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.

1. 1. Bartlett MJ
   2. Steeves TE
   3. Gemmell NJ
   4. Rosengrave PC

   (2017) Data from: Sperm competition risk drives rapid ejaculate adjustments mediated by seminal fluid

   Available at Dryad Digital Repository under a CC0 Public Domain Dedication.

   https://doi.org/10.5061/dryad.kr137

1. 1. Avila FW
   2. Sirot LK
   3. LaFlamme BA
   4. Rubinstein CD
   5. Wolfner MF

   (2011) Insect seminal fluid proteins: identification and function

   Annual Review of Entomology 56:21–40.

   https://doi.org/10.1146/annurev-ento-120709-144823

   • PubMed
   • Google Scholar
2. 1. Bailey J
   2. Alanäuräu A
   3. Bräunnäus E

   (2000) Methods for assessing social status in Arctic charr

   Journal of Fish Biology 57:258–261.

   https://doi.org/10.1111/j.1095-8649.2000.tb00792.x

   • Google Scholar
3. 1. Bates D
   2. Mächler M
   3. Bolker B
   4. Walker S

   (2015) Fitting linear mixed-effects models using lme4

   Journal of Statistical Software 67:1–48.

   https://doi.org/10.18637/jss.v067.i01

   • Google Scholar
4. 1. Bencic DC
   2. Krisfalusi M
   3. Cloud JG
   4. Ingermann RL

   (1999) Maintenance of steelhead trout (Oncorhynchus Mykiss) sperm at different in vitro oxygen tensions alters ATP levels and cell functional characteristics

   Fish Physiology and Biochemistry 21:193–200.

   https://doi.org/10.1023/A:1007880426488

   • Google Scholar
5. 1. Berejikian BA
   2. Van Doornik DM
   3. Endicott RC
   4. Hoffnagle TL
   5. Tezak EP
   6. Moore ME
   7. Atkins J

   (2010) Mating success of alternative male phenotypes and evidence for frequency-dependent selection in Chinook salmon, Oncorhynchus tshawytscha

   Canadian Journal of Fisheries and Aquatic Sciences 67:1933–1941.

   https://doi.org/10.1139/F10-112

   • Google Scholar
6. 1. Billard R

   (1983a) A quantitative analysis of spermatogenesis in the trout, Salmo trutta fario

   Cell and Tissue Research 230:495–502.

   https://doi.org/10.1007/BF00216195

   • PubMed
   • Google Scholar
7. 1. Billard R

   (1983b) Spermiogenesis in the rainbow trout (Salmo gairdneri)

   Cell and Tissue Research 233:265–284.

   https://doi.org/10.1007/BF00238295

   • Google Scholar
8. Book

   1. Birkhead TR
   2. Hosken DJ
   3. Pitnick SS

   (2009)

   Sperm Biology: An Evolutionary Perspective

   Burlington, MA: Academic press.

   • Google Scholar
9. Book

   1. Birkhead TR
   2. Møller AP

   (1998)

   Sperm Competition and Sexual Selection

   London: Academic Press.

   • Google Scholar
10. 1. Birkhead TR
    2. Martínez JG
    3. Burke T
    4. Froman DP

    (1999) Sperm mobility determines the outcome of sperm competition in the domestic fowl

    Proceedings of the Royal Society B: Biological Sciences 266:1759–1764.

    https://doi.org/10.1098/rspb.1999.0843

    • PubMed
    • Google Scholar
11. 1. Birkhead TR
    2. Pizzari T

    (2002) Evolution of sex: postcopulatory sexual selection

    Nature Reviews Genetics 3:262–273.

    https://doi.org/10.1038/nrg774

    • Google Scholar
12. Book

    1. Bolker BM

    (2015)

    Linear and generalized linear mixed models

    In: Fox GA, Negrete-Yankelevich S, Sosa VJ, editors. Ecological Statistics: Contemporary Theory and Application (1st ed). Oxford University Press. pp. 309–333.

    • Google Scholar
13. 1. Boschetto C
    2. Gasparini C
    3. Pilastro A

    (2011) Sperm number and velocity affect sperm competition success in the guppy (Poecilia reticulata)

    Behavioral Ecology and Sociobiology 65:813–821.

    https://doi.org/10.1007/s00265-010-1085-y

    • Google Scholar
14. 1. Burness G
    2. Casselman SJ
    3. Schulte-Hostedde AI
    4. Moyes CD
    5. Montgomerie R

    (2004) Sperm swimming speed and energetics vary with sperm competition risk in bluegill (Lepomis macrochirus )

    Behavioral Ecology and Sociobiology 56:65–70.

    https://doi.org/10.1007/s00265-003-0752-7

    • Google Scholar
15. 1. Butts IA
    2. Litvak MK
    3. Trippel EA

    (2010) Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

    Theriogenology 73:873–885.

    https://doi.org/10.1016/j.theriogenology.2009.11.011

    • PubMed
    • Google Scholar
16. 1. Cameron E
    2. Day T
    3. Rowe L

    (2007) Sperm competition and the evolution of ejaculate composition

    The American Naturalist 169:E158–E172.

    https://doi.org/10.1086/516718

    • PubMed
    • Google Scholar
17. 1. Christen R
    2. Gatti JL
    3. Billard R

    (1987) Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement

    European Journal of Biochemistry 166:667–671.

    https://doi.org/10.1111/j.1432-1033.1987.tb13565.x

    • PubMed
    • Google Scholar
18. 1. Cornwallis CK
    2. Birkhead TR

    (2006) Social status and availability of females determine patterns of sperm allocation in the fowl

    Evolution 60:1486–1493.

    https://doi.org/10.1111/j.0014-3820.2006.tb01227.x

    • PubMed
    • Google Scholar
19. 1. Cornwallis CK
    2. Birkhead TR

    (2007a) Changes in sperm quality and numbers in response to experimental manipulation of male social status and female attractiveness

    The American Naturalist 170:758–770.

    https://doi.org/10.1086/521955

    • PubMed
    • Google Scholar
20. 1. Cornwallis CK
    2. Birkhead TR

    (2007b) Experimental evidence that female ornamentation increases the acquisition of sperm and signals fecundity

    Proceedings of the Royal Society B: Biological Sciences 274:583–590.

    https://doi.org/10.1098/rspb.2006.3757

    • PubMed
    • Google Scholar
21. 1. Cornwallis CK
    2. O'Connor EA

    (2009) Sperm: seminal fluid interactions and the adjustment of sperm quality in relation to female attractiveness

    Proceedings of the Royal Society B: Biological Sciences 276:3467–3475.

