Modeling genetic benefits and financial costs of integrating biobanking into the conservation breeding of managed marsupials.

Managed breeding programs are an important tool in marsupial conservation efforts but may be costly and have adverse genetic effects in unavoidably small captive colonies. Biobanking and assisted reproductive technologies (ARTs) could help overcome these challenges, but further demonstration of their potential is required to improve uptake. We used genetic and economic models to examine whether supplementing hypothetical captive populations of dibblers (Parantechinus apicalis) and numbats (Myrmecobius fasciatus) with biobanked founder sperm through ARTs could reduce inbreeding, lower required colony sizes, and reduce program costs. We also asked practitioners of the black-footed ferret (Mustela nigripes) captive recovery program to complete a questionnaire to examine the resources and model species research pathways required to develop an optimized biobanking protocol in the black-footed ferret. We used data from this questionnaire to devise similar costed research pathways for Australian marsupials. With biobanking and assisted reproduction, inbreeding was reduced on average by between 80% and 98%, colony sizes were on average 99% smaller, and program costs were 69- to 83-fold lower. Integrating biobanking made long-standing captive genetic retention targets possible in marsupials (90% source population heterozygosity for a minimum of 100 years) within realistic cost frameworks. Lessons from the use of biobanking technology that contributed to the recovery of the black-footed ferret include the importance of adequate research funding (US$4.2 million), extensive partnerships that provide access to facilities and equipment, colony animals, appropriate research model species, and professional and technical staff required to address knowledge gaps to deliver an optimized biobanking protocol. Applied research investment of A$133 million across marsupial research pathways could deliver biobanking protocols for 15 of Australia's most at-risk marsupial species and 7 model species. The technical expertise and ex situ facilities exist to emulate the success of the black-footed ferret recovery program in threatened marsupials using these research pathways. All that is needed now for significant and cost-effective conservation gains is greater investment by policy makers in marsupial ARTs.

Los programas de reproducción controlada son una herramienta importante para los esfuerzos de conservación de marsupiales, aunque pueden resultar costosos y tener efectos genéticos adversos en las colonias cautivas incapaces de aumentar en tamaño. Los biobancos y las tecnologías de reproducción asistida (TRA) podrían ayudar a superar estos problemas, pero es necesario seguir demostrando su potencial para mejorar su adopción. Utilizamos modelos genéticos y económicos para analizar si la introducción de esperma fundador proveniente de biobancos mediante tecnologías de reproducción asistida a poblaciones cautivas hipotéticas de los marsupiales Parantechinus apicalis y Myrmecobius fasciatus podría reducir la endogamia, disminuir el tamaño efectivo de las colonias y reducir el costo de los programas. También pedimos a los profesionales del programa de recuperación en cautiverio del hurón de patas negras (Mustella nigripes) que respondieran un cuestionario para analizar los recursos y los métodos de investigación de las especies modelo necesarias para desarrollar un protocolo de biobanco optimizado para el hurón de patas negras. Utilizamos los datos de este cuestionario para diseñar métodos de investigación con costos similares para los marsupiales australianos. Con el biobanco y la reproducción asistida, la endogamia se redujo en promedio entre un 80 y un 98%, el tamaño de las colonias fue en promedio un 99% más pequeño y los costos del programa entre 69 y 83 veces menores. La integración del biobanco posibilitó los objetivos de retención genética en cautiverio a largo plazo en marsupiales (90% de heterocigosidad de la población de origen durante un mínimo de 100 años) dentro de un marco realista de costos. Entre el aprendizaje extraído del uso de la tecnología de biobancos que contribuyó a la recuperación del hurón de patas negras figuran la importancia de una financiación adecuada de la investigación (4.2 millones de dólares), colaboraciones profundas que faciliten el acceso a instalaciones y equipos, colonias de animales, especies modelo adecuadas para la investigación y el personal profesional y técnico necesario para abordar las lagunas de conocimiento y ofrecer un protocolo optimizado para los biobancos. Una inversión en investigación aplicada de 133 millones de dólares australianos para la investigación de los marsupiales podría proporcionar protocolos de biobancos para 15 de las especies de marsupiales australianos en mayor riesgo y 7 especies modelo. Existen los conocimientos técnicos y las instalaciones ex situ para emular el éxito del programa de recuperación del hurón de patas negras en marsupiales amenazados utilizando estas vías de investigación. Ahora sólo se necesita una mayor inversión por parte de los responsables políticos de las TRA para marsupiales para obtener beneficios de conservación significativos y rentables.

