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From the * School of Animal Studies and
School of Agronomy and Horticulture, The
University of Queensland, Gatton, Queensland, Australia; and the
Environmental Protection Agency, Queensland
Government, Bellbowrie, Queensland, Australia.
| Correspondence to: Stephen D. Johnston, School of Animal Studies, The University of Queensland, Gatton, Queensland 4343, Australia (e-mail: s.johnston1{at}uq.edu.au). |
| Received for publication April 3, 2006; accepted for publication June 2, 2006. |
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Abstract |
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Key words: Marsupial, hormone stimulation test
McFarlane (1990) previously reported that treatment with 2 µg of natural sequence GnRH did not induce an increase in testosterone secretion in koalas. It is possible that the dose of GnRH used in the latter study was below the threshold required to induce LH and testosterone responses. This may have been related, in part, to rapid clearance of natural sequence GnRH from circulation. In the present study, a GnRH agonist with a longer half-life in circulation than natural sequence GnRH and higher affinity for the GnRH receptor was used to ascertain whether a testosterone response could be elicited with exogenous hormone in male koalas. Given that the testosterone response to GnRH relies on LH release from the pituitary, direct stimulation of the testes with hCG was also evaluated as a potential testosterone stimulation test in koalas.
The specific aims of the present study were to examine fluctuations in endogenous testosterone secretion in the koala (n = 6) over a 24-hour period and then to characterise testosterone secretion after injection of the GnRH agonist buserelin or hCG, in order to establish a reliable index of the prevailing testosterone biosynthetic capacity of the koala testis.
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Materials and Methods |
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Anesthesia and Venipuncture Procedure
Venipuncture was conducted on anaesthetised koalas using a combination of
chemical and gaseous agents (Blanshard,
1994; McGowan et al, 1994). Briefly, anesthesia was initially
induced for the first blood sample by intramuscular injection of 20 µg of
Zoletil 100 (tiletamine/zolazepam; Virbac, Peakhurst, Australia; Animal Health
Australia, Canberra, Australia); subsequent blood samples for each experiment
where recovered by induction with 2%–4% gaseous isoflurane (Attane;
Pharmtech, Hornsby, Australia; Animal Health Australia) at a flow rate of 1.5
L of oxygen/minute.
Blood was collected from the cephalic vein using a 25 gauge winged infusion set (Miniset; Baxter Healthcare, Toongabbie, Australia) and 3-mL syringe (Terumo; Terumo Corporation, Binan, Philippines). Approximately 1 mL of blood was recovered at each venipuncture and dispensed into a 1.3-mL lithium-heparin micro tube (Sarstedt, Nümbrecht, Germany). Heparinised samples were maintained at room temperature for 5 minutes and then stored at 4°C until the end of sampling when they were centrifuged. To facilitate venipuncture, the forearm was shaved and a tourniquet applied at the elbow during sampling. Blood was collected from alternate arms and a cold pack was applied to the venipuncture site after each collection to minimise the risk of hematoma. Heparinized blood samples were centrifuged at 3000 x g for 10 minutes and plasma samples frozen at –20°C.
Experiment 1: Changes in Testosterone Secretion Over 24 Hours
In Experiment 1, each koala (K1–K6, n = 6) was bled at 0800, 1200,
1600, 2000, 0000, 0400, and again at 0800 hours. The objective in this
experiment was to obtain a measure of the variability of the prevailing
testosterone secretory status of individual koalas before undertaking
stimulation tests with a GnRH agonist and hCG.
Experiment 2: GnRH Agonist Stimulation Test
The GnRH agonist test was conducted over 2 days, in which the KSP koalas (n
= 3) were assessed on the first day and MKH koalas on the second day (n = 3).
After the initial blood sample (T0) each koala received a 4-µg injection IM
of the GnRH agonist buserelin (Receptal; Intervet, Bendigo, Australia). Other
blood samples were taken at 30, 60, 90, 120, 180, and 240 minutes after
injection of GnRH.
Experiment 3: hCG Stimulation Test
Experiment 3 was the same design as Experiment 2 except that 1000 IU human
chorionic gonadotrophin (Chorulon; Intervet) were injected IM. MKH koalas (n =
3) were bled on the first day and the KSP koalas (n = 3) on the second.
Hormone Analysis
Plasma samples were assayed for testosterone concentration using
SpectriaTestosterone RIA kits (Orion Diagnostica, Espoo, Finland). The assay
was validated for koala plasma by demonstrating parallelism between dilutions
of pooled plasma and the standard curve (F6, 28 = 1.87, P = .121).
