**3.1 Improvements in boar semen freezing**

Over the past decade, the cryobiology of boar semen has diminished its empiric approach towards a more experimental one. Major areas of research have involved: (i) the determination of *in vivo* features (particularly regarding seminal plasma (SP) and the presence of characteristic fractions of the ejaculate), (ii) the action of specific additives and different CPA, (iii) the use of automated freezers and of directional gradient freezing and, (iv) the use of novel containers adapted for the freezing of concentrated spermatozoa. Fertility post-AI is nowadays substantially better, closer to AI with liquid semen, even when using lower sperm numbers and alternative sites of sperm deposition, such as deeply intra-utero (Roca et al 2011).

**Specific additives and different CPA´s:** Glycerol, a small, poly-hydroxylated solute highly soluble in water since it interacts with it by hydrogen bonding, and able to permeate across the

Table 1. Fertility after conventional (cervical) AI in field trials (>100 sows) with frozen-

by a country or breeding organization before use. The challenge is there, undoubtedly.

and alternative sites of sperm deposition, such as deeply intra-utero (Roca et al 2011).

Over the past decade, the cryobiology of boar semen has diminished its empiric approach towards a more experimental one. Major areas of research have involved: (i) the determination of *in vivo* features (particularly regarding seminal plasma (SP) and the presence of characteristic fractions of the ejaculate), (ii) the action of specific additives and different CPA, (iii) the use of automated freezers and of directional gradient freezing and, (iv) the use of novel containers adapted for the freezing of concentrated spermatozoa. Fertility post-AI is nowadays substantially better, closer to AI with liquid semen, even when using lower sperm numbers

**Specific additives and different CPA´s:** Glycerol, a small, poly-hydroxylated solute highly soluble in water since it interacts with it by hydrogen bonding, and able to permeate across the

Therefore, it seems -at first sight- unlikely that deep frozen semen will replace the use of fresh semen on an extensive basis even if the fertility levels were similar. It is too expensive considering that the current cryopreservation protocols barely yield half of the doses produceable per ejaculate. Since the amount of spermatozoa per dose is minimum twice that of liquid-stored semen, such equation is simply undependable from a commercial point of view both in production costs for AI-doses and the sub-optimal boar use. However, having a reliable cryopreservation method for boar semen would (a) allow selection of genetics from all over the world, (b) enable planned, essential AIs´ at the top of the breeding pyramid and so (c) facilitate preservation of top quality genetic lines for ongoing or future breeding programmes and/or (d) offer an extra health safeguard, by allowing completion of any health test specified

thawed boar semen (modified from Roca et al 2006).

