Membrane integrity and development of immature murine cumulus–oocyte complexes following slow cooling to −60 °C: The effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling

Cryopreservation of murine germinal vesicle (GV) stage cumulus–oocyte complexes (COCs) has been shown to result in poor development and cumulus cell damage. In an attempt to determine the stage of the cryopreservation protocol at which damage occurs, three cooling profiles were compared: slow-cooling (0.3 °C/min) to −60 °C (protocol A); slow-cooling to −60 °C and plunging to −196 °C (protocol B); or slow-cooling to −60 °C followed by further cooling at 10 °C/min to −150 °C, then plunging to −196 °C (protocol C). GV-stage COCs were collected from hormone-primed mice by repeated puncturing of ovarian follicles. COCs were exposed to 1.5 M Me2SO prior to cooling to −60 or −196 °C. Membrane integrity was assessed immediately after thawing using carboxy fluorescein and propidium iodide. A greater proportion of cumulus cells were damaged following protocol B than protocol A. Damage was less extensive following protocol C than following protocol B. For assessment of development, COCs were matured and fertilised in vitro. Morphological normality was significantly reduced following cooling to −60 or −196 °C compared with non-cryopreserved controls. Fertilisation of oocytes assessed as normal post-treatment was not significantly different between any of the groups. Development to blastocyst was least from oocytes exposed to protocol B, being significantly worse than for oocytes exposed to protocol A, but not significantly different to protocol C. A protocol comprising two stages of controlled-rate cooling decreased damage to the membranes of cumulus cells but did not significantly improve embryo development.