    https://doi.org/10.1098/rspb.2009.0807

    • PubMed
    • Google Scholar
22. 1. Darszon A
    2. Guerrero A
    3. Galindo BE
    4. Nishigaki T
    5. Wood CD

    (2008) Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

    The International Journal of Developmental Biology 52:595–606.

    https://doi.org/10.1387/ijdb.072550ad

    • PubMed
    • Google Scholar
23. 1. den Boer SP
    2. Baer B
    3. Boomsma JJ

    (2010) Seminal fluid mediates ejaculate competition in social insects

    Science 327:1506–1509.

    https://doi.org/10.1126/science.1184709

    • PubMed
    • Google Scholar
24. 1. Egeland TB
    2. Rudolfsen G
    3. Nordeide JT
    4. Folstad I

    (2015) On the relative effect of spawning asynchrony, sperm quantity, and sperm quality on paternity under sperm competition in an external fertilizer

    Frontiers in Ecology and Evolution 3:77.

    https://doi.org/10.3389/fevo.2015.00077

    • Google Scholar
25. 1. Esteve M

    (2005) Observations of spawning behaviour in salmoninae: salmo, oncorhynchus and salvelinus

    Reviews in Fish Biology and Fisheries 15:1–21.

    https://doi.org/10.1007/s11160-005-7434-7

    • Google Scholar
26. 1. Evans JP
    2. Rosengrave P
    3. Gasparini C
    4. Gemmell NJ

    (2013) Delineating the roles of males and females in sperm competition

    Proceedings of the Royal Society B: Biological Sciences 280:20132047.

    https://doi.org/10.1098/rspb.2013.2047

    • Google Scholar
27. 1. Evans JP
    2. Sherman CD

    (2013) Sexual selection and the evolution of egg-sperm interactions in broadcast-spawning invertebrates

    The Biological Bulletin 224:166–183.

    https://doi.org/10.1086/BBLv224n3p166

    • PubMed
    • Google Scholar
28. 1. Fedorka KM
    2. Winterhalter WE
    3. Ware B

    (2011) Perceived sperm competition intensity influences seminal fluid protein production prior to courtship and mating

    Evolution 65:584–590.

    https://doi.org/10.1111/j.1558-5646.2010.01141.x

    • PubMed
    • Google Scholar
29. 1. Fellows I

    (2012) Deducer: a data analysis GUI for R

    Journal of Statistical Software 49:1–15.

    https://doi.org/10.18637/jss.v049.i08

    • Google Scholar
30. 1. Fitzpatrick JL
    2. Lüpold S

    (2014) Sexual selection and the evolution of sperm quality

    Molecular Human Reproduction 20:1180–1189.

    https://doi.org/10.1093/molehr/gau067

    • PubMed
    • Google Scholar
31. 1. Flannery EW
    2. Butts IAE
    3. Słowińska M
    4. Ciereszko A
    5. Pitcher TE

    (2013) Reproductive investment patterns, sperm characteristics, and seminal plasma physiology in alternative reproductive tactics of Chinook salmon (Oncorhynchus tshawytscha)

    Biological Journal of the Linnean Society 108:99–108.

    https://doi.org/10.1111/j.1095-8312.2012.01980.x

    • Google Scholar
32. 1. Gage MJ
    2. Macfarlane CP
    3. Yeates S
    4. Ward RG
    5. Searle JB
    6. Parker GA

    (2004) Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success

    Current Biology 14:44–47.

    https://doi.org/10.1016/S0960-9822(03)00939-4

    • PubMed
    • Google Scholar
33. 1. Gasparini C
    2. Peretti AV
    3. Pilastro A

    (2009) Female presence influences sperm velocity in the guppy

    Biology Letters 5:792–794.

    https://doi.org/10.1098/rsbl.2009.0413

    • PubMed
    • Google Scholar
34. 1. Gasparini C
    2. Simmons LW
    3. Beveridge M
    4. Evans JP

    (2010) Sperm swimming velocity predicts competitive fertilization success in the green swordtail Xiphophorus helleri

    PLoS One 5:e12146.

    https://doi.org/10.1371/journal.pone.0012146

    • PubMed
    • Google Scholar
35. 1. Gombar R
    2. Pitcher TE
    3. Lewis JA
    4. Auld J
    5. Vacratsis PO

    (2017) Proteomic characterization of seminal plasma from alternative reproductive tactics of Chinook salmon (Oncorhynchus tswatchysha)

    Journal of Proteomics 157:1–9.

    https://doi.org/10.1016/j.jprot.2017.01.019

    • PubMed
    • Google Scholar
36. 1. González-Cadavid V
    2. Martins JA
    3. Moreno FB
    4. Andrade TS
    5. Santos AC
    6. Monteiro-Moreira AC
    7. Moreira RA
    8. Moura AA

    (2014) Seminal plasma proteins of adult boars and correlations with sperm parameters

    Theriogenology 82:697–707.

    https://doi.org/10.1016/j.theriogenology.2014.05.024

    • PubMed
    • Google Scholar
37. 1. Hajirezaee S
    2. Amiri BM
    3. Mirvaghefi AR

    (2010) Changes in sperm production, sperm motility, and composition of seminal fluid in caspian brown trout, Salmo trutta caspius, over the course of a spawning season

    Journal of Applied Aquaculture 22:157–170.

    https://doi.org/10.1080/10454431003736482

    • Google Scholar
38. 1. Haugland T
    2. Rudolfsen G
    3. Figenschou L
    4. Folstad I

    (2009) Sperm velocity and its relation to social status in Arctic charr (Salvelinus alpinus)

    Animal Reproduction Science 115:231–237.

    https://doi.org/10.1016/j.anireprosci.2008.11.004

    • PubMed
    • Google Scholar
39. 1. Healey MC
    2. Prince A

    (1998) Alternative tactics in the breeding behaviour of male Coho Salmon

    Behaviour 135:1099–1124.

    https://doi.org/10.1163/156853998792913564

    • Google Scholar
40. 1. Hervé M

    (2016) RVAideMemoire: diverse basic statistical and graphical functions

    RVAideMemoire: diverse basic statistical and graphical functions, R package v 0.9-55, http://CRAN.R-project.org/package=RVAideMemoire.

    http://CRAN.R-project.org/package=RVAideMemoire

    • Google Scholar
41. 1. Hoysak DJ
    2. Liley NR

    (2001) Fertilization dynamics in sockeye salmon and a comparison of sperm from alternative male phenotypes

    Journal of Fish Biology 58:1286–1300.

