**2.5 The success of cryopreservation on mammalian embryos**

The first mammalian embryo that survived cryopreservation was the mouse embryo 40 years ago by slow-cooling (Whittingham et al., 1972). More than 50% of preimplantation embryos survived after thawing and about 40% of the surviving embryos developed to full-term after being transferred to foster females. A similar protocol was applied successfully on cow embryos (Wilmut & Rowson, 1973), sheep embryos (Willadsen, 1977) and many other domestic mammals (Saragusty & Arav, 2011), suggesting that slow cooling is basically applicable to other mammalian embryos, provided the cooling rate can be optimized and well controlled. By using 1.5M DMSO as a cryoprotectant, sheep morulae and blastocysts were cooled at a rate of 0.1°C per minute. A survival rate of 80% can be achieved. With a similar protocol developed from mouse experiments, the first pregnancy after transfer of a cryopreserved human 8-cell embryo was recorded in 1983 (Trounson & Mohr, 1983). The pregnancy terminated at 24-weeks due to premature rupture of the membrane and *Streptomyces agalactiae* infection (Trounson & Mohr, 1983). A year later, Zeilmaker et al. (1984) described the first live birth after the transfer of cryopreserved human embryos. More reports of live births of cryopreserved human embryos were reported in the following year (Cohen et al., 1985; Downing et al., 1985) and in the years after that.

With the extensive characterization of embryo-cryoprotectant interactions, the feasibility of embryo cryopreservation by vitrification was demonstrated by Rall and Fahy (1985). The next challenges were to further lower the concentration of the cryoprotecting agent to be used and to increase the cooling rate. Increasing the cooling rate not only guarantees the absence of ice formation but also allows a further decrease in the amount of cryoprotecting agents being used, thus minimizing the potential toxic effects on the embryos. It can be illustrated by a recent report describing the use of a highly conductive micro-capillary to increase the cooling rate up to about 4,000C per second. The cryoprotectant propylene glycol concentration can be reduced to 1.5M, compared to the normally used 4M or above (Lee et al., 2010). Over the past decade, a variety of holding devices have been developed to allow fast transmission of heat (thus a high cooling rate) from the sample to the coolants (reviewed in detail in Tsang and Chow, 2010). Some researchers have made use of containers with thinner walls, such as pulled-straw (Vajta et al., 1998). Others have made use of an open property of the devices to hold the sample on a surface to allow direct heat transfer between the samples and the coolant. These include the electronic microscopic metal grid (Martino et al., 1996), cryoloop (Lane et al., 1999), nylon mesh (Matsumoto et al., 2001), hemi-straw (Vanderzwalmen et al., 2003), cryotop (Kuwayama et al., 2005), vitrification spatula (Tsang & Chow, 2009) and plastic blade (Sugiyama et al., 2010). Using most of these open-systems, cryo-survival rates of above 80% are usually obtained. The remaining challenge is to select the right tool by considering the microbial surveillance requirement (a closed-storage system versus an open-storage system), the convenience factor and economic considerations in a routine facility operation.

Cryopreservation of Embryos from Model Animals and Human 269

Vitrification is now well accepted as a reliable means for cryopreserving mammalian embryos because of its simplicity; it does not require a controllable cooler. One factor responsible for the acceptance of this technology for cryopreservation of mammalian embryos has been the intensive studies on the interaction between different cryoprotectants and embryos, i.e. permeability and toxicity, at different developmental stages. Luckily, the most permeating cryoprotectants are not very toxic to the embryos. It allows the use of the cryoprotectants at a high concentration, yet below the lethal dose, to promote vitrification in

Nematoda belong to the ecdysozoa, sharing the same clade with arthropoda. The members range from free living species to parasitic species in plants and animals. The life cycle of the nematode is generally divided into five morphological stages. Each successive larval stage is preceded by a molting process to remove the collagenous cuticle from the former larval stage. At hatching, the first-stage larva (Figure 3 A) consisting of 558 cells is under the protection by a cuticular layer. The animal grows in size after each hatching. In adulthood, a reproductive hermaphrodite is about 1mm long, 0.06mm in diameter, containing about 1,000 somatic cells. In comparison, a first-stage larva is about 0.37mm in length and

