta africana). Journal of Chemical Ecology 4, 17-25.
Abstract: The temporal glands of African elephants were examined microscopically and histologically, and were found to be modified apocrine sweat glands. The secretion from thse glands was analyzed by gas chromotography and mass spectrometry, and some of the major volatile components have been identified as phenol and m- and p-cresol.

Cmelik, S.H.W., Ley, H., 1978. Neutral lipids from the temporal gland of the African elephant (Loxodonta africana). Lipids 13, 195-198.

Bentley, P.J., 1976. Comparative vertebrate endocrinology. Cambridge University Press, New York.

Buss, I.O., Estes, J.A., Rasmussen, L.E., Smuts, G.L., 1976. The role of stress and individual recognition in the function of the African elephant's temporal gland. Mammalia 40, 437-451.
Abstract: Biochemical measurements were made from a sample of temporal gland secretion from each of five wild African elephant bulls (23 to 38 years of age) collected in Kruger National Park, South Africa between November 1974 and April 1975. Total protein content was high (26-57 mg/ml), acid phosphatase ranged between 1.9 and 6.3 mM/h/mgm protein, and lactic dehydrogenase levels were undetectable. Total lipid content in the secretion averaged 80 mg% and ranged from 75 to 87 mg%. Triglycerides were just detectable, varying from 2 to 8 mg%, and phospholipids ranged from 9 to 11 mg% (ave. 10 mg%). Cholesterol content was surprisingly high, measuring 12, 19, 26, 36, and 70 mg% for five samples of secretion. Field observations indicated that stress triggers liberation of temporal gland secretion. Among 116 elephants collected in Uganda, secretory activity of their temporal glands was more frequent during dry (probably more stressful) than during wet seasons. Among 62 elephants driven by helicopter to roadways for collection in Kruger National Park, 23 driven relatively far and fast were in prominent musth; most of those driven slower and shorter distances showed no evidence of musth. The matriarchal leader of an elephant family near Lake Albert, Uganda developed very prominent temporal gland activity after an hour and 45 minutes of vigorously defending three of her family members. Chemical individuality of cholesterol levels in temporal glands of five adult bulls suggests a pheromone-producing function which serves for individual recognition by the African elephant. Direct observations of wild elephants also suggest that the temporal gland functions as a scent gland helping to recognize other members of the group or to find them.

Estes, J.A., Buss, I.O., 1976. Microanatomical structure and development of the African elephants' temporal gland. Mammalia 40, 429-436.
Abstract: The microanatomical structure of temporal glands from thirteen adult African elephants and one fetus, approximately five months old, were examined. The temporal gland consists of compound tubular alveoli interspersed within a loose connective tissue matrix, which is separated into numerous lobes by septa of dense connective tissue. Structurally the gland is nearly identical with the temporal gland of the Asiatic elephant. Metachromatic granules are numerous around the lumenal border of glandular cells suggesting an acid mucopolysaccharide secretory product. Fibroblasts and macrophages are numerous along with occasional plasma cells and mast cells in the loose connective tissue matrix. Mallory's azan indicated that glandular cytoplasm adjacent to the basement membrane was acidophilic whereas the secretion was always basophilic. Frozen sections of temporal glands stained with oil red-O confirmed the presence of lipids throughout the glandular cytoplasm. The presence of myoepithelial cells was suggested, but not confirmed. Histogenesis of the temporal gland at the five month fetal stage indicates epithelial primordia forming downward projections into the connective tissue. Development at this point is similar to that of mammary gland further confirming a common origin with apocrine sweat glands.

Pillay, K.R., 1976. A note on the diagnosis of pregnancy in elephants. Indian Veterinary Journal 53, 19-21.

Gombe, S., Heap, R.B., Sale, J.B., 1975. Endocrinology of pregnancy in the hyrax; plasma progesterone concentration and erythrocyte metabolism. Proc. Physiol. Soc. September, 13P-14P.

Heap, R.B., Gombe, S., Sale, J.B., 1975. Pregnancy in the hyrax and erthrocyte metabolism of progesterone. Nature 257, 809-811.

Joasoo, A., Murray, I.P.C., Parkin, J., 1975. Comparative studies of thyroid function in mammals. General and Comparative Endocrinology 26, 135-138.