The intraassay and interassay coefficients of variation were 5.3% (n = 20;
mean = 4.3 ng/mL) and 12.0% (n = 10; mean = 6.0 ng/mL), respectively. A
recovery of 97.5% was obtained when testosterone was added to a plasma sample
before assay. The detection limit of the assay was 0.07 ng/mL. All samples
were assayed in duplicate. The testosterone antibody cross reacted with
testosterone 100%, dihydrotestosterone 4.5% and <1% with all other steroids
tested (Spectria specifications).
Statistical Analyses
Changes in plasma concentrations of testosterone over 24 hours and after
injection of hCG or buserelin were analysed by a single-factor
repeated-measures ANOVA, with an ante-dependence error structure
(Wang and Goonewardene, 2004).
Analysis was carried out using the MIXED procedure in version 8.2; SAS (Cary,
NC). Following the analyses of the individual experiments, Spearman's rank
correlation coefficient (Conover,
1999) was used to test for association between the average
testosterone concentrations over 24 hours and the maximum observed
testosterone concentration in each of the stimulation experiments.
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Results |
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Changes in plasma testosterone concentrations of individual koalas after the injection of buserelin and hCG are depicted in Figures 2 and 3, respectively, and the data are summarised in the Table. Koala K6 was not included in the analyses, as this animal did not show testosterone concentrations above the sensitivity of the assay. Both hormones induced an increase (P < .05) in plasma testosterone, with maximum concentrations (buserelin, 5.8 ± 2.2 ng/mL; hCG, 7.6 ± 2.9 ng/mL) occurring at 90 and 120 minutes after the injection of buserelin and hCG, respectively. Testosterone tended to decline beyond 90 minutes after treatment with buserelin, but this was not significant. In contrast, maximal concentrations of testosterone were maintained for 240 minutes after the injection of hCG.
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[T]; where [T]
= [T]Tx – [T]T0) when calculated over all time
periods (time 30 to 240 minutes) after injection of hCG and GnRH was 4.69
± 0.52 ng/mL and 2.87 ± 0.53 ng/mL, respectively.
A Spearman's rank correlation revealed a significant association between the average testosterone concentration over 24 hours of all 6 koalas and the maximum observed testosterone concentration in the hCG stimulation experiment (r = 1.0; P < .01). There was also a positive correlation between the average testosterone concentration over 24 hours and the maximum observed testosterone concentration following the GnRH stimulation test (r = .772; P = .07), but this was not significant at P < .05.
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Discussion |
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There was no evidence in this study of a diurnal rhythm in testosterone
secretion either for individual koalas or when data were pooled for 6 animals.
It is possible that the failure to observe diurnal secretion of testosterone
may have been associated with the use of repeated anesthesia and/or a
consequence of the relatively long interval between blood sampling (4 hours)
used in this study. With regard to possible effects of repeated anesthesia,
this did not prevent the occurrence of large fluctuations in testosterone
which presumably would have been preceded by pulses of LH and therefore GnRH.
The random, large fluctuations in koala plasma testosterone concentrations
emphasized the importance of a practical stimulation test that would allow the
viable and reliable assessment of prevailing testosterone secretory
capacity.
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Both the GnRH agonist buserelin and hCG induced testosterone secretion in male koalas. The initial phase of the testosterone response curve was similar for buserelin and hCG, and near maximal testosterone concentrations typically occurred around 60 minutes after injection. There were positive relationships between average testosterone secretion during 24 hours and the maximal concentration achieved after injection of buserelin (P = .07) and hCG (P < .01). For example, Koala K5 had the highest testosterone concentrations during 24 hours and also showed the greatest testosterone response after the injection of both buserelin and hCG. Koalas K1 and K6, on the other hand, had low plasma testosterone during 24 hours, and Koala K1 showed the lowest testosterone responses to both buserelin and hCG, while Koala K6 did not show a response to either exogenous hormone. Koalas K2, K3, and K4 had intermediate concentrations of testosterone during 24 hours and showed intermediate responses to buserelin and hCG. It is concluded from these findings that a GnRH agonist and hCG can be used in a testosterone stimulation test to gain an accurate index of prevailing testosterone secretion for individual male koalas. The failure to induce a testosterone response with natural sequence GnRH in an earlier study in male koalas (McFarlane, 1990) may have been due to an insufficient dose to generate an adequate LH response and/or the rapid clearance of natural sequence GnRH from circulation. The testosterone response to buserelin after 90 minutes in this study may be explained by either continued downregulation of GnRH receptors or the relatively rapid clearance of buserelin from the circulation.