**3.1 Improvements in boar semen freezing** 

plasma membrane, at a low rate, is by far the mostly used CPA for boar semen conventional freezing. Since glycerol disturbs cell metabolism at body temperature, boar spermatozoa are usually exposed to this CPA at ~5°C, which –unfortunately- further slows its low rate permeation. Mixed with the other solutes of the extender in solution, it depresses their freezing point and ameliorates the rise in sodium chloride concentration during dehydration. Moreover, glycerol increases viscosity with lowering temperatures to more than 100,000 cP by -55°C (Morris et al 2006), leading to a retardation of both ice crystal growth and of dehydration speed on a kinetic basis. Moreover, glycerol eliminates eutectic phase changes of the extender (Han & Bischof 2004b), making it a very suitable CPA when added at 2-3% rates. While such interval does not affect cryosurvival in "good-freezer" boars, those considered moderate or bad freezers benefit from a minimum of 3% glycerol (Hernandez et al 2007a). A broad range of other solutes (mostly alcohols, sugars, diols and amides) have also been tested for CPA capacity (Fuller 2004, Buranaamnuay et al 2011), but boar spermatozoa react variably. Alcohols and diols can induce membrane blebbing. Sugars (such as the dissacharides sucrose, raffinose or trehalose which both increase viscosity and stabilise the membrane by interacting with phospholipids) are not better than glycerol, regarding cryosurvival (Hu et al 2008), but shows synergistic effects (Gutierrez-Perez et al 2009, Hu et al 2009). On the other hand, replacing glycerol with amides (formamide; methyl- or dimethylformamide, MF- DMF; acetamide; methyl- or dimethylacetamide (MA- DMA) at ~5% concentration, has proven beneficial for cryo-susceptible boars, probably because the amide permeates the plasma membrane more effectively than glycerol, thus causing less osmotic damage during thawing (Bianchi et al 2008). Other additives enhance cryosurvival of boar spermatozoa, such as Lglutamine (de Mercado et al 2009) or low rates (<0.1%) of N-acetyl-D-glucosamine (Yi et al 2002a), the latter possibly interacting with the surfactant OEP (Yi et al 2002b). Laurylsulphate, albeit its mode of action is yet unexplained in detail regarding interaction with egg yolk and the sperm plasma membrane, has repeatedly proven valuable (Karosas & Rodriguez-Martinez 1993, Buranaamnuay et al 2009). Use of low-density lipoproteins (LDLs), isolated from eggyolk from different species (Jiang et al 2007), has proven beneficial for sperm function postthaw, particularly for DNA-integrity. Similarly, sperm cryosurvival has been enhanced by the addition of antioxidants (Peña et al 2003, 2004a, Roca et al 2005, Jeong et al 2009, Kaeoket et al 2010), hyaluronan (Peña et al 2004b), or platelet-activating factor (PAF, Bathgate et al 2007), although the beneficial effects vary, particularly when different sperm sub-populations are used. Cryosurvival of several cold-shock susceptible species, of which the porcine is one, has been found to improve when cholesterol-loaded cyclodextrins (CLC) are used as additives before cooling (Zeng & Terada 2001, Mocé et al 2010). Cyclodextrins can encapsulate hydrophobic compounds, such as cholesterol, and transfer the cholesterol into membranes down a concentration gradient (Zidovetzki & Levitan 2007). However, it is yet to determine if the effects are substantial and not only individually-related (Waterhouse et al 2006).

**Automated freezers and directional gradient freezing:** Controlled freezing using programmed freezers improves cryosurvival by use of "optimal" cooling (and thawing) rates e.g. those that substantially diminish the period during which heat is released/absorbed in the sample when water changed phase (i.e. ice was formed/melt). Interestingly enough, experimentallydetermined optimal rates of the range 30-50°C/min (Thurston et al 2003, Medrano et al 2009, Juarez et al 2011) have been theoretically predicted (Devireddy et al 2004, Woelders & Chaveiro 2004) and confirmed by use of novel procedures such as directional freezing where the thermal gradient is monitored by modifying the velocity at which the liquid-ice interface grows so that the size and shape of the ice crystals is maintained within optimal limits. In this methodology,

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 241

Fig. 4. Cryo-SEM micrographs at low magnification of cross sectioned frozen mini-straw (a) and a MiniFlatPack™ (b) depicting major differences in the orientation and size frozen water lakes/extender veins. In (c) a higher magnification of (b) showing morphologically well preserved boar spermatozoa from the sperm-peak portion of the ejaculate (Courtesy of

Dr Hans Ekwall).

derived from the principle of seeding, the biological material is moved through a linear temperature gradient, so that both the freezing rate and the ice front propagation are controlled (Arav et al 2002, Woelders et al 2005, Saragusty et al 2007). The method can be advantageously applied both to large samples, frozen in large containers moving along a rim of seeding or to highly concentrated samples, in smaller (i.e. mini-straws) containers.

