Variation pattern of sperm quality traits in two gobies with alternative mating tactics


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  • 1Theoretical models predict that ejaculate expenditure will positively covary with the level of sperm competition but trade-offs between either different measures of sperm quality or sperm quality traits and sperm number are expected. However, empirical results have generally failed to find trade-offs between sperm number, size, velocity and longevity or viability.
  • 2We analyzed the sperm traits of the grass goby, Zosterisessor ophiocephalus, and the black goby, Gobius niger, two fish characterized by alternative male reproductive tactics, long lasting egg deposition and intra-specific variability in ejaculate characteristics. Indeed, in both species sneaker males release ejaculates with greater sperm number and lower seminal fluid content than territorial ones.
  • 3We showed that sperm of grass goby males have a similar quality, whereas in the black goby sneakers have faster swimming speeds, greater viability and higher ATP content than those of territorial males. The effectiveness of territorial male mate guarding differs in the two goby species and might account for the observed difference in the intra-specific variability of sperm quality.
  • 4The pattern of variation that we found in the investment of sperm numbers and sperm quality in goby territorial and sneaker males, supports the results found in other species of fish with alternative mating tactics, suggesting that ejaculate quality traits are usually not traded off one against the other when ejaculate effort is increased in response to increased levels of sperm competition.


Sperm competition, occurring whenever the sperm from different males compete for access to the same eggs (Parker 1970), is a widespread and powerful evolutionary force shaping male behaviour, morphology and physiology (Birkhead & Møller 1998; Birkhead & Pizzari 2002). An almost universal response to sperm competition in males is an increase in testis investment and in the number of sperm released at mating (Birkhead & Møller 1998). The strongest evidence of this comes from comparative studies (e.g. on insects, fish, birds, mammals) and from species with alternative male mating tactics in which dominant males often have smaller testes and lower sperm reserves than sneakers (Birkhead & Møller 1998; Simmons 2001; Birkhead & Pizzari 2002). However, the scenario is more complicated than just variation in testis size and sperm number. Indeed, according to theoretical models, the whole ejaculate expenditure, defined as the product of sperm number and any ejaculate property that is related to quality, is expected to covary with the level of sperm competition (Ball & Parker 1996). Empirical studies have recently shown that, in addition to sperm number, sperm quality traits, such as size, velocity, motility and longevity, are important in fertilization performances and hence play a substantial role in paternity contests (Vladić & Järvi 2001; Snook 2005). Sperm quality traits appear to vary among species, but how they vary intra-specifically in response to different levels of sperm competition is just beginning to be investigated (Snook 2005; Locatello et al. 2006; Minoretti & Baur 2006; Rudolfsen et al. 2006; Stoltz & Neff 2006).

In promiscuous species, ejaculate functions as an integrated unit both to ensure fertilization and to maximize the paternity of a male by out-competing sperm from other males (Birkhead & Møller 1998) or by manipulating female reproduction (Peng et al. 2005). Investment in sperm number must be maintained at some minimum requirement to secure fertility but, with limited resources available to reproduction, trade-offs are expected, either between different measures of sperm quality or sperm quality and number (Parker 1993; Ball & Parker 1996; Moore et al. 2004). However, empirical evidence has generally failed to support the expected trade-off between different ejaculate traits, such as sperm number, length, speed and longevity (see Snook 2005 for a review).

Fish with alternative male mating tactics have been a favoured subject for investigating how sperm traits vary in response to different sperm competition levels. By contrast to theoretical expectations, most studies have found that males respond to higher sperm competition by increasing sperm number without decreasing sperm quality or, in some instances, even by increasing it (de Fraipont et al. 1993; Gage et al. 1995; Uglem et al. 2001; Vladić & Järvi 2001; Rudolfsen et al. 2006, but see also Burness et al. 2004). This discrepancy between theory and empirical evidence could be due to several factors. First, in most studies only one or two sperm quality traits have been analyzed (de Fraipont et al. 1993; Uglem et al. 2001; Rudolfsen et al. 2006) and the trade-off may involve other, unconsidered, sperm quality traits. Second, in external fertilizers, in which fertilization occurs rapidly following gametes’ release and most of the eggs are fertilized before sperm die, the expected trade-off between sperm longevity and sperm velocity (Ball & Parker 1996) may be obscured. By contrast, this trade-off may become apparent when egg deposition lasts for a long time and fertilization can occur at any time during female spawning. Third, there is increasing evidence that ejaculate performance can be influenced by seminal fluid characteristics (e.g. Scaggiante et al. 1999; Rasotto & Mazzoldi 2002), and a trade-off might also occur at this level. A better comprehension of the evolutionary trajectories of the ejaculate phenotypes associated with increasing sperm competition therefore requires consideration of all traits that can potentially contribute to the overall quality of the ejaculate.

