**4. Cryopreservation of oocytes and embryos**

The slow freezing technique developed for oocytes and embryos in the 1970´s (Willadsen et al 1978) has been thoroughly established by the increasing repertoire of CPA where they were gradually exposed to. Cultured cells/embryos are exposed to relatively low concentration of permeating CPA´s (glycerol, DMSO, EG or PG at 1-1.5 M (oocytes) or 1.3-1.5 M (embryos) alternatively non-permeating CPA in the culture medium, loaded into mini-straws and cooled at -5 to -7◦C, equilibrated for some minutes followed by seeding of extracellular ice nucleation, to be thereafter slowly cooled at ̴ 0.3-0.5◦C/minute to -40/-65◦C and final plunge in LN2 for

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 245

(>20,000ºC/min) can nowadays be reached using using cryo-loops (Lane et al 1999) or with straws with a smaller inner diameter and wall thickness (the Superfine Open Pulled Straws: SOPS; Isachenko et al 2003), and by applying immersion in LN2-slush, which allowed for the

Vitrification of untreated morulae and blastocysts has resulted in high survival rates after warming (Berthelot et al 2003), especially when re-warming after SOPS is done in one stage (direct warming, a very practical solution for ET, Cuello et al 2004b), yielding live litters (Cuello et al 2005). For blastocysts (See **Figure 5**), use of the SOPS waived the need for centrifugation (dislocation of lipids) or microtubule stabilization, thus making the method a very practical one and indicating the procedure is now reaching maturity for commercial application (Cameron et al 2004, Beebe et al 2005, Martinat-Botté et al 2006, Cuello et al 2008, 2010, Sanchez-Osorio et al 2009, 2010). Cryopreservation of in IVP-pig embryos -owing to differences in the cytoskeleton and the distribution of the lipid deposits- has been, until recently (Esaki et al 2004), considered as more difficult than for *in vivo*-developed, but the birth of piglets resulting from ET of IVP, transgenic pig embryos, has modified this view opening for the commercialization of highly valuable, modified genetic material (Li et al 2006, Kawagami et al 2008). Despite peri-hatching blastocyst stage embryos are the ones best sustaining vitrification and warming with continued *in vitro* development (Dobrinsky 2001),

this particular embryo stage can not be commercially used since there is no ZP.

Fig. 5. Laser scanning confocal microphotographs of grade I (A and D), grade II (B and E) or grade III (C and F) in vivo-derived fresh (A-C) and superfine open pulled straws (SOPS) vitrified (D-F) porcine blastocysts, following uploading of Hoescht H-33342 (blue, cell nuclei), phalloidin-Alexa Fluor 488 (green, actin filaments) and wheat germ agglutinin-Alexa fluor 594 (red, lectin reactive membrane elements). Note the high degree of

morphological intactness even after rewarming compared to fresh controls (Reprinted from

Cuello et al 2010, with permission).

use of lower concentrations of toxic cryoprotectant.

storage of the now carefully dehydrated and vitrified germplasm (for a comprehensive review see Saragusty & Arav 2011). However, pig oocytes, zygotes and cleavage embryos are rich in cytoplasmic lipids, and very sensitive to temperatures below 15ºC (Wilmut 1972), a sensitivity that decreases -along with the amount of lipids- with development, towards peri-hatching blastocysts (Niimura and Ishida 1980). Offspring has been obtained after embryo transfer (ET) of slow-frozen and thawed 2-4 cell pig embryos where these cytoplasmic lipids were removed in vitro (de-lipation) before cooling (Hayashi et al 1989, Nagashima et al 1994, 1995, 1996) and thereafter the technique, albeit cumbersome, has been thoroughly applied (Yoneda et al 2004). The results enhanced when the cytoskeleton was preserved from damage using exogenous chemicals (Shi et al 2006).

Over the past years, vitrification (Rall and Fahy 1985) appeared as a better alternative for long-term storage of pig oocytes and embryos. One one hand, the small size of the material to process provided another dimension: vitrification could be modulated via size of sample (10 µL in most cases) so that neither cooling rate nor CPA-amounts ruled so that the method was more practical and less risky. Samples could be handled and carried/stored through either "surface" methods (e.g on liquid loops, mesh of different materials etc) or "tubing" carriers (thin straws, cyopipettes, ultrathin tubing etc). Both yield high cooling rates but while the surface type has the highest warming rates, the other is much easier to handle and, safer (Saragusty & Arav 2011).

