Variations in epithelial Na(+) transport and epithelial sodium channel localisation in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, during the oestrous cycle.

The fluid in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, is copious at ovulation when it may be involved in sperm transport or maturation, but is rapidly reabsorbed following ovulation. We have used the Ussing short-circuit current (Isc) technique and measurements of transcript and protein expression of the epithelial Na(+) channel (ENaC) to determine if variations in electrogenic Na(+) transport are associated with this fluid absorption. Spontaneous Isc (<20µAcm(-2) during anoestrus, 60-80µAcm(-2) in cycling animals) was inhibited by serosal ouabain. Mucosal amiloride (10µmolL(-1)), an inhibitor of ENaC, had little effect on follicular Isc but reduced luteal Isc by ~35%. This amiloride-sensitive Isc was dependent on mucosal Na(+) and the half-maximal inhibitory concentration (IC50)-amiloride (0.95µmolL(-1)) was consistent with ENaC-mediated Na(+) absorption. Results from polymerase chain reaction with reverse transcription (RT-PCR) indicate that αENaC mRNA is expressed in anoestrous, follicular and luteal phases. However, in follicular animals αENaC immunoreactivity in epithelial cells was distributed throughout the cytoplasm, whereas immunoreactivity was restricted to the apical pole of cells from luteal animals. These data suggest that increased Na(+) absorption contributes to fluid absorption during the luteal phase and is regulated by insertion of ENaC into the apical membrane of cul-de-sac epithelial cells.

Energy substrate utilization in the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests.

The aim of this study was to investigate the energy substrate requirements of the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests for glucose, alanine, and propionate in five adult male and female animals under standardized conditions. Significant differences (p<0.01) were observed for fasting blood glucose values between males (6.3±0.16 mmol L(-1)) and females (4.8±0.13 mmol L(-1)), and males had a significantly (p<0.001) increased response to glucose. All animals returned to fasting glucose levels within 120 min after the glucose challenge. No significant change in blood glucose levels was observed for either the alanine or propionate tolerance tests (p>0.05). However, following propionate administration, there was a highly significant (p<0.001) decrease in blood lactate concentrations over 120 min. There was no evidence of ketone formation using ß-hydroxybutyrate as a biomarker during any of the tests, indicating that there was no significant switch to lipolysis. In conclusion, the study provides new information on energy substrate utilization in this species and has identified that a gluconeogenic response normally identified in other species is not apparent in the common brushtailed possum.

Molecular and functional characterization of the cystic fibrosis transmembrane conductance regulator from the Australian common brushtail possum, Trichosurus vulpecula.

Unlike eutherian mammals, the colon of the Australian common brushtail possum, Trichosurus vulpecula, a metatherian mammal, is incapable of electrogenic Cl(-) secretion and has elevated levels of electrogenic Na(+) absorption, while the ileum secretes HCO (3) (-) rather than Cl(-). In eutherian mammals, the cystic fibrosis transmembrane conductance regulator (CFTR) is essential for both Cl(-) and HCO (3) (-) secretion and the regulation of Na(+) absorption. Therefore, we have sequenced possum (p)CFTR, described its distribution and characterized the properties of cloned pCFTR expressed in Fischer rat thyroid (FRT) cells. pCFTR (GenBank accession No. AY916796) has a 1,478 amino acid open reading frame, which has >90% identity with CFTR from other marsupials and >80% identity with non-rodent eutherian mammals. In pCFTR, there is a high level of conservation of the transmembrane and nucleotide binding domains although, with the exception of other marsupials, there is considerable divergence from other species in the R domain. FRT cells transfected with pCFTR express mature CFTR protein which functions as a small Cl(-) channel activated by cAMP-dependent phosphorylation. In whole-cell recordings it has a linear, time and voltage-independent conductance, with a selectivity sequence P(Br) > P(Cl) > P(I) > P(HCO)(3) >> P(Gluconate). pCFTR transcript is present in a range of epithelia, including the ileum and the colon. The presence of pCFTR in the ileum and its measured HCO (3) (-) permeability suggest that it may be involved in ileal HCO (3) (-) secretion. Why the possum colon does not secrete Cl(-) and has elevated electrogenic Na(+) absorption, despite the apparent expression of CFTR, remains to be determined.