Plotka, E.D., Seal, U.S., Schobert, E.E., Schmoller, G.C., 1975. Serum progesterone and estrogens in elephants. Endocrinology 97, 485-487.
Abstract: Serum progesterone and estrogens were measured by radioimmunoassay in the serum of immature, mature, and pregnant African and Asian elephants. Progesterone was elevated from 26 to 215 pg/ml in nonpregnant animals and up to 480pg/ml in late pregnancy animals. No relationship to reproductive state was evident for the low levels of estrogens, which ranged from 9 to 37 pg/ml.

Cmelik, S., Ley, H., 1974. Fatty acid composition of some cellular and subcellular elements of the elephant adrenal gland. Hoppe-Seyler's Z. Physiol. Chem. 355, 797-802.
Abstract: Lipids from the cortex and medulla of the elephant adrenal gland were extracted with chloroform/methanol, and purified over Sephadex G-25. The total lipids, excluding gangliosides were fractionated by a combination of Unisil columns and preparative silica gel G plates and various fractions analyzed by gas-liquid chromatography for their fatty acid components. Results show that nearly 70% of the total lipids in both parts of the gland consist of cholesterol esters differing in the content of polyunsaturated fatty acids. Phospholipids from the cortex and medulla show an identical fatty acid pattern and are characterized by a considerable amount of arachidonic acid. Mitochondrial and microsomal lipids of the whole adrenal gland were extracted, purified and fractionated in the same way. 35% of the mitochondrial lipids consisted of cholesterol esters with C18:1, C16:0 and C20:3(omega 6) as predominant fatty acids. Triglycerides did not contain any higher polyunsaturated fatty acids. In the phospholipids the predominant fatty acids were C18:0, C18:1, C20:4(omega 6) and C16:0. Microsomal lipids yielded 13% cholesterol esters with very few polyunsaturated fatty acids and a considerable amount of C12 and C14 acids. Unlike the mitochondria, microsomes did not contain any triglycerides. Phospholipids of the microsomes were rich in arachidonic acid and contained polyunsaturated acids not present in other fractions. The presence of larger quantities of C16:1 and C16:2 acids in the adrenal gland suggests the possible existence of the omega 7 pathway, although no other metabolites were identified. The fatty acid pattern excludes the possibility of an essential fatty acid deficiency.

Cmelik, S., Ley, H., 1974. Composition of the lipids from the cell sap of the elephant adrenal cortex. Hoppe-Seyler's Z. Physiol. Chem. 355, 1463-1465.

Ogle, T.F., Braach, H.H., Buss, I.O., 1973. Fine structure and progesterone concentration in the corpus luteum of the African elephant. Anatomical Record 175, 707-724.
Abstract: Corpora lutea from one recently post-partum and six pregnant African elephants (Loxodonta africana) were fixed for electron microscopy. Progesterone concentration was determined for five of these corpora lutea utilizing the competitive protein binding technique. Lutein cell size and progesterone concentration increased from one and two months of pregnancy to a maximum at three months (P< 0.01). After eight months of pregnancy, lutein cell size decreased to the one-month level. Maximum lutein progesterone was 4.1 ng/mg, much lower than that reported for other mammals. Lutein cell fine structure exhibited characteristics typical of lutein cells from other species but with some variation. Unusual features include peripheral distribution of organelles, large stores of lipid and lipofuscin throughout pregnancy, and mitochondria sparsely populated with lamellar cristae. The data indicate that the corpus luteum of the African elephant has significant but limited steroidogenic capabilities.

Jainudeen, M.R., Katongole, C.B., Short, R.V., 1972. Plasma testosterone levels in relation to musth and sexual activity in the male Asiatic elephant, Elephas maximus. Journal of Reproduction and Fertility 29, 99-103.
Abstract: Testosterone was measured in the peripheral blood plasma of eleven male Asiatic elephants, using a competitive protein-binding assay. When the animals showed no signs of musth, the testosterone levels were low (<0.2 to 1.4 ng/ml); as they began to come into musth and the temporal glands started to enlarge, the testosterone levels rose (4.3 to 13.7 ng/ml), and when the animals were in full musth, with discharging temporal glands and an aggressive temperment, the levels were extremely high (29.6 to 65.4 ng/ml). Musth may therefore be comparable to the rutting behavior of some seasonally breeding mammals, although, in the elephant, there is some indication that it may be induced by sexual activity.

