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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

The ejaculated semen of mammals comprises spermatozoa derived from the testes and seminal plasma derived from the accessory sex glands, testes and epididymides. Whilst initial observations suggested that seminal plasma acted as a vehicle for the transport of spermatozoa into and within the female reproductive tract, further investigations highlighted an important role of seminal plasma in sperm function, integrity and fertilizing ability. Understanding the components of seminal plasma and the role that these components have in sperm function has progressed the development of assisted reproductive technologies (ARTs) such as sperm cryopreservation and artificial insemination (AI) in numerous species including sheep, goats and cattle (Maxwell et al. 2007). Conversely in camelids, which include alpacas, llamas, vicunas, guanacos and dromedary and Bactrian camels, very little is known about the components of the seminal plasma and the role that the seminal plasma has in sperm function and integrity. This review outlines the implications for seminal plasma components associated with the development of ARTs in camelids and compares these components with those in the seminal plasma of other species to hypothesize their role in sperm function and fertility.

Semen Characteristics

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

Whilst semen encompasses spermatozoa and seminal plasma, which generally comprises proteins and organic and inorganic compounds, the concentration of spermatozoa, seminal plasma proteins present and the abundance of each component vary widely amongst species. This can be, in part, attributed to the relative contribution of the testes and accessory sex glands to the ejaculate. In bulls, more than 50% of the ejaculate originates from the vesicular glands with limited secretions from the bulbourethral glands and prostate, which are relatively small in size (Mann 1964). The accessory sex glands in the ram are anatomically similar to those in the bull, comprising large vesicular glands and small or disseminated prostate and bulbourethral glands (Maxwell et al. 2007). Owing to the small size of the glands, their contribution to the final ejaculate is low, and thus, ejaculates in the bull and ram are of relatively low volume with high sperm concentration (bull: 2–10 ml, 1000 × 106 spermatozoa/ml (Mann 1964); ram: 0.7–2 ml, 3000 × 106 spermatozoa/ml) (Evans and Maxwell 1987). Conversely in the boar, the bulbourethral glands, prostate and vesicular glands are large and therefore contribute a large proportion of the ejaculate that can be up to 300 ml in volume with 100 × 106 spermatozoa/ml (Mann 1964). In boars, the bulbourethral (Badia et al. 2006) and vesicular glands (Boursnell et al. 1970) are also responsible for secreting the gelatinous material that forms the gel fraction of the ejaculate.

In camelids, the vesicular glands are absent and seminal plasma is derived from the relatively small bulbourethral and prostate glands. Camelids consequently have a low-volume ejaculate that averages 1–2 ml in alpacas and llamas (Garnica et al. 1993; Lichtenwalner et al. 1996; Bravo et al. 1997; Vaughan et al. 2003) and 4–8 ml in camels (Mosaferi et al. 2005; Morton et al. 2008; Wani et al. 2008). Unlike the ram and bull where approximately 30% of the ejaculate is spermatozoa (Mann 1964), in camelids approximately 85% of the ejaculate is seminal plasma and the remaining 15% sperm cells (Garnica et al. 1993; Bravo et al. 1997). In alpacas, the testes average 3–5 cm in length and 15–18 g in weight, which is equivalent to 0.02–0.03% body weight compared with 0.18% in bulls and 1.4% in rams (Vaughan et al. 2003). Consequently, camelids produce fewer sperm daily compared with rams and bulls. The semen therefore has a low concentration of 30–150 × 106 spermatozoa/ml in alpacas and llamas and 80–300 × 106 spermatozoa/ml in camels despite the low seminal plasma volume (Deen et al. 2003; Wani et al. 2008).

Another characteristic of camelid semen is the highly viscous seminal plasma. Unlike boars and rodents, but similar to humans and monkeys, the high viscosity is present upon ejaculation and the viscous material is distributed throughout the ejaculate rather than being a gel fraction or gel plug. The viscosity of camelid seminal plasma has been routinely assessed using the thread test method, in which semen is pipetted onto a glass slide, the pipette lifted vertically and the length of thread that forms measured (Bravo et al. 1999, 2000a; Giuliano et al. 2010; Kershaw-Young et al. 2012). Recent findings suggest that thread formation is not correlated with structural viscosity, but is correlated with coefficient of consistency and viscosity at high shear rates. Therefore, the thread test can be used as a measure of the rheological properties of the seminal plasma, but not as a measure of structural viscosity (Casaretto et al., 2012). In contrast to the human (Lilja and Laurell 1984) and baboon (Amboka and Mwethera 2003) where natural liquefaction of the seminal plasma coagulum is observed within 15 min after ejaculation, alpaca semen liquefies on average 23 h after ejaculation, ranging from 8 to 48 h (Garnica et al. 1993). Camel semen tends to liquefy more quickly with partial liquefaction within 20–30 min (Skidmore and Billah 2006; Niasari-Naslaji et al. 2007) and complete reduction of viscosity, as measured using the thread test, after 1.5 h after ejaculation (Wani et al. 2008). Whilst in the boar (Boursnell et al. 1970) and human (Lilja and Laurell 1984; Lilja et al. 1987), the vesicular glands are involved in the formation of the coagulum, the absence of the vesicular glands in camelids indicates that another mechanism of seminal plasma viscosity is present. The cause and source of this viscosity has not been documented.

