Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free‐living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.
GENERAL FIELD PROCEDURES
This study was conducted in the vicinity of Queen's University Biological Station, Ontario, Canada, and in southern Norway during the breeding seasons (i.e., April–July) of 2006 and 2007. Free‐living males were captured using mist‐nets and playback, or box‐traps. To avoid pseudo‐replication (i.e., that an individual was sampled twice), the males were ringed with a uniquely numbered aluminum band provided by the Canadian Wildlife Service (for birds banded in Canada) and the Norwegian Bird Ringing Centre at Stavanger Museum (for birds banded in Norway). We gently massaged the cloacal protuberance to obtain an ejaculate and then released the birds. The ejaculate was immediately diluted in 20–100 μl, depending on the size of the ejaculate, preheated (40°C) Dulbecco's Modified Eagle Medium (Advanced D‐MEM, Invitrogen, Carlsbad, CA). Within 30 sec after ejaculation, about 3–5 μl of the diluted sperm was pipetted onto a preheated standard microscopic count slide (20 μm depth two‐chamber, Leja, Nieuw‐Vennep, The Netherlands) mounted on a MiniTherm slide warmer (Hamilton Thorne Inc, Beverly, MA) kept at a constant temperature of 40°C. Sperm swimming speed was then recorded for up to 90 sec using a digital video camera (HDR‐HC1E, PAL, Sony, Tokyo, Japan) mounted on an upright microscope (CX41, Olympus, Japan) equipped with a 4× objective. We recorded multiple independent frames for each slide to increase the number of sperm measured for each male.
Sperm samples were fixed in a 5% formalin solution and a droplet of this solution was placed on a microslide and air dried. We captured high‐resolution digital images at microscope magnifications of 200× or 320× using a digital camera (DFC420, Lieca Microsystems, Heerbrugg, Switzerland) mounted onto a digital light microscope (DM6000 B, Leica Microsystems). Leica Application Suite (version 2.6.0 R1) was used to measure (± 0.1 μm) length (head, midpiece, and tail) of 10 intact spermatozoa for each individual. Total length was calculated as the sum of head, midpiece, and tail length, and flagellum length as the sum of midpiece and tail length. Measuring 10 spermatozoa provides a representative estimate of a male's mean sperm total length (Laskemoen et al. 2007). We have previously shown that repeatability (sensu Lessells and Boag 1987) of the measurements of total length of individual spermatozoa is high (r = 0.98; Kleven et al. 2008). Sperm lengths were logarithm (ln) transformed prior to analysis to normalize the distribution.