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

Sperm competition occurs when sperm from more than one male compete for fertilizations. This form of post-copulatory sexual selection is recognized as a significant and widespread force in the evolution of male reproductive biology and as a key determinant of differential male reproductive success. Despite its importance, however, detailed mechanisms of sperm competition at the gamete level remain poorly understood. Here, we use natural variation in spermatozoal traits among wild Atlantic salmon (Salmo salar), a species naturally adapted to sperm competition, to examine how the relative influences of sperm (i) number, (ii) velocity, (iii) longevity, and (iv) total length determine sperm competition success. Atlantic salmon fertilize externally, and we were therefore able to conduct controlled in vitro fertilization competitions while concurrently measuring spermatozoal traits within the aqueous micro-environment to which salmon gametes are naturally adapted. Microsatellite DNA fingerprinting revealed that a male's relative sperm velocity was the primary determinant of sperm competition success. There was no significant relationship between fertilization success and either relative sperm number or total length; sperm longevity showed an inverse relationship with competition success. These relationships were consistent for two experimental repeats of the in vitro fertilization competitions. Our results therefore show, under the natural microenvironment for salmon gametes, that relative sperm velocity is a key spermatozoal component for sperm competition success. Atlantic salmon sperm can be considered to enter a competition analogous to a race in which the fastest sperm have the highest probability of success.     

Fertilisation and sperm competition dynamics in salmon

Fishes have evolved an enormous range of mating patterns and sperm competition is important in the majority of these. Detailed mechanisms of fertilization and sperm competition dynamics at the gamete level are poorly understood. We have been using salmon models to conduct in vitro fertilization trials that: (1) allow us experimental control, (2) eliminate confounding effects from the whole organism, and (3) enable detailed measures of sperm form and function in the natural fertilization medium to which gametes are adapted. Using both Atlantic and sockeye salmon, we present results on experiments exploring: (1) the importance of relative sperm number, size, % motility, longevity, and velocity for fertilization and sperm competition success, (2) the importance of natural variation in egg size for 'fertilizability' under sperm limited conditions and (3) the influence of ovarian fluid in fertilization dynamics.     

Relationships between sperm morphometry and sperm motility in the Atlantic salmon

Relationships between spermatozoal design and swimming behaviour were investigated using the significant natural variance in sperm traits in Atlantic salmon Salmo salar. In vitro motility and fertilization experiments were conducted with 86 Atlantic salmon to measure sperm form and function under natural fertilization conditions. Spermatozoal traits of Atlantic salmon showed narrow variance within individuals but differed extensively between samples: mean sperm length varied from 32·3 to 39·5 μm, mean velocity ranged from 18 to 127 μm s⁻¹, and ejaculate longevity varied from 18 to 78 s. In addition to variation in sperm morphometry between fish, a negative relationship was also found between sperm head length and flagellum length. This natural variation in sperm form and function between males is counter-intuitive since measures are from a single Atlantic salmon population where all males are adapted to a common fertilization environment. No evidence was found that longer sperm, or sperm with longer flagella, achieved faster swimming velocities. Also no evidence was found for a trade-off between mean sperm velocity and ejaculate longevity. There were significant negative associations, however, between sperm total and flagellum length and ejaculate longevity, so that males with longer sperm had shorter-lived gametes. This finding has previously been reported in a study across fish species, supporting the theory that increased hydrostatic forces generated by longer flagella may trade against sperm cell longevity.     

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.     

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

Sockeye salmon Oncorhynchus nerka sperm maintained high fertility for 10 s after being exposed to water but fertility dropped sharply after that. Eggs also showed limited longevity after being immersed in water, with a large drop in fertility occurring after 20 s of exposure to water. The association between eggs and sperm occurs very rapidly. 80% fertilization success was achieved with 5 s of sperm-egg mixing and c. 25% was achieved with <0·5 s of sperm-egg mixing. Sperm from different male age classes differed little in motility and did not differ at all in their propensity to fertilize eggs. The results have important implications for mate choice and sperm competition.     

