Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 66, 98-105 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Sperm Quality as Reflected Through Morphology in Salmon Alternative Life Histories1

Tomislav V. Vladic2,a,b, Björn A. Afzeliusc, and Gennady E. Bronnikov3,c

a Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden b Institute of Freshwater Research, National Board of Fisheries, SE-178 93 Drottningholm, Sweden c Arrhenius Laboratories F3, SE-106 91 Stockholm, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

behavior, fertilization, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertilization in the Atlantic salmon (Salmo salar L.) is external, and the spermatozoa have an aerobic metabolism [1]. Sperm movement results from the viscous interactions of sperm flagella with the medium [2]. Gray and Hancock [3] have shown that the viscous drag of the sperm head is small relative to the viscous drag of the flagellum itself. Thus, the sperm head is not involved in locomotion. The main portion of the sperm flagellum has a "9+2" microtubular structure: the axoneme. Dyneins, or axonemal molecular motors, are large ATPases that interact with microtubules to produce mechanical work (reviewed in [4, 5]). The ATP is used by the dynein arms of the axoneme as a source of energy for sperm motility [6]. Because dynein arms produce a self-oscillatory bending behavior of the flagellar axoneme [7], the sperm flagellum is decisive for sperm fertilizing capacity. Therefore, size of the sperm flagellum is expected to confer a fitness advantage to a male during competition between rival ejaculates to fertilize a female's eggs (i.e., "sperm competition" [8]).

The role of sperm competition in the evolution of great diversity in sperm morphology within a single ejaculate is a strongly debated issue [9–11]. The sperm competition theory predicts that increased sperm numbers should lead to a reduction in sperm size if the resources allocated to sperm production are fixed [12]. However, if a greater flagellar length confers an adaptive advantage in terms of sperm competitiveness at the expense of survival, then selection should favor a greater sperm length [13]. It was suggested that sperm competition selects for longer spermatozoa in mammals, thereby influencing sperm survival through the effect of the associated energetic costs of a long flagellum that provides an increased propulsive force [13, 14]. Available evidence from interspecific studies in fish suggests that longer spermatozoa have a shorter life span as the sperm competition intensity increases across fish species [15]. This cost of sperm length may, therefore, affect the viability of the spermatozoa surrounding the eggs in fishes, with the consequence that some of the eggs are unfertilized at the time when all sperm become immotile (i.e., "adaptive infertility" [16]). It is generally predicted that, during the fertilization process, an increased number of sperm in the ejaculate will yield a greater chance for success in a scramble competition [12].

Dimorphism of spermatozoa from one ejaculate has been reported in several animal species [17]. An adaptive explanation for this phenomenon was suggested in the mechanism of functional partitioning of spermatozoa within an ejaculate, or the so-called "Kamikaze sperm hypothesis" of Baker and Bellis [9]. Those authors suggest that only a small proportion of spermatozoa within an ejaculate are capable of fertilizing an egg, whereas a majority of sperm from the ejaculate function to incapacitate rival spermatozoa. It is suggested that nonfertilizing "soldier" spermatozoa are more likely to be smaller than the reproductive sperm [18].

