Microtubulin configuration and mitochondrial distribution after ultra‐rapid cooling of bovine oocytes

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.