Intrauterine development after diapause in the marsupial Macropus eugenii

Three-quarters of adult female Macropus eugenii carry a diapausing blastocyst for up to 10 months of the year. For the first half of the year the diapause is due to the presence of a pouch young, but it continues through the subsequent anestrus. Spontaneous resumption occurs 1–15 days after the summer solstice. Development can be initiated experimentally during the first half of the year by removing the pouch young (RPY) and during anestrus by 10 daily injections of 10 mg progesterone.All blastocysts completed development after RPY, but only about half successfully completed intrauterine development after progesterone treatment. After RPY the corpus luteum grew and influenced growth of the endometrium and blastocyst. After day 13, however, the endometrium was further stimulated by the presence of the embryo or fetal membranes, probably the latter. Most of the failure after progesterone treatment occurred during the first 10 days and after the blastocyst had resumed development. It is suggested that these embryos failed to reach the stage where they could stimulate the endometrium directly. The later failure of progesterone-treated animals to give birth to full term fetuses was probably due to lack of stimulation from the anestrous ovaries.     

Mammogenesis and changing prolactin receptor concentrations in the mammary glands of the tammar wallaby (Macropus eugenii)

All 4 mammary glands of the tammar wallaby showed a steady increase in weight and prolactin receptor concentration during the luteal phase of the oestrous cycle to reach a peak at oestrus. Removal of the corpus luteum abolished this mammogenesis , while pregnancy, which in this species is a day or so shorter than the oestrous cycle, had no effect. This provides an explanation for the previous finding that pregnancy is not a necessary pre-requisite for lactation in marsupials and that nonpregnant animals will lactate very successfully, provided the suckling stimulus is applied at the correct stage of the oestrous cycle. During lactation, only the gland supplying the teat to which the pouch young was attached developed and showed any further increase in prolactin receptors; the other 3 glands remained small and inactive. These results indicate the importance of the suckling stimulus and milk withdrawal on the initiation and maintenance of lactation.     

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.     

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.     

Evidence from plasma progesterone concentrations for male-induced ovulation in the brush-tailed bettong, Bettongia penicillata.

Female brush-tailed bettongs, Bettongia penicillata, were housed with either an intact or vasectomized male or isolated from males in the peripartum period. Development of the quiescent corpus luteum formed at the post partum oestrus was initiated by removing the pouch young. Blood samples for analysis of plasma progesterone were collected from the females 2 days before removal of pouch young, daily for 5 or 6 days and then 2-3 times each week until 19 days after removal of pouch young. Plasma progesterone profiles were similar in pregnant and nonpregnant cycles. There was an early progesterone peak (1206 +/- 121 pg ml-1, mean +/- SEM; n = 16) between days 2 and 5 after removal of pouch young, and a second period of high concentrations (greater than 800 pg ml-1) before birth on day 17.4 +/- 0.2 (n = 16). The interval between the early peak and birth was 14 or 15 days. On five of 34 occasions, no increases in plasma progesterone concentrations occurred after removal of pouch young. On 12 of 15 occasions for 13 females that had been isolated from males post partum, plasma progesterone concentrations also remained low (less than 100 pg ml-1) and did not change after removal of pouch young. Females that showed no increases in plasma progesterone concentration after removal of pouch young had significantly lower (P less than 0.001) plasma progesterone concentrations while lactating than those females that did undergo a cycle after removal of pouch young (60 +/- 4 pg ml-1, n = 17 and 225 +/- 23 pg ml-1, n = 30, respectively). Females isolated from males post partum, and monitored until day 12 after removal of the pouch young, and that showed no increases in progesterone in this period, had ovaries that contained no corpus luteum, only corpora albicantia and numerous atretic or developing follicles. We conclude that brush-tailed bettongs are induced ovulators, a characteristic described for only one other marsupial, Monodelphis domestica, from South America.     

The critical period for the maternal recognition of pregnancy in pony mares.

Two experiments were performed to deterine the critical time at which the equine blastocyst must be present within the uterus of the mare to prevent regression of the corpus luteum, and thus establish the critical time for the maternal recognition of pregnancy. A non-surgical blastocyst collection technique was developed to study this relationship between the blastocyst and the maternal ovary. Results from these experiments demonstrated that the cyclic life-span of the corpus luteum is not affected by the presence of the blastocyst within the mare's uterus until after Day 14 after ovulation. Luteal function was prolonged when blastocysts were removed on Day 15 or later. The critical period for the maternal recognition of pregnancy in the Pony mare appears to be confined to the period between Days 14 and 16 after ovulation.     

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.     

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.     

