The effects of dietary inclusion of dried Leucaena leucocephala leaves (DLL) on nutrient digestibility, fermentation parameters, microbial rumen population, and production of enteric methane (CH₄) in crossbred heifers were evaluated. Four heifers were used in a 4 × 4 Latin square design consisting of four periods and four levels of inclusion of DLL: 0%, 12%, 24%, and 36% of dry matter (DM) intake. Results showed that DM intake (DMI), organic matter intake, and gross energy intake (GEI) were similar (p > 0.05) among treatments. Apparent digestibility of organic matter, neutral detergent fiber, and energy decreased with increasing levels of DLL in the ration (p < 0.05). In contrast, digestible crude protein (CP) was higher (p < 0.05) in treatments with 12% and 24% DM of DLL. The inclusion of DLL did not affect (p > 0.05) rumen pH and total volatile fatty acids. Rumen microbial community was not affected (p > 0.05) by treatment. There was a linear reduction (p < 0.05) in CH₄ emissions as the levels of DLL in the ration were increased. Results of this study suggest that an inclusion of 12% DM of ration as DLL enhances digestible CP and reduces daily production of enteric CH₄ without adversely affecting DMI, rumen microbial population, and fermentation parameters.

In order to meet consumer needs, the livestock industry is increasingly seeking natural feed additives with the ability to improve the efficiency of nutrient utilization, alternatives to antibiotics, and mitigate methane emissions in ruminants. Chitosan (CHI) is a polysaccharide with antimicrobial capability against protozoa and Gram-positive and -negative bacteria, fungi, and yeasts while naringin (NA) is a flavonoid with antimicrobial and antioxidant properties. First, an in vitro gas production experiment was performed adding 0, 1.5, 3.0 g/kg of CHI and NA under a completely randomized design. The substrate containing forage and concentrate in a 70:30 ratio on a dry matter (DM) basis. Compounds increased the concentration of propionic acid, and a significant reduction in methane production was observed with the inclusion of CHI at 1.5 g/kg in in vitro experiments (p < 0.001). In a dry matter rumen degradability study for 96 h, there were no differences in potential and effective degradability. In the in vivo study, six crossbred heifers fitted with rumen cannulas were assigned to a 6 × 6 Latin square design according to the following treatments: control (CTL), no additive; chitosan (CHI1, 1.5 g/kg DMI); (CHI2, 3.0 g/kg DMI); naringin (NA1, 1.5 g/kg DMI); (NA2, 3.0 g/kg DMI) and a mixture of CHI and NA (1.5 + 1.5 g/kg DMI) given directly through the rumen cannula. Additives did not affect rumen fermentation (p > 0.05), DM intake and digestibility of (p > 0.05), and enteric methane emissions (p > 0.05). CHI at a concentration of 1.5 g/kg DM in in vitro experiments had a positive effect on fermentation pattern increasing propionate and reduced methane production. In contrast, in the in vivo studies, there was not a positive effect on rumen fermentation, nor in enteric methane production in crossbred heifers fed a basal ration of tropical grass.