Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula)

The present study investigated the effects of slow-release implants containing the gonadotrophin-releasing hormone (GnRH) agonist deslorelin on reproduction in the common brushtail possum (Trichosurus vulpecula). Captive female brushtail possums were assigned to control (placebo implant), low dose (4.7 mg deslorelin) or high dose (9.4 mg deslorelin) groups; males were assigned to control or high dose (9.4 mg deslorelin) groups. The acute effects of deslorelin treatment at the level of the pituitary gland were similar between the two sexes, where a transient rise in luteinising hormone concentration was induced over the first 24 h. In females, this was associated with the disruption of the normal oestrous cycle and mating within 2-10 days in some treated individuals, but no young were subsequently detected. By 3 weeks after treatment, treated females became anoestrus and remained infertile for at least one breeding season. The effects of treatment were reversible in a subset of females that had their implants removed, although the time taken to produce offspring was variable. Paradoxically, male brushtail possums remained fertile during chronic deslorelin exposure. Despite significant declines in basal follicle-stimulating hormone and testosterone concentrations, as well as an inability to respond to a GnRH challenge, treated males sired as many offspring as control males and there was no evidence of testicular regression. In conclusion, there is potential to control reproduction in female brushtail possums by using chronic GnRH agonist treatment.     

A simple glycerol-based freezing protocol for the semen of a marsupial Trichosurus vulpecula, the common brushtail possum

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)     

Seasonal variation in ovarian response to pregnant mares serum gonadotrophin in the brushtail possum (Trichosurus vulpecula)

This study examined the seasonal variation in ovarian response to the exogenous hormone pregnant mares serum gonadotrophin (PMSG) in the brushtail possum (Trichosurus vulpecula). Ovarian stimulation was achieved by administration of a single intramuscular injection of 15 IU PMSG. Animals (n = 165) responded to this treatment in a variable manner depending on the month of the year. The maximum response (measured by the total number of large (>2 mm) follicles) was 15.8 +/- 1.5 follicles per animal (n = 12) in May and the minimum response was 4.9 +/- 1.6 follicles per animal (n = 7) in January. The response throughout the rest of the year closely paralleled the birth seasonal distribution observed in wild possums in published reports. In the summer months (December, January and February) when few females carried pouch young, 55%, 42% and 17% of PMSG-primed animals, respectively, exhibited a nil response to PMSG (i.e. no follicles >2 mm were found on the ovaries of treated animals). In addition, when wild possums were in lactational anoestrus after the autumnal breeding peak, possums with a nil response to PMSG accounted for 17% (July) and 6% (August) of those treated, but the other animals gave near maximal numbers of large follicles (July, 5-21; August, 2-25). Whether the ovaries of non-responders are downregulated in some manner outside the breeding season or they become insensitive to gonadotrophin remains to be investigated. These observations pinpoint times of the year when the highest productivity can be expected following PMSG treatment, and together with information on the timing of ovulation provide critical data for the development of artificial breeding strategies for the possum.     

Changes in milk composition during lactation in the common brushtail possum, Trichosurus vulpecula (Marsupialia: Phalangeridae)

The milk constituents of Trichosurus vulpecula, a folivorous marsupial, showed marked quantitative and qualitative changes during the course of lactation. The milk produced in the early stages of lactation was dilute, with about 9-13% (w/w) solids during the first 3 weeks, comprising mostly carbohydrate and protein (35-40%). At 20 weeks, about three-quarters of the way through lactation, the milk was much more concentrated, about 28% solids, with lipid the predominant fraction (30-35%), after a marked decline in carbohydrate content (20-25%). Concentrations of the electrolytes sodium and potassium also underwent marked changes. The changes in milk composition of T. vulpecula during the first three-quarters of lactation were similar to those described for a range of herbivorous, insectivorous and carnivorous marsupials. In the last quarter of lactation, however, brushtail possum milk maintained a relatively stable composition, with higher levels of carbohydrate and lower levels of lipid than for other marsupials. There appears to be a uniform pattern of changes in milk composition throughout the Marsupialia over most of lactation, with family differences evident only in the latter stages.     

