The intestinal barrier plays a critical role in maintaining gastrointestinal health and nutrient utilization in yaks. This study investigated the effects of dietary RPG level and RPT supplementation on colonic barrier function and microbial composition in yaks. Twenty-eight healthy male yaks (3 years old; 192.7 ± 4.52 kg) were assigned to a 2 × 2 factorial design with two dietary rumen-protected glucose (RPG) levels (1.0% or 3.0% of dietary DM) and two rumen-protected taurine (RPT) supplementation levels (5 or 20 g/animal/day) for 63 days. High-level RPG impaired colonic physical barrier function by reducing tight junction protein expression and microbial diversity, whereas high-level RPT mainly compromised chemical and immune barrier function by decreasing diamine oxidase activity, mucin-2, and secretory immunoglobulin A, accompanied by alterations in the colonic microbial community. Significant interactions between RPG and RPT were observed for several barrier- and microbiota-related indices. Overall, the effects of RPG and RPT on colonic health were dose-dependent, and moderate supplementation, particularly the combination of 1.0% dietary RPG and 5 g/day RPT, was the most effective in maintaining colonic barrier integrity and microbial homeostasis in yaks.

Yaks (Bos grunniens) on the Qinghai–Tibetan Plateau face severe nutritional limitations during the dry season due to dependence on highly lignified, low-quality roughage. Identifying safe and effective rumen regulators capable of enhancing fiber utilization in this species is therefore of great practical importance. This study employed a two-pronged approach integrating in vitro mechanistic investigation and in vivo validation to evaluate the effects of the amphoteric surfactant cocamidopropyl betaine (CAPB) on rumen fermentation, the micro-spatial distribution of digestive enzymes, apparent total tract digestibility, and the macroscopic growth performance of yaks. In the in vitro fermentation trial (Experiment 1), a randomized block design was employed where a straw-based high-forage diet was used as the substrate and supplemented with 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% CAPB (based on substrate dry matter, DM) for a 48 h batch culture. The results showed that as the CAPB supplementation level increased, cumulative gas production, the degradation rates of DM and neutral detergent fiber (NDF), and the yields of total volatile fatty acids and microbial protein all exhibited significant quadratic responses (p < 0.05), peaking at the 0.5–1.0% supplementation levels. Concurrently, CAPB significantly promoted the transfer and release of carboxymethyl cellulase and xylanase into the free liquid phase (p < 0.01). In the in vivo validation trial (Experiment 2), 24 healthy growing male yaks (initial body weight 131.2 ± 8.4 kg) were allocated in a completely randomized design to four groups and fed a basal diet supplemented with 0, 0.5, 1.0, or 2.0% CAPB for 44 days. The results indicated that, while maintaining a stable DM intake, the addition of 0.5% CAPB significantly increased the average daily gain (ADG) of yaks (p < 0.05), improved the feed-to-gain ratio, and significantly enhanced the apparent total tract digestibility of NDF and ether extract (p < 0.05). However, when the supplementation dose exceeded the safety threshold (≥2.5% in vitro and ≥2.0% in vivo), both fermentation parameters and growth advantages declined. In conclusion, under the present experimental conditions, 0.5% CAPB improved roughage fermentation efficiency, putatively through an ‘enzyme elution’ mechanism, and was associated with macroscopic improvements in NDF and EE apparent digestibility and ADG in growing yaks. These findings identify 0.5% CAPB as a promising candidate rumen regulator for improving roughage utilization in growing yaks; broader generalization will require larger-scale and longer-duration trials.