The viscous nature of the seminal plasma hinders semen assessment in camelids as it entraps the spermatozoa causing them to move in an oscillatory manner with limited progressive motility (Garnica et al. 1993; Deen et al. 2003). In addition, the highly viscous seminal plasma is currently the major impediment to the development of ARTs in camelids as it impedes the homogenous mixing of semen with extender, thereby limiting contact between the sperm cell membrane and cryoprotective compounds during cryopreservation. The post-thaw motility of frozen-thawed camelid spermatozoa is approximately 20% (Santiani et al. 2005; Niasari-Naslaji et al. 2006, 2007), which is not commercially viable. Pregnancy rates after AI with frozen-thawed semen in camelids are also low. In dromedary camels, only 1/13 camels were diagnosed pregnant following insemination with frozen-thawed spermatozoa (Deen et al. 2003), whereas in llamas, pregnancy rates of 8% were obtained using frozen-thawed sperm (Aller et al. 2003 cited in Miragaya et al. 2006), and in alpacas, no pregnancies were obtained using AI with frozen-thawed sperm (Vaughan et al. 2003). The best success using frozen-thawed semen in llamas and alpacas was achieved after reducing the viscosity of the spermatozoa. Thus, when the viscosity of llama and alpaca semen was reduced using collagenase, then cryopreserved and used for AI of alpacas, 5 of 19 females (26%) gave birth to live cria (Bravo et al. 2000b). The findings of Bravo et al. (2000b) highlight the advantage of reducing seminal plasma viscosity prior to cryopreservation on the development of ARTs in camelids. To develop reliable, effective ART protocols in camelids, it is essential to gain an understanding of the seminal plasma components, including those involved in seminal plasma viscosity, and to determine the role that these components play in reproductive function.

Biochemical Components and Cryopreservation

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

The biochemical composition of camelid seminal plasma has been described and includes, amongst other things, chloride, calcium, protein, lipids and glucose at concentrations similar to those observed in ram and bull semen (Table 1). The concentration of fructose and citric acid is much lower in alpaca and camel seminal plasma compared with that of the ram and bull, most likely due to the absence of the vesicular glands in camelids, which are the main source of fructose and citric acid in other livestock species. As the biochemical components of camelid seminal plasma are similar to other domestic livestock species, it is reasonable to suggest that semen extenders used for cryopreservation of spermatozoa in species such as the ram and bull would be suitable for the storage of camelid semen. However, despite attempts using lactose-, sucrose-, citrate- and fructose-based buffers in addition to the commercially available extenders manufactured for other livestock species such as Green buffer® (IMV, L'Aigle, France), Biladyl®, Androhep® and Triladyl® (Minitube, Tiefenbach, Germany), the post-thaw motility of frozen-thawed camelid spermatozoa averages 20% (von Baer and Hellemann 1999; Deen et al. 2003; Vaughan et al. 2003; Santiani et al. 2005) and is rarely >40% (Bravo et al. 2000b; Niasari-Naslaji et al. 2007; El-Bahrawy et al. 2010). Given that in sheep and goats ejaculates with <40% of progressively motile spermatozoa post-thaw are not suitable for AI (Evans and Maxwell 1987), these post-thaw motility rates of camelid spermatozoa are unlikely to be commercially viable or adequate for AI. Post-thaw motility rates are higher (up to 40%) when the viscous seminal plasma is reduced by either mechanical (Niasari-Naslaji et al. 2007) or enzymatic (Bravo et al. 2000b) means highlighting the detrimental effect of the seminal plasma viscosity on cryopreservation of spermatozoa in camelids.

Table 1.   Biochemical composition of alpaca, camel, bull and ram semen. Concentrations are mg/dl except chloride and sodium in alpaca and camel, which are expressed as mEq/l
ComponentAlpacaCamelBullRam
Citric acid4.3a9.8b720c137c
Fructose5.0a23.5b540c247c
Glucose5.0d35.8e  
Chloride404d97.9e371c87c
Sodium 158–163f109c103c
Phosphorus 2.9e82c357c
Calcium18d8.2e34c9c
Total nitrogen647d 756c875c
Total lipids95d87b  
Total protein4000d2200e7300–9300g3500h

Glycosaminoglycans

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

The source of the viscosity of camelid seminal plasma has not been documented, although it has been postulated that it is caused by glycosaminoglycans (GAGs) (Perk 1962; Ali et al. 1976). The concentration of GAGs in alpaca seminal plasma is 15 times higher than in that of the ram (Kershaw-Young et al. 2012) and almost three times higher than in human seminal plasma (Binette et al. 1996). More than 85% of the total GAG content in alpaca seminal plasma is keratan sulphate, and the concentration of keratan sulphate is correlated with viscosity (Kershaw-Young et al. 2012). Despite these findings, enzymes that degrade GAGs, including keratanase that specifically degrades keratan sulphate, do not completely reduce alpaca seminal plasma viscosity within 2 h of incubation (C. M. Kershaw-Young, unpublished), suggesting that GAGs are not the cause of viscosity in camelid seminal plasma.