    https://doi.org/10.1111/j.1095-8649.2001.tb02286.x

    • Google Scholar
42. 1. Jodar M
    2. Sendler E
    3. Krawetz SA

    (2016) The protein and transcript profiles of human semen

    Cell and Tissue Research 363:85–96.

    https://doi.org/10.1007/s00441-015-2237-1

    • PubMed
    • Google Scholar
43. 1. Joseph PN
    2. Sharma RK
    3. Agarwal A
    4. Sirot LK

    (2015) Men ejaculate larger volumes of semen, more motile sperm, and more quickly when exposed to images of novel women

    Evolutionary Psychological Science 1:195–200.

    https://doi.org/10.1007/s40806-015-0022-8

    • Google Scholar
44. 1. Juyena NS
    2. Stelletta C

    (2012) Seminal plasma: an essential attribute to spermatozoa

    Journal of Andrology 33:536–551.

    https://doi.org/10.2164/jandrol.110.012583

    • PubMed
    • Google Scholar
45. 1. Kaupp UB
    2. Solzin J
    3. Hildebrand E
    4. Brown JE
    5. Helbig A
    6. Hagen V
    7. Beyermann M
    8. Pampaloni F
    9. Weyand I

    (2003) The signal flow and motor response controling chemotaxis of sea urchin sperm

    Nature Cell Biology 5:109–117.

    https://doi.org/10.1038/ncb915

    • PubMed
    • Google Scholar
46. 1. Kilgallon SJ
    2. Simmons LW

    (2005) Image content influences men's semen quality

    Biology Letters 1:253–255.

    https://doi.org/10.1098/rsbl.2005.0324

    • PubMed
    • Google Scholar
47. 1. Kinnison MT
    2. Bentzen P
    3. Unwin MJ
    4. Quinn TP

    (2002) Reconstructing recent divergence: evaluating nonequilibrium population structure in New Zealand chinook salmon

    Molecular Ecology 11:739–754.

    https://doi.org/10.1046/j.1365-294X.2002.01477.x

    • PubMed
    • Google Scholar
48. 1. Kuznetsova A
    2. Bruun Brockhoff P
    3. Haubo Bojesen Christensen R

    (2016) lmerTest: tests in linear mixed effects models

    lmerTest: tests in linear mixed effects models, v 2.0-33, https://CRAN.R-project.org/package=lmerTest.

    https://CRAN.R-project.org/package=lmerTest

    • Google Scholar
49. 1. Lahnsteiner F
    2. Berger B
    3. Weismann T
    4. Patzner RA

    (1996) Motility of spermatozoa of Alburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism

    Fish Physiology and Biochemistry 15:167–179.

    https://doi.org/10.1007/BF01875596

    • PubMed
    • Google Scholar
50. 1. Lahnsteiner F
    2. Berger B
    3. Weismann T
    4. Patzner RA

    (1998) Determination of semen quality of the rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoal metabolism

    Aquaculture 163:163–181.

    https://doi.org/10.1016/S0044-8486(98)00243-9

    • Google Scholar
51. 1. Lahnsteiner F
    2. Berger B
    3. Weismann T

    (1999) Sperm metabolism of the telost fishes Chalcalburnus chalcoides and Oncorhynchus mykiss and its relation to motility and viability

    Journal of Experimental Zoology 284:454–465.

    https://doi.org/10.1002/(SICI)1097-010X(19990901)284:4<454::AID-JEZ12>3.0.CO;2-O

    • PubMed
    • Google Scholar
52. 1. Lahnsteiner F
    2. Patzner RA
    3. Weismann T

    (1993) Energy resources of spermatozoa of the rainbow trout Oncorhynchus mykiss (Pisces, Teleostei)

    Reproduction Nutrition Development 33:349–360.

    https://doi.org/10.1051/rnd:19930404

    • Google Scholar
53. 1. Lewis JA
    2. Pitcher TE

    (2017) The effects of rival seminal plasma on sperm velocity in the alternative reproductive tactics of Chinook salmon

    Theriogenology 92:24–29.

    https://doi.org/10.1016/j.theriogenology.2016.12.032

    • PubMed
    • Google Scholar
54. 1. Liley NR
    2. Tamkee P
    3. Tsai R
    4. Hoysak DJ

    (2002) Fertilization dynamics in rainbow trout (Oncorhynchus mykiss): effect of male age, social experience, and sperm concentration and motility on in vitro fertilization

    Canadian Journal of Fisheries and Aquatic Sciences 59:144–152.

    https://doi.org/10.1139/f01-202

    • Google Scholar
55. 1. Liljedal S
    2. Folstad I

    (2003) Milt quality, parasites, and immune function in dominant and subordinate Arctic charr

    Canadian Journal of Zoology 81:221–227.

    https://doi.org/10.1139/z02-244

    • Google Scholar
56. 1. Liljedal S
    2. Rudolfsen G
    3. Folstad I

    (2008) Factors predicting male fertilization success in an external fertilizer

    Behavioral Ecology and Sociobiology 62:1805–1811.

    https://doi.org/10.1007/s00265-008-0609-1

    • Google Scholar
57. 1. Locatello L
    2. Poli F
    3. Rasotto MB

    (2013) Tactic-specific differences in seminal fluid influence sperm performance

    Proceedings of the Royal Society B: Biological Sciences 280:20122891.

    https://doi.org/10.1098/rspb.2012.2891

    • Google Scholar
58. 1. Malo AF
    2. Garde JJ
    3. Soler AJ
    4. García AJ
    5. Gomendio M
    6. Roldan ER

    (2005) Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa

    Biology of Reproduction 72:822–829.

    https://doi.org/10.1095/biolreprod.104.036368

    • PubMed
    • Google Scholar
59. 1. Nelson RJ
    2. Beacham TD

    (1999) Isolation and cross species amplification of microsatellite loci useful for study of Pacific salmon

    Animal Genetics 30:228–229.

    https://doi.org/10.1046/j.1365-2052.1999.00404-4.x

    • PubMed
    • Google Scholar
60. 1. Nynca J
    2. Arnold GJ
    3. Fröhlich T
    4. Otte K
    5. Flenkenthaler F
    6. Ciereszko A

    (2014) Proteomic identification of rainbow trout seminal plasma proteins

    Proteomics 14:133–140.

    https://doi.org/10.1002/pmic.201300267

    • PubMed
    • Google Scholar
61. 1. Parker GA
    2. Ball MA
    3. Stockley P
    4. Gage MJ

    (1997) Sperm competition games: a prospective analysis of risk assessment

    Proceedings of the Royal Society B: Biological Sciences 264:1793–1802.