Sperm competition,but not major histocompatibility divergence,drives differential fertilization success between alternative reproductive tactics in Chinook salmon

Post‐copulatory sexual selection processes, including sperm competition and cryptic female choice (CFC), can operate based on major histocompatibility (MH) genes. We investigated sperm competition between male alternative reproductive tactics [jack (sneaker) and hooknose (guard)] of Chinook salmon (Oncorhynchus tshawytscha). Using a full factorial design, we examined in vitro competitive fertilization success of paired jack and hooknose males at three time points after sperm activation (0, 15 and 60 s) to test for male competition, CFC and time effects on male fertilization success. We also examined egg‐mediated CFC at two MH genes by examining both the relationship between competitive fertilization success and MH divergence as well as inheritance patterns of MH alleles in resulting offspring. We found that jacks sired more offspring than hooknose males at 0 s post‐activation; however, jack fertilization success declined over time post‐activation, suggesting a trade‐off between sperm speed and longevity. Enhanced fertilization success of jacks (presumably via higher sperm quality) may serve to increase sneaker tactic competitiveness relative to dominant hooknose males. We also found evidence of egg‐mediated CFC (i.e. female × male interaction) influencing competitive fertilization success; however, CFC was not acting on the MH genes as we found no relationship between fertilization success and MH II β₁ or MH I α₁ divergence and we found no deviations from Mendelian inheritance of MH alleles in the offspring. Our study provides insight into evolutionary mechanisms influencing variation in male mating success within alternative reproductive tactics, thus underscoring different strategies that males can adopt to attain success.     

Sperm quality in the alternative reproductive tactics of Atlantic salmon: the importance of the loaded raffle mechanism.

The outcome of sperm competition in species with alternative male reproductive strategies may be determined by fair or loaded raffle mechanisms. The sperm production and quality of male Atlantic salmon using alternative reproductive tactics were investigated in order to determine the relative importance of sperm quality for male reproductive success. Sexually mature resident parr males produced greater numbers of spermatozoa per millilitre of ejaculate and invested more in their gonads as a percentage of body mass than their anadromous counterparts. Parr males had greater proportions of motile spermatozoa and a greater sperm ATP content as compared with anadromous males. Parr males invested relatively more in sperm quality and sperm numbers after the effect of body size was accounted for. In fertilization experiments, parr males fertilized greater proportions of eggs than anadromous males. A polynomial model exhibited a trade-off between testes mass and ejaculate expenditure and explained 60% of the variation. These results establish that, in sperm competition with dominant males, parr males may compensate for behavioural subordinance by producing physiologically superior spermatozoa.     

A one‐step rectification of sperm cell targeting ensures the success of double fertilization

Successful fertilization in animals depends on competition among millions of sperm cells, whereas double fertilization in flowering plants usually involves just one pollen tube releasing two immobile sperm cells. It is largely a mystery how the plant sperm cells fuse efficiently with their female targets within an embryo sac. We show that the initial positioning of sperm cells upon discharge from the pollen tube is usually inopportune for gamete fusions and that adjustment of sperm cell targeting occurs through release and re-adhesion of one sperm cell, while the other connected sperm cell remains in stagnation.This enables proper adhesion of each sperm cell to a female gamete and coordinates the gamete fusions. Our findings reveal inner embryo sac dynamics that ensure the reproductive success of flowering plants and suggest a requirement for sperm cell differentiation as the basis of double fertilization.     