The Cause of Camelid Seminal Plasma Viscosity

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

In contrast to the minimal effect of GAG enzymes on seminal plasma viscosity, collagenase, fibrinolysin and trypsin all reduce, but do not completely eliminate, the viscosity of llama and alpaca seminal plasma within 5 min of treatment (Bravo et al. 2000a). In addition, the proteases papain (Morton et al. 2008; C. M. Kershaw-Young, unpublished) and proteinase K (C. M. Kershaw-Young, unpublished) completely eliminate alpaca seminal plasma viscosity within 20–40 min of treatment. Similar effects are observed in hyperviscous human seminal plasma in which the enzymes trypsin (Pattinson et al. 1990; Mendeluk et al. 2000), α-amylase (Mendeluk et al. 2000), and chymotrypsin, subtilisin and papain (Pattinson et al. 1990) all reduce viscosity. The cause of viscosity in human seminal plasma is the protein semenogelin, which is secreted by the vesicular glands (Lilja and Laurell 1984; Lilja et al. 1987) and semenogelin is degraded by prostate-specific antigen that is secreted by the prostate gland (Lilja et al. 1987). This cause of viscosity and method of liquefaction are unlikely to be the same in camelid semen because of their lack of vesicular glands. However, the similar effect of proteases on camelid and human seminal plasma viscosity, in addition to the limited effect of GAG enzymes on alpaca seminal plasma viscosity suggest that proteins, not GAGs, are the cause of viscosity in camelids.

The protein composition of camelid seminal plasma, and thus the protein responsible for viscosity, has not been reported. Mass spectrometry and iTRAQ analysis of alpaca seminal plasma with either high or low viscosity identified four proteins that differed in abundance between samples (Table 2, C. M. Kershaw-Young, X. Druart and W. M. C. Maxwell, unpublished). Three of these proteins, monocyte differentiation antigen CD14 (CD14), phosphatidylethanolamine-binding protein (PEBP) and tissue alpha-l-fucosidase (FUCA1) were approximately two times more abundant in low viscosity samples compared with those of high viscosity. Conversely, mucin 5B was more than five times more abundant in seminal plasma samples with high viscosity compared with those with low viscosity.

Table 2.   Relative abundance of proteins detected by iTRAQ and LC MS/MS that differ between low viscosity and high viscosity alpaca seminal plasma samples
ProteinLow viscosity (mean ± SEM)High viscosity (mean ± SEM)Ratioap-Value
  1. aAverage abundance in high viscosity divided by protein in low viscosity.

Mucin 5B1.29 ± 0.297.91 ± 1.205.840.04
Monocyte differentiation antigen CD140.96 ± 0.040.46 ± 0.020.480.01
Phosphatidylethanolamine-binding protein0.95 ± 0.050.62 ± 0.030.650.03
Tissue alpha-l-fucosidase1.03 ± 0.030.68 ± 0.000.660.007

Mucin 5B (MUC5B, also termed MG1) is a member of the mucin protein family, which contains, to date, 17 genes including MUC 1-4, 5AC, 5B, 6-13, 15-17, 19 and 20. There are two classes of mucins: membrane associated and secreted. The secreted mucins are further classified into large gel-forming mucins (MUC 2, 5AC, 5B, 6 and 19) and small soluble mucins (MUC 7 and 9) (Russo et al. 2006). Consequently, mucin 5B is defined as a large gel-forming protein that is secreted by glandular epithelial cells. Although not previously reported in camelid seminal plasma, mucin 5B is present in human seminal plasma (Russo et al. 2006) and the gel fraction of boar semen (Boursnell et al. 1970).

It is believed that the deposition of the gel fraction in the sow reproductive tract helps prevent sperm loss following mating. It is likely that a similar mechanism exists in camelids, in that mucin 5B creates a viscous ejaculate that remains in the female reproductive tract following mating and prevents sperm loss. This is a favourable mechanism given the relatively low concentrations of spermatozoa in each ejaculate. Moreover, the viscous seminal plasma probably degrades slowly once deposited in the female reproductive tract, allowing small numbers of spermatozoa to be released over time and increasing the likelihood of fertile cells being present at the time of ovulation, which occurs approximately 28–30 h post-coitum (Vaughan and Tibary 2006). This is beneficial, as camelids are induced to ovulate following mating by a seminal plasma protein termed ovulation inducing factor (Chen et al. 1985; Pan et al. 2001; Adams et al. 2005). The predominant source of mucin 5B in camelid seminal plasma is the bulbourethral gland (C. M. Kershaw-Young, X. Druart and W. M. C. Maxwell, unpublished) as has been reported in the boar (Badia et al. 2006) and human (Piludu et al. 2009).

