The reproductive biology of Bennett's wallaby (Macropus rufogriseus rufogriseus) ranging free at Whipsnade Park

Between August 1981 and October 1982, 76 female and 50 male of the estimated 400 Bennett's wallabies at Whipsnade were caught and examined. Sixty-four of the adult females inspected (84%) were carrying pouch young. A small captive breeding colony was established in the laboratory. Weight and growth curves were established for captive born pouch young and these curves were used to determine the approximate age of pouch young examined at Whipsnade. Eighty-four percent of births occurred in August and September, exactly a six month difference from births reported for this species in Tasmania. The mean length of pouch life of wild living wallabies was 247 days with a range of185–284 days. Young wallabies were observed accompanying their mothers for up to 204 days after emerging permanently from the pouch. Sixty-three percent of young vacated the pouch in May. Sixty-three percent of the total (104) pouch young examined in the course of this study, from animals caught or from post mortem records, were male, but teat selection showed a random distribution. The gestation period from removal of pouch young to day of birth was 27·3 days. The Bennett's wallaby showed precise seasonal breeding with embryonic diapause that may extend for up to 11 months.     

The onset of seasonal quiescence in the female Bennett's wallaby (Macropus rufogriseus rufogriseus)

The breeding season of the non-lactating Bennett's wallaby terminates when animals enter the state of seasonal quiescence. To examine this transition, pouch young were removed from females at intervals which were 3, 4 or 8 weeks (6, 11 and 8 animals respectively) after the winter solstice. Within 48 days, 3, 1 and 1 females gave birth respectively, indicating that these animals were not in seasonal quiescence when pouch young were removed. Those animals which did not give birth were either in seasonal quiescence or had undergone a non-pregnant cycle. To differentiate between the 2 possibilities, techniques which would ensure the detection of pregnant and non-pregnant cycles were assessed in 8 females during the breeding season. As has been previously reported for the wallaby, changes in peripheral progesterone concentrations and the vaginal smear occurred during pregnant and non-pregnant reproductive cycles. In addition, mating was detected by marking the male with a mixture of coloured crayon and paraffin wax. It was concluded that reproductive cycles in female wallabies could be monitored by collecting blood samples 2 times each week for progesterone determination and daily examination of females for mating marks. These techniques were then used to study the onset of seasonal quiescence in 9 females. All animals continued to show reproductive cycles after the winter solstice and it was not until 10 weeks after the winter solstice that all animals were in seasonal quiescence. This represents an increase in the duration of the breeding season over that previously reported for this species.     

The role of refractorinesss to long daylength in the annual reproductive cycle of the female Bennett's Wallaby (Macropus rufogriseus rufogriseus)

The beginning of the breeding season of the female Bennett's wallaby occurs when seasonal quiescence terminates 1-2 months after the summer solstice. In this study, the role of photoperiod in terminating seasonal quiescence was examined. One week before the summer solstice, five non-lactating wallabies were transferred from natural to artificial summer solstice daylength for 5 months. The beginning of the breeding season in these animals as indicated by births, matings, and peripheral progesterone profiles was not different from that of five control animals maintained on natural photoperiod. The following year, three animals were transferred from natural to summer solstice daylength on February 25 and were held on the artificial photoperiod until September 30. Changes in plasma progesterone concentrations indicative of the beginning of the breeding season occurred on June 12-30 (range), which was significantly (P less than 0.01) advanced by 29 days when compared with six control animals. These results indicate that the decrease in daylength that occurs after the summer solstice is not required to induce the termination of seasonal quiescence at the beginning of the breeding season. Further, the beginning of the breeding season can be advanced by transferring animals to long daylength early in seasonal quiescence. Photorefractoriness to long daylengths may therefore be important in the initiation of the breeding season in this species. In further experiments, groups of six animals were transferred from natural to artificial summer solstice daylength on September 26 and December 9 and pouch young were removed 7 days after the transfer. In September, reactivation of the quiescent corpus luteum followed soon after removal of pouch young (RPY) indicating that exposure to long daylength had not induced a transition into seasonal quiescence. In December, RPY was not followed by reactivation of the quiescent corpus luteum indicating that animals were in seasonal quiescence. These results suggest that the female Bennett's wallaby may need to experience a period of shortening days after the summer solstice before exposure to long days can again initiate seasonal quiescence.     