Releasing small ejaculates slowly increases per‐gamete fertilization success in an external fertilizer: Galeolaria caespitosa (Polychaeta: Serpulidae)

The idea that male reproductive strategies evolve primarily in response to sperm competition is almost axiomatic in evolutionary biology. However, externally fertilizing species, especially broadcast spawners, represent a large and taxonomically diverse group that have long challenged predictions from sperm competition theory—broadcast spawning males often release sperm slowly, with weak resource‐dependent allocation to ejaculates despite massive investment in gonads. One possible explanation for these counter‐intuitive patterns is that male broadcast spawners experience strong natural selection from the external environment during sperm dispersal. Using a manipulative experiment, we examine how male reproductive success in the absence of sperm competition varies with ejaculate size and rate of sperm release, in the broadcast spawning marine invertebrate Galeolaria caespitosa (Polychaeta: Serpulidae). We find that the benefits of Fast or Slow sperm release depend strongly on ejaculate size, but also that the per‐gamete fertilization rate decreases precipitously with ejaculate size. Overall, these results suggest that, if males can facultatively adjust ejaculate size, they should slowly release small amounts of sperm. Recent theory for broadcast spawners predicts that sperm competition can also select for Slow release rates. Taken together, our results and theory suggest that selection often favours Slow ejaculate release rates whether males experience sperm competition or not.     

Long sperm fertilize more eggs in a bird

Sperm competition, in which the ejaculates of multiple males compete to fertilize a female''s ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch (Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition.     

Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets

The cryopreservation of salmonid sperm is a complex process involving the interplay of many factors. Although cryopreservation protocols can be evaluated through a range of responses at various stages in the process, the number of progeny is the ultimate indicator of success. We compared reproductive success from freezing Atlantic salmon (Salmo salar L.) sperm using the eight combinations of (1) the penetrating cryoprotectants, 10% dimethyl sulfoxide (DMSO) or methanol (MeOH); (2) the nonpenetrating cryoprotectants glucose (0.3 M) or sucrose (0.6 M), and freezing in 0.1 mL pellets or 0.25 mL straws. All cryodiluents were supplemented with 10% (v/v) of hen's egg yolk. Response variables were the percentage and degree of motility of thawed and activated sperm using computer assisted sperm analysis (CASA), and rates of eyed embryos, hatch and egg sac larvae. Growth rates of alevins were assessed to two months post hatch. Atlantic salmon milt cryopreserved in straws had higher spermatozoa motility and fertilization success than milt cryopreserved in pellets (P < 0.05). Type of sugar tested did not significantly affect the response variables. In the MeOH treatment, thawed spermatozoa achieved higher speed and a higher fertilization rate evaluated at the eyed embryo stage than spermatozoa subjected to the DMSO treatment. Higher mortality rate (especially before hatching) of MeOH offspring than DMSO offspring led to equal numbers of progeny for the two treatments from the swimming stage to the end of the study. Moreover, during feeding fish from the MeOH group produced significantly lower weight larvae than the DMSO and control groups. Even so, the weight of the MeOH group was satisfactory. Length and the condition factors did not differ significantly among the larvae groups. Significant positive correlations were found between fertilization success (measured in number of eyed eggs) and both motility (rs = 0.81), and velocity (rs = 0.49). Freezing in straws gave betters results than freezing in pellets for cryopreservation of salmon milt; whereas type of sugar tested (glucose vs sucrose) did not have significant effects. Penetrating cryoprotectants DMSO and MeOH differed in their effect on post-thawed sperm velocity, fertilization rate and mortality rate of progeny, suggesting the need for further research on the influence of these cryoprotectants on frozen sperm and and post-fertilization devopmental processes.     

Does egg competition occur in marine broadcast‐spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.     