    https://doi.org/10.1098/rspb.1997.0249

    • PubMed
    • Google Scholar
62. 1. Parker GA
    2. Pizzari T

    (2010) Sperm competition and ejaculate economics

    Biological reviews of the Cambridge Philosophical Society 85:897–934.

    https://doi.org/10.1111/j.1469-185X.2010.00140.x

    • PubMed
    • Google Scholar
63. 1. Parker GA

    (1970) Sperm competition and its evolutionary consequences in the insects

    Biological Reviews 45:525–567.

    https://doi.org/10.1111/j.1469-185X.1970.tb01176.x

    • Google Scholar
64. 1. Parker GA

    (1990) Sperm competition games: sneaks and extra-pair copulations

    Proceedings of the Royal Society B: Biological Sciences 242:127–133.

    https://doi.org/10.1098/rspb.1990.0115

    • Google Scholar
65. Book

    1. Parker GA

    (1998)

    Sperm competition games

    In: Birkhead T. R, Møller A. P, editors. Sperm Competition and Sexual Selection. London: Academic Press. pp. 3–48.

    • Google Scholar
66. 1. Perry JC
    2. Sirot L
    3. Wigby S

    (2013) The seminal symphony: how to compose an ejaculate

    Trends in Ecology & Evolution 28:414–422.

    https://doi.org/10.1016/j.tree.2013.03.005

    • PubMed
    • Google Scholar
67. Software

    1. Pinheiro J
    2. Bates D
    3. DebRoy S
    4. Sarkar D
    5. R Core Team

    (2015) nlme: linear and nonlinear mixed effects models, version v 3.1-121

    R Package.

    http://CRAN.R-project.org/package=nlme
68. 1. Pizzari T
    2. Cornwallis CK
    3. Froman DP

    (2007) Social competitiveness associated with rapid fluctuations in sperm quality in male fowl

    Proceedings of the Royal Society B: Biological Sciences 274:853–860.

    https://doi.org/10.1098/rspb.2006.0080

    • PubMed
    • Google Scholar
69. 1. Pizzari T
    2. Cornwallis CK
    3. Løvlie H
    4. Jakobsson S
    5. Birkhead TR

    (2003) Sophisticated sperm allocation in male fowl

    Nature 426:70–74.

    https://doi.org/10.1038/nature02004

    • PubMed
    • Google Scholar
70. 1. Poiani A

    (2006) Complexity of seminal fluid: a review

    Behavioral Ecology and Sociobiology 60:289–310.

    https://doi.org/10.1007/s00265-006-0178-0

    • Google Scholar
71. Software

    1. R Core Team

    (2016) R: a language and environment for statistical computing

    R Foundation for Statistical Computing, Vienna, Austria.

    http://www.R-project.org/
72. 1. Ramm SA
    2. Edward DA
    3. Claydon AJ
    4. Hammond DE
    5. Brownridge P
    6. Hurst JL
    7. Beynon RJ
    8. Stockley P

    (2015) Sperm competition risk drives plasticity in seminal fluid composition

    BMC Biology 13:87.

    https://doi.org/10.1186/s12915-015-0197-2

    • PubMed
    • Google Scholar
73. 1. Ramm SA
    2. Oliver PL
    3. Ponting CP
    4. Stockley P
    5. Emes RD

    (2008) Sexual selection and the adaptive evolution of mammalian ejaculate proteins

    Molecular Biology and Evolution 25:207–219.

    https://doi.org/10.1093/molbev/msm242

    • PubMed
    • Google Scholar
74. 1. Rosengrave P
    2. Gemmell NJ
    3. Metcalf V
    4. McBride K
    5. Montgomerie R

    (2008) A mechanism for cryptic female choice in chinook salmon

    Behavioral Ecology 19:1179–1185.

    https://doi.org/10.1093/beheco/arn089

    • Google Scholar
75. 1. Rosengrave P
    2. Montgomerie R
    3. Gemmell N

    (2016) Cryptic female choice enhances fertilization success and embryo survival in chinook salmon

    Proceedings of the Royal Society B: Biological Sciences 283:20160001.

    https://doi.org/10.1098/rspb.2016.0001

    • Google Scholar
76. 1. Rosengrave P
    2. Montgomerie R
    3. Metcalf VJ
    4. McBride K
    5. Gemmell NJ

    (2009b) Sperm traits in Chinook salmon depend upon activation medium: implications for studies of sperm competition in fishes

    Canadian Journal of Zoology 87:920–927.

    https://doi.org/10.1139/Z09-081

    • Google Scholar
77. 1. Rosengrave P
    2. Taylor H
    3. Montgomerie R
    4. Metcalf V
    5. McBride K
    6. Gemmell NJ

    (2009a) Chemical composition of seminal and ovarian fluids of chinook salmon (Oncorhynchus tshawytscha) and their effects on sperm motility traits

    Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 152:123–129.

    https://doi.org/10.1016/j.cbpa.2008.09.009

    • PubMed
    • Google Scholar
78. 1. Rudolfsen G
    2. Figenschou L
    3. Folstad I
    4. Tveiten H
    5. Figenschou M

    (2006) Rapid adjustments of sperm characteristics in relation to social status

    Proceedings of the Royal Society B: Biological Sciences 273:325–332.

    https://doi.org/10.1098/rspb.2005.3305

    • PubMed
    • Google Scholar
79. 1. Rudolfsen G
    2. Serrano JV
    3. Folstad I

    (2015) Own, but not foreign seminal fluid inhibits sperm activation in a vertebrate with external fertilization

    Frontiers in Ecology and Evolution 3:92.

    https://doi.org/10.3389/fevo.2015.00092

    • Google Scholar
80. 1. Sørum V
    2. Rudolfsen G
    3. Folstad I
    4. Figenschou L

    (2011) Spawning behaviour of Arctic charr (Salvelinus alpinus): risk of sperm competition and timing of milt release for sneaker and dominant males

    Behaviour 148:1157–1172.

    https://doi.org/10.1163/000579511X596615

    • Google Scholar
81. Book

    1. Sarkar D

    (2008)

    Lattice: Multivariate data visualization with R

    New York: Springer.