Organogenesis of the pituitary, adrenal, and lung at birth in the wallaby, Macropus rufogriseus

The ultrastructure of the pituitary, the adrenal, and the lung was examined in the newborn wallaby, Macropus rufogriseus. Tissue from six wallaby neonates (less than 8 hr of age), two near-term fetuses (26 days after removal of suckling pouch young [RPY]), and a two-day-old pouch young was examined; and tissue levels of cortisol in the adrenal glands of five neonates and a near-term fetus (26 days) were measured by radioimmunoassay. At birth the adenohypophysis comprised the bulk of the pituitary gland. The pars distalis was well vascularized and many cells contained electron-dense, membrane-bound granules. The adrenal glands lacked specific zones but comprised two distinct populations of cells. The cytoplasm of one cell type contained electron-dense, membrane-bound granules, similar to those observed inside catecholamine-secreting cells of the adrenal medulla; the other cell type possessed large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. These features are characteristic of cells which are actively synthesizing steroid hormones. The concentration of cortisol was 0.58 ng/adrenal in the wallaby at birth. The fetal lungs near term were at the glandular stage of development, and epithelial differentiation of type I and type II pneumocytes was imminent although attenuation was not evident. The canalicular neonatal lung did not contain true alveoli, but type II pneumocytes contained osmiophilic lamellar inclusions of surfactant. The fetal pituitary and adrenal are functional at birth and are thus capable of initiating parturition and of influencing lung maturation in the fetus.     

The effect of pregnant and oestrous females on male testosterone and behaviour in the tammar wallaby

Tammar wallaby females (Macropus eugenii) are seasonally breeding marsupials with a post-partum oestrus after a highly synchronised birth period when testosterone concentrations rise in males. Chemical communication appears to be important for mating, as males show checking behaviour, sniffing the urogenital opening (UGO) and the pouch of females. This study investigates whether the presence of pregnant and oestrous females directly influences testosterone in males and if oestrous odours or secretion from the pouch or UGO are attractive. Concentrations of plasma testosterone were measured in males housed with pregnant and oestrous females during two consecutive cycles in the breeding season, and an artificially induced cycle in the non-breeding season. Males were also tested for their interest in swabs taken from the urogenital opening (UGO) or pouch of oestrous females. Testosterone increased sharply in males in the presence of pregnant and oestrous females during all cycles in both seasons, but there was no change when males were exposed to non-cycling females in lactational or seasonal diapause. Males had no preference for either oestrous or non-oestrous samples taken from the pouch or from the UGO from oestrous females. This study confirms that the increase in plasma testosterone in tammar males can be induced through the presence of pregnant and oestrous females, regardless of season and that the increase began when the females were in late-pregnancy. This confirms that the male's reproductive state is dependent on a signal from females and is not blocked through seasonal effects.     

The effect of pregnancy on the interval between one oestrus and the next in the tammar wallaby, Macropus eugenii.

In females mated to vasectomized males the interval between oestrous periods was 30.6 +/- 1.17 (s.d.) days but when mated to intact males and pregnancy intervened, it was 29. +/- 1.26 days (P less than 0.0025). After removal of pouch young, females carrying diapausing blastocysts gave birth 26.2 days later and came into oestrus 26.4 +/- 0.57 (s.d.) days later. When post-partum fertilization was prevented, removal of pouch young was followed by oestrus 30.4 +/- 0.99 days later (P less than 0.0005). These results indicate an influence of the conceptus upon the oestrous cycle of Macropus eugenii.     

Immunological aspects of gestation in the tammar wallaby, Macropus eugenii.

