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Contents

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

Encapsulation of boar semen is a novel technique that allows insemination to be performed as a single intervention without the need to dilute the semen. The research reviewed in this paper shows that spermatozoa encapsulated in alginate are able to achieve the same fertility as two or three inseminations per oestrus using standard techniques and unencapsulated cells. The use of encapsulated spermatozoa is currently limited by the need for longer semen processing time and wastage of disposable material (catheters, plastic bottles, etc.). In this review, the advantages, the drawbacks and the future possibilities for artificial insemination with encapsulated spermatozoa in the sow are discussed, with the aim of applying this promising new methodology for the optimization of sow reproductive performance.


Introduction: Towards Sperm Encapsulation

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

Domestic swine has reached high levels of prolificacy and fertility compared with wild swine strains: these results are the non-algebraic sum of several innovative aspects and technologies achieved over time to enhance the productivity. Artificial insemination techniques undoubtedly gave a strong boost to these reproductive parameters, but a number of aspects of pig reproduction still need to be enhanced. Several problems arise from sow reproductive physiology, which is characterized by a high variability in the length of oestrus, the interval from oestrus to ovulation and the time span of ovulation, all of which remain under a strong seasonality (Belstra et al. 2004). These factors limit the fertility achievable from a single insemination intervention per heat.

It is well known that a limited proportion of ejaculated boar spermatozoa survive after preservation in a liquid or frozen state. Generally speaking, the survival of sperm cells is greater after liquid storage than after freezing. The proportion of surviving spermatozoa steeply diminishes after the temperature is lowered, paralleled by a loss of membrane integrity and alterations in biochemical support. Johnson et al. (2000) reported that more than 99% of artificial inseminations worldwide are made with a dose of same-day diluted semen or with semen stored at 15–20°C for 1–5 days, limiting to <1% the inseminations performed with frozen semen. The advantages of artificial insemination in pigs are limited by a general sensitivity of the spermatozoa of this species to the chilling/dilution process. Boar spermatozoa, in fact, suffer from ‘cold shock’ when the temperature falls below 15°C even if pre-dilution, mainly in large volumes (100 ml), can attenuate this phenomenon (Johnson et al. 2000). Perhaps, these spermatozoa also suffer from a ‘dilution effect’ due to the fact that several proteins and antioxidant molecules are dispersed in the aqueous medium. The washing effect caused by the extenders can trigger a chain of events that alter the plasma membranes of the spermatozoa. The stripping effect of manipulation and handling of sperm cells can cause capacitation-like events, which normally only occur in the female reproductive tract (Holt and Fazeli 2010). These events lead to the formation of membrane lipid and protein rafts, which involve mainly zona-binding proteins (Tsai et al. 2007, 2010), and are undesirable if they commence before their normal physiological time.

Other problems in boar sperm preservation arise from the freeze-thawing (F/T) processes. Leahy and Gadella (2011) classified four main types of damage caused by F/T procedures in boar spermatozoa: (i) decoating of seminal plasma components (i.e. lipids and proteins) and substitution with proteins and lipids from the extender; (ii) phase separation of lipids and reordering of membrane components; (iii) alterations in ion/water permeability; and (iv) weakening of the ‘stress primed’ cells that often are unable to stand further stress. A number of different methods have been developed to overcome some of these drawbacks, mainly those linked to the dilution effect. The aim of these methods is to ensure a regular controlled release of spermatozoa in the female reproductive tract, preserving as much as possible of their physiological extracellular environment.

One valid technique to achieve this is sperm encapsulation or microencapsulation. This involves the ‘wrapping’ of an undiluted sperm droplet in a biodegradable polymer. The polymer acts either as a releaser of spermatozoa over time or as a molecular sieve, allowing the passage of nutritive elements and wastage and halting the passage of immuno-competent molecules and cells through the capsule. The encapsulation method can be modified in order to adapt to the needs and the targets of breeders. Furthermore, the gradual leakage of spermatozoa from the capsules allows a regular flow of sperm cells towards the fertilization site.

In the following paragraphs, the practical advantages and disadvantages of existing microencapsulation methods will be reviewed. We will also describe a new method that aims to ameliorate the efficiency of sow artificial insemination and to optimize the procedures when applied in large-scale breeding centres.

Targeting Spermatozoa Through Controlled Release

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

The idea of targeting protected spermatozoa towards the site of fertilization was borrowed from pharmaceutical technology. The main advantages of the controlled release of molecules or cells are (i) to allow the molecules (or cells) to reach their site of action protected from external, potentially harmful agents and (ii) to modulate their release over time by tuning their polymeric envelope. Therefore, the adoption of an optimal encapsulation technique in the sow should ensure a slow, constant release of healthy, motile spermatozoa towards the site of fertilization, avoiding the decay of sperm cell function. Furthermore, sperm encapsulation should avoid the ‘dilution effect’, with minimal dispersion of protective molecules. In most encapsulation systems, the volume of the polymer membranes increases the total semen volume without lowering the concentration of extracellular matrix components. In describing this beneficial effect, we have proposed the term ‘virtual dilution’ to indicate the increase in semen volume, without dispersion of spermatozoa, which occurs with microencapsulation (Faustini et al. 2004).