Proteins in Camelid Seminal Plasma

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

In other livestock species including the bull, ram and boar, numerous proteins have been identified that play an integral role in sperm function, binding with the sperm membrane and initiating capacitation and the acrosome reaction (Maxwell et al. 2007; Mogielnicka-Brzozowska and Kordan 2011) as well as enhancing sperm-oocyte binding. Mass spectrometry analysis of pooled alpaca seminal plasma identified 89 proteins, of which 28 have previously been reported in the seminal plasma of other species and have been associated with sperm function, integrity and fertilizing ability (Table 3, C. M. Kershaw-Young, X. Druart and W. M. C. Maxwell, unpublished).

Table 3.   Alpaca seminal plasma proteins as determined by mass spectrometry of pooled seminal plasma from six male alpacas
Protein
Alpha-2-macroglobulin protease inhibitor
Apomucin
Aspartylglucosaminidase
Beta 1,4-galactosyltransferase
Beta-nerve growth factor
Cathepsin D
Cathepsin L
Chaperonin containing TCP1
Clusterin
Cysteine-rich secretory protein 1
Epididymal secretory protein E1
Epididymal sperm-binding protein
Heat shock 70 kDa protein
Heat shock protein HSP 90-alpha
Insulin-like growth factor-binding protein 5
Lactoferrin
l-lactate dehydrogenase
Mannosidase
Monocyte differentiation antigen CD14
Mucin 5B
Mucin 5AC
Phosphatidylethanolamine-binding protein 1
Serum albumin
Sulfated glycoprotein-1/prosaposin
Sulfhydryl oxidase 1
Tissue alpha-l-fucosidase
Triosephosphate isomerase 1
Zinc alpha-2-glycoprotein 1

Mammalian seminal plasma proteins are generally divided into three major protein types: spermadhesins or heparin-binding proteins, those proteins that contain fibronectin II (Fn-2) domains, and cysteine-rich secretory proteins (CRISPs) (Rodriguez-Martinez et al. 2011). These proteins have been well-characterized in the boar, bull, ram and stallion and named based on their size and binding affinities (Maxwell et al. 2007). Whilst mass spectrometry analysis of alpaca seminal plasma did not provide information regarding the size and binding properties of the identified proteins, a number of proteins that fall into these groups were identified. The epididymal protein CRISP 1 was present in alpaca seminal plasma and is involved in sperm binding to the zona pellucida (Cohen et al. 2008). In addition, the spermadhesin epididymal sperm-binding protein 1 and the heparin-binding proteins clusterin and lactoferrin (also referred to as lactotransferrin) were present (Table 3). Clusterin is the major protein of epididymal fluid in the human (Dacheux et al. 2006) and is involved in sperm protection (Bailey and Griswold 1999). Lactoferrin, also secreted by the epididymis (Dacheux et al. 2006) and a major heparin-binding protein present in human seminal plasma (Kumar et al. 2008), is thought to be involved in sperm capacitation and the acrosome reaction because of its heparin-binding properties (Mogielnicka-Brzozowska and Kordan 2011). Epididymal sperm-binding protein 1 is a spermadhesin and therefore, like other spermadhesins, most likely binds to phosphatidylcholine and phosphatidylethanolamine phospholipids on the sperm membrane upon ejaculation stabilizing the sperm membrane and preventing capacitation (Maxwell et al. 2007; Mogielnicka-Brzozowska and Kordan 2011).

Phosphatidylethanolamine-binding protein was also detected in alpaca seminal plasma (Table 3). It is present in human epididymal fluid (Dacheux et al. 2006) and is expressed on the membrane of bull (D’Amours et al. 2010) and mouse spermatozoa (Nixon et al. 2006). Phosphatidylethanolamine-binding protein binds phospholipids and may have an inhibitory effect on sperm capacitation (Nixon et al. 2006) or act as a decapacitation factor (Moffit et al. 2007). The expression of PEBP is higher in frozen-thawed bull spermatozoa that exhibit high fertilizing ability compared with those with low fertility (D’Amours et al. 2010), suggesting that higher concentrations of PEBP reduce the number of spermatozoa that become capacitated during cryopreservation. Phosphatidylethanolamine-binding protein is less abundant in highly viscous alpaca seminal plasma compared with ejaculates with low viscosity (C. M. Kershaw-Young, X. Druart and W. M. C. Maxwell, unpublished) and may reflect the lower fertilizing ability of frozen-thawed spermatozoa derived from viscous semen.

Mucin 5B, mucin 5AC and apomucin were all present in alpaca seminal plasma (Table 3). Apomucin is the protein core of mucin proteins, and both mucin 5B and mucin 5AC are large gel-forming proteins and thus the viscosity of alpaca seminal plasma is most likely attributed to these proteins, especially as highly viscous seminal plasma contains up to five times more mucin 5B than low viscosity seminal plasma.

