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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Seminal plasma can both inhibit and stimulate sperm function, making its use as a supportive medium somewhat contradictory. These effects are directed by the multifunctional action of numerous inorganic and organic components, but it is the direct association of seminal plasma proteins with the sperm membrane that is thought to exert the most significant response. In vitro handling of spermatozoa in preparation for artificial insemination may involve washing, dilution, cooling, freezing, re-warming and sex-sorting. These processes can alter proteins of the sperm surface and reduce seminal plasma in the sperm environment. This, among other factors, may destabilize the sperm membrane and reduce the fertilizable lifespan of spermatozoa. Such handling-induced damage may be prevented or reversed through supplementation of seminal plasma, but the effectiveness of this technique differs with species, and the source and subsequent treatment of both spermatozoa and seminal plasma. Seminal plasma appears to act as a protective medium during in vitro processing of ram spermatozoa, but this does not appear to be the case for bull spermatozoa. The reasons for this divergent effect will be discussed with particular emphasis on the influence of the major proteins of ruminant seminal plasma, known as BSP proteins. The biochemical and biophysical properties of these proteins are well documented, and this information has provided greater insight into the signalling pathways of capacitation and the protective action of extender components.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Ejaculation results in the confluence of spermatozoa from the tail of the epididymis with the protein-rich plasma of the accessory sex glands. Seminal plasma provides a buffered fluid vehicle for the deposition of spermatozoa, which thereafter swim free and interact with the fluids and cells of the female tract. Because spermatozoa are only in contact with seminal plasma for a relatively short period, the substance was initially thought to serve exclusively as a transport medium, but it is now recognized to have long-lasting effects on many aspects of mammalian reproductive physiology including sperm movement (Maxwell et al. 2007), regulation of capacitation (Manjunath et al. 2007; Leahy and Gadella 2011), sperm storage in the female reproductive tract (Talevi and Gualtieri 2010) and modulation of the female immune response to tolerate spermatozoa and the conceptus (Robertson, 2007).

As information on the function of seminal plasma has accumulated, it has become clear that dilution of seminal plasma during semen processing for artificial breeding (e.g. sex-sorting and cryopreservation) may in some part explain the altered function and fertile status of processed spermatozoa. As a result, recent decades have seen considerable investigation of the effect of seminal plasma supplementation on the survival and function of spermatozoa processed for controlled breeding. This will be the main area of interest discussed herein, with particular emphasis placed on the influence of seminal plasma proteins, particularly those containing a fibronectin type-2 domain (Fn-2; also termed BSP proteins). The situation in the bull and ram will be compared and contrasted with occasional reference to other domestic species (particularly the pig and horse) where appropriate.

Seminal Plasma Characteristics of the Ram and Bull

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Seminal plasma composition is highly variable between species; this is largely owing to differences in the relative contribution of the accessory sex glands (Mann 1964). The vesicular gland is large in the ram and bull, but the prostate, ampulla and bulbourethral glands are relatively small or disseminated, producing a concentrated ejaculate of low volume (Maxwell et al. 2007). As the ram and bull are vaginal depositors, a low-volume ejaculate reduces semen backflow, which helps ensure that a sufficient number of spermatozoa are able to negotiate the cervix. When semen deposition occurs closer to the site of fertilization, such as in the uterus of the mare or sow, the prostate and bulbourethral glands are larger in size and secretions are deposited sequentially, delivering an ejaculate that is of larger volume, lower concentration and which contains sperm-rich and sperm-poor fractions (Rodriguez-Martinez et al. 2009).

The major inorganic constituents of ruminant seminal plasma were identified through biochemical analyses (Mann 1964) and include fructose, citric acid and sodium. These components help to buffer the sperm environment and maintain sperm metabolism and osmolarity and are still the base ingredients for standard semen extenders used today (Evans and Maxwell 1987). However, the major component of seminal plasma (by weight) is protein, and it is this constituent that is considered to be the dominant modulator of sperm function.

