Distribution of active mitochondria in canine oocytes is related to reproductive cycle stage but can be damaged during IVM culture

Aim We investigated the effect of the reproductive cycle stage on the distribution of active mitochondria in canine oocytes examined 1) at collection and 2) after in vitro maturation (IVM). Methods Cumulus-oocyte complexes (ooplasmic size >120 µm in diameter) were recovered from 20 bitches divided into five groups based on their reproductive status: anestrous (A, n=4), follicular phase (F, n=4), ovulation (O, n=2), early luteal phase (until 15 days after ovulation, EL, n=7) and mid/late luteal phase (MLL, n=3). IVM culture was performed in TCM199 with 10% estrous canine serum (72h, 5% CO2). Oocyte mitochondrial (mt) distribution pattern was revealed after 30’ incubation in 280 nM MitoTracker Orange CMTM Ros and confocal laser scanning microscopy. Data were analyzed by Chi-square Test. Results In oocytes examined at collection, three mt patters were found: I) small granules diffused throughout the cytoplasm; II) diffused tubular networks; III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II and III) were obtained from bitches in F (21/28, 75%) and in O (23/24, 96%) compared with bitches in A (4/13, 31%; F vs A: P<0.05; O vs A: P<0.001), in EL (27/44, 61%; F vs EL: NS; O vs EL: P<0.01), or in MLL (0/9, 0%; F vs MLL: P<0.05; O vs MLL: P<0.001). In cultured oocytes, the maturation rates (MII+PB) did not differ statistically among reproductive stages (1%, 1/72 vs 6%, 12/189 vs 2% 1/58 vs 4% 2/47 vs 9%, 5/53, for oocytes from bitches in A, F, O, EL and MLL, respectively). The only oocyte from an A bitch had reticular mt pattern. The 12 oocytes from bitches in F showed tubular networks (3/12, 25%) or granular mt distribution (9/12, 75%). The only oocyte from an O bitch and all oocytes from bitches in EL (n=2 oocytes) and from bitches in MLL (n=5 oocytes) showed granular mt distribution. Conclusions Our data demonstrate that the distribution of active mt in canine immature oocytes is related to cycle stage. However, the used IVM culture system did not yield high MII rates as well as mt distribution patterns expressive of full cytoplasmic maturation. This compromised oocyte energy status may be one of the factors responsible for overall reported low MII rates in this species.