The properties required for a good polymer

The choice of a suitable polymer is a crucial step for good fertilization success after encapsulation. A number of different polymers are available for sperm encapsulation, but, in our opinion, there is not an ideal one. The main requirements for a suitable polymer are as follows: (i) biodegradability; (ii) adhesiveness to the uterine mucosa and to the other capsules to prevent semen backflow; (iii) limited toxicity to spermatozoa; (iv) the ability to limit changes in the environment of the spermatozoa; (v) no or low interactions with immuno-competent molecules and cells; and (vi) low cost.

Alginates are one of the polymers that fulfil many of these requirements. They are unbranched polysaccharides extracted from algae, mainly brown algae (Phaeophyceae), formed by a backbone of guluronic (G) and mannuronic acid (M). The primary structure of these molecules is organized either in brief homo-polymeric sequences or ‘blocks’, made of -GGGG-, or -MMMM-, and called G-blocks or M-blocks, respectively, or in heteropolymers (-GMGMGMGM-, called GM-blocks). The molecular axial orientation of G-blocks forms molecular ‘pockets’ that can be occupied by cross-linking di- and trivalent ions, (e.g. Ca2+, Sr2+, Ba2+, Al3+), forming a three-dimensional network able to entrap water molecules. The capacity to form stiffer or softer gels, and many other chemical/physical properties, depends on the G/M ratio and the length of G-blocks: G-rich alginates having long G-blocks yield stronger gels than M-rich gels. Approximately 200 different alginates are being manufactured (Tønnesen and Karlsen 2002), and the proportion of guluronate generally varies from 14% to 31%, although the alginate extracted from Laminaria hyperborean stems can contain up to 60% of guluronate (Qin 2008). On the basis of the considerations above, divalent ions (calcium or barium) cross-linked with alginate capsules are at present considered to be the best candidates for sperm encapsulation. Alginate capsules can be easily tailored to different needs and have high biocompatibility, as evidenced in several biomedical fields (Lee and Mooney 2011), preserving the membrane structure and function of various cell types. In the uterine environment, they have good adhesive properties, thus preventing the backflow of semen during standard artificial insemination procedures. Alginate capsules are hyaline, smooth and soft and flow freely along the common sponge catheters when mildly dispersed in a simple semen extender.

The evolution of the method

The first attempts to encapsulate spermatozoa date back to 1985 when Nebel et al. (1985) obtained optimal results, in terms of sperm motility and intact acrosomes, through a three-step technique to encapsulate bull spermatozoa in poly-l-lysine capsules. The three-step encapsulation process started with the suspension of semen in a sodium alginate solution. The suspension was then dripped through a needle into a CaCl2-HEPES solution. Once the semen/alginate droplets reached the calcium solution, they immediately formed calcium alginate spheres. These spheres were removed from the calcium solution, rinsed and transferred into a second polymer solution so that a semi-permeable membrane was formed around the droplet. Poly-l-lysine, protamine sulphate and polyvinylamine were described as the most successful polymers for this outer coating of gel droplets (Nebel et al. 1993). Finally, in the third step, the alginate droplet was dissolved by adding a sodium citrate solution to the capsules.

The application of this technique to boar spermatozoa resulted in a good percentage of normal acrosomes but a rapid reduction in sperm motility (Esbenshade and Nebel 1990). In swine, therefore, some changes in the encapsulation technique were required, starting from the process and the polymer employed. The first trials conducted by our research group on boar spermatozoa tested the use of calcium alginate for capsule preparation in a single-step procedure with the aim of avoiding sperm damage caused by over manipulation. Furthermore, we observed that the motility and speed of spermatozoa were severely compromised after encapsulation, with a decrease from 95% to 35–40% and from 65 to 45 μm/s, respectively (Torre et al. 2000).

To avoid such problems, Ba2+ was substituted for Ca2+, to prevent precocious capacitation and hyperactivation of spermatozoa because it interferes with ion channel-driven membrane processes (Munoz-Garay et al. 2001; Zhang and Gopalakrishnan 2005). Perhaps, barium resulted in stiffer gels than calcium at the same ion concentration: at 30 mm, the strength of capsules determined with a texture analyser by applying a compression force of 50 g for 30 min was 1.80 ± 1.54 and 4.99 ± 2.67 for calcium and barium ions, respectively (Villani et al. 2008).