Semen Processing and Supplementation with Seminal Plasma

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Despite considerable research to optimize procedures associated with assisted reproduction, sperm storage and sex-sorting still inflict significant lethal and sub-lethal damage on spermatozoa [for detailed review please refer to the following (Parks and Graham 1992; Salamon and Maxwell 1995; Vishwanath and Shannon 2000; Leahy and Gadella 2011)]. This damage is primarily to the plasma membrane, which contributes to the advanced membrane state [similar to capacitation (Bailey et al. 2003)] of processed spermatozoa that shortens their fertilizing lifespan.

Part of this damage, at least in simple media, is thought to be due to excessive dilution, which is known to result in cessation of sperm motility, metabolic activity and fertilizing potential. This phenomenon was termed the ‘dilution effect’ (Mann 1964) and can be reversed by the addition of seminal plasma (Maxwell and Johnson 1999). As assisted reproductive technologies usually involve sperm dilution and the removal or reduction of seminal plasma components, there has been considerable interest in the possibility of utilizing seminal plasma, or seminal components, to prevent handling-induced sperm damage.

Supplementation of Seminal Plasma in the Ram

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

In the ram, seminal plasma has been shown to negate the dilution effect (Ashworth et al. 1994) and even increase the resistance of spermatozoa to stress (Maxwell et al. 2007; Muino-Blanco et al. 2008). Seminal plasma proteins have been shown to prevent and/or reverse cold-shock damage, improving the viability and heterogeneity (assessed by centrifugal counter current distribution) of the sperm sample and reducing protein tyrosine phosphorylation (Garcia-Lopez et al. 1996; Barrios et al. 2000; Perez-Pe et al. 2001a, 2002). Seminal plasma supplementation has also been noted to improve characteristics of frozen-thawed ram spermatozoa including motility, capacitation status and ability to penetrate cervical mucus in vitro (Graham 1994; Maxwell et al. 1999; El-Hajj Ghaoui et al. 2007a,b; Leahy et al. 2010c).

The inclusion of ram seminal plasma in sex-sorting protocols has also been successful. Early studies showed supplementation of the staining and collection medium improved the viability and motility of the final sorted product (Maxwell et al. 1996; Catt et al. 1997). Latter studies compared the addition of seminal plasma across various time points in the sorting and freezing process and found seminal plasma supplementation prior to freezing was of most benefit (Leahy et al. 2009). This effect is most likely related to the protection of the spermatozoa during freeze–thawing. However, seminal plasma supplementation may also be protecting sorted spermatozoa from oxidative stress as this sperm type has been shown to be particularly susceptible, and seminal plasma proteins have been shown to protect sex-sorted ram spermatozoa from direct oxidative challenge (Leahy et al. 2010a).

While these results combined appear to clearly demonstrate the positive influence of seminal plasma during ram semen processing, this is unfortunately not the complete story. In fact, the response of ram spermatozoa to seminal plasma has proved to be an inordinately complex interaction dependent on a number of factors. Of primary concern is that the amount and chemical nature of seminal plasma is influenced by physiological, pathological and exogenous (e.g. season) conditions (Maxwell et al. 2007; Muino-Blanco et al. 2008). Post-collection handling further influences the final outcome with noted differences in effect of seminal plasma owing to the washing of spermatozoa (Ollero et al. 1997; Perez-Pe et al. 2001b), the point at which it is introduced and the protein concentration applied (Leahy et al. 2009, 2010c). This variability is thought to account for reports of seminal plasma having no influence on ram sperm survival [chilled storage (Morrier et al. 2003)] or even being detrimental [epididymal (Dott et al. 1979) sex-sorted frozen-thawed (de Graaf et al. 2007)]. Moreover, such inherent variability contributes to the inconsistent positive effect of seminal plasma supplementation on the in vivo fertility of frozen-thawed ram spermatozoa (Maxwell et al. 1999; El-Hajj Ghaoui et al. 2007b; O’Meara et al. 2007; Leahy et al. 2010b).

