Males of many animal species fight to establish social dominance and control access to females so that they have more opportunities to reproduce than their competitors. Males with lower social status will struggle to directly compete for mates, thus they attempt to mate with females by stealth. This often leads to more than one male mating with the same female so that the sperm from each male end up competing to fertilise that female’s eggs, a phenomenon known as sperm competition.

Males suspend their sperm in a fluid to make a mixture known as semen. It has been shown that, compared to high status males, low status males will produce higher quality semen that contains greater numbers of faster swimming sperm, giving them an advantage in sperm competition. Growing evidence from several species indicates that males can quickly adjust how fast their sperm swim in response to social cues that signal changing risks of sperm competition. However, how these rapid adjustments occur remains largely unknown, and whether they alter a male’s reproductive success against a competitor has seldom been examined.

Chinook salmon usually live in the North Pacific Ocean but they swim up rivers in North America and Asia to reproduce. They have also been introduced to several other countries including New Zealand where they are farmed commercially. The fish are highly prized by sport fishermen and are also of cultural significance to certain groups of indigenous people in North America. Barlett et al. studied the semen of chinook salmon, undertaking a series of experiments in which males switched between high and low social status. The experiments show that, as predicted, the sperm of males that changed from high to low social status started to swim faster. These changes in speed were caused by the fluid in the semen and altered the number of eggs that the male’s sperm fertilised when competing against sperm from another male.

In their natural range some populations of chinook salmon are declining due to overfishing combined with habitat loss and alteration. The findings of Barlett et al. contribute to a better understanding of how this fish species reproduces, which may lead to the introduction of measures that help natural populations to recover or help to improve commercial farming. Improved knowledge of how the fluid in semen affects sperm activity may also have important consequences for our wider understanding of male fertility in humans and other animals.

Sperm competition (Parker, 1970) occurs commonly across many invertebrate and vertebrate taxa and is a potent evolutionary force influencing male reproductive biology (Birkhead and Møller, 1998; Birkhead and Pizzari, 2002; Simmons and Fitzpatrick, 2012). Sperm competition theory predicts that males will trade-off between energy expended making high-quality ejaculates with obtaining mating opportunities and that males will invest differentially in ejaculates with respect to sperm competition risk (Parker, 1990; Parker et al., 1997; Parker, 1998; Wedell et al., 2002; Birkhead et al., 2009; Parker and Pizzari, 2010). In agreement with these predictions, males of many species can make rapid adjustments to ejaculate quality within days (Rudolfsen et al., 2006; Pizzari et al., 2007; Thomas and Simmons, 2007; Gasparini et al., 2009; Smith and Ryan, 2011), hours (Cornwallis and Birkhead, 2007a) and even minutes (Kilgallon and Simmons, 2005; Joseph et al., 2015) of exposure to a new social cue that signals changing sperm competition risk, such as the presence of a female, or a male competitor. For example, in fowl (Gallus gallus), males of dominant social status strategically allocate sperm, ejaculating more and faster swimming sperm in initial copulations and to females of higher quality (Pizzari et al., 2003; Cornwallis and Birkhead, 2006; Cornwallis and Birkhead, 2007a; Cornwallis and Birkhead, 2007b), and alter their allocation strategy accordingly when changing social status (Cornwallis and Birkhead, 2007a). While males of several vertebrate species ranging from fish (Rudolfsen et al., 2006; Gasparini et al., 2009; Smith and Ryan, 2011) to humans (Kilgallon and Simmons, 2005; Joseph et al., 2015) can strategically alter the quality of their ejaculate in response to social cues, the mechanism behind such rapid adjustments is as yet unknown.