Polymeric nanoparticles as an oral delivery system for biocontrol agents for the brushtail possum (Trichosurus vulpecula).

AIM: To investigate polymeric nanoparticles as an oral delivery system for protein biocontrol agents for control of the brushtail possum, Trichosurus vulpecula. METHODS: Insulin-loaded poly(ethyl 2-cyanoacrylate) (PECA) nanoparticles were prepared using interfacial polymerisation, and characterised for size, zeta potential, and efficiency of encapsulation of insulin. In-vitro release of insulin-loaded PECA nanoparticles was quantified using reverse-phase high-pressure liquid chromatography (HPLC). The in-vivo pharmacokinetics of insulin in PECA nanoparticles was investigated following I/V administration, and when injected directly into the caecum alone or in conjunction with the permeation enhancer EDTA. Blood samples were collected at intervals from -5 to 180 minutes, and the concentration of insulin in plasma was quantified using a radioimmunoassay (RIA) validated for possum plasma. RESULTS: Poly(ethyl 2-cyanoacrylate) nanoparticles were produced with a uniform particle size of 200-300 nm, and the mean entrapment of insulin was 78%. In-vitro release of insulin from the PECA nanoparticles was controlled, although incomplete, and approximately 30% of the insulin remained entrapped. The bioavailability of insulin when administered in a PECA nanoparticulate formulation injected directly into the caecum was <1%, and was not increased by addition of the permeation enhancer. CONCLUSIONS: The nanoparticulate formulations investigated as part of this study resulted in low bioavailability of the peptide insulin in the brushtail possum.

Enhancing absorption of fluorescein and LHRH across hindgut epithelia in a marsupial, the common brushtail possum Trichosurus vulpecula.

We have identified differences in transport properties of intestinal epithelia in the marsupial brushtail possum, compared to eutherian mammals. To determine whether differences in its permeability to hydrophilic compounds also occur, the absorption of sodium fluorescein and luteinizing hormone releasing hormone (LHRH) was assessed in vitro and the ability of chemical enhancers and a metabolic inhibitor to promote their absorption investigated. The apparent permeability of colonic and caecal tissues to fluorescein and LHRH and transepithelial resistance (Rt) in the absence or presence of ethylenediamine tetra-acetic acid (EDTA), sodium deoxycholic acid (SDA), dithiothreitol (DTT), polyacrylic acids (PAA), or the inhibitor bacitracin were determined. The effects of SDA and/or DTT on adherent mucus and the release of lactate dehydrogenase (LDH) were also assessed. In the absence of treatment, both tissues had comparable amounts of adherent mucus, Rt and low permeabilities to fluorescein and LHRH. All chemical enhancers increased fluorescein permeability, but SDA at concentrations >0.5 mM also induced LDH release. DTT alone and in combination with SDA reduced the amount of adherent mucus. Bacitracin inhibited LHRH metabolism and increased LHRH permeability. These data indicate that the possum hindgut epithelium represents a significant barrier to the uptake of hydrophilic compounds, similar to that in eutherians.

Gastrointestinal transit in the common brushtail possum measured by gamma scintigraphy.