    • Google Scholar
82. 1. Scaggiante M
    2. Mazzoldi C
    3. Petersen CW
    4. Rasotto MB

    (1999) Sperm competition and mode of fertilization in the grass goby Zosterisessor ophiocephalus (Teleostei: Gobiidae)

    Journal of Experimental Zoology 283:81–90.

    https://doi.org/10.1002/(SICI)1097-010X(19990101)283:1<81::AID-JEZ9>3.0.CO;2-9

    • Google Scholar
83. 1. Schulz RW
    2. de França LR
    3. Lareyre JJ
    4. LeGac F
    5. Chiarini-Garcia H
    6. Nobrega RH
    7. Miura T

    (2010) Spermatogenesis in fish

    General and Comparative Endocrinology 165:390–411.

    https://doi.org/10.1016/j.ygcen.2009.02.013

    • PubMed
    • Google Scholar
84. 1. Simmons LW
    2. Fitzpatrick JL

    (2012) Sperm wars and the evolution of male fertility

    Reproduction 144:519–534.

    https://doi.org/10.1530/REP-12-0285

    • PubMed
    • Google Scholar
85. 1. Simmons LW
    2. Lovegrove M

    (2017) Socially cued seminal fluid gene expression mediates responses in ejaculate quality to sperm competition risk

    Proceedings of the Royal Society B: Biological Sciences 284:20171486.

    https://doi.org/10.1098/rspb.2017.1486

    • Google Scholar
86. 1. Sirot LK
    2. Wolfner MF
    3. Wigby S

    (2011) Protein-specific manipulation of ejaculate composition in response to female mating status in Drosophila melanogaster

    PNAS 108:9922–9926.

    https://doi.org/10.1073/pnas.1100905108

    • PubMed
    • Google Scholar
87. 1. Small MP
    2. Beacham TD
    3. Withler RE
    4. Nelson RJ

    (1998) Discriminating coho salmon (Oncorhynchus kisutch) populations within the Fraser River, British Columbia, using microsatellite DNA markers

    Molecular Ecology 7:141–155.

    https://doi.org/10.1046/j.1365-294x.1998.00324.x

    • Google Scholar
88. 1. Smith CC
    2. Ryan MJ

    (2011) Tactic-dependent plasticity in ejaculate traits in the swordtail Xiphophorus nigrensis

    Biology Letters 7:733–735.

    https://doi.org/10.1098/rsbl.2011.0286

    • PubMed
    • Google Scholar
89. 1. Thomas ML
    2. Simmons LW

    (2007) Male crickets adjust the viability of their sperm in response to female mating status

    The American Naturalist 170:190–195.

    https://doi.org/10.1086/519404

    • PubMed
    • Google Scholar
90. 1. Tremblay A
    2. Ransijn J

    (2015) LMERConvenienceFunctions: model selection and post-hoc analysis for (G)LMER models

    LMERConvenienceFunctions: model selection and post-hoc analysis for (G)LMER models, R package v 2.10, http://CRAN.R-project.org/package=LMERConvenienceFunctions.

    http://CRAN.R-project.org/package=LMERConvenienceFunctions

    • Google Scholar
91. 1. Unwin MJ
    2. Quinn TP
    3. Kinnison MT
    4. Boustead NC

    (2000) Divergence in juvenile growth and life history in two recently colonized and partially isolated chinook salmon populations

    Journal of Fish Biology 57:943–960.

    https://doi.org/10.1111/j.1095-8649.2000.tb02203.x

    • Google Scholar
92. 1. Vaz Serrano J
    2. Folstad I
    3. Rudolfsen G
    4. Figenschou L

    (2006) Do the fastest sperm within an ejaculate swim faster in subordinate than in dominant males of Arctic char?

    Canadian Journal of Zoology 84:1019–1024.

    https://doi.org/10.1139/Z06-097

    • Google Scholar
93. 1. Vladić T
    2. Forsberg LA
    3. Järvi T

    (2010) Sperm competition between alternative reproductive tactics of the Atlantic salmon in vitro

    Aquaculture 302:265–269.

    https://doi.org/10.1016/j.aquaculture.2010.02.024

    • Google Scholar
94. 1. Vojtech L
    2. Woo S
    3. Hughes S
    4. Levy C
    5. Ballweber L
    6. Sauteraud RP
    7. Strobl J
    8. Westerberg K
    9. Gottardo R
    10. Tewari M
    11. Hladik F

    (2014) Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions

    Nucleic Acids Research 42:7290–7304.

    https://doi.org/10.1093/nar/gku347

    • PubMed
    • Google Scholar
95. 1. Walsh PS
    2. Metzger DA
    3. Higuchi R

    (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material

    BioTechniques 10:506–513.

    https://doi.org/10.2144/000113897

    • PubMed
    • Google Scholar
96. 1. Wedell N
    2. Gage MJG
    3. Parker GA

    (2002) Sperm competition, male prudence and sperm-limited females

    Trends in Ecology & Evolution 17:313–320.

    https://doi.org/10.1016/S0169-5347(02)02533-8

    • Google Scholar
97. Book

    1. Wickham H

    (2009)

    ggplot2: Elegant Graphics for Data Analysis

    New York: Springer-Verlag.

    • Google Scholar
98. 1. Wigby S
    2. Sirot LK
    3. Linklater JR
    4. Buehner N
    5. Calboli FC
    6. Bretman A
    7. Wolfner MF
    8. Chapman T

    (2009) Seminal fluid protein allocation and male reproductive success

    Current Biology 19:751–757.

    https://doi.org/10.1016/j.cub.2009.03.036

    • PubMed
    • Google Scholar
99. 1. Winberg S
    2. Nilsson GE
    3. Olsén KH

    (1991) Social rank and brain levels of monoamines and monoamine metabolites in Arctic charr, Salvelinus alpinus (L.)

    Journal of Comparative Physiology A 168:241–246.

    https://doi.org/10.1007/BF00218416

    • Google Scholar
100. 1. Yeates S
     2. Searle J
     3. Ward RG
     4. Gage MJG

     (2007) A two-second delay confers first-male fertilization precedence within in vitro sperm competition experiments in Atlantic salmon

     Journal of Fish Biology 70:318–322.

     https://doi.org/10.1111/j.1095-8649.2006.01294.x

     • Google Scholar
101. 1. Zilli L
     2. Schiavone R
     3. Zonno V
     4. Storelli C
     5. Vilella S

     (2004) Adenosine triphosphate concentration and β-D-glucuronidase activity as indicators of sea bass semen quality

     Biology of Reproduction 70:1679–1684.

     https://doi.org/10.1095/biolreprod.103.027177

     • PubMed
     • Google Scholar

Thank you for submitting your article "Sperm competition risk drives rapid ejaculate adjustments mediated by seminal fluid" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ian Baldwin as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Matthew Gage (reviewer #1); Tom Pizzari (reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Two independent reviews have now been submitted for this manuscript; one is extremely positive, and the other contains more caveats. The less positive reviewer sees value in the work, but concludes that general presentation of the manuscript and referencing to previous work needs further work. Both reviewers suggest a number of specific routes to improvement, which we see as being easily surmountable.