The testosterone response curves after the injection of buserelin and hCG showed subtle differences. After injection of buserelin, testosterone typically peaked around 60–90 minutes, and this was followed by a decline to 240 min. It is most likely that injection of buserelin induced an acute release of LH from the pituitary, after which the gonadotroph cells became refractory to further stimulation by buserelin. A classical desensitisation of the gonadotrophs occurs in response to both endogenous and exogenous natural sequence GnRH and also GnRH agonists (Conn et al, 1999). Following injection of buserelin, therefore, the Leydig cells would have been exposed to a transient peak of LH similar to that which normally occurs in males. In contrast, after injection of hCG, the Leydig cells would have been exposed to chronic stimulation by hCG, which has a relatively long half-life in circulation (Birken et al, 1999). The response to hCG indicated that the Leydig cells in koalas have the capacity to sustain elevated testosterone secretion when exposed to chronic trophic hormone stimulation.
In summary, the present study has demonstrated that a GnRH agonist and hCG can be used to obtain an accurate index of testosterone biosynthetic capacity in male koalas. A practical testosterone stimulation test would involve the injection of exogenous hormone and collection of a single blood sample between 60 and 90 minutes after injection. The removal of the need for multiple blood samples to determine testosterone status makes the testosterone stimulation test particularly applicable in longitudinal studies of reproductive function in male koalas. This will allow changes in reproductive function including behavior to be more accurately related to changes in testicular endocrine activity. In this regard, a relationship was reported between the testosterone response to GnRH and social status in the sugar glider, which is also a marsupial (Bradley and Stoddart, 1997).
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Blanshard W. Medicine and husbandry of koalas. In: Wildlife. Proceedings 233, University of Sydney Post Graduate Committee in Veterinary Science; 1994: 547 –623.
Bradley AJ, Stoddart DM. Plasma androgen, social position, and response to GnRH in the marsupial sugar glider Petaurus breviceps (Marsupialia: Petauridae). J Zool (Lond). 1997; 241: 579 –587.
Carrick FN and Cox RI. Testicular endocrinology of marsupials and monotremes. In: Calaby JH, Tyndale-Biscoe CH, eds. Reproduction and Evolution. Canberra: Australian Academy of Science, 1977 : 137–141.
Carrick FN, McFarlane JR, Bancroft BJ, Brown AS. Androgen secretion in the koala. In: Proceedings of the Lone Pine Koala Symposium 21–22 August 1981. Brisbane: Department of Veterinary Anatomy, University of Queensland and Lone Pine Koala Sanctuary; 1981; 5.
Cleva GM, Stone GM, Dickens RK. Variation in reproductive parameters in the captive male koala (Phascolarctos cinereus). Reprod Fertil Dev. 1994; 6: 713 –719.[CrossRef][Medline]
Conn PM, Jennes L, Janovick JA. GnRH (gonadotropin-releasing hormone). In: Knobil E, Neill JD, eds. Encyclopedia of Reproduction. Vol 2. Sydney: Academic Press, 1999; 464–477.
Conover WJ. Practical Nonparametric Statistics. 3rd ed. New York, NY: Wiley; 1999 .
Handasyde KA, McDonald IR, Than KA, Michaelides J, Martin RW. Reproductive hormones and reproduction in the koala. In: Lee Anthony K, Handasyde KA, Sanson GD, eds. Biology of the Koala. Chipping Norton, Australia: Surrey Beatty & Sons; 1990: 203 –210.
Kauschansky A, Dickerman Z, Phillips M, Weintrob N, Strich D. Use of GnRH agonist and human chorionic gonadotrophin tests for differentiating constitutional delayed puberty from gonadotropin deficiency in boys. Clinical Endocrinology. 2002; 56: 603 –607.[CrossRef][Medline]
McFarlane JR. Endocrine Factors Influencing Reproduction in Male Non-Eutherian Mammals [PhD thesis]. Brisbane, Australia: University of Queensland; 1990.
McGowan MR, Tribe A, Johnston SD, Carrick FN, Van Hensbroek L. The use of isoflurane to anaesthetise koalas (Phascolarctos cinereus). Aust Vet J. 1995; 72: 472 –473.[Medline]
Parlevliet JM, Bevers MM, van de Broek J, Colenbrander B. Effect of GnRH and hCG administration on plasma LH and testosterone concentrations in normal stallions, aged stallions and stallions with lack of libido. Vet Q. 2001;2: 84 –87.
Ptaszynska M. Compendium of Animal Reproduction. 7th ed. The Netherlands: Intervet International; 2002 .
Stocco DM. Testosterone biosynthesis. In: Knobil E and Neill JD, eds. Encyclopedia of Reproduction. Vol 4 . San Diego, California: Academic Press; 1999; 784–789.
Wang Z, Goonewardene LA. The use of MIXED models in the analysis of animal experiments with repeated measures data. Can J Anim Sci. 2004;84: 1 –11.
Wildt DE. Male reproduction: assessment, management, and control of fertility. In: Kleiman DG, Allen ME, Thompson KV, Lumpkin S, eds. Wild Animals in Captivity: Principles and Techniques. Chicago: University of Chicago Press; 1996: 429 –450.
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