Background and Aim: Resistant starch (RS) is difficult to digest in the digestive tract. This study aimed to evaluate the effects of heat-moisture treatment (HMT) on RS in cassava and examined its impact on rumen fermentation. Materials and Methods: Cassava flour was used as a raw material and used in a randomized block design with four different cycles of HMT as the treatments and four different rumen incubations in vitro as blocks. Treatments included: HMT0: without HMT (control), HMT1: one HMT cycle, HMT2: two HMT cycles, and HMT3: three HMT cycles. Heat-moisture treatment processes were performed at 121°C for 15 min and then freezing at -20°C for 6 h. Analyzed HMT cassava starch characteristics included components, digestibility, and physicochemical properties. In in vitro rumen fermentation studies (48 h incubation) using HMT cassava, digestibility, gas production, methane, fermentation profiles, and microbial population assessments were performed. Results: Heat-moisture treatment significantly reduced (p < 0.05) starch, amylopectin, rapidly digestible starch (RDS), and slowly digestible starch levels. In contrast, amylose, reducing sugars, very RDS, RS, and protein digestion levels were significantly increased (p < 0.05). Additionally, a reduced crystallinity index and an increased amorphous index were observed in starch using Fourier-transform infrared analyses, while a change in crystalline type from type A to type B, along with a reduction in crystallinity degree, was observed in X-ray diffraction analyses. Heat-moisture treatment significantly (p < 0.05) reduced rumen dry matter (DM) degradation, gas production, methane (CH4 for 12 h), volatile fatty acid (VFA), and propionate levels. In addition, acetate, butyrate, and acetate/propionate ratios, as well as population of Streptococcus bovis and Bacteroides were significantly increased (p < 0.05). However, pH, ammonia, and organic matter digestibility were unaffected (p < 0.05) by HMT. Conclusion: Cassava HMT altered starch characteristics, significantly increased RS, which appeared to limit rumen digestion activity, decreased rumen DM degradation, gas production, VFAs, and CH4 production for 12 h, but increased S. bovis and Bacteroides levels. Keywords: heat-moisture treatment, in vitro, rumen fermentation, starch modification.

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