Chronic treatment of female tammar wallabies with deslorelin implants during pouch life: effects on reproductive maturation.

The present study reports on attempts to delay puberty in a model marsupial species using the gonadotrophin-releasing hormone (GnRH) agonist deslorelin. Female tammar wallaby pouch young received deslorelin (5 mg) or placebo implants (n=8/group) when they were 193±2 days old. Sexual maturity was significantly delayed in deslorelin-treated animals, with the first successful production of offspring in treated and control animals occurring at 813±62 and 430±42 days of age, respectively. This delay was associated with a period of retarded pouch and teat development. Progesterone concentrations remained at basal levels throughout the first breeding season, indicating the absence of luteal cycles in treated females. Recovery and maturation of the hypothalamic-pituitary axis was a gradual process. Treated animals failed to respond to GnRH challenge at 12 months of age and had a reduced LH response at 18 months of age, before attaining full responsiveness by 24 months of age. Despite this apparent pituitary recovery by 24 months of age, as evidenced by complete teat eversion and LH responsiveness to GnRH, the time to first parturition was significantly delayed beyond this time in three females. This suggests that there may be longer-lasting effects at the level of the ovary and/or on FSH secretion. The significant delay in the onset of sexual maturation in response to chronic GnRH agonist treatment in this model marsupial species may be of practical significance to the management of fertility in captive and semi-free range marsupial populations.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.

Retrospective study of association between choice of vasopressor given during spinal anaesthesia for high-risk caesarean delivery and fetal pH.

BACKGROUND: Phenylephrine given during spinal anaesthesia for low-risk caesarean section is associated with higher fetal pH than ephedrine. However, there is little evidence on the effects of ephedrine and phenylephrine in complicated pregnancies. The aim of this study was to compare umbilical artery pH with phenylephrine and ephedrine given during spinal anaesthesia where caesarean section was performed because of an increased risk of fetal compromise. METHODS: We reviewed the case notes of all women at our hospital from 2000-2003 who had undergone high-risk caesarean section under spinal anaesthesia, where umbilical artery and venous pH had been recorded at delivery. Umbilical artery pH was compared by choice of vasopressor and multiple regression analysis was used to investigate the effects of other possible confounding variables. RESULTS: One hundred and fifteen patients received no vasopressor, 122 ephedrine (group E) and 148 phenylephrine (group P). The median umbilical artery pH was 7.26 (IQR 7.21-7.30) for the no-vasopressor group, 7.27 (7.22-7.30) for group E and 7.28 (7.22-7.32) for group P (P=0.21). Using multiple regression analysis, the only variable associated with altered umbilical artery pH was a non-reassuring fetal heart trace. CONCLUSIONS: Umbilical artery pH was similar whether ephedrine or phenylephrine was used to maintain maternal arterial pressure, which contrasts with studies of low-risk caesarean section.

Molecular detection of hybridization between sympatric kangaroo species in south-eastern Australia.

Introgressive hybridization has traditionally been regarded as rare in many vertebrate groups, including mammals. Despite a propensity to hybridize in captivity, introgression has rarely been reported between wild sympatric macropodid marsupials. Here we investigate sympatric populations of western (Macropus fuliginosus) and eastern (Macropus giganteus) grey kangaroos through 12 autosomal microsatellite loci and 626 bp of the hypervariable mitochondrial DNA (mtDNA) control region. M. fuliginosus and M. giganteus within the region of sympatry corresponded, both genetically and morphologically, to their respective species elsewhere in their distributions. Of the 223 grey kangaroos examined, 7.6% displayed evidence of introgression, although no F1 hybrids were detected. In contrast to captive studies, there was no evidence for unidirectional hybridization in sympatric grey kangaroos. However, a higher portion of M. giganteus backcrosses existed within the sample compared with M. fuliginosus. Hybridization in grey kangaroos is reflective of occasional breakdowns in species boundaries, occurring throughout the region and potentially associated with variable conditions and dramatic reductions in densities. Such rare hybridization events allow populations to incorporate novel diversity while still retaining species integrity.

Effect of intravenous vasopressor on spread of spinal anaesthesia and fetal acid-base equilibrium.

