271 VIABILITY OF OOCYTES FROM CANINE OVARIES GRAFTED IN THE PROXIMAL PORTION OF THE BODY SURFACE

Abstract

Numerous studies have reported that follicles in ovarian tissue are not only able to survive ovarian grafting but also to grow from the early to advanced stages of follicular development. The site of grafting is very important for tissue survival and follicle development after transplantation. Several grafting techniques have been reported, basically differing only in the location to which the ovarian grafts have been transplanted, such as the bursal cavity, kidney capsule, and subcutaneous sites. The objective of the present study was to determine the viability of canine oocytes from ovaries transplanted in the proximal portion of the body surface. Bilateral ovariectomy was performed using a ventral midline abdominal approach with routine techniques and materials. After excision of ovaries from 10 bitches (4–7 y of age), each ovary of the pairs was cut approximately in half using a scalpel blade. The hemi-ovaries were kept in physiological saline, maintained at 38°C, and then transplanted to the animal of origin in each body site. The ovaries were transplanted under the fascia of the left quadriceps femoris muscle, the left thoracolumbar fascia, and the deltoid muscle in left scapular region. As control, a remaining hemi-ovary was used to examine the morphology and viability of oocytes from fresh ovaries without transplantation. All grafted ovaries were recovered from animals at 28 to 31 days after transplantation under the anesthesia. After collection, each ovary was sliced repeatedly to release oocytes. All oocytes collected from ovaries were examined microscopically for morphological quality and categorized by the classification as previously described (Hewitt et al. 1998 Theriogenology 49, 1083–1101). Grade A oocytes were darkly pigmented and surrounded by one or more layers of cumulus cells, Grade B oocytes were lightly pigmented with incomplete layers of cumulus cells, and Grade C oocytes were pale coloured and irregularly shaped without any cumulus cells. Only Grade A oocytes were cultured in maturation medium for 72 h at 38.5°C in a humidified atmosphere of 5% CO₂ in air. After incubation for 72 h, the oocytes were denuded, fixed, and stained by Hoechst 33342 to examine the nuclear maturation. After grafting for 28 to 31 days, a total of 5, 7, and 4 oocytes were recovered, respectively, from hemi-ovaries transplanted into quadriceps femoris fascia, thoracolumbar fascia, and scapular deltoid muscle, of which 1 and 2 oocytes from quadriceps femoris fascia and thoracolumbar fascia were classified into Grade A, respectively. After maturation culture of 3 oocytes, one oocyte reached the metaphase II stage. In the control group, a total number of 183 oocytes were collected from fresh ovaries, and 20.8% of these oocytes were classified into Grade A, of which 6.1% of oocytes reached the metaphase II stage after maturation culture. These results indicate that some oocytes from grafted ovaries can develop and maintain meiotic competence even when the ovaries are transplanted under the fascia in the proximal portion of the body surface.