This paper reports an example of the application of pharmaceutical technology to wildlife management, specifically the design of an oral delivery system for the common brushtail possum in New Zealand. Designing an oral delivery system requires a knowledge of the time taken for particulates to reach target sites within the gastrointestinal tract (GIT). The transit time for fluid and indigestible particles of two different size ranges was determined in the common brushtail possum (Trichosurus vulpecula). Technetium-labelled (99mTc) anion exchange resin particles (75-125 or 500-700 microm diameter) or solution (99mTc-labelled diethylenetriamine pentaacetic acid, 99mTc-DTPA) was administered orally. At predetermined times after dosing (3, 6, 12, 24 or 32 h), the distribution of radioactivity throughout excised gastrointestinal tracts was determined by gamma scintigraphy. The transit profile was similar for the three formulations investigated. Unlike other closely related hindgut fermenting marsupials, there was no evidence to support the presence of a colonic separating mechanism in the common brushtail possum. Gastrointestinal transit was independent of body mass, gender and time of day that the dose is given. To target the hindgut for oral delivery of protein and peptide biocontrol agents, the formulation would need to protect the bioactive for approximately 12 h prior to release.

An in situ single-pass perfusion model for assessing absorption across the intestinal mucosa of the brushtail possum.

AIM: To develop an in situ animal model for assessing absorption of molecules across the intestinal mucosa of possums. METHODS: A surgical preparation was used to perfuse known concentrations of reference compounds (fluorescein and luteinising hormone-releasing hormone; LHRH) through measured sections of selected regions (jejunum, caecum, proximal colon) of the intestinal tract of 19 possums, over a 2-h period. Plasma concentrations of the compounds, which were perfused either with or without co-administration of a permeation enhancer (sodium deoxycholic acid; SDA), were determined in the perfusion effluent, peripheral and in some instances in the pre-hepatic circulation by spectrofluorometry (fluorescein) or radioimmunoassay (LHRH). Pharmacokinetic parameters of both compounds in the possum were determined over a period of up to 4 h in a further 30 animals (fluorescein, n = 6; LHRH n = 24), from their plasma profiles following intravenous (I/V) administration of a bolus dose. RESULTS: In animals perfused with 25 mg/ml fluorescein (Perfusion Experiment (PE) 1), the mean plasma concentration was 2.8 (SE 0.12) microg/ml in post-hepatic blood samples. When possums were perfused with 2.5 mg/ml fluorescein and 7 microg/ml LHRH (PE 2), mean plasma concentrations were 0.3 (SE 0.01) and 7.8 (SE 1.64) microg/ml fluorescein and 0.1 (SE 0.02) and 6.3 (SE 0.45) ng/ml LHRH, in the absence and presence of permeation enhancer, respectively. There was a poor correlation between pre-hepatic and post-hepatic concentrations. CONCLUSIONS: The single-pass in situ perfusion technique provided a useful model for investigating basic information on the absorption of biocontrol agents across the intestinal tract of possums, but had limitations that must be recognised.

Effect of steroid hormones on tissue remodelling and progesterone receptors in the uterus of seasonally anoestrous brushtail possums (Trichosurus vulpecula).

This study describes progesterone receptor (PR) location within uterine cells and associated morphological changes to the uterine luminal and glandular epithelium in seasonally anoestrous brushtail possums treated with oestradiol and/or progesterone. Twenty-four adult female possums (n = 6/group) were treated with oestradiol, progesterone, oestradiol followed by progesterone or with the oil vehicle alone for 6-day periods. Uterine tissue was recovered, weighed and processed for light and transmission electron microscopy and for immunohistochemistry for PRs. Stereological techniques were used to quantify epithelial cell and constituent volumes for both luminal and glandular tissues. Plasma concentrations of oestradiol and progesterone were determined by radioimmunoassay. Mean uterine wet weights were significantly heavier (P < 0.001) following oestradiol/progesterone treatment and maximum gland dilation, cellular and stromal growth, maximum cell height, and cell and constituent volumes were recorded after this treatment regimen. Cell nuclei and debris were commonly observed in gland lumina, and nuclear PRs were found predominantly in stromal cells following oestradiol-only treatment. Sequential treatment with oestradiol and then progesterone caused a decline in the number of positively stained cells. Epithelial cells contained extensive secretory organelles and degenerating cells were common within the glands. Oestradiol treatment induced cell and cell constituent growth and promoted PR formation in anoestrous possum uterine tissue. Subsequent exposure to progesterone stimulated uterine tissues to reach maximum wet weights and led to the cellular maturation necessary to remodel the uterus in preparation for pregnancy.

