Osmometric Behavior, Hydraulic Conductivity, and Incidence of Intracellular Ice Formation in Bovine Oocytes at Different Developmental Stages

Abstract

Bovine oocytes that were immature (IMM), matured in vitro (IVM) or in vivo (MAT), or matured and fertilized in vitro (IVF) were studied using a microscope diffusion chamber to estimate osmotic parameters and a cryomicroscope to characterize intracellular ice formation (11F). Linear Boyle van't Hoff relationships were observed with all four types of oocytes between 0.265 and 0.799 osm NaCl. At 20°C, estimates of hydraulic conductivity ( L p ) were significantly higher for IVM oocytes than IMM and MAT oocytes (0.84 μm/(min · atm) vs 0.45 and 0.47, respectively). IVM oocytes also tended to have higher L p values than IVF oocytes (0.55 μm/(min · atm)). At 5°C, the L p of IVM oocytes decreased to 0.36 μm/min · atm) corresponding to an Arrhenius activation energy of 7.84 kcal/mol. The incidence of IIF in MAT oocytes suspended in salt solution and subjected to linear cooling to - 60°C was 45% at 4°C/min, 75% at 8°C/min, and 93% at 16°C/min; with IVF oocytes, the incidence of IIF was 40% at 4°C/min. 92% at 8°C/min, and 100% at 16°C/min. Comparisons involving median IIF temperatures (THF 50s) and the distributions of the observed IIF temperatures for IMM (Myers et al., Cryo-Lett. 8, 260), IVM (Chandrasekaran et al., Cryobiology 27, 676), MAT and IVF oocytes indicated that the IIF incidence in IMM oocytes cooled at 4°C/min was greater than that of oocytes at the other developmental stages cooled at the same rate. The THF 50s of IVM and IVF oocytes were lowered by equilibration in 1.5 M ethylene glycol (EG), glycerol, or propylene glycol (PG) prior to cooling, with EG tending to lower the THF 50s more than glycerol or PG. For all three cryoprotectants, the THF 50s and IFF temperature distributions were cooling-rate dependent. The Weibull probability distribution was fitted to the distributions of the IIF temperatures of oocytes suspended in salt solutions with and without cryoprotectants yielding R 2 values ranging from 0.70 to 0.98.