**1. Introduction**

Preservation of germplasm (e.g. a term hereby applied to collectively gather spermatozoa, oocytes or early embryos whose use would –eventually- lead to offspring) for research, repository building and propagation of genetic material using Assisted Reproductive Techniques (ART) has been a long lasting priority (Mazur et al 2008). The first approaches, besides those historically anecdotic (see Flowers 1999) were directed to the application of artificial insemination (AI) of domestic species (Foote 1999) pertaining dissemination of genetics to a general population of, particularly, production animals. Positive effects for simple cryo-protectant agents (CPA) such as glycerol on animal sperm cryoprotection were demonstrated already by the end of the 1930`s (Bernschtein & Petropavloski 1937) and a decade later it became apparent that spermatozoa could be cooled, frozen and thawed in solutions containing egg yolk and glycerol (Polge et al 1949). For some species, such as bovine, the fact that bull semen could be easily frozen with an acceptable sperm survival post-thaw and accompanied by acceptable fertility after intra-uterine AI led to the rapid development of such primary reproductive biotechnology (Rodriguez-Martinez & Barth 2007). Attempts in other species of domesticated animals followed, and it was soon realised that the success seen with bovine could not be reached, primarily due to low sperm survival, difficulties in attaining an optimal deposition or proper timing towards spontaneous ovulation. Differences in survival and fertility varied not only among species but also between individuals of a given species or even ejaculates within sires (Holt 2000).

Porcine male germplasm freezing started already by the 1950´s (Polge 1956) but their postthaw fertility was not reassured using cervical AI until a decade later (Crabo and Einarsson 1971, Graham et al 1971, Pursel & Johnson 1971), which revealed major constrains when applying cryopreservation on boar spermatozoa. Today, despite documented efforts to reach acceptable fertility and prolificacy after AI (Eriksson et al 2002, Roca et al 2011), the cryosurvival of boar spermatozoa is still consistently low in comparison to other species, owing to damage during a processing that is time-consuming, costly and yields few doses per ejaculate (see Rath et al 2009, Rodriguez-Martinez & Wallgren 2011). Number of piglets born is lower than for cooled or neat semen implying that sperm lifespan, deposition site and closeness to ovulation are yet significant hurdles to be overcome (Roca et al 2006b, Wongtawan et al 2006). Preservation of male genetics can also be performed by freezing of

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 233

entire process basically concerns the way we handle the presence of water in and around the cells and whether its freezing is allowed (conventional cryopreservation, slow equilibrium

In the first method, which is the one traditionally used in biomedicine, particularly for sperm preservation, cells are subjected to slow cooling to temperatures below zero, with freezing rates of 0.5-100 ◦C/min). The method allows ice to form and solute to concentrate alongside the change in water phase. Both ice and high solute concentrations can cause direct (either initial or eutectic, Han & Bischof 2004a), respectively secondary damage, jeopardizing cell survival or handicapping vital cell functions post-thaw. At some moment during the process, water freezes to form ice, primarily extracellular, but even intracellular. Ice grows and becomes over time surrounded by an increasing amount of solutes which move to the areas where water did not yet changed phase. Cells balance ion concentrations at either side of the plasma membrane thus keeping proper osmotic pressure. Depending on the relative amounts of free and bound water, such a change of phase (either formation or dissolution of ice and de/rehydration phenomena) implies changes in ionic concentration caused by directional movement of water across the membrane, disturbing the homeostatic osmotic pressure of the cell/s (Pegg 2007). Cells respond by allowing water to leave the intra-cellular compartment, to compensate the increasing hyper-osmotic extra-cellular compartment caused by the progressive formation of ice. Those water movements lead both to cell dehydration and to a toxic hyper-concentration of solutes intracellular which, ultimately, affects cells viability (Watson & Fuller 2001). Freezing injury can then be related to high electrolyte concentration effects (solute effects), presence of intracellularly ice (formed direct or eutectic) and also the pressure of large extracellular crystals on the veins of concentrated (i.e. vitrified) extender and cells (Saragusty et al 2009). See **Figures 1and 2** for

