| Summary: | The objective of this study was to compare the effects of Crabtree-negative ruminal yeast and Crabtree-positive yeast in ensiled rice straw (RS) on the ensilage quality, nutritive value, and microorganism composition, including the evaluation of the ensiled RS using the in vitro gas production technique. The experiment was conducted in a 4 × 3 factorial arrangement in a randomized complete design. Factor A was yeast species with no inoculant, Crabtree-negative yeasts (<i>Pichia kudriavzevii</i> KKU20 and <i>Candida tropicalis</i> KKU20), and Crabtree-positive yeast (<i>Saccharomyces cerevisae</i>), whereas factor B was ensilage times (7, 14, and 21 days). The rate of growth was revealed to be lower in Crabtree-positive yeasts than the other Crabtree-negative yeast strains (<i>p</i> < 0.01). RS ensiled with <i>S. cerevisiae</i> showed decreased dry matter (DM) content by 9.0% when compared to the sample without a yeast inoculant. In addition, organic matter (OM) content was greater (<i>p</i> < 0.01) for <i>P. kudriavzevii</i> KKU20 than <i>C. tropicalis</i> KKU20 and without an inoculant. Neutral detergent fiber (NDF) content was significantly decreased (<i>p</i> < 0.01) by yeast inoculants by about 2.75% when compared to the control group. Lactic acid bacteria (LAB) and aerobic bacteria were low (<i>p</i> < 0.05) when yeasts were added. However, no interaction was found between yeast and ensilage times on the quality of ensiled RS (<i>p</i> > 0.05). The <i>P. kudriavzevii</i> KKU20 addition was associated with the highest value (<i>p</i> < 0.01) of gas produced—an insoluble fraction (b), potential extent of gas production (a + b), and cumulative gas production at 96 h—when compared with <i>S. cerevisiae</i> or the control group. The highest in vitro dry matter digestibility and in vitro neutral detergent fiber digestibility was observed in RS ensiled with <i>P. kudriavzevii</i> KKU20 for 14 days (<i>p</i> < 0.01, <i>p</i> < 0.05). The maximum total volatile fatty acids (VFAs) at 4 and 8 h of incubation and the mean value were observed in RS ensiled with <i>P. kudriavzevii</i> KKU20 (<i>p</i> < 0.01). RS ensiled with all yeast strains showed an increased propionate concentration at 8 h (<i>p</i> < 0.01). In conclusion, ensiling RS with isolated Crabtree-negative ruminal yeasts could benefit feed digestion and in vitro gas production more than Crabtree-positive yeast does. <i>P. kudriavzevii</i> KKU20, an isolated Crabtree-negative ruminal yeast used to treat RS, had the highest potential for increasing cumulative gas production and enhancing in vitro digestibility.
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