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《Sperm collection in Blacklegged Kittiwakes and characterization of sperm velocity and morphology》

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M e a n，s t a n d a r d d e v i a t i o n（SD）a n d s a m p l e s i z e（N）o f s p e r m s w i m m i n g t r a i t s m e a s u r e d i n DMEM，s p e r m m o r p h o l o g i c a l t r a i t s m e a s u r e d f r o m s m e a r s p r e s e r v e d i n f o

Apart from the addition of a sperm extender，field conditions preclude any sample preparation，and sperm samples cannot be washed and cleaned from unwanted cells or debris.The presence of such cells or debris in sperm samples may compromise the accuracy of the measures performed by the CASA plugin.Here，most particles were removed using the threshold function in ImageJ and by setting a range of size（in pixels）for sperm cells.Yet，we are aware that not all particles could be removed this way.However，this should not affect the estimation of sperm speed or rectitude in sperm trajectory，because immotile particles or particles moved by drift having a size within that of a sperm cell were excluded from the analysis by our criteria on VCL and VAP（see“Methods”section）.Nevertheless，it should be noted that such particles lead to underestimating the percentage of motile sperm.