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.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

Ovulation and luteal characteristics following removal of the ovine corpus luteum (lutectomy) at four times during the oestrous cycle

The life span of the corpus luteum (CL) may depend on follicular development. To provide evidence relating to this hypothesis, each of 32 ewes was randomly assigned to have its CL removed on day 2, 3, 4 or 10 after oestrus. Twenty ewes were treated with 1000 IU of human chorionic gonadotrophin (hCG) 36 h after CL removal to induce ovulation; the other 12 ewes were not treated with hCG. Blood samples were collected daily to monitor the ovulatory response and the characteristics of the next cycle at the first sign of oestrus and up to day 21 after surgery or hCG administration. Every animal ovulated within 7 days of hCG administration, regardless of when its CL had been removed. It was concluded that the follicles found in the ovary as early as the second day after oestrus respond to endogenous or exogenous ovulatory stimuli affecting the life span of resulting CL.     

Reproductive biology of the Red-necked wallaby (Macropus rufogriseus banksianus) and Bennett's wallaby (M. r. rufogriseus) in captivity

Bennett's wallaby ( Macropus r. rufogriseus ) of Tasmania give birth from late January to early August in marked contrast to the Red-necked wallaby ( M. r. banksianus ) of mainland south-eastern Australia which produced young in all months. Within the breeding season however, the lengths of the oestrous cycle and gestation period are similar in the two forms and did not differ by more than 0.5 days. The gestation period of about 30 days extended to almost the length of the oestrous cycle of approximately 33 days. Birth was closely followed by mating which normally resulted in fertilization and subsequent embryonic diapause. Renewed blastocyst development was initiated by removal or loss of a pouch young and birth followed about 27 days later.
Unlike other macropodids with a similar breeding pattern, birth, as a result of renewed blastocyst development near the end of a large young's pouch life, did not occur within a day or two of the permanent emergence of the young, but followed 16 to 29 days later. In M. r. rufogriseus , young that left the pouch permanently in the non-breeding period were not replaced by new young until the beginning of the next breeding season two to four months later, and blastocysts resulting from mating of females without pouch young at the end of the breeding season remained quiescent until the next breeding season five to eight months later.
Females of both subspecies first mated at an age of about 14 months, and males were producing mature spermatozoa by about 19 months.
Young first left the pouch for short periods at about 230 days of age and permanently at about 280 days.
Observations are also given on reproductive behaviour, interpretation of vaginal smears, sex ratio of young, selection of teat by pouch young, and development of morphological features in known-age young that may be used as an aid in age determination.     

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.     

Effects of corpus luteum removal on progesterone, estradiol-17 beta and LH in early pregnancy of the tammar wallaby, Macropus eugenii

The quiescent corpus luteum of female tammars was reactivated by removal of the pouch young (RPY). The reactivated corpus luteum was ablated 3 days after RPY. Plasma progesterone and oestradiol concentrations were measured by radioimmunoassay in these and in sham-operated controls. Excision of the CL abolished the rise in progesterone seen at Day 5-6 in the sham-operated animals (130.7 +/- 56.6 vs 452.4 +/- 176.0 pg/ml, mean +/- s.d.). By contrast, oestradiol-17 beta values increased within 6-16 h of CL excision to 16.3 +/- 6.9 pg/ml and remained high for 1-3 days while in the sham-operated animals there were less sustained and more variable peaks of 10-20 pg/ml between Days 3 and 5 (mean 12.0 +/- 3.6 pg/ml at Day 4-5). We conclude that the early transient increase in peripheral plasma of progesterone is of luteal origin but the source of the oestradiol remains unknown.     

Growth of the corpus luteum and its progesterone content during pregnancy in the tammar wallaby, Macropus eugenii

Corpora lutea were obtained from wallabies at different stages of pregnancy, following removal of pouch young to initiate embryonic development. Progesterone was present at a concentration of 11.3 ng/mg in quiescent corpora lutea from lactating animals. Progesterone values rose with increasing corpus luteum weight to reach a maximum of 40--50 ng/mg in corpora lutea weighing 50--60 mg. Total progesterone reached a maximum of 1500 +/- 300 (s.e.m.) ng at Days 21--23 after removal of pouch young, but fell markedly at Days 24 and 25 (900 +/- 150 ng) immediately before parturition.     

