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Acute myocardial infarction used to be defined by criteria based on symptoms, changes in electrocardiograms and the concentrations of cardiac enzymes, as recommended by the World Health Organization. (1) Specific [...]

A protocol was developed for pellet-freezing spermatozoa of the tammar wallaby and the brushtail possum. Seren was collected by electro-ejaculation and wallaby spermatozoa were washed by 'swim-up' into phosphate-buffered saline (PBS), whereas possum spermatozoa were not washed. Wallaby spermatozoa were screened for toxicity in diluents containing a range of cryoprotectants (0-10%): dimethyl sulfoxide (DMSO), ethylene glycol and propanediol. Possum spermatozoa were tolerant of diluents containing 17.5% glycerol. Wallaby and possum spermatozoa were diluted 1:1 with the most promising cryoprotective diluents (final concentrations in PBS: possum, 17.5% glycerol; wallaby, 7.5% glycerol + 10% DMSO) and, after 5 min equilibration at room temperature, were pellet-frozen. Pellets were thawed (35 degrees C) and wallaby spermatozoa were washed by centrifugation (200 g for 5 min) and resuspended in PBS to minimize cryoprotectant toxicity. A high proportion of possum spermatozoa was recovered after freezing (67.5%), having good progressive motility (3.6 on a 0-5 scale). The progressive motility of frozen-thawed wallaby spermatozoa was also high (3.0), but only 10% of motile spermatozoa were recovered. The pellet-freezing method in conjunction with the post-thaw washing procedure (wallaby) may produce a viable population of cryopreserved marsupial spermatozoa suitable for use in assisted-breeding techniques such as in vitro fertilization and artificial insemination.

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.

Although yet to be established definitively, it appears that marsupial spermatozoa require a process of capacitation and that the mechanisms involved may be quite different between the Australian and American species. For Australian species, failure to induce this functional event in culture has meant that in vitro fertilization (IVF) is yet to be achieved. However, in the American species with paired spermatozoa, IVF and subsequent embryo development have been obtained under quite simple culture conditions. Our understanding of the interactions of marsupial spermatozoa with the female tract, and in particular the oviduct, the most likely site of capacitation, is discussed. Although the acrosome reaction (AR) is an equally critical event in marsupial fertilization it appears to be regulated quite differently. The uniquely stable character of the marsupial acrosome is examined as well as our current understanding of the regulation of the marsupial sperm AR in vivo and in vitro.

Techniques to manipulate reproduction and productivity are well established features of the husbandry of domestic animals and the treatment of human infertility. Similar approaches are feasible in marsupials, but little work has been done to apply our considerable basic knowledge of marsupial reproduction to produce practical artificial breeding. If the reproduction and productivity of marsupials could be more effectively controlled it would greatly aid research and profoundly change both current practice in the management of zoo animals, and the strategies employed for the breeding and conservation of rare or endangered species. The present paper sets out the likely agenda, reports progress to date and discusses short and long-term prospects for the artificial breeding of marsupials. Topics discussed include: induced ovulation and superovulation, oocyte and embryo collection, semen collection and the frozen storage of sperm, artificial insemination, in vitro fertilization and embryo transfer, frozen storage of embryos, and the use of surrogate mothers in supporting both pregnancy to term, and the development of young in the pouch.

Despite many fundamental similarities between the gametes of marsupials and placental mammals, the regulation and timing of prefertilization gamete maturation are quite different. The marsupial acrosome is remarkably stable and an acrosome reaction (AR) is not induced by reagent effective for the sperm of placental mammals. The ultrastructure of the marsupial sperm AR is essentially similar to that of placental mammals, however, whether an equatorial segment (ES) persists to serve as the site of sperm-egg membrane fusion is unclear. Diacylglycerol induction of the AR suggests that the sperm of Australian species lack an ES, yet an ES-like region appears to be involved in fertilization in the opossum Monodelphis. The marsupial oocyte, unlike those of placentals, continues to grow throughout follicular life and major cytoplasmic maturation events occur late in oocyte development. Cortical granules only become evident shortly before ovulation and mature dark granules may only appear after ovulation. Further, the zona pellucida (ZP) changes in character and function during the peri-ovulatory period. In vitro fertilization has been achieved for an opossum but not for any Australian marsupial, owing to failure of sperm-ZP binding. Requirement for a sperm maturation process is likely, but capacitation treatments used for placental sperm in vitro have been ineffective. Since it is now feasible to experimentally manipulate marsupial gametes in vitro major advances in our understanding of their function can be expected.