BACKGROUND: We previously found rostral spread of spinal plain levobupivacaine to be less with prophylactic i.v. phenylephrine than with ephedrine during Caesarean delivery. This study investigated whether rostral spread of spinal hyperbaric bupivacaine is also less with phenylephrine than with ephedrine. METHODS: The study was randomized and double blind. It compared phenylephrine 100 microg ml-1 (phenylephrine group, n=27), and ephedrine 4.5 mg ml-1 (ephedrine group, n=27), given by infusion during spinal anaesthesia for Caesarean delivery. Block height was assessed to cold and light touch sensation at 15, 30, 60, and 90-min after the spinal injection of 2.8 ml of hyperbaric 0.5% w/v bupivacaine, combined with 0.4 ml diamorphine (1 mg ml-1). Umbilical blood gas values were monitored during the study. RESULTS: Block height was similar for both groups at all of the assessment times. Umbilical artery pH was higher with phenylephrine [median 7.32 (IQR 7.28-7.34)] than with ephedrine [7.20 (7.10-7.28)] (P<0.0001). There was a strong negative correlation between umbilical artery pH and spinal-delivery interval, but only with ephedrine: phenylephrine group, r2=0.09 (P=0.17), and ephedrine group, r2=0.53 (P<0.0001). Five-minute Apgar scores were higher with phenylephrine [10 (9-10)] than ephedrine [9 (9-9)] (P=0.009). CONCLUSIONS: In contrast to its effect on spinal plain levobupivacaine, we did not find rostral spread of spinal hyperbaric bupivacaine to be less with prophylactic phenylephrine than with ephedrine. We observed an unexpectedly high incidence of fetal acidosis with ephedrine and found evidence that longer spinal-delivery intervals increase the risk of fetal acidosis developing with ephedrine, but not phenylephrine.

Chronic treatment of male tammar wallabies with deslorelin implants during pouch life: effects on development, puberty, and reproduction in adulthood.

The present study evaluated the effects of chronic GnRH agonist (deslorelin) treatment on sexual maturation in the male tammar wallaby. Slow-release deslorelin or placebo implants were administered to male pouch young (n = 10/group) when they were between 180 and 200 days old, to determine if disruption of the pituitary-testicular axis during development altered the timing of sexual maturation or had long-term effects on adult reproductive function. Deslorelin treatment caused retardation of testicular growth and reduced the serum FSH and testosterone concentrations between 12 and 24 mo of age. Maturation of the hypothalamic-pituitary-testicular axis was also delayed in treated animals at 13 and 19 mo of age. Despite these alterations in the pattern and timing of neuroendocrine development, sexual maturation was not permanently blocked in these animals and deslorelin-treated animals reached sexual maturity at the same age as treated animals, as evidenced by a fully functional pituitary-testicular axis and proven fertility at 25 mo of age. The ability of the treated animals to reach puberty at the same time as control animals, despite delayed maturation of the hypothalamic-pituitary-testicular axis, suggests that puberty in the male tammar wallaby is additionally regulated by other, gonadotropin-independent factors.

Detection of CAG repeats in pre-eclampsia/eclampsia using the repeat expansion detection method.

Pre-eclampsia/eclampsia is a serious disorder of human pregnancy that has a worldwide incidence of 2-10% and carries a severe morbidity and mortality risk for both mother and child. Its precise cause remains unknown. However, there is increasing evidence of an underlying complex maternal genetic susceptibility. Its high population incidence in the face of strong negative selection pressure suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. Thus, the aim of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia/eclampsia. We have used the repeat expansion detection (RED) method, which was developed to directly identify clinically significant repeat expansions, to analyse genomic DNA from an Australian and New Zealand population. The maximal CAG repeat length for each individual was recorded and the Mann-Whitney U and Wilcoxon rank sum test for independent samples were used to compare distributions for CAG/CTG repeats between two populations. There were no statistically significant differences between the distribution of CAG repeats in normotensive (n = 59) and severe pre-eclampsia (n = 69) (Mann-Whitney U = 1732; P = 0.14), and normotensive (n = 59) and eclamptic (n = 15) populations (Mann-Whitney U = 417, P = 0.726). Therefore, these RED results do not support a role for a large CAG expansion in pre-eclampsia/eclampsia. However, these data do not preclude the possibility that a small CAG expansion is associated with the disorder nor do they negate the hypothesis that a highly mutable gene contributes to the genetic component of pre-eclampsia/eclampsia.

Long-term effects of deslorelin implants on reproduction in the female tammar wallaby (Macropus eugenii).

The contraceptive and endocrine effects of long-term treatment with implants containing the GnRH agonist deslorelin were investigated in female tammar wallabies (Macropus eugenii). Fertility was successfully inhibited for 515 +/- 87 days after treatment with a 5 mg deslorelin implant (n = 7), while control animals gave birth to their first young 159 +/- 47 days after placebo implant administration (n = 8). The duration of contraception was highly variable, ranging from 344 to 761 days. The strict reproductive seasonality in the tammar wallaby was maintained once the implant had expired. This inhibition of reproduction was associated with a significant reduction in basal LH concentrations and a cessation of oestrous cycles, as evidenced by low progesterone concentrations. There was evidence to suggest that some aspect of either blastocyst survival, luteal reactivation, pregnancy or birth may be affected by deslorelin treatment in some animals. These results show that long-term inhibition of fertility in the female tammar wallaby is possible using slow-release deslorelin implants. The effects of deslorelin treatment were fully reversible and there was no evidence of negative side effects. Slow-release GnRH agonist implants may represent a practicable method for reproductive management of captive and semi-wild populations of marsupials.

