Mitochondria play a critical role in oocyte developmental competence by providing energy to the oocyte through oxidative phosphorylation. We hypothesised that oxygen consumption, a measure of mitochondrial function, would be lower in the oocytes of old versus young mares as a potential mechanism of reduced developmental competence associated with equine aging. To test this hypothesis, we used a novel electrochemical sensor device to compare basal and maximal oxygen consumption rates of oocytes obtained from mares aged 6 to 12 years (Young, n=8 from 7 mares) and=20 years (Old, n=12 from 8 mares). Cumulus-oocyte complexes (COC) were collected by transvaginal, ultrasound-guided follicular aspirations of dominant follicles (35mm) during oestrus and at 16±2h after induction of follicular maturation (2000IU of hCG and 0.75mg of deslorelin acetate). Recovered COC were incubated in medium [TCM-199; Life Technologies, Grand Island, NY, USA) with 10% fetal calf serum, 25mg mL−1 gentamicin, and 0.2mM pyruvate] at 38.5°C in 5% CO2 and air for 26±2h. After maturation, COC were stripped of cumulus cells and checked for the presence of an extruded polar body before electrochemical measurements using amperometry. A microchamber with a 3-electrode system (working, counter, and reference electrodes) was filled with 120µL of medium (G-MOPS™, Vitrolife, Englewood, CO, USA) and overlaid with 120µL of paraffin oil (OVOIL™, Vitrolife). The baseline current was measured before adding the oocyte through the oil layer and over the centre of the working electrode. Oxygen consumption was monitored as the linear decrease in the oxygen reduction current over time, which was converted to femtomoles of O2 consumed per second (fmol s−1) using a calibration curve for the sensor. Basal (nonstimulated) oxygen consumption was measured for 5min, followed by assessment of maximal noncoupled respiratory capacity obtained by titrating 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 8-min intervals. The highest value observed during CCCP titrations was considered the maximal oxygen consumption. Two-tailed Student’s t-tests were used to analyse data. Oocytes from young mares had higher basal oxygen consumption compared with old mares, respectively (mean±s.e.m., 1.74±0.18 and 1.27±0.09 fmol s−1; P=0.04) as well as higher maximal oxygen consumption (2.67±0.19 and 2.05±0.12 fmol s−1; P=0.016). Mitochondrial efficiency (maximal/basal oxygen consumption) was not different for Young and Old, respectively (1.62±0.13 and 1.65±0.07; P=0.9). In summary, we used a novel electrochemical sensor device to measure basal and maximal oxygen consumption of oocytes and to confirm our hypothesis that aging impairs mitochondrial respiratory capacity in equine oocytes, which may contribute to age-associated changes in equine fertility.
Tongue Rehabilitation Device for Dysphagic Patients