**Cryobiologically best-suited packaging containers:** use of cryobiologically adequate packaging systems for the extended spermatozoa, showed a direct improvement of cryosurvival. Boar spermatozoa has been processed in plastic straws of different volumes (0.25 to 5 mL, Johnson et al 2000), in flattened 5 mL straws (Weitze et al 1987), in metal (Fraser & Strzezek 2007) or in plastic bags of various types and constitution (Bwanga et al 1991, Mwanza & Rodriguez-Martinez 1993, Ortman & Rodriguez-Martinez 1994; Eriksson & Rodriguez-Martinez 2000, Eriksson & Rodriguez-Martinez 2000, Saravia et al 2005). The latter developed, denominated "FlatPacks™" proven equally good or better than 0.25 mL straws in terms of sperm cryosurvival despite of the fact that they held 5 mL of semen (an entire dose for cervical AI, 5 billion spermatozoa), thus waiving the need of pooling innumerable straws when thawing. Fertility after conventional cervical AI of FlatPack™ frozen-thawed semen yield acceptable farrowing rates and litter sizes (Eriksson et al 2002). See **Figure 3** for a schematic description of the differences between containers. The FlatPack™ was considered as cryobiologically convenient (very thin and with a large surface) to dissipate heat during cooling and warm rapidly, as those small containers tested. It is important to remember that the freezing in these containers, with high heat dissipation, inflicts less damage to the cells by intra-container mechanical pressure (Saragusty et al 2009). **Figure 4** shows cryological differences in shape and size of frozen water lakes between mini-straws and FlatPack™.

Fig. 3. Schematic representation of the major differences between plastic 0.25 mL ministraws, with single and Multiple FlatPack™ (the latter also named "MiniFlatPack™, see adjacent photograph of a filled and sealed MiniFlatPack™)(Diagramme/photograph: courtesy of Dr Fernando Saravia).

derived from the principle of seeding, the biological material is moved through a linear temperature gradient, so that both the freezing rate and the ice front propagation are controlled (Arav et al 2002, Woelders et al 2005, Saragusty et al 2007). The method can be advantageously applied both to large samples, frozen in large containers moving along a rim of seeding or to

**Cryobiologically best-suited packaging containers:** use of cryobiologically adequate packaging systems for the extended spermatozoa, showed a direct improvement of cryosurvival. Boar spermatozoa has been processed in plastic straws of different volumes (0.25 to 5 mL, Johnson et al 2000), in flattened 5 mL straws (Weitze et al 1987), in metal (Fraser & Strzezek 2007) or in plastic bags of various types and constitution (Bwanga et al 1991, Mwanza & Rodriguez-Martinez 1993, Ortman & Rodriguez-Martinez 1994; Eriksson & Rodriguez-Martinez 2000, Eriksson & Rodriguez-Martinez 2000, Saravia et al 2005). The latter developed, denominated "FlatPacks™" proven equally good or better than 0.25 mL straws in terms of sperm cryosurvival despite of the fact that they held 5 mL of semen (an entire dose for cervical AI, 5 billion spermatozoa), thus waiving the need of pooling innumerable straws when thawing. Fertility after conventional cervical AI of FlatPack™ frozen-thawed semen yield acceptable farrowing rates and litter sizes (Eriksson et al 2002). See **Figure 3** for a schematic description of the differences between containers. The FlatPack™ was considered as cryobiologically convenient (very thin and with a large surface) to dissipate heat during cooling and warm rapidly, as those small containers tested. It is important to remember that the freezing in these containers, with high heat dissipation, inflicts less damage to the cells by intra-container mechanical pressure (Saragusty et al 2009). **Figure 4** shows cryological differences in shape and size of frozen water lakes between mini-straws and FlatPack™.

Fig. 3. Schematic representation of the major differences between plastic 0.25 mL ministraws, with single and Multiple FlatPack™ (the latter also named "MiniFlatPack™, see adjacent photograph of a filled and sealed MiniFlatPack™)(Diagramme/photograph:

courtesy of Dr Fernando Saravia).

highly concentrated samples, in smaller (i.e. mini-straws) containers.

Fig. 4. Cryo-SEM micrographs at low magnification of cross sectioned frozen mini-straw (a) and a MiniFlatPack™ (b) depicting major differences in the orientation and size frozen water lakes/extender veins. In (c) a higher magnification of (b) showing morphologically well preserved boar spermatozoa from the sperm-peak portion of the ejaculate (Courtesy of Dr Hans Ekwall).

