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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

Sperm quality can be variable in morphometric and physiological attributes between males of different species, between males within species subtypes reared under different environmental conditions, between ejaculates of the same male or even between sperm populations within an ejaculate. Clinical semen evaluation is based on evaluation of whole ejaculates, which is not a chemically or physiologically well-defined entity, rather a collection of heterogeneous subpopulations giving different measurements and possessing different fertilizing potential. Identification of subpopulations with different motility patterns is important as well as characterizing the subtle structural changes underlying the motility differences observed. The ability to identify populations of sperm responding rapidly or failing to progress through the capacitation process may have clinical applications. Studies of lipid-phase fluidity of sperm membranes, mathematical modelling of membrane ion transport, role of modifying components and detergent-resistant microdomains are of particular interest. When customizing extenders to ejaculates from cryosensitive males or species, a thorough knowledge of species sperm membrane physiology and an assessment of the individual ejaculate's sperm populations are necessary. Structural differences have been found in sperm membranes between fox species with different cryosurvival potential of their spermatozoa. Supplementation of lipids and detergents in cryoextenders may influence membrane fluidity of the surviving spermatozoa in a species-dependent manner and influence capacitation. Immobilization of sperm prior to cryopreservation with subsequent slow release of sperm in the female genital tract may be a way to prolong the fertile life of sperm. In canids with a long oocyte maturation time, delayed capacitation may be beneficial.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

In the commercial setting of the production animal AI industry, males whose ejaculates do not meet the minimum criteria for processing through the routine preservation procedure or freezing protocol are discarded. Although males reared to puberty represent an economic loss if culled, the consequences of culling a male or the occasional ejaculate are, albeit costly, not devastating. In the scenario of a particularly appreciated dog or horse, or a genetically unique individual of an endangered species, the consequences of not being able to use a particular sperm sample or even the male at all may be large, although not necessarily based on economic considerations. Spermatozoa carrying the male genome are essential in preserving genetic variation within a species. Artificial insemination with superior or unique semen is currently the most powerful genetic tool which can be combined with a variety of reproductive technologies. Rescuing semen samples by customizing preservation protocols to individuals to make use of their genetics is therefore a great challenge, even recognized as economically significant by the horse industry (Loomis and Graham 2008).

Sperm quality varies in morphometric and physiological attributes between males of different species (Miller et al. 2005), between males within species subtypes reared under different environmental conditions (Butts et al. 2011), between ejaculates of the same male depending on, for example, age, time of year, physical condition and sexual stimulus; or even between sperm populations within an ejaculate. It is now well documented and widely accepted that sperm from a given ejaculate is not one homogenous population, rather a collection of heterogeneous subpopulations giving different measurements and possessing different fertilizing potential (Martinez-Pastor et al. 2005; Núñez-Martínez et al. 2006; Núñez-Martinez et al. 2007). Semen evaluation and short- or long-term preservation of sperm must deal with these differences.

Furthermore, in some cases relating to endangered canids, when sperm availability, concentration or morphology is the bottleneck, or there are limitations to capturing females for AI, single deposition of semen may be the only option. Sperm management must then be adapted to what you sample in the ejaculate with regards to sperm morphology and sperm concentration. In such cases, the vitality and longevity of the spermatozoa both during transport to the female if they are not located at the same premises and after deposition into the female genital tract are crucial. The possibility to select potentially vital and long-living sperm and to be able to influence longevity in the female tract is an important aspect for success.

In this review, some of the aspects of semen preservation challenges for different species, different ejaculates and even for subpopulations of fertile vs. non-fertile spermatozoa within ejaculates will be discussed, and some points of focus for sperm preservation beyond the state of the art will be highlighted.

Sperm Selection and Competition in the Oviduct

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

When screening males for semen quality in the laboratory before adding extender for fresh or frozen preservation, the accuracy of prediction of fertility of the current ejaculate is highly variable; only a few methods used to assess sperm samples have been shown to have some predictive value. This may partly depend on the interaction with the female genital tract in vivo.

The female genital tract, the oviduct in particular, is capable of sorting out single spermatozoa with superior sperm quality from the rest through a series of selective gateways. This capacity seems well conserved among mammalian species and can be observed in different species of animals such as cattle, pigs and poultry. If mechanisms of sperm selection are similar in these animal species, they are relevant to determine fertility and hence, would assist in improving semen evaluation as well as semen conservation in the laboratory. So what could they be?

