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Artificial insemination of brushtail possums (Trichosurus vulpecula) is being developed as an assisted breeding model for endangered marsupials, as well as a bioassay for testing fertility control vaccines to manage overabundant populations. Procedures were optimised in animals superovulated with pregnant mare serum gonadotrophin (PMSG) and porcine luteinising hormone (pLH). Of three intervals examined, yields were maximal following uterine insemination at 27-29.5 h after pLH treatment (four eggs, two to three embryos per female). Compared with no insemination, uterine-inseminated animals ovulated 30-36 h rather than 28-34 h after pLH treatment. For the vaginal route, yields were maximal following insemination at 10-13 h after pLH treatment (six to seven eggs, four embryos per female) than at five other intervals, and when using acclimatised females during the autumn breeding season. This protocol was suitable for testing fertility control vaccines in April-June and was influenced by the housing location of animals, the presence of an active corpus luteum and PMSG batch, but not other factors (year of trial, Freund's adjuvant treatment, changes in bodyweight, dose of PMSG kg(-1)). Embryos developed to the eight- to 16-cell or unilaminar blastocyst stage after uterine or vaginal insemination, respectively. With the timing of artificial insemination optimised, new methods to synchronise or induce oestrus and ovulation are required to achieve year-round testing of fertility control vaccines or birth of offspring.

This study looked at the feasibility of targeting the zona pellucida for a contraceptive vaccine as a possible alternative method of control for overabundant macropods. Tammar wallabies, as a model for other macropods, were immunized with porcine zonae pellucidae (PZP) and were found to achieve significant concentrations of antibody to PZP in sera and reproductive tract fluids. Wallabies immunized with PZP exhibited lower ovarian weight with reduced numbers of antral follicles when compared with control animals. Wallabies were placed in a natural mating trial followed by an artificial insemination trial. None of the PZP-immunized wallabies produced offspring in the natural mating trial compared with 67% of control animals. To further assess fertility, a sub-sample of the wallabies were superovulated and artificially inseminated. This resulted in all control wallabies producing fertilized ova and all PZP-immunized wallabies failing to ovulate. These results suggest that immunocontraception based on targeting antigens of the zona pellucida may be an effective strategy for fertility reduction in macropods.

Monovulatory brushtail possums (Trichosurus vulpecula) were stimulated with exogenous hormones during seasonal anoestrus to overcome ovarian insensitivity and induce ovulation. Seasonal ovarian insensitivity to pregnant mare serum gonadotrophin (PMSG) was overcome by a new porcine follicle-stimulating hormone/porcine luteinizing hormone (pFSH/pLH) protocol. This protocol was refined because the original treatment produced oocytes with abnormal morphology. Possums (n = 12 per group) received eight injections of pFSH of 1.5, 3.0 or 6.0 mg per injection (at 12-h intervals for 4 consecutive days). Ovulation was induced 12 h after the final pFSH injection with a 4-mg injection of pLH. Control animals were treated with the established protocol of a single injection of 15 IU of PMSG, followed 48 h later with an injection of 4 mg of pLH. All females responded to pFSH/pLH treatment, although optimal stimulation occurred in those receiving 8 x 3 mg pFSH, with 13-14 ovulations and recovery of 11-12 oocytes per female (8 x 1.5 mg pFSH: 13 ovulations, 8-9 oocytes; 8 x 6 mg pFSH: 7-8 ovulations, 4-5 oocytes). In contrast, only seven of 12 females responded to PMSG/pLH and, of those responding, only 2-3 ovulations occurred and only 1-2 oocytes per female were recovered. However, around 80% of oocytes recovered after PMSG/pLH treatment had undergone nuclear maturation (metaphase II/1st polar body) compared with around 60% of oocytes from pFSH/pLH-treated animals. In possums killed from 27 to 39 h after pLH treatment, ovulation onset was first observed from 30 h and by 31.5 h, all animals had completed ovulation. Laparoscopic artificial insemination (LAI) was performed on pFSH/pLH-treated animals to determine whether the oocytes produced were capable of fertilization. Uterine LAI performed 27.5-28.5 h after pLH treatment yielded 11/26 fertilized oocytes (up to 4-cell stage), whereas vaginal LAI performed 13-14 h after pLH treatment yielded 21/53 fertilized oocytes. A proportion of oocytes generated from the refined pFSH/pLH protocol are thus properly mature and capable of fertilization. Further refinement of the protocol is now needed to improve the yield of fully matured oocytes.

Previous studies have demonstrated that co-culture of brushtail possum epididymal spermatozoa with oviduct epithelial cell monolayers prolongs sperm survival and results in the re-orientation of the sperm head and tail to the T-shape (thumbtack) configuration. Transformation of sperm to thumbtack orientation is believed to be associated with marsupial sperm capacitation. Here we report that incubation in oviduct-conditioned media also significantly prolongs sperm survival and results in the transformation of sperm to the thumbtack orientation. The major objective of the current study was to examine the proteins present in the conditioned media, to determine whether any of these proteins specifically bound to sperm and the relationship between these proteins and sperm survival and thumbtack orientation. Co-culturing brushtail possum sperm with biotin-labeled proteins in conditioned media (CM) from ampulla, isthmus and uterine explants demonstrated strong binding of two proteins of molecular mass 230 and 61 kD and weak binding of nine proteins of molecular mass 200, 180, 120, 140, 55, 52, 48, 34, 30 kD to sperm within 30 min. The binding of the 61-kD protein from the conditioned media appeared specific as increasing concentrations of non-labeled oviduct proteins, but not serum proteins, inhibited the binding of labeled proteins. The binding of oviduct and uterine proteins in the conditioned media significantly prolonged sperm survival and percentage motility and also transformed a large number of sperm to a thumbtack orientation. The implication of binding of these proteins is discussed in the context of sperm survival and capacitation in this species.

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.

The motility, acrosome integrity and fertility of ram spermatozoa were examined after treatment with five compounds (caffeine, pentoxifylline, cAMP, 2-deoxyadenosine and kallikrein). Semen was extended with a Tris-based medium and frozen in pellet form. The compounds were added at various concentrations to the semen before freezing or after thawing. Motility and acrosome characteristics were assessed over a 6-h post-thaw period of incubation at 37 degrees C. Of the five compounds examined, only pentoxifylline, when added after thawing, stimulated motility but had not effect on the acrosome integrity of spermatozoa. Pregnancy rates for ewes inseminated in the uterus with semen treated after thawing with Dulbecco's phosphate-buffered saline (PBS, control) or PBS containing pentoxifylline (15 mM in the thawed semen), were 16/39 (41%) and 22/42 (52%) respectively. Motility characteristics of spermatozoa assessed by image analysis (Hamilton-Thorne analyser) were initially better after treatment of thawed semen with pentoxifylline than with PBS ("percent motility', "percent progressive motility', "percent rapid', "percent moderate' and "percent static', P < 0.01 or P < 0.001), but there was a greater deterioration in these characteristics during post-thaw incubation in semen treated with pentoxifylline than in the controls. The deterioration in motility characteristics occurred within 4 h of thawing and this may have been responsible for the modest improvement in fertility of spermatozoa treated with pentoxifylline.