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 243

contained in the first 10 mL of the SRF (also called sperm-peak portion or P1, where about ¼ of all spermatozoa in the SRF are) were more resilient to handling (from extension to cooling) and cryopreservation that the spermatozoa contained in the rest of the ejaculate (Peña et al 2003, Rodriguez-Martinez et al 2009, Saravia et al 2007, 2010, Rodriguez-Martinez et al 2008). It appeared that it was actually the SP in this sperm-peak P1 portion that was beneficial for spermatozoa, either because of its higher contents of cauda epididymal fluid and specific proteins, or its lower amounts of seminal plasma spermadhesins, bicarbonate or zinc levels (Rodriguez-Martinez et al 2011), compared to other fractions of the ejaculate

In an attempt to simplify the freezing protocol, only the P1-spermatozoa were frozen in concentrated form for eventual use with deep-intrauterine AI. These spermatozoa were firstly kept in their SP for 30 min, and thereafter, without centrifugation (i.e. without removal of the SP) they were mixed with lactose-egg yolk (LEY) extender and cooled down to +5°C within 1.5 h, before being mixed with LEY+glycerol (3%) and OEP and packed into MiniFlatPack™´s for customary freezing using 50°C/min cooling rate. This "simplified" entire procedure (SF), lasted 3.5 h compared to the "conventional freezing" (CF) that was used as control procedure, which lasted 8 h. As controls, spermatozoa from the SRF were compared to P1-spermatozoa. Cryosurvival was, as seen in **Table 2**, equally good (above 60% of the processed cells (Saravia et al 2010, Pimenta-Siqueira et al 2011). Moreover, the spermatozoa in the sperm-peak-fraction of the boar ejaculate showed a maintained plasmalemmal intactness and fluidity and a lower flow of Ca2+ under capacitation conditions post-thaw, which might account for their higher

There are several advantages of using this simplified, shorter protocol, namely the exclusion of the customary primary extension and the following removal of this conspicuously beneficial SP-aliquot by centrifugation. As well, it waives the need of expensive refrigerated centrifuges. Moreover, inter-boar variation was minimised by use of P1-spermatozoa which, not only were the "best" spermatozoa to be cryopreserved, but uses a portion of the spermrich fraction where the documented "fertility-associated" proteins are present (Rodriguez-Martinez et al 2011). Finally, the procedure frees the rest of the collected spermatozoa (75% of the total sperm count) for additional processing of liquid semen AI-doses. This simpler protocol ought thus to be an interesting alternative for AI-studs to –using the one and the same ejaculate- freeze boar semen (P1) for gene banking or for repopulation or commercial distribution, along with production of conventional semen doses for AI with liquid semen, using the rest of the ejaculate. Such procedures would not disturb routine handling of boars or their ejaculates. Inseminations in the field (deep intra-utero) have shown acceptable

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

membrane stability after cryopreservation (Hossain et al 2011).

figures for farrowing and litter size (Wallgren, personal communication).

**4. Cryopreservation of oocytes and embryos** 

(Saravia et al 2010).

However, doses with such large sperm numbers conspire against the best use of the ejaculates and, with the introduction of intrauterine deposition of semen, it opened for the use of smaller containers with high numbers of spermatozoa to contain a single AI-dose. Recently, boar spermatozoa have been frozen, highly concentrated, in small volumes (0.5-0.7 mL) in novel containers, the so-called "MiniFlatPack™" (Saravia et al 2005, 2010, Pimenta-Siqueira et al 2011), as 1-2 billion spermatozoa/mL. Interestingly, cryosurvival (see **Table 2**) was equal or higher than for 0.5 mL plastic straws, suggesting the shape maintained the cryobiological advantages of the FlatPack™ (Ekwall et al 2007), including fertility when using deep-intrauterine AI (Wongtawan et al 2006).