Sperm competition has been associated with postcopulatory competition between different males. Sperm competition also exists between and within sperm population of the same ejaculate. Factors regulating how spermatozoa survive and maintain their fertility in the female genital tract and sperm selection occurs in the oviduct are not known, but may lie in simple changes in physical properties of the oviduct, such as pH gradients or local osmolarity changes. Motility dynamics in sperm populations while moving within the tract towards the site of fertilization, or structural alterations in individual sperm as a response to oviductal signals, may modify the effects of physiochemical changes in the microenvironment. Mimicking some of these changes in the laboratory before and after cryopreservation may give an opportunity to evaluate the competitive ability of sperm.

Associations between sperm and its binding proteins and receptors (e.g. sperm progesterone (P4) receptors) help build sperm reservoirs from which pools of sperm are released (Ignotz et al. 2007). Regions of the reproductive tract contain receptors to which sperm associate before fertilization, such as the caudal portion of the oviduct in cows, which is enriched with sperm-associating annexins. Phosphatidyl serine (PS) translocation is an indicator of asymmetry alterations of plasma membrane associated with apoptosis in somatic cells and with capacitation of sperm in humans. Assessment of degree of PS translocation, that is PS exposed cells, can be performed by using Annexin V conjugated to a fluorochrome such as FITC (de Vries et al. 2003; Cheuquemán et al. 2011).

Gene Expression and Sperm Structure

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

Moreover, sperm responses connected to structural changes which alter gene expression and membrane surface protein composition or distribution may influence their competitive ability. Spermatozoa themselves, for instance, as a response to physical stimuli from either oviduct epithelium or seminal fluids or extenders may undergo genomic shifts (change of methylation patterns) resulting in transcription of specific proteins. Surface proteins which change the surface qualities of individual spermatozoa can influence sperm selection as well as capacitation time. Changes in gene expression and global methylation patterns of spermatozoa have been shown to influence sperm concentration, sperm motility and the outcome of IVF in humans (Benchaib et al. 2005; Marques et al. 2008).

Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

When customizing extenders for ejaculates from different males or species, a thorough knowledge of sperm membrane physiology of the species, of the individual ejaculate's sperm populations and of their motility pattern, is necessary.

Identification of subpopulations with different motility patterns is important as is characterizing the subtle structural changes underlying the motility differences observed. Hyperactivation observed during capacitation of mammalian sperm is typically observed as high-velocity symmetrical flagellar beating.

Manipulation of Ca2+ transport across membranes modulates motility patterns of spermatozoa. Extenders that contain substances that either influence calcium leakage from the membrane, or increase intracellular calcium may be used to prolong the longevity of sperm. For example, chelating agents that bind free calcium (e.g. EDTA) added to extenders for fresh uncooled sperm were used for a number of years to prolong the life span of spermatozoa used for AI in foxes and pigs.

Ca2+ is believed to be the primary second messenger that triggers hyperactivated motility. Cytoplasmic Ca2+ levels in the flagellum must increase to allow hyperactivation. The major mechanism for providing Ca2+ to the flagellum (studies from mouse sperm) are CatSper channels in the plasma membrane of the principal piece of the flagellum. Mathematical analysis of Ca2+ dynamics has been used to develop a model for Ca2+ clearance and for CatSper-mediated Ca2+ dynamics. Mathematical models may also be used to understand how Ca2+ patterns produce flagellar bending patterns of sperm in fluids of low and high viscosity and elasticity. Mathematical modelling of Ca2+ dynamics measured by specific extracellular signals such as membrane depolarization, neurotransmitters, calcium channels and intracellular stores of Ca2+ may become a practical tool to predict how sperm may react to capacitating conditions in the laboratory, and buffering of ions can be an useful tool to influence the level of free Ca2+ (Olson et al. 2011).

From this follows that the ability to identify and then manipulate sperm populations which respond to specific capacitating stimuli or fail to progress through the capacitation process may also have clinical applications. Apart from being a diagnostic tool for predicting a semen population's fertilizing potential or a male's reproductive fitness, it may be used to artificially delay capacitation in the female tract by withholding sperm from capacitating conditions. One way to do this is to keep sperm in an immobilized state in chemical substances prior to cryopreservation. In cattle biopolymer, microencapsulation facilitating controlled release of sperm in the female genital tract has been patented and commercialized by SpermVital™, Hamar, Norway (http://www.biokapital.no/Startside/About-us1/SpermVital/). The company claims this immobilization preserves energy and enables a controlled release of spermatozoa in the uterus after insemination over an extended period of time. This makes timing of insemination less critical with regards to ovulation in the female and increases the odds of fertilization success. Microencapsulation techniques have also been studied in fresh canine semen (Shah et al. 2010).