Intrinsic post‐ejaculation sperm ageing does not affect offspring fitness in Atlantic salmon

Post‐meiotic sperm ageing, both before ejaculation and after ejaculation, has been shown to negatively affect offspring fitness by lowering the rate of embryonic development, reducing embryonic viability and decreasing offspring condition. These negative effects are thought to be caused by intrinsic factors such as oxidative stress and ATP depletion or extrinsic factors such as temperature and osmosis. Effects of post‐ejaculation sperm ageing on offspring fitness have so far almost exclusively been tested in internal fertilizers. Here, we tested whether intrinsic post‐ejaculation sperm ageing affects offspring performance in an external fertilizer, the Atlantic salmon Salmo salar. We performed in vitro fertilizations with a split‐clutch design where sperm were subjected to four post‐ejaculation ageing treatments. We varied the duration between sperm activation and fertilization while minimizing extrinsic stress factors and tested how this affected offspring fitness. We found no evidence for an effect of our treatments on embryo survival, hatching time, larval standard length, early larval survival or larval growth rate, indicating that intrinsic post‐ejaculation sperm ageing may not occur in Atlantic salmon. One reason may be the short life span of salmon sperm after ejaculation. Whether our findings are true in other external fertilizers with extended sperm activity remains to be tested.     

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

Sperm velocity has been shown to be a major determinant of fertilization success of external fertilizers. Yet, sperm velocity varies both within and between ejaculates and only a small number of fast sperm cells within an ejaculate are likely to have the potential of fertilizing the eggs. Having such fast cells should be of special importance during sperm competition, particularly for subordinate males that may release their sperm later or further away from eggs, than dominants. We examined ejaculates of dominant and subordinate male Arctic charr (Salvelinus alpinus), a species with sperm competition. Yet, rather than examining just average sperm velocity values, the aim was to examine whether the fastest fraction of sperm cell from dominant and subordinate males differed in velocity. While there was no difference in the average sperm velocity between dominant and subordinate males, analysis of the fastest swimming sperm cells show that subordinate males have significantly higher initial sperm velocity than dominant males within the 10, 5 and 1% fastest sperm cells. That is, the difference in sperm velocity between dominant and subordinate charr is most predominant among the fastest sperm cells. In sum, this study emphasizes the importance of studying the fastest sperm cells in the ejaculates, as status-dependent differences in sperm velocity may not be detected using average values.     

Fertilization in landlocked sea lamprey: storage of gametes, optimal sperm: egg ratio, and methods of assessing fertilization success

Ovulated, unfertilized eggs of sea lamprey Petromyzon marinus could be stored for 1 day at 15° C without significant loss of fertilizing ability. After 2 days storage most eggs could still be fertilized. Lamprey semen could be stored up to 1 day. Thereafter, a decrease in sperm fertilizing ability occurred, accompanied with a decrease in sperm motility. Unlike teleost fish, sea lamprey eggs could still be fertilized after 1 h contact with water. This extended time of gamete fertility after release into water may help to account for the reproductive success of this species. Maximal fertilization rates were obtained at a sperm: egg ratio of 50 000, a ratio recommended for studies on fertility of individual males. Assessing fertilization success 3 min after fertilization (at cytoplasmic bleb stage) or 5 h after fertilization (at two–cell embryo) was strongly correlated ( r =0·92 and 0·98) with estimation and fertilization success at hatching. These results offer improvement in artificial fertilization techniques under laboratory conditions and provide new information on the biology of fertilization in sea lamprey.     

Conspecific sperm precedence in two species of tropical sea urchins

Conspecific sperm precedence occurs when females are exposed to sperm from males of multiple species, but preferentially use sperm of a conspecific. Conspecific sperm precedence and its mechanisms have been documented widely in terrestrial species, in which complex female behaviors or reproductive tract morphologies can allow many opportunities for female choice and sperm competition, however, the opportunity for conspecific sperm precedence in free spawning marine invertebrates has been largely ignored. Two sea urchin species, Echinometra oblonga and E. sp. C, have high levels of interspecific fertilization in no-choice lab crosses, but no natural hybrids have been found. We performed competitive fertilization assays to test for conspecific sperm precedence and found that eggs of both species showed a marked preference for conspecific sperm when fertilized with heterospecific sperm mixtures. Strong rejection of heterospecific sperm would not have been predicted from no-choice assays and helps explain the lack of natural hybrids. We also found significant variation in hybridization success among crosses. Conspecific sperm precedence in free spawning invertebrates shows that the simple surfaces of eggs and sperm provide ample opportunity for egg choice and sperm competition even in the absence of intricate behavior or complex reproductive morphologies.     