Proteins that are postulated to be involved in sperm–oocyte interactions and zona pellucida binding were also detected in alpaca seminal plasma. These include: tissue alpha-l-fucosidase, which is also present bull (Jauhiainen and Vanha-Perttula 1986) and human semen (Venditti et al. 2007); triophosphate isomerise (Auer et al. 2004) present in human epididymal fluid (Dacheux et al. 2006), beta 1,4-galactosyltransferase (Lyng and Shur 2007); sulphated glycoprotein-1 (also termed prosaposin), which is present in the sertoli cells and epididymal fluid of the rat (Sylvester et al. 1989); chaperone-containing TCP1, which is present on the membrane of capacitated spermatozoa (Dun et al. 2011); and mannosidase, which is present on sperm cell membranes (Cornwall et al. 1991).

Numerous enzymes that have been linked with sperm function were also identified including: cysteine proteases cathepsin D and cathepsin L; oxidoreductase l-lactate dehydrogenase, which is present in human epididymal fluid (Dacheux et al. 2006); and sulfhydryloxidase-1, which is expressed by the testes and involved in spermatogenesis (Aumüller et al. 1991).

Monocyte differentiation antigen CD14 (CD14) was also detected and, as stated previously, was less abundant in high viscosity than in low viscosity alpaca seminal plasma. CD14 is involved in the innate immune response and is present in human seminal plasma and on the sperm membrane and is produced by the epididymis (Harris et al. 2001; Malm et al. 2005). It is postulated that the presence of CD14 in semen helps prevent infection of the male genital tract and may also protect spermatozoa when deposited in the female reproductive tract (Harris et al. 2001).

Epididymal secretory protein 1 was present in alpaca seminal plasma and has been reported in the epididymal fluid of the bull (Moura et al. 2010). This protein may be involved in the regulation of the lipid composition of sperm membranes during maturation in the epididymis (Manjunath and Therien 2002; Moura et al. 2010).

An unexpected protein that was detected in alpaca seminal plasma was the 27 kDa homodimer beta-nerve growth factor (β-NGF). Beta-nerve growth factor mRNA is expressed in the reproductive tract of the mouse, rat and guinea-pig (MacGrogan et al. 1991). One-dimensional SDS-PAGE indicted that β-NGF was extremely abundant in alpaca seminal plasma as a 14 kDa protein owing to the conditions in the gel that reduce β-NGF to two 14 kDa dimers (Kershaw-Young et al. in press). Nerve growth factor has been implicated in the control of ovarian function, particularly ovulation (Dissen et al. 1996), and a 14 kDa protein has been identified as the ovulation inducing factor in llama seminal plasma (Ratto et al. 2011), suggesting that β-NGF is ovulation inducing factor. When female alpacas were scanned for the presence of a dominant follicle (diameter 6–10 mm) then injected intramuscularly with either 1 mg β-NGF, 2 ml seminal plasma, 4 μg buserelin or 2 ml 0.9% saline, ovulation was induced in 4/5, 4/5, 4/5 and 0/5 female alpacas, respectively, as determined by ultrasonography and plasma progesterone profiles 8 days following treatment (Kershaw-Young et al. in press). This provides evidence that the ovulation inducing factor in camelids that has previously remained unidentified in seminal plasma is β-NGF. This finding is significant in that β-NGF may provide an alternative mechanism for the induction of ovulation in alpacas for ARTs.

Of the proteins identified in alpaca seminal plasma (Table 3), some including cathepsin D and alpha-l-fucosidase (Moura et al. 2006) are more abundant in animals with high fertility, whereas others including sulphated glycoprotein-1 (Amann et al. 1999) improve the fertilizing ability of cryopreserved spermatozoa. During sperm processing, dilution of semen can induce negative or positive effects owing to the dilution of seminal plasma components and, at times, these effects can be reversed by the addition of seminal plasma or seminal plasma proteins (Maxwell et al. 2007). When alpaca semen is diluted to a final seminal plasma concentration of 10% (1 : 9 dilution) sperm motility, acrosome integrity and viability are maintained for longer than when incubated in 0, 25, 50 or 100% seminal plasma (Kershaw-Young and Maxwell 2011). This highlights the role that seminal plasma components play in sperm function and integrity and suggests, as observed in other species, that the addition of seminal plasma proteins, or the retention or addition of seminal plasma, as part of the process for cryopreservation of alpaca spermatozoa may improve their post-thaw motility and fertility.

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

Camelid semen is highly viscous with a low volume and low concentration of spermatozoa. The viscous seminal plasma is a major impediment to the development of semen cryopreservation and other ARTs. Glycosaminoglycans, which were postulated to be the cause of viscosity, are abundant in alpaca seminal plasma. However, GAG enzymes do not reduce viscosity, whereas proteases do, indicating that proteins are responsible for the viscosity observed in camelid seminal plasma. Our work has demonstrated that mucin 5B is the main protein responsible for the viscosity of alpaca seminal plasma. We have also identified β-NGF as the ovulation inducing factor protein in camelid seminal plasma and this may lead to new methods for the induction of ovulation in camelids. Other proteins identified in alpaca seminal plasma are similar to those reported for other species and include those involved in sperm function, fertility and oocyte binding. The role of seminal plasma proteins in sperm function in camelids requires further research. In particular, seminal plasma proteins, added to spermatozoa destined for cryopreservation, may improve their post-thaw motility as observed in the ram and bull and therefore warrants further investigation.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References

