The Fertility of Frozen Boar Sperm When used for Artificial Insemination

Introduction

The swine industry has tried for many years to determine conditions for practical and economic use of cryopreserved boar sperm. Its value has been modelled on success in other species to dramatically increase the number of sperm that can be frozen and stored as individual insemination doses from each ejaculate. In addition, in swine and other species, it is greatly advantageous to be able to extend the storage time for sperm by weeks and months, distribute sperm over great distances and shipping conditions, allow for precision mating when females are fertile, preserve and bank valuable and unique genetics, provide time for screening sires and semen for pathogens, have on farm semen in case of natural disaster and serve as a gateway technology for use of sex-sorted sperm (Bailey et al. 2008; Knox 2011). In swine, production and use of cryopreserved sperm has been ongoing for 25 years, although it has generally been limited to use by genetic suppliers with needs to support export markets and a desire to create sperm banks for valuable sires (Hofmo and Grevle 2000). Reports suggest that cryopreservation of sperm in most species results in loss and damage to sperm and a reduction in overall fertility when performing AI (Bailey et al. 2000; Watson and Green 2000). In swine, cryopreservation has not allowed an increase in the number of doses of sperm that can be frozen and stored. Further, the sperm that do survive the freeze and thaw process are damaged more so than in other species (Rath et al. 2009). The reasons for these phenomena are likely related to the large volume of the boar ejaculate and the fragile nature of the sperm membrane (Johnson et al. 2000; Techakumphu et al. 2013). The freeze–thaw process causes dramatic changes in the boar sperm membrane that involve removal, lateral displacement or replacement of proteins and lipids that can affect cell permeability and function (Leahy and Gadella 2011). The higher ratio of polyunsaturated to saturated fatty acids in the membrane and the increase in ROS as a result of freezing and thawing increases rates of lipid peroxidation and alters sperm activation, capacitation and acrosome exocytosis (Bailey et al. 2000; Bathgate 2011). Cryopreservation of boar sperm has also been associated with alterations in DNA condensation and de-condensation and fragmentation (Fraser et al. 2011). Sperm damage can also result from ice crystal formation and thawing and osmotic stress occurring during the freezing and thawing processes which could affect membrane integrity, mitochondria and sperm motility, viability, and number of sperm cell functions required for fertility (Watson 2000).

Since the 1970s, evidence of fertility was demonstrated with cryopreserved boar sperm. These early studies set the stage for research into methods to develop the technology for practical use (Johnson 1985). Yet even today, large differences exist in the costs and time required to prepare a dose of liquid semen compared to a dose of frozen boar sperm. The production of high-quality liquid semen has been reported (Colenbrander et al. 1993), and 24 doses of semen can be produced by a boar from each collection when using 3.0 billion sperm per AI dose (Knox et al. 2008). In newer production systems using low dose AI, this could translate into 48 AI doses if 1.5 billion viable sperm are used. This figure is based on 80 billion sperm produced per ejaculate with approximately 90% of the sperm viable. On average, production of frozen boar sperm results in a loss of 60% of the viable sperm and allows for the production of 5–10 doses of four to six billion sperm. It is not uncommon today to review studies with considerable variation in overall post-thaw sperm survival in the range of 25 to 60%. Higher survival rates for boar sperm are more common when boar selection and specified cryopreservation and thawing procedures are employed (Hernandez et al. 2007b; Rodriguez-Martinez and Wallgren 2011). With many studies, while there is evidence of treatment effects for freezing and thawing methodology for boar sperm, in most cases, there appears to be more variation among experiments in post-thaw sperm survival and fertility than within individual experiments. This is likely due to the dominant effects of the boar on cryosurvival (Waterhouse et al. 2006; Hernandez et al. 2007b) and as a result makes it difficult to choose a single procedure that will work for all boars for cryopreservation.

Despite the significant limits to production of cryopreserved boar sperm doses, the remaining advantages for use of cryopreserved boar sperm are still important. Yet it is known that the sperm that survive cryopreservation have reduced fertility when used for AI (Johnson 1985), and this has been associated with damage that alters sperm motility, viability, capacitation, DNA, membrane structure and acrosome integrity (Holt and Medrano 1997; Fraser and Strzezek 2007). Collectively, this extensive damage could help explain why fertility is difficult to compensate for when using increased sperm numbers alone. The wide array of possible damage to sperm could affect the processes related to sperm survival in the female reproductive tract, establishment of the sperm reservoir, fertilization of ova and embryo survival. What is not clear is whether certain strategies for insemination of cryopreserved boar sperm could help to improve the fertility of the compromised sperm.

