Abstract
Few studies have investigated the effects of L-valine (L-Val) supplementation on in vitro rumen fermentation parameters and methane (CH(4)) production in low-nitrogen diets for ruminants. Therefore, we examined the impact of L-Val supplementation in low-protein diets on in vitro rumen fermentation parameters, CH(4) production, and microbial community structure. Two crude protein (CP) levels and 4 L-Val levels were tested as follows: CON group (Control group with 14.05% CP), LD group (low-nitrogen diets with 11.26% CP), LVA group (LD group + 0.25% L-Val), LVB group (LD group + 0.5% L-Val), LVC group (LD group + 0.75% L-Val), and LVD group (LD group + 1% L-Val). The experiment was conducted at five time points (2 h, 4 h, 8 h, 12 h, 24 h), with three replicates per treatment at each time point. Results indicated the following: (1) Fermentation pH decreased overall with time; at each time point, the LVB group exhibited the highest pH, significantly higher than the LD, LVC, and LVD groups (p < 0.05). (2) Ammonia nitrogen (NH(3)-N) concentration increased over time, with LVA~LVD groups showing higher levels than the LD group at 24 h, while showing no difference compared to the CON group (p > 0.05). (3) Microbial protein (MCP) trends aligned with NH(3)-N, with the LVB group exhibiting higher MCP than the LD group, while showing no difference compared to the CON group (p > 0.05). (4) Compared to the LD group, adding 0.5~1% L-Val increased acetic acid, total VFA (TVFA), and isobutyric acid concentrations at 4 h, 8 h, and 24 h fermentation (p < 0.05). (5) The LVB group exhibited higher proportions of protozoa and Fibrobacter succinogenes (F. succinogenes) compared to the LD group (p < 0.05). The proportion of F. succinogenes showed no significant difference from the CON group (p > 0.05), while the proportion of Butyrivibrio fibrisolvens (B. fibrisolvens) decreased when L-Val addition exceeded 0.5%. (6) Correlation analysis revealed positive correlation between protozoa and TVFA (R = 0.512, p = 0.030). Isobutyric acid showed positive correlations with protozoa, B. fibrisolvens, and F. succinogenes (p < 0.05). In summary, under the present experimental conditions, the addition of 0.5% L-Val to a low-nitrogen diet did not affect predicted CH(4) production, but improved other in vitro rumen fermentation parameters, including acetate, isobutyrate and MCP. Meanwhile, it favored the growth and proliferation of the fibrolytic bacteria (B. fibrisolvens and F. succinogenes). This provides a theoretical basis for the rational formulation of low-nitrogen diets for sheep.