A condition of "the loaded raffle" [19] applies to situations in which the competitor's spermatozoa do not count equally because of an apparent asymmetry in the role that a given male has in mating. Atlantic salmon exhibit alternative male reproductive behaviors: courting and sneaking. The latter tactic is exhibited by the precociously maturing juveniles (parr), whereas the former is exhibited by older and bigger, anadromous adults. The two strategies are conditional [20, 21], with a contributing genetic component [22]. Fish that have better growing conditions during the juvenile period have a higher probability to mature precociously next season and, thereby, to become sneaker opportunists into the spawning of anadromous fish. Older adult males are anadromous, and a greater amount of food in the sea is used for prolonged body growth, making these males late-maturing at a large size (up to 90 cm) [23]. They behave as typical guarders of the female at the spawning ground. Parr males are relatively much smaller than the anadromous males (the difference in body size may be greater than 99%) and are unattractive to females, which are features that make them typical sneakers in the spawning of the adult fish [24]. Because of this size disparity, salmon is a particularly suitable species for studies of the sperm competition theory. Ejaculate characteristics and sperm morphology may possibly be affected by the intensity of sperm competition at the salmon spawning ground, because sperm production and motility are reportedly enhanced in Atlantic salmon parr relative to the anadromous males [24]. The loaded raffle that occurs in species with different male mating strategies could give rise to the evolution of different sperm size optima that correspond to spawning conditions in the competing male tactics [25]. Although a study on sperm dimensions in alternative male reproductive strategies has been performed [24], no attempts have been made in these male phenotypes to elucidate the possible interdependence between sperm morphological parameters and adenine nucleotide amount, on which the quality of sperm cells depends.

Based on this theoretical background, we investigated three hypotheses. First, that alternative mating strategies express different sperm size optima, which are tuned by the intensity of sperm competition that the two alternative male tactics experience [25]. Second, that long spermatozoa are superior to short spermatozoa, with an associated cost on sperm longevity [13, 16, 26]. Third, that because of their behaviorally disfavored role in mating, parr males are selected to produce relatively greater densities of high-quality spermatozoa than their anadromous counterparts [24]. To test these hypotheses, we examined the size of the sperm flagellum (i.e., tail length including the terminal end piece) and of the adjacent midpiece as well as the sperm production and fertility in anadromous and parr males.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of Fish and Collection of Semen

At the peak of the spawning season in the river Dalälven, between 4 and 19 November 1997, eight sexually mature anadromous males and eight females were caught at the spawning run, and six precocious juveniles (parr) were obtained from the National Board of Fisheries hatchery at Älvkarleby, Sweden. Anadromous adults were sea-ranched fish that were caught in an underwater trap by the hydroelectric power plant-dam in Älvkarleby when migrating upstream to spawn. Precocious parr were 2-yr-old, hatchery-reared males that were held under the natural photoperiod. These precocious males belonged to the stock used for artificial propagation of the salmon population in the river after the hydroelectric power plant cut off the natural spawning routes for returning salmonids in 1915. Anadromous males were absolutely bigger than precocious parr (Kruskal-Wallis ANOVA, H = 9.60, P < 0.01; anadromous, µ = 4837.5 ± 1570.2 g [mean ± SD]; parr, µ = 90.2 ± 18.6 g). The fish were stripped for milt by pressuring the abdomen of the fish under tricaine methane sulfonate, MS-222 (Thomson and Joseph Ltd., Norwich, U.K.) anesthesia. Care was taken to avoid contamination of the milt with urine or feces. Semen samples were stored at 1–2°C for 3 h before the fertilization experiments and fixation. The number of sperm cells (i.e., spermatocrit) in the ejaculate was determined as the percentage of sperm cells per milliliter of fluid by centrifuging undiluted milt samples for 5 min in a microhematocrit centrifuge (2000 x g) and by reading sperm cell density against a calibrated scale. Spermatocrit has been assessed as a reliable and sufficient method for estimating sperm density in Atlantic salmon ejaculates [27].

Electron Microscopy

Aliquots of spermatozoa were fixed in 2.5% (v/v) glutaraldehyde in a 0.05 M phosphate buffer. Samples were deposited on 300-mesh copper grids covered with Formvar (Agar, Stansted, U.K.) support films and examined with a JEOL 100 S electron microscope (Tokyo, Japan) at 1000x magnification. In total, 141 adult and 91 parr spermatozoa were measured. The size of the sperm midpiece and the terminal end piece of the sperm tail were measured in addition to the sperm tail length. Mean values of the sperm parameters for each individual were used, so that individual males were used as a level of independence. Sperm morphological traits were separately measured by one investigator (T.V.) without knowledge of the individual's strategy. The Leica Q500IW (Cambridge, U.K.) software was used for sperm morphological measurements.