Liquid and Cryopreserved Boar Semen Insemination

A general comparison of AI procedures used for liquid and frozen boar sperm helps to illustrate some critical points of value to the swine industry. In liquid form, semen is preserved in extender for storage at 15 to 17°C and for delivery to a breeding farm once to twice each week. The semen can be prepared for use in conventional AI (CAI) or for intrauterine insemination (IUI). For CAI, the semen is packaged to provide ~3 billion motile sperm in 60–80 mL of an extender that can sustain sperm fertility for 3–7 days while in storage (Johnson et al. 2000; Knox et al. 2013). Semen doses are most often packaged in 100-mL disposable plastic tubes, flat bags or bottles. Insemination can occur immediately out of storage, a disposable AI catheter is inserted partway into the cervix, and the semen deposited into the cervix with volume flow into the uterus. The process requires minimal time to insert the catheter and then 3–4 min to facilitate insemination. The IUI approach may also require insertion of a CAI rod into the cervix and then passage of a thin rod into the cervix and into the uterine body. The time to insert the intrauterine rod may require up to a minute or two, but semen deposition requires only a few seconds. Smaller packaging systems in the low volume doses are in use, but the extenders used are the same as for CAI. Various studies have been performed to evaluate a range of sperm numbers (0.25 to 4 billion) used in each AI dose with volumes tested from 25 to 100 mL. Among the different studies, reduced numbers of sperm and volumes are required compared to CAI, but variation is evident in the minimum required to match the fertility with CAI (Watson and Behan 2002; Rozeboom et al. 2004; Behan and Watson 2006; Hernández-Caravaca et al. 2012; Sbardella et al. 2014). The majority of IUI catheters cannot be passed successfully in most gilts and young sows, but new catheter designs have been shown to allow successful passage in mature gilts (Mezalira et al. 2005; Behan and Watson 2006). In comparison with liquid semen, frozen–thawed sperm are most commonly stored on farm in plastic 5.0 or 0.5 mL straws or flat paks in liquid nitrogen tanks (Johnson 1985; Eriksson et al. 2002). Preparation of doses requires thawing of single large or multiple smaller packages. Thawing methods are variable and have been performed in water baths at 37 to 70°C for 10 to 50 s depending upon packaging method and choice. Immediately after thawing, the sperm are expelled from the package into 60–80 mL of liquid extender in plastic bottles held at 22 to 30°C. Insemination typically occurs within 10–30 min after thawing using AI procedures described for liquid sperm.

Quality of the Cryopreserved Sperm Sample

Post-thaw sperm motility and viability assessed within 30 min of thawing has been an important measure used to determine the number of sperm required for insemination. However, extended durations of 120 and 240 min are perhaps more indicative of fertility for cryopreserved sperm (Yeste et al. 2014). Large differences in post-thaw motility are evident and have been associated with boar (Pelaez et al. 2006; Gil et al. 2008), ejaculate (Park and Yi 2002) and the cryopreservation process (Roca et al. 2006; Juarez et al. 2011). New approaches for selecting boars and ejaculates for freezing identify ejaculate parameters that correlate with the ability to survive freeze and thawing. Heat shock protein, Cu and Zn SOD (Casas et al. 2009, 2010), acrosin binding protein (Vilagran et al. 2013) as well as fibronectin 1 in seminal plasma (Vilagran et al. 2015) have each been associated with good freezing and improved progressive motility and viability for up to 4 h after thawing. In other studies, certain traits of the ejaculate samples after freezing have been associated with fertility (Safranski et al. 2011; Daigneault et al. 2014). Studies indicate that sire and not breed is most related to post-thaw fertility. Other more specific sperm attributes that are related to cryosurvival include the amount of long-chain polyunsaturated fatty acids in the sperm membrane (Waterhouse et al. 2006). Others have also noted that in boar collections over seasons, season had significant effects on quality after thawing (Barranco et al. 2013). Other indicators such as sperm capacitation status which have been used as a predictors for litter size for liquid semen (Oh et al. 2010) might also be valuable for cryopreserved boar sperm as sperm response to capacitating stimuli changes with cold stress (Schmid et al. 2013).

