Elephant
Bibliographic
Database

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Elephant Bibliographic Database
www.elephantcare.org

References Updated October 2007

     1.    Brown J.L., Somerville M., Riddle H.S. et al. 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.

     2.    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.

     3.    Wallis M. 2007. 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 Epub ahead of print.
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.

     4.    Wittemyer G., Ganswindt A. and 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.

     5.    Yon L., Kanchanapangka S., Chaiyabutr N. et al. 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.

     6.    Yon L., Kanchanapangka S., Chaiyabutr N. et al. 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.

     7.    Yon L., Chen J., Moran P. and 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.

     8.    Ball R. and Fad O. 2006. Serum cortisols in captive Asian elephants (Elephas maximus) in different management systems at Busch Gardens Tampa Bay.   2006 Proceedings American Association of Zoo Veterinarians, pp. 177-180.
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. ……………………………………………………………………………

     9.    Ball R.L. and Brown J. 2006. Preliminary results of a cabergoline trial in captive elephants with hyperprolactinemia.  Proceedings International Elephant Conservation & Research Symposium., 2006, pp. 26-28.

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

   11.    Ball R.L. and Brown J. 2006. Preliminary results of a cabergoline trial in captive elephant with hyperprolactinemia.   2006 Proceedings American Association of Zoo Veterinarians, pp. 174-176.
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

   12.    Bertschinger H., Delsink A., Kirkpatrick J.F. et al. 2006. Management of elephant populations in private South African game reserves with porcine zona pellucida vaccine.   2006 Proceedings American Association of Zoo Veterinarians, pp. 283-285.
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.

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

   14.    Keay J.M., Singh J., Gaunt M.C. and 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.

   15.    Bonar C.J., Lewandowski A.H., Arafah B. and Capen C.C. 2005. Pheochromocytoma in an aged female African elephant (Loxodonta africana).Journal of Zoo and Wildlife Medicine 36: 719-723.

   16.    Delves P.J. and Roitt I.M. 2005. Vaccines for the control of reproduction--status in mammals, and aspects of comparative interest.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.

   17.    Ganswindt A., Rasmssen H.B., Heistermann M. and 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.

   18.    Ganswindt A., Heistermann M. and Hodges K. 2005. Physical, physiological, and behavioral correlates of musth in captive African elephants (Loxodonta africana).Physiological and Biochemical Zoology 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.

   19.    Glickman S.E., Short R.V. and 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.

   20.    Rasmussen L.E.L., Krishamurthy V. and 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.

   21.    Steinetz B.G., Brown J.L., Roth T.L. and Czekala N. 2005. Relaxin concentrations in serum and urine of endangered species.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.

   22.    Brown J.L., Walker S.L. and Moeller T. 2004. Comparative endocrinology of cycling and non-cycling Asian (Elephas maximus) and African (Loxodonta africana) elephants.General and Comparative Endocrinology 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, hyperpro-lactinemia 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.

   23.    Buchanan K.L. and 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.

   24.    Carter A.M. and 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.

   25.    Hermes R., Hildebrandt T.B. and 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.

   26.    Meyer J.M., Walker S.L., Freeman E.W., Steinetz B.G. and Brown J.L. 2004. Species and fetal gender effects on the endocrinology of pregnancy in elephants.General and Comparative Endocrinology 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.

   27.    Wilson M.L., Bloomsmith M.A. and 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.

   28.    Dehnhard M., Hatt J.M., Eulenberger K., Ochs A. and 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

   29.    Ganswindt A., Palme R., Heistermann M., Borragan S. and 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.

   30.    Hunt K.E. and 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.

   31.    Rajaram A. and 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.

   32.    Rasmussen L.E. and 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

   33.    Wilson J.D., Leihy M.W., Shaw G. and 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.

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

   35.    Allen W.R., Mathias S.S., Wooding F.B., Skidmore J.A. and 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.

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

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

   38.    Forsyth I.A. and 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

   39.    Goodwin T.E., Brown F.D., Counts R.W. et al. 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.

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

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

   42.    Rasmussen L.E., Riddle H.S. and 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.

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

   44.    Welsch U., Unterberger P., Hofter E., Cuttitta F. and 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.

   45.    Wielebnowski N.C. and 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.

   46.    Dehnhard M., Hildebrand T., Rohleder M. et al. 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.

   47.    Dehnhard M., Hesitermann M., Goritz F. et al. 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.

   48.    Ganswindt A., Heistermann M. and Hodges J.K. 2001. 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, 2001, p. 258. Schuling Verlag, Vienna, Austria.

   49.    Ganswindt A., Heistermann M., Borragan S. and 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.

   50.    Lamps L.W., Smoller B.R., Rasmussen L.E.  et al. 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.

   51.    Pucher H.E., Stremme C., Tu N.C. et al. 2001. 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, 2001, p. 283. Schuling Verlag, Vienna, Austria.

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

   53.    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

   54.    Suedmeyer W.K. 2001. Serum hydrocortisone levels in a manually restrained African elephant (Loxodonta africana)  pre- and post- semen collection. In: Kirk Baer C and Wilmette MW (eds), 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, September 18, 2001-September 23, 2001, pp. 388-389.  American Association of Zoo Veterinarians.

   55.    Wilson M.L., Bloomsmith M.A., Crane M. and Maple T.L. 2001. 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, pp. 147-149. Schuling Verlag, Vienna, Austria.