M.C.J. Paris

Cryopreservation of ovarian tissue: An emerging technology for female germline preservation of endangered species and breeds

Many hundreds of exotic species and domestic animal breeds have been lost over the course of the last few decades. In order to avoid a similar fate to other animals threatened with extinction, it is crucial to develop and apply rescue strategies to ensure their survival for the future. One option as a safeguard measure is the cryopreservation of the main source of female gametes enclosed within the ovary: the primordial follicles. So far, there are three options to cryopreserve small ovarian follicles: whole ovary, ovarian cortical tissue or isolated follicles, with the use of slow freezing or vitrification methods. After cryopreservation, the harvested material can be transplanted or cultured, with the aim to produce mature fertilizable oocytes. The objective of this review is to summarize the current status of the cryopreservation of ovarian tissue in domestic species and non-endangered wild mammals as model for threatened and endangered species and breeds, and to provide new insights into techniques that can be applied in the future.

Xenotransplantation: a tool for reproductive biology and animal conservation?

The transplantation of reproductive organs, including ovaries and ovarian tissue, was pioneered over 100 years ago. In the 1960s, ovarian grafting was used as a tool to investigate ovarian function, but with the recent development of more effective cryopreservation protocols for ovarian tissue, germline preservation and propagation have now become realistic goals. This review describes progress in ovarian banking and ovarian tissue transplantation, with emphasis on how fresh and frozen ovarian tissue can be used in assisted reproduction for both humans and animals. This paper focuses most closely on the potential value of xenotransplantation, the transplantation of gonads from one species to another, to conserve rare and endangered species. Specific attention is drawn to the use of xenotransplantation as a strategy for generating viable gametes that can be used to produce live fertile offspring. Other upcoming xenogeneic technologies that may be of potential significance in animal conservation, such as transplantation of whole ovaries or isolated growing follicles, and even male germ cells, are discussed.

Faecal progesterone metabolites and behavioural observations for the non-invasive assessment of oestrous cycles in the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons)

Wombats belong to Australias unique marsupial species. Two of the three remaining species, the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons) are abundant. The third species, the northern hairy-nosed wombat (Lasiorhinus krefftii) has only about 115 individuals left in the wild. This study aimed to gain further insight into the basic reproductive biology of wombat species and evaluate the value of faecal progesterone metabolites and behavioural patterns as a means for non-invasive monitoring of the oestrous cycle in common and the southern hairy-nosed wombats. In an initial study, three different faecal steroid assays showed that 20alpha-OH-pregnanes were the main progesterone metabolites. These metabolites were examined in captive female common wombats (n = 5) and southern hairy-nosed wombats (n = 2). In one female common wombat 11.7 days with a follicular phase of 25.6 +/- 6.3 days and a luteal phase of 28.2 +/- 12.7 days. The data for faecal pregnanes obtained in the southern and in one male common wombat oestrous related behavioural data were obtained. Individual cycling females exhibited a significant relationship between plasma progesterone and faecal pregnanes. In the common wombat, the values for faecal pregnanes showed an oestrous cycle length of 55.1 +/- hairy-nosed wombat during the breeding season gave an oestrous cycle length of 41.1 +/- 12.8 days with a follicular phase of 27.9 +/- 12.3 days and a short luteal phase of 13.3 +/- 1.1 days. The behavioural data show that the faecal sniffing behaviour of the male, tended to increase around the time that oestrous was found. In conclusion, monitoring of 20alpha-OH-pregnanes in wombat faeces could be a useful methodology to monitor reproductive cycles in the wombat, and can possibly be applied to monitor the endangered northern hairy-nosed wombat.

Ovarian and testicular tissue xenografting: its potential for germline preservation of companion animals, non-domestic and endangered species

In the present paper we aim to review the development of both ovarian and testicular xenografting with specific emphasis on its usage for companion animals, non-domestic and endangered species. Ovarian and testicular tissue xenografting has been used successfully across a variety of species for the harvesting of mature gametes and subsequent fertilisation. It has become a novel and promising tool to explore various aspects of testicular development and function and was useful for determination of gonadotoxic treatments on xenografted gonads. In rodent animal models live births have been reported using gametes from xenografted gonadal tissue. Live births were also reported after grafting of human ovarian tissue. We envisage that it will not be long before the first live births across other species, including companion animals, non-domestic and endangered species, will be achieved.