Modelling Genetic Benefits and Financial Costs of Integrating Biobanking into the Captive Management of Koalas.

Zoo and wildlife hospital networks are set to become a vital component of Australia's contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive colonies. Emerging evidence suggests that biobanking and associated assisted reproductive technologies could address these economic and genetic challenges. We present a modelled scenario, supported by detailed costings, where these technologies are optimized and could be integrated into conservation breeding programs of koalas across the established zoo and wildlife hospital network. Genetic and economic modelling comparing closed captive koala populations suggest that supplementing them with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower the required colony sizes of conservation breeding programs, and greatly reduce program costs. Ambitious genetic retention targets (maintaining 90%, 95% and 99% of source population heterozygosity for 100 years) could be possible within realistic cost frameworks, with output koalas suited for wild release. Integrating biobanking into the zoo and wildlife hospital network presents a cost-effective and financially feasible model for the uptake of these tools due to the technical and research expertise, captive koala colonies, and ex situ facilities that already exist across these networks.

Resetting the paradigm of reproductive science and conservation.

In the application of reproductive science to conservation breeding, it has long been assumed that artificial insemination using frozen thawed sperm would be the default technology. This has always been problematic considering the wide range of tolerance to freeze thawing among vertebrate sperm. Furthermore, those providing leadership for genome banking should be proactive to preserve maximum genetic diversity, however, for many species there is little or no sperm motility after thawing of cryopreserved sperm. In this review article, there is the contention that a much wider range of tissues should be banked, and the range of evolving advanced reproductive and developmental technologies should be considered for conservation breeding programs, to realize the maximum opportunities of genome banking to contribute to conservation of animal species.

Integrating biobanking could produce significant cost benefits and minimise inbreeding for Australian amphibian captive breeding programs.

Captive breeding is an important tool for amphibian conservation despite high economic costs and deleterious genetic effects of sustained captivity and unavoidably small colony sizes. Integration of biobanking and assisted reproductive technologies (ARTs) could provide solutions to these challenges, but is rarely used due to lack of recognition of the potential benefits and clear policy direction. Here we present compelling genetic and economic arguments to integrate biobanking and ARTs into captive breeding programs using modelled captive populations of two Australian threatened frogs, namely the orange-bellied frog Geocrinia vitellina and the white bellied frog Geocrinia alba . Back-crossing with frozen founder spermatozoa using ARTs every generation minimises rates of inbreeding and provides considerable reductions in colony size and program costs compared with conventional captive management. Biobanking could allow captive institutions to meet or exceed longstanding genetic retention targets (90% of source population heterozygosity over 100 years). We provide a broad policy direction that could make biobanking technology a practical reality across Australia's ex situ management of amphibians in current and future holdings. Incorporating biobanking technology widely across this network could deliver outcomes by maintaining high levels of source population genetic diversity and freeing economic resources to develop ex situ programs for a greater number of threatened amphibian species.

Marsupials the alternative therians - From gametes to birth.

Marsupial reproduction shares many common features with the more familiar eutherian mammals but things are often done differently, in alternative ways. Like the eutherians marsupials are placental but the period and degree of development supported in the uterus is much shorter and the long growth phase of development is supported by lactation. But these different ways of achieving often similar outcomes are also seen in gamete formation and function, fertilization and early development. This review presents an overview of marsupial reproductive biology with an emphasis on gamete biology.