A promising candidate mechanism for rapid adjustment of sperm velocity may be found in the non-sperm component (seminal fluid and its constituents) of the ejaculate, particularly if such adjustments occur more rapidly than spermatogenesis (Cameron et al., 2007; Perry et al., 2013; Fitzpatrick and Lüpold, 2014). Seminal fluid is a complex medium containing a great diversity of molecules (Poiani, 2006; Juyena and Stelletta, 2012) and is known to influence sperm velocity and motility in vertebrates (Lahnsteiner et al., 1998; Lahnsteiner et al., 1996; Poiani, 2006; Locatello et al., 2013; González-Cadavid et al., 2014). For example, research using an externally fertilising fish, the grass goby (Zosterisessor ophiocephalus), compared males for which sperm competition strategy is determined by age/size and found large males that adopt a guarding strategy have a greater concentration of the seminal fluid glycoprotein mucin (Scaggiante et al., 1999). Furthermore, by separating and recombining seminal fluid and sperm from different males, research using the same species found seminal fluid had a tactic-specific effect on sperm velocity, with seminal fluid from sneak males decreasing the velocity of rival guard male sperm and seminal fluid from guard males increasing the velocity of sneak male sperm (Locatello et al., 2013).

However, only one study to date has investigated the role that seminal fluid plays as a mediator of short-term plastic sperm performance in a vertebrate species using fowl and the results were inconsistent with theoretical expectation: Cornwallis and O'Connor, 2009 found that while ejaculates produced by male fowl that were allocated to females of higher quality contained faster sperm, seminal fluid from those ejaculates reduced the velocity of sperm from the same male allocated to females of lower quality. To be consistent with the prediction that seminal fluid mediates changes in sperm velocity, seminal fluid from ejaculates allocated to higher quality females should increase, not decrease the speed of sperm isolated from ejaculates allocated to lower quality females. Thus, although there is evidence that seminal fluid can influence sperm velocity, evidence that seminal fluid mediates the rapid plastic adjustment of an ejaculate’s motile performance consistent with theoretical expectation is lacking.

We use an ideal model species, chinook salmon (Oncorhynchus tshawytscha), to examine patterns of ejaculate plasticity in response to changes in male social status and the reproductive consequences of these changes. In salmonids, fertilisation occurs externally and sperm competition occurs in the majority of spawnings (Berejikian et al., 2010; Sørum et al., 2011). Male chinook salmon adopt Alternative Reproductive Tactics (ARTs) situationally, as ‘hooknose’ males fight to establish social dominance (Esteve, 2005). Only dominant males guard spawning females thus obtaining priority in mating position, while subdominant males that lose contests attempt to sneak fertilisations by invading spawning pairs and releasing their sperm (Esteve, 2005). The social status of male salmon is subject to change over the course of a spawning season; for example, in coho salmon (O. kisutch), 22% of observed contests between hooknose males resulted in displacement of the previous dominant male (Healey and Prince, 1998). Therefore, in this mating system, females mate with multiple males in a dynamic social environment that results in intense levels of fluctuating sperm competition risk.

Previous research has shown that when males engage in sperm competition, sperm swimming speed is the primary predictor of fertilisation success in chinook salmon (Evans et al., 2013; Rosengrave et al., 2016) and other salmonids (Gage et al., 2004; Liljedal et al., 2008; Egeland et al., 2015). Sperm competition theory therefore predicts subdominant males, which have greater sperm competition risk, will invest in ejaculates with faster swimming sperm than dominant males and males changing social status should adjust their investment accordingly (Parker, 1990; Parker et al., 1997; Parker, 1998; Wedell et al., 2002; Birkhead et al., 2009; Parker and Pizzari, 2010). Indeed, several studies that experimentally manipulated social status using Arctic charr (Salvelinus alpinus) have found that subdominant males produce ejaculates with more sperm and faster swimming sperm than dominant males (Liljedal and Folstad, 2003; Rudolfsen et al., 2006; Vaz Serrano et al., 2006; Haugland et al., 2009). Furthermore, Rudolfsen et al. (2006) demonstrated that following a social challenge, both sperm concentration and velocity decreased over a 4-day period compared with pre-trial levels in dominant males, and observed an increase in sperm concentration but no change in sperm velocity for subdominant males. However, Rudolfsen et al. (2006) did not evaluate male social status prior to the social challenge, so it is unknown if these males actually changed or simply retained the same status through the course of the experiment. Recent research shows that ejaculates from subdominant Arctic charr sire the same number of eggs when in competition with ejaculates from dominant males if their sperm were released after the average delay observed under natural conditions (Egeland et al., 2015). These results suggest that salmonid males in disfavoured mating positions can compensate by producing more competitive ejaculates than dominant males, but whether males changing social status adjust their sperm velocity, and if such adjustments to ejaculates are mediated by sperm or non-sperm components of the ejaculate, is yet to be determined.