Supplementation of Seminal Plasma in the Bull

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Unlike the ram, the hypothesis that seminal plasma acts as a protective medium for spermatozoa is not well supported in the bull (Vishwanath and Shannon 1997). Motility of bull spermatozoa, inactivated by dilution (Mann 1964) or Ficoll washing (Harrison 1976), can be restored by seminal plasma (Dott et al. 1979; Baas et al. 1983), but it reduces motile lifespan (Baas et al. 1983). Accelerated cell death was also noted when highly diluted ejaculated (12.5 × 106/ml) bull spermatozoa were supplemented with seminal plasma at low temperatures [5°C (Shannon 1965)] and when epididymal bull spermatozoa were co-incubated with seminal plasma at higher temperatures [30°C; (Way et al. 2000)]. However, seminal plasma did provide some compensatory protection against dilution when bull spermatozoa were frozen in a highly diluted form [2–20 × 106/ml; (Garner et al. 2001)], but no effect was observed on the motility of washed frozen-thawed ejaculated and epididymal spermatozoa from co-incubation with seminal plasma (Graham 1994).

These equivocal, often negative results have meant the use of seminal plasma as a protective supplement during the sex-sorting of bull spermatozoa has not been widely explored. Maxwell et al. (1996) found the presence of seminal plasma in simple sorting diluents had no effect during Hoechst staining, but reported supplementation of the collection medium to be beneficial. However, more recent studies (on high speed sorters and in more complex media) reported decreases in the number of viable and correctly orientated spermatozoa when seminal plasma (5–20% v/v) was present in the staining medium (Burroughs 2011), which caused a reduction in the speed at which spermatozoa could be sex-sorted. In addition, this detrimental effect of seminal plasma prior to sorting was evident after sorting (where membrane-damaged sperm are removed) and freezing, indicating a persistent destabilization of the sperm membrane.

Reasons for Variable Supplementation Efficacy of Seminal Plasma

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Although significant differences in the effect of seminal plasma supplementation exist between the two ruminant species outlined above, one characteristic that the seminal plasma of both cattle and sheep do share is the inherent variability of their effect.

Such variability has been largely attributed to differences in the relative abundance of seminal plasma proteins (Maxwell et al. 2007; Muino-Blanco et al. 2008), with either an inhibitory or stimulatory effect on capacitation (Chang 1957). During natural mating, it is thought that these proteins interact with molecules present on the surface of the spermatozoa, and in its immediate environment, in an attempt to synchronize capacitation with the arrival of spermatozoa at the oocyte (Bedford 2004). Furthermore, it is thought that in vitro processing of spermatozoa disrupts this natural balance of inhibition and stimulation causing loss of sperm function and fertility (Leahy and Gadella 2011).

Characterization of these stimulatory and inhibitory factors would lead to better sperm handling techniques and the potential identification and exploitation of protective factors. In addition, the use of heterologous seminal plasma in semen extenders is a serious animal health issue and the isolation and production of synthetic protective factors would remove risks associated with the transmission of seminal fluid-borne micropathogens. Such motivation has driven the separation and characterization of seminal plasma proteins based on various physio-chemical properties including size, binding ability and charge (Mogielnicka-Brzozowska and Kordan 2011). One family of proteins, particularly well characterized and highly abundant in the bull, will be discussed in depth for the remainder of this review. They are small acidic proteins that originate in the seminal vesicles and are designated by the presence of two tandemly repeated fibronectin type 2 (Fn-2) modules. Historically known as bovine seminal plasma (BSP) proteins, they were soon discovered in numerous mammalian species where they were often named according to species of origin and the molecular weight of protein [e.g. ram seminal plasma proteins in sheep; (Manjunath et al. 2009)]. The term ‘BSP’ has now been re-named binder of sperm (BSP) proteins to incorporate all mammalian genes in this protein family, but for simplicity, in this review, the original descriptors will be used and bull Fn-2 proteins will be referred to as BSPs and ram Fn-2 proteins will be designated RSPs.

Biological Function of Bull BSPs

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Fibronectin-2 proteins are the major proteins of bull seminal plasma, comprising approximately 65% of total proteins (Nauc and Manjunath 2000). In the bull, these include BSP-A1, BSP-A2, BSP-A3 and BSP-A5 [previously termed BSP-30 kDa; (Manjunath et al. 2009)]. BSP-A1 and BSP-A2 have an average mass of 15 kDa and differ only in glycosylation forms. Together, they are designated BSP1 (formerly PDC-109).