This is a strong and valuable piece of work that could easily be improved to a very high standard by addressing both reviewers' comments. After this, the work would constitute a major contribution to our understanding in reproductive biology and evolution.

The work combines a series of complementary findings within one study that sum to an important advance. There are three well-executed experimental manipulations which allow clear findings that: a) male dominance status influences sperm behaviour and this can change over short time periods; b) seminal fluid biochemistry is a primary driver behind this change in sperm behaviour; and c) the change in sperm behaviour as a consequence of seminal fluid biochemistry translates into male reproductive success. From this work, which uses an ideal model system, we can conclude that males are sensitive to proximate risks of competition for fertilisations, and they modulate sperm function over short time periods to match the risk of competition using seminal fluid. Altogether, these are important findings that allow novel insights into the surprising extents to which males and their sperm are shaped by the struggle to reproduce.

Essential revisions:

The authors should refer to the two separate reviews in order to satisfy their concerns about the current state of the manuscript.

The more 'problematic' issues raised by reviewer 3 are also readily addressed as they concern originality, presentation style, and statistics.

In reverse order: reviewer 3 asks for clarity concerning statistical analysis of the switch in social status and effects on VAP, clarity which would be helpful for readers.

In terms of presentation, it would be helpful to use original references that reviewer 3 is concerned about, and to flesh out some of the theory on sperm competition.

Considering originality, which links to the presentation and overstatement concerns of reviewer 3: I agree that Locatello et al., 2013 work with goby males also used the clever transfer of sperm and seminal fluid between morphs to show that fluid can indeed influence sperm function, however the current study did refer to this work in the introduction. Despite this study, conclusive empirical findings of the consequence of seminal fluid changes, and that these can happen in the short term in relation to dominance status have been so far non-existent. I therefore see this as the strength of the current work, and it would strengthen the manuscript to ensure both Locatello et al., and Scaggiante et al., studies are mentioned clearly in the Introduction.

Finally, reviewer 3 is concerned that the seminal fluid compositional changes were not measured in tandem with sperm behaviour changes and the consequences for fertilisations success. I believe, with reviewer 2, that this is beyond the scope of the current work, and may be a huge task given the diversity of compounds within male seminal fluids. However, I believe it would strengthen the manuscript to include wider discussion of how seminal fluid might influence sperm behaviour, as suggested by reviewer 3. An additional thought to consider is whether seminal fluid acts as a signal for sperm behavioural switching, or whether it contains exogenous nutrients that 'fed' and then changed sperm motility? This can be considered in the light of what is known about exogenous and endogenous metabolism in the sperm of external fertilisers. I believe the consensus is that there is rather little evidence for metabolism of exogenous nutrients in the short term following spermatozoal activation – but it may be different following incubation. It would be worth considering a brief discussion on this point, including reference to reviewer 3's question about possible RNA signalling, and the finding of glycoprotein variation in goby seminal fluid composition (Scaggiante et al., 1999). It also relates to the strong work on fowl where rival male ejaculates have had different and interesting impacts on sperm behaviour under male dominance variation. If seminal fluid contains signals, for example, which sperm react to, then the shift in velocity may well trade against other activity components such as longevity, with changes to behaviour possibly influenced by both signaller and receiver. If, on the other hand, seminal fluid contains nutrients for exogenous metabolism by sperm, then some discussion about sugars/glycoproteins as well as SFPs would be helpful.

Reviewer #2:

This study investigates experimentally: (a) the influence of seminal fluid in patterns of variation in sperm swimming velocity, a key driver of fertilising efficiency, and (b) the extent to which such seminal fluid-driven changes in sperm swimming velocity impact the outcome of sperm competition. The possibility that seminal fluid compounds, primarily accessory gland products, influence the competitive efficiency of an ejaculate, and have largely evolved for their role in sperm competition, has been around for decades but direct evidence in support of this has been lacking. This is not surprising given the technical difficulties involved in testing this idea under controlled experimental conditions (as way of reference, my own research group has been working on these very questions and using similar approaches for years now, and we haven't yet submitted for publication because our data are not yet as clear and as complete as these presented here). The present study of a chinook salmon population presents so far the most convincing piece of evidence that I have seen in support of the notion that seminal fluid drives changes in sperm fertilising efficiency with tangible impact on sperm competition. Importantly, these results also suggests that males are able to strategically change the composition of their seminal fluid in order to respond to changes in the risk of sperm competition associated with rapid changes in social status. Specifically, by presenting males with a double social challenge the study shows that males that lost the first contest (SS + SD) produce faster swimming sperm than males that won the first contest (DD + DS), and that -after controlling for multiple comparisons- the sperm of males that decreased in status over the two challenges (DS) increased in swimming velocity. Given that these changes occurred rapidly (~48hrs) and males were experimentally sperm depleted, it is parsimonious to assume that they are due to seminal fluid changes rather than sperm phenotype. This is then confirmed by a subsequent experiment in which the sperm of a focal male is incubated in the seminal fluid of a competitor male, and the change in the focal male's sperm velocity is then shown to be predicted by the social status of the competitor male. Critically, when the relative sperm swimming velocity of the competitor male is entered in the model, this becomes the significant predictor while status becomes non-significant, suggesting that male status explains changes in the sperm velocity only to the extent to which it predicts the relative swimming velocity of the competitor male's sperm. This demonstrates that the seminal fluid of the competitor male has a causal effect in the swimming velocity of the focal male. Finally, the study performs a series of in vitro competitive fertilisation trials to show that when the sperm of a focal male are exposed to the seminal fluid of his respective rival, changes in fertilisation success are best predicted by the changes in sperm swimming velocity due to exposure to the rival's seminal fluid. Together, these results present a robust case for socially subordinate males compensating through preferential investment in seminal fluid components which increase the fertilising efficiency of their ejaculates, confirming previous suggestions of alternative mating tactics in this and other fish species with similar mating systems. Overall, I think that this is an excellent study. The experimental work is conducted meticulously and impressive in scale. The manuscript is written clearly and with appropriate consideration of previous work. The conclusions drawn are justified and discussed in adequate depth.

Reviewer #3:

This study investigates how male social status (i.e. dominant versus subordinate) affects sperm performance in male chinook salmon. They paired males up and let them establish a hierarchy and collected sperm after 48 hours, male were then re-paired by social status (dominant with dominant and subordinate with subordinate) forcing half of the ales to switch status. The authors tested how seminal fluid affects sperm performance by separating sperm from seminal fluid and re-suspending sperm in the seminal fluid of a different male. They found subordinate males to produce generally faster swimming sperm than dominant males. Furthermore, they show that sperm from dominant males in subordinate seminal fluid became faster and vice versa and that the resulting sperm velocity was a strong predictor of fertilisation success.

