Cross-fostering is the rearing of young by a surrogate mother of a different taxon. In marsupials this technique has been used to study lactation as well as pouch young growth and development. Recently, cross-fostering and short-term pouch young isolation have been used by wildlife agencies and zoos to increase female reproductive rates and fecundity, and to manage the genetics of captive and wild animals as part of wider conservation and management strategies for macropodoid marsupials. Data on cross-fostering are now available for six potoroid and 13 macropodid species. Success of cross-fostering studies between donor and foster species varies widely from complete failure to an eight-fold increase in the production of pouch young annually. Factors implicated in successful cross-fostering include relative size of donor and surrogate females, size of pouch young at weaning, differences in length of pouch life between species and size differences between donor young and those of the surrogate species at transfer. Studies have shown that females regulate milk composition and production irrespective of pouch young age, and that transfer of donor young to species with more immature or advanced mammary glands will result in a slowing or an acceleration of pouch young growth and development, and affect the duration of pouch life. Small pouch young tolerate short-term isolation from the pouch at a range of temperatures, provided high humidity is maintained throughout the period of isolation. Maintenance of pouch young at temperatures lower than those that occur in the pouch (23 degrees C compared with 37 degrees C) during isolation reduces the pouch young's basal metabolic rate, oxygen consumption and evaporative water loss and thus improves survival rates of very small pouch young. The success of these techniques in managing population genetics and accelerating breeding in donor species within the Macropodoidea are enhanced by post-partum oestrus and mating after the removal of pouch young, and the reactivation and birth of the diapausing embryo. This chapter reviews the available data on cross-fostering and pouch young isolation in macropodoid marsupials, and examines use of these techniques in the conservation and management of species in this group.

Gilbert's potoroo (Potorous gilbertii) was rediscovered in 1994 after having been presumed extinct for 120 years. Estimates indicate fewer than 40 individuals remain at Two Peoples Bay Nature Reserve on the south coast of Western Australia although a translocated population of approximately 20 animals has recently been established on nearby Bald Island. A captive breeding facility has been established adjacent to the mainland population but few young have been produced (8 since 1995). Faecal levels of oestradiol-17 beta (E(2)) were monitored over a 2-year period in an effort to determine cyclic reproductive activity, and faecal cortisol levels were also monitored to gauge whether chronic stress may be a factor limiting breeding in captivity.Faecal steroids were monitored in six captive females, and four captive male potoroos, and four wild females. The only captive births recorded after 1998 were one in August 1999 and one in February 2001, both to the same female. Peaks in E(2) concentration, up to 10 ng g(-1) of dried faecal mass were measured and results to date suggest the main breeding period to be November-December based on elevated E(2) levels at this time. Clear patterns of reproductive activity in the captive females, however, were not evident. Analysis of epithelial cell counts from urinogenital swabs and faecal E(2) and progestagen (PM) levels from a single female kept at the Perth Zoo, suggest that Gilbert's potoroo has an oestrous cycle of approximately 39 days. Faecal cortisol levels in captive females were significantly lower than those in wild-caught individuals and thus there is no indication that elevated cortisol levels per se inhibited reproduction in captive females. (C) 2009 Elsevier Inc. All rights reserved.

Growth hormone is essential for post-natal growth and its actions are triggered by the upregulation of hepatic growth hormone receptors (GHRs) after birth in eutherian mammals. In utero, fetal growth is largely driven by the availability of nutrients that control the relative levels of insulin and insulin-like growth factors (IGFs). This study examined expression of hepatic GHR, IGF-1, IGF-2 and IGF binding protein-3 (IGFBP-3) from late fetal stages of pregnancy through pouch life to weaning using the tammar wallaby. The advantage of the tammar wallaby is that it gives birth to an altricial young that is only 0.1% of adult female size, essentially an exteriorized fetus. After 9 months of pouch life the young exits the pouch, a time equivalent to birth in precocial eutherian mammals. We cloned GHR, IGF-1, IGF-2 and IGFBP-3 using RT-PCR and used quantitative PCR to determine the relative change in expression of these genes in male and female fetuses one day before birth and young at 15, 45, 70, 100, 150 and 250 days (d) post-partum (pp) and adults (n=7-10 per stage). There was significant protein homology between tammar genes and those of mouse and man (62 and 68% for GHR, 79 and 85% for IGF-1, 63 and 65% for IGF-2, 69 and 71% for IGFBP-3 respectively). Hepatic GHR and IGF-1 expression increased gradually in both males and females from birth to d150pp, after which levels reached a plateau (ANOVA P<0.0001).IGFBP-3 and IGF-2 expression rose to a peak about d70-100pp then fell at later ages of lactation and in adults, with generally lower levels in females than males (ANOVA P<0.01 for age and P<0.05 for sex). The age-related changes in expression of these genes probably reflect a conserved role in development, while the sex-specific differences may correlate with some of the growth and developmental differences, observed between males and females especially in the phallus.

