Anim Sci J. 2021;92:e13530. wileyonlinelibrary.com/journal/asj | 1 of 7 https://doi.org/10.1111/asj.13530 © 2021 Japanese Society of Animal Science 1 | INTRODUCTION Soluble sugars are important carbohydrates, similar to starch, cel- lulose, and hemicellulose, for forage science and ruminant nutri- tion. To preserve forage as silage, sufficient amounts of soluble sugars in forage are necessary to lower the pH of silage by lactic acid and acetic acid (McDonald et al., 1991). As nutrients for rumi- nants, soluble sugars are fermented rapidly in the rumen and used as an energy source for microbial growth and protein synthesis (Hall & Herejk, 2001). The soluble sugars content in feeds has been analyzed in various fields of research and extension, such as rumi- nant nutrition, breeding and cultivation of forage, and silage pro- duction. Several colorimetric methods such as the phenol-sulfuric acid (PSA) assay, anthrone-sulfuric acid assay, reducing sugar assay, and enzymatic assay are routinely performed (Hall, 2014; Pereira et al., 2019; Rivero et al., 2019; Zhao et al., 2020; Zhou et al., 2019). The PSA assay is a relatively simple and easy colorimetric method for measuring soluble sugars in feeds (Hall, 2014). However, quantifica- tion of soluble sugars content from a large number of samples is neces- sary in forage science and ruminant nutrition research. It is desirable to improve the method when handling a large number of samples and to reduce the amounts of deleterious chemicals used in the assay for the safety of analysts and environmental aspects. The amounts of concen- trated sulfuric acid and phenol using the PSA method with test tubes are not small. As an example of a common procedure, 1 ml of 50 g/L phenol solution, and 2.5 or 5 ml of concentrated sulfuric acid were used in a reaction (Chow & Landhäusser, 2004; Dubois et al., 1956). To resolve this issue, a PSA method in microplates was reported for pure chemical compounds and extracted juice from sorghum internodes (Li et al., 2018; Masuko et al., 2005; Wang et al., 2017). A scaled-down method using microplates requires approximately one-tenth of the chemicals compared to the method using test tubes. The PSA method in a microplate format has dual benefits: handling of a large number Received: 23 September 2020 | Revised: 29 January 2021 | Accepted: 4 February 2021 DOI: 10.1111/asj.13530 ORIGINAL ARTICLE Validation of a phenol-sulfuric acid method in a microplate format for the quantification of soluble sugars in ruminant feeds Makoto Kondo 1 | Randi Mulianda 1,2 | Masaya Matamura 1 | Toshiyuki Shibata 3 | Takashi Mishima 3 | Anuraga Jayanegara 2 | Naoto Isono 3 1 Department of Sustainable Resource Sciences, Graduate School of Bioresources, Mie University, Tsu, Japan 2 Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia 3 Department of Life Sciences and Chemistry, Graduate School of Bioresources, Mie University, Tsu, Japan Correspondence Makoto Kondo, Faculty of Bioresources, Mie University, Tsu, Japan. Email: makok@bio.mie-u.ac.jp Funding information This research was supported by JSPS KAKENHI Grant Number 18K05943. Abstract Soluble sugars in feeds are important for ruminant production; however, performing numerous sugar analyses within a short period is a laborious task. Here, we devel- oped a phenol-sulfuric acid (PSA) assay in a microplate format to quantify soluble sugars in ruminant feeds. This method is easy and quick and requires only a small quantity of harmful reagents. We found that assay measurements were not affected by the representative organic acids and sugar alcohol contained in feeds. The treat- ment of activated charcoal with ethanol extract prior to the PSA assay was effec- tive in removing interfering compounds for a more accurate determination of soluble sugars in certain feeds. Furthermore, the inter-day and intra-day repeatability of the present method was acceptable. Hence, we conclude that the method developed in this study is suitable for routine analysis of soluble sugars content in ruminant feeds. KEYWORDS feed, phenol-sulfuric acid, ruminant, soluble sugars