While the study is solid and results seem convincing (although more statistics are needed), the manuscript could be improved by tightening the writing style and making the arguments and more concrete. The writing style is often loose and ambivalent, which this is also true for the methods section. In addition, the use of references is not always satisfying as it would be great to see references to original papers rather than reviews for example when introducing the theoretical predictions about sperm competition. In general, the manuscript would benefit from careful revision to make it tighter and more accurate.

For one, the authors claim that they have been using a novel experimental approach to disentangle the effect of sperm versus seminal fluid on sperm velocity. However, the same approach has been used in a study by Locatello et al., (2013) published a few years ago showing a similar effect of seminal fluid on sperm performance between males adopting alternative mating tactics in a goby. Furthermore, previous studies showed that sneaker and territorial male ejaculates in a Mediterranean goby differ in the composition and amount of polysaccharides present in the seminal fluid (e.g. Scaggiante et al., 1999). The authors should mention these studies in the Introduction (and not only in the Discussion) and refer to these previous results when introducing their current study. In other words, a more accurate coverage of the literature in the relevant places would help to put this present study into context.

Furthermore, the authors argue that sperm velocity is affected by a change in composition in the seminal fluid based on the fact that males changing from dominant to subordinate did not produce significantly more sperm over the span of 48 hours. Unfortunately, they performed no test – not even a simple test for polysaccharides or general protein content – to actually verify a change in composition of the seminal fluid. There are several alternative explanations for a change in sperm swimming velocity, such as RNAs injected into sperm through vesicles immediately prior to ejaculate release that alter sperm metabolic pathways to name but one, which should also be discussed.

Finally, the fact that males experience a rapid turnover in treatments where males are kept in each social status for three days in total needs a bit more attention. The sequence of the social status clearly has an effect on sperm traits as the main effect on VAP was found in males switching from dominant to subordinate. I am therefore wondering how these issues were taken into account when running the statistical analyses. The authors write that they used either two levels (D and S) when analysing the stages separately or four when analysing stage 2. This is an ambivalent description and should be clarified; for example in Table 3, it is not clear how the statistical model looked like and no interaction terms are presented whereas Figure 4 suggests a clear interaction between treatments (DD and the other three). I am generally wondering about the role of interaction terms between the social status during stage 1 and stage 2 as none are presented in the current version of the manuscript.

In summary, while I can see the value of this study, the general presentation should be improved and the novelty of the findings is overstated.

Essential revisions:

The authors should refer to the two separate reviews in order to satisfy their concerns about the current state of the manuscript.

The more 'problematic' issues raised by reviewer 3 are also readily addressed as they concern originality, presentation style, and statistics.

In reverse order: reviewer 3 asks for clarity concerning statistical analysis of the switch in social status and effects on VAP, clarity which would be helpful for readers.

In terms of presentation, it would be helpful to use original references that reviewer 3 is concerned about, and to flesh out some of the theory on sperm competition.

We have expanded on our explanation of sperm competition theory and include additional empirical references, including highly relevant papers by Parker and colleagues that originally explored sperm competition modelling under different scenarios (Introduction).

Considering originality, which links to the presentation and overstatement concerns of reviewer 3: I agree that Locatello et al., 2013 work with goby males also used the clever transfer of sperm and seminal fluid between morphs to show that fluid can indeed influence sperm function, however the current study did refer to this work in the introduction. Despite this study, conclusive empirical findings of the consequence of seminal fluid changes, and that these can happen in the short term in relation to dominance status have been so far non-existent. I therefore see this as the strength of the current work, and it would strengthen the manuscript to ensure both Locatello et al., and Scaggiante et al., studies are mentioned clearly in the Introduction.

Finally, reviewer 3 is concerned that the seminal fluid compositional changes were not measured in tandem with sperm behaviour changes and the consequences for fertilisations success. I believe, with reviewer 2, that this is beyond the scope of the current work, and may be a huge task given the diversity of compounds within male seminal fluids. However, I believe it would strengthen the manuscript to include wider discussion of how seminal fluid might influence sperm behaviour, as suggested by reviewer 3. An additional thought to consider is whether seminal fluid acts as a signal for sperm behavioural switching, or whether it contains exogenous nutrients that 'fed' and then changed sperm motility? This can be considered in the light of what is known about exogenous and endogenous metabolism in the sperm of external fertilisers. I believe the consensus is that there is rather little evidence for metabolism of exogenous nutrients in the short term following spermatozoal activation – but it may be different following incubation. It would be worth considering a brief discussion on this point, including reference to reviewer 3's question about possible RNA signalling, and the finding of glycoprotein variation in goby seminal fluid composition (Scaggiante et al., 1999). It also relates to the strong work on fowl where rival male ejaculates have had different and interesting impacts on sperm behaviour under male dominance variation. If seminal fluid contains signals, for example, which sperm react to, then the shift in velocity may well trade against other activity components such as longevity, with changes to behaviour possibly influenced by both signaller and receiver. If, on the other hand, seminal fluid contains nutrients for exogenous metabolism by sperm, then some discussion about sugars/glycoproteins as well as SFPs would be helpful.

Reviewer #2:

