Requirements for cryopreservation of in vitro-produced bovine embryos by a standard method of vitrification

In relation to assisted mammalian reproduction, the goal of cryopreservation is to preserve without significant loss of viability a stock of gametes and/or embryos with a view to thawing those cells for use in in vitro reproduction treatments. There are numerous cryopreservation protocols, which vary in terms of cryoprotectant used, storage temperature, freezing and thawing rates, and the particular cells that they are suitable for preserving. Although slow freezing has become a standard method for in vivo bovine embryo cryopreservation, it seems not to be efficient for preserving in vitro-produced (IVP) bovine embryos. Over the past decade, vitrification, a process of glass-like solidification, has become the cryopreservation method of choice for human oocytes and embryos. This is because it is often less time-consuming than slow freezing, does not require expensive ‘slow rate’ freezing machines, and has been proven to increase survival rates compared to slow frozen embryos. In contrast to clinical applications, in the cattle industry vitrification presents shortcomings, especially when applied to IVP embryos. A high concentration of cryoprotectant in solution is needed to induce vitrification. However, if left too long with metabolizing embryos, cryoprotectants can become toxic. Failure to standardize vitrification protocols leads to inconsistent results between laboratories, making application less practical in field settings. Therefore, determination of the most suitable vitrification method is important to advance its routine commercial use. Moreover, simplification of the vitrification procedure through development of an in-straw dilution without the use of a microscope may help to expand the use of vitrification methods on the farm.