Induction of synchronous oestrus but not ovulation after pre-treatment with the GnRH agonist, Lucrin® Depot, in the tammar wallaby.

We investigated the capacity for pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce synchronous oestrus and ovulation in the tammar wallaby (Notamacropus eugenii) after follicular suppression with Lucrin® Depot, a one-month GnRH agonist. On Day 0 pouch young were removed (RPY) to reactivate a normal oestrous cycle and then two groups were treated with vehicle (Control; n = 5 and Superovulation (SOvn); n = 5) and two groups were treated with 7.5 mg of Lucrin Depot (Lucrin; n = 6; Lucrin+SOvn, n = 6). On Day 20 RPY the SOvn and Lucrin+SOvn Group received 20 IU of PMSG, which was followed on Day 23 RPY with 500 IU of hCG. The Lucrin+SOvn females underwent a more synchronous oestrus with 5 of 6 mating on Day 26 RPY while the SOvn (n = 5/5) and Control (n = 5/5) Groups copulated over two days, between Day 25-27 RPY and Day 27-29 RPY respectively. Mating plugs were not detected in any of the females in the Lucrin Group by Day 31 RPY. Autopsy on Day 31 RPY confirmed all females in each treatment group had undergone a reactivated cycle as evidenced by the presence of a large corpus luteum (CL) in one ovary. At autopsy the females in the Lucrin+SOvn Group had highly stimulated reproductive tracts, and their ovaries contained many follicles >3 mm; 14 ± 2.1 and 15.3 ± 2.1 follicles >3 mm in the CL-bearing ovary and contralateral ovary respectively. Similarly, females in the SOvn Group had 11.4 ± 2.4 and 17.4 ± 1.9 follicles >3 mm in each respective ovary. Uterine flushing and ovarian histology confirmed that females in Lucrin+SOvn and SOvn Groups had not ovulated, but normal oocytes were present in the follicles. By comparison, the Control Group had ovulated with a single embryo being recovered from the uterus of 4 of 5 females. In contrast to all groups, females in the Lucrin Group showed follicular suppression (all follicles <1.5 mm) and an unstimulated reproductive tract. We conclude that a suppression plus stimulation regimen using Lucrin Depot followed by PMSG and hCG has the capacity to synchronise oestrus, and that 20 IU of PMSG stimulates the development of antral follicles >3 mm in both ovaries. However, a single 500 IU treatment of hCG on Day 23 RPY was not able to induce ovulation in the tammar wallaby.

Marsupials: Progress and Prospects.

The chapter provides a review of the application of reproductive science to technologies for marsupial conservation and population management and discusses prospects for the future. This includes the status of technologies such as sperm freezing, artificial insemination, and exogenous hormone treatments to stimulate ovarian activity and cycling in the female. Fertility-based population management for introduced pest species and over-abundant native marsupials remain an elusive goal. Immune-contraceptive approaches, despite demonstration of basic effectiveness, have not progressed to field deliverable agents. Emerging genetic technologies such as gene drives offer great promise, but gene modifications face major challenges to be broadly accepted both socially and politically. A main theme is the potential advantages, both genetic and economic, of integrating frozen stored genomic material, such as sperm, into the captive breeding component of threatened species strategies. However, the sperm of many marsupial species display no or very poor recovery of motility on thawing. For this reason, it is proposed that the traditional assisted breeding paradigm for conservation-cervical artificial insemination with thawed frozen sperm, based on cattle breeding-is not a viable default strategy. Rather, techniques such as sperm injection and emerging stem cell technologies that utilize stored frozen cells, and in the case of sperm, immotile cells, are better candidates for the development of a more generic approach. In addition, this change in focus encourages wide scale proactive genome storage when genetic diversity is greatest, without the need to demonstrate success in traditional sperm cryopreservation and thawing. However, the promise of the potential of reproductive science to conservation and non-lethal population management is problematic without far greater recognition of, and investment in, the needs of wildlife by society.