Growth hormone receptor (GH-R) plays a critical role in the control of growth and metabolism in all vertebrates. GH-R consists of 9 coding exons (2–10) in all eutherian mammals, while the chicken only has 8 coding exons, and does not have an orthologous region to eutherian exon 3. To further understand the evolutionary origins of exon 3 of the GH-R we have cloned the full-length GH-R sequence in a marsupial, the tammar wallaby to determine whether exon 3 was present or absent in marsupial liver cDNA. There was no evidence for the presence of an exon 3 containing mRNA in sequence of tammar pouch young and adult livers. We next examined the genomes of the platypus (a monotreme mammal) and the grey short-tailed opossum (another marsupial). Like the tammar, the GH-R gene of neither species contained an exon 3. GH receptor can obviously function in the absence of this exon, raising speculation about the function of this domain, if any, in eutherians. A comparison of exon 3 protein sequences within 16 species of eutherian mammals showed that there was ~75% homology in the domain despite only 3 residues being identical (Leu12, Gln13 and Pro17). Interestingly, we detected greater evolutionary divergence in exon 3 sequences from species that have variants of GH or prolactin (PRL) in their placentas. These data show that exon 3 was inserted into the GH-R after the divergence of marsupial and eutherian lineages at least 130 million years ago.

Lactation is a defining feature of mammals. The production of milk by the mammary gland during lactation is generally regarded as a mechanism required to supply nutrition to the neonate before it is able to digest other types of food. Increasing evidence suggests that components of milk have additional functions in addition to nutrition. Monotremes, marsupials and eutherians are all members of the class mammalia, but monotremes and marsupials have evolved a lactation pattern that is very different from eutherians. In eutherians, the gestation period is long relative to its lactation period. However, in marsupials and monotremes the gestation period is relatively short. As a result, the newborn is relatively small and undeveloped and most of the early development occurs postnatally. Eutherians produce milk of a constant composition after the expression of the initial colostrum, whereas marsupials such as the tammar wallaby (Macropus eugenii), have a short gestation, and give birth to a highly immature young and then commence a long lactation during which the milk production and composition progressively change in composition to suit the needs of the developing young. The comparative study of monotreme, marsupial and eutherian milk is of significant interest and may reveal bioactives required for developmental processes. These factors may have been either lost, down-regulated or altered/modified in eutherians since most development of eutherian young occurs in utero with nutrition and developmental signaling provided by the amniotic fluid and supplemented from the mother via the placenta.

The growth and timing of female puberty in a seasonally breeding marsupial, the tammar wallaby, was examined in wild and captive animals. Puberty, defined as the time of first estrus and ovulation, can occur at any time of the year. Sixty percent of young wild females went through puberty in late October-November, 3 mo before the normal adult mating season in late January-February, but puberty was delayed in captive animals kept with a low ratio of males to females. During initial cycles, 19% of these captive animals were infertile as judged by failure to conceive. In the wild, puberty occurred well before the animals were fully grown (body weight 2.0 +/- 0.3 kg [mean +/- SD], n = 23; adult females, 4.7 +/- 0.6 kg; n = 34). Only 3% of animals with a body weight below 1.5 kg had ovulated. Thus, attainment of a minimum body weight was a key prerequisite associated with puberty. Progesterone concentrations in the peripheral plasma of prepubertal females were not significantly different from those of adult females during the nonbreeding season (prepubertal, 142 +/- 121 pg/ml, n = 34; adult, 194 +/- 105 pg/ml, n = 32, p > 0.05). However, there was a significant increase in progesterone (322 +/- 242 pg/ml, n = 32, p < 0.05) in the postpubertal females (ovulating but still < 3.5 kg body weight) even though the corpus luteum was quiescent after its formation. There was no increase in plasma progesterone before the first estrus. These data confirm that estrus does not require a change in the progesterone:estradiol ratio, and that a "silent" ovulation does not precede the first estrus in this species, so that the onset of puberty coincides with the first behavioral estrus and ovulation, when the animals have reached a body weight of 2 kg. Although adult female tammars are strict seasonal breeders, with 6 mo of seasonal quiescence from the winter to the summer solstice, young females can go through puberty at any time of the year. The unique feature of the female tammar wallaby is that it does not become a seasonally breeding mammal until after puberty, when it has acquired a corpus luteum.

