**3. Cryopreservation of boar semen: State-of-the-art**

Porcine AI with liquid-stored semen where either the entire ejaculate or only the sperm-rich fraction (SRF) of the ejaculate is collected, the spermatozoa are re-suspended at low concentrations in chemically defined extenders and stored at 16–20°C for several days before use, most often for up to 3 days) has increased exponentially in the past 25 years. Globally, AI is practiced in 75% of sows (range 10-99%) using >160 million semen doses, with countries in Europe and the Americas having basically all sows under AI (Riesenbeck 2011). Fertility rates are similar to those obtained after natural mating (for a review, see Rodriguez-Martinez 2007a). Liquid semen is therefore used both for production breeding and for genetic improvement at national or regional level, with some export countries having a major international trade. However, the limited shelf-life of liquid semen, its decline in fertility over transit time, and risks of damage due to temperature, pressure or handling changes, all call for alternatives, with a focus on frozen-thawed semen.

Boar spermatozoa are still being "best" cryopreserved (in terms of cryosurvival) using protocols originally devised in the mid 1970´s (Westendorf et al 1975) with modifications (most often empirically introduced). Most methods use standard lactose-egg yolk (or LDL) based cooling and freezing media, following the removal of most of the seminal plasma by extension in chelate-containing (often EDTA) buffers and centrifugation. The freezing media most often include a surfactant (often laurylsulphate, Orvus es Paste-OEP) and glycerol as CPA (2-3% final concentration added at +5◦C). Spermatozoa are further cooled beyond the eutectic temperature at 30 to 50°C/min. Thawing is done at 1,000-1,800 °C/min. The entire procedure takes most often 8-9 hours from semen collection to storage of the frozen doses in LN2, being still tedious (many different steps) and, inconvenient, producing few AI-doses

by slow freezing, but the time elapsing is not short enough to avoid the toxicity that the solute concentration exerts on the cells, either leading to cell death or dysfunction. If the rewarming is too slow, ice (intracellular in particular) can damage organelles and the cytoskeleton. Rapid rewarming diminishes these risks since the toxic solutes or CPA are

For either method listed, the CPA has to gain access to all areas of the cell/tissue/organ. Traditional cooling and re-warming rates affect the fluidity of the membranes of the cell and the organelles through the rearrangement of structural proteins and the dislocation of constituent lipids. If these changes affect diffusion and/or osmosis, they can jeopardize -by causing changes in the viscosity of fluids or inducing osmotic inbalance- the proper distribution of the CPA, its introduction and removal and ultimately, the freezing and the thawing process (Morris 2006, Morris et al 2007). Cooling can disrupt the integrity of the cytoskeleton and of the chromatin structure, including DNA damage (Watson & Fuller 2001, Fraser et al 2011). In cells in suspension, such as spermatozoa, both the form and volume of the sample to be cooled/re-warmed, and the concentration of the contained cells play major roles during the most damaging interval in the process, i.e. during the changes in phase of the extra-cellular water, when heat is either dissipated (during cooling) or incorporated (during re-warming) (Mazur & Cole 1989, Morris et al 1999). It is therefore obvious that samples (cells, tissues, organs) have to pass cooling and re-warming under conditions where

Porcine AI with liquid-stored semen where either the entire ejaculate or only the sperm-rich fraction (SRF) of the ejaculate is collected, the spermatozoa are re-suspended at low concentrations in chemically defined extenders and stored at 16–20°C for several days before use, most often for up to 3 days) has increased exponentially in the past 25 years. Globally, AI is practiced in 75% of sows (range 10-99%) using >160 million semen doses, with countries in Europe and the Americas having basically all sows under AI (Riesenbeck 2011). Fertility rates are similar to those obtained after natural mating (for a review, see Rodriguez-Martinez 2007a). Liquid semen is therefore used both for production breeding and for genetic improvement at national or regional level, with some export countries having a major international trade. However, the limited shelf-life of liquid semen, its decline in fertility over transit time, and risks of damage due to temperature, pressure or

handling changes, all call for alternatives, with a focus on frozen-thawed semen.

