**7. Acknowledgements**

The studies of the author have been made possible by grants from the Swedish Research Council Formas, the Swedish Farmer´s Foundation for Agricultural Research (SLF), and The Swedish Research Council (VR), Stockholm, Sweden.

#### **8. References**

246 Current Frontiers in Cryobiology

Freezing of ovarian tissue in humans relate primarily, but not only, to a dramatic measure to warrant availability of oocytes in cases of oncotherapy, when sterility is foreseen, similar to the ongoing sperm banking prior to onco- or hormonal therapy. Rescue of oocytes from frozen samples of ovarian cortex is then feasible for ART (Shaw & Trounson 2002). Both slow freezing and directional freezing had been assayed with acceptable results (Arav & Natan 2009), opening possibilities for the cryopreservation of large samples and even of whole ovary for autografting purposes and possibly evolving in oocyte banking as an insurance against childlessnes. Adult testicular samples (aspiration or biopsy) are mainly issued during biopsy for recovery of spermatids for ICSI (Keros et al 2005, Curaba et al 2011). However, the strongly ongoing research in adult stem cells shall be based on the absolute need of properly cryopreserving pre-pubertal testicular tissues. Transplantation of other organs or tissues (uterine in particular) is also within the scope of not-far, albeit

Regarding the porcine species, although there is no obvious rationale for most of the above considerations in human, it provides an excellent animal model for experimental reproductive medicine, particularly considering transplantation surgery. Porcine whole uteri were arterially perfused with CPA (DMSO) prior to slow controlled freezing. Rewarmed tissues were able to present live cells 7 h post rewarming (Dittrich et al 2006) and even to demonstrate contractility *in vitro* 60 min post-rewarming (Dittrich et al 2010). As such, comparative analyses of equilibrium freezing and vitrification procedures have involved pig ovarian fragments (Gandolfi et al 2006, Borges et al 2009), or whole ovaries (Imhof et al 2004). These attempts were all done using slow freezing, but evidence is now provided that vitrification of thin slices of ovarian cortex is feasible and that rewarmed primordial follicles from these samples were able to develop (albeit slower than controls) in murine xenografts (Moniruzzaman et al 2009). Further development in this area is expected.

Vitrification as a method for cryopreservation in porcine applies thus far to small samples that can be managed at high cooling and rewarming rates without need of applying permeating CPA of potential toxicity. Therefore, the technique has developmental potential for oocytes, COCs and embryos for IVF and ET. Boar spermatozoa are yet to follow this path, and although there is a potential breach for vitrifying limited volumes of sperm suspensions, such approach is yet solely academic in nature. Semen for breeding ought to be frozen conventionally, albeit with a focus on increased cell lifespan, and managing concentrated semen doses for deep intrauterine AI. There is much yet to be learned from the ejaculate and the relationships between specific components of the seminal plasma and

The studies of the author have been made possible by grants from the Swedish Research Council Formas, the Swedish Farmer´s Foundation for Agricultural Research (SLF), and The

**5. Cryopreservation of genital tissues** 

discussable, scenarios (Bredkjaer & Grudzinskas 2001).

**6. Conclusions** 

sperm function.

**7. Acknowledgements** 

Swedish Research Council (VR), Stockholm, Sweden.


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**10** 

 *Hong Kong* 

**Cryopreservation of Embryos from** 

*Division of Life Science, The Hong Kong University of Science and Technology* 

Diploidic germplasms such as embryos, compared to haploidic gametes, are theoretically a better choice for preservation of an animal species. However, there are significant challenges in cryopreservation of multicellular materials due to their size and physical complexity which affect the permeation of cryoprotectants and water, sensitivity to chilling and toxicity of cryoprotectants. While cryopreservation technologies are well developed and found feasible in embryos/larvae of some species, embryos of other species such as zebrafish failed to be cryopreserved. In addition, cryopreservation in many other emerging model organisms have not been developed at all. Hence, the limited cryopreservation technology has become a bottleneck in the development of various research areas, especially those relying on molecular genetics of emerging model organisms. Thorough understanding of the embryonic development and critical stages tolerant to cryopreservation needs to be identified so as to facilitate expansion of model systems available for specific biological and

Classical animal models, including species that represent major branches of the tree of life, are being used in biological studies. They include *Caenorhabditis elegans* (a nematode), *Drosophila melanogaster* (an arthropod), *Danio rerio* (a teleost fish), *Gallus gallus* (an avian), and *Mus musculus* (a mammal). They have been widely used in scientific research, primarily due to the ease of maintenance and specific features that facilitate experimental manipulations, genetic study and observation. As knowledge from these models has accumulated over the years, they offer important insights into the overall organization and functional composition of the general form of life. However, a comprehensive picture of variations of mechanistic innovation in the vast diversity of species in the Animal Kingdom is not available. Greater understanding of these organisms in different branches of the phylogenetic tree is in demand, in order to fill the gaps of existing findings. To meet this demand, more model organisms are emerging to provide unique perspectives of animal development and specific biological functions not yet uncovered in the study of other classical models. Emerging animal models include the brine shrimp *Artemia sinica*, starlet sea anemone *Nematostella vectensis*, non-parasitic flatworm *Planaria*, amphioxus *Branchiostoma floridae*, sea squirt *Ciona intestinalis*, sea lamprey *Petromyzon marinus*, Japanese

**1. Introduction** 

experimental interrogations.

**1.2 Traditional and emerging animal models** 

**Model Animals and Human** 

Wai Hung Tsang and King L. Chow