Effect of oestradiol treatment on mast cell populations and microflora in the vaginal cul-de-sac of seasonally anoestrous brushtail possums (Trichosurus vulpecula).

Mast cell populations in the vaginal cul-de-sac of female brushtail possums do not appear to be related to microbial invasion but changes in their density occur at oestrus, indicating a hormonal influence. The present study examined the effect of treatment with oestradiol on microflora and on mast cell numbers and their spatial location in cul-de-sac tissue of seasonally anoestrous brushtail possums. Tissue was collected from seasonally anoestrous brushtail possums (n = 6 per group) that were either untreated (anoestrous group) or were subjected to 6 days of treatment with oestradiol (oestradiol group) administered via subcutaneous implants or with the oil vehicle alone (control group). Tissue was collected aseptically for microbiological procedures and the fractionator and optical disector were used to quantify mast cell populations. Microflora populations were low (< 4.0 x 10(4) organisms g(-1)) and numbers of mast cells were similar in all groups. Mast cell density was greatest in epithelial and connective tissues from seasonally anoestrous and control animals and lowest in oestradiol-treated possums, in which there was a significant increase in cul-de-sac mass and volume. There is an inverse relationship between circulating oestrogen concentrations and mast cell density in possum cul-de-sac tissue, which is probably the result of an increase in tissue volume.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula).

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula).

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.

Electrogenic ion transport in the intestine of the Australian common brushtail possum, Trichosurus vulpecula: indications of novel transport patterns in a marsupial.

In this study, electrogenic ion transport in the intestine of the Australian common brushtail possum, Trichosurus vulpecula was investigated. In the ileum, a Na(+)-dependent, phloridzin- and amiloride-insensitive short-circuit current ( Isc) was present. Mucosal glucose stimulated a further phloridzin-sensitive, dose-dependent increase in Isc. A Na(+)-dependent, ouabain-sensitive Isc was also present in the caecum and colon. In the proximal and distal colon, amiloride (100 micro mol l(-1), mucosal) inhibited this Isc by 81+/-4% and 65+/-3%, respectively and the Ki for amiloride (approximately 1 micro mol l(-1)) was consistent with the inhibition of a classical epithelial Na(+) channel. In the caecum, 50% of the Isc was inhibited by amiloride (100 micro mol l(-1), mucosal). The amiloride-insensitive Isc in the colon was not due to electrogenic Cl(-) secretion, as serosal bumetanide (100 micro mol l(-1)) had no effect on the Isc. Furthermore, the secretagogues forskolin (10 micro mol l(-1)), carbachol (100 micro mol l(-1)) and dibutyryl-cAMP or dibutyryl-cGMP (100 micro mol l(-1)) did not stimulate electrogenic Cl(-) secretion by the colon. These results indicate that the transport properties of the hindgut of the possum differ significantly from those of eutherian mammals and may be associated with different functions of the hindgut of possums when compared to eutherian mammals.

Aldosterone does not regulate amiloride-sensitive Na(+) transport in the colon of the Australian common brushtail possum, Trichosurus vulpecula.

Plasma aldosterone concentrations and the magnitude of amiloride-sensitive Na(+) transport in the proximal and distal colon of newly caught wild possums, and of possums maintained on either low or high Na(+) diets, were determined to evaluate their role in the regulation of salt content of faeces in common brushtail possums. Wild animals had a plasma aldosterone concentration of 439+/-115 pmol l(-1) and high levels of amiloride-sensitive Na(+) transport in both the proximal and distal colon. Animals maintained on a low Na(+) diet for 14 days excreted very little Na(+) in the urine and had an elevated aldosterone concentration (840+/-246 pmol l(-1)), but levels of amiloride-sensitive Na(+) transport in the proximal and distal colon were similar to those in wild animals. Conversely, while provision of a high Na(+) diet (7 days or 14 days) resulted in increased urinary Na(+) excretion and a fall in aldosterone (approximately 250 pmol l(-1)), there was no reduction in the amount of amiloride-sensitive Na(+) transport in either the proximal or distal colon. These data strongly indicate that aldosterone does not regulate amiloride-sensitive Na(+) transport in the colon of possums and that the colon is not involved in the regulation of salt content of the faeces in this species.