The one-step encapsulation process begins with the suspension of the sperm-rich fraction of the boar ejaculate in a saturated solution of barium chloride. The resulting suspension is then dripped into a sodium alginate solution of medium viscosity under continuous stirring. Barium reacts immediately with alginate, leading to the growth of a barium alginate wall. The inner barium ions diffuse through the growing membrane, cross-link at the interface with alginate and thicken the capsule wall. This process stops when all barium has diffused out of the semen droplet (Fig. 1). Once in the reproductive tract, the polymer wall absorbs water (swelling process) and degrades by naturally replacing the divalent cross-linking ions with Na+ ions. This process allows the release of spermatozoa, and the time of this release depends on the type of ion and the concentration of cross-linking ions.

image

Figure 1.  Encapsulation of boar spermatozoa by the one-step alginate capsule method

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The Results that Can Be Achieved With Controlled Sperm Release

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

The aim of sperm encapsulation is mainly biological: the preservation of sperm quality without compromising membrane integrity. Furthermore, controlled release implies several advantages over standard insemination, because the process has a low cost and can be undertaken, with polymers like alginate, directly in the breeding centre. In addition, the encapsulation process enables a single insemination per heat and, therefore, can sensibly reduce the labour costs and the amount of disposable material required for insemination.

We performed a thorough examination of the effects exerted in vitro by barium alginate capsules on boar spermatozoa. Encapsulated spermatozoa had more non-reacted acrosomes (NRA) less secondary anomalies when compared to free spermatozoa (12.95 ± 7.54% vs 20.35 ± 9.29%, respectively; Torre et al. 2002). We also demonstrated an improvement in NRA when free or encapsulated spermatozoa were preserved up to 72 h at 18°C (96% vs 85% at 4 h and 77% vs 55% at 72 h, respectively; Faustini et al. 2004). Furthermore, the percentage of NRA remained high also when in vitro released spermatozoa were assessed after a period of 24 h (Torre et al. 2002). The activity of the key intracellular enzymes lactate dehydrogenase (LDH) and cytochrome c oxidase (COX), assessed by in situ microdensitometry, decreased steeply after 72 h in unencapsulated compared with encapsulated spermatozoa (Faustini et al. 2004). Because LDH decrease is usually caused by damage to the plasma membrane, and the reduction in COX activity is be related to the integrity of mitochondrial membranes, our results suggest capsules have the capacity to stabilize the sperm membrane.

In a large-scale in vivo trial, we found that a single insemination with spermatozoa encapsulated in barium alginate yielded the same fertility as two or three inseminations with unencapsulated semen (Vigo et al. 2009). Furthermore, our results showed that using encapsulated spermatozoa had no detrimental effect on the number of piglets born while significantly improving both pregnancy and delivery rate. Furthermore, these promising results can still be improved because we also identified some weak points that, if cleared, could significantly enhance the results. One such problem is represented by the difficulty in calculating the length of time during which spermatozoa are released in the female reproductive tract. Although in vitro trials gave us some indicative parameters (Torre et al. 2002), we observed that the time of release of spermatozoa in vivo from alginate capsules can vary over a broad time range. This is caused by a number of different physical/chemical variables acting on the integrity of the polymer wall once the capsules are deposited into the uterine environment. Mechanical forces exerted by the movement of the sow, contractions of the myometrium, body temperature and the degree of gel swelling can all modify the degradation process of the polymer wall. The pH and viscosity of semen can combine to determine the strength of the gel and, in turn, influence the characteristics of the capsules (Vigo et al. 2002). Furthermore, the strong seasonal effect on the quality of both semen and oocytes must play an important role in determining sperm release, as evidenced by the high variation observed throughout the year (Vigo et al. 2002). Therefore, based on our experience, we believe that rather than using a fixed formulation, the preparation of the alginate capsules should be modified taking into consideration the different parameters already indentified and others that will become evident in the future. This should not be a problem given the wide range of polymers with different physical/chemical characteristics currently available.

Use of Encapsulation in Combination With Other Reproductive Technologies

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

Sperm encapsulation could open new opportunities and improve results in artificial breeding of pigs if used in combination with sexed or frozen semen. Sex sorting results in appreciable damage to the spermatozoa, although different from that caused by freezing-thawing procedures. The intense hydrodynamic forces, UV laser stimulation (Guthrie and Welch 2005) and high dilution rates (Maxwell and Johnson 1997) used in sex-sorting procedures lead to precocious acrosome reactions, loss of fertilizing ability (Bailey et al. 2000) and capacitation-like changes (Spinaci et al. 2006).