Here, we use a comprehensive experimental approach to determine if changes in sperm velocity observed in response to an individual’s social position are the result of alterations to the gametes or to seminal fluid and if such responses actually alter a male’s reproductive success against a sperm competitor. Specifically, we examine whether ejaculate quality is phenotypically plastic in response to changes in sperm competition risk over 48 hr periods, using a two-stage challenge to manipulate social status (Cornwallis and Birkhead, 2007a; Pizzari et al., 2007) and collected ejaculates at each stage of the experiment. In the second stage, males either retained or were forced to change their social status, creating four social phenotypes with varying sperm competition risk (Figure 1). We found that subdominant males, which have greater sperm competition risk, invest more in both sperm concentration and sperm velocity compared to socially dominant males. Additionally, we find males that change from dominant to subdominant social status, thus elevated their sperm competition risk, increased their sperm velocity as predicted by sperm competition theory (Parker, 1990; Parker et al., 1997; Parker, 1998; Wedell et al., 2002; Birkhead et al., 2009; Parker and Pizzari, 2010). We also separated sperm from seminal fluid and created reciprocal combinations both within and between rival males, finding that males can make rapid adjustments to sperm velocity by producing seminal fluid that enhances sperm function. We then used in vitro fertilisation trials and found the seminal fluid effects on sperm swimming speed influences male reproductive success under sperm competition. Our combined experimental results provide compelling evidence that seminal fluid is the mediator of rapid strategic adjustment of sperm velocity, thus bringing us a critical step closer to identifying the underlying molecular mechanism that enables plasticity of ejaculate performance in dynamic social environments.

Figure 1

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In this study, we experimentally manipulated social status to produce four social phenotypes with differing levels of sperm competition risk, and in accordance with sperm competition theory (Parker, 1990; Parker et al., 1997; Parker, 1998; Wedell et al., 2002; Birkhead et al., 2009; Parker and Pizzari, 2010), found males with the highest risk of sperm competition produced ejaculates with both higher sperm concentration and faster swimming sperm. We also found males can make rapid adjustments to sperm velocity in a strategic response to changes in social position that signal increased sperm competition risk. While seminal fluid is often implicated to harbour the unknown mechanism behind plastic sperm performance in vertebrates (Perry et al., 2013; Fitzpatrick and Lüpold, 2014), our combined results for the first time, unequivocally demonstrate that seminal fluid acts as a mediator of rapid strategic adjustment to sperm velocity. Furthermore, we demonstrate strategic adjustments of sperm velocity mediated by seminal fluid directly impact male fitness, highlighting the adaptive significance of plastic ejaculate performance.

Sperm competition theory predicts that males should strategically adjust ejaculates in response to changing sperm competition risk (Wedell et al., 2002; Parker and Pizzari, 2010). In chinook salmon, relative sperm velocity among males is the primary determinant of fertilisation success (Evans et al., 2013; Rosengrave et al., 2016). We show males forced to change from dominant to subdominant social status, and therefore exposed to increased sperm competition risk, responded by increasing the quality of their ejaculate, in this case sperm velocity, within 48 hr (Figure 4). While we predict that males forced to change from subdominant to dominant social status, therefore exposed to decreased sperm competition risk, would respond by decreasing their ejaculate quality, we did not see a significant change in sperm velocity for these males. However, subdominant males that later became dominant had a relatively low mean sperm velocity that was more similar to dominant males than those from the other subdominant phenotype in the first stage of the experiment (Figure 4). In this case, these subdominant males may have attempted to adopt a guarding tactic even after losing in the first social challenge, as males that lose contests can either sneak or fight for dominance elsewhere (Esteve, 2005).

Males should also strategically adjust sperm concentration in response to changing sperm competition risk (Wedell et al., 2002; Parker and Pizzari, 2010). Accordingly, we found subdominant males produced ejaculates with greater sperm concentration than dominant males. However, our results show that there was no significant increase in sperm concentration for any of the social phenotypes over a 48-hr period. The exact time taken for spermatogenesis in salmonids is unknown; however, the process almost certainly takes more than 48 hr (Billard, 1983a; Billard, 1983b; Schulz et al., 2010). Therefore, these results suggest that the observed changes in sperm velocity are mediated by a component of the ejaculate that modifies the competitiveness of existing sperm, rather than simply via the production of new sperm.