Sensitization to male histocompatibility antigens and repeated pregnancy to the same male were found to have little effect on fertility or length of gestation in the tammar wallaby, M. eugenii. However, in some sensitized females a long interval occurred between removal of pouch young and the next birth. In addition to studies on fertility, the immunological response of female tammars to their mate has been examined by one-way mixed leucocyte culture (MLC) carried out at the beginning and end of one breeding season. In virgin females, examined at the beginning of the breeding season, the MLC response to the prospective mate peaked on day 6. In contrast, by the end of the season, MLC responses were much lower and peaked earlier, on day 3 to day 5.     

Proteins in cerebrospinal fluid and plasma of the pouch young tammar wallaby (Macropus eugenii) during development

The concentrations of total protein and albumin in cerebrospinal fluid (CSF) and plasma of tammar wallaby pouch young (Macropus eugenii) from birth until leaving the pouch have been measured. Total protein in CSF increased from birth (about 240 mg/100 ml) to 15-20 days postnatal (about 400 mg/100 ml) after which it declined. Albumin showed a proportionately greater increase from around 40 mg/100 ml to over 130 mg/100 ml, followed by decline after 75 days. Total protein and albumin in plasma increased throughout the period studied. Other proteins identified in CSF and plasma were: fetuin, alpha 2-macroglobulin, transferrin, alpha-lipoprotein, beta-lipoprotein, immunoglobin G and fibrinogen. One protein was only present in early pouch young (up to about 40 days) and was presumed to be the tammar equivalent of alpha-fetoprotein.     

Reproduction in the agile wallaby Macropus agilis (Gould) in the tropical lowlands of the Northern Territory: opportunism in a seasonal environment

The agile wallaby, Macropus agilis, abounds on the tropical lowlands of the Northern Territory (12° 30′-13°S). The climate is markedly monsoonal and has an annual mean rainfall of 135 cm. During the rainy season, much of the wallaby's major habitat, the extensive coastal plains adjacent to the rivers, becomes flooded. The agile wallaby breeds continuously in captivity but not necessarily so in the wild, although fecundity was very high indeed. Reproduction was more successful on fertilized, nitrogenous pastures of farmland than on grasses of lower nutritive value. Breeding declined least (5%) in the dry season droughts as pastures dwindled and dried out, and most (> 45% in one year) during the rainy season in bushland where the dry season feeding grounds were inundated. The more quickly that the open riverine plains were flooded after the rains began, the higher the loss of breeding and the mortality of pouch-young. The mortality rates of young were similar on both improved and native pastures, however, with only about 35% of new-born surviving to 7 months in the pouch. Wallabies on the improved pastures were at higher densities than on the native pastures which may have influenced that mortality. That females became anoestrous during pregnancy, subsequently gave birth but did not have the usual post-partum oestrus makes the agile wallaby similar to the desert red kangaroo, M. rufus, in its method of entering anoestrus. Also as in that species, anoestrous lactating females can return to oestrus later. The agile wallaby is only the second macropodid marsupial for which these adaptations have been described. Such an adaptation in a desert species has a clear reproductive advantage which is not obvious in this tropical macropodid. Unlike the agile wallaby, the Asiatic waterbuffalo, (Babulis babulus) and domestic cattle, (Bostaurus and B. indicus) which have been introduced to its range breed and give birth on the rains. Such is the reproductive pattern in the tropical ruminants of East Africa also. Seasonal breeding is phylogenetically possible in macropodid marsupials because southern species. M fuliginosus, M. Eugenii, M. r. rufogriseus and Setonix brachyunis, do so in the mediterranean environment there. They time their breeding so that young leave the pouch when pastures are flush after the winter rains. The suggested reason for these differences in breeding of macropodids is seasonal unpredictability. Despite the enormous difference in rainfalls, weekly variability in pasture growth throughout the year is actually the same in monsoonal northern Australia as in the desert, and greater than in southern Australia. Environmental unpredictability may therefore be as important as the overall level of pasture growth in determining reproductive strategies of macropodid marsupials. For the tropical agile wallaby, the unpredictability can cause an absolute shortage or absolute abundance of food in the usual dry season depending on the length of the rainy season and on unseasonal cyclonic weather. It can also cause a relative shortage of food in the rainy season due to flooding of the best feeding grounds. It is concluded that reproduction in the wild mainly depends on the quantity and quality of the food supply.     