Comparison of the effects of in vitro and in situ storage on the viability of mouse ovarian tissue collected after death

Ovarian tissues, collected or salvaged from endangered species at the time of gonadectomy or following their death, are being transported to genebanks for storage with the assumption that they will (subsequently) yield sufficient numbers of germ cells to help preserve the species. The present study aimed to quantify the impact of delays in collecting and/or processing ovarian tissue on the number of follicles in this tissue that remained normal after grafting. The study compared the viability of ovarian tissue stored in vitro (in phosphate-buffered saline) versus in situ (in the body) either on ice or at room temperature for 0 (non-stored fresh grafts), 3, 6, 12, 24 or 48 h. The conditions of storage had significant effects on the total number of morphologically normal follicles, with significantly more follicles in grafts developing from in vitro-stored tissue than in situ-stored tissue. Storage temperature and duration of storage, but not the storage temperature alone, influenced follicle survival. Tissue that was grafted immediately after collection (0 h) was best, but normal follicles were recovered in grafts stored in vitro (on ice or at room temperature) or in situ (on ice only) for up to 48 h before grafting. The rate of follicle loss over time was very rapid, with approximately 50% fewer follicles in grafts derived from tissue stored for only 3 h compared with non-stored fresh grafts (0 h). The results show that viable ovarian tissue can be salvaged from animals up to 48 h after death; however, in order to best protect the follicle population, the ovaries should be removed from the animals body as soon as possible.

Sex, stress and social status: patterns in fecal testosterone and glucocorticoid metabolites in male Ethiopian wolves.

Ethiopian wolves, Canis simensis, live in large multi-male family packs, where males are philopatric and do not disperse. Within a pack, mating and breeding is largely monopolized by the dominant male and female, although extra-pack copulations are common, and subordinate males may sire pups in neighboring packs. Regardless of paternity, all males in a pack help rear the pups. We non-invasively studied patterns in fecal testosterone and glucocorticoid metabolite concentrations using radioimmunoassays of fecal samples collected from nine wild male Ethiopian wolves between August 2007 and February 2008. We tested the predictions of the Challenge Hypothesis, namely that fecal testosterone metabolite concentrations would be higher during the annual mating season, which is the portion of the reproductive cycle when mating and increased aggression typically occur, and lower when there were pups in the pack for which to care. Contrary to the predictions of the Challenge Hypothesis, we did not detect patterns in fecal testosterone metabolite concentrations associated with reproductive stage during our study period. Similarly, we found no patterns associated with reproductive stage in male fecal glucocorticoid metabolite concentrations. Dominant males had higher average fecal testosterone and glucocorticoid metabolite concentrations than did subordinates, which may be related to higher rates of aggression and mate guarding in dominant males of group-living canids, a pattern also reported in African wild dogs, Lycaon pictus.

Oestrous cycle of the common wombat, Vombatus ursinus, in Victoria, Australia.

Wild-caught female common wombats from Victoria, Australia, were studied in captivity to investigate the oestrous cycle by assessing vaginal cytology and peripheral plasma progesterone concentrations. Eight wombats, five adults (21-29 kg) and three subadults (19-23 kg), which were held for between 2 weeks and 11 months did not cycle in captivity. Their progesterone concentrations were consistently low (< or = 6.9 nmol L(-1)) and vaginal smears contained predominantly superficial epithelial cells. Three wombats (21-27 kg), held in captivity for >1 year, regularly cycled (when bodyweights exceeded 23.5 kg). Information gathered from four consecutive cycles in each of these three wombats revealed a follicular phase with low progesterone concentrations (< or = 6.9 nmol L(-1)) and vaginal smears with a high percentage of superficial epithelial cells alternating with periods of high progesterone concentrations (range 41.6-123.8 nmol L(-1)) and smears in which parabasal-intermediate epithelial cells predominated. The average length of the monitored oestrous cycles was 47.2 days (35-60 days). The follicular phase lasted ~19 days and the luteal phase lasted ~28 days. In conclusion, wombats can cycle regularly in captivity even under conditions of intensive monitoring.