Changes in milk carbohydrates during lactation in the common brushtail possum, Trichosurus vulpecula (Marsupialia:Phalangeridae)

Milk samples (186) were obtained at various stages of lactation from 27 common brushtail possums (Trichosurus vulpecula). Qualitative and quantitative changes in the milk carbohydrates during early and mid-lactation were similar to those previously seen in other marsupials; the principal carbohydrate was lactose early in lactation and higher oligosaccharides in mid-lactation, and the hexose concentration reached a peak during mid-lactation. However, the late-lactation milk was unusual in that the carbohydrate was mainly lactose and its concentration remained relatively high (3.5 to 5.5%). In contrast to earlier findings on the milk of the tammar wallaby (Macropus eugenii), little or no nucleotide pyrophosphatase, beta-galactosidase and alkaline phosphatase activities were detected late in lactation.     

Uterine and vaginal insemination optimised in brushtail possums (Trichosurus vulpecula) superovulated with pregnant mare serum gonadotrophin and porcine luteinising hormone

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.     

Cycle of the seminiferous epithelium in a marsupial species, the brushtail possum (Trichosurus vulpecula), and estimation of its duration

We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail possum (Trichosurus vulpecula), using semithin sections of seminiferous tubules embedded in Spurr's resin. Using 14 steps of spermatid development as markers, we were able to class tubular cross-sections into 10 well-defined stages of the seminiferous epithelial cycle. The duration of one cycle was 13.5 days, as determined by injections of [(3)H]-thymidine and autoradiographic examination of the most advanced sperm cells at 2 h and 17 days after injection. The durations of stages I-X were 21.4, 66.4, 54.1, 47.0, 29.8, 28.5, 25.3, 25.0, 12.0 and 15.9 h, respectively, estimated by the relative percentage of occurrence of each stage. It was estimated that the life spans of the main germ cells were as follows: type B spermatogonia, 5.4 days; primary spermatocytes, 16.7 days; secondary spermatocytes, 0.7 days; and spermatids, 21.4 days. The results suggest that the kinetics of spermatogenesis in marsupials show a similar pattern to that in eutherians.     

Secretory proteins from the female reproductive tract of the brushtail possum (Trichosurus vulpecula): binding to sperm and effects on sperm survival in vitro

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.     

Ontogeny and pathway of formation of 5alpha-androstane-3alpha,17beta-diol in the testes of the immature brushtail possum Trichosurus vulpecula

The testicular androgen 5alpha;-androstane-3alpha,17beta-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with (3)H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Delta(4)-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5alpha-reduced androgen (androsterone) were synthesised by testes from Day 87-200 males and these appeared to be formed from the 5alpha-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, (3)H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.     

Expression of mRNAs encoding oestrogen receptor (ER) alpha and ERbeta, androgen receptor and progesterone receptor during gonadal and follicular development in the marsupial brushtail possum (Trichosurus vulpecula)

The objective of the present study was to determine which ovarian cells express mRNAs for oestrogen (ERalpha and ERbeta), androgen (AR) and progesterone (PR) receptors during ovarian and follicular development in the brushtail possum. Expression of ERalpha and/or ERbeta mRNA was observed from birth, initially in cells of the blastema, then in the medullary cords from Day 20. ERalpha was expressed in the oocytes and granulosa cells of secondary and antral follicles. Preovulatory follicles did not express ERalpha mRNA, although their oocytes were not examined for any gene. ERbeta mRNA was observed in oocytes at all follicular stages examined, but was not consistently observed in granulosa or theca cells. Expression of AR mRNA before Day 40 was very faint; thereafter, expression was observed in the medullary cords, peaking between Days 60 and 120. Oocytes, granulosa cells and theca of secondary and antral, but not preovulatory, follicles expressed AR mRNA. PR mRNA was expressed throughout the gonad by Day 20. Granulosa cells of some secondary and antral follicles and theca of antral follicles expressed PR mRNA. Thus, the expression of mRNAs encoding steroidogenic receptors in a time- and cell-specific manner supports a role for steroids in the process of ovarian follicular formation and growth.     

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula)

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.     

