John C. Rodger

Reproductive science and integrated conservation

CONSERVATION and reproduction are inextricably linked, for reproduction is the science of the transmission of life, and conservation is the ability to maintain that life. The theme of this book is that reproductive science in all its multiple sub-disciplines is an essential (but often overlooked) component for all successful conservation. The very first statement in the volume makes this message clear “Therefore, the study of reproduction is fundamental to conserving species, populations and, indirectly, the vitality of entire ecosystems”. The overriding message is just how little we know, and for only a handful of vertebrate species. The lack of basic research is of great concern, for, as Short (1985) so succinctly stated, “we cannot conserve until we comprehend”. This compendium of papers from world experts makes a start at redressing our ignorance.

Why so many mammalian spermatozoa - a clue from marsupials ?

Mammals generally ejaculate many more spermatozoa than seem to be needed for fertilization. This apparent profligacy has not been explained, but observations made in marsupials may shed light on it. The Virginia opossum, Didelphis virginiana, inseminates only about three million spermatozoa, a very low number. As a corollary, relatively few (ca. 13 x 106) are stored in each cauda epididymidis. However, some 5% of the spermatozoa that the opossum ejaculates populate the oviduct about 12 h later when ovulation can be anticipated - a success rate in the female orders of magnitude greater than in eutherian mammals. I t is not certain what determines the unusually efficient transport to and the high survival rate of spermatozoa in the oviduct of Didelphis, but two unusual features suggest themselves as possible contributors. Didelphis (and all other American marsupial) spermatozoa undergo a head-to-head pairing in the epididymis by the acrosomal face; this serves to isolate the acrosome of ejaculated spermatozoa from the female milieu until the pairs separate in the oviduct. Secondly, spermatozoa are housed in special crypts in the isthmus of the oviduct. Australian marsupials, which usually lack such features, store spermatozoa in the epididymis in numbers more close to those in comparably sized eutheriam mammals. Exceptions which store very low sperm numbers there can be seen in one Australian Family, the Dasyuridae. The spermatozoa of dasyurids are not paired, but the species examined possess distinctive sperm storage crypts in the oviducal isthmus similar to those in the opossum. The present findings suggest that where mechanisms exist that could protect the acrosome and, or, the whole spermatozoon in the female tract, a much lower level of sperm production can be maintained without compromising fertility. While the number ejaculated typically by any one species is probably determined ultimately by several interacting factors, it therefore seems likely that a most important one in this respect relates to conditions spermatozoa face in the female tract.

Lack of a requirement for a maternal humoral immune response to establish or maintain successful allogeneic pregnancy.

In general, breeding pairs are not major histocompatibility complex (MHC)-compatible, and therefore the fetoplacental unit can be considered as a natural allograft. In many mammals pregnancy leads to the production of nonlytic antibodies of antipaternal MHC specificity. It has been suggested that these protect the semiallogeneic fetus from rejection by acting as blocking or enhancing factors—or, alternatively, that they are part of a humoral response involved in the establishment of normal pregnancy. These hypotheses were tested in allomated mice made B cell deficient by continuous treatment with αIgM

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

mU antiserum. The status of the maternal immune system was assessed by in vivo antibody production, in vitro mitogen responses, and allograft rejection. By these criteria B cell function could not be demonstrated in αIgM

Ultrastructural evidence of the onset of testicular pathological conditions in the cryptorchid human testis within the first year of life.

mU treated female mice, but T cell responses were unaffected. Allogeneic pregnancy, however, was not compromized by this humoral immune system dysfunction—litter size and neonatal survival being the same in the αgM

Spermiogenesis and spermiation in a marsupial, the tammar wallaby (Macropus eugenii)

mU and control serum-treated groups. These results indicate that a maternal humoral immune response is not essential for the establishment of pregnancy or the survival of the semiallogeneic: fetus.

Artificial insemination in marsupials

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.

Manipulation of the fertility of marsupials for conservation of endangered species and control of over-abundant populations

Pathological fibrosis of testicular intertubular tissues may be evident in the cryptorchid testis of children 1 year old. A characteristic feature of such testes is massive collagen deposition, which disrupts the regular order of the interstitium, in particular the peritubular layer of cells, by the time the child is 4 years old. These conclusions are based on ultrastructural examination of testicular biopsies collected at orchiopexy from 13 boys with cryptorchidism between 1 and 13 years old. These findings persuade us that orchiopexy should be performed in children about 1 year old and, certainly, no later than 2 years. Otherwise, eventual normal testicular function is highly unlikely.

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro

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 principal 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.

Origin of Glycerylphosphorylcholine, Inositol, N-Acetylaminosugar, and Prostaglandins in Human Seminal Plasma and Their Effects on Sperm Metabolism

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.