**3.2.2 Isachenko Method**

12 Will-be-set-by-IN-TECH

bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization

Bovine oocytes matured in vitro are transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose inTCM 199 and 20% FBS). A metal cube covered with aluminum foil is partially submersed into liquid nitrogen (Fig. 10): the surface reaches the temperature of -150◦. Microdrops of vitrification solution, containing the oocytes, are dropped onto the cold upper surface of the metal cube and are instantaneously vitrified. The vitrified

CryoTip consists of a plastic straw with a thin part (250 *μm* inner diameter, 20 *μm* wall thickness and 3 cm length) connected to a thick part (2000 *μm* inner diameter and 150 *μm* wall thickness, 4.5 cm length) and equipped with a movable protective metal sleeve (Fig. 11)

Fig. 11. The CryoTip is a finely pulled straw designed for holding gametes or embryos

and developmental rates in vitro and in vivo (Kuwayama, Vajta, Ieda & Kato, 2005).

Embryos are loaded in approximately 1 *μl* solution into the narrow part of the CryoTips without any air bubbles by aspiration of medium. Subsequently, the straw is heat-sealed at both ends, the protective sleeve is pulled over the narrow part and the device is plunged into liquid nitrogen. The time required for loading, sealing, adjustment of the sleeve and plunging does not exceed 90 s. The use of the closed CryoTip system eliminates potential embryo's contamination during cryopreservation and storage without compromising survival

microdrops are then stored in liquid nitrogen (Dinnyés et al., 2000).

Fig. 10. The solid surface vitrification (SSV) device

(Kuwayama, Vajta, Ieda & Kato, 2005).

and nuclear transfer.

**3.2 Closed supports**

**3.2.1 Cryotip**

In the *Isachenko Method* (Isachenko et al., 2005), embryos are located inside a open-pulled straws (OPS). The OPS is placed inside a sterile insemination straw (indicative size 90-mm), manufactured from standard 0.5-mL insemination straws. One end of sterile insemination straw is previously sealed using a hand-held sealer. The open end is hermetically closed by a metal ball and this container (OPS and sterile insemination straw) is plunged into liquid nitrogen ("straw in straw" vitrification). The *Isachenko Method*, applied to biopsied mouse pronuclear embryos is resulted efficient as conventional vitrification, guaranteeing a complete isolation of embryos from liquid nitrogen and avoiding potential contamination by pathogenic microorganisms.