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1.8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose-response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus, Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum, Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

Oestrus and ovulation occur spontaneously in the majority of marsupials, with behavioural oestrus usually occurring 1-2 days before ovulation. The hormone changes that occur at this time have been described in the most detail for the monovular tammar wallaby Macropus eugenii. The respective roles of the Graafian follicle, corpus luteum and the pituitary in the events leading up to oestrus and ovulation in this species are also reviewed. Recently, various protocols have been developed for superovulation of marsupials, including Australian species, such as the brush-tailed possum, fat-tailed dunnart, brush-tailed bettong and tammar wallaby, and the American laboratory opossum, Monodelphis domestica. These protocols provide an opportunity for studying the regulation of ovarian activity and for the collection of larger quantities of material for the study of gamete maturation, in vitro fertilization and embryonic development.

The oxytocic peptide mesotocin was measured in plasma samples collected throughout pregnancy in the conscious tammar wallaby, Macropus eugenii. Plasma mesotocin and the prostaglandin metabolite 13,14-dihydro-15-oxo-prostaglandin F2 alpha were also assessed immediately prepartum and during parturition. A radioimmunoassay for mesotocin was validated in the tammar and this assay allowed direct measurement in 50 microliters unextracted plasma with a sensitivity of 12.5 pmol l-1. Plasma concentrations of mesotocin remained basal (approximately 15 pmol l-1) at all stages of pregnancy, including prepartum. A significant (P < 0.05) increase in plasma mesotocin was observed only immediately after delivery of the neonate and this increase was maintained for at least 15 min postpartum. Mesotocin concentrations returned to basal values 2 h after birth. Peak concentrations of mesotocin of 516.7 +/- 108.1 pmol l-1 were measured within 2 min of birth. This peak coincided with a short-lived peak in concentration of prostaglandin F2 alpha metabolite immediately after birth (2.1 +/- 0.4 nmol l-1) which decreased to less than 0.3 nmol l-1 within 2 h postpartum. These data demonstrate that mesotocin is released during, or immediately after, delivery and appears to parallel the profile of circulating prostaglandin F2 alpha metabolite in this marsupial.

Dexamethasone treatment induces premature birth in tammar wallabies. Treatment was administered at one of three times between 1200 h on Day 24 and 0930 on Day 25, and birth occurred 22.8 +/- 0.5 h later, significantly earlier than the time of birth for controls, which was 47.7 +/- 2.3 h after treatment. The neonates from treated females were significantly lighter than control neonates (360 +/- 9 vs. 413 +/- 5 mg), and 60% of these died within 12 h of birth, suggesting that premature birth can lead to neonatal mortality. None of the control neonates died. The patterns of secretion of prolactin, prostaglandin F2 alpha-metabolite (PGFM), and progesterone of control and treated animals around the time of birth were similar. A transient pulse of PGFM was coincident with birth while prolactin levels in plasma increased before, and progesterone concentrations fell steeply immediately after, parturition in both groups of animals. The only difference between control and treated animals was in the timing of the hormonal events, which, along with birth, was significantly advanced by the treatment. We conclude that cortisol may play a role in triggering parturition in this marsupial species.