Retaining the non-capacitated state may not always be an advantage, depending on the timing of sperm entry to the site of fertilization in relation to the time when mature ova are present there. Rather, the ability to swiftly react to capacitating conditions may be essential if the time window is narrow, as shown in semen from high-fertility bulls exposed to swim up post-thaw, compared with low-fertility (LF) bulls. Sperm from LF bulls showed a reduced ability to develop hyperactivation under capacitating conditions, which may affect their ability to effectively reach the mature oocyte.

Delay of capacitation for extended time periods may be beneficial in canid species because of the long maturation period of the canine oocyte and the reduced longevity of frozen–thawed sperm, or of compromised fresh sperm.

The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

The reduced ability to respond with a hyperactivated motility pattern after having been exposed to capacitation permissive conditions may be due to an inherent deficiency at the structural level. Studies of lipid-phase fluidity of dog sperm membranes (Alhaider and Watson 2009), the role of membrane modifying components and detergent-resistant microdomains, are of particular interest (Nixon et al. 2011).

On the basis of experimental data in canids, we have found that there are structural differences in membrane fatty acids between fox species with different cryosurvival potential (Miller et al. 2005) and recently that supplementation of detergents such as sodium lauryl sulphate (Equex STM paste) may influence membrane fluidity of the surviving spermatozoa in a species-dependent manner. In a small trial, we tested the original EYT extender (with 3–4% (v/v) glycerol in the final dilution, respectively) versus a two-step EYT extender containing Equex STM paste [Uppsala Equex (UE) extender with 5% glycerol (v/v)] in the final dilution processed according to Linde-Forsberg (2002) for freezing of ejaculates from males of three different canid species, blue fox (Vulpes lagopus), silver fox (Vulpes vulpes) and dog (Canis familaris). The Equex addition and/or the two-step dilution protocol significantly improved post-thaw sperm plasma membrane fluidity of the viable sperm population in both fox species, but not of the viable sperm population of dog semen. Although experiments were not conclusive with regards to the effect of Equex, the different response of sperm from three different species to identical treatment indicated that species adaptive measures can be useful (K.E. Waterhouse, R. Thomassen, C Kielland and W. Farstad, unpublished observations).

While the above is an example of differences between species, examples also exist on differences in sperm attributes between ecotypes within the same species, and that this can be detected for a variety of sperm attributes.

Interestingly, in cod (Gadus morhua) swimming velocities of wild cod sperm were significantly faster than those of cultivated males. Wild males had larger spermatozoa and less circular head morphology than cultivated males. The fatty acid composition of the membranes, the content of monounsaturated fatty acids and the ratio of the different unsaturated fatty acids were also different (Butts et al. 2011). Studies of sperm membrane components in different species of canids are therefore of interest, followed by membrane response studies to homologous seminal plasma proteins or added lipids or lipoproteins in the extenders. Hence, this is an area in which important clues may lie regarding cryopreservation adaptation for cryosensitive species and even individuals.

Studies of lipid-phase fluidity of sperm membranes, the role of membrane modifying components and detergent-resistant microdomains, are of particular interest (Post et al. 2010; Nixon et al. 2011). Also here Ca2+ plays a pivotal role. The plasma membrane Ca2+ -ATPase (PMCA) restores Ca2+ balance in sperm. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function is to release calcium to the extracellular compartment. Particularly, the PMCA isoform 4 in bovine sperm has an essential function for fertility contributing to sperm hyperactivated motility. PMCA activity is influenced by its lipid environment. Sperm membranes have lipid microdomains or detergent-resistant membrane domains (DRM), rich in sphingolipids and cholesterol, forming specialized functional areas. During capacitation characterized by cholesterol efflux, the lipid and protein composition of these special functional areas (DRM) changes (Post et al. 2010).

In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched DRM domains. Sperm-associated proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Homologous seminal plasma regulates sperm cell membrane fluidity. DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival, attachment and detachment within the female reproductive tract and to facilitate recognition, binding and penetration of the oocyte (Girouard et al. 2008).

