Effect of pre-weaning glycyl-glutamine feeding on diarrhea rate, hindgut fermentation, antioxidant stress capability, and fecal microbiota in calves

Glycyl-glutamine (Gly-Gln, GQ) is formed by the dehydration-condensation reaction of glycine and glutamine. Different studies have shown a positive effect of GQ on gut health and growth performance in early neonatal animals. We evaluated oral GQ effects in Holstein calves on growth (body weight, average dairy gain), fecal score, diarrhea rate, serum biomarkers (antioxidants/inflammation/biochemistry/amino acids), and hindgut parameters (fermentation and microbiota). Twenty-two neonatal Holstein heifer calves (1-5 d of age; 36.93 ± 1.052 kg) from multiparous dams were randomly divided into control (CON, n = 11) or GQ-treated (GQ, n = 11) groups. All calves received colostrum within 2 h of birth and pasteurized milk (days 1-28), followed by replacement by milk replacer. Groups received oral gavages every other day for 28 d: CON received 50 mL phosphate-buffered saline (PBS), while GQ received PBS containing GQ (100 mg/mL). Gavages were administered 2 h before morning feeding. Calf growth was monitored for 365 d post-trial. The GQ treatment period lasted for 28 d and calf growth was monitored for 365 d. Statistical analysis used mixed linear models with treatment, day, and their interaction as fixed effects, and individual calf as random effect for repeated measure. Gly-Gln supplementation did not affect body weight or average daily gain but reduced diarrhea incidence and fecal scores (indicating firmer stool consistency; P < 0.001), and increased serum concentrations of albumin, globulin, total protein, glucose, triglycerides, and alanine transaminase (P < 0.05), along with elevated amino acids (Asp, Glu, Ala, Val, and Trp; P < 0.05). Calves receiving Gly-Gln also showed tendencies toward increased glutathione peroxidase and total antioxidant capacity (P < 0.1) in serum. After lipopolysaccharide challenge, GQ did not influence Claudin-1 and glutathione peroxidase 4 relative expression of bovine intestinal epithelial cell in vitro (P < 0.05). Gut microbiota analysis showed higher complexity and increased abundance of beneficial taxa (Fournierella and Erysipelatoclostridium; P < 0.05). Glycyl-glutamine improved gastrointestinal health and altered lipid and glucose metabolism without altering short-chain fatty acid molar proportions. In conclusion, GQ supplementation mitigates early-life diarrhea in calves, possibly by enhancing gut microbiota stability and antioxidant capacity, while reversing diarrhea-induced metabolic declines. These findings highlight GQ as a promising dietary intervention to improve neonatal calf health.