Background: The influence of sperm competition upon sperm size has been a controversial issue during the last 20 years which remains unresolved for mammals. The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory results from both theoretical and empirical studies. In addition, the debate has extended to which sperm components should increase in size: the midpiece to accommodate more mitochondria and produce more energy to fuel motility, or the principal piece to generate greater propulsion forces.Results: In this study we examined the influence of sperm competition upon sperm design in mammals using a much larger data set (226 species) than in previous analyses, and we corrected for phylogenetic effects by using a more complete and resolved phylogeny, and more robust phylogenetic control methods. Our results show that, as sperm competition increases, all sperm components increase in an integrated manner and sperm heads become more elongated. The increase in sperm length was found to be associated with enhanced swimming velocity, an adaptive trait under sperm competition.Conclusions: We conclude that sperm competition has played an important role in the evolution of sperm design in mammals, and discuss why previous studies have failed to detect it.

Mouse sperm produce enough ATP to sustain motility by anaerobic glycolysis and respiration. However, previous studies indicated that an active glycolytic pathway is required to achieve normal sperm function, and identified glycolysis as the main source of ATP to fuel the motility of mouse sperm. All the available evidence has been gathered for the lab mouse, while comparative studies including closely related mouse species have revealed (a) a wide range of variation in sperm motility and ATP production, and (b) that the lab mouse shows comparatively low values in these traits. In this study, we compared the relative reliance on the usage of glycolysis or oxidative phosphorylation as ATP sources for sperm motility between mouse species that exhibit significantly different sperm performance parameters. We found that the sperm of species with higher oxygen consumption/lactate excretion rate ratio were able to produce higher amounts of ATP, achieving higher swimming velocities. Additionally, we show that the species with higher respiration/glycolysis ratio have a higher degree of dependence upon active oxidative phosphorylation. Moreover, we characterize for the first time, two mouse species in which sperm depend on functional oxidative phosphorylation to achieve normal performance. Finally, we discuss that sexual selection through sperm competition could promote adaptations in sperm energetic metabolism tending to increase the usage of oxidative phosphorylation in relation to glycolysis, as this would constitute a more efficient pathway for the generation of ATP (and faster sperm).

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.

Sperm competition favours an increase in sperm swimming velocity that maximises the chances that sperm will reach the ova before rival sperm and fertilise. Comparative studies have shown that the increase in sperm swimming speed is associated with an increase in total sperm size. However, it is not known which are the first evolutionary steps that lead to increases in sperm swimming velocity. Using a group of closely related muroid rodents that differ in levels of sperm competition, we here test the hypothesis that subtle changes in sperm design may represent early evolutionary changes that could make sperm swim faster. Our findings show that as sperm competition increases so does sperm swimming speed. Sperm swimming velocity is associated with the size of all sperm components. However, levels of sperm competition are only related to an increase in sperm head area. Such increase is a consequence of an increase in the length of the sperm head, and also of the presence of an apical hook in some of the species studied. These findings suggest that the presence of a hook may modify the sperm head in such a way that would help sperm swim faster and may also be advantageous if sperm with larger heads are better able to attach to the epithelial cells lining the lower isthmus of the oviduct where sperm remain quiescent before the final race to reach the site of fertilisation.

Sperm competition often leads to increase in sperm numbers and sperm quality, and its effects on sperm function are now beginning to emerge. Rapid swimming speeds are crucial for mammalian spermatozoa, because they need to overcome physical barriers in the female tract, reach the ovum, and generate force to penetrate its vestments. Faster velocities associate with high sperm competition levels in many taxa and may be due to increases in sperm dimensions, but they may also relate to higher adenosine triphosphate (ATP) content. We examined if variation in sperm ATP levels relates to both sperm competition and sperm swimming speed in rodents. We found that sperm competition associates with variations in sperm ATP content and sperm-size adjusted ATP concentrations, which suggests proportionally higher ATP content in response to sperm competition. Moreover, both measures were associated with sperm swimming velocities. Our findings thus support the idea that sperm competition may select for higher ATP content leading to faster sperm swimming velocity.

Little is known about the genetic basis of evolutionary changes in sperm phenotype. Postcopulatory sexual selection is associated with differences in protamine gene sequences and promoters and is a powerful force acting on sperm form and function, although links between protamine evolution and sperm phenotype are scarce. Protamines are involved in sperm chromatin condensation, and protamine deficiency negatively affects sperm morphology and male fertility, thus suggesting that they are important for sperm design and function. We examined changes in protamine genes and sperm phenotype in rodents to understand the role of sexual selection on protamine evolution and sperm design. We performed a genotype-phenotype association study using root-to-tip dN/dS (nonsynonymous/synonymous substitutions rate ratio) to account for evolutionary rates and phylogenetic generalized least squares analyses to compare genetic and morphometric data. Evolutionary rates of protamine 1 and the protamine 2 domain cleaved off during chromatin condensation correlated with head size and elongation. Protamine 1 exhibited restricted positive selection on some functional sites, which seemed sufficient to preserve its role in head design. The cleaved-protamine 2, whose relaxation is halted by sexual selection, seems to ensure small, elongated heads that would make sperm more competitive. No association existed between mature-protamine 2 and head phenotype, suggesting little involvement during chromatin condensation and a likely role maintaining the condensed state. Our results suggest that evolutionary changes in protamines could be related to complex developmental modifications in the sperm head. This represents an important step toward understanding the role of changes in gene coding sequences in the divergence of germ cell phenotype.