Two gobiid species, the grass goby Zosterisessor ophiocephalus (Pallas) and the black goby Gobius niger L., with long lasting egg deposition, alternative male mating tactics, long living sperm and intra-specific variability in seminal fluid content (Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002), provide excellent models to study the pattern of covariance of sperm quality traits in response to sperm competition level. In both species, large territorial males monopolize females, defend a nest and provide egg parental care, while smaller and younger males attempt to parasitize parental male spawning. Territorial males lay sperm trails, bands of mucins that slowly release active sperm into the water. The duration of a sperm trail is positively correlated with its mucin content and can last several hours (Scaggiante et al. 1999; Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002). Sneaker males produce sperm trails poorer in mucins and richer in sperm than those of territorial males (Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002). Thus, while territorial male trails release a low but steady supply of sperm for hours, sneakers’ sperm can outnumber those of territorial males (on an average 5·2 : 1 in the grass goby and 10·4 : 1 in the black goby), even if it is only for a short time (Scaggiante et al. 1999; Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002). However, whether or not the greater investment in sperm number represents a trade-off with other sperm quality traits is not known. Here, we examined differences in sperm size, velocity, viability and ATP content between territorial and sneaker males.

Materials and methods

sampling and handling

Males of both species were collected in the Venetian Lagoon during their breeding season and kept separate in stock tanks with artificial light (14 L : 10 D), daily change of water (20 °C) and fresh feeding. Before the analyses, each male was anaesthetized in a water solution of MS 222, the standard length (SL: distance between the snout and the base of the tail) measured and the male assigned to parental or sneaker category, based on the development of secondary sexual traits and the characteristics of sperm trails, as previously described (Mazzoldi et al. 2000; Mazzoldi & Rasotto 2002). Ejaculate was obtained through a gentle pressure on the abdomen of the male. All males were used for a single trial and then released in their natural habitat. None of them died as a consequence of the treatment.

sperm viability

Sperm viability was measured as the proportion of sperm live at stripping, and 1 and 3 h later using the Eosin-Y staining test (Lin et al. 1998). Sperm were stripped from 26 black goby males (territorials: SL range = 7·5–11·3 cm, n = 13; sneakers: SL range = 4·9–6·8 cm, n = 13) and from 30 grass goby males (territorials: SL range = 4·2–17·0 cm, n = 15; sneakers: SL range = 6·0–11·1 cm, n = 15). Each sample was immediately diluted in 100 µL of filtered sea water and centrifuged at 18 000g for 3 min to remove the supernatant containing the mucins in which the sperm are embedded. Sperm cells were then re-suspended in filtered sea water and this solution was maintained at 20 °C until all the measures were performed. Sperm counts were performed by mixing, on a microscope slide, 5 µL of the sample solution with 5 µL of Eosin-Y stain (0·5% wt/vol). The stain penetrates the plasma membrane of dead cells which appear pink, whereas live cells are colourless. After 2 min, the slide was covered with a coverslip and examined under a light microscope (×1000 magnification with oil immersion). The dilution resulted in c. 20 sperm per visual field. For each male, two slides were prepared, using the above described method, three times, that is, at stripping, 1 and 3 h later. The proportion of live sperm was calculated per 100 sperm per slide, using the two slides for each male and the mean value was used for the analyses.