Both motility and viability decline after thawing, but significant declines are noted 2 h following thawing. New analyses suggest that single and multiple sperm measures over time can help explain variation for in vivo fertility (Gil et al. 2005; Willems et al. 2010; McNamara and Knox 2013; Daigneault et al. 2014). Data on the fertility of thawed boar sperm that can remain motile and viable for hours in vitro when stored to close to body temperature is limited but could be a useful indicator of their ability to survive and bind to oviduct cells to form a reservoir and fertilize eggs. It is unclear if this measure may be useful for liquid and cryopreserved sperm as well, but it is clear that sperm function is limited for both liquid and frozen–thawed sperm when held at 37° C in vitro. Although the issue of quality and cryopreserved sperm fertility is complex, there is data to suggest that cryopreservation success may be related to sperm exposure to seminal plasma and modification of the sperm membrane. In some studies, pre-freeze exposure to seminal plasma has been shown to reduce vitro motility and fertilizing capacity (Rath and Niemann 1997). While exposure of sperm to seminal plasma is common for freezing sperm rich ejaculates and has led to much success, it could still help explain some of the origins for post-thaw variation in sperm quality (Hernandez et al. 2007a), the boar effect for cryosurvival (Roca et al. 2006) and the different effects for success for freezing the different phases of the ejaculates (Rodriguez-Martinez and Wallgren 2011). In contrast, there are clear examples where addition of seminal plasma to frozen–thawed sperm can help improve in vitro and in vivo sperm fertility (Garcia et al. 2010). In boars with semen classified as poor freezers, motility and viability are low. It has been shown that addition of the seminal plasma from the good freezer boars to the thawing extender improves in vitro sperm fertility (Hernandez et al. 2007a). The type of seminal plasma and the concentration appears important as concentrations that are too high (>50%) could damage sperm whereas when too low (<15%) may have limited to no effect (Kirkwood et al. 2008) and in the optimal ranges will have the most beneficial effect on sperm fertility at extended time intervals after thawing (Kaeoket et al. 2011).

Artificial Insemination with Cryopreserved Boar Sperm

Fertility studies with cryopreserved boar sperm in weaned sows have been reviewed to illustrate the conditions for use up to 1985 (Johnson 1985) and also studies since that time (Table 1). Years of research indicate that fertility could be achieved with boar sperm frozen in multiple forms and with variable numbers of sperm, inseminations and when used in sows and gilts. The fertility results were variable across methods but based on conventional insemination using four to six billion total sperm, there appeared little effect of using more than six billion total sperm and surprisingly, more than a single insemination. Up to the time of 1985, pregnancy rates were variable but with an average of 65% and a litter size of 8.5. Since that time, numerous studies have been performed with cryopreserved sperm. From a review of the publications, it appears that little has changed from the procedures used and the variability in the fertility responses. Yet it is difficult not to notice that the frequency of studies with improved in vitro sperm quality and higher pregnancy rates and litter sizes have been published. This observation could be related to the significant advancements that have occurred with breeding herd management, insemination technology and boar evaluation for fertility. However, with respect to use of cryopreserved boar sperm, improvements in sperm quality and fertility from insemination may reside in sire selection for post-thaw motility, and procedures that reduce the extent of sperm damage during cryopreservation. This would be supported by reviews that report the ability to improve consistency and control of the cryopreservation and thawing processes for boar sperm can be achieved using selected technologies (Rath et al. 2009; Rodriguez-Martinez and Wallgren 2011). A recent study also indicated that farrowing rates and litter sizes could approach commercial expectations with high sperm motility, 81% farrowing rates and 13 total born pigs when using frozen–thawed boar sperm in three inseminations of two billion motile sperm in each AI (Didion et al. 2013). This study was also important because it showed a significant improvement in farrowing rates over a 4-year period that was attributed to improvements in the procedures for semen cryopreservation, AI and handling frozen–thawed sperm.