Fujimoto, E., Koto, N., Imori, T., Nakama, S., 1971. Gonadotropin in the urine of a pregnant Indian elephant -- a case report. Zoologica 55, 73.
Abstract: In 1963, at Takarazuka Zoo, Japan, a young female Indian elephant became pregnant, and in May, 1965, she gave birth to a very large stillborn calf (weighing 133.3 kg, male). The time of conception was problematical, but it was assumed as April or May of 1963, hence the gestation period may have been 24 or 25 months, a little longer than average. Pregnancy diagnosis was attempted during the early and middle gestation period. For exploration, an urinary gonadotropin was checked by the Friedman and Aschheim-Zondek tests on the whole urine samples collected twice in August 1963. Results showed apparently positive results in both tests. However, the samples collected in May and September, 1964, showed negative in three tests, including a male frog (Rana) reaction which was subjected to the concentrated urine samples. So, probably a gondotropic substance many have been excreted in urine of this elephant at some time of the early pregnancy, and this may be more like FSH than LH in its activity.

Jainudeen, M.R., Eisenberg, J.F., Tilakertne, N., 1971. Oestrous cycle of the Asiatic elephant, Elephas maximus, in captivity. Journal of Reproduction and Fertility 27, 321-328.
Abstract: During the course of a programme to breed the Asiatic elephant, Elephas maximus, in captivity, the oestrous cycles of eleven adult females were studied. Two methods were used for detecting oestrus: (1) daily testing with a male elephant, and (2) urogenital smear cytology. Overt signs of oestrus were not observed but "standing" oestrus was observed in ten animals. The duration of oestrus ranged from 2 to 8 days with a mode of 4 days. Oestrous cycles in six animals ranged from 18 to 27 days with a mean of 22 days. Urogenital smear cytology failed to indicate accurately the onset of behavioural oestrus but increases in the number of cornified cells may occur before, during and slightly after behavioural oestrus. Considerable mating activity occurred during oestrus. A description of mating behaviour is presented. The findings are discussed in relation to breeding elephants in captivity and to the phenomenon of temporal gland activity.

Estes, J.A. Observations on the temporal gland of the African elephant. 1970. Washington, USA, Washington State University.
Ref Type: Thesis/Dissertation

Krumrey, W.A., Buss, I.O., 1969. Observations on the adrenal gland of the African elephant. Journal of Mammalogy 50, 90-101.
Abstract: Forty-nine female and 32 male African elephants (Loxodonta africana) were collected from July 19558 to May 1959 in Bunyoro District, Uganda. Reproductive status, body weight, and measurements were recorded and ages estimated. Gross morphology and histology of the adrenal gland are described: histology of the elephant adrenal corresponds with generalized descriptions for those of other eutherian mammals. The fetal cortex is well developed in an elephant 2 months of age, but gradually degenerates and is completely absorbed by the fourth year of postnatal life without giving rise to another transitory zone. No significant increase in relative adrenal weight related to Uganda's December-to-March dry season was detected. Relative adrenal weight of elephants is higher in females than males of comparable age, in immature males that mature males, and in immature females than nonpregnant or nonlactating females. Pronounced increase in relative adrenal weight is related to pregnancy and lactation.

Smith, J.G., Hanks, J., Short, R.V., 1969. Biochemical observations on the corpora lutea of the African elephant, Loxodonta africana. Journal of Reproduction and Fertility 20, 111-117.

Buss, I.O., Johnson, O.W., 1967. Relationships of Leydig cell characteristics and intratesticular testosterone levels to sexual activity in the African elephant. Anatomical Record 157, 191-196.
Abstract: Histological characteristics of testis tissues from 25 African elephants (Loxodonta africana) collected in Uganda, showed no consistent relationships among the following variables: Leydig cell size, cytoplasmic characteristics, and abundance; testicular testosterone content; and age. From these findings, plus field observations of sexual behavior, emerges the hypothesis that individual cyclicity in Leydig cell function was inherent in the elephant population studied. Testosterone content of testes from 32 elephants (including the 25 studied histologically) suggested that lone bulls were not of a senile nature since they contained relatively large quantities of testosterone and were relatively young (from about 12 to 25 years of age). Also, lone bulls were observed searching out estrous females. Among bulls collected from family units and herds, testosterone levels and behavior differed conspicuously. Behavior appeared to be directly related to testosterone content in several instances. Non-aggressive behavior among members of bull herds, plus the high proportion of such individuals with low testosterone content, suggest that some of these animals were in a depressed phase of sexual activity whereas others were undergoing pubertal development.