High microsatellite diversity and differential structuring among populations of the introduced common brushtail possum, Trichosurus vulpecula, in New Zealand.

An understanding of genetic variation and structure of pest populations has the potential to improve the efficiency of measures to control them. Genetic analysis was undertaken at five microsatellite loci in four native Australian and 14 introduced New Zealand populations of the common brushtail possum Trichosurus vulpecula in order to document these parameters. Genetic variation in New Zealand populations, and phylogenetic relationships among Australian and New Zealand populations, were largely predicted by the recorded introduction history. Populations on the two main islands of New Zealand had only slightly lower genetic diversity than did Australian populations, except that allelic richness on the South Is. was significantly lower. Diversity was higher in North Is. than in South Is. populations (although not significantly so) and mainland New Zealand populations as a group were significantly more diverse than offshore islands that represented secondary population size bottlenecks. In phylogenetic analyses South Is. and offshore island populations grouped with Tasmania, while North Is. populations grouped either with mainland Australia or were intermediate between the two Australian sources. This scheme was supported by admixture coefficients showing that North and South Is./offshore island populations were largely mainland Australian and Tasmanian in origin, respectively. Population structure differed markedly between the North and South Islands: populations were typically more genetically differentiated on the former than the latter, which also showed significant isolation-by-distance. Substantial linkage disequilibrium in most sampled New Zealand but no Australian population between microsatellite loci Tv16 and Tv27 suggests they may be physically linked.

Effect of deslorelin implants on follicular development, parturition and post-partum oestrus in the tammar wallaby (Macropus eugenii).

The effect of treatment with slow release implants containing the GnRH agonist, deslorelin, was investigated in female tammar wallabies. Pouch young were removed from 16 wallabies presumed to be carrying quiescent blastocysts. Eight received a 5 mg deslorelin implant and eight received a placebo implant. Animals were caught daily from day 25 to day 30 and their pouches inspected for newborn young and their urogenital sinus checked for a copulatory plug. Treatment with deslorelin did not affect reactivation of a dormant blastocyst and subsequent birth in 4/8 animals, but post-partum mating was inhibited in these animals. Five control and five treated animals were killed within 0-48 h post partum and their reproductive tracts analysed. At autopsy, all five control animals had large preovulatory follicles but only one deslorelin-treated animal showed signs of follicular development. These differences were also reflected in the weights of the lateral vaginae, with treated animals showing no evidence of oestrogenic stimulation. The remaining three control and three treated animals were monitored for approximately 2 years. The long-term contraceptive effects of a single 5 mg deslorelin implant lasted for just under one year. These results indicate that slow release deslorelin implants inhibit follicular development in the female tammar wallaby for extended periods of time and may have potential application in reproductive management of captive marsupials in the kangaroo family.

Effects of a gonadotropin-releasing hormone agonist implant on reproduction in a male marsupial, Macropus eugenii.

This study evaluated the potential of slow-release GnRH agonist (deslorelin) implants to inhibit reproductive function in the male tammar wallaby. The specific aim was to measure the effects of graded dosages of deslorelin on testes size and plasma LH and testosterone concentrations. Adult male tammar wallabies were assigned to four groups (n = 6 per group) and received the following treatment: control, placebo implant; low dose, 5 mg deslorelin; medium dose, 10 mg; high dose, 20 mg. All dosages of deslorelin induced acute increases (P < 0.001) in plasma LH and testosterone concentrations within 2 h, with concentrations remaining elevated during the first 24 h but returning to pretreatment levels by Day 7. Thereafter, there was no evidence of a treatment-induced decline in plasma testosterone concentrations. There was no detectable difference in basal LH concentrations between treated and control animals, nor was there a significant change in testes width or length (P > 0.05). These results suggest that the male tammar wallaby is resistant to the contraceptive effects of chronic GnRH agonist treatment. Despite the maintenance of testosterone secretion, the majority of male tammars (10 of 17) failed to respond to a GnRH challenge with a release of LH between Days 186 and 197 of treatment. The failure of animals to respond to exogenous GnRH suggests a direct effect of deslorelin on the pituitary, resulting in a level of desensitization that was sufficient to inhibit a LH surge but insufficient to inhibit basal LH secretion. The variation between animals is believed to result from earlier recovery of some individuals, in particular those that received a lower dose, or individual resistance to the desensitization process.