Quantification of mast cells and microflora in the vaginal cul-de-sac of the brushtail possum (Trichosurus vulpecula).

Mast cells are typically associated with biological responses to intracellular parasitic or bacterial invasion in gut and pulmonary tissues. Large numbers of these cells are present in the vaginal cul-de-sac, a component of the reproductive tract in female brushtail possums (Trichosurus vulpecula). The presence of mast cells in this area may reflect microbial presence or indicate a role for mast cells in reproductive function in this marsupial. In this study, the number and spatial location of mast cells in vaginal cul-de-sac tissues at different reproductive stages was determined, and related to the presence of microflora. Vaginal cul-de-sac tissue was collected from juvenile (n = 5), lactationally anoestrous and cyclic adult brushtail possums (n = 6 per group). Oestrous cyclicity was induced by removal of pouch young and reproductive status was confirmed by laparoscopy. Tissue was collected aseptically for microbiological procedures. The fractionator and optical disector stereological methods were used to quantify mast cell populations. In all groups, microflora populations were low (< 5 x 10(5) organisms g(-1)). Mast cell density in epithelial tissue was highest in lactationally anoestrous and luteal phase brushtail possums and lowest in follicular phase animals. No correlation between the numbers of bacteria isolated and mast cell density was apparent. Gram stained tissue showed no evidence of bacterial invasion. Mast cell density was significantly lower in epithelial tissue from follicular phase animals, corroborating previous studies in which an increase in mast cell degranulation coincided with high oestrogen concentrations.

Variation in antral follicle development during the follicular phase of the oestrous cycle in red deer (Cervus elaphus) hinds.

The aim of this study was to quantify antral follicle populations in cyclic red deer hinds and to monitor follicle development leading to ovulation. Oestrus was synchronized with exogenous progesterone and ovaries were recovered approximately 0, 12, 24 or 36 h (follicular phase) or 10 days (luteal phase) after progesterone withdrawal (n = 5 per group). All follicles > or = 2 mm in diameter were dissected out, health status was assessed, follicular fluid oestradiol content was measured, granulosa cells were harvested and their capacity for oestradiol and cAMP production was determined. The time of oestrus and the preovulatory LH surge were monitored in five control hinds. Deer ovaries contained 26.6 +/- 3.45 (mean +/- SEM) follicles > or = 2 mm in diameter (range 4-81), with at least one large antral follicle (diameter: 8.3 +/- 0.38 mm) per hind. There was a strong correlation between follicle size and granulosa cell population (r(2) = 0.676). Approximately half (50.7%) of the follicles were classified as healthy, with the percentage classified as atretic decreasing with increasing follicle size. Neither the total number of antral follicles nor their size distribution differed significantly among groups. There were significantly more (P < 0.05) healthy follicles at 24 h after progesterone withdrawal than at 0 h, when large oestrogenic follicles had fewer granulosa cells, lower follicular fluid oestradiol concentrations and lower aromatase activity (P < 0.05) than did those from other groups. In summary, antral follicle development in red deer is similar to that in other monovulatory ruminants, and at least one large follicle is present at all stages of the oestrous cycle.

Gonadotrophin-induced follicle development in red deer hinds during the breeding and non-breeding seasons.

The effect of exogenous equine chorionic gonadotrophin (eCG) and endogenous (GnRH-treatment) gonadotrophins in promoting antral follicle development in red deer hinds was assessed during the breeding and non-breeding seasons. After progesterone pretreatment, hinds received no further treatment, a single injection of 300 iu equine chorionic gonadotrophin or infusion of GnRH (1.0 microg h(-1)) for up to 4 days. Ovaries were recovered (n = 5 per group) at the time of, or 36 h after, progesterone removal. All follicles > or = 2.0 mm in diameter were dissected out, their health status assessed and follicular fluid oestradiol content measured. Granulosa cells were counted and their capacity for oestradiol and cAMP production assessed in vitro. Oestrus, the preovulatory LH surge and ovulation rates were monitored in additional groups of hinds that had been treated identically (n = 5 per group). During the breeding season, all monitored animals ovulated, but five of 15 (one eCG, three GnRH, one progesterone alone) did not exhibit oestrus, and three (all eCG) had luteinized follicles. During seasonal anoestrus, four hinds (all eCG-treated) displayed oestrus, but only two ovulated. Two non-ovulating hinds (one eCG, one progesterone alone) had luteinized follicles. The total number of follicles, or of large (> or = 4 mm) follicles, did not differ significantly between seasons. There were proportionately more healthy follicles during seasonal anoestrus (P < 0.05). Treatment with GnRH, and to a lesser extent eCG, increased the number of oestrogenic follicles and their follicular fluid oestradiol content. In summary, exogenous and endogenous gonadotrophins affected antral follicle development similarly in both the breeding and non-breeding seasons, although the response was variable among animals and often associated with failure of oestrous expression, ovulation and with follicle luteinization.

Role of ovarian failure in reproductive senescence in aged red deer (Cervus elaphus) hinds.

Physiological and endocrine factors associated with reproductive senescence were assessed in a group of 19 ageing red deer hinds. Reproductive success, defined as the percentage of hinds weaning a calf successfully, decreased gradually from 89% at 6-7 years of age to 50% at 17 years, and subsequently decreased markedly; only one hind reared a calf at 19-20 years of age. When the 12 surviving hinds were approaching 21 years of age, they were compared with ten mature 7-year-old females over the onset of the breeding season. All hinds were subsequently killed, the reproductive tracts were recovered and antral (>/= 2 mm in diameter) and preantral follicle populations were determined by dissection (n = 7 hinds per age group) or stereological analysis (n = 2 ovaries per age group), respectively. Cyclical ovarian activity (plasma progesterone) was evident in fewer aged hinds compared with mature hinds (3/12 versus 10/10, P < 0.001) and mean plasma LH concentrations were higher in aged animals than in mature animals (0.57 +/- 0.05 and 0.20 +/- 0.05 ng ml(-1), P < 0.001). Mean uterine (44.2 +/- 4.5 and 75.4 +/- 4.2 g; P < 0.001) and ovarian masses (0.88 +/- 0.11 and 1.52 +/- 0.12 g; P < 0.001) were lower in the aged hinds, which also had fewer antral follicles than did mature hinds (0.89 +/- 0.35 and 23.5 +/- 4.5 follicles per hind, respectively; P < 0.001). Only one primordial follicle was observed in one of the ovaries of the aged hinds, compared with 7000-21 000 in the ovaries of mature hinds. The high gonadotrophin concentrations, paucity of primordial and antral follicles and failure of ovulation indicate collectively that waning reproductive performance after 17 years of age is primarily due to ovarian failure.

Follicle development in cyclic, anoestrous and FSH-treated brushtail possums (Trichosurus vulpecula).

The common brushtail possum (Trichosurus vulpecula) is a pest of considerable economic importance in New Zealand. Attempts to develop methods of suppressing reproduction in this species are currently hampered by the lack of reliable methods to synchronise oestrus and ovulation in this species. The objective of this study was to compare antral follicle populations in anoestrous and cyclic brushtail possums and to assess the efficacy of exogenous FSH to induce follicle development in anoestrous animals. Ovaries were recovered from anoestrous possums after administration of either exogenous FSH (1.0 mg/injection) or the saline vehicle alone (0.5 ml/injection) at 12-h intervals for 3 days (n = 6/group), and from cyclic animals (n = 6) that were euthanised in mid-follicular phase (5 days after removal of their pouch young). All antral follicles > or =1.0 mm in diameter were dissected free of extraneous tissue, incubated in vitro to measure oestradiol production, and then processed for histological assessment of health status. Mean weight of ovaries and vaginal cul-de-sac tissues were both significantly greater (P<0.001) in FSH-treated anoestrous females (24.2+/-5.1 mg and 6.50+/-1.34 g, respectively), but did not differ significantly between saline-treated anoestrous possums (12.4+/-3.0 mg and 1.31+/-0.27 g) and cyclic animals (13.5+/-1.6 mg and 2.62+/-0.95 g). Mean uterine weights in both cyclic (889+/-161 mg) and FSH-treated (1098+/-184 mg) animals were significantly heavier(P<0.001) than those of anoestrous possums (414+/-61 mg). The mean number of follicles (> or =1.0-mm diameter) present was significantly greater (P<0.001) in FSH-treated, than in cyclic and anoestrous possums (38.0+/-4.4, 23.2+/-3.2 and 10.7+/-3.4 follicles/animal, respectively). Cyclic animals had significantly more (P<0.01) follicles than anoestrous possums. The proportion of follicles that were classified as healthy, was significantly lower (P<0.01) in cyclic possums(38%) than in anoestrous (69%) and FSH-treated (88%) animals. The mean diameter of the largest healthy follicle present was 2.5+/-0.41, 2.1+/-0.08, and 3.1+/-0.15 mm for cyclic, anoestrous and FSH-treated animals, respectively. None of the follicles harvested from saline-treated anoestrous possums produced measurable levels of oestradiol in vitro, whereas 7% and 59% of those from cyclic and FSH-treated animals did so. In summary, cyclic possums had more antral follicles present than anoestrous animals, but a lower percentage of these follicles were healthy. Less than 10% of healthy follicles from cyclic possums, and none of those from anoestrous animals, were capable of producing oestradiol when incubated in vitro. Treatment with ovine FSH promoted follicle development in anoestrous possums, to significantly increase the number of follicles present, the proportion that were healthy and the percentage capable of producing oestradiol.

Plasma gonadotropin concentrations in the cyclic female brushtail possum (Trichosurus vulpecula).

Changes in plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and their relationship to antral follicle development and ovulation, were determined in female brushtail possums (Trichosurus vulpecula) in experiments in which pouch young were removed (RPY) from lactating females to promote ovarian activity. In Experiment 1 (n = 8), the development of preovulatory follicles and ovulation was monitored by laparoscopy. In Experiment 2 (n = 15) estrus and mating were monitored by cytology of urine. Ovulation occurred in 4/8 (Experiment 1) and 9/16 (Experiment 2) possums, and in these animals, plasma FSH concentrations fell progressively over the period of preovulatory follicle development and returned to pretreatment levels after ovulation. With the exception of samples taken at the time of the preovulatory gonadotropin surge, mean plasma LH levels remained basal. In those possums that failed to ovulate, plasma FSH concentrations were elevated while plasma LH concentrations were low; these patterns remained unchanged throughout the sampling period. It was not possible to distinguish between animals that would ovulate and those that would not ovulate after RPY on the basis of gonadotropin profiles at the time of RPY. A further group of possums (Experiment 3, n = 10) were blood-sampled at hourly intervals for 48 h to characterize preovulatory gonadotropin surges, using laparoscopy to monitor preovulatory follicular development and predict ovulation. A preovulatory LH surge (max. conc. 10.2-43.5 ng/ml, duration 7-9 h) was recorded in 4 animals, with a coincident preovulatory FSH surge (max. conc. 1.4-21.4 ng/ml, duration 3-11 h) observed in 3 of these possums. The patterns of gonadotropin secretion in the cycling brushtail possum conform to those reported for eutherians that ovulate spontaneously and appear to be regulated by similar mechanisms.