This study aimed to develop a superovulation protocol for the monovulatory tammar wallaby (Macropus eugenii), and examined the regulation of ovarian activity which leads to alternate ovulation in this marsupial. The most effective stimulatory treatment was 20 I.U. pregnant mare serum gonadotrophin (PMSG) given intramuscularly (i.m.) 20 days after the activation of an oestrous cycle by the removal of a sucking pouch young (RPY). Bromocriptine treatment was given at the time of RPY if the animal was in early seasonal quiescence. Mating had generally occurred when animals were examined 2 days after PMSG treatment on the morning of Day 22 RPY. Ovulation occurred only if the animal was treated on Day 22 or 23 (i.e. 2 or 3 days after PMSG) with gonadotrophin releasing hormone (GnRH) to induce a luteinizing hormone (LH) surge. Three 30-micrograms injections of GnRH (in 0.2 mL olive oil) were delivered as i.m. injections at 3-h intervals. Radioimmunoassay confirmed that the PMSG dose used did not elevate circulating steroid hormone concentrations beyond those found in normal cycles and that the GnRH protocol led to an LH surge of at least 6 h. Although multiple ovulation was achieved, the number of ovulations was low (2 or 3 per female). A major factor influencing the low ovulation rate was that generally only one ovary responded. Fertilized eggs and cleaving embryos were obtained. However, the fertility of induced ovulations has not yet been examined systematically. Laparoscopic examination through successive natural cycles confirmed that follicle growth and ovulation in the tammar wallaby alternates between the right and left ovary. Inhibition of follicle development in the corpus luteum (CL)-bearing ovary was also seen in females treated with the exogenous gonadotrophin (PMSG/GnRH) superovulation protocol. Follicle development was inhibited during the first half of the cycle in the non-CL-bearing ovary and during the entire cycle in the CL-bearing ovary. This inhibition seemed to occur at the follicular level because exogenous gonadotrophin was unable to initiate a response during periods of inhibition and the response to gonadotrophin differed in the two ovaries. The number of follicles growing in the non-CL-bearing ovary in response to an exogenous gonadotrophin stimulus was inversely related to the weight of the growing CL for the first 19 days after RPY.(ABSTRACT TRUNCATED AT 400 WORDS)

In this study of the unique stability of the marsupial acrosome, experiments were carried out on the acrosomes of spermatozoa of the tammar wallaby (Macropus eugenii), common brushtail possum (Trichosurus vulpecula) and grey short-tailed opossum (Monodelphis domestica). Light microscopy showed that 4% of opossum and 15% of possum and wallaby spermatozoa lost their acrosomes after freeze-thawing. Electron microscopy revealed that freeze-thawing also induced changes in the acrosomal matrix of some acrosome intact spermatozoa. In both possum and wallaby, freeze-thawing increased the number of spermatozoa with vesiculation of the acrosomal matrix. Freeze-thawing disrupted the plasma membrane of spermatozoa but the acrosomal membranes remained intact. Immediately on addition of high concentrations of TX-100 (0.02% and 0.04%) there was significant loss of acrosomes and motility in possum and wallaby spermatozoa. Lower concentrations of TX-100 (< or = 0.01%) did not affect motility for up to 30 min in all three species, and there was no significant loss of acrosomes. Although loss of acrosomes did not occur under mild detergent treatment, 56% of wallaby and 70% of possum spermatozoa had altered acrosomes after 30 min in 0.01% TX-100. Electron microscopy revealed that acrosomes were undergoing a vesiculation process similar to that seen after freeze-thawing. Often the plasma membrane of detergent-treated spermatozoa was disrupted and had formed plasma membrane vesicles. However, the acrosomal membranes remained intact despite major changes to the acrosomal matrix. The study confirmed the remarkable stability of the marsupial acrosome and suggested that this is probably based in the acrosomal membranes.

Sminthopsis crassicaudata is a small (approximately 16 g) polyovulatory dasyurid marsupial which has the potential to become an important model species. This study examined the use of exogenous hormone treatment to manipulate the breeding of S. crassicaudata and as a means to obtain timed developmental stages for further study. Two thirds (21/32) of the females treated with 1.0 or 5.0 I.U. of pregnant mare serum gonadotrophin (PMSG) had ovulated when the contents of their reproductive tracts were examined 5 or 6 days later. Only one of eight females treated with 0.2 I.U. PMSG had ovulated in the same period. Although a similar proportion of animals treated with 1.0 I.U. and 5.0 I.U. ovulated, the ovulation rate was significantly lower when the higher dose was administered (mean of 10.5 ovulations per female v. 3.8 ovulations per female). In addition, the ovaries of 6/8 of the animals treated with 5.0 I.U. PMSG had luteinized follicles with degenerating oocytes, evidence of over-stimulation. Follicular luteinization also occurred in 4/8 animals treated with 1 I.U. PMSG. Oocyte maturation and ovulation occurred following PMSG stimulation without injection of synthetic gonadotrophin releasing hormone (GnRH). Treatment with a 10-micrograms dose of GnRH following PMSG seemed to have no effect on the outcome. Of the females that had ovulated by Day 6, three quarters had mated and some had fertilized eggs and two-cell embryos in the oviducts and uteri. In a further series of experiments the subsequent development of embryos conceived after PMSG treatment was assessed. Two thirds of treated females mated within 7 days of treatment and 60% of these matings yielded embryos when examined 11 days after PMSG. However, full-term development was only achieved in one animal. Gonadotrophin treatment of S. crassicaudata thus may have application as a means to obtain mature or maturing oocytes, cleavage stage embryos and blastocysts, but at this stage it appears not to offer promise as a method to achieve full-term development.