Estimates of Sperm Quality

The longevity of sperm motility was determined by microscopic observations under the Nikon phase-contrast microscope (Tokyo, Japan) at 100x magnification in triplicates within 3 h after milt collection. No cover-slide glass was applied. The motility was initiated by suspending an ejaculate drop in the motility activation buffer (20 mM Tris-HCl [pH 8.0] and 125 mM NaCl) [28] in a dilution ratio of 1:500 at 4°C. This temperature was previously determined as being optimal for Atlantic salmon sperm motility [29]. Sperm longevity was defined as the time period until all spermatozoa in the sample observed under the microscope had stopped the forward movements characteristic of motile spermatozoa. Simply vibrating spermatozoa beginning the end of propulsive sperm motility were assessed as being immotile. Zero sperm motility in a given ejaculate commenced after the last spermatozoon in the visual field had started to vibrate and the other spermatozoa were immotile. Duration of sperm motility was assessed by using a stopwatch, starting at contact of semen with the river water in the Bürker chamber. The mean of three measurements was used to assign each individual's sperm longevity.

The ATP-ADP cycle is the fundamental mode of energy exchange in biological systems, and ATP is the main source of chemical energy for sperm motility [1]. The enzyme adenylate kinase (also known as myokinase), which is broadly present in all cell types, catalyzes the reaction

indicating that the three adenosine phosphates are interconvertible. Therefore, the effective mole fraction of ATP that describes the "metabolic energy-state" of the cell was employed and defined as described by Atkinson [30]:

The energy charge value varies between zero and one, where the value of zero represents all adenylate as AMP and the value of one represents all adenylate as ATP. Sperm quality was determined by measuring these adenosine phosphates. The adenosine phosphate assay was conducted by the luminometric method as described by Hampp [31] using the firefly luciferin-luciferase system at pH 7.75. Phosphoenolpyruvate and pyruvate kinase for ADP determination and myokinase for AMP determination were obtained from Sigma-Aldrich AB (Steinheim, Germany). The ATP monitoring reagent, containing firefly luciferase and D-luciferin, and ATP standard were obtained from Bio-Orbit (Turku, Finland).

Samples of stripped semen were fixed with 6% trichloracetic acid (TCA) and then immediately frozen. Before measurement, the samples were thawed, centrifuged at 11 600 x g for 5 min, and diluted with Hepes buffer (pH 7.75) at a ratio of 1:100. Each day before measuring adenosine nucleotide concentrations from the biogenic samples, a calibration of the luminometer was performed. The calibration curve never deviated from the linearity for more than 1% after subsequent addition of arithmetically increasing quantities of the ATP standard solution (3, 6, 9, and 12 µl). After measurement of light emission from the sample, an internal ATP standard (10 µl) was added after 10 sec. In calculation of the sample nucleotides, the following expression was used:

where A represents the light emission recorded from the sample before addition of the internal standard, B represents the light emission after addition of the internal standard, and C represents the amount of ATP added (10-12 mole). Concentrations of ADP and AMP were measured after their conversion to ATP [31]. The TCA extracts of sperm specimens diluted with Hepes buffer were preincubated either with phosphoenolpyruvate and pyruvate kinase only or additionally with myokinase to convert ADP and AMP, respectively, to ATP. During calculation of ADP and AMP concentrations, the light signal resulting from the ATP concentration for ADP signal or from the ATP plus ADP concentration for the AMP signal were extracted, respectively. All measurements were performed at room temperature in duplicates, and the mean value of the two measurements was used.