Manipulation or delay of dissociation by allowing proteins to remain DRM-associated may be one method to delay capacitation. Premature capitation and subsequent acrosome reaction render sperm incapable of fertilization. In the dog, sperm data on variables associated with membrane function have been published, showing that motility and acrosome membrane integrity were highly correlated. Motility was positively and acrosome membrane integrity was negatively correlated with PS translocation of membranes (Cheuquemán et al. 2011). However, in a later study by this group, the addition of sperm plasma to the extender (TEY) helped preserve the integrity of plasma and acrosome membranes but seemed to compromise motility in 72 h-chilled canine spermatozoa (Treulen et al. 2012).

Conventional light microscopic qualitative assessment of sperm is gradually replaced by more quantitative and sophisticated methods of assessing structural sperm attributes in the laboratory, such as computer aided sperm analysis (CASA) technology, Nucleocounter (ChemoMethec, Allerød, Denmark) and flow cytometry. These procedures which allow evaluation of a large number of cells (e.g. from 10.000-300.000 cells per minute in a flow cytometer depending on the flow rate of the cytometer)*, facilitate examination of whole ejaculates, as well as within ejaculate sperm populations. Furthermore, different sperm populations exposed to different treatment protocols can be assessed quantitatively. Data obtained by CASA suggest correlations between some semen motility parameters and fertility after AI, for instance, in different breeds of cattle (Kathiravan et al. 2011). Such data are still scarce in canids, because only very few laboratories have published data on fertility of frozen–thawed semen from AI of any substantial number of females.

A number of functional characteristics of sperm may be obtained through flow cytometry (Cheuquemán et al. 2011). Based on large data sets obtained through these measurements, treatments may be customized to species as well as to individuals, within individual and within ejaculates.

Several research groups have identified and characterized sperm populations in dog ejaculates by using CASA. By this technology, subpopulations of sperm in individual ejaculates may be detected based on motility patterns (sperm velocity, forward progression and lateral tail displacement etc. are detected.) Núñez-Martínez et al. (2006) proposed that sperm cryosurvival could be predicted to a great extent by studying the sperm subpopulation structure.

The sperm head comprises the sperm DNA. It has been suggested that subtle changes in sperm head morphology may be related to abnormal DNA content. Computerized sperm morphometry (ASMA) and sperm chromatin structure assay have been applied for dog sperm to detect subpopulations of sperm differing in head morphology and DNA integrity. Because the sperm head area influences water and heat exchange, which are important processes during cryopreservation, such morphometric differences may permit individually adapted protocols for freezing (Núñez-Martinez et al. 2007). By use of molecular fluorescent probes such as YO-PRO-1 (penetrates cells when damage occurs to the membrane) and propidium iodide, early cryodamage could be discriminantly detected in dog sperm.

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

In conclusion, new knowledge on how the sperm membrane is organized into microdomains with interaction between lipids and proteins has been described. A comparative approach to studying structural aspects of sperm morphometry, motility and capacitation in canid ejaculates would be beneficial to bring cryopreservation methods further. Identifying sperm subpopulations, applying mathematical modelling of Ca2+ movement and cholesterol efflux across membranes and using substances that temporarily arrest sperm motility both before freezing and after AI may offer new tools to influence post-thaw sperm longevity after deposition in the female genital tract. In canids with a long oocyte maturation time, delaying capacitation by temporarily arresting sperm motility and providing slow release of sperm in the female genital tract may be beneficial if sperm source and AI opportunities are limited, as well as in situations of samples with few or slow-moving sperm.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

The author acknowledges funding from the Division of Research and Ethics (UFE) at the Norwegian School of Veterinary Science and the Research Council of Norway. The sponsors took neither part in the experimental set up or in any intellectual contribution to the study. The author is grateful to Karin E. Waterhouse for her valuable discussions on sperm function and flow cytometry assessment.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References

The paper is an invited contribution to the 15th International Symposium on Canine and Feline Reproduction. The author has worked with sperm assessment and sperm cryobiology in various species and planned and wrote the paper.(As stated under acknowledgement K.E. Waterhouse has read the paper prior to submission with particular reference to paragraph on flow cytometer).

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Sperm Selection and Competition in the Oviduct
  5. Gene Expression and Sperm Structure
  6. Understanding the Molecular Basis for Sperm Capacitation Opens New Possibilities
  7. The Sperm Membrane Plays a Crucial Role in Freezing and Thawing of Spermatozoa
  8. Conclusion
  9. Acknowledgements
  10. Conflicts of interests
  11. Author contributions
  12. References