Requirements for Cryopreservation of In vitro-Produced Bovine Embryos by a Standard Method of Vitrification

Do Vh, Taylor-Robinson Aw
Journal of Veterinary Science and Animal Husbandry   unpublished
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
more » ... 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. According to Leibo [1], cryopreservation is an essential component of the embryo transfer industry in cattle that, as Hasler points out [2] , is now an established commerce on a global scale. The standard method of bovine embryo cryopreservation, slow freezing, was refined over a period of 40 years of research whereas the alternative vitrification procedure has developed more recently and more rapidly [2]. Pregnancy rates of recipients implanted with in vivo frozen embryos are not inferior to those of recipients receiving fresh embryos [3, 4] . Nevertheless, several studies have highlighted the inefficiency of this conventional method of cryopreserving in vitro-produced (IVP) bovine embryos [4] [5] [6] [7] [8] [9] . These possibly contain more lipid droplets in their cytoplasm than do their in vivo counterparts [10] [11] [12] , leading to their greater susceptibility to the freezing process [13, 14] . In vitro production of bovine embryos has progressed rapidly and they are now relatively cheap to generate in large quantity [4] . Although oocyte numbers from different donors are variable, the in vivo ovum pick-up method coupled with in vitro fertilization and culture can produce an average of 50 calves from repeated aspiration of oocytes of a single donor cow per year [15] . While the yield of IVP embryos may be sizeable, a corresponding number of recipients are often not available [16] . Thus, valuable unused embryos are frequently discarded in the laboratory. Since vitrification is now the main cryopreservation method to store human oocytes and embryos [17] , this procedure has also been recommended to assist in the provision of stocks of IVP bovine embryos [2, 4, 18] . Vitrification is technically simple [19] , and does not require a programmable freezer [2, 20] . Laboratory experiments have shown that vitrified IVP bovine embryos achieve a better survival rate than do those cryopreserved by slow freezing [4, 9, 21, 22] . As a proof of principle, calves were born successfully after transfer to recipients of vitrified IVP embryos [23, 24] ; moreover, the pregnancy rates of recipients that are implanted with vitrified IVP embryos may be acceptable in commercial conditions [25] .