Ovarian tissue xenografting may be applied to increase the population size of rare or endangered animals. However, optimal grafting conditions, such as graft position and recipient hormonal status, are yet to be established. The present study, using common wombat ovarian tissue, showed that development of xenografted ovarian tissue to the antral follicle stage can be achieved irrespective of graft position. However, increased graft recovery rates and follicle survival were evident after grafting under the kidney capsule compared with grafting to subcutaneous sites. No increase in follicle development was observed after placing grafts both under the kidney capsule and subcutaneously in the one recipient compared with grafts placed under the kidney capsule alone or subcutaneously alone. Removal of the recipient's own ovaries at the time of grafting accelerated graft follicle development, with antral follicles seen by Week 12 after grafting compared with by Week 16 in recipients that retained their own ovaries. More oocytes were collected from xenograft recipients receiving hormonal stimulation before collection compared with non-stimulated recipients. No oocytes were mature (extruded a polar body) at the time of collection or after a subsequent period of in vitro maturation. This is the first study to demonstrate that antral follicle development can occur and oocytes can be collected from xenografted common wombat ovarian tissue.
Developmentally competent oocytes can be collected from xenografted ovarian tissues; however, optimal xenograft conditions need to be established for this technique to be of use in assisted reproduction. In the present study, common wombat ovarian tissue was xenografted under the kidney capsule of nude mice to clarify the role of recipient gonadal status and donor tissue age on graft establishment, follicle development and oocyte recovery. Eighty-nine per cent of all grafts were recovered; of these, 78% contained growing follicles. In female graft recipients, follicle development to the antral stage occurred earlier in ovariectomised recipients compared with intact graft recipients. Similarly, follicle development occurred earlier in recipients of pouch young ovarian tissue grafts when compared with subadult xenografts. Follicle development proceeded to the antral stage in subadult grafts placed under the kidney capsule of male recipient mice, albeit at a slower rate than subadult grafts placed in female recipients. Oocytes were collected from grafts placed in female and male recipients, but no mature oocytes were observed at the time of collection, nor could these oocytes be matured in vitro. The present study demonstrated that common wombat pouch young tissue xenografted to female recipient mice, and subadult ovarian tissue xenografted to male recipient mice, can develop to the antral stage and can therefore facilitate oocyte collection. However, mature oocytes were not obtained using the current protocol.
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 animal's body as soon as possible.
Assisted reproductive techniques, such as in vitro oocyte maturation in conjunction with in vitro fertilisation, may be used as a tool to manipulate reproduction. Using the common wombat as a model for the critically endangered northern hairy-nosed wombat, the present study examined whether oocyte maturation could be achieved under field conditions. At the time of collection, no oocytes were at the metaphase II (MII) stage (0/42). After 60 h culture using the submarine incubation system, 34% of oocytes (24/70) matured to telophase/MII, as indicated by the presence of a polar body. The proportion of oocytes that reached MII was higher for oocytes collected from follicles >2 mm in diameter compared with follicles <2 mm (40% v. 22%, respectively). The presence of cumulus cells alone did not influence the maturation potential. Oocytes without cumulus cells collected from follicles >2 mm in diameter had the highest maturation rate (58%). Maturation was not affected by the reproductive status of the common wombat or a delay of up to 5 h before oocyte collection. In conclusion, the present study demonstrated that oocytes collected from non-stimulated common wombats can mature to MII in culture.
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