Unlike eutherian mammals which secrete only oxytocin (OT), marsupials secrete the typically reptilian mesotocin (MT) and/or OT as their oxytocic peptides. Our laboratories have been conducting research on various aspects of the roles of OT-like peptides in three marsupials, the brushtail possum, the northern brown bandicoot and the tammar wallaby. By providing information on the functions of OT-like peptides in these species we hope to provide some clues as to the evolution of neurohypophysial hormones in marsupials. Brain and peripheral distributions of OT-like peptides have been studied in the possum and bandicoot. As in eutherian mammals, OT-like peptides are distributed throughout the brain and are present in the testis, corpus luteum, prostate and adrenal glands. Studied on the regulation of release of MT into plasma in the possum show that it is regulated by similar mechanisms to OT release in eutherian mammals. OT receptors have been characterized and localized in the possum and the tissue distributions and pharmacological characteristics of the receptor are similar to both the sheep and rat OT receptors. The marsupial OT receptor shows no pharmacological specificity for MT over OT which is reflected in the similar potency of these peptides in eliciting contractions of the uterus of the tammar wallaby in vitro. MT seems to play an important but not essential role in parturition in the tammar. MT concentrations are increased immediately after delivery in the tammar but infusion of an OT antagonist before expected birth delays but does not prevent parturition. The presence of OT receptors in the marsupial mammary gland and the sensitivity of the gland to exogenous OT and stimulation of mesotocinergic neurones demonstrates that these peptides are important for marsupial lactation. Our data suggest that the presence of MT with or without OT in marsupials is a result of a neutral mutation rather than functional evolution.

Female tammar wallabies were treated prepartum with the prostaglandin synthase inhibitor indomethacin, with or without the dopamine agonist bromocriptine, to suppress the peripartum pulses of plasma prostaglandin and prolactin. The animals were observed continuously to detect birth, and a series of blood samples taken to define the hormonal profiles before and immediately after parturition. Birth was observed in ten of twelve control animals but not in the six animals treated with indomethacin alone or the six animals treated with indomethacin and bromocriptine. Indomethacin disrupted the normal profile of PGF2 alpha metabolite 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) concentrations, and in the females treated with bromocriptine plus indomethacin the pulse of prolactin normally seen at parturition was completely abolished. Plasma progesterone concentrations fell slowly in treated animals, whereas in control animals they fell steeply immediately after parturition. Postpartum oestrus was delayed or absent in treated and most control animals, suggesting that the frequent blood sampling and disturbances in the peripartum period interfered with these endocrine processes. We conclude that prostaglandin is essential for normal birth. Prolactin, in the apparent absence of a prostaglandin peak, does not induce birth or rapid luteolysis. Prostaglandin release may synchronize the rapid fall in progesterone concentrations associated with birth, but in the absence of this signal, the corpus luteum undergoes a less rapid, autonomous decline.

In Exp. 1 non-pregnant female tammars were injected, on Day 26 (the day parturition would normally occur) after removal of pouch young, with saline, 200 micrograms ovine prolactin or 5 mg PG and changes in plasma concentrations of progesterone, prolactin, PGF-2 alpha metabolite (PGFM), oestradiol-17 beta and LH were determined. Luteolysis occurred in females treated with prolactin alone, while treatment with PG first induced a rapid rise in prolactin and subsequently a significant decrease in plasma progesterone. After prolactin treatment the oestradiol peak, oestrus and the LH surge were advanced significantly compared to the saline-treated females. In Exp. 2 the effects of the same treatments as used in Exp. 1 were determined on Day 23 and again on Day 26 after removal of pouch young in non-pregnant females. On Day 23 both prolactin and PG induced significant elevations in plasma progesterone, but luteolysis did not occur. On Day 26 the treatments initially induced significant elevations in plasma progesterone but these were followed by luteolysis within 8-12 h after treatment. PG treatment induced parturient behaviour in the non-pregnant females within 3-21 min and this persisted during the period that plasma concentrations of PGFM were elevated. The results show that PG induces birth behaviour and the release of prolactin, while prolactin first induces an elevation of plasma progesterone concentrations and, in the mature CL on Day 26, subsequently induces luteolysis.