Under favorable conditions, the development of the animal continues through the first- to the fourth-larval stage and finally to the reproductive adults. In many parasitic species such as the entomopathogenic species, the third-stage larvae are juveniles that are infective to their hosts. Under unfavorable conditions, i.e. outside the host body, a second-stage larva develops into the third-stage infective juvenile but retains the cuticle from the last larval stage to form a sheath. The entire animal is enclosed in the sheath until a suitable host is

The simplest method for nematode cryopreservation was reported on ruminant nematodes. Infective juveniles were cooled directly in liquid nitrogen vapor after being unsheathed by sodium hypochlorite, and suspended in physiological saline (Campbell and Thomson, 1973; Van Wyk et al., 1977). For example, infective juveniles of sheep nematodes (*Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum columbianum*) and the bovine nematodes (*Haemonchus placei, Ostertagia ostertagi, Nematodirus helvetianus, Oesophagostomum radiatum, Cooperia pectinata and Cooperia punctata*) survived these simple cryopreservation procedures. James (1985) suggested that the presence of natural cryoprotectants plays a role in the cryosurvival of domestic animal parasitic nematodes. Bai et al. (2004) demonstrated in *Steinernema carpocapsae* and *Heterorhabditis bacteriophora* that the cryo-survival rates of infective juveniles are positively correlated with worm concentration during the cryoprotectant glycerol incubation step. The survival rates ranged from about 20% to 100%, which was proportional to worm concentration of 120-12,000 per ml (Bai et al., 2004). Infective juveniles indeed produce cryoprotecting molecules such as trehalose and glycerol,

response to a convenient cooling rate in most laboratories.

**3.1 Development of the nematode** 

0.025mm in diameter.

infected.

**3. Cryopreservation of larvae of nematodes and platyhelminthes** 

**3.2 Cryopreservation of nematode without cryoprotectant additive** 

Fig. 4. Morphological changes of mammalian (mouse) preimplantation embryos in response to cryoprotectant treatments (for vitrification) and rehydration. Panel A-E: zygote (1-cell); panel F-J, pre-compact morula (8-cell); panel K-O, compact morula (8-cell); panel P-T, expanded blastocyst. Individual embryos were held by a glass micropipette by a slight suction under physiological isotonic medium (A, F, K and P). Vitrification solutions containing low concentration of permeating cryoprotectants and high concentration of permeating cryoprotectants plus non-permeating cryoprotectants were applied to the surroundings of the embryos, sequentially. About 30 seconds after the application of solution containing low concentration of permeating cryoprotectants, the embryos osmotically shrunk to minimal volumes (panel B, G, L and Q). The embryos (except the blastocyst) later re-expanded to a size closer to the original volumes, after an additional 30 seconds, when cryoprotectants and water re-entered the cells passively (panel C, H, M and R). After addition of the final vitrification solution containing high concentration of permeating cryoprotectants and non-permeating cryoprotectants, the embryos were further dehydrated by osmosis and shrunk without reexpansion. (panel D, I, N and S) High concentration of intracellular permeating cryoprotectants was achieved. Rapid cooling is normally done at this stage to vitrify the embryos but such cooling was not done in this demonstration. Step-wise rehydration of the embryos was done after dehydration to imitate the recovery steps after thawing the embryos from vitrification. After rehydration, the embryos re-expanded to their original size and regained normal morphologies (E, J, O and T).

Vitrification is now well accepted as a reliable means for cryopreserving mammalian embryos because of its simplicity; it does not require a controllable cooler. One factor responsible for the acceptance of this technology for cryopreservation of mammalian embryos has been the intensive studies on the interaction between different cryoprotectants and embryos, i.e. permeability and toxicity, at different developmental stages. Luckily, the most permeating cryoprotectants are not very toxic to the embryos. It allows the use of the cryoprotectants at a high concentration, yet below the lethal dose, to promote vitrification in response to a convenient cooling rate in most laboratories.
