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. 2020 Aug 7;10:13360. doi: 10.1038/s41598-020-70302-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Immunocytochemistry of GM-CSF in the pig mature spermatozoa. Top images (a and c) show spermatozoa with low/absent GM-CSF expression and bottom images show spermatozoa viable and non-viable (DAPI stain). The sperm samples were incubated with anti-GM-CSF antibody (orb6090, Biorbyt) to identify GM-CSF expression (composite in red in top images) and incubated with DAPI staining to identify viable from non-viable (DAPI positive in blue in bottom images) spermatozoa. Sperm in (a) and (c), were included in cluster 1 (fluorescence intensity 4.45 ± 0.17 RU; ranging from 0.0 to 15.33 RU) while spermatozoa displaying high GM-CSF expression were included in cluster 2 (fluorescence intensity 29.81 ± 1.82 RU; ranging from 15.67 to 84.0 RU, b and d). Scale bar: 10 μm. Cropped image of immunocytochemistry capture (see Supplemental Information, Fig S5 for full image).