Fertility studies using cryopreserved boar sperm have been performed using gilts as models for sow fertility to help explore early fertilization and embryo development following insemination. The use of gilts allows routine recovery of the reproductive tract for assessment of ovulation, fertilization and embryo survival rates (Gadea 2005). However, the data can be difficult to compare as some studies induce and breed pre-pubertal gilts while in others use mature gilts for breeding. In the gilt studies, the insemination conditions tested were often similar to those for sows based on sperm numbers, number of inseminations and the timing of the inseminations. In the gilt models, pregnancy rates were often in the 60–70% range with 9–10 fertilized eggs and embryos recovered. From the published work when using frozen–thawed sperm in both sows and gilts, it is not clear whether the use of cryopreserved sperm resulted in reduced fertilization rates and higher extents of embryo loss and pregnancy failures. From the available data, it appears that in many cases of cryopreserved sperm use, pregnancy rates and farrowing rates differ by 5–10%. Yet this difference may not only be attributed to cryopreserved sperm use because differences in pregnancy and farrowing rates with liquid semen often include 5–7% changes. In fact, research suggests that there is no difference between fresh and frozen sperm on the capability of cryopreserved sperm to fertilize eggs, induce embryo development and establish pregnancy (Bertani et al. 1997) and is supported by in vitro and in vivo fertilization and embryo production systems (Almiñana et al. 2010). With cryopreserved sperm use in gilts, fertilization and embryo recovery rates have been reported at 60–70%, which are similar to those for data in sows and for fertility when using fresh semen (Bwanga et al. 1991; McNamara and Knox 2013; Ringwelski et al. 2013). Yet despite some evidence that cryopreserved and fresh semen have similar fertility capabilities, there are numerous reports that show sperm damage and reduced in vitro (Johnson 1985; Córdova et al. 2002; Guthrie and Welch 2005; Fraser and Strzezek 2007; Flores et al. 2009) and in vivo fertility (Woelders 1997; Abad et al. 2007; Estrada et al. 2014). There is also evidence that excessive sperm damage, such as that occurring during the sorting and then cryopreservation process, can lead to increased rates of early pregnancy loss (Bathgate et al. 2008). This is interesting as there are reports that when comparing fresh and cryopreserved sperm, there are overall differences in the number of sperm transported through the uterus following insemination, but little effect on numbers of sperm in the reservoirs at 8 h following AI. Yet differences in the number of normal, fertile sperm are evident, and these differences persist and become more pronounced as time from insemination progresses (Pursel et al. 1978). These data might suggest a capability of a critical number of cryopreserved sperm to occupy the limited number of binding spots in the oviductal reservoir, but as the numbers of normal sperm are limited or become more limited with time, higher rates of abnormal fertilization and embryo loss may occur.

Alternative Insemination Approaches

A technology that attracts much attention in the swine breeding industry is the alternative insemination methods. These methods are of great interest for limiting the number of cryopreserved sperm required for fertility (Rath et al. 2009). The intrauterine insemination (IUI) technology has been successful for reducing the requirement for fresh sperm numbers by 1/3rd. However, with frozen boar sperm, IUI allowed establishment of fertility but did not provide any clear advantage to fertility (Abad et al. 2007). This appears to be supported by other studies using two billion motile sperm, which resulted in a farrowing rate of 76% and 9.0 pigs born but with no clear advantage to using IUI (Chanapiwat et al. 2014). On the other hand, deep uterine AI (DUI) has been shown to be able to reduce numbers of cryopreserved sperm from 6 to 1 billion sperm with acceptable pregnancy rates and litter sizes occurring (Roca et al. 2003). Yet the comparisons of methods alone may not give a clear picture as sperm numbers, and many other factors may affect fertility. For example, one study demonstrated that intrauterine AI using two billion frozen–thawed sperm established a 66% pregnancy rate with an average of 13 embryos, while DUI with only 1 billion frozen–thawed sperm established a 33% pregnancy rate with nearly seven embryos (Buranaamnuay et al. 2011b). The use of IUI and DUI both appear to hold promise, but to achieve the benefits of their use, perhaps the conditions for use and value for frozen sperm fertility will reside in control of many other variables.

Timing of Inseminations Relative to Ovulation

One of the key factors associated with fertility of frozen boar sperm involves the timing of interval from insemination to ovulation. Variation in onset of oestrus to ovulation occurs in swine (Weitze et al. 1994), and the interval from insemination to ovulation has clear effects on fertility (Flowers and Esbenshade 1993). Due to the inability to predict time of ovulation and optimize timing for AI, multiple inseminations are performed to increase the chances that an AI occurs at the optimal interval for fertility. The timing of insemination for liquid semen is targeted to occur within 24 h before ovulation (Soede et al. 1995) but for cryopreserved boar may need to occur at intervals closer to time of ovulation (Waberski et al. 1994). As such, multiple inseminations (Martin et al. 2000) or timed inseminations following induced ovulation (Garcia et al. 2007) are often used to increase chances of success. Interestingly, the number of inseminations with frozen–thawed sperm has not always shown significant effects. In cases where two or more inseminations occurred during oestrus, results were not able to demonstrate an advantage in fertility. Timing of insemination relative to ovulation is sometimes difficult as observations are not continuous, and there are other factors which may affect fertility. Further, in AI studies with frozen–thawed sperm, multiple inseminations can mask timing effects. Yet with single AI studies, and in studies with multiple inseminations but applying genotyping of offspring, the majority of the pregnancies that are established occur with an AI occurring at -12 h before to 12 h after ovulation. Fertility rates clearly decline with insemination intervals of frozen–thawed sperm extending before or after this period. The litter size data are not as clear because many pregnancies fail to establish and limited observations occur out of the windows. However, the greatest frequency of litter observations occurs in the similar window to that for optimal pregnancy rates (Spencer et al. 2010; McNamara and Knox 2013; Ringwelski et al. 2013). This fertility window is supported by other studies that show no differences in timing for cryopreserved sperm when inseminations occurred within a −12 to −2 h window relative to ovulation (Abad et al. 2007). Others have also reported timing of ovulation on cryopreserved boar sperm fertility (Bolarín et al. 2006), and there is a report that suggests higher embryo viability when AI is performed before as opposed to after ovulation (Bertani et al. 1997). Overall, the studies are in general agreement in that the fertility of cryopreserved boar sperm has optimal fertility within a close interval to ovulation and declines away from that time. The explanation for time effects is likely tied to the number of normal fertile sperm in the reservoir. In two studies using multiple inseminations, genotyping indicated that a 1st AI occurring at -12 h before ovulation, sired 60% of the litter while the 2nd AI occurring 0 h, sired the remainder. Further, there were clear effects of an insemination accounting for 100% or 0% of paternity that was not related to boar. If true, this might suggest a reservoir limitation for fertility.