**Sperm vitrification**: Boar spermatozoa packed in 0.12 mm thick film plastic bags were frozen ultra-rapidly at various stages of conventional freezing-thawing and besides survival, samples were explored ultrastructurally, for presence of ice damage. Survival was minimal and ice presence was detected, indicating that cooling rates, although high, were not enough to handle the volumes assayed (Bwanga et al 1991b). Non-penetrating sugars have either been used for vitrification (Meng et al 2010) and also for empirical improvement of slow freezing (Malo et al 2010). There is, *a priori*, nothing against the use of vitrification for freezing small suspensions of boar spermatozoa (for instance for intracytoplasmic sperm injection, ICSI, Meng et al 2010) but there is no practical use for breeding, since the amounts needed are too large to achieve ultra-rapid cooling and thawing rates.


Table 2. Cryosurvival (Computer Assisted Sperm Analysis, CASA, mean±SEM), as percentages of motile spermatozoa, 30 min post-thaw at 38 °C) of ejaculated boar spermatozoa from the sperm-peak portion (P1, first 10mL of the sperm-rich fraction, SRF) or the entire SRF subjected to a simplified (SF, 3.5h) or a conventional freezing (CF, 8h) and an equal thawing (35°C for 20 seconds) (Modified from Saravia et al 2010).

**Learning from the ejaculate:** Boar SP is a composite, heterogeneous fluid composed by fractioned secretions of the epididymal caudae and the accessory sexual glands. *In vivo*, spermatozoa contact some of these fractions but not necessarily all, and different effects (sometimes deleterious, sometimes advantageous) have been recorded *in vitro* when removing (Fraser et al 2007) alternatively keeping boar spermatozoa in its own SP, depending on the fraction used (Guthrie & Welch 2005, Rodriguez-Martinez et al 2009, 2011). The SP or the sperm-rich fraction (SRF) might not be necessary for cryosurvival or fertility, since spermatozoa from boars that were semino-vesiculectomised were able to sustain freezing and thawing equally well as spermatozoa bathing in seminal vesicular proteins (Moore et al 1977). However, we have recently determined that boar spermatozoa

However, doses with such large sperm numbers conspire against the best use of the ejaculates and, with the introduction of intrauterine deposition of semen, it opened for the use of smaller containers with high numbers of spermatozoa to contain a single AI-dose. Recently, boar spermatozoa have been frozen, highly concentrated, in small volumes (0.5-0.7 mL) in novel containers, the so-called "MiniFlatPack™" (Saravia et al 2005, 2010, Pimenta-Siqueira et al 2011), as 1-2 billion spermatozoa/mL. Interestingly, cryosurvival (see **Table 2**) was equal or higher than for 0.5 mL plastic straws, suggesting the shape maintained the cryobiological advantages of the FlatPack™ (Ekwall et al 2007), including fertility when

**Sperm vitrification**: Boar spermatozoa packed in 0.12 mm thick film plastic bags were frozen ultra-rapidly at various stages of conventional freezing-thawing and besides survival, samples were explored ultrastructurally, for presence of ice damage. Survival was minimal and ice presence was detected, indicating that cooling rates, although high, were not enough to handle the volumes assayed (Bwanga et al 1991b). Non-penetrating sugars have either been used for vitrification (Meng et al 2010) and also for empirical improvement of slow freezing (Malo et al 2010). There is, *a priori*, nothing against the use of vitrification for freezing small suspensions of boar spermatozoa (for instance for intracytoplasmic sperm injection, ICSI, Meng et al 2010) but there is no practical use for breeding, since the amounts

using deep-intrauterine AI (Wongtawan et al 2006).

needed are too large to achieve ultra-rapid cooling and thawing rates.