On the other hand, vitrification of oocytes and embryos differ in degree of difficulty. As already mentioned, oocytes are more sensitive than embryos, particularly morulas or blastocysts since oocytes have a high cytoplasmic lipid contents (chilling sensitive). Moreover, oocytes have easily disrupted submembranous actin microtubules (which decreases plasmalemman robustness) and fragile meiotic spindle and cytoskeleton, which complicates the resumption of development. Lastly, the process of freezing and thawing can increase the risk for ROS-attack and the premature emptying of cortical granules, thus changing the structure of the zona pellucida (ZP) (Gajda 2009). Therefore, chemical stabilization of the cytoskeleton (Esaki et al 2004) and the use of increased pressure following vitrification (Du et al 2008) had been successfully applied, obtaining development post-rewarming towards the fetal stage (Ogawa et al 2010). Other measures, such as induction of osmotic stress (by exposure to NaCl) has shown to improve developmental competence after vitrification (Lin et al 2008). Centrifugation (lipid depot relocation) for vitrification appears detrimental for *in vitro*-matured oocytes, but not in zygotes or later stages (Somfai et al 2008)

Vitrification of in vivo-developed, ZP-intact pig embryos, where lipids were polarized by centrifugation of the blastomeres, by delipation and/or treatment with cytochalasin for cytoskeleton stabilization, has resulted after rewarming and ET, in piglets (Dobrinsky 1997, Dobrinsky et al 2000, 2001, Kobayashi et al 1998, Berthelot et al 2000, 2003, Cameron et al 2000). Blastocysts were also developed by *in vitro* fertilization (IVF) of follicular oocytes vitrified as cumulus-oocyte complexes from offal porcine follicles (Somfai et al 2010).

Recently, piglets were even obtained following vitrification of delipated 4-8 cell stages of in vitro produced (IVP), parthenogenetic embryos and ET (Nagashima et al 2007). Vitrification, usually done within 0.25 mL plastics-straws, yield better embryo survival post-warming when Open Pulled Straws (OPS; Vajta et al 1997), which increases the cooling rate achievable in 0.25 mL straws (2,500ºC/min) by almost 8-fold (Cuello et al 2004a-b), were used, again resulting in piglets born (Berthelot et al 2000; 2001). Higher cooling-rates

storage of the now carefully dehydrated and vitrified germplasm (for a comprehensive review see Saragusty & Arav 2011). However, pig oocytes, zygotes and cleavage embryos are rich in cytoplasmic lipids, and very sensitive to temperatures below 15ºC (Wilmut 1972), a sensitivity that decreases -along with the amount of lipids- with development, towards peri-hatching blastocysts (Niimura and Ishida 1980). Offspring has been obtained after embryo transfer (ET) of slow-frozen and thawed 2-4 cell pig embryos where these cytoplasmic lipids were removed in vitro (de-lipation) before cooling (Hayashi et al 1989, Nagashima et al 1994, 1995, 1996) and thereafter the technique, albeit cumbersome, has been thoroughly applied (Yoneda et al 2004). The results enhanced when the cytoskeleton was preserved from damage using exogenous

Over the past years, vitrification (Rall and Fahy 1985) appeared as a better alternative for long-term storage of pig oocytes and embryos. One one hand, the small size of the material to process provided another dimension: vitrification could be modulated via size of sample (10 µL in most cases) so that neither cooling rate nor CPA-amounts ruled so that the method was more practical and less risky. Samples could be handled and carried/stored through either "surface" methods (e.g on liquid loops, mesh of different materials etc) or "tubing" carriers (thin straws, cyopipettes, ultrathin tubing etc). Both yield high cooling rates but while the surface type has the highest warming rates, the other is much easier to handle and,