The biochemical and biophysical properties of BSP proteins have been extensively investigated. They can bind to a variety of molecules including heparin, gelatin, choline phospholipids, glycosaminoglycans and lipoproteins (Manjunath et al. 2007), and these diverse binding properties impart numerous biological functions. When spermatozoa are mixed with seminal plasma at ejaculation, BSP proteins bind tightly to choline phospholipids of the sperm membrane in a rapid (half-time less than 1 s) and specific manner (Desnoyers and Manjunath 1992; Muller et al. 1998). This association does not only involve the solvent exposed choline group, but results in partial insertion of BSP1 into the hydrophobic environment of the external leaflet of the lipid bilayer (Muller et al. 1998; Greube et al. 2001). This association restricts mobility of the lipid phase, which is thought to result in a rigidification of the sperm membrane that provides stability during sperm transit through the reproductive tract. Upon reaching the oviduct, BSP proteins may also assist in the establishment of a bovine oviductal sperm reservoir. Evidence for this function is found in reports that in vitro supplementation of BSP proteins to epididymal spermatozoa enhances sperm–oviductal epithelial cell binding (Gwathmey et al. 2003, 2006). It is proposed that when the oocyte arrives, secondary messengers in the oviductal fluid, such as high-density lipoprotein and heparin-like glycosaminoglycans, may bind to BSP proteins to coordinate the onset and progression of capacitation (Lane et al. 1999).

Under the artificial conditions that occur during semen processing, BSPs can be detrimental to bull spermatozoa. The extended association of bull spermatozoa and BSP proteins in vitro causes efflux of phospholipid and cholesterol from the sperm membrane in a dose and time-dependent manner (Therien et al. 1998, 1999). This destabilizes the sperm membrane and renders it more susceptible to stress (Therien et al. 1997; Manjunath and Therien 2002; Manjunath et al. 2002; Bergeron and Manjunath 2006). Fortunately, the common use of egg yolk and skim milk in cryoprotective diluents appears to have unintentionally afforded partial protection from this detrimental effect as low-density lipoproteins and casein micelles present in these diluents preferentially bind BSP proteins, reducing the number available to bind to the sperm membrane (Manjunath et al. 2002).

Further information on the function of BSPs in the bull can be found through the investigation of the large breeding datasets brought about by widespread AI in the cattle industry. Comparison of proteins from the sperm plasma membrane of bulls of high or low fertility showed the relative abundance of BSP1 (D’Amours et al. 2010) and BSP3 (Roncoletta et al. 2006) was positively associated with low fertility. Another study showed the concentration of BSP5 in seminal fluid to have a quadratic association with fertility following artificial insemination of frozen-thawed bull spermatozoa, with both high and low concentrations of BSP proteins associated with low fertility (Moura et al. 2006). This latter study reflects the aforementioned positive and negative effects of BSPs during in vivo and in vitro studies in which a certain level of BSP proteins would promote fertilization (e.g. through improved sperm–OEC binding) and an excessive amount would be detrimental (e.g. greater contact during in vitro processing). However, it is worth remembering that while these field studies give us some insights into the correlation between protein composition and reproductive outcomes, such data are typically collected from insemination with frozen-thawed semen. As such, these correlations do not assess the inherent fertility of the bulls (and BSPs) per se but rather reflect the ability of their semen to survive dilution, freezing and thawing (Van doormaal 1993). It would be interesting to determine the relationship between BSP concentration (on sperm membrane and/or in seminal plasma) and the outcome of a natural mating or artificial insemination of fresh spermatozoa.

Biological Function of Ram RSPs

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

Fibronectin type-2 (Fn-2) proteins have been reported in a wide range of species and appear highly conserved (Fan et al. 2006). Their role is thought to be ubiquitous (Calvete and Sanz 2007), but their function in species other than the bull is not well defined. Despite ram seminal plasma containing a large proportion of RSP proteins [∼20% of total proteins (Bergeron et al. 2005)], the biochemical and biophysical properties of these proteins are not well described. Ram seminal plasma contains four Fn-2 proteins designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa and RSP-24 kDa (Bergeron et al. 2005). Like their bovine counterparts, RSP proteins bind gelatin, hen’s egg yolk low-density lipoprotein and heparin, although affinity for the latter is higher for RSP-22 kDa and RSP-24 kDa than RSP-15 kDa and RSP-16 kDa (Bergeron et al. 2005). However, this is where the similarities between the two species end.

