Mitotic index and aneuploidy variation in groving day 2- to day 7- IVP bovine embryos of the Agerolese breed of cattle

The present study reports on the variation of the mitotic index in growing day 2- to day 7- IVP bovine embryos of the Agerolese breed of cattle. After IVM (24 hours), COCs were transferred into 25/well with 300 μl IVF-TALP and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45-80%). The pellet was diluted in IVF medium and added to the COCs at the concentration of 1 x 106 sperm/mL. Gametes were co-incubated for 20-22 h at 39°C, in 5% CO2 in air. After co-incubation, presumptive zygotes were vortexed to remove cumulus cells and randomly allocated in six groups, each into 400 μl of SOF medium, with 30 μl/ml essential amino acids, 10 μl/ml non-essential amino acids, 0.34 mM tri-sodium citrate, 2.77 mM myo-inositol, and 5% BS. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2 and 88% N2 in air at 39°C for 20-22 hours. Starting on Day 0 ( IVF day), embryos were taken out of the incubator at day 2(48 h),3 (72h),4 (96h), 5(120h), 6(144h) and 7(168h), examined under a stereomicroscope, treated in a lysing buffer (0.01 N HCl, 0.1% Tween 20) for 30 sec, transferred in a small droplet to a precleaned slide and fixed with methanol-acetic acid (3:1). Out of 178 embryos (3,100 cells) analyzed, the mitotic index was 19.6 % (28/143 cells) at day 2, 18.6 % (41/221 cells) at day 3, 10.3 % (27/263 cells) at day 4, 7.1 % (26/367 cells) at day 5, 0.7 % (5/681 cells) at day 6 and 0.6 (9/1425 cells) at day 7. FISH analysis is undergoing and will be reported elsewhere.