Moore, J.H., Sikes, S.K., 1967. The serum and adrenal lipids of the African elephant (Loxodonta africana). Comparative Biochemistry and Physiology [A] 20, 779-792.
Abstract: 1. The serum and adrenal lipids of the African elephant were fractionated by chromatography on columns of silicic acid into cholesterol esters, cholesterol, triglycerides, unesterified fatty acids and phospholipids. The fatty acid compositions of the various lipid fractions were determined by gas-liquid chromatography. 2. The results obtained from the African elephant were compared with the results reported in the literature for other species of mammals. In many respects the composition of the serum lipids of the African elephant was similar to that of the rat and rabbit but was markedly different from that of the ox and man. 3. Unlike the serum cholesterol esters and phospholipids of other animals, these two lipid fractions in the serum of these elephants contained appreciable concentrations of delta-8,11,14-eicosatrienoic acid. 4. The total lipid content of the African elephant adrenal galnd was particularly high (63 per cent of the dry tissue). Cholesterol esters accounted from almost half of the adrenal lipid. Delta-8,11,14-eicosatrienoic acid was present in substantial amounts in the adrenal cholesterol esters and phospholipids.

O'Donoghue, P.N., Sikes, S.K., Turvey, A., 1967. Notes on the adrenal of the African elephant. Journal of Zoology (Lond) 152, 281-286.
Abstract: Sections of the adrenals of two male specimens of Loxodonta africana were examined. Apart from the dimensions of the gland and the amount and disposition of connective tissue in it, the most characteristic features were the very high lipid content of the cortical secretory cells, the large size of these cells, and the occurrence of dark cells in the zona fasciculata.

Short, R.V., Mann, T., Hay, M.F., 1967. Male reproductive organs of the African elephant, Loxodonta africana. Journal of Reproduction and Fertility 13, 517-536.

Krumrey, W.A. Age estimation and observations on the adrenal gland of the female African elephant. -69pp. 1966. Pullman, WA, USA, Washington State University.
Ref Type: Thesis/Dissertation

Short, R.V., Buss, I.O., 1965. Biochemical and histological observation on the corpora lutea of the African elephant (Loxodonta africana). Journal of Reproduction and Fertility 9, 61-67.
Abstract: Progesterone determinations were carried out on eleven corpora lutea obtained from six African elephants in various stages of the oestrous cycle and pregnancy. In no case was it possible to detect any progesterone whatsoever, even when using an extremely sensitive gas chromatographic technique, coupled with an internal isotope standard of ⁴C₁₄-progesterone to correct for extraction losses. The histological appearance of many of the corpora lutea in these elephants suggested that they might be degenerate structures. It is therefore postulated that the functional life of the elephant's corpus luteum may be relatively short, even though it persists structurally for long periods of time. However, we cannot entirely exclude the possibility that the elephant's corpus luteum never secretes progesterone at all.

Johnson, O.W. Histological and quantitative characteristics of the testes, observations on the teeth and pituitary gland, and the possibility of reproductive cyclicity in the African elephant (Loxodonta africana). 1-124. 1964. Pullman, Washington, USA, Washington State University.
Ref Type: Thesis/Dissertation

Fernando, S.D.A., Jayasinghe, J.B., Panabokke, R.G., 1963. A study of the temporal gland in an Asiatic elephant (Elephas maximus). Ceylon Veterinary Journal 11, 108-111.

Hanstrom, B., 1953. The hypophysis in some South African insectivora, carnivora, hyracoidae, proboscidea, artiodactyla and primates. Arkiv foer Zoologi (Stockholm) 4, 187-294.

Bourne, G.H., 1949. The mammalian adrenal gland. Oxford at the Clarendon Press, Oxford.

Wislocki, G.B., 1940. The topography of the hypophysis of the elephant, manatee, and hyrax. Anatomical Record 77, 427-445.

Wislocki, G.B., 1939. Note on the hypophysis of an adult Indian elephant. Anatomical Record 74, 321-328.

Eales, N.B., 1925. External characteristics, skin, and temporal gland of a foetal African elephant. Procedings of the Zoological Society of London 2, 445-456.

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