Feasibility of a Nylon-Mesh Holder for Vitrification of Bovine Germinal Vesicle Oocytes in Subsequent Production of Viable Blastocysts1

Abstract

To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.

The Nuclear Maturation and Embryo Development of Mice Germinal Vesicle Oocytes with and without Cumulus Cell after Vitrification

“…The injury to the cells in the process of cryopreservation can be due to osmotic effects accompanying saturation with permeable cryoprotectants [14]. The reduction in subsequent developmental competence caused by exposure to cryoprotectant solutions has been shown to be more severe in the GV oocytes than MII oocytes [15][16].…”

“…Ohboshi, et al, [34] reported that vitrification of bovine blastocysts, and with two-step exposure to the cryoprotectants showed less damage compared with the single-step procedure. Abe, et al, [16] reported survival, fertilization, maturation and developmental rates to blastocyst of bovine GV-COCs, using Nylon-Mesh and expoxer with stepwise cryoprotctant, were significantly higher compared with the single-step vitrification. Aono et al, [34][35] reported higher survival, maturation and blastocysts rates, using ultra rapid vitrification accompanied by step-wise equilibration in mouse GV oocytes than single step vitrified group [35][36].…”

Cryopreservation of Cattle, Pig, Inobuta Sperm and Oocyte after the Fukushima Nuclear Plant Accident

“…As a result, total 493 of morphologically normal COCs were recovered, and 40 blastocysts were yielded from 9 donors following in vitro embryo production. The bovine blastocysts produced were cryopreserved by vitrification using a nylon mesh method described in the previous report [19] (Table 2). On the other hand, pig and inobuta ovaries were transported to the laboratory at 37°C.…”

Oocyte and embryo cryopreservation – state of art and recent developments in domestic animals

“…However, a bigger pore size is helpful for decreasing the volume of solution containing oocytes, which is the case with the EM grid (55 µm mesh size) and nylon mesh (60 µm mesh size). The EM grid has been used for vitrification of bovine cumulus-oocyte complexes (COCs) (Martino et al, 1996), bovine blastocysts (Park et al, 1999), and human zygotes ; nylon mesh may be applicable to other mammalian oocytes and embryos (Matsumoto et al, 2001;Abe et al, 2005). Given that a nylon mesh can easily handle a large number of oocytes or embryos, using this holder for the vitrification of GV bovine oocytes, for example, should facilitate gamete storage for further application in assisted reproductive technologies, such as in vitro fertilization, cloning, and stem cell biology.…”