sperm velocity and length

The analyses were performed on 24 black gobies (territorials: SL range = 8·9–11·6 cm, n = 12; sneakers: SL range = 5·3–6·7 cm, n = 12) and on 28 grass goby males (territorials: SL range = 12·8–18·5 cm, n = 14; sneakers: SL range = 6·8–10·2 cm, n = 14). The ejaculate was diluted in 100 µL of an extender inactivating medium (3·5 g L−1 NaCl, 0·11 g L−1 KCl, 0·39 g L−1 CaCl2, 1·23 g L−1 MgCl2, 1·68 g L−1 NaHCO3, glucose 0·08 g L−1, pH 7·7; Fauvel 1999), centrifuged (see above), re-suspended in the inactivating medium and maintained at 3 °C –5 °C until analysis. Sperm were activated by adding filtered sea water at 24 °C  ± 1 °C containing 2 mg/mL of BSA. Sperm velocity was measured with an IVOS Sperm Tracker (Hamilton Thorne Research, Beverly, MA), immediately after activation and again 30 min later. Mean speed measurements were based on 91 ± 17·1 SE (black goby) and 111 ± 21·5 SE (grass goby) sperm tracks per sample immediately after activation and on 60 ± 13·5 SE (black goby) and 70 ± 15·2 SE (grass goby) sperm tracks per sample after 30 min. We measured average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL), which are the progressive velocity measures that usually correlate with fertilization rate in fish (Rurangwa et al. 2004). The threshold values defining static cells were predetermined at 20 µm/s for VAP and VCL and 15 µm/s for VSL. Sperm concentration (sperm/field) was 37 ± 6·1 SE and 47 ± 4·9 SE in black and grass gobies, respectively, and did not correlate with any of the sperm speed measurements (all r < 0·05, all P > 0·7, Pearson correlation coefficient). The sperm speed measure was repeated on two grass goby subsamples (for which the ejaculate stripped was sufficient), yielding repeatability values of 0·87, 0·82 and 0·72 for VAP, VSL and VCL, respectively.

Tail length and total sperm length were measured (15 sperm from each male) on digital images taken at 1000× magnification using Image Tool (black goby: 16 territorials, SL range = 7·5–12·2 cm, 18 sneakers, SL range = 4·9–6·8 cm; grass goby: 21 territorials, SL range = 13·2–17·2 cm, 26 sneakers, SL range = 6·0–8·2 cm).

atp measurement

The ATP content of sperm was determined by a luminometric method (Hampp 1985) with a LKB Wallac 1250 luminometer on 16 black gobies (territorials: SL range = 9·0–11·0 cm, n = 6; sneakers: SL range = 5·4–6·3 cm, n = 9) and on 22 grass gobies (territorials: SL range = 13·3–17·8 cm, n = 11; sneakers: SL range = 7·2–9·9 cm, n = 11). ATP monitoring reagents (luciferase) and ATP standards were provided by Bioluminescence Assay kit CLS II (Roche Diagnostics). Analyses were repeated on three subsamples per male ejaculate and the three ATP content measures (picomoles of ATP per 106 sperm) were averaged. Within-male repeatability was 0·85 and 0·99 for the black and the grass goby, respectively.

statistical analysis

Statistical tests were performed using statistica 7·0. Data were tested for normality using the Kolmogorov–Smirnov test and proportions were arcsine transformed prior to analysis. Data on sperm viability and velocity obtained at different times were analyzed using a repeated measure anova (Sokal & Rohlf 1997).


In the black goby the proportion of live sperm was significantly different between sneaker and territorial males, and decreased significantly over time for both type of males (repeated measures anova: male type F1,24 = 16·13, P < 0·001; time F2,48 = 224·77, P < 0·001; male type × time F2,48 = 6·39, P < 0·01), but more rapidly in territorial. Indeed the proportion of live sperm was lower in territorial males than in sneakers both 1 and 3 h after stripping (Newman–Keuls post-hoc test: P = 0·002 and P = 0·012 for 1 and 3 h, respectively) (Fig. 1). In the grass goby, the proportion of live sperm decreased with time but did not differ between male types (repeated measures anova: male type F1,28 = 0·05, P = 0·82; time F2,56 = 135·42, P < 0·001; male type × time F2,56 = 0·12, P = 0·89) (Fig. 1).

Figure 1.

 Mean ( ± SE) percentage of alive sperm in territorial (○) and sneaker (•) males of (a) G. niger and (b) Z. ophiocephalus immediately after stripping, 1 and 3 h later.

Black goby sneaker sperm proved faster than that of territorial males immediately after activation (Table 1; Newman–Keuls post-hoc test: VAP time 0/sneaker × VAP time 0/territorial, P = 0·004; VSL time 0/sneaker × VSL time 0/territorial, P = 0·001; VCL time 0/sneaker × VCL time 0/territorial, P = 0·006). However, sperm speed declined at 30 min, when sperm speed no longer differed between male types (Newman–Keuls post-hoc test: in all comparisons P > 0·05) (Fig. 2). Sperm speed in the grass goby also declined significantly over 30 min, but we did not find any significant difference between male types (Table 1; Fig. 2).

