Oocytes, recovered from the oviduct or from ripe follicles, and spermatozoa flushed from the oviduct of opossums mated under laboratory conditions, were used to study the characteristics of marsupial gamete interaction and sperm incorporation. Opossum oocytes lose all granulosa cells before ovulation and are invested only by a thin zona pellucida when ovulated. The spermatozoa unpair in the oviduct but the motile population flushed at about the time of ovulation includes paired, separating and single spermatozoa. Separation of paired spermatozoa was associated with changes in the acrosome, the amorphous matrix being displaced by membrane-bound vacuoles. Fertilization is normally monospermic. Spermatozoa associated with the zona of unfertilized eggs by the flat acrosomal face of the head, whereas those trapped in mucoid eventually laid down around the zona were orientated at random. In penetrating the zona, the spermatozoon created a relatively large uneven hole that contrasts with the discrete penetration slit seen in the thicker zona of eutherian oocytes. Spermatozoa appeared to associate and fuse with the oolemma by the acrosomal face of the head. Ultrastructurally the sperm head, in oocytes fixed immediately after incorporation into the ooplasm, was devoid of surrounding membranes. This pattern of gamete interaction resembles the mode seen in non-mammalian vertebrates and invertebrates, and not that in eutherian mammals. Sperm penetration by tubal sperm samples occurred readily in vitro within 1 h of placing ripe follicular oocytes with the oviducal flushings containing spermatozoa. None of the oocytes penetrated in vivo or in vitro displayed extra perivitelline spermatozoa. This suggests that the oocyte is able to mount a block to polyspermy at the zona surface, although the mucoid deposited on the zona pellucida may be involved in vivo. The results indicate that the complex mode of sperm incorporation seen in eutherian mammals is unique to the infraclass and not shared by their closest relatives the marsupials. differences between their gametes suggest that this may also be the case for the mode of

Captive breeding is an integral part of global conservation efforts despite high costs and adverse genetic effects associated with unavoidably small population sizes. Supplementing captive-bred populations with biobanked founder sperm to restore genetic diversity offers a solution to colony size, costs and inbreeding, yet is rarely done, partly due to a lack of concrete examples or awareness amongst the conservation community of the huge potential benefits. We present a model system of the cost and genetic benefits achieved by incorporating biobanking into captive breeding of Oregon spotted frogs (Rana pretiosa). Backcrossing with frozen sperm every generation resulted in very large reductions in required programme expenditure compared to traditional captive breeding. This model supports the view that integration of biobanking into captive breeding would make longstanding and previously unachievable genetic diversity retention targets feasible (90% source population heterozygosity for a minimum of 100 years) at much reduced costs. This study suggests that the credibility of captive breeding as a conservation strategy would be enhanced by integrating genome storage and assisted breeding to produce far larger numbers of animals of higher genetic quality. This innovation would justify increased public and agency support for captive breeding.

The reproductive tracts of males from eight species of Australian marsupial were examined (Macropus eugenii, Potovous tridactyhs, Sminthopsis crassicaudata, Antechinus stuartii, Pseudocheirus peregrinus, Trichosurus vulpecula, Isoodon macrourus, and Perameles nasuta). The prostate glands of these species were found to be of two shapes, carrot-like or heart-like. From one to three pairs of Cowper's glands were observed; these were mostly bulbous in shape but some were kidney-shaped. Both prostate and Cowper's glands were tubular in structure with the glandular tubules lined by a simple columnar epithelium. The glandular tubules of Cowper's glands were of much larger diameter than those of the prostate. The prostate glands were segmented, and this segmentation was usually shown by variations in the height and staining reactions of the tubular epithelium and in the volume of connective tissue between glandular tubules. Differences in microanatomy between pairs of Cowper's glands were far less than those between prostate segments. Mucosubstance appeared to be the major contribution of the prostate to the seminal plasma. This mucosubstance was mainly neutral, with glycogen largely absent. The present results indicate that the Cowper's glands secrete mucus but that various glands also contributed lipid and glycogen.

Rewilding is emerging as a major issue in conservation. However, there are currently a dozen definitions of rewilding that include Pleistocene rewilding, island rewilding, trophic rewilding, functional rewilding and passive rewilding, and these remain fuzzy, lack clarity and, hence, hinder scientific discourse. Based on current definitions, it is unclear how the interventions described under the rewilding umbrella differ from those framed within the long-standing term 'restoration'. Even projects held up as iconic rewilding endeavours invariably began as restoration projects (e.g., Oostvaaderplassen; Pleistocene Park; the return of wolves to Yellowstone, etc.). Similarly, rewilding organisations (e.g., Rewilding Europe) typically began with a restoration focus. Scientific discourse requires precise language. The fuzziness of existing definitions of rewilding and lack of distinction from restoration practices means that scientific messages cannot be transferred accurately to a policy