Delayed return to estrus following treatment with the gonadotrophin-releasing hormone agonist, Lucrin® Depot, in the tammar wallaby.

Gonadotrophin-releasing hormone agonists that induce a reversible contraceptive state in several marsupials have the potential to be used to synchronize estrus. We used a model macropod, the tammar wallaby (Notamacropus eugenii), to investigate whether Lucrin Depot (AbbVie), a GnRH agonist microsphere preparation, could (1) inhibit follicular development and estrus in a cycle reactivated by removal of pouch young (RPY) and (2) facilitate a synchronous return to estrus. Our results show that females reactivated with bromocriptine and RPY in early seasonal quiescence (July 2015) were inhibited by Lucrin Depot (0.125-0.5 mg kg -1, n = 9) and unlike control females (n = 3), did not copulate before Day 32 RPY. During the next breeding season (February 2016), the return to estrus after RPY was not delayed in animals treated with Lucrin Depot (≤0.20 mg kg -1; n = 12), and copulation occurred in treated and control females within the expected natural period after RPY (Day 26-33 RPY). In the following breeding season (March 2017), estrus was delayed in animals treated with Lucrin Depot (1.25 mg kg -1) on either Day 0 (Group A, n = 6) or Day 10 (Group B, n = 6) after RPY compared to control females (n = 6). Estrus was detected in Group A between 39 and 66 days (55 ±â€¯4.8d) and in Group B between 43 and 71 days (55.2 ±â€¯3.9d) after RPY. In contrast, all control females underwent estrus and copulated as expected by Day 30 RPY. We conclude Lucrin Depot can inhibit ovarian follicular activity after RPY but as a standalone treatment does not result in a highly synchronous return to estrus in the tammar wallaby.

Breeding in the fat-tailed dunnart following ovarian suppression with the gonadotrophin-releasing hormone agonist Lucrin® Depot.

Lucrin Depot (AbbVie), a 1-month microsphere gonadotrophin-releasing hormone (GnRH) agonist preparation, was investigated as a potential agent to synchronise cycling in the fat-tailed dunnart (Sminthopsis crassicaudata). Forty-eight randomly selected females were treated with 5 or 10mgkg-1 Lucrin Depot (n=24 per dose). Eighteen females per treatment had their reproductive activity scored at 4, 8, 12 and 16 weeks using two ovarian (Graafian follicle and corpus luteum status) and two reproductive tract (uterine and vaginal muscularity and vascularity) parameters that formed a reproductive activity score. Six females per treatment were paired with a male at 4 weeks. Fertility was assessed between 8 and 16 weeks by pouch check, and thereafter by dissection. The effects of the 5 and 10mgkg-1 doses were statistically equivalent. Females showed suppression at 4-8 weeks, an increase in reproductive activity at 8-12 weeks and all were cycling normally at 16 weeks. Six pouch young were born at 12 weeks to two females treated with the 5mgkg-1 dose. Nine embryos were recovered at 16 weeks from two females treated with the 10mgkg-1 dose. In conclusion, Lucrin Depot can suppress breeding, and fertile mating can occur in subsequent cycles in the dunnart. There is potential for Lucrin Depot to be used as an assisted breeding tool, but it may need to be combined with ovarian stimulation treatment to achieve practical levels of synchronisation in the fat-tailed dunnart.

Recent advances in tools and technologies for monitoring and controlling ovarian activity in marsupials.