Effect of oestradiol benzoate given after prostaglandin at two stages of follicle wave development on oestrus synchronisation,the LH surge and ovulation in heifers

Oestrus synchronization following prostaglandin-induced luteolysis is variable and dependent on follicle wave status in cattle. Oestradiol benzoate (ODB) has been used following prostaglandin to reduce the interval to oestrus and ovulation, but the effect of follicle wave status at the time of ODB administration is not clear. The aim of this study was to characterize the endocrine and follicular responses following ODB after luteolysis at different stages of the follicle wave. Prostaglandin was administered at either emergence or dominance of the second follicle wave. Twenty-four hours later animals received either 0.5mg ODB in oil or a control oil injection. Follicular development was monitored daily by ultrasonography, oestrous behavior was determined and blood samples were collected. In animals treated with ODB at emergence, there was a reduction (P<0.05) in the maximum diameter of the ovulatory follicle (11.7+/-1.2 mm versus 13.1+/-0.1 mm) and in the interval from prostaglandin to oestrus (52.0+/-2.3 h versus 88.0+/-9.6h), to the LH surge (53.3+/-3.5 h versus 89.1+/-6.5 h) and to ovulation (96+/-0.0 h versus 129.6+/-9.6h), compared with controls. In animals treated with ODB at dominance, there was a reduction (P<0.05) in the interval from prostaglandin to the LH surge (54.0+/-3.1 h versus 70.9+/-4.8 h), but not in the interval from prostaglandin to oestrus (53.3+/-2.7 h versus 65.7+/-4.5 h; P=0.11), to ovulation (96.0+/-0.0 h versus 110.4+/-4.8 h; P=0.12) or the maximum diameter of the ovulatory follicle (12.7+/-0.3 mm versus 13.6+/-0.4 mm; P=0.12), compared with controls. Treatment did not affect (P>0.05) the length of the subsequent oestrous cycle or corpus luteum size. In conclusion, the use of ODB advanced, but did not alter the temporal relationships among oestrus, the LH surge and ovulation, regardless of stage of follicle development at treatment.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.     

Ram-induced ovulation in seasonally anovular merino ewes: Effect of oestradiol on the frequency of ovulation,oestrus and short cycles

Seasonally anovular Merino ewes can be induced to ovulate by introducing rams. This ovulation is rarely accompanied by oestrus, and the resulting corpus luteum may regress prematurely. Oestradiol (100 μg i.m.) delayed and depressed the ovulatory response (33/45 vs 33/34 for controls), but had no effect on the expression of oestrus (10/33 vs 7/33 in controls) or the frequency of short cycles (16/33 vs 15/33 for controls). The ovulation following premature regression of the CL was not accompanied by oestrus. It seems unlikely that the lack of oestrus and the formation of a CL with short life span are due to a deficiency of oestradiol.     

Growth and role of the corpus luteum throughout delayed gestation in the potoroo, Potorous tridactylus

The growth cycle of the corpus luteum (CL) of the potoroo is similar to that of other macropodids. During delayed gestation, the post-partum CL remains quiescent until it is reactivated by removal of the sucking pouch young. The CL then undergoes a period of growth, rapid from Day 6 until Day 12, followed by a gradual decline from Day 21 to Day 27. Excision of the CL before Day 6 of pregnancy either inhibited embryonic development or failed to support it. Excision of the CL between 6 and 21 days after removal of pouch young did not prevent embryos developing to full term but interfered with parturition. Excision on Day 25 after removal of pouch young allowed birth but impaired lactation, neonates dying within 2 days. By Day 27, the CL appeared to be no longer essential for embryonic development, birth or neonate survival. It is suggested that the CL of the potoroo is required for a slightly greater proportion of pregnancy than in most larger kangaroos because the birth canal must be prepared before each parturition.     

Hormonal control of reproduction in small ruminants

Reproduction of small ruminants can be controlled by several methods developed in recent decades. Some of these involve administration of hormones that modify the physiological chain of events involved in the sexual cycle. Methods which utilise progesterone or its analogues are based on their effects in the luteal phase of the cycle, simulating the action of natural progesterone produced by the corpus luteum after ovulation, which is responsible for controlling LH secretion from the pituitary. Use of prostaglandins is an alternative method for controlling reproduction by eliminating the corpus luteum and inducing a subsequent follicular phase with ovulation. Finally, the discovery of the properties of melatonin in photoperiod-dependent breeding animals opened up a new methodology to control reproduction in these species, inducing changes in the perception of photoperiod and the annual pattern of reproduction. Use of hormones to induce oestrus has allowed increased use of artificial insemination in small ruminants, a very useful management tool, considering the difficulty of detecting oestrus in these species. At commercial level, synchronisation of oestrus allows control of lambing and kidding, with subsequent synchronisation of weaning of young animals for slaughter. Also, it allows more efficient use of labour and animal facilities. Multiple ovulation and embryo transfer programmes are also possible with the use of oestrus synchronisation and artificial insemination. Finally, hormonal treatments have also been used to induce puberty in ewe-lambs and doelings.