Sperm Viability Experiments

Sperm viability was tested in the laboratory by employing fertilization experiments. Six parr and eight adult male ejaculates were used to fertilize batches consisting of 50 eggs from the eight females in a fertilization channel (length, 18 cm; width, 10 cm) under a 0.2 m/sec flow of river water at 4–5°C. Eggs were positioned at the proximate end of the spawning channel, and ejaculates were applied by a laboratory pipette at the rear end of the spawning channel. Sperm quantity was calculated as the product of the spermatocrit and ejaculate volume applied during the fertilization experiment and compared by paired t-test for dependent samples. To mimic a natural disparity in the ejaculate size between males, the following expression was used in calculating the proportion of ejaculate volume applied by the pipette (volume, 20–1000 µl):

where V(x) is the volume of a given individual ejaculate applied in the experiment, SES(x) is the stripped milt weight from an individual (g), and sperm(ref) is the spermatocrit value of the reference parr individual (63%). Consequently, parr had approximately only 20% of the sperm quantity of the adults in the fertilization test (anadromous, µ = 52670.8 ± 28154.3; parr, µ = 10669.2 ± 5285.5; t = -3.65; df = 13; P < 0.01). Ten minutes after the fertilization experiment, the eggs were collected, carefully rinsed with Buffodine (Evans Vanodine Int. PLS, Preston, U.K.) to prevent bacterial infection, and transferred into incubation holders containing running groundwater at 6–7°C. Fertilized eggs were monitored weekly during development, and the alevines were killed in March 1998.

Statistical Analysis

Frequency distributions of all variables were tested for normality by the Kolmogorov-Smirnov test. The hypotheses were tested by one-tailed tests. The first hypothesis was tested by Kruskal-Wallis ANOVA. The second hypothesis was tested by a multiple regression and a forward stepwise selection in multiple regression. In the regressions, variation in sperm longevity was explained by the three sperm morphological traits, and fertilization success was explained by the three sperm morphological traits measured, ATP concentration, energy charge controlled for spermatocrit, and sperm quantity applied in the fertilization experiments. The "F-to-enter" value was set at 1.50. The third hypothesis was tested by comparing regression lines between sperm quantity and fertilization success of alternative male phenotypes by an analysis of covariance (ANCOVA). Statistical significance was set at P <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sperm Tail Morphology

Whole mounts of the salmon sperm showed the tail flagellum to have a long main piece and a short end piece, as illustrated by Retzius [32], Felix [33], and others. A short transition region is found between these two regions (Fig. 1, arrows). Most of the main piece consists of the well-known 9+2 microtubular pattern surrounded by a cell membrane, which projects to form two side fins (Fig. 2). The main difference between the two tail regions is that the nine microtubular doublets in the main piece carry outer and inner dynein arms, whereas in the end piece, the microtubules (usually singlets) are devoid of dynein arms. The end piece has a variable number of microtubules, although the two central microtubules always seem to be present as they extend to the very tip of the end piece.



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  		FIG. 1. Whole mount of spermatozoa from a salmon parr. Arrows indicate the transition region between main piece and the end piece of the tail flagellum. x2200.FIG. 2. Section through three sperm tails from an adult salmon. The transected main piece (bottom) has nine microtubular doublets carrying inner and outer dynein arms; the cell membrane forms two side fins. The end piece near the upper side fin contains only the two inner microtubules. However, the one at the upper side contains 8+2 singlets; probably, it is close to transition region. x80 000

Sperm Dimensions in Alternative Males

None of the morphological characters measured differed between adult and parr males. Thus, no difference was found in the size of the main piece, end piece of the sperm tail, or sperm midpiece (Table 1). Individual sperm length was normally distributed in the two strategies; the Kolmogorov-Smirnov test showed no departure from normality (P > 0.10). No association existed between sperm tail length and midpiece (r2 = 0.02, F1.12 = 0.26, P = 0.62) or between sperm tail length and end piece length (r2 = 0.02, F1.12 = 0.25, P = 0.63). No correlation between end piece and midpiece was detected (r2 = 0.02, F1.12 = 0.28, P = 0.61). No significant correlations between individual sperm morphological parameters and sperm longevity were observed (multiple regression, r2 = 0.34, F3.10 = 1.72, P = 0.11). Thus, we found no evidence that sperm morphology influences the ejaculate longevity. In addition, no significant correlations between sperm morphological parameters and spermatocrit were observed (multiple regression, r2 = 0.054, F3.10 = 0.189, P = 0.901).