Our research group recently evaluated the effect of encapsulating sex-sorted boar spermatozoa. In our preliminary results, encapsulation significantly reduced the in vitro fertilizing capacity of sorted spermatozoa, but, at the same time, the proportion of polyspermic oocytes was reduced. The proportions of normospermic oocytes fertilized with sorted-encapsulated spermatozoa increased from 31.29% to 84.7% after 24 h and from 49.4% to 84.6% after 48 h of storage compared with sorted non-encapsulated spermatozoa. These first results are in agreement with other data reported by our laboratory (Faustini et al. 2010), which showed a significant reduction in polyspermic oocytes using encapsulated spermatozoa for in vitro fertilization. Our current research is trying to clarify the effects of encapsulation on the membrane characteristics of sex-sorted spermatozoa and to design strategies to overcome the drawbacks associated with these procedures.

Freezing of encapsulated spermatozoa has already been performed in the human (Herrler et al. 2006) and the dog (Shah et al. 2011) with promising results. Therefore, the prospect for freezing boar spermatozoa in capsules is appealing. Yet, some difficulties have emerged from the well-known sensitivity of boar sperm membranes to freezing and thawing. After the application of a freezing-thawing protocol (Peña et al. 2007) on encapsulated spermatozoa, performed by our group, the majority were dead (>90%), and the consistency of the capsules was severely compromised, significantly below the data reported by Villani et al. (2008). These difficulties might be overcome by the addition of cryoprotectants and/or seminal plasma to the spermatozoa or by adding specific non-heparin binding proteins, such as the spermadhesin PSPI/PSP II heterodimer or the PSP I monomer that can have a prolonged protective effect (Caballero et al. 2006). It remains to be assessed whether these protocols can be employed economically in large-scale artificial insemination programs.

Future Perspectives

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

One of the drawbacks of encapsulation, the inability to time the release of spermatozoa, could be solved by the use of a mix of capsules with different mechanical/release characteristics. For example, the preparation of two or three different kinds of alginate capsule, using different starting ion concentrations, could extend the timing of sperm release, mimicking a pulse-acting system. More elegant methods for controlling the release of spermatozoa could include the use of multilayered capsules (Anal 2007) loaded with spermatozoa between the layers. In this system, the disruption of the wall of the capsule in an intermittent fashion would free the spermatozoa in timed batches, acting as a ‘wave releaser’ (Fig. 2a).

image

Figure 2.  Patterns of release of spermatozoa in multilayered (a) and in smart polymer (b) capsules

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The use of the so-called smart polymers could also open ways to tailor the release of spermatozoa from the capsules. Smart polymers are substances that change their physico-chemical properties (strength, disruption and release rates) in response to environmental changes such as pH, temperature or ionic strength, even if in small ranges (Kumar et al. 2007). In this case, small changes in the physiological condition of the receiver, like modifications of physiological fluids in the reproductive tract in response to the changing endocrine environment approaching ovulation, could be used to trigger the degradation of capsules (Fig. 2b). Degradation of capsules containing bovine spermatozoa triggered by physiological stimuli was attempted by Kemmer et al. (2011) by inducing the rupture of cellulose-based capsules through the release of cellulase from engineered cells co-encapsulated with spermatozoa. The release of cellulase was triggered in response to the pre-ovulatory LH peak. Such approaches appear somewhat articulate, but they are appealing for their capacity to adapt to the biological condition of the receiver, particularly in the case of the sow, given the prolonged ovulation time span and its seasonal variation.

Finally, one must consider the huge multitude of polymers on the market. New natural or synthetic polymers with different properties (and prices) could help to modulate the characteristics of the capsules. In addition to alginate, hydroxyethylmethacrylate, methylmethacrylate, chitosan, and other hydrogels or their combinations are suitable polymers for cell encapsulation (Prakash and Soe-Lin 2004), and their use could widen the possibilities to adapt the release patterns of spermatozoa in response to different biological conditions and needs.

Concluding Remarks

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

Sperm encapsulation is an appealing strategy in an attempt to boost the management and the performances of pig reproduction. Many promising results have already been achieved, but this technology still requires improvement (storage, transport, making and use of doses, scheduling, etc.), particularly in combination with other reproductive strategies, before it can be widely applied in swine breeding practice. Future research will need to integrate different knowledge in a multidisciplinary way, to develop an efficient and reliable method of sperm encapsulation, ready to satisfy the complex needs of a modern pig breeding centre.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References

The authors are indebted with Prof. Chis Maxwell, Faculty of Veterinary Science, University of Sydney, NSW, Australia, and Prof. Fulvio Gandolfi, Faculty of Veterinary Medicine, Milan University, Italy, for the revision of the text and their helpful suggestions.

References

  1. Top of page
  2. Contents
  3. Introduction: Towards Sperm Encapsulation
  4. Targeting Spermatozoa Through Controlled Release
  5. The Results that Can Be Achieved With Controlled Sperm Release
  6. Use of Encapsulation in Combination With Other Reproductive Technologies
  7. Future Perspectives
  8. Concluding Remarks
  9. Acknowledgements
  10. Conflicts of interest
  11. References