The authors thank Dr Jane Vaughan, Dr Xavier Druart, Dr Svetlana Mactier and Cassie Stuart for scientific input and technical assistance and Associate Professor Peter Thomson for assistance with statistical analysis. This research was funded by the Rural Industries Research and Development Corporation (RIRDC), Australia.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Semen Characteristics
  5. Biochemical Components and Cryopreservation
  6. Glycosaminoglycans
  7. The Cause of Camelid Seminal Plasma Viscosity
  8. Proteins in Camelid Seminal Plasma
  9. Conclusion
  10. Acknowledgements
  11. Conflicts of interest
  12. Author contributions
  13. References
  • Adams GP, Ratto MH, Huanca W, Singh J, 2005: Ovulation-inducing factor in the seminal plasma of alpacas and llamas. Biol Reprod 73, 452457.
  • Ali HA, Moniem KA, Tingari MD, 1976: Some histochemical studies on the prostate, urethral and bulbourethral glands of the one-humped camel (camelus dromedarius). Histochem J 8, 565578.
  • Amann RP, Seidel GE, Brink ZA, 1999: Exposure of thawed frozen bull sperm to a synthetic peptide before artificial insemination increases fertility. J Androl 20, 4246.
  • Amboka JNO, Mwethera PG, 2003: Characterization of semen from olive baboons. J Med Primatol 32, 325329.
  • Auer J, Camoin L, Courtot AM, Hotellier F, De Almeida M, 2004: Evidence that p36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase. Mol Reprod Dev 68, 515523.
  • Aumüller G, Bergmann M, Seitz J, 1991: Immunohistochemical distribution of sulfhydryl oxidase in the human testis. Cell Tissue Res 266, 2328.
  • Badia E, Briz MD, Pinart E, Sancho S, Garcia N, Bassols J, Pruneda A, Bussalleu E, Yeste M, Casas I, Bonet S, 2006: Structural and ultrastructural features of boar bulbourethral glands. Tissue Cell 38, 718.
  • von Baer L, Hellemann C, 1999: Cryopreservation of llama (lama glama) semen. Reprod Domest Anim 34, 9596.
  • Bailey R, Griswold MD, 1999: Clusterin in the male reproductive system: localization and possible function. Mol Cell Endocrinol 151, 1723.
  • Binette JP, Ohishi H, Burgi W, Kimura A, Suyemitsu T, Seno N, Schmid K, 1996: The content and distribution of glycosaminoglycans in the ejaculates of normal and vasectomized men. Andrologia 28, 145149.
  • Boursnell JC, Hartree EF, Briggs PA, 1970: Studies of the bulbo-urethral (cowper’s)-gland mucin and seminal gel of the boar. Biochem J 117, 981988.
  • Bravo PW, Flores U, Garnica J, Ordonez C, 1997: Collection of semen and artificial insemination of alpacas. Theriogenology 47, 619626.
  • Bravo PW, Pacheco C, Quispe G, Vilcapaza L, Ordonez C, 1999: Degelification of alpaca semen and the effect of dilution rates on artificial insemination outcome. Arch Androl 43, 239246.
  • Bravo PW, Ccallo M, Garnica J, 2000a: The effect of enzymes on semen viscosity in llamas and alpacas. Small Rumin Res 38, 9195.
  • Bravo PW, Skidmore JA, Zhao XX, 2000b: Reproductive aspects and storage of semen in camelidae. Anim Reprod Sci 62, 173193.
  • Casaretto C, Martinez Sarrasague M, Giuliano S, Rubin de Celis E, Gambarotta M, Carretero M, Miragaya M, 2012: Evaluation of lama glama semen viscosity with a cone-plate rotational viscometer. Andrologia, E-pub ahead of print.
  • Chen BX, Yuen ZX, Pan GW, 1985: Semen-induced ovulation in the bactrian camel (camelus bactrianus). J Reprod Fertil 73, 335339.
  • Cohen DJ, Busso D, Da Ros V, Ellerman DA, Maldera JA, Goldweic N, Cuasnicu PS, 2008: Participation of cysteine-rich secretory proteins (crisp) in mammalian sperm-egg interaction. Int J Dev Biol 52, 737742.
  • Cornwall GA, Tulsiani DR, Orgebin-Crist MC, 1991: Inhibition of the mouse sperm surface alpha-d-mannosidase inhibits sperm-egg binding in vitro. Biol Reprod 44, 913921.
  • Dacheux JL, Belghazi M, Lanson Y, Dacheux F, 2006: Human epididymal secretome and proteome. Mol Cell Endocrinol 250, 3642.
  • D’Amours O, Frenette G, Fortier M, Leclerc P, Sullivan R, 2010: Proteomic comparison of detergent-extracted sperm proteins from bulls with different fertility indexes. Reproduction 139, 545556.
  • Deen A, Vyas S, Sahani MS, 2003: Semen collection, cryopreservation and artificial insemination in the dromedary camel. Anim Reprod Sci 77, 223233.