Summary. Ovulation in the tammar wallaby alternates between the ovaries. The genital duct of each side enters the median vaginal culs-de-sac separately. Post-partum oestrus occurred 0\m=.\4days after birth and ovulation 1 day later. After a single copulation spermatozoa were found in both cervical canals at 0\m=.\5h and extended to the oviduct on the non-parturient side only by 8 h. Very few spermatozoa were found in sections of the post-partum uterus or its associated oviduct at any time. Spermatozoa were recovered by flushing from both sides but the numbers were 2\p=n-\20times greater in the non\x=req-\ parturient than in the post-partum side: the greatest difference occurred in the cervical canals 2\p=n-\5h after copulation. In females which had undergone a previous infertile cycle, spermatozoa were abundant in both cervices and both uteri. It is concluded that the differential distribution of spermatozoa in post-partum animals was (1) due to failure of transport in the recently pregnant side of the tract, rather than attraction of spermatozoa to the ovulation side, and (2) established at the cervix which, on the ovulation side, provides a reservoir of spermatozoa for 24 h after copulation.

Fertilization has been achieved in superovulated brushtail possums and tammar wallabies after laparoscopic intrauterine artificial insemination. Various superovulation protocols and insemination times were examined but a maximum of 2-5 eggs including 1-2 embryos per possum were recovered. The female possums were superovulated by treatment with 15 iu pregnant mares' serum gonadotrophin and then either GnRH (4 x 50 micrograms, at intervals of 90 min) or 4 mg LH, 3 days later. Inseminations were performed within 6 h before or 4-10 h after (pregnant mares' serum gonadotrophin-GnRH group only) the expected onset of ovulation using epididymal spermatozoa. Superovulation in wallabies was achieved by treatment with FSH (8 x 6 mg, at intervals of 12 h for 4 days) followed by 4 mg LH on day 5. Inseminations were performed 4-6 h before the expected onset of ovulation using ejaculated spermatozoa, which resulted in the recovery of 7-8 eggs including 3-4 embryos per female. All embryos recovered were from possums and wallabies examined 1-2 days after insemination and included fertilized eggs, two-cell and four-cell embryos. Motile spermatozoa were recovered from the oviducts and uteri but only immotile spermatozoa were found in the vaginal complex. Five to thirty per cent of spermatozoa recovered from the oviducts of possums examined 2-6 h after insemination had thumbtack morphology, which is thought to be correlated with capacitation. Although embryo yields per female were low, this study has established that intrauterine artificial insemination after superovulation is a feasible assisted breeding strategy for marsupials with implications for species conservation and population control.

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii ), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis : the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm-egg fusion site proposed for Monodelphis domestica (Taggart et al. 1993). The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principle piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.Key words : Marsupial spermiogenesis ; spermatogenesis ; spermatids ; testicular spermatozoa ; acrosome ; tammar wallaby. Spermiogenesis, the remarkable morphological and biochemical transformation of the round spermatid into the elongate spermatozoon, is one of the most complex cell differentiations found in animals. During this process of spermatozoon formation the spermatid reduces its cell volume more than a hundred times, radically changes its shape and produces a number of morphologically elaborate organelles, including the acrosome, the midpiece and the flagellum. The biochemical transformation of the spermatid is equally complex. During this period the proteins of the unique cytoskeletal el...

Of 14 lactating opossums maintained in laboratory conditions, 13 mated 4.7-8.5 days after removal of pouch young. The time between this removal and onset of receptive oestrus was negatively correlated with the age of the pouch young. Mating generally occurred between 24:00 and 06:00 h, with ovulation following between 13:00 and 16:00 h. Each animal ovulated a mean of 29.6 eggs (range 19-40), approximately equal numbers coming from both ovaries. Spermatozoa were absent from the uterus and were present only in the oviducts during the periovular period. Those not cleared by flushing (1-160 x 103/oviduct) remained incarcerated in isthmic crypts lined by a simple cuboidal epithelium. Spermatozoa in crypts were paired, separating or single. The progressively motile cells flushed from the oviduct presented a similar pattern to that in the crypts, about 30% of spermatozoa were firmly paired, the others either loosely associated or single. Only single spermatozoa attached to ova. Monospermic fertilization followed shortly after ovulation, and no supplementary spermatozoa were present in the perivitelline space. Deposition of the mucoid layer on the zona pellucida then began, often before incorporation of the fertilizing spermatozoon by the vitellus was complete. The oviducal epithelium was formed throughout by ciliated and secretory cells. In the ampulla and upper isthmus, the secretory cells produced the mucoid material which formed a thick coat over the egg surface. Ovum transit through the oviduct was rapid, in one animal eggs had reached the uterus and acquired a shell within 15-20 h of ovulation.