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. 2020 Jun 1;10:8862. doi: 10.1038/s41598-020-65931-z

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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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Effect of sorting on sperm fertility and developmental ability. Sperm were pre-incubated for 60 min, and sperm suspensions were diluted using mHTF to adjust the flow rate to approximately 400 events/s. Then, cell sorting was performed using forward scattered light (FSC) and side scattered light (SSC). After sorting, the sorted sperm suspensions were collected from the sorting reservoir and used as in vitro fertilisation (IVF) medium. Oocytes were introduced into the IVF medium. After IVF, the two-cell embryos were either cultured in KSOM/AA medium or transferred into the oviducts of pseudopregnant ICR females (10 embryos/oviducts). (B) The fertilisation rate was calculated as the number of two-cell embryos (A) divided by the number of inseminated oocytes x 100. (G) The developmental rate was calculated as the number of embryos at several stages— 4-cell (D), morulae (E) and blastcysts (F)—divided by the number of two-cell embryos (C) x 100. (H) Two-cell embryos developed normally into live pups. I) the birth rate was calculated as the (number of live pups divided by the number of transferred two-cell embryos) x 100. Values are given as the mean ± SD (n = 3-6). *p < 0.05 compared with sort (−).