Our results clearly demonstrate the observed plasticity of sperm velocity in chinook salmon, a key determinant of fertilisation success in several vertebrate species (Birkhead et al., 1999; Malo et al., 2005; Gasparini et al., 2010; Boschetto et al., 2011) including salmonids (Gage et al., 2004; Liljedal et al., 2008; Evans et al., 2013; Egeland et al., 2015; Rosengrave et al., 2016), is mediated by seminal fluid. We found sperm from the same male, when incubated in seminal fluid from different males, had significantly different sperm velocities, and the direction of this effect could be predicted by social status. For example, when sperm from dominant males were incubated in seminal fluid from subdominant males we found that on average their sperm velocity increased compared to the baseline measures in their own seminal fluid, and found the opposite effect when sperm from subdominant males were incubated in seminal fluid from dominant males (Figure 5). Contrary to Cornwallis and O'Connor, 2009, for which seminal fluid from higher quality ejaculates decreased the velocity of sperm from lower quality ejaculates in fowl, our findings are consistent with the prediction that seminal fluid from ejaculates with faster swimming sperm will enhance the speed of sperm from ejaculates with slower sperm. The disparity between our findings and those in fowl (Cornwallis and O'Connor, 2009) possibly reflect differences in the reproductive biology of these species; including internal and external modes of fertilisation and differences in the structure and formation of social hierarchies and associated sperm competition risk.

Ejaculate allocation in fowl is also influenced by factors other than sperm competition risk, including female quality and the probability of future mating opportunities (Pizzari et al., 2003; Cornwallis and Birkhead, 2006; Cornwallis and Birkhead, 2007a; Cornwallis and Birkhead, 2007b); whether such factors influence ejaculate allocation strategies in salmonids is unknown. It is also possible that seminal fluid in fowl has evolved to interact with sperm from rivals, as observed in some insect species (den Boer et al., 2010) and reported for the grass goby (Zosterisessor ophiocephalus) (Locatello et al., 2013). Fertilisation occurs rapidly in salmonids, with the majority of eggs fertilised within 10 s post ejaculation (Hoysak and Liley, 2001; Liley et al., 2002; Yeates et al., 2007). Such rapid time frames may allow for little interaction between seminal fluid and sperm from different males during spawning. This is supported by research using Arctic charr that found the activation of sperm with a solution containing seminal fluid from another male had no effect on sperm velocity (Rudolfsen et al., 2015). However, a recent experiment that separated and recombined ejaculates from precocious chinook salmon males (obligate sneakers) and adult hooknose males report similar results to those found in the grass goby, with seminal fluid from precocious males significantly decreasing the velocity of hooknose male sperm (Lewis and Pitcher, 2017). Our results suggest chinook salmon seminal fluid has not evolved a targeted effect on sperm from males adopting a different tactic within the same age/size class, as regardless of social status, males that have faster recorded sperm velocities produced seminal fluid that increases the velocity of sperm from other males with slower speeds, and likewise males with slower sperm velocity produced seminal fluid that decreases the velocity of sperm from males with faster speeds (Figure 6).

In addition to demonstrating that seminal fluid influences sperm competitiveness, our in vitro sperm competition trials show the influence seminal fluid has on sperm velocity translates to having an effect on male fitness. We measured the fertilisation success within the same male x male x female combinations across trials, and compared those males across unmanipulated and recombined ejaculate treatments, finding changes in the relative sperm velocity between competitors were significantly correlated with the change in the proportion of eggs sired by each male (Figure 7). That is, the change in sperm velocity due to the seminal fluid in which sperm were incubated had a significant influence on the proportion of eggs sired by those sperm, in some cases completely reversing the ‘winner’ of sperm competition within the same male-female group. We now need further investigation to determine the component of seminal fluid that is strategically adjusted by males in response to sperm competition risk.

