Cryopreservation of animal oocytes will permit germplasm of valuable or unique females to be preserved for extended times. The objective of this research was to derive a procedure to cryopreserve bovine oocytes by vitrification to be used as recipients for somatic cell nuclear transfer (SCNT). Caprine oocytes vitrified by the same procedure were assayed by cytological examination of microtubules.
In the first two of three experiments, bovine oocytes matured in vitro were vitrified in a mixture of ethylene glycol (EG), dimethylsulfoxide (Me2SO) and trehalose, and then subjected to in vitro fertilization (IVF) or SCNT. For vitrification, oocytes were first exposed to increasing concentrations of EG + Me2SO, placed into the vitrification solution composed of 2.8 M Me2SO + 3.6 M EG + 0.65 M trehalose for 20 sec, immediately loaded onto 20-ìm cryoloops, and finally plunged directly into liquid nitrogen (LN2). Vitrified oocytes were warmed by direct immersion of cryoloops into 0.25 M trehalose prepared in TCM-199 medium at 37°C, rinsed briefly, and then assayed. In Experiment I, of 327 bovine oocytes subjected to IVF after being vitrified, 267 cleaved and 32 (9.8%) formed blastocysts, compared to 32.1% blastocysts for control oocytes. In Experiment II, of 266 bovine oocytes enucleated after vitrification and subjected to SCNT, 248 formed couplets, 152 of which cleaved and 31 (12.5%) developed into blastocysts, compared to 33.0% blastocysts for controls. During the course of Experiment II, 20 of 31 blastocysts derived by SCNT of somatic cells from a Brahman cow into vitrified oocytes were transferred into recipients, resulting in three pregnancies and the birth of one Braham calf that has survived to adulthood. In Experiment III, cytological analysis of caprine oocytes vitrified by the same procedure used for bovine oocytes demonstrated that their microtubules were normal, suggesting that this same procedure can also be used for the former species.
The results demonstrate that bovine oocytes can be successfully vitrified and warmed, yielding normal embryos after fertilization or SCNT. Additional research is needed to verify that caprine oocytes vitrified by this method can also develop into kids.