The gestational period is a critical developmental window where maternal nutrition programs offspring growth and long-term health. This study evaluated dietary rumen-protected glutamine (RP-Gln) supplementation during late gestation on lamb growth performance, and its regulatory effects on metabolic, immune, and microbial parameters in ewes and offspring. Compared with controls, moderate RP-Gln significantly improved lamb growth, increasing birth weight, 15-day body weight, and average daily gain (p < 0.01). In ewes, RP-Gln elevated serum GGT activity (p < 0.05), improved lipid metabolism, and reduced offspring muscular stress. Antioxidant capacity was enhanced in both ewes and lambs, with higher antioxidant enzyme activities and lower MDA levels (p < 0.05). RP-Gln also remodeled gut microbiota by promoting beneficial genera like Lactobacillus and Faecalibacterium, and reinforcing dam–offspring microbial transmission. Metabolomics revealed elevated polyunsaturated fatty acids, vitamin C, and tyrosine derivatives in ewes, and enhanced aromatic amino acid biosynthesis in lambs. In summary, glutamine supplementation in late-gestation ewes enhances maternal antioxidant and anti-inflammatory capacity, optimizes gut microbiota, and stimulates n-3 PUFA synthesis. These benefits are transmitted to the fetus, improving offspring antioxidant function, immune regulation, and intestinal health. Glutamine also promotes aromatic amino acid synthesis while inhibiting the tryptophan–kynurenine pathway, collectively supporting lamb growth and health.

The optimal timing for initiating starter feeding in lambs remains controversial, warranting a systematic evaluation of its effects across multiple indicators. This study investigated the effects of initiating starter feeding at 7, 14, or 21 days of age on growth performance, rumen fermentation, serum immunity, gastrointestinal development, and microbiota in Hu lambs. Forty-five newborn lambs were randomly assigned to three groups (n = 15 per group) and fed starter until slaughter at weaning (60 days). The 14-day group exhibited significantly higher body weight (BW) at 49 and 60 days compared with the 7-day group (p < 0.05), and greater average daily gain (ADG) during 28–35 days than the 21-day group. Rumen ammonia nitrogen (NH₃-N), acetate, propionate, valerate, and total volatile fatty acids (VFAs) were higher in the 7-day and 14-day groups than in the 21-day group (p < 0.05). The 14-day group showed lower pro-inflammatory cytokines (IL-1β, TNF-α) and higher immunoglobulins (IgA, IgG, IgM) and anti-inflammatory cytokines (IL-2, IL-4) (p < 0.05). This group also displayed improved rumen papilla width, jejunal villus dimensions, and reduced crypt depth. Beneficial microbes such as Christensenellaceae_R-7_group and Butyrivibrio were enriched in the 14-day and 21-day groups. In conclusion, initiating starter feeding at 14 days of age optimizes growth, rumen function, immune response, and colonization of beneficial microbiota in Hu lambs.