Unlike in the bull, the supplementation of RSPs during the processing of ram semen is of benefit to sperm function. As previously mentioned, ovine seminal plasma is considered by many investigators to be a supportive medium that can be used to partly attenuate freeze–thaw damage in ram spermatozoa. Size exclusion fractionation of ram seminal plasma has shown the main protective component to be a 14 kDa Fn-2 protein (likely RSP-15) and a unidentified 20 kDa protein (Muino-Blanco et al. 2008). Co-incubation of washed diluted spermatozoa with this protein fraction has been shown to partly prevent (Perez-Pe et al. 2001a) or revert (Barrios et al. 2000) cold-shock damage. It is worthy of note that these ram spermatozoa were supplemented with high concentration of BSP proteins without cryoprotectants, and thus no protection from overexposure to BSPs was afforded from the scavenging properties of egg yolk or skim milk (as has been shown to be the case in cattle). Further investigation revealed the RSP protein reduced tyrosine phosphorylation (Perez-Pe et al. 2002), a well-known parameter associated with capacitation. Thus, in the ram, Fn-2 proteins appear to stabilize the sperm membrane during processing, resulting in reduced handling-induced damage, unlike the situation in the bull where BSP proteins destabilize the sperm membrane and decrease the resistance of spermatozoa to in vitro stressors. This protective effect may be due to a rigidification of the sperm membrane in a similar fashion as described for bull spermatozoa, but in a less aggressive form (even at high concentrations or long exposure to RSPs) which does not result in lipid efflux and membrane destabilization.

Reasons for Species Differences in the Function of BSPs and RSPs

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

The reason for the striking difference in the action of Fn-2 proteins between sheep and cattle is not clear, particularly when both species are closely related and share a number of common reproductive attributes (e.g. chemical composition of SP, reproductive strategies). Analysis of the sperm membrane composition and signal transduction pathways of capacitation in these species, as well as the biochemical and biophysical properties of BSP and RSP proteins, may provide potential explanations for this species variation.

One factor that could account for the apparently opposing effects of Fn-2 proteins is the different source of spermatozoa often used for bull and ram studies. Most of the studies reporting the effect of BSP proteins in the bull were performed with epididymal spermatozoa, while the majority of those in the ram were conducted with ejaculated, washed spermatozoa. These two sperm types have markedly different extracellular matrices, which may influence their response to exogenous protein supplementation. For example, prior exposure to seminal plasma may mask binding sites of detrimental proteins as whole seminal plasma has been reported to negate the effect of toxic proteins (Lusignan et al. 2007; Kumar et al. 2008).

Distinct biological activity between BSP and RSP proteins could also be due to differences in the relative concentration of these proteins and/or subtle variations in their primary structure, expression or post-translational modification (e.g. glycosylation). For example, the equine protein HSP-1/2 (SP-1/2) binds lipid membranes in a similar fashion to its bovine homologue (BSP1), but this association does not appear to be as disruptive as BSP1 because it does not result in the extraction of membrane lipids (Greube et al. 2004). Interestingly, proteins of this heterodimer have been both negatively (HSP1) and positively (HSP2) correlated with the ability of stallion spermatozoa to survive freezing (Jobim et al. 2011), and the functional significance of this effect is unclear.

It is also possible that bull BSP proteins have a greater affinity for cholesterol compared to other species. It is still unknown whether cholesterol efflux is a direct result of BSP proteins binding to the sperm membrane or an indirect consequence of phospholipid loss. Early studies indicated the latter (Desnoyers and Manjunath 1992; Muller et al. 2002), but recent evidence showing BSP1 decreases the rotational mobility of cholesterol within lipid vesicles suggests BSP proteins may exert a direct influence on cholesterol (Scolari et al. 2010). A recognition site, the cholesterol recognition/interaction amino acid consensus (CRAC) region, identified in BSP1, has been proposed. Interestingly, sequence analysis across a range of species showed high variability in CRAC patterns and that the bull contained a significantly higher frequency than the Fn-2 proteins from other species studied (Scolari et al. 2010).

