Bioenergetic/oxidative balance in feline morulae and blastocysts produced in vitro after temporary oocyte holding at room temperature versus cold ovary storage.

Maternal aging is associated with an increase in embryonic aneuploidy and early miscarriage, as a result of errors in chromosome segregation [1]. Failure to achieve correct alignment of the chromosomes on the spindle is an important factor contributing to mis-segregation errors in oocytes [2,3]. Since little data is available in horses, the aim of this study was to evaluate the effect of maternal aging on spindle morphology and the incidence of chromosomes misalignment. Cumulus oocyte complexes (COCs) were recovered from slaughtered mares, divided into groups depending on mare age (young, < 15 years; old, ≥15 years) and matured in vitro for 26h. After maturation and denudation, only the oocytes that had reached MII were used and these were further subdivided into Control and Nocodazole groups (total = 4 groups; n=20 per group). Oocytes in the Nocodazole groups were incubated for 10 min in medium containing 20 µM Nocodazole, to induce tubulin depolymerization, washed and then incubated for 2 hrs in maturation medium. In order to destabilize any tubulin fibers not properly attached to the kinetochores samples were subjected to cold shock, fixed and stored at 4˚C prior to immunofluorescent staining for DNA and alphatubulin. Spindle morphology and the incidence of chromosome misalignment were evaluated by confocal microscopy and 3D image analysis (Imaris 8.3). Spindle morphology was scored as normal (fusiform, bipolar) or abnormal (tri- or tetra-polar, severely misshapen), chromosome misalignment was scored as absent, mild (1-5 misaligned chromosomes) or severe (>5 misaligned chromosomes). Oocytes from aged mares showed higher rates of mild and severe chromosome misalignments when compared to those from young mares, both in normal condition (mild 37 vs 5%; severe 11 vs 0%) and after Nocodazole treatment (mild 42 vs 15%; severe 21 vs 0%). Moreover, oocytes from old mares were more likely to show abnormal spindle morphology both under control conditions (5 vs 0%) and after Nocodazole treatment (10 vs 0%). Although nocodazole treatment did not result in a significant increase in chromosome misalignment and spindle abnormalities, the incidence of chromosomal misalignments increased numerically in both aged and young groups (total % of misalignment without treatment 47.4 and 4.5% vs 63.2 and 15% after nocodazole treatment). We suggest that the compromised ability to form a normal meiotic spindle and correctly align the chromosomes observed in MII oocytes from aged mares might reflect an impaired function of the spindle assembly check point components, and explain the age-related reduction in oocyte developmental competence.