  • Dissen GA, Mayerhofer A, Ojeda SR, 1996: Participation of nerve growth factor in the regulation of ovarian function. Zygote 4, 309312.
  • Dun MD, Smith ND, Baker MA, Lin M, Aitken RJ, Nixon B, 2011: The chaperonin containing tcp1 complex (cct/tric) is involved in mediating sperm-oocyte interaction. J Biol Chem 286, 3687536887.
  • El-Bahrawy KA, El-Hassanein ESE, Kamel YM, 2010: Comparison of gentamycin and ciprofloxacin in dromedary camels’ semen extender. World J Agric Sci 6, 419424.
  • El-Manna MM, Tingari MD, Ahmed AK, 1986: Studies on camel semen ii. Biochemical characteristics. Anim Reprod Sci 12, 223231.
  • Evans G, Maxwell WMC, 1987: Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Sydney.
  • Garnica J, Achata R, Bravo PW, 1993: Physical and biochemical characteristics of alpaca semen. Anim Reprod Sci 32, 8590.
  • Garnica J, Flores E, Bravo PW, 1995: Citric acid and fructose concentrations in seminal plasma of the alpaca. Small Rumin Res 18, 9598.
  • Giuliano S, Carretero M, Gambarotta M, Neild D, Trasorras V, Pinto M, Miragaya M, 2010: Improvement of llama (lama glama) seminal characteristics using collagenase. Anim Reprod Sci 118, 98102.
  • Harris CL, Vigar MA, Rey Nores JE, Horejsi V, Labeta MO, Morgan BP, 2001: The lipopolysaccharide co-receptor cd14 is present and functional in seminal plasma and expressed on spermatozoa. Immunology 104, 317323.
  • Jauhiainen A, Vanha-Perttula T, 1986: Alpha-l-fucosidase in the reproductive organs and seminal plasma of the bull. Biochim Biophys Acta 880, 9195.
  • Kershaw-Young CM, Maxwell WMC, 2011: The effect of seminal plasma on alpaca sperm function. Theriogenology 76, 11971206.
  • Kershaw-Young CM, Evans G, Maxwell WMC, 2012: Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram. Reprod Fertil Dev 24, 362369.
  • Kershaw-Young CM, Druart X, Vaughan J, Maxwell WMC, 2012: Beta nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas. Reprod Fertil Dev, E-pub ahead of print.
  • Kumar V, Hassan I, Kashav T, Singh TP, Yadav S, 2008: Heparin-binding proteins of human seminal plasma: purification and characterization. Mol Reprod Dev 75, 17671774.
  • Lichtenwalner AB, Woods GL, Weber JA, 1996: Seminal collection, seminal charateristics and pattern of ejaculation in llamas. Theriogenology 46, 293305.
  • Lilja H, Laurell CB, 1984: Liquefaction of coagulated human semen. Scand J Clin Lab Invest 44, 447452.
  • Lilja H, Oldbring J, Rannevik G, Laurell CB, 1987: Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J Clin Invest 80, 281285.
  • Lyng R, Shur BD, 2007: Sperm-egg binding requires a multiplicity of receptor-ligand interactions: new insights into the nature of gamete receptors derived from reproductive tract secretions. Soc Reprod Fertil Suppl 65, 335351.
  • MacGrogan D, Despres G, Romand R, Dicou E, 1991: Expression of the b-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig. J Neurosci Res 28, 567573.
  • Malm J, Nordahl EA, Bjartell A, Sørensen OE, Frohm B, Dentener MA, Egesten A, 2005: Lipopolysaccharide-binding protein is produced in the epididymis and associated with spermatozoa and prostasomes. J Reprod Immunol 66, 3343.
  • Manjunath P, Therien I, 2002: Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. J Reprod Immunol 53, 109119.
  • Mann T, 1964: The Biochemistry of Semen. Methuan and Co. Ltd., London.
  • Marco-Jime′nez F, Vicente JS, Viudes-de-Castro MP, 2008: Seminal plasma composition from ejaculates collected by artificial vagina and electroejaculation in guirra ram. Reprod Domest Anim 43, 403408.
  • Maxwell WM, de Graaf SP, Ghaoui Rel H, Evans G, 2007: Seminal plasma effects on sperm handling and female fertility. Soc Reprod Fertil Suppl 64, 1338.
  • Mendeluk G, Gonza FL, Flecha L, Castello PR, Bregni C, 2000: Factors involved in the biochemical etiology of human seminal plasma hyperviscosity. J Androl 21, 262267.
  • Miragaya M, Chaves MG, Aguero A, 2006: Reproductive biotechnology in south american camelids. Small Rumin Res 61, 299310.
  • Moffit JS, Boekelheide K, Sedivy JM, Klysik J, 2007: Mice lacking raf kinase inhibitor protein-1 (rkip-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury. J Androl 28, 883890.