Patterns of scat deposition by brown hyaenas Hyaena brunnea in a mountain savannah region of South Africa

Abstract The brown hyaena Hyaena brunnea is a near threatened large carnivore inhabiting sub-Saharan Africa. Like many other species of terrestrial carnivores, brown hyaenas often and repeatedly deposit scats at specific latrine sites as a means of olfactory communication. However, previous studies on brown hyaena latrine use have been constrained to the arid Kalahari region in southern Africa, an area of low resource abundance. To improve our understanding of geographic variation in the biology of this species, we monitored patterns of brown hyaena scat deposition in the Waterberg of northern South Africa, an area of higher net productivity than previous areas for published brown hyaena studies. Defecation rates at latrine sites were low in our study area (median: < 1 defecation in 30 days), but brown hyaenas visited sites significantly more often than they defecated at them (median: 2.6 visits/30 days). The temporal patterns of activity at defecation sites were significantly related to the overall temporal activity patterns of brown hyaenas on the roads within the reserve, and generally confirmed a nocturnal activity pattern in the species. Our result on brown hyaena scat deposition in the Waterberg region indicates a geographic variation in latrine use, and we suggest that such a variation could be linked to resource-driven variation in social and spatial organisation.

The physiology of cooperative breeding in a rare social canid; sex, suppression and pseudopregnancy in female Ethiopian wolves

Ethiopian wolves, Canis simensis, differ from other cooperatively breeding canids in that they combine intense sociality with solitary foraging, making them a suitable species in which to study the physiology of cooperative breeding. The reproductive physiology of twenty wild female Ethiopian wolves (eleven dominant and nine subordinate) in Ethiopias Bale Mountains National Park was studied non-invasively through the extraction and assaying of estradiol, progesterone and glucocorticoids in collected fecal samples using enzyme and radioimmunoassays. All dominant females showed increased estradiol concentrations and/or mating behavior during the annual mating season. In contrast, none of the subordinate females showed increased estradiol concentrations or mating behavior during the mating season. However, two subordinate females came into estrus outside of the mating season. Both dominant and subordinate females had higher average progesterone concentrations during the dominant females pregnancy than at other times of the year, and two subordinate females allosuckled the dominant females pups. No statistically significant differences in glucocorticoid concentrations were found between dominant and subordinate females. These results suggest that subordinate females are reproductively suppressed during the annual mating season, but may ovulate outside of the mating season and become pseudopregnant. No evidence was found to suggest that reproductive suppression in subordinate females was regulated through aggressive behaviors, and no relationship was found between fecal glucocorticoids and dominance status.

Validation of Noninvasive Monitoring of Adrenocortical Endocrine Activity in Ground-Feeding Aardwolves (Proteles cristata): Exemplifying the Influence of Consumption of Inorganic Material for Fecal Steroid Analysis

Biologically inert material in feces may confound interpretations of noninvasive fecal endocrine data, because it may induce variance related to differences in foraging behavior rather than to differences in endocrine activity. We evaluated two different enzyme immunoassays (EIAs) for the noninvasive evaluation of adrenocortical activity in ground-feeding aardwolves (Proteles cristata) and tested the influence of soil content in aardwolf feces on the interpretation of fecal glucocorticoid metabolite data. Using adrenocorticotropic hormone (ACTH) challenges for validation, we successfully identified a cortisol EIA suitable for assessing adrenocortical activity in aardwolves. An alternatively tested 11-oxoetiocholanolone EIA failed to detect a biologically relevant signal after ACTH administration. Although the proportion of inorganic content in aardwolf feces did not alter qualitative conclusions from the endocrine data, the data related to mass of organic content had a larger amount of variance attributed to relevant biological contrasts and a lower amount of variance attributed to individual variation, compared with data related to total dry mass of extracted material. Compared with data expressed as dry mass of extracted material, data expressed as mass of organic content may provide a more refined and statistically powerful measure of endocrine activity in species that ingest large amounts of indigestible material.