Gestational length in the koala, Phascolarctos cinereus

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male.

These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus.     

Evidence from the oestrous cycle for male-induced ovulation in Bettongia penicillata (Marsupialia).

Female brush-tailed bettongs (Bettongia penicillata) housed in a breeding group of one male and one to three females had an average gestation period of 21.2 days (n = 58) and parturition was followed within 24 h by oestrus and mating. If a young was not born as a result of the mating, oestrus recurred after about 21.7 days (n = 12). From removal of pouch young to birth was 17.5 days, on average (n = 85), in females that had mated post partum, but most females that were isolated from a male before parturition returned to oestrus about 6.6 days after simultaneous removal of pouch young and return to the male (n = 9). Females housed in female-only groups appeared not to come into oestrus or to do so irregularly. These females, when returned to the male, usually came into oestrus within 10 days. These data provide evidence that, in most females of B. penicillata, ovulation does not occur in the absence of a male and that previously isolated females return to oestrus within 10 days of return to the male.     

The effect of melatonin on the seasonal embryonic diapause of the Bennett's wallaby (Macropus rufogriseus rufogriseus)

In seasonally breeding mammals, the hormone melatonin, produced at night by the pineal gland, is known to be important in transducing the effect of photoperiod in timing reproduction. In the Bennett's wallaby, an unimplanted unilaminar blastocyst is held in a state of seasonal diapause from mid-winter to mid-summer. Here we show that an implant of the hormone melatonin rapidly terminates seasonal diapause in this species. Blastocyst reactivation is not accompanied by a significant reduction in levels of the hormone prolactin, thereby refuting earlier suggestions that this hormone is responsible for maintaining seasonal embryonic diapause.     

Evidence that the seasonally breeding Bennett's wallaby (Macropus rufogriseus rufogriseus) does not exhibit short-day photorefractoriness

Adult female Bennett's wallabies (N = 6) were maintained in artificial winter solstitial daylengths commencing 3 weeks before the winter solstice for 16 or 42 weeks. Such treatment effectively prevented the normal establishment of seasonal reproductive quiescence with animals continuing to exhibit reproductive cycles beyond the time of the normal termination of the breeding season. Animals maintained in natural photoperiods or simulated natural changes in daylength after the winter solstice all entered reproductive quiescence by early February. In the Bennett's wallaby, therefore, the breeding season does not terminate as a result of refractoriness to short daylengths. Our results indicate that the relatively small increases in photoperiod shortly after the winter solstice provide the environmental signal responsible for initiating the onset of seasonal reproductive quiescence which normally occurs 5-8 weeks after the solstice. These results contrast with the effect of fixed artificial summer solstitial daylengths on the onset of the breeding season in which breeding begins spontaneously at the normal time of year as a result of long-day photorefractoriness.     

Reproductive biology of the Tasmanian Bettong (Bettongia gaimardi: Macropodidae)

The reproductive biology of the Tasmanian Bettong ( Bettongia gaimardi ) conforms in many respects to that found in related kangaroos. Gestation and the oestrous cycle are of similar duration (21–22 days), and a postpartum oestrus may result in the formation of a blastocyst that remains quiescent (embryonic diapause) throughout most of pouch life. Oestrous cycles including a gestation are shorter (by 1·5 days) than those cycles without a pregnancy. This 'maternal recognition of pregnancy' is extended into pouch life as the furred young always vacates the pouch when a new young is born. Pouch life is relatively short (106 days) and, as the Bettong breeds throughout the year, several young may be reared/year. Both males and females become mature before one year.     