Finally, the membrane makeup of ram spermatozoa may make it more resistant than bull spermatozoa to modulation by Fn-2 proteins. Cholesterol loss is a hallmark of the capacitation process, but the capacitation pathway is incredibly species specific and Colas et al. (2008) showed that ram spermatozoa are relatively insensitive to cholesterol-depleting reagents (e.g. BSA) that provoke tyrosine phosphorylation and capacitation in other species. Such resistance may be a protective mechanism as ram sperm membranes have a low cholesterol: phospholipid ratio (Darin-Bennett and White 1977) and low proportion of lipid rafts (Colas et al. 2008) compared to other species.

Whatever the reason, ram spermatozoa do appear to respond well to RSP supplementation and seem to have an incredibly stabile membrane during processing. This may account for differences in the ability of ram spermatozoa to resist the stressors of sex-sorting and storage compared to bull spermatozoa (de Graaf et al. 2009). In any event, further study of the mode of action of Fn-2 proteins on ram spermatozoa is warranted to ensure a greater understanding of its true effect on the membrane and the differences observed between the bull and ram.

Future Directions in Seminal Plasma Research

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

In the bull, BSPs have been shown to be largely responsible for promoting capacitation (though other unidentified proteins could be involved), but it is yet to be determined what proteins may play the converse role of suppressing capacitation to prevent inappropriate acrosomal exocytosis. Many potential candidates, termed ‘decapacitation factors’, have been identified in other species [mainly murine; for recent review see (Leahy and Gadella 2011)], but only a few (e.g. caltrin, also known as seminalplasmin, PYY2) have been described in the bull (Sitaram and Nagaraj 1995). With proteomic techniques evolving at a rapid rate and novel high throughput off-gel methods increasing protein coverage, it is hoped that future biochemical studies will further clarify the proteins responsible for regulation of capacitation and that this information may eventually be used as a tool to enhance sperm preservation.

In the ram, future work should utilize similar proteomic techniques to further characterize ovine seminal plasma and the multitude of unidentified proteins contained within. Even the RSPs are relatively poorly studied in comparison with BSPs in the bull, and both proteomic and functional studies are required to understand their apparently beneficial effects. For sheep, processing such as freezing not only affects motility and viability, but also the ability of sperm to penetrate the cervix. As has been previously alluded to, factors within seminal plasma have been shown to inconsistently improve the fertility of frozen-thawed spermatozoa following cervical AI (Maxwell et al. 1999; O’Meara et al. 2007; Leahy et al. 2010b). This would suggest the impairment of function is related to the proteins present on the sperm surface (as is the case in transit through the utero-tubal junction) and that such an impairment is reversible given identification of the specific protein responsible or factors required for cervical transit. This issue warrants considerable further investigation as a solution would facilitate widespread artificial insemination of frozen semen without the need for expensive laparoscopic surgery.

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References

The studies outlined in this review show the seminal plasma of the major ruminant species to be highly variable in its effect on spermatozoa, particularly within the context of in vitro supplementation during processing for semen storage and sperm sexing. Current knowledge suggests whole seminal plasma will only have a beneficial effect (and even then inconsistently) when supplemented during processing of ram semen. This is not only attributable to variations in seminal plasma composition between the species as even homologous Fn-2 proteins of the ram and bull contain subtle differences, which impart distinct biological effects. The reasons for these differences are yet to be determined, as is the complete role of seminal plasma in the biological function of the spermatozoon. It is clear that extensive further characterization of the seminal plasma and sperm surface proteome of each species is required if we are to fully understand the purpose, function and potential of seminal plasma.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Seminal Plasma Characteristics of the Ram and Bull
  5. Semen Processing and Supplementation with Seminal Plasma
  6. Supplementation of Seminal Plasma in the Ram
  7. Supplementation of Seminal Plasma in the Bull
  8. Reasons for Variable Supplementation Efficacy of Seminal Plasma
  9. Biological Function of Bull BSPs
  10. Biological Function of Ram RSPs
  11. Reasons for Species Differences in the Function of BSPs and RSPs
  12. Future Directions in Seminal Plasma Research
  13. Conclusion
  14. Acknowledgements
  15. Conflicts of interest
  16. References
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