Characterisation of and immunity to the aerobic bacteria found in the pouch of the brushtail possum Trichosurus vulpecula.

The bacterial composition of the brustail possum (Trichosurus vulpecula) pouch was characterized throughout the reproductive cycle using brushtails from an Australian captive breeding colony (45 swabs) and a wild population in New Zealand (26 swabs). Gram-positive coccal species predominate throughout the reproductive cycle. Enteric Gram-negative rods, particularly Escherichia coli, were most prevalent when a pouch young was present and was most likely the result of faecal contamination from the pouch young. As culturing is only able to detect a proportion of bacteria present in a particular environment, molecular 16S rDNA sequencing was carried out on DNA extracted from a pouch wash of a female carrying a pouch young to gain a more accurate assessment of the pouch microflora. This approach identified approximately five times the number of bacterial species when compared to culture results. The majority detected were Gram negative rods or most closely related to Gram-negative rods species. Brushtails are immunologically immature at birth yet survive in a pouch colonised with potentially pathogenic bacteria. A haemagglutination assay was used to determine whether antibodies to a frequently isolated bacterium (Klebsiella pneumoniae) were transferred via milk from mother to pouch young. IgG antibodies were detected in maternal serum, milk and pouch young serum. In young over 70 days, antibody titres were significantly higher than those found in maternal serum, suggesting that the young is capable of producing adult type antibodies to pouch bacteria at this time.

Fine mapping and SNP analysis of positional candidates at the preeclampsia susceptibility locus (PREG1) on chromosome 2.

Genome scans in Icelandic, Australian and New Zealand, and Finnish families have localized putative susceptibility loci for preeclampsia/ eclampsia to chromosome 2. The locus mapped in the Australian and New Zealand study (designated PREG1) was thought to be the same locus as that identified in the Icelandic study. In both these studies, two distinct quantitative trait locus (QTL) regions were evident on chromosome 2. Here, we describe our fine mapping of the PREG1 locus and a genetic analysis of two positional candidate genes. Twenty-five additional microsatellite markers were genotyped within the 74-cM linkage region defined by the combined Icelandic and Australian and New Zealand genome scans. The overall position and shape of the localization evidence obtained using nonparametric multipoint analysis did not change from that seen previously in our 10-cM resolution genome scan; two peaks were displayed, one on chromosome 2p at marker D2S388 (107.46 cM) and the other on chromosome 2q at 151.5 cM at marker D2S2313. Using the robust two-point linkage analysis implemented in the Analyze program, all 25 markers gave positive LOD scores with significant evidence of linkage being seen at marker D2S2313 (151.5 cM), achieving a LOD score of 3.37 under a strict diagnostic model. Suggestive evidence of linkage was seen at marker D2S388 (107.46 cM) with a LOD score of 2.22 under the general diagnostic model. Two candidate genes beneath the peak on chromosome 2p were selected for further analysis using public single nucleotide polymorphisms (SNPs) within these genes. Maximum LOD scores were obtained for an SNP in TACR1 (LOD = 3.5) and for an SNP in TCF7L1 (LOD = 3.33), both achieving genome-wide significance. However, no evidence of association was seen with any of the markers tested. These data strongly support the presence of a susceptibility gene on chromosome 2p11-12 and substantiate the possibility of a second locus on chromosome 2q23.

Intraspecific variation, sex-biased dispersal and phylogeography of the eastern grey kangaroo (Macropus giganteus).

Genetic information has played an important role in the development of management units by focusing attention on the evolutionary properties and genetics of populations. Wildlife authorities cannot hope to manage species effectively without knowledge of geographical boundaries and demic structure. The present investigation provides an analysis of mitochondrial DNA and microsatellite data, which is used to infer both historical and contemporary patterns of population structuring and dispersal in the eastern grey kangaroo (Macropus giganteus) in Australia. The average level of genetic variation across sample locations was one of the highest observed for marsupials (h=0.95, HE=0.82). Contrary to ecological studies, both genic and genotypic analyses reveal weak genetic structure of populations, where high levels of dispersal may be inferred up to 230 km. The movement of individuals was predominantly male-biased (average Nem=22.61, average Nfm=2.73). However, neither sex showed significant isolation by distance. On a continental scale, there was strong genetic differentiation and phylogeographic distinction between southern (TAS, VIC and NSW) and northern (QLD) populations, indicating a current and/or historical restriction of gene flow. In addition, it is evident that northern populations are historically more recent, and were derived from a small number of southern founders. Phylogenetic comparisons between M. g. giganteus and M. g. tasmaniensis indicated that the current taxonomic status of these subspecies should be revised as there was a lack of genetic differentiation between the populations sampled.