Female tammar wallabies were treated with the dopamine agonist bromocriptine at the end of pregnancy to suppress the peripartum pulse of plasma prolactin. The animals were subsequently observed, and a series of blood samples taken to define the hormonal profiles before and immediately after parturition. Birth was observed in 4/5 control animals and occurred in 8/9 bromocriptine-treated animals. The peripartum peak in plasma PGFM concentrations was not affected by bromocriptine although the pulse of prolactin normally seen at parturition was completely abolished. The timing of luteolysis was apparently unaffected, as plasma progesterone concentrations fell similarly in both treated and control animals immediately after parturition. However, all of the neonates of the bromocriptine-treated animals died within 24 h, possibly because of a failure to establish lactation. Subsequent onset of post-partum oestrus was delayed or absent both in control and in bromocriptine-treated animals, suggesting that the frequent blood sampling and disturbances in the peripartum period interfered with these endocrine processes. It is concluded that both prolactin and prostaglandin can induce luteolysis in the pregnant wallaby, but that the normal sequence of events results from a signal of fetal origin inducing a prostaglandin release from the uterus, which in turn releases a pulse of prolactin that induces a progesterone decline.

Testicular growth and maturation of the hypothalamic-pituitary-testicular axis were assessed in male tammars from 12 to 25 months of age to establish the time of sexual maturity. The testicular dimensions and body weights of 20 male tammars, approximately 12 months of age at the beginning of the study, were measured monthly for 1 year. Groups of 3 animals were castrated at 13, 19 and 25 months of age and their testes sectioned for histological examination. Testicular volume increased between 12 and 24 months of age and was highly correlated with body weight (r = 0.91). In the 13-month group the seminiferous tubules were closed with few mitotic figures. Spermatogenesis had begun in 2 of the 19-month animals. All stages of spermatogenesis were present in the other 19-month male, and in all of the 25-month males. Basal FSH concentrations increased with the age of the animal (21.0 +/- 32.48, 94.40 +/- 55.18 and 193.05 +/- 40.21 ng/ml (mean +/- s.d.) at 19, 20 and 25 months respectively) while basal LH concentrations were similar at 20 months and 25 months (0.43 +/- 0.18 and 0.58 +/- 0.25 ng/ml respectively). Basal testosterone concentrations were also similar 0.11 +/- 0.04, 0.35 +/- 0.16 and 0.22 +/- 0.10 ng/ml in 13-, 19- and 25-month-old animals. LHRH injection in tammars at 13, 19 and 25 months of age induced release of both LH and testosterone 10-30 min after injection. The hormone concentrations increased in both magnitude and duration with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)

A heterologous double antibody radioimmunoassay has been validated for measurement of LH in plasma of a dasyurid marsupial. Basal concentrations in the oestrous cycle of female kowaris were in the range of 0.5-2.0 ng NIH-oLH-S19 mL-1. Many animals showed elevated LH concentrations (3.0-12.0 ng mL-1) between 8 and 15 days before oestrous but no pre-ovulatory surge was detected. Gonadectomy resulted in greatly increased concentrations of LH, and decreases in testosterone and progesterone concentrations in male and female respectively. In the female LH values ranged from 10-50 ng mL-1 but in the male LH values were greater than 50 ng mL-1.

Female kowaris are seasonal breeders (May-November) with a gestation of 32.5 +/- 1.8 days (mean +/- s.d., n = 35). The oestrus cycle including pregnancy is 59.0 +/- 6.7 days in duration (mean +/- s.d., n = 12) and is not significantly different from the non-pregnant cycle of 60.3 +/- 7.1 days (mean +/- s.d., n = 15) (P greater than 0.1). Animals maintained on a fixed diet show an increase and fall in body weight before oestrus and again at the end of the oestrous cycle or a pregnancy; these changes correlate closely with progesterone concentration. Plasma progesterone concentration and body weight were measured in six kowaris undergoing an oestrous cycle then a pregnancy in the breeding season of 1981. The basal concentration (mean +/- s.d.) of progesterone prior to the breeding season was 0.35 +/- 0.21 ng mL-1. There were mean rises of 1.84 +/- 1.1 and 2.47 +/- 1.5 ng mL-1, respectively, before the first and second oestrous periods which had declined to near basal levels by the day of oestrus. Peak values of 9.9-11.5 ng mL-1 were measured between days 23 and 30 of oestrous cycle and pregnancy. The pattern of changes in body weight and plasma progesterone concentration was similar in pregnant and non-pregnant animals but the decline in weight and progesterone occurred earlier in pregnant kowaris.