Authors
  • Laskemoen, Terje
  • Kleven, Oddmund
  • Fossøy, Frode
  • Robertson, Raleigh J.
  • Rudolfsen, Geir
  • Lifjeld, Jan
Universities
  • University of Oslo

Summary

Sperm competition is widespread among animal taxa and considered a major force in sperm evolution. Recent comparative studies have indicated that sperm competition selects for high sperm production capacity and long and fast-swimming spermatozoa across species. Here, we examine the role of sperm quantity and quality for fertilization success of individual males in a Canadian population of tree swallows Tachycineta bicolor, a socially monogamous, but highly promiscuous passerine. Male fertilization success (the sum of withinpair and extrapair young) was significantly associated with the size of the cloacal protuberance (a proxy for sperm quantity), but not with sperm size or in vitro sperm swimming speed. In a multivariate analysis, both cloacal protuberance volume and relative sperm midpiece size (i.e. high mitochondrial loading) had significant effects on male fertilization success. However, relative sperm midpiece size was not associated with fertilization success in a simple regression. Further, both cloacal protuberance volume and relative midpiece size had significant effects on sperm velocity, both in simple regressions and in a multivariate analysis. The finding that males with large relative midpiece size had both higher fertilization success and faster swimming sperm, suggests an indirect link between sperm morphology and male fertility mediated through sperm velocity. In conclusion, both quantitative and qualitative sperm traits seem to affect male fertilization success in tree swallows.

Methodology

Study site and study species

The fieldwork was carried out from April to July 2006 at Queen's University Biological Station (44°34′ N, 76°20′ W), Ontario, Canada. The tree swallows breeding at grids and solitarily in nest-boxes at this site have been intensively studied for more than three decades. Detailed information about the study area and general field procedures can be found elsewhere (e.g. Robertson and Rendell 2001). Briefly, adult individuals were captured in their nest-boxes, standard biometric measures were taken, blood was sampled by brachial venipuncture and the birds were banded with a Canadian Wildlife Service aluminium band. We measured the dimensions of the male cloacal protuberance, which is swollen as a result of the growth of the seminal glomera (the coiled distal end of the vas deferens where sperm are stored before ejaculation). With a set of calipers, we measured the height and two diameters, d1 and d2, perpendicular to each other, as the cloaca appears ellipsoid from a ventral view. The cloacal protuberance volume was calculated according to the formula of an ellipsoid cylinder: height × π × 0.5d1 × 0.5d2. All measurements of cloacal protuberance size were performed by one person to remove inter-observer effects. As cloacal protuberance volume may vary across the breeding season, we tested for an association between male sample date and cloacal protuberance volume. However, we found no such relationship (r = 0.03, P = 0.87, n = 43 males). A small amount of blood was sampled from nestlings, and we collected unhatched eggs and dead young for later genetic analyses. We classified adult males as second year (SY) or after second year (ASY) on the basis of wing length (Rendell, W.B., Kempenaers, B., Robertson R. J., unpublished data). Briefly, males with wing length ≤119 mm are classified as SY, wing length between 119 mm and 120 mm are classified as after first year and wing length >120 mm are classified as ASY. As a conservative approach, we classified all males with wing length ≤120 mm as SY and wing length >120 mm as ASY. We confirmed age estimates from wing length measurements when possible using banding records from previous years. All measurements of wing length were performed by the same person.

Sperm motility recordings

We gently massaged the cloacal protuberance of males to obtain an ejaculate (cf. Kleven et al. 2009a), and then released the birds. The ejaculate was immediately diluted in 20–100 µl, depending on the volume of the ejaculate, pre-heated (40°C) Dulbecco's modified Eagle medium (Advanced D-MEM, Invitrogen, USA). Within 30 s after ejaculation, about 3–5 µl of the diluted sperm was pipetted onto a pre-heated standard microscopic count slide (20 µm-depth 2-chamber, Leja, The Netherlands), mounted on a MiniTherm slide warmer (Hamilton Thorne Biosciences, USA) and kept at a constant temperature of 40°C. Sperm motility was then recorded for up to 90 seconds using a digital video camera (HDR-HC1E, PAL, Sony, Japan) mounted on an upright microscope (CX41, Olympus, Japan), with a total magnification of 400×. We recorded multiple independent frames (mean: 10.0 ± 2.2 SD) for each slide to increase the number of sperm measured for each male.

Location