This study investigates experimentally: (a) the influence of seminal fluid in patterns of variation in sperm swimming velocity, a key driver of fertilising efficiency, and (b) the extent to which such seminal fluid-driven changes in sperm swimming velocity impact the outcome of sperm competition. The possibility that seminal fluid compounds, primarily accessory gland products, influence the competitive efficiency of an ejaculate, and have largely evolved for their role in sperm competition, has been around for decades but direct evidence in support of this has been lacking. This is not surprising given the technical difficulties involved in testing this idea under controlled experimental conditions (as way of reference, my own research group has been working on these very questions and using similar approaches for years now, and we haven't yet submitted for publication because our data are not yet as clear and as complete as these presented here). The present study of a chinook salmon population presents so far the most convincing piece of evidence that I have seen in support of the notion that seminal fluid drives changes in sperm fertilising efficiency with tangible impact on sperm competition. Importantly, these results also suggests that males are able to strategically change the composition of their seminal fluid in order to respond to changes in the risk of sperm competition associated with rapid changes in social status. Specifically, by presenting males with a double social challenge the study shows that males that lost the first contest (SS + SD) produce faster swimming sperm than males that won the first contest (DD + DS), and that -after controlling for multiple comparisons- the sperm of males that decreased in status over the two challenges (DS) increased in swimming velocity. Given that these changes occurred rapidly (~48hrs) and males were experimentally sperm depleted, it is parsimonious to assume that they are due to seminal fluid changes rather than sperm phenotype. This is then confirmed by a subsequent experiment in which the sperm of a focal male is incubated in the seminal fluid of a competitor male, and the change in the focal male's sperm velocity is then shown to be predicted by the social status of the competitor male. Critically, when the relative sperm swimming velocity of the competitor male is entered in the model, this becomes the significant predictor while status becomes non-significant, suggesting that male status explains changes in the sperm velocity only to the extent to which it predicts the relative swimming velocity of the competitor male's sperm. This demonstrates that the seminal fluid of the competitor male has a causal effect in the swimming velocity of the focal male. Finally, the study performs a series of in vitro competitive fertilisation trials to show that when the sperm of a focal male are exposed to the seminal fluid of his respective rival, changes in fertilisation success are best predicted by the changes in sperm swimming velocity due to exposure to the rival's seminal fluid. Together, these results present a robust case for socially subordinate males compensating through preferential investment in seminal fluid components which increase the fertilising efficiency of their ejaculates, confirming previous suggestions of alternative mating tactics in this and other fish species with similar mating systems. Overall, I think that this is an excellent study. The experimental work is conducted meticulously and impressive in scale. The manuscript is written clearly and with appropriate consideration of previous work. The conclusions drawn are justified and discussed in adequate depth.

Reviewer #3:

This study investigates how male social status (i.e. dominant versus subordinate) affects sperm performance in male chinook salmon. They paired males up and let them establish a hierarchy and collected sperm after 48 hours, male were then re-paired by social status (dominant with dominant and subordinate with subordinate) forcing half of the ales to switch status. The authors tested how seminal fluid affects sperm performance by separating sperm from seminal fluid and re-suspending sperm in the seminal fluid of a different male. They found subordinate males to produce generally faster swimming sperm than dominant males. Furthermore, they show that sperm from dominant males in subordinate seminal fluid became faster and vice versa and that the resulting sperm velocity was a strong predictor of fertilisation success.

While the study is solid and results seem convincing (although more statistics are needed), the manuscript could be improved by tightening the writing style and making the arguments and more concrete. The writing style is often loose and ambivalent, which this is also true for the methods section. In addition, the use of references is not always satisfying as it would be great to see references to original papers rather than reviews for example when introducing the theoretical predictions about sperm competition. In general, the manuscript would benefit from careful revision to make it tighter and more accurate.

We have carefully revised our manuscript in accord with the constructive criticisms received. We are confident that it is now stronger as a consequence of the review process and we thank the Editor and all the reviewers for their insightful feedback.

For one, the authors claim that they have been using a novel experimental approach to disentangle the effect of sperm versus seminal fluid on sperm velocity. However, the same approach has been used in a study by Locatello et al., (2013) published a few years ago showing a similar effect of seminal fluid on sperm performance between males adopting alternative mating tactics in a goby. Furthermore, previous studies showed that sneaker and territorial male ejaculates in a Mediterranean goby differ in the composition and amount of polysaccharides present in the seminal fluid (e.g. Scaggiante et al.,). The authors should mention these studies in the Introduction (and not only in the Discussion) and refer to these previous results when introducing their current study. In other words, a more accurate coverage of the literature in the relevant places would help to put this present study into context.

We have now included a clear explanation of Locatello et al., (2013) and Scaggiante et al., (1999) results (Introduction). We have also changed the description of our experimental approach from “innovative” to “comprehensive” (Introduction) to reflect that while each of the methods used here has been previously implemented singularly or partially in other study systems, we have been able to gain novel insight by using these methods in a comprehensive series of experiments in an ideal study system. We have also expanded our discussion of targeted seminal fluid effects on rival male sperm to refer to some recent relevant research in salmonids on this topic (Discussion).

Furthermore, the authors argue that sperm velocity is affected by a change in composition in the seminal fluid based on the fact that males changing from dominant to subordinate did not produce significantly more sperm over the span of 48 hours. Unfortunately, they performed no test – not even a simple test for polysaccharides or general protein content – to actually verify a change in composition of the seminal fluid. There are several alternative explanations for a change in sperm swimming velocity, such as RNAs injected into sperm through vesicles immediately prior to ejaculate release that alter sperm metabolic pathways to name but one, which should also be discussed.

We agree that a wider discussion of how seminal fluid might influence sperm behaviour strengthens our manuscript. We have expanded our discussion on this topic, addressing the potential for seminal fluid to fuel differential energy metabolism in sperm, and the potential for seminal fluid to contain signalling molecules (either RNAs or peptides) that influence sperm behaviour (Discussion). We have also referred to Scaggiante et al., (1999) and a recent study by Gombar et al., (2017) with respect to variation in SFP composition in males with different fixed reproductive tactics (Discussion). Finally, we adjusted our focus on SFPs at the end of this discussion to a broader statement (Discussion).

Finally, the fact that males experience a rapid turnover in treatments where males are kept in each social status for three days in total needs a bit more attention. The sequence of the social status clearly has an effect on sperm traits as the main effect on VAP was found in males switching from dominant to subordinate. I am therefore wondering how these issues were taken into account when running the statistical analyses. The authors write that they used either two levels (D and S) when analyzing the stages separately or four when analyzing stage 2. This is an ambivalent description and should be clarified; for example, in Table 3 it is not clear how the statistical model looked like and no interaction terms are presented whereas Figure 4 suggests a clear interaction between treatments (DD and the other three). I am generally wondering about the role of interaction terms between the social status during stage 1 and stage 2 as none are presented in the current version of the manuscript.

As stated above for reviewer 2’s first comment, we have clarified the methods used for group comparisons in stage 2 (Materials and methods). We have also clarified the methods used for the statistical analysis of change in sperm velocity and sperm concentration from stage 1 to 2 (Materials and methods). Our original analyses used separate models for each of the four social status groups (DD,SD,DS,SS) and simply compared sperm velocity or concentration between stages, with no ability to test for an interaction effect. We have now included an additional analyses for both sperm velocity and concentration in which a single model is run for each response variable with data for all four social status groups to test for an interaction effect between social group and stage. We found that the results for this additional analysis were equivalent to those from our previous analyses and have included them in the manuscript (Results; Materials and methods). We have also updated the supplementary file to include the R code and output for these new analyses.

In summary, while I can see the value of this study, the general presentation should be improved and the novelty of the findings is overstated.

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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