Frozen storage of semen and embryos is now a well established part of the breeding of many eutherian mammals but it has not been applied to marsupials. This paper reports the first successful technique for the frozen preservation of marsupial spermatozoa. Semen was collected by electroejaculation under anaesthesia from a pool of five brushtail possums. The ejaculated semen was diluted 1:1 with Krebs Henseleit Ringer, centrifuged at 800 g for 5 min, resuspended in the test cryoprotectant media at 1, 2 and 5 x 10(6) spermatozoa mL-1 and 7, 10.5, 14 and 17.5% glycerol and then drawn up into 0.25 mL plastic straws. The spermatozoa were rapidly frozen in the vapour phase, 6 cm above liquid nitrogen, for 30 min before the straws were plunged into the liquid. Sperm motility was assessed blind for coded straws by phase-contrast microscopy on a warmed stage (35 degrees C), before freezing and after rapid thawing in a water bath at 37 degrees C (10 s). The highest recovery of both percentage motility (around 50-60%) and progressive motility (around 0.5-1 unit lower than prefreeze) occurred when spermatozoa were frozen and thawed in the presence of 17.5% glycerol. Recovery of motility was greater at the higher sperm concentrations (2 and 5 x 10(6) mL-1). There was no evidence of acrosomal damage or loss after freezing and thawing in high concentrations of glycerol. The only defect detected in spermatozoa subjected to the protocol was a variable tendency to bending of the neck region. This ranged from heads inclined at a slight angle to the tail through to complete flexure.(ABSTRACT TRUNCATED AT 250 WORDS)

The reproductive tracts of males from eight species of Australian marsupial were examined (Macropus eugenii, Potovous tridactyhs, Sminthopsis crassicaudata, Antechinus stuartii, Pseudocheirus peregrinus, Trichosurus vulpecula, Isoodon macrourus, and Perameles nasuta). The prostate glands of these species were found to be of two shapes, carrot-like or heart-like. From one to three pairs of Cowper's glands were observed; these were mostly bulbous in shape but some were kidney-shaped. Both prostate and Cowper's glands were tubular in structure with the glandular tubules lined by a simple columnar epithelium. The glandular tubules of Cowper's glands were of much larger diameter than those of the prostate. The prostate glands were segmented, and this segmentation was usually shown by variations in the height and staining reactions of the tubular epithelium and in the volume of connective tissue between glandular tubules. Differences in microanatomy between pairs of Cowper's glands were far less than those between prostate segments. Mucosubstance appeared to be the major contribution of the prostate to the seminal plasma. This mucosubstance was mainly neutral, with glycogen largely absent. The present results indicate that the Cowper's glands secrete mucus but that various glands also contributed lipid and glycogen.

The vaginal mucus of T. vulpecula is secreted by the simple columnar epithelium of the vaginal cul-de-sac and upper portion of the lateral vaginal canals. Vaginal mucus was present in measurable quantities for a period of only 3-4 days: from the commencement of oestrus (as judged by vaginal smears) until about half a day after ovulation. Over this period the volume of mucus in the cul-de-sac rose rapidly from practically nil to about 3.5 ml at ovulation. Following ovulation, the volume very soon returned to its former level. Treatment of anoestrous animals with doses of oestradiol benzoate at 40ug/kg body weight daily for 2 or 4 days, and 20 or 40 ug/kg body weight for 8 days, resulted in secretion of mucus in similar quantities to that found in oestrous animals. Preliminary studies of the physical properties (density, percentage dry matter, viscosity, and "stickiness") of the vaginal mucus of T. vulpecula revealed few trends that could be causally related to follicle development, oestrus, and ovulation. However, the pH appeared to follow a trend with regard to follicle development. It was found to fall from 7-8 to 6 at the end of follicle growth. The histochemical findings suggest that the vaginal mucus of T. vulpecula is a carbohydrate-protein com- plex, containing neutral sugars and acidic mucopolysaccharides. The acidic mucopolysaccharides are mainly carboxylated; however, low concentrations of sulphated mucopolysaccharide are also evident. The presence of appreciable quantities of free vaginal mucus in the vaginal cul-de-sac between oestrus and ovulation suggests that it is likely to be functionally important in the maintenance of a sperm reservoir.