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  		TABLE 1. Sperm morphological characters in the anadromous and parr Atlantic salmon males.a

Sperm Production and Quality

No difference in sperm longevity between adult and parr ejaculates was observed (one-way ANOVA, F1.12 = 0.49; P = 0.25; adult longevity, µ = 291.2 ± 78.5 sec; parr longevity, µ = 323.4 ± 93.2 sec). Interestingly, a positive association between sperm midpiece and sperm ATP concentrations was detected, mirroring more active mitochondria in a longer midpiece (multiple regression, r2 = 0.41, F1.12 = 8.327, P < 0.02) (Fig. 3). The energy charge was positively correlated with the spermatocrit (linear regression, F1.12 = 8.078, r2 = 0.402, P < 0.02), indicating that more dense ejaculates are of better quality (Fig. 4a). Therefore, we took residuals of charge on spermatocrit values to control for this effect. Flagellar length was positively correlated with sperm energy charge, indicating that spermatozoa with a long flagellum have a greater effective ATP mole fraction available for enabling the spermatozoon to swim toward the egg than do spermatozoa with a short flagellum (multiple regression, r2 = 0.511, F2:11 = 5.754, P < 0.01) (Fig. 4b). This result was further supported by the finding that a shorter sperm flagellum had higher ADP concentrations per spermatozoon (linear regression, F1:12 = 25.170, r2 = 0.68, P < 0.001). Precocious males did not have greater energy charge values (one-way ANOVA, F = 3.86, df = 1.12, P = 0.07; anadromous, µ = 0.620 ± 0.09; parr, µ = 0.752 ± 0.16). This result was consistent after spermatocrit was taken into account (one-way ANOVA, F = 0.00, df = 1.12, P = 0.50; anadromous charge residuals, µ = 0.001 ± 0.11; parr charge residuals, µ = -0.001 ± 0.11).



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  		FIG. 3. Length of sperm midpiece versus ATP concentration. Solid circles indicate adults; open circles indicate parr (multiple regression, r2 = 0.41, F1.12 = 8.327, P < 0.015, {Upsilon} = -3.494 + 9.774 X)



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  		FIG. 4. a) Energy charge versus spermatocrit (linear regression, r2 = 0.40, F1.12 = 8.078, P < 0.015, {Upsilon} = 0.481 + 0.004 X). b) Length of the sperm flagellum versus energy charge. Energy charge residuals on spermatocrit are used (multiple regression, r2 = 0.51, F2.11 = 5.754, P < 0.02, {Upsilon} = -1.286 + 0.031 X). Solid circles indicate adults; open circles indicate parr

Parr males had greater spermatocrit values than adults (one-way ANOVA, F = 19.66, df = 1.12, P < 0.001; adult, µ = 38.6 ± 16.6; parr, µ = 76.0 ± 14.1). Despite having only 20% of the adult male sperm number, parr spermatozoa fertilized an equal proportion of eggs in the fertilization experiments (ANCOVA, within-cells regression, F1.11 = 0.311, P = 0.30) (Fig. 5). Interestingly, the only parameter that explained a significant proportion of variation in fertilization success was the end piece of the tail, suggesting that spermatozoa with a longer end piece have a higher probability of fertilizing an egg (multiple regression, F1.12 = 6.439, r2 = 0.35, P < 0.02) (Fig. 6). Sperm tail and midpiece length, as well as ATP, charge residuals of spermatocrit, and sperm quantity, explained no additional variation in fertilization success. We compared regression lines between the end piece of the tail and fertilization success of the alternative males by ANCOVA, and within-cells regression was significant (F1.11 = 6.148, P < 0.02).