Table 2. Cryosurvival (Computer Assisted Sperm Analysis, CASA, mean±SEM), as percentages of motile spermatozoa, 30 min post-thaw at 38 °C) of ejaculated boar

equal thawing (35°C for 20 seconds) (Modified from Saravia et al 2010).

spermatozoa from the sperm-peak portion (P1, first 10mL of the sperm-rich fraction, SRF) or the entire SRF subjected to a simplified (SF, 3.5h) or a conventional freezing (CF, 8h) and an

**Learning from the ejaculate:** Boar SP is a composite, heterogeneous fluid composed by fractioned secretions of the epididymal caudae and the accessory sexual glands. *In vivo*, spermatozoa contact some of these fractions but not necessarily all, and different effects (sometimes deleterious, sometimes advantageous) have been recorded *in vitro* when removing (Fraser et al 2007) alternatively keeping boar spermatozoa in its own SP, depending on the fraction used (Guthrie & Welch 2005, Rodriguez-Martinez et al 2009, 2011). The SP or the sperm-rich fraction (SRF) might not be necessary for cryosurvival or fertility, since spermatozoa from boars that were semino-vesiculectomised were able to sustain freezing and thawing equally well as spermatozoa bathing in seminal vesicular proteins (Moore et al 1977). However, we have recently determined that boar spermatozoa contained in the first 10 mL of the SRF (also called sperm-peak portion or P1, where about ¼ of all spermatozoa in the SRF are) were more resilient to handling (from extension to cooling) and cryopreservation that the spermatozoa contained in the rest of the ejaculate (Peña et al 2003, Rodriguez-Martinez et al 2009, Saravia et al 2007, 2010, Rodriguez-Martinez et al 2008). It appeared that it was actually the SP in this sperm-peak P1 portion that was beneficial for spermatozoa, either because of its higher contents of cauda epididymal fluid and specific proteins, or its lower amounts of seminal plasma spermadhesins, bicarbonate or zinc levels (Rodriguez-Martinez et al 2011), compared to other fractions of the ejaculate (Saravia et al 2010).

In an attempt to simplify the freezing protocol, only the P1-spermatozoa were frozen in concentrated form for eventual use with deep-intrauterine AI. These spermatozoa were firstly kept in their SP for 30 min, and thereafter, without centrifugation (i.e. without removal of the SP) they were mixed with lactose-egg yolk (LEY) extender and cooled down to +5°C within 1.5 h, before being mixed with LEY+glycerol (3%) and OEP and packed into MiniFlatPack™´s for customary freezing using 50°C/min cooling rate. This "simplified" entire procedure (SF), lasted 3.5 h compared to the "conventional freezing" (CF) that was used as control procedure, which lasted 8 h. As controls, spermatozoa from the SRF were compared to P1-spermatozoa. Cryosurvival was, as seen in **Table 2**, equally good (above 60% of the processed cells (Saravia et al 2010, Pimenta-Siqueira et al 2011). Moreover, the spermatozoa in the sperm-peak-fraction of the boar ejaculate showed a maintained plasmalemmal intactness and fluidity and a lower flow of Ca2+ under capacitation conditions post-thaw, which might account for their higher membrane stability after cryopreservation (Hossain et al 2011).

There are several advantages of using this simplified, shorter protocol, namely the exclusion of the customary primary extension and the following removal of this conspicuously beneficial SP-aliquot by centrifugation. As well, it waives the need of expensive refrigerated centrifuges. Moreover, inter-boar variation was minimised by use of P1-spermatozoa which, not only were the "best" spermatozoa to be cryopreserved, but uses a portion of the spermrich fraction where the documented "fertility-associated" proteins are present (Rodriguez-Martinez et al 2011). Finally, the procedure frees the rest of the collected spermatozoa (75% of the total sperm count) for additional processing of liquid semen AI-doses. This simpler protocol ought thus to be an interesting alternative for AI-studs to –using the one and the same ejaculate- freeze boar semen (P1) for gene banking or for repopulation or commercial distribution, along with production of conventional semen doses for AI with liquid semen, using the rest of the ejaculate. Such procedures would not disturb routine handling of boars or their ejaculates. Inseminations in the field (deep intra-utero) have shown acceptable figures for farrowing and litter size (Wallgren, personal communication).