Reproduction in Red pandas, Ailurus fulgens (Carnivora: Ailuropodidae)

Several aspects were studied of the reproductive biology of the Red panda, Ailurus fulgens , in captivity at the National Zoological Park (NZP), Washington, D.C. over a six-year period. This species exhibits marked mating and birth seasons and produces litters averaging 1·7 with a sex ratio of 17·2 males to one female. Sexual maturity is reached in the second breeding season after birth or when animals are approximately 18 months of age. Growth, development and mortality of young are discussed. This study noted a wide range in observed gestation periods, averaging 131 days, which may be due to one or a combination of factors. Breeding behaviour is briefly noted and compared to that in other, related species. Predictions about reproductive and other life strategies of Red pandas in nature are drawn by fitting captive data into accepted ecological models.     

The effects of corpus luteum removal during gestation on parturition in the tammar wallaby (Macorpus eugenii).

Removal of the corpus luteum of pregnancy of the tammar wallaby interfered with successful parturition if carried out before Day 17 of the 27-day pregnancy. After removal at Days 17 and 21, 40% of animals gave birth but pouch young died with 24 h; if performed at Days 23 or 25, pouch young survived. However, surgery also affected sham-operated controls if performed between Days 15 and 21. In control animals, sodium pentobarbitone followed by halothane anaesthesia was the least disruptive anaesthetic procedure. This study shows that the corpus luteum has an essential role in parturition and subsequent survival of the neonate in the pouch.     

Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons)

The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat.     

Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation.

The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of the reactivated corpus luteum after embryonic diapause in the marsupial,Macropus rufogriseus banksianus

Summary During embryonic diapause in the red-necked wallaby, M. r. banksianus, both the corpus luteum and uterine blastocyst remain dormant, and are reactivated following removal of the suckling pouch young (RPY). The morphology of dormant and reactivated corpora lutea has been studied throughout the 26.5 days of delayed gestation. Corpora lutea at 0, 2 1/2, 4, 9, 14, 21 and 25 days after RPY were fixed by perfusion. From day 4 to day 14 after RPY there was a progressive increase in the amount of smooth endoplasmic reticulum and the numbers of mitochondria. However there was a decrease in mitochondrial size from 1–2 m in diameter (0 days after RPY) to 0.5–1 m (14 days after RPY). Densely-staining granules (approximately 0.2 m in diameter) were first observed in the luteal cells at 4 days after RPY. The maximum density of granules was observed at 21 days after RPY. Shortly before birth (25 days after RPY) the number of secretory granules had significantly decreased and the features of cellular regression were evident. As with the eutherian mammals, the wallaby luteal cells have all the structural organelles associated with steroid hormone production. The numbers of densely-staining granules are greatest at 21 days after RPY and may reflect the luteal progesterone content since similar granules in the sheep and cow have been shown to be associated with elevated levels of progesterone.     

Studies of the oestrous cycle, oestrus and pregnancy in the koala (Phascolarctos cinereus)

As an integral part of the development of an artificial insemination programme in the captive koala, female reproductive physiology and behaviour were studied. The oestrous cycle in non-mated and mated koalas was characterized by means of behavioural oestrus, morphology of external genitalia and changes in the peripheral plasma concentrations of oestradiol and progestogen. The mean (+/- SEM) duration of the non-mated oestrous cycle and duration of oestrus in 12 koalas was 32.9 +/- 1.1 (n = 22) and 10.3 +/- 0.9 (n = 24) days, respectively. Although the commencement of oestrous behaviour was associated with increasing or high concentrations of oestradiol, there were no consistent changes in the morphology or appearance of the clitoris, pericloacal region, pouch or mammary teats that could be used to characterize the non-mated cycle. As progestogen concentrations remained at basal values throughout the interoestrous period, non-mated cycles were considered non-luteal and presumed anovulatory. After mating of the 12 koalas, six females gave birth with a mean (+/- SEM) gestation of 34.8 +/- 0.3 days, whereas the remaining six non-parturient females returned to oestrus 49.5 +/- 1. 0 days later. After mating, oestrous behaviour ceased and the progestogen profile showed a significant increase in both pregnant and non-parturient females, indicating that a luteal phase had been induced by the physical act of mating. Progestogen concentrations throughout the luteal phase of the pregnant females were significantly higher than those of non-parturient females. Parturition was associated with a decreasing concentration of progestogen, which was increased above that of basal concentrations until 7 days post partum.