  • Mogielnicka-Brzozowska M, Kordan W, 2011: Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals. Pol J Vet Sci 14, 489499.
  • Morton KM, Vaughan J, Maxwell WM, 2008: Continued Development of Artificial Insemination Technology in Alpacas. Rural Industries Research and Development Corporation, Kingston, ACT Australia.
  • Mosaferi S, Niasari-Naslaji A, Abarghani A, Gharahdaghi AA, Gerami A, 2005: Biophysical and biochemical characteristics of bactrian camel semen collected by artificial vagina. Theriogenology 63, 92101.
  • Moura AA, Chapman DA, Koc H, Killian GJ, 2006: Proteins of the cauda epididymal fluid associated with fertility of mature dairy bulls. J Androl 27, 534541.
  • Moura AA, Souza CE, Stanley BA, Chapman DA, Killian GJ, 2010: Proteomics of cauda epididymal fluid from mature holstein bulls. J Proteomics 73, 20062020.
  • Nauc V, Manjunath P, 2000: Radioimmunoassays for bull seminal plasma proteins (bsp-a1/-a2, bsp-a3, and bsp-30-kilodaltons), and their quantification in seminal plasma and sperm. Biol Reprod 63, 10581066.
  • Niasari-Naslaji A, Mosaferi S, Bahmani N, Gharahdaghi AA, Abarghani A, Ghanbari A, Gerami A, 2006: Effectiveness of a tris-based extender (shotor diluent) for the preservation of bactrian camel (camelus bactrianus) semen. Cryobiology 51, 1221.
  • Niasari-Naslaji A, Mosaferi S, Bahmani N, Gerami A, Gharahdaghi AA, Abarghani A, Ghanbari A, 2007: Semen cryopreservation in bactrian camel (camelus bactrianus) using shotor diluent: effects of cooling rates and glycerol concentrations. Theriogenology 68, 618625.
  • Nixon B, MacIntyre DA, Mitchell LA, Gibbs GM, O’Bryan M, Aitken RJ, 2006: The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors. Biol Reprod 74, 275287.
  • Pan G, Chen Z, Liu X, Li D, Xie Q, Ling F, Fang L, 2001: Isolation and purification of the ovulation-inducing factor from seminal plasma in the bactrian camel (camelus bactrianus). Theriogenology 55, 18631879.
  • Pattinson HA, Mortimer D, Curtis EF, Leader A, Taylor PJ, 1990: Treatment of spermagglutination with proteolytic enzymes. I. Sperm motility, vitality, longevity and successful disagglutination. Hum Reprod 5, 167173.
  • Perk K, 1962: Seasonal changes in the glandula bulbo-urethralis of the camel. Bull Res Counc Isr Sect E Exp Med 10, 3744.
  • Piludu M, Hand AR, Cossu M, Piras M, 2009: Immunocytochemical localization of mg1 mucin in human bulbourethral glands. J Anat 214, 179182.
  • Ratto MH, Delbaere LTJ, Leduc YA, Pierson RA, Adams GP, 2011: Biochemical isolation and purification of ovulation-inducing factor (oif) in seminal plasma of llamas. Reprod Biol Endocrinol 9, 2431.
  • Rodriguez-Martinez H, Kvist U, Ernerudh J, Sanz L, Calvete JJ, 2011: Seminal plasma proteins: what role do they play? Am J Reprod Immunol 66, 1122.
  • Russo CL, Spurr-Michaud S, Tisdale A, Pudney J, Anderson D, Gipson IK, 2006: Mucin gene expression in human male urogenital tract epithelia. Hum Reprod 21, 27832793.
  • Santiani A, Huanca W, Sapana R, Huanca T, Sepulveda N, Sanchez R, 2005: Effects on the quality of frozen-thawed alpaca (lama pacos) semen using two different cryoprotectants and extenders. Asian J Androl 7, 303309.
  • Skidmore JA, Billah M, 2006: Comparison of pregnancy rates in dromedary camels (camelus dromedarius) after deep intra-uterine versus cervical insemination. Theriogenology 66, 292296.
  • Sylvester SR, Morales C, Oko R, Griswold MD, 1989: Sulfated glycoprotein-1 (saposin precursor) in the reproductive tract of the male rat. Biol Reprod 41, 941948.
  • Vaughan JL, Tibary J, 2006: Reproduction in female south american camelids: a review and clinical observations. Small Rumin Res 61, 259281.
  • Vaughan J, Galloway D, Hopkins D, 2003: Artificial Insemination in Alpacas (lama pacos). Rural Industries Research and Development Corporation, Kingston, ACT, Australia.
  • Venditti JJ, Donigan KA, Bean BS, 2007: Crypticity and functional distribution of the membrane associated alpha-l-fucosidase of human sperm. Mol Reprod Dev 74, 758766.
  • Wani NA, Billah M, Skidmore JA, 2008: Studies on liquefaction and storage of ejaculated dromedary camel (camelus dromedarius) semen. Anim Reprod Sci 109, 309318.