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Measuring sperm swimming performance in birds: effects of dilution, suspension medium, mechanical agitation, and sperm number

  • Emily R. A. CramerEmail author
  • Melissah Rowe
  • Fabrice Eroukhmanoff
  • Jan T. Lifjeld
  • Glenn-Peter Sætre
  • Arild Johnsen
Original Article

Abstract

Sperm swimming performance, including swimming speed and the proportion of motile cells, may strongly affect fertilization success. However, little is known about how methodological factors affect in vitro measurement of these parameters. We compare the swimming performance of sperm from House Sparrows (Passer domesticus) and Spanish Sparrows (Passer hispaniolensis) in two standard suspension media, at two different dilutions, and with different degrees of cell agitation. Further, we conduct a resampling analysis to investigate sample size effects. Sperm performance was generally reduced when sperm were diluted, or when suspended in phosphate-buffered saline (PBS) rather than a medium containing additional nutrients. Sperm performance was particularly low when they were diluted more and suspended in PBS, suggesting that seminal fluid may provide compounds that enhance performance but that these are less available following dilution. Mechanical agitation of the cells by vigorous pipetting increased the proportion of motile cells. Between-male repeatability, assessed on a single sample measured in multiple conditions, was moderate and significant, suggesting that similar results may be obtained regardless of the methodology used to assess sperm motion. We found no evidence of biased results when low numbers of cells per male were used in analysis, though precision increased substantially as sample size increased from five to 20 or more cells per male. We recommend using the same suspension media and similar sperm concentrations and levels of agitation, to the greatest degree possible, and including as many individuals as possible in analyses, even when some individuals are represented by few sperm cells.

Keywords

Postcopulatory sexual selection Sperm competition Cryptic female choice Experimental design Sperm behavior 

Zusammenfassung

Messung der Schwimmfähigkeit von Spermien bei Vögeln: Verdünnungseffekte, Suspensionsmedium, mechanische Durchmischung und Spermienzahl Die Schwimmfähigkeit der Spermien, einschließlich der Schwimmgeschwindigkeit und dem Anteil an beweglichen Zellen, könnte den Befruchtungserfolg stark beeinflussen. Jedoch ist wenig darüber bekannt, wie die methodischen Faktoren die in vitro-Messungen dieser Parameter beeinflussen. Wir vergleichen die Schwimmfähigkeit der Spermien von Haus- und Weidensperlingen (Passer domesticus und P. hispaniolensis) in zwei verschiedenen Standardsuspensionen, in zwei verschiedenen Verdünnungen und unter unterschiedlich starker Durchmischung (Agitation) der Zellen. Weiterhin führten wir eine wiederholte Probenanalyse durch, um von der Stichprobengröße abhängige Effekte zu untersuchen. Die Spermienleistung reduzierte sich grundsätzlich bei der Verdünnung der Spermien oder bei Zugabe einer phosphatgepufferten Salzlösung (PBS; engl. phosphate-buffered saline) anstelle eines zusätzlich mit Nährstoffen angereicherten Mediums (DMEM). Die Spermienleistung war besonders gering bei einer stärkeren Verdünnung oder bei der Zugabe von PBS, was vermuten lässt, dass die Samenflüssigkeit bestimmte Komponenten zur Steigerung der Schwimmfähigkeit beinhaltet, welche jedoch nach einer Verdünnung nur noch im geringeren Maße zur Verfügung stehen. Eine mechanische Agitation der Zellen durch starkes Pipettieren erhöhte den Anteil an beweglichen Zellen. Die Wiederholbarkeit der Messungen zwischen den untersuchten Männchen, basierend auf einzelnen Proben, welche unter verschiedenen Bedingungen gemessen wurden, war moderat und signifikant. Dies lässt vermuten, dass gleichartige Ergebnisse unabhängig von der für die Messung der Spermienmobilität angewandten Methode erzielt werden können. Wir haben keine Anhaltspunkte dafür gefunden, dass eine geringe Spermienanzahl pro Männchen die Analyseergebnisse beeinflusst. Jedoch nahm bei der Erhöhung der Stichprobengröße von fünf auf 20 oder mehr Zellen pro Männchen auch die Präzession zu. Wir empfehlen - sofern möglich - die Anwendung des gleichen Suspensionsmediums, einer gleichartigen Spermienkonzentration und einen gleichbleibenden Durchmischungsgrad. Weiterhin sollten so viele Individuen wie möglich in einer Analyse berücksichtigt werden, auch wenn einige Individuen nur durch wenige Spermienzellen repräsentiert werden.

Notes

Acknowledgments

Jo Hermansen, Stein Are Sæther, and Fredrik Haas helped to set up the initial aviary populations. Silje Larsen Rekdal, Silje Hogner, Even Stensrud, and Camilla Lo Cascio Sætre assisted with the experiments. Tim Schmoll provided useful comments on an earlier version of this manuscript. Video recordings and associated sperm morphological samples are archived at the Natural History Museum of Oslo. Funding was provided by the Research Council of Norway (grant 213592 to A. J.; 196554/V40 to M. R. and J. T. L.; 204523 to G. P. S.; and 230434 to M. R.) and the Swedish Research Council (F. E.). Ethical permission for the study was given by the Norwegian Animal Research Authority (FOTS ID 2394). The experiments complied with the current laws of Norway. Datasets used in the study are available online at  https://doi.org/10.5061/dryad.662kb6m.

Supplementary material

10336_2019_1672_MOESM1_ESM.docx (130 kb)
Supplementary material 1 (DOCX 130 kb)

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Copyright information

© Deutsche Ornithologen-Gesellschaft e.V. 2019

Authors and Affiliations

  1. 1.Natural History MuseumUniversity of OsloOsloNorway
  2. 2.Premedical Education DepartmentWeill Cornell Medicine QatarDohaQatar
  3. 3.Department of Biosciences, Center for Ecological and Evolutionary SynthesisUniversity of OsloOsloNorway

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