On the other hand, vitrification of oocytes and embryos differ in degree of difficulty. As already mentioned, oocytes are more sensitive than embryos, particularly morulas or blastocysts since oocytes have a high cytoplasmic lipid contents (chilling sensitive). Moreover, oocytes have easily disrupted submembranous actin microtubules (which decreases plasmalemman robustness) and fragile meiotic spindle and cytoskeleton, which complicates the resumption of development. Lastly, the process of freezing and thawing can increase the risk for ROS-attack and the premature emptying of cortical granules, thus changing the structure of the zona pellucida (ZP) (Gajda 2009). Therefore, chemical stabilization of the cytoskeleton (Esaki et al 2004) and the use of increased pressure following vitrification (Du et al 2008) had been successfully applied, obtaining development post-rewarming towards the fetal stage (Ogawa et al 2010). Other measures, such as induction of osmotic stress (by exposure to NaCl) has shown to improve developmental competence after vitrification (Lin et al 2008). Centrifugation (lipid depot relocation) for vitrification appears detrimental for *in* 

Vitrification of in vivo-developed, ZP-intact pig embryos, where lipids were polarized by centrifugation of the blastomeres, by delipation and/or treatment with cytochalasin for cytoskeleton stabilization, has resulted after rewarming and ET, in piglets (Dobrinsky 1997, Dobrinsky et al 2000, 2001, Kobayashi et al 1998, Berthelot et al 2000, 2003, Cameron et al 2000). Blastocysts were also developed by *in vitro* fertilization (IVF) of follicular oocytes

Recently, piglets were even obtained following vitrification of delipated 4-8 cell stages of in vitro produced (IVP), parthenogenetic embryos and ET (Nagashima et al 2007). Vitrification, usually done within 0.25 mL plastics-straws, yield better embryo survival post-warming when Open Pulled Straws (OPS; Vajta et al 1997), which increases the cooling rate achievable in 0.25 mL straws (2,500ºC/min) by almost 8-fold (Cuello et al 2004a-b), were used, again resulting in piglets born (Berthelot et al 2000; 2001). Higher cooling-rates

vitrified as cumulus-oocyte complexes from offal porcine follicles (Somfai et al 2010).

*vitro*-matured oocytes, but not in zygotes or later stages (Somfai et al 2008)

chemicals (Shi et al 2006).

safer (Saragusty & Arav 2011).

(>20,000ºC/min) can nowadays be reached using using cryo-loops (Lane et al 1999) or with straws with a smaller inner diameter and wall thickness (the Superfine Open Pulled Straws: SOPS; Isachenko et al 2003), and by applying immersion in LN2-slush, which allowed for the use of lower concentrations of toxic cryoprotectant.

Vitrification of untreated morulae and blastocysts has resulted in high survival rates after warming (Berthelot et al 2003), especially when re-warming after SOPS is done in one stage (direct warming, a very practical solution for ET, Cuello et al 2004b), yielding live litters (Cuello et al 2005). For blastocysts (See **Figure 5**), use of the SOPS waived the need for centrifugation (dislocation of lipids) or microtubule stabilization, thus making the method a very practical one and indicating the procedure is now reaching maturity for commercial application (Cameron et al 2004, Beebe et al 2005, Martinat-Botté et al 2006, Cuello et al 2008, 2010, Sanchez-Osorio et al 2009, 2010). Cryopreservation of in IVP-pig embryos -owing to differences in the cytoskeleton and the distribution of the lipid deposits- has been, until recently (Esaki et al 2004), considered as more difficult than for *in vivo*-developed, but the birth of piglets resulting from ET of IVP, transgenic pig embryos, has modified this view opening for the commercialization of highly valuable, modified genetic material (Li et al 2006, Kawagami et al 2008). Despite peri-hatching blastocyst stage embryos are the ones best sustaining vitrification and warming with continued *in vitro* development (Dobrinsky 2001), this particular embryo stage can not be commercially used since there is no ZP.

Fig. 5. Laser scanning confocal microphotographs of grade I (A and D), grade II (B and E) or grade III (C and F) in vivo-derived fresh (A-C) and superfine open pulled straws (SOPS) vitrified (D-F) porcine blastocysts, following uploading of Hoescht H-33342 (blue, cell nuclei), phalloidin-Alexa Fluor 488 (green, actin filaments) and wheat germ agglutinin-Alexa fluor 594 (red, lectin reactive membrane elements). Note the high degree of morphological intactness even after rewarming compared to fresh controls (Reprinted from Cuello et al 2010, with permission).

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 247

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