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  		FIG. 5. Sperm quantity applied in experiments versus fertilization success (ANCOVA; test of parallelism, F1.10 = 0.751, P = 0.41; within-cells regression, F1.11 = 0.311, P = 0.30). Solid circles indicate adults; open circles indicate parr



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  		FIG. 6. Sperm tail end-piece length versus fertilization success (ANCOVA; test of parallelism, F1.10 = 1.474, P = 0.25; within-cells regression, F1.11 = 6.148, P < 0.02). Solid circles indicate adults; open circles indicate parr


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The first hypothesis predicts that differing intensities of sperm competition faced by alternative mating male strategies might select for differing sperm tail optima, assuming that increased sperm length has a competitive advantage [25]. Our results showed no evidence for differences in sperm size between ejaculates from adult and parr salmons, despite the fact that they must experience different conditions of sperm competition intensity. An extended length of the flagellum, which could give a greater competitive ability due to a greater propulsive force [13], thus does not seem to be affected by a disruptive selection on spermatozoa with different size optima in the alternative reproductive strategies in Atlantic salmon. The spermatozoa have the same dimensions in males of both life histories. This finding is in agreement with the results of Gage et al. [24], who reported no difference in sperm flagellar length between salmon parr and adult males.

The second hypothesis predicts that long spermatozoa are superior to short ones, assuming that an increased sperm length adversely affects sperm longevity due to associated energetic costs of long flagellum. This prediction was generated from comparative studies on mammals [13, 14] and from subsequent theoretical models [16]. Our study is, to our knowledge, the first one that tests this prediction in an intraspecific study. Our results indicate that the capacity for ATP synthesis by mitochondrial oxidative phosphorylation is greater in a longer midpiece, as illustrated by a positive correlation between the size of the sperm midpiece and sperm ATP concentrations. This finding reflects the fact that ATP produced by mitochondrial respiration is the main energy source for sperm motility. Long spermatozoa had higher energy charge values than short spermatozoa after sperm density had been taken into consideration. Our results thus imply that long spermatozoa have more ATP available to be used for sperm motility. We found no evidence that the mean sperm dimensions influence the mean sperm longevity. Thus, although this effect was suggested in previous modeling [16] and from interspecific comparative work on fishes [15], we did not find in this intraspecific study any evidence that the mean sperm size has an effect on sperm survival within an ejaculate, either negatively or positively. This is plausible, because in nature, teleosts spawn in close proximity so that fertilization occurs within a few seconds, and the first spermatozoon that reaches the aperture on the chorionic egg membrane (i.e., micropyle) effectively seals the egg [34]. Hence, selection is not expected to favor long sperm motility [35].

The third hypothesis predicts that parr males are selected for an increased production of spermatozoa with greater quality than their adult counterparts, due to the parr's behaviorally disfavored role in mating [24]. In this study, parr males had greater sperm density per ejaculate unit than the adults, a finding in agreement with the results of Gage et al. [24]. Sperm longevity, however, did not differ between anadromous and precocious juvenile ejaculates. This result stands in contrast to previously reported data [24, 36, 37].

A positive correlation between sperm energy charge and spermatocrit implies that individuals producing more sperm-dense ejaculates (parr) are more likely to produce physiologically superior spermatozoa as well. No indices that energy charge differs between alternative life histories were found; however, the results from the fertilization experiments suggest a higher sperm quality in parr males. Despite having fivefold greater sperm numbers in the fertilization test, adult males were unable to fertilize a greater proportion of eggs in the batch. Adenosine monophosphate is not a significant regulator of mitochondrial respiration [38]. Because the axonemal molecular motor, dynein ATPase, powers sperm motility by hydrolyzing ATP to ADP and Pi [39], the phosphorylation potential, or [ATP]/[ADP] x [Pi], may regulate sperm energy metabolism [40]. It was suggested that a phosphocreatine shuttle mediates energy transport between ATP production (i.e., mitochondrion) and ATP utilization (i.e., flagellar axoneme) sites in trout spermatozoon, thereby maintaining relatively high concentrations of ADP in the vicinity of the mitochondria, which is necessary for a maximal respiration rate [41]. Precocious juveniles may have a more effective phosphocreatine shuttle system, which then could account for the observed advantage in parr sperm quality. This effect is possible, because both a simple diffusion of ATP to axonemal dynein ATPase and a phosphocreatine shuttle may participate in trout sperm motility [41]. With a larger sample, it was shown that Atlantic salmon parr, indeed, produce sperm cells that contain greater ATP content than those of anadromous adults [42].

