This experiment investigated the effects of rumen-protected leucine (RP-Leu) supplementation on growth performance, rumen fermentation parameters, and rumen microbiota of fattening sheep. Forty fattening sheep (19.03 ± 0.38 kg) were randomly divided into four groups. The four groups were fed the basal diet supplemented with RP-Leu at 0 (L-0), 0.5 (L-0.5), 1.0 (L-1.0), or 1.5 (L-1.5) g/d. The experimental period lasted 67 d. The results demonstrated that compared with the L-0 group, the L-1.0 and L-1.5 groups significantly increased the average daily gain (ADG) by 22.2% and 18.0%, respectively (p < 0.05), and reduced the feed-to-gain ratio (F:G ratio) by 20.0% and 13.4% (p < 0.05); the L-1.5 group significantly increased the concentrations of propionate and isovalerate (p < 0.05) and significantly decreased the acetate to propionate ratio (A:P) by 25.7% (p < 0.05); and the L-1.0 group significantly decreased the concentrations of free branched-chain amino acids (BCAAs) in rumen (p < 0.05). A microbiota analysis showed that RP-Leu enriched the abundance of fiber-degrading bacteria. A correlation analysis showed that acetic acid and A:P were positively correlated with norank_f_F082 and norank_f_UCG-011 (p < 0.05). Phenylalanine, isoleucine, valine, and leucine were negatively correlated with norank_o_Bacteroidales (p < 0.05). In conclusion, RP-Leu improved the growth performance of fattening sheep by changing the rumen fermentation parameters and patterns; the optimal supplementation level is 1.0 g/d.

Cottonseed meal (CSM), an important protein-rich feed ingredient, faces limited utilization in livestock diets due to the presence of free gossypol (FG)—a potent antinutritional toxin. This study aimed to isolate FG-degrading bacteria from the cotton bollworm, Helicoverpa armigera, and to evaluate their potential as probiotics in vitro. Eleven gossypol-tolerant strains were isolated from the gut of Helicoverpa armigera larvae using a screening medium containing gossypol as the sole carbon source. Among these, four lactic acid bacteria strains—Pediococcus acidilactici GM-NP, Pediococcus acidilactici GM-P, Enterococcus faecalis GM-6, and Weissella confusa GM-2—were selected for further investigation of their gossypol degradation capacity and probiotic potential. Probiotic characterization revealed that all strains exhibited tolerance to gastrointestinal fluids and bile salts, safe γ-hemolysis, and strong auto-aggregation, cell surface hydrophobicity, and antimicrobial activity. Solid-state fermentation of CSM with these strains reduced FG content by more than 50%, increased crude protein by over 6%, and elevated acid-soluble protein content by more than 70%, thereby effectively enhancing the nutritional quality of CSM. This study is the first to demonstrate that bacterial isolates from the gut of Helicoverpa armigera possess concurrent high-efficiency gossypol degradation and probiotic properties, providing a theoretical foundation for developing novel probiotic resources and promoting the safe utilization of CSM.