Boar spermatozoa are still being "best" cryopreserved (in terms of cryosurvival) using protocols originally devised in the mid 1970´s (Westendorf et al 1975) with modifications (most often empirically introduced). Most methods use standard lactose-egg yolk (or LDL) based cooling and freezing media, following the removal of most of the seminal plasma by extension in chelate-containing (often EDTA) buffers and centrifugation. The freezing media most often include a surfactant (often laurylsulphate, Orvus es Paste-OEP) and glycerol as CPA (2-3% final concentration added at +5◦C). Spermatozoa are further cooled beyond the eutectic temperature at 30 to 50°C/min. Thawing is done at 1,000-1,800 °C/min. The entire procedure takes most often 8-9 hours from semen collection to storage of the frozen doses in LN2, being still tedious (many different steps) and, inconvenient, producing few AI-doses

cell injury can be minimized (Morris 2006, Morris et al 2007).

**3. Cryopreservation of boar semen: State-of-the-art** 

only momentarily present.

per ejaculate (5-8). For examples of current protocols see Eriksson & Rodriguez-Martinez (2000), Saravia et al (2005), Parrilla et al (2009) or Rath et al (2009) and methods cited therein.

This general current protocol fits most boars but considering the large variation between ejaculates and –particularly- among boars for their capacity to sustain cryopreservation (Roca et al 2006a), the protocol has to be modified to accommodate those with sub-optimal sperm freezability (the so-called bad freezers), particularly regarding glycerol concentration and warming rates (Hernandez et al 2007a). Those changes usually allow for minimum acceptable cryosurvival (i.e. around 40%). However, it clearly shows that the methodology is still sub-optimal. Current semen cryopreservation techniques are technically demanding and expensive, both in terms of labour- and laboratory equipment costs, as well as timeconsuming (rev by Roca et al 2006b, 2011). Last but not least, there is a lack of reliable laboratory tests for the accurate assessment of semen quality in vitro, that limits our capacity to properly monitor the methods used to freeze-thaw boar semen and, particularly, its relationship to AI-fertility (Rodriguez-Martinez, 2007b). This is critical, since despite having acceptable post-thaw survival (even above 60%) this cryosurvival is not reflected in fertility after AI. Thus, boar spermatozoa are considered one of the most demanding cell types with respect to sustaining viability during freezing and thawing, with a large proportion of the spermatozoa not surviving these procedures (Penfold & Watson 2001). Moreover, those surviving spermatozoa are usually a mixture of cells, some of which survive well while others show modified motility and a shortened lifespan, factors which compromise their fertilising ability. Insemination with such spermatozoa leads, ultimately, to lowered pregnancy rates and fewer piglets born, compared with AI using liquid-stored semen (Knox 2011). In sum, although freezing methods are nowadays rather stable in many laboratories and yield above 50% of sperm survival post-thaw, fertility after AI is extremely variable (Parrilla et al 2009). The major constrain is not only the inherent difficulties to freeze spermatozoa from this species (Holt, 2000a,b), but -within the species- the sire-dependent cryosurvivability to the current procedures (Eriksson et al 2002, Holt et al 2005, Gil et al 2005, Waterhouse et al 2006, Hernandez et al 2006, 2007a, Roca et al., 2006a, Parrilla et al 2009, Roca et al 2011).

This variation is usually compensated by the AI of excessive sperm numbers (at least 5x109 spermatozoa per AI-dose), i.e. double the numbers of total spermatozoa present in liquid semen doses. Fertility post-AI is nowadays substantially better, closer to AI with liquid semen (Eriksson et al 2002). See **Table 1** for an overview of fertility after conventional (cervical) AI with frozen-thawed boar semen. Fertility with lower sperm numbers is also becoming acceptable when deep intrauterine AI is practiced, although data are still restricted in numbers (Bathgate et al 2006, Roca et al 2006b, 2011). But, even with these huge sperm numbers, overall fertility (as farrowing rates) and prolificacy (as litter size) are still lower than for liquid semen (around 10-30 % lower farrowing rates, and 1-3 less piglets), indicating that other factors are limiting, such as the timing of insemination respective to spontaneous ovulation (Bolarín et al 2006, Wongtawan et al 2006). This implies that we are far from reaching the goals set up by the industry for the use of frozen-thawed semen: 85% of conception rates and a litter size of 11 piglets (Knox 2011). So, frozen-thawed boar semen is still basically limited to research, genetic banking or the export of semen for selected nuclei lines, constituting barely above 1% of all AIs.

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art 239

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,


Table 1. Fertility after conventional (cervical) AI in field trials (>100 sows) with frozenthawed boar semen (modified from Roca et al 2006).

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