Cryopreserved Sperm Additives

A promising approach that has been reviewed for improving cryopreserved boar sperm fertility has been the use of additives included prior to cooling, during processing for freezing, and when thawing. Substance such as antioxidants, hormones, sugars, proteins and lipids have been included to improve the ability of sperm to retain normal cell characteristics important to fertility. The boar sperm membrane is high in PUFAs, and these are observed to decrease due to lipid peroxidation by ROS during the freeze and thaw process. Further, sperm that undergo freezing and thawing generate increased ROS that damage sperm. Antioxidants have been used to protect boar sperm from ROS damage to unsaturated fatty acids in the sperm membrane and to nuclear DNA (Großfeld et al. 2008; Bathgate 2011). Several studies have shown moderate to quite dramatic effects of a variety of antioxidants on in vitro sperm quality. Antioxidants such as BHT (Roca et al. 2004), polysaccharides (Hu et al. 2013), rice bran oil (Kaeoket et al. 2012), rosemary extract (Malo et al. 2010) and fennel extract (Malo et al. 2012) were able to improve most measures of post-thaw sperm fertility. Others have added the target molecules of the ROS and certain PUFAs in the freezing extenders to improve post-thaw sperm quality (Kaeoket et al. 2010b). A wide array of antioxidants, including estradiol 17β, alpha tocopherol, GSH and ascorbic acid, has been shown to reduce lipid peroxidation and improve most measures of in vitro sperm fertility for hours following thawing (Breininger et al. 2011; Giaretta et al. 2015). Even the addition of precursors to endogenous ROS scavengers, such as L-cysteine in the GSH pathway, improves post-thaw sperm measures (Kaeoket et al. 2010a). This may be a critical component because GSH concentrations show notable differences between boars classified as good and bad freezers (Yeste et al. 2014). Inclusion of antioxidants such as glutathione (Estrada et al. 2014) or butylated hydroxytoluene (Trzcińska et al. 2015) in the freezing media have had dramatic effects for protecting sperm in vitro, and this effect remains when used for insemination. Excellent fertility (>90% farrowing rate and 13.0 total born) was obtained with glutathione addition when using a total of three billion frozen–thawed sperm in a double IUI. However, not all antioxidants have worked as well and might be attributed to the concentration and the freezing conditions (Buranaamnuay et al. 2011a), or perhaps while improving in vitro sperm motility and viability and IVF (Luño et al. 2014) have only minimal effects on in vivo fertility (Luño et al. 2015). Similarly, not all sperm additives, such as caffeine (Yamaguchi et al. 2009) or prostaglandin (Knox and Yantis 2014) have worked well to improve fertility when used with AI. Collectively, these data suggest that the conditions for improving the yield and fertility of frozen–thawed boar sperm is possible with the use of specific additives under defined conditions. The potential for improved fertility of cryopreserved boar sperm through the use of additives to protect against cell damage appears the most promising approach to advance this technology for practical use.

Summary

These data strongly suggest that to take advantage of the opportunities for use of cryopreserved boar sperm, selection of sires for post-thaw sperm quality must be taken into account. Screening boars for success in cryopreservation and the strategic use of antioxidants should greatly increase the doses of cryopreserved sperm produced per ejaculate. Controlling time of ovulation must be applied as a priority for reducing variation in AI timing and for facilitating single insemination approaches for efficient use of frozen boar sperm.