Table 1.  Results of repeated measures anova on sperm velocity parameters of sneaker and territorial males of G. niger and Z. ophiocephalus. Significant values are in bold
Gobius nigerVAPVSLVCL
Between subjects
  Male type 1 3620·1 7·98< 0·001 12178·9112·15< 0·01 1 4030·6 5·81< 0·05
  Error22  453·5  22 179·32  22  694  
Within subjects
  Time 115912·364·96< 0·001 14049·8537·93< 0·001 113771·941·65< 0·001
  Male type × time 1 1318·3 5·38< 0·05 1 725·41 6·79< 0·05 1  811·4 2·45    0·13
  Error22  245  22 106·77  22  330·6  
Zosterisessor ophiocephalusd.f.MSFPd.f.MSFPd.f.MSFP
Between subjects
  Male type 1  122·6 0·140·71 1 162·67 0·640·43 1   12·6 0·010·92
  Error26  453·5  26 253·1  26 1111·4  
Within subjects
  Time 112161·477·79< 0·001 13480·6551·1< 0·001 112721·875·21< 0·001
  Male type × time 1  117·7 0·750·39 1  24·42 0·360·55 1  136·0 0·800·38
  Error26  156·3  26  68·11  26  169·1  
Figure 2.

 Mean ( ± SE) sperm velocity in territorial (○) and sneaker (•) males of G. niger and Z. ophiocephalus immediately after activation and 30 min later. (a) Average path velocity; (b) straight line velocity; (c) curvilinear velocity.

Sperm length (mean ±  SE: black goby, total sperm length = 31·43 ± 0·19 µm, tail length = 27·33 ±  0·17 µm; grass goby, total sperm length = 32·9 ± 0·22 µm, tail length = 29·0 ± 0·29 µm) did not differ between male types (black goby: total length, t = 1·37, d.f. = 32, P = 0·18, tail length t = 1·86, d.f. = 32, P = 0·07; grass goby: total length t = 0·75, d.f. = 45, P = 0·46; tail length t = 0·62, d.f. = 45, P = 0·54). Sperm length did not correlate with any parameter of sperm velocity either in the black or in the grass goby (all r < 0·55, all P > 0·1 N = 26, Pearson correlation coefficient).

Black goby sneaker males’ sperm had a significantly higher ATP content than the territorial males’ perm (t-test: t = 2·69, d.f. = 13, P < 0·05). By contrast, we found no difference in the ATP content between the two male tactics in the grass goby (t-test: t = 0·022, d.f. = 20, P = 0·98) (Fig. 3).

Figure 3.

Mean ( ± SE) sperm ATP content in territorial and sneaker males of (a) G. niger and (b) Z. ophiocephalus.


Our results show that grass goby males adopting different mating tactics did not differ in their sperm quality investment, whereas black goby sneakers have sperm with higher ATP content (about twice that of territorial males), and greater speed and viability than the sperm of territorial males. We can therefore conclude that, in these two gobiids, the increased production of sperm associated with sneaking tactics does not occur at the expense of any of the sperm quality traits measured. Our results are in agreement with most studies on species with male alternative mating tactics (de Fraipont et al. 1993; Gage et al. 1995; Uglem et al. 2001; Vladić & Järvi 2001, but see also Burness et al. 2004). Thus, if a general pattern can be derived from the results obtained so far it is that sneaker males respond to increased sperm competition by increasing both their sperm numbers and their sperm quality (or, at least, by increasing the number of sperm without reducing their quality), in line with the pattern observed at an inter-specific level (Stockley et al. 1997; Snook 2005).

In the sperm competition game an increased sperm longevity (potentially traded off with sperm speed) may be favourable when there is a continuous introduction of eggs, or when the timing of sperm release differs between the two morphs (Ball & Parker 1996; Taborsky 1998; Uglem et al. 2001; Snook 2005). Even though gamete release is asynchronous, egg release is long lasting and eggs remain fertilizable for several hours in both the grass goby and the black goby (Mazzoldi 1999; Scaggiante et al. 1999), an intra-specific difference in sperm viability is present only in the black goby. In both species, the timing and coordination of gamete release might have selected for great absolute sperm longevity, compared to other fish with external fertilization (Stockley et al. 1997), whereas the intra-specific variability may be explained by considering that sneaking attempts observed in the field are higher and more prolonged in time in grass goby than in black goby (Mazzoldi 1999; Mazzoldi & Rasotto 2002). The grass goby nest, a muddy chamber dug under sea grass rhizomes, has multiple entrances, enabling sneaker males to overcome territorial male guard, to remain inside the nest, where the level of water mixing is low and the dilution of sperm is slow, and to release sperm in close proximity to the female (Mazzoldi et al. 2000). By contrast, black goby territorial males, nesting in rocky caves or under stones, may constantly control the single nest entrance, forcing sneakers to release sperm at a greater distance from the females entailing a rapid sperm dilution (Mazzoldi 1999). Indeed, absolute sperm longevity is higher and absolute sperm number is lower in the grass goby than in the black goby, as expected if the latter's sperm are dispersed more rapidly (Scaggiante et al. 1999; Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002).