Previous studies have found that natural variation in several seminal fluid metrics was not correlated with sperm velocity in chinook salmon, including pH, osmolality and ion composition (Rosengrave et al., 2009a; Flannery et al., 2013). It is possible that seminal fluid contains different levels of available nutrients therefore fuelling differential energy production in sperm. In the short term following activation of motility in salmonids, sperm utilise ATP as the energy source for flagellar movement (Christen et al., 1987) using both stored ATP reserves and increasing ATP production significantly via aerobic respiration (Lahnsteiner et al., 1993, Lahnsteiner et al., 1999). Sperm ATP levels have been positively correlated with sperm velocity (Lahnsteiner et al., 1998; Bencic et al., 1999; Burness et al., 2004) and fertilisation success (Zilli et al., 2004; Vladić et al., 2010) in external fertilisers. Exposure to different levels of exogenous nutrients in seminal fluid while sperm are immotile in the testis may influence energy metabolism, for example altering available energy reserves or stored nutrient reserves, influencing sperm velocity post activation (Lahnsteiner et al., 1999). Alternatively, seminal fluid may contain peptide or RNA signalling molecules, that alter sperm behaviour. For example, chemotaxis in several marine invertebrates is controlled by signalling pathways that are initiated by chemoattractant peptides released by ova (Kaupp et al., 2003; Darszon et al., 2008; Evans and Sherman, 2013). Evidence is also accruing that proteins and RNAs in seminal fluid exosomes may play critical roles in regulating sperm development and fertilisation (Vojtech et al., 2014; Jodar et al., 2016).

Several Seminal Fluid Proteins (SFPs) have been associated with sperm velocity in vertebrate species (Lahnsteiner et al., 1996, 1998; Poiani, 2006) and are therefore likely candidates for modifying rapid adjustment of sperm velocity (Simmons and Fitzpatrick, 2012). Differences in SFP composition have been documented among males adopting different reproductive tactics in externally fertilising fish (Scaggiante et al., 1999; Gombar et al., 2017). Additionally, a growing body of empirical work has demonstrated that males can tailor SFP composition in response to sperm competition risk (Wigby et al., 2009; Fedorka et al., 2011; Ramm et al., 2015; Simmons and Lovegrove, 2017) and the mating status of females (Sirot et al., 2011). The role of SFPs in sperm competition, with the exception for some insect species (den Boer et al., 2010; Avila et al., 2011) and specific proteins in mammals (Ramm et al., 2008), is generally poorly understood. While the activity of SFPs associated with energy metabolism and respiration have been correlated with sperm velocity in a Cyprinid species (Lahnsteiner et al., 1996) and rainbow trout (O. mykiss) (Lahnsteiner et al., 1998), total protein concentration as well as the activity of lactate dehydrogenase, anti-trypsin and superoxide dismutase enzymes were not correlated with sperm velocity in chinook salmon (Flannery et al., 2013). However, these SFPs represents only a small fraction of the enzymatic activity likely to occur in fish seminal fluid (Gombar et al., 2017; Nynca et al., 2014). The critical next step in determining the molecular mechanism(s) involved will be to link variation in seminal fluid components to sperm velocity, and confirm these results experimentally.

In conclusion, as predicted by sperm competition theory (Parker, 1990; Parker et al., 1997; Parker, 1998; Wedell et al., 2002; Birkhead et al., 2009; Parker and Pizzari, 2010), we find male chinook salmon can make rapid adjustment to sperm velocity in response to social cues that signal changing sperm competition risk and such changes have a significant impact on the outcome of sperm competition and therefore male fitness. We further demonstrate that seminal fluid, even in a species with external fertilisation, plays a key role in mediating the strategic rapid adjustment of sperm velocity and for the first time provide strong evidence the mechanism behind plasticity in sperm velocity lies within the non-sperm component of the ejaculate. Our results support plastic adjustment of ejaculate quality in response to changing sperm competition risk is an effective evolutionary strategy in systems with dynamic social environments and we show seminal fluid mediates such adjustments.

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Author details

1. Michael J Bartlett

   School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   For correspondence

   michael.bartlett@pg.canterbury.ac.nz

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4253-6589

2. Tammy E Steeves

   School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

   Contribution

   Supervision, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

3. Neil J Gemmell

   Department of Anatomy, University of Otago, Dunedin, New Zealand

   Contribution

   Conceptualization, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0671-3637

4. Patrice C Rosengrave

   Department of Anatomy, University of Otago, Dunedin, New Zealand

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8421-3094

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