Components of assisted reproduction technologies (ART), such as sperm cryopreservation, artificial insemination, superovulation and pouch young surrogacy, have been developed for a range of Australian and American marsupials. However, methods to effectively control ovarian function, arguably the key limiting factors in applying and integrating ART as a practical tool in conservation management, remain poorly developed. This is largely due to unique characteristics of the marsupial corpus luteum and its failure to respond to agents used to synchronize ovarian function in eutherian mammals. This paper presents an overview of relevant aspects of marsupial reproductive biology across marsupial taxonomic groups including information on the long-established technique of removal of suckling young to activate ovarian cycles. Ovarian monitoring tools for marsupials are reviewed and their usefulness for ART assessed (laparotomy, hormone cycling, vaginal cytology, laparoscopy and ultrasonography). We also discuss promising recent work examining the potential of manipulating hypothalamic-pituitary function using GnRH agonists and antagonists as the basis of ovarian control (female synchronization) strategies.

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.

Mammalian hair as an accumulative bioindicator of metal bioavailability in Australian terrestrial environments.

The current study represents the first investigation of the suitability of marsupial and eutherian mammalian hair as indicator tissue for metal exposure and accumulation within contaminated Australian terrestrial ecosystems. A soil metal contamination gradient was established across 22 sites at increasing distances from a decommissioned Lead/Zinc smelter in NSW, Australia. Within each site, soil and small mammal populations were sampled. An Australian native marsupial, the insectivorous Brown Antechinus, Antechinus stuartii: Dasyuridae, and introduced rodents, the omnivorous Brown or Norway Rat, Rattus norvegicus: Muridae and the Black Rat, Rattus rattus: Muridae were assessed for hair concentrations of Cadmium (Cd), Copper (Cu), Lead (Pb) and Zinc (Zn). Metals in soil were most elevated at sites within close proximity to the smelter, with soil metal concentrations decreasing with distance from the smelter. The non-essential metals Pb and Cd were accumulated in hair, both metals exhibiting positive linear relationships with environmental exposure (soil metal concentrations). When the variables of weight and snout-vent length were considered, no further contribution in terms of explaining the variability in hair Cd or Pb was observed for all species examined. The essential metals Cu and Zn were regulated in hair, remaining similar across the metal contamination gradient. A significant negative correlation between snout-vent length and hair Cu concentration was found for the Brown Rat; greater hair Cu concentrations were found in smaller individuals of this species. Accumulation of Pb to hair was similar among species while concentrations of Cd in Brown Rat hair were higher than both Black Rat and Brown Antechinus hair. As each of the three aforementioned species exhibit similar bioaccumulation relationships for Pb, we suggest that sampling hair from introduced rodents (pest species) may provide a suitable proxy for the assessment of Pb bioavailability for a range of small mammals within Australian urban remnants.

Artificial insemination in marsupials.

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.

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.

Characterisation of an epitope shared by an acrosomal acrosin-like protein and the surface of tammar wallaby (Macropus eugenii) spermatozoa.

Monoclonal antibodies (mAb) have been raised against marsupial sperm proteins to provide insights into the molecular nature of marsupial spermatozoa, and the proteins that mediate sperm maturation and interaction with the oocyte. This study reports the production of a mAb, designated WSA-1, which bound acrosomal and surface determinants on tammar wallaby spermatozoa. The acrosomal antigen was first detected in the wallaby testis; however, ejaculated spermatozoa demonstrated whole cell WSA-1 immunoreactivity as a result of binding an epididymal protein. Ultrastructural and agglutination analyses localised the WSA-1 epitope to the acrosomal matrix and the whole sperm plasmalemma. The WSA-1 mAb bound three polypeptides with relative molecular weights of 35, 31 and 15 kDa on western blots under reducing conditions. The N-terminal amino acid sequence obtained for the 35 kDa wallaby sperm polypeptide demonstrated identity with the eutherian acrosomal protein acrosin. The 31 kDa polypeptide was of epididymal origin and will be the subject of a separate study. Further studies of the WSA-1 antigens are likely to provide useful insights into the function and maturation of marsupial sperm since proacrosin has a number of putative roles in eutherian fertilisation, and epididymal proteins are thought to mediate sperm maturation and storage.

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums.

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.