Freezing injury during slow freezing can be minimized. Intracellular freezing is generally lethal but can be avoided by sufficiently slowing the rate of cooling. Solute-caused damage, which is the dominating feature under conventional slow freezing especially in cells in liquid suspension, can be minimized by the addition of CPA. Most CPA´s (as glycerol, dimethyl sulfoxide (DMSO), ethylenglycol (EG), propyleneglycol (PG)) are highly soluble, permeating compounds of low-to-medium toxicity, whose primary role is to reduce the amount of ice formed at any given sub-zero temperature, by simply increasing the total concentration of all solutes in the system, thus defining the concept of slow equilibrium freezing (Pegg 2002 2007). Introduction of sufficient CPA would eventually avoid freezing and a glassy of vitreous state could be produced instead. Such concept is the theoretical rationale for the second method listed above: rapid nonequilibrium vitrification. Vitrification is the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures (often to -120 to -130 ◦C) to finally solidify into a metastable glass, without undergoing crystallization at a practical, high speed cooling rate (i.e. dipping onto LN2). Use of ultra-high speed voids the need of penetrating CPA, open for using non-penetrating CPA (such as sucrose, fructose, glucose), but demands the use of small (5-50µL) suspension droplets. The glassy state is defined by its viscosity reaching 10-13 poises, sufficient for the aqueous material to behave as a solid, without any water crystallization. Once again, this waives the above listed sources for cell injury: ice crystals and increased/ill distributed solute

freezing) or totally prevented (vitrification).

an illustration of these events.

epidydimal spermatozoa (retrieved by biopsy of the cauda) or by tissue sampling through testicular biopsies (Keros et al 2005, Curaba et al 2011). However, these approaches are not relevant for porcine breeding. Cauda epididymal spermatozoa are easier to slow freeze than ejaculated spermatozoa (Rath & Niemann 1997) and testicular biopsies are not advisable in boars owing to their highly vascularized testicular capsule (Ohanian et al 1979).

Preservation of female genetics can be done either by freezing of germplam (e.g. oocytes or embryos) or of ovarian tissue (slices or whole ovary), from which oocytes can thereafter be harvested for ART. Germplasm freezing in pigs has also followed a tortuous road, with deceiving results for decades, particularly related to the high sensitivity of pig oocytes and early embryos to chilling, similarly to other species containing large deposits of intracellular lipids (Zhou & Li 2009), in contrast to blastocysts where the lipid amounts were lower. Delipation (or side-dislocation of lipid depots by ultracentrifugation) was soon shown to increase the survival of oocytes/embryos subjected to freezing (Nagashima et al 1995), survival that could be enhanced if the cytoskeleton could be preserved from damage using exogenous chemicals (Shi et al 2006). Use of alternative methods such as vitrification instead of slow cooling led to better survival (see Massip 2001) including the birth of offspring (Berthelot et al 2000). However, large variation was seen among methods, sources and laboratories (Holm et al 1999, Cuello et al 2007, Somfai et al 2008, Ogawa et al 2010), including the method used for intrauterine deposition (Rodriguez-Martinez 2007b, Roca et al 2011).

Cryopreservation of ovarian tissue (or even of whole ovaries) has been tested in several species including human (Isachenko et al 2009) pertaining the recovery of follicular oocytes for ART or ultimate autographs (Kim et al 2010). Procedures for porcine ovarian samples have followed methods tested in other species (Imhof et al 2004, Borges et al 2009) with promising results, albeit yet at an academic level, pertaining the advancement of xenografting (Moniruzzaman et al 2009). As well, experimental models using the porcine species have been developed for cryopreservation of genital tissues, particularly the uterus (Dittrich et al 2004, 2006) paving the way for human transplantation procedures (Diaz-Garcia et al 2011).

Thus, interest has been large to attempt routine cryopreservation of porcine gametes, embryos and genital tissues, yet with various degrees of success. Therefore, the present review aims to summarize the state-of-the-art regarding established and emerging methods for the cryopreservation of porcine gametes and embryos as germplasm, intending a critical revision of the underlying problems that still constrain their application for establishing repositories, their use in reproductive biotechnologies and, ultimately, for breeding. As well, it intends to describe our level of knowledge when attempting cryogenics of gonads and other genital tissues for comparative research, particularly on human regenerative medicine. The review is not exhaustive and focus on methodological aspects of procedures.