Sperm traits in farmed and wild Atlantic salmon Salmo salar

Differences in sperm metabolism and morphology between wild and non‐local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.     

Sperm quality as reflected through morphology in salmon alternative life histories.

Male salmon exhibit alternative mating strategies, as both older anadromous adults and precocious juveniles (parr) participate in the spawning of a single female. This study tested the following hypotheses: 1) different intensities of sperm competition may reflect different sperm tail optima; 2) long spermatozoa are superior to short ones, with an associated cost on sperm longevity; and 3) a disfavored role in sperm competition selects for parr investing more in sperm quality. Comparisons included sperm morphological traits, whereas sperm quality was investigated by motility duration observations, measurement of the sperm adenylate system, and fertilization experiments. No evidence of different adaptive sperm dimensions between the male types was found. Positive association between spermatocrit and energy charge was, however, detected. Sperm length parameters correlated positively with ATP, energy charge, and fertilization success, whereas no evidence for an effect of sperm morphology on longevity was found. Male parr had greater spermatocrit than adults and fertilized equal proportions of eggs as adults despite a pronounced numerical subordinance in the fertilization experiments. It is concluded that a long sperm tail and midpiece may be selected to optimize energetic demands under conditions of increased sperm competition intensity.     

Sperm competition, alternative mating tactics and context-dependent fertilization success in the burying beetle, Nicrophorus vespilloides

Fertilization success in sperm competition is often determined by laboratory estimates of the proportion of offspring sired by the first (P1) or second (P2) male that mates. However, inferences from such data about how sexual selection acts on male traits in nature may be misleading if fertilization success depends on the biological context in which it is measured. We used the sterile male technique to examine the paternity of the same male in two mating contexts in the burying beetle, Nicrophorus vespilloides, a species where males have alternative mating strategies based on the presence or absence of resources. We found no congruence in the paternity achieved by a given male when mating under different social conditions. P2 estimates were extremely variable under both conditions. Body size was unrelated to success in sperm competition away from a carcass but, most probably through pre-copulatory male-male competition, influenced fertilization success on a carcass. The contribution of sperm competition is therefore dependent on the conditions under which it is measured. We discuss our findings in relation to sperm competition theory and highlight the need to consider biological context in order to link copulation and fertilization success for competing males.     

THE QUICK AND THE DEAD? SPERM COMPETITION AND SEXUAL CONFLICT IN SEA

Our view of sperm competition is largely shaped by game-theoretic models based on external fertilizers. External fertilization is of particular interest as it is the ancestral mode of reproduction and as such, relevant to the evolution and maintenance of anisogamy (i.e., large eggs and tiny, numerous sperm). Current game-theoretic models have been invaluable in generating predictions of male responses to sperm competition in a range of internal fertilizers but these models are less relevant to marine broadcast spawners, the most common and archetypal external fertilizers. Broadcast spawners typically have incomplete fertilization due to sperm limitation and/or polyspermy (too many sperm), but the effects of incomplete (<100% fertilization rates) fertilization on game-theoretic predictions are unclear particular with regards to polyspermy. We show that incorporating the effects of sperm concentration on fertilization success changes the predictions of a classic game-theoretic model, dramatically reversing the relationship between sperm competition and the evolutionarily stable sperm release strategy. Furthermore, our results suggest that male and female broadcast spawners are likely to be in conflict at both ends of the sperm environment continuum rather than only in conditions of excess sperm as previously thought. Across the majority of the parameter space we explored, males release either too little to too much sperm for females to achieve complete fertilization. This conflict could result in a coevolutionary race that may have led to the evolution of internal fertilization in marine organisms.