Intestinal lactase (beta-galactosidase) and other disaccharidase activities of suckling and adult common brushtail possums, Trichosurus vulpecula (Marsupialia:Phalangeridae)

Small-intestinal disaccharidase activities of eight suckling T. vulpecula, aged from 34 to 150 days, and of two adult animals were investigated. Intestinal maltase, isomaltase and sucrase activities increased with age, whereas lactase activities decreased. Trehalase activities were relatively high in all animals and showed no obvious age-related changes. Three separate beta-galactosidase activities, one neutral and two acid, acted on lactose. The neutral beta-galactosidase activity appeared to be due to a brush border enzyme similar to that of eutherian mammals, whereas the acid beta-galactosidases were soluble and probably of lysosomal origin. One of these, acid beta-galactosidase-1, had similar properties to the sole intestinal beta-galactosidase of macropodid marsupials, whereas the other, acid beta-galactosidase-2, has not previously been described. Galactosyl oligosaccharides isolated from macropodid milk were readily hydrolysed by both acid beta-galactosidases but not by the neutral beta-galactosidase. The total intestinal lactase activity in animals aged up to 125 days was due mainly to acid beta-galactosidase-1, whereas in older animals it was due mostly to the neutral beta-galactosidase; this suggests that late in lactation the young T. vulpecula change from a macropodid mode of digestion of galactosyl oligosaccharides to a eutherian mechanism for the digestion of lactose. These findings may have implications for the hand-rearing of orphaned T. vulpecula.     

Reduced spillover transmission of Mycobacterium bovis to feral pigs (Sus scofa) following population control of brushtail possums (Trichosurus vulpecula)

In New Zealand, bovine tuberculosis (bTB) is present in domestic cattle and deer herds primarily as the result of on-going disease transmission from the primary wildlife host, the brushtail possum (Trichosurus vulpecula). However, bTB is also present in other introduced free-ranging mammalian species. Between 1996 and 2007, we conducted a series of studies to determine whether poison control of possum populations would have any effect on the prevalence of Mycobacterium bovis infection in sympatric feral pigs (Sus scrofa). We compared trends in the prevalence of bTB infection in feral pigs in six study areas: possum numbers were reduced in three areas, but not in the other three, effectively providing a thrice-replicated before-after-control-intervention design. Before possum control, the overall prevalence of culture-confirmed M. bovis infection in feral pigs was 16.7-94.4%, depending on area. Infection prevalence varied little between genders but did vary with age, increasing during the first 2-3 years of life but then declining in older pigs. In the areas in which possum control was applied, M. bovis prevalence in feral pigs fell to near zero within 2-3 years, provided control was applied successfully at the whole-landscape scale. In contrast, prevalence changed much less or not at all in the areas with no possum control. We conclude that feral pigs in New Zealand acquire M. bovis infection mainly by inter-species transmission from possums, but then rarely pass the disease on to other pigs and are end hosts. This is in contrast to the purported role of pigs as bTB maintenance hosts in other countries, and we suggest the difference in host status may reflect differences in the relative importance of the oral route of infection in different environments. Despite harbouring M. bovis infection for a number of years, pigs in New Zealand do not sustain bTB independently, but are good sentinels for disease prevalence in possum populations.     

Mixed Function Oxidases in an Australian Marsupial, the Brushtail Possum (Trichosurus vulpecula)

Investigation of the mixed function oxidase system of the brushtail possum was undertaken to provide fundamental information about this detoxication enzyme system in a marsupial. Brushtail possum hepatic cytochrome P450, cytochrome b ₅ and NADPH-cytochrome c reductase levels, 7-ethoxyresorufin O-deethylase and (EROD) 7-ethoxycoumarin O-deethylase (ECOD) activities were in the range of values reported for eutherian mammals. Hepatic cyrochrome P450 content was significantly greater (p < 0.01) in brushtail possums from a non-urban population in comparison to an urban population, as was ECOD activity (p < 0.0001). EROD activity was significantly greater in female brushtail possums in comparison to males (p < 0.01). The factors potentially influencing the population- and sex-specific expression of cytochrome P450 isoenzymes in the brushtail possum are discussed and include exogenous dietary xenobiotics and endogenous hormonal alterations influenced by reproductive status. Received: 15 June 1996/Revised: 1 November 1996     

Induction of ovulation in the pregnant rat with luteinizing hormone-releasing hormone (LRH).

Injection of luteinizing hormone-releasing hormone (LRH) induced ovulation in the pregnant rat on days 4 through 22. Only rats autopsied on days 2 and 3 failed to ovulate. Reduced responsiveness occurred between days 12 and 19; the least sensitive day was 16. Based on the number of ova recovered, day 21 was the most sensitive to LRH stimulation.     

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein

The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L′ membrane-anchored protein (ZP3-L′) or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage ?X174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals.