Changes in the food and drink consumption patterns of Australian women during the COVID-19 pandemic.

OBJECTIVE: This paper uses data from the seventh fortnightly Coronavirus (COVID-19) Survey sent to women in the Australian Longitudinal Study on Women's Health to investigate the relationship between the COVID-19 pandemic and the food and drink consumption of women born in 1946-51, 1973-78 and 1989-95. METHODS: A survey about changes in fruit, vegetable, discretionary food, takeaway and sugary drink consumption during the pandemic was emailed on 22 July 2020 to 28,709 women in three cohorts of the Australian Longitudinal Study on Women's Health. Thematic qualitative analysis was conducted on comments about changes in consumption, and basic quantitative analysis was included for context. RESULTS: There were significant associations between age and all categories of food and drink consumption. Women wrote of lifestyle changes and choices during lockdowns, comfort and emotional eating, and access to food and drink changing their consumption behaviours. CONCLUSIONS: The COVID-19 pandemic and interventions had both positive and negative impacts on the food and drink consumption behaviours of Australian women. IMPLICATIONS FOR PUBLIC HEALTH: These findings can be used to directly influence practice around healthy food and drink consumption, highlighting enablers, including being at home, and barriers, including mental health, that should be considered.

Considerations and strategies for effective online assessment with a focus on the biomedical sciences.

The COVID-19 pandemic in 2020 caused many universities to rapidly transition into online learning and assessment. For many this created a marked shift in design of assessments in an attempt to counteract the lack of invigilation of examinations conducted online. While disruptive for both staff and students, this sudden change provided a much needed reconsideration of the purpose of assessment. This review considers the implications of transitioning to online assessment providing practical strategies for achieving authentic assessment of students online, while ensuring standards and accountability against professional accrediting body requirements. The case study presented demonstrates that an online multiple choice assessment provides similar rigor in assessment to invigilated examination of the same concepts in human physiology. Online assessment has the added benefit of enabling rapid and specific feedback to large cohorts of students on their personal performance, allowing students to target their weaker areas for remediation. This has implications for improving both pedagogy and efficiency in assessment of large cohorts where the default is often to assess basic recall knowledge in a multiple choice assessment. This review examines the key elements for implementation of online assessments including consideration of the role of assessment in teaching and learning, the rationale for online delivery, accessibility of the assessment from both a technical and equity perspective, academic integrity as well as the authenticity and structure of the assessment.

Use of tricyclic antidepressants and other anticholinergic medicines by older Aboriginal Australians: association with negative health outcomes.

BACKGROUND: Aboriginal and Torres Strait Islander Australians have a relatively high prevalence of multimorbidity requiring treatment with medications. This study examines medication use and anticholinergic burden (ACB) among a cohort of older Aboriginal and Torres Strait Island people. METHOD: This cross-sectional study involving five Aboriginal communities (two in metropolitan Sydney and three on the mid-north coast of New South Wales) used a structured interview process to assess cognition, depression, and activities of daily living for a cohort of older adults (aged 60 years and over). Participants also reported on their health status, medical history, and prescription medications during the interview. ACB was calculated, and its association with adverse health outcomes including cognitive impairment, falls, hospitalization, and depressive symptoms were examined. RESULTS: Most participants (95%) were taking at least one regular medication with polypharmacy (≥5 medications) observed in 43% of participants; 12.2% had a significant ACB (≥3) with antidepressants being a major contributor. Anticholinergic medication use was associated with cognitive impairment, recent hospitalization (past 12 months), and depressive symptoms. After controlling for age, sex, and comorbidity, only the presence of depressive symptoms remained significantly associated with the use of anticholinergic medication (odds ratio 2.86; 95% confidence interval 1.48-5.51). CONCLUSIONS: Clinically significant ACB was common in older Aboriginal Australians and was largely attributable to inappropriate use of tricyclic antidepressants. Greater awareness of medication-related risk factors among both health care professionals and Aboriginal communities can play an important role in improving health and quality of life outcomes.

Mobile memory clinic: implementing a nurse practitioner-led, collaborative dementia model of care within general practice.

The limited capacity of secondary health services to address the increasing prevalence of dementia within the community draws attention to the need for an enhanced role for nurses working collaboratively with GPs in diagnosing and coordinating post-diagnostic care for patients with dementia. This study investigated the feasibility and acceptability of a nurse practitioner-led mobile memory clinic that was embedded within general practice and targeted to caring for patients and their carers in areas of socioeconomic disadvantage with poor access to specialist health services. Over the period from mid-2013 to mid-2014, 40 GPs referred 102 patients, with the nurse practitioner conducting assessments with 77 of these patients in their homes. Overall, there was a strong interest in this model of care by general practice staff, with the assessment and care provided by the nurse practitioner evaluated as highly acceptable by both patients and their carers. Nonetheless, there are financial and structural impediments to this model of care being implemented within the current Australian health service framework, necessitating further research investigating its cost-effectiveness and efficacy.

Stability of anticholinergic load in Australian community-dwelling older people: a longitudinal analysis.

BACKGROUND: It is recommended that anticholinergic medication is avoided in older people, especially those with cognitive impairment. OBJECTIVE: To investigate anticholinergic load (ACL) over time in older primary care patients with and without cognitive impairment. METHODS: Community-dwelling general practice patients at baseline (n = 1768), at year one (n = 1373) and a restricted cohort (with possible or definite cognitive impairment) at year two (n = 370) had medication regimens documented by a research nurse during a home visit. Anticholinergic medicines were categorized as levels 1-3 (low-high potency) and summed for each participant as a measure of their ACL. RESULTS: Most participants had no change in ACL over time, but there was some turnover in the anticholinergic medications used. The mean change in ACL was 0.012 ± 0.99 from baseline to 12 months and -0.04 ± 1.3 from baseline to 24 months. Cardiovascular drugs were the most commonly used level 1 anticholinergics, followed by antidepressants and opioids. Antidepressants and urologicals were the most commonly used level 3 anticholinergics. The rate of anticholinergic deprescribing was equivalent to the rate of anticholinergic initiation, and commonly involved the level 1 drugs warfarin, furosemide and temazepam, and the level 3 drugs amitriptyline and oxybutynin. People with dementia had a higher ACL at baseline and year one compared with other participants. CONCLUSION: ACL of community-dwelling older people was very stable over time. This may represent lost opportunities for deprescribing as well as potentially inappropriate prescribing, particularly in those with cognitive impairment.

Effectiveness of a peer-mediated educational intervention in improving general practitioner diagnostic assessment and management of dementia: a cluster randomised controlled trial.

OBJECTIVE: Test effectiveness of an educational intervention for general practitioners (GPs) on quality of life and depression outcomes for patients. DESIGN: Double-blind, cluster randomised controlled trial. SETTING: General practices in Australia between 2007 and 2010. PARTICIPANTS: General practices were randomly allocated to the waitlist (n=37) or intervention (n=66) group, in a ratio of 1:2. A total of 2030 (1478 intervention; 552 waitlist) community-dwelling participants aged 75 years or older were recruited via 168 GPs (113 intervention; 55 waitlist). INTERVENTIONS: A practice-based academic detailing intervention led by a peer educator that included: (1) training in use of the GP assessment of cognition dementia screening instrument; (2) training in diagnosis and management based on Royal Australian College of General Practitioners Dementia Guidelines; (3) addressing GPs' barriers to dementia diagnosis; and (4) a business case outlining a cost-effective dementia assessment approach. OUTCOME MEASURES: Primary outcome measures were patient quality of life and depression; secondary outcome measures were: (1) sensitivity and specificity of GP identification of dementia; (2) referral to medical specialists and/or support services; (3) patient satisfaction with care; and (4) carer quality of life, depression and satisfaction with care. RESULTS: The educational intervention had no significant effect on patient quality of life or depression scores after 12 months. There were however improvements in secondary outcome measures including sensitivity of GP judgement of dementia (p=0.002; OR 6.0, 95% CI 1.92 to 18.73), satisfaction with GP communication for all patients (p=0.024; mean difference 2.1, 95% CI 0.27 to 3.93) and for patients with dementia (p=0.007; mean difference 7.44, 95% CI 2.02 to 12.86) and enablement of carers (p=0.0185; mean difference 24.77, 95% CI 4.15 to 45.40). CONCLUSION: Practice-based academic detailing did not improve patient quality of life or depression scores but did improve detection of dementia in primary care and patient satisfaction with GP communication. TRIAL REGISTRATION NUMBER: ACTRN12607000117415; Pre-results.

An evaluation of the additional benefit of population screening for dementia beyond a passive case-finding approach.

OBJECTIVE: General practitioners (GPs) fail to identify more than 50% of dementia cases using the existing passive case-finding approach. Using data from the "Ageing in General Practice" study, we sought to establish the additional benefit of screening all patients over the age of 75 for dementia beyond those patients already identified by passive case-finding. METHOD: Patients were classified as "case-finding" (n = 425) or "screening" (n = 1006) based on their answers to four subjective memory related questions or their GP's clinical judgement of their dementia status. Cognitive status of each patient was formally assessed by a research nurse using the Cambridge Cognition Examination (CAMCOG-R). Patients then attended their usual GP for administration of the GP assessment of Cognition (GPCOG) dementia screening instrument, and follow-up care and/or referral as necessary in light of the outcome. RESULTS: The prevalence of dementia was significantly higher in the case-finding group (13.6%) compared to the screening group (4.6%; p < 0.01). The GPCOG had a positive predictive value (PPV) of 61% in the case-finding group and 39% in the screening group; negative predictive value was >95% in both groups. GPs and their patients both found the GPCOG to be an acceptable cognitive assessment tool. The dementia cases missed via case-finding were younger (p = 0.024) and less cognitively impaired (p = 0.020) than those detected. CONCLUSION: There is a very limited benefit of screening for dementia, as most people with dementia could be detected using a case-finding approach, and considerable potential for social and economic harm because of the low PPV associated with screening.

Screening for Dementia in Primary Care: A Comparison of the GPCOG and the MMSE.

BACKGROUND/AIMS: The General Practitioner Assessment of Cognition (GPCOG) is a brief cognitive test. This study compared the GPCOG to the Mini-Mental State Examination (MMSE), the most widely used test, in terms of their ability to detect likely dementia in primary care. METHODS: General practitioners across three states in Australia recruited 2,028 elderly patients from the community. A research nurse administered the GPCOG and the MMSE, as well as the Cambridge Examination for Mental Disorders of the Elderly Cognitive Scale-Revised that we used to define likely dementia. RESULTS: Overall, the GPCOG and the MMSE were similarly effective at detecting likely dementia. The GPCOG, however, had a higher sensitivity than the MMSE when using published cutpoints. CONCLUSION: The GPCOG is an effective screening tool for dementia in primary care. It appears to be a viable alternative to the MMSE, whilst also requiring less time to administer.

Impact of multiple low-level anticholinergic medications on anticholinergic load of community-dwelling elderly with and without dementia.

BACKGROUND: Elderly people, particularly those with dementia, are sensitive to adverse anticholinergic drug effects. This study examines the prevalence of anticholinergic medication, and anticholinergic load and its predictors, in community-dwelling elderly patients (aged 75 years and older) in Australia. METHODS: A research nurse visited the home of each participant (n = 1,044), compiled a list of current medications, and assessed participants' cognitive status using a subsection of the revised Cambridge Examination for Mental Disorders of the Elderly (CAMCOG-R). Anticholinergic load was determined for each patient using the Anticholinergic Drug Scale (ADS). RESULTS: Multivariate analysis identified several patient factors that were associated with higher anticholinergic burden, including polypharmacy (i.e. taking five or more medications) (p < 0.001), increasing age (p = 0.018), CAMCOG-R dementia (p = 0.003), depression (p = 0.003), and lower physical quality of life (p < 0.001). The dementia group (n = 86) took a significantly higher number of medications (4.6 vs. 3.9; p = 0.04), and had a significantly higher anticholinergic load (1.5 vs. 0.8; p = 0.002) than those without dementia (n = 958). Approximately 60% of the dementia group and 40% of the non-dementia group were receiving at least one anticholinergic drug. This difference was due to the higher proportion of dementia patients taking level 1 (potentially anticholinergic) (p = 0.002) and level 3 (markedly anticholinergic) (p = 0.005) drugs. CONCLUSIONS: There is considerable scope for the improvement of prescribing practices in the elderly, and particularly those with dementia. Importantly, level 1 anticholinergics have been identified as major contributors to the anticholinergic load in people with dementia. Longitudinal studies are required to determine the effects of increased and decreased anticholinergic load on cognitive function and other clinical outcomes for people with dementia.

Predictors of agreement between general practitioner detection of dementia and the revised Cambridge Cognitive Assessment (CAMCOG-R).

BACKGROUND: Dementia is a complex and variable condition which makes recognition of it particularly difficult in a low prevalence primary care setting. This study examined the factors associated with agreement between an objective measure of cognitive function (the revised Cambridge Cognitive Assessment, CAMCOG-R) and general practitioner (GP) clinical judgment of dementia. METHODS: This was a cross-sectional study involving 165 GPs and 2,024 community-dwelling patients aged 75 years or older. GPs provided their clinical judgment in relation to each of their patient's dementia status. Each patient's cognitive function and depression status was measured by a research nurse using the CAMCOG-R and the 15-item Geriatric Depression Scale (GDS), respectively. RESULTS: GPs correctly identified 44.5% of patients with CAMCOG-R dementia and 90% of patients without CAMCOG-R dementia. In those patients with CAMCOG-R dementia, two patient-dependent factors were most important for predicting agreement between the CAMCOG-R and GP judgment: the CAMCOG-R score (p = 0.006) and patient's mention of subjective memory complaints (SMC) to the GP (p = 0.040). A higher CAMCOG-R (p < 0.001) score, female gender (p = 0.005), and larger practice size (p < 0.001) were positively associated with GP agreement that the patient did not have dementia. Subjective memory complaints (p < 0.001) were more likely to result in a false-positive diagnosis of dementia. CONCLUSIONS: Timely recognition of dementia is advocated for optimal dementia management, but early recognition of a possible dementia syndrome needs to be balanced with awareness of the likelihood of false positives in detection. Although GPs correctly agree with dimensions measured by the CAMCOG-R, improvements in sensitivity are required for earlier detection of dementia.

Investigation of the human disease osteogenesis imperfecta: a research-based introduction to concepts and skills in biomolecular analysis.

A blended approach encompassing problem-based learning (PBL) and structured inquiry was used in this laboratory exercise based on the congenital disease Osteogenesis imperfecta (OI), to introduce commonly used techniques in biomolecular analysis within a clinical context. During a series of PBL sessions students were presented with several scenarios involving a 2 year old child, who had experienced numerous fractures. Key learning goals related to both the theory and practical aspects of the course, covering biomolecular analysis and functional genomics, were identified in successive PBL sessions. The laboratory exercises were conducted in 3 hour blocks over six weeks, focused firstly on protein analysis, followed by nucleic acids. Students isolated collagen from normal and OI affected fibroblast cultures. Analysis by SDS-PAGE demonstrated α1 and α2 of collagen Type I chains at approximately 95 kDa and 92 kDa, respectively. Subtle differences in protein mobility between the control and OI samples were observed by some students, but most considered it inconclusive as a diagnostic tool. The nucleic acid module involved isolation of RNA from OI affected fibroblasts. The RNA was reverse transcribed and used as template to amplify a 354 bp COL1A1 fragment. Students were provided with the sequence of the OI affected COL1A1 PCR product aligned with the normal COL1A1 sequence, allowing identification of the mutation, as the substitution of Arg for Gly(976) of the triple helical region. Our experience with student cohorts over several years is that presentation of this laboratory exercise within a relevant clinical context, and the opportunity for active engagement with the experimental procedures via PBL sessions, supported the learning of basic theory and practical techniques of biomolecular analysis.

Diagnosis and disclosure of a memory problem is associated with quality of life in community based older Australians with dementia.

BACKGROUND: Identification of factors associated with quality of life (QoL) in people having dementia will help develop strategies for maintenance and improvement of patient QoL. This study examined the predictors of QoL in a community-dwelling population aged 75 years and over, with or without dementia. METHODS: This was a cross-sectional study involving 169 GPs and 2,028 patients. Patients were interviewed to collect information on personal circumstances. Several instruments were administered including the WHOQOL-BREF (quality of life outcome measure), Geriatric Depression Scale, GPAQ (satisfaction with GP care), and the CAMCOG-R (cognitive function). Patients with a CAMCOG-R score < 80 were allocated to the dementia group. GPs provided an independent clinical judgment of cognitive function for each of their participating patients. RESULTS: The dementia group had significantly lower QoL scores in all four domains of the WHOQOL-BREF (all p ≤ 0.002). The GDS score was negatively correlated with all four domains in the non-dementia group and with physical, psychological, and environmental QoL in the dementia group (all p < 0.001). Satisfaction with GP communication was positively associated with psychological QoL in the dementia group and all domains in the non-dementia group. Participants in the dementia group who had been given a diagnosis of a memory problem had significantly higher physical (2.05, 95% CI 0.36 to 3.74) and environmental (2.18, 95% CI 0.72 to 3.64) QoL. CONCLUSIONS: Satisfaction with GP communication is associated with a higher QoL in their older patients. Diagnosis and disclosure of memory problems is associated with better QoL in people with dementia. Clinicians should not be deterred from discussing a memory diagnosis and plans for the future with patients.

Ageing in general practice (AGP) trial: a cluster randomised trial to examine the effectiveness of peer education on GP diagnostic assessment and management of dementia.

BACKGROUND: Dementia is increasing in prevalence as the population ages. An earlier rather than later diagnosis allows persons with dementia and their families to plan ahead and access appropriate management. However, most diagnoses are made by general practitioners (GPs) later in the course of the disease and are associated with management that is poorly adherent to recommended guidelines. This trial examines the effectiveness of a peer led dementia educational intervention for GPs. METHODS: The study is a cluster randomised trial, conducted across three states and five sites. All GPs will complete an audit of their consenting patients aged 75 years or more at three time points - baseline, 12 and 24 months. GPs allocated to the intervention group will receive two educational sessions from a peer GP or nurse, and will administer the GPCOG to consenting patients at baseline and 12 months. The first education session will provide information about dementia and the second will provide individualised feedback on audit results. GPs in the waitlist group will receive the RACGP Guidelines by post following the 12 month audit OUTCOMES: Primary outcomes are carer and consumer quality of life and depression. Secondary outcomes include: rates of GP identification of dementia compared to a more detailed gold standard assessment conducted in the patient's home; GP identification of differential diagnoses including reversible causes of cognitive impairment; and GP referral to specialists, Alzheimers' Australia and support services. A "case finding" and a "screening" group will be compared and the psychometrics of the GPCOG will be examined. SAMPLE SIZE: Approximately 2,000 subjects aged 75 years and over will be recruited through approximately 160 GPs, to yield approximately 200 subjects with dementia (reducing to 168 by 24 months). DISCUSSION: The trial outlined in this paper has been peer reviewed and supported by the Australian National Health and Medical Research Council. At the time of submission of this paper 2,034 subjects have been recruited and the intervention delivered to 114 GPs. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12607000117415.

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein.

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Towards a ZP-based contraceptive for marsupials: comparative analysis and developmental expression of marsupial ZP genes.

Fertility control in the form of a zona pellucida (ZP)-based immunocontraceptive has shown potential as a humane form of control for overabundant marsupials including the brushtail possum and macropods. Further refinement and development of a ZP-based vaccine requires detailed knowledge of the protein structure and expression in order to ensure maximum efficacy and specificity. Sequencing and comparative analysis of the ZP3 protein from three marsupial orders in this study found a high overall level of conservation; within order Diprotodontia, the ZP3 protein is 86.9-98.9% identical. ZP3 identity falls to 56.6-57.2%, when the grey, short-tailed opossum (a Didelphimorphian) is compared to dasyurid and diprotodontan marsupials. This is similar to its amino acid identity with ZP3 from eutherian species (50.7-52.8%). Comparison of a 21 amino acid epitope in marsupial ZP3 that has shown contraceptive effects, reveals 95-100% identity between the four macropodid species, 81-86% amino acid identity between brushtail possum and the macropods and 67-71% identity between the diprotodontans and the fat-tailed dunnart (a dasyurid). This is comparable to the level of identity between related eutherian mammals. The expression pattern of three ZP genes during brushtail possum and tammar wallaby pouch young development was examined by RT-PCR. This analysis of ZP gene expression has confirmed that ZP mRNA transcription begins in the ovary during pouch young development by about 51 days of age. The presence of ZP transcripts at this stage in pouch young development suggests that marsupial ZP gene transcription begins before the onset of follicular development.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial, the brushtail possum (Trichosurus vulpecula).

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E. coli) strain JM109, M15, SG13009, or BL21 codon plus. Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3. Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3. Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence. Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only. The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials.

Timing and ultrastructure of events following intracytoplasmic sperm injection in a marsupial, the tammar wallaby (Macropus eugenii).

The aim of the present study was to determine the timing of oocyte activation, sperm decondensation and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the tammar wallaby and to determine the fate of sperm structures at an ultrastructural level. Metaphase II-stage tammar wallaby oocytes were injected with spermatozoa and cultured for 1 (n = 15), 2 (n = 24), 4 (n = 30), 6 (n = 14), 8 (n = 32), 10 (n = 25), 12 (n = 29) or 19 h (n = 12). Oocytes were assessed using light, fluorescence and electron microscopy. The timing of oocyte activation and sperm decondensation after ICSI in the tammar wallaby is relatively similar to that of some eutherian species. Resumption of meiosis II was observed from 1 h and the first female pronucleus was seen 6 h after ICSI. Most oocytes (88%) possessed a female pronucleus by 10 h. Intact acrosomes persisted with intact sperm heads up to 2 h after ICSI. At 10 h, 80% of oocytes possessed a male pronucleus. The sperm tail had undergone considerable degeneration by 10 h after ICSI, including breakdown of the fibrous sheath dense fibres. The identification of sperm tail and midpiece remnants adjacent to pronuclei confirms that the events observed in wallaby oocytes after ICSI are not due to parthenogenetic activation.