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. 2024 Jul 10;20:306. doi: 10.1186/s12917-024-04166-w

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Measurement of alpha diversity indices (observed species, Shannon diversity) for DNA and cDNA-based analysis. G1 = small (300 µl or 1.5 ml) aliquots with saline only buffer; G2 = 1-liter aliquot with saline only buffer; G3 = small aliquots with saline plus glycerol buffer; G4 = 1-liter aliquot with saline plus glycerol buffer. X-axis shows the day of storage (Day 0 or fresh, 30, 60, or 90). The different colors represent the storage temperature (fresh, frozen at -20 °C, or frozen at -80 °C). Each box and whisker plot shows results for 3 samples (1 from each horse), as represented by the individual data points. Alpha diversity indices for cDNA-based analysis showed much less variation between day and temperature of storage (P > 0.05) than for DNA-based analysis (P < 0.001 for fixed effect of day for both − 20 °C and − 80 °C), and the frozen samples were overall more similar to fresh samples than for DNA-based analysis. There was less variation in alpha diversity between horse for cDNA-based analysis when using saline plus glycerol buffer as compared to saline only buffer. For the 1-liter aliquot size, less variation was seen when using saline plus glycerol buffer as opposed to saline only buffer (only DNA-based analysis performed)