When mate guarding is effective, territorial males are predicted to reduce their allocation to sperm production, whereas when mate guarding is less effective territorial males are expected to invest more in sperm production (as in the grass goby, Alonzo & Warner 2000). However, territorial males of both these goby species respond to the presence of sneakers only by increasing mate-guarding and not by adjusting their ejaculate effort (Pilastro et al. 2002; Scaggiante et al. 2005), even though territorial black gobies are probably more effective in mate guarding than territorial grass gobies. When shifting from sneaky to territorial tactics (Mazzoldi et al. 2000; Rasotto & Mazzoldi 2002; Immler et al. 2004; Scaggiante et al. 2004), black goby males might successfully invest in traits favouring mate acquisition at the expense of both sperm number and quality, whereas grass goby territorial males, despite reducing their sperm number, might be constrained to maintain sperm quality at the same level as in sneakers by the lower effectiveness of mate guarding.

The deviation of our results from theoretical predictions that sperm quality traits should be traded off at the intra-specific level (Parker 1993; Ball & Parker 1996) could have several explanations. First, the models assume a constant overall allocation to post-copulatory traits (Parker 1993; Ball & Parker 1996), which is obviously not the case in most species with alternative mating tactics in which sneaker males usually have larger testes than dominant, territorial ones (Taborsky 1998). Second, the machinery controlling sperm quality may not be flexible enough to respond to varying levels of sperm competition, forcing males to increase (or decrease) their sperm production at constant sperm quality. This seems to be the case at least for sperm morphology, which shows little intra-specific variation. However, this likely does not apply to the other traits (sperm speed, viability and ATP content), considering that sneaker black gobies do vary both sperm quality and number, even if in opposing directions to those predicted by theory. Third, when both tactics face a high level of sperm competition (as in the case of the two gobies studied here and probably of most fishes with alternative mating tactics), selection for high sperm quality should be similar in both type of males, or even stronger in sneaker males. If a decrease in sperm quality drastically decreases fertilization success, we expect sperm quality not to vary too much among males. Thus, sneaker males, which probably have greater overall investment in post-copulatory traits than do territorial males, are able to invest in both sperm number and quality.

In conclusion, the pattern of co-variation we found in investment in sperm numbers and sperm quality in the two types of male gobies (territorial and sneakers), parallels the results found in most species of fish with alternative mating tactics (Gage et al. 1995; Uglem et al. 2001; Vladić & Järvi 2001; Rudolfsen et al. 2006), and suggests that ejaculate quality traits are usually not traded off one against the other when ejaculate effort is increased in response to increased levels of sperm competition. By contrast, it appears evident that increased investment in pre-copulatory traits (i.e. dominance and/or mate guarding) is often accompanied by a reduction of sperm production and quality, as has been reported in other vertebrates (e.g. Froman et al. 2002; Pizzari et al. 2002; but see Koyama & Kamimura 1999). It must be noted, however, that grass and black goby males release sperm trails in which sperm number and mucin content are negatively correlated (Scaggiante et al. 1999; Rasotto & Mazzoldi 2002). The function of mucins in territorial males’ ejaculate is to allow a constant sperm release while the territorial male guards the female and patrols the nest. Little is known about the relative cost of producing mucins compared to the cost of producing free-swimming sperm, and it is therefore difficult to say whether sperm and ejaculate fluids are really traded off or if a mucin-rich ejaculate is just cheaper and/or allows sperm trail to perform more advantageously for territorial males, such as exhibiting an higher viability (Scaggiante et al. 1999). Certainly, the importance of seminal fluid in the context of post-copulatory sexual selection has been shown in other taxa (Simmons 2001; Ramm et al. 2005) and the role that seminal fluids play in the sperm competition game certainly merits further investigation.


We thank Dr Giuseppe Tambuscio and the Dipartimento di Scienze Ginecologiche e della Riproduzione Umana for access to the sperm tracker and for technical assistance and advice. This study was carried out in accordance with current Italian regulations for the use of animals in scientific procedures.