Particularly interesting is the finding that the length of the end piece of the sperm tail may be a predictor of the sperm fertilization ability. An attenuated end piece of the sperm tail is universal in the animal kingdom [32, 43] and influences the undulatory movements of the sperm tail, possibly as an adaptation to viscous resistances [44]. It has also been named the whiplash or the sperm terminal filament, and it consists of a variable number of microtubules, which are devoid of dynein arms and surrounded by the cell membrane. Omoto and Brokaw [44] have shown that it is an essential component of the tail; if removed, the flagellar undulations show a clear "end effect" rather than propagating smoothly off the end of the flagellum. This sperm tail region experiences increased viscous drag relative to the rest of the sperm flagellum [44]. Our data thus suggest that individuals producing a long end piece are capable of achieving an increased fertilization success, probably because of its resistance to the viscous drag of the surrounding media. Because the ANCOVA discovered a significant difference between within-cells regression of parr and anadromous males, this finding indicates that the increased sperm quality of parr as compared to that of anadromous males is partly due to the attenuated sperm flagellar end piece.

In conclusion, this study suggests that no adaptation for different sperm size optima occurs in salmon males that employ alternative reproductive tactics. Sperm longevity was the same for both types of males, and none of the sperm morphological traits measured correlated with mean sperm longevity. Thus, it would appear that the mean lengths of the sperm tail and of the adjacent midpiece are not confounding factors to salmon mean ejaculate longevity. Precocious parr, however, produced a greater sperm density per milliliter of ejaculate and fertilized the same proportion of eggs in the batch, despite having much less spermatozoa than the anadromous adults in the fertilization experiments. Together with the positive association between spermatocrit and sperm energy charge, this finding implies that parr are capable of producing physiologically superior spermatozoa. In addition, long sperm tails had higher energy charge values than short sperm tails. These findings have at least two consequences. First, sperm length has its cost at the metabolic level, and second, long spermatozoa are the superior ones in Atlantic salmon. The advantage of an increased sperm length is maintained through a long sperm flagellum with an elaborate axonemal dynein ATPase machinery, and a long sperm midpiece with mitochondria, capable of an intense oxidative respiration. Sperm fertilizing ability is influenced by the size of the thin sperm end piece, suggesting an adaptation to the viscosity of surrounding media. We conclude that these sperm traits are those that may be selected to optimize sperm energetic demands under conditions of increased sperm competition intensity.


   ACKNOWLEDGMENTS
 
We thank Bjarne Ragnarsson and the staff of the National Board of Fisheries' Research Station in Älvkarleby for help with handling the fish. We also thank Janet Jansson and Riccardo Tombolini from the Department of Biochemistry, Stockholm University, for hospitality in their laboratory while measuring sperm nucleoside phosphates. Bertil Borg, Torbjörn Järvi, and two anonymous referees are acknowledged for valuable comments on previous drafts of the paper.


   FOOTNOTES
 
First decision: 11 June 2001.

1 The experimental work in the study complies with the standards and procedures laid down by the Swedish Ministry of Agriculture (permission license no. 34 3632/92). Back

2 Correspondence. FAX: 46 8 759 03 38; tomislav.vladic{at}fiskeriverket.se Back

3 Current address: Puschino Moscow Region, D-1-80, Russia 142292. Back

Accepted: August 16, 2001.

Received: May 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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