Distinct performance and ruminal adaptations to ethyl-cellulose rumen-protected methionine and guanidinoacetic acid during a heat stress challenge in lactating Holstein cows.

Thermal stress alters ruminal function and physiology, compromising health and productivity. Methionine enhances endogenous antioxidant synthesis, whereas guanidinoacetic acid (GAA) supports cellular ATP production. Because methionine serves as a methyl donor for the conversion of GAA to creatine, both nutrients are linked to cellular metabolism and antioxidant defense. The objective was to determine effects of rumen-protected methionine (RPM; Mepron®, Evonik) and GAA (GuanAMINO®, ≥ 97% GAA, ∼1% starch, ∼1% moisture; Evonik), fed alone or in combination during heat stress (HS) on production, thermoregulation, ruminal biopolymer-degrading enzymes and microbiota, ruminal papillae gene expression and cystathionine β-synthase (CBS) activity, and plasma biomarkers. Milk, blood, ruminal fluid and papillae were collected to evaluate lactational, metabolic, microbial, and molecular responses. Ten multiparous Holstein cows (5 intact and 5 with a ruminal cannula; 122 ± 28 DIM, 41.8 ± 3.7 kg milk/d) were used in a replicated 5 × 5 Latin square with 23-d periods (16 d thermoneutral, 7 d HS) and 5 treatments: thermoneutral control (CON-TN), HS control (CON-HS), RPM-HS (RPM at 0.10% of DM), GAA-HS (25 g GAA/d), MIX-HS (RPM at 0.10% of DM plus 25 g GAA/d). Electric heat blankets (EHB) operating at the highest setting (∼40°C) were used to induce HS. Data were analyzed using PROC MIXED (SAS v9.4) with preplanned contrasts: CON-HS vs. CON-TN, RPM-HS vs. CON-HS, RPM-HS vs. GAA-HS, and MIX-HS vs. CON-HS. Compared with CON-TN, CON-HS cows had greater rectal (+0.32°C) and vaginal (+0.34°C) temperatures and respiration rate (+11 beats/min). These variables did not differ among HS treatments. Compared with CON-TN, CON-HS cows had lower milk yield (-3.9 kg/d), milk protein concentration (-0.22 percentage units), and milk protein yield (-0.11 kg/d), whereas MUN was greater (+2.4 mg/dL). Compared with CON-HS, RPM-HS increased milk protein (+0.28 percentage units), casein (+0.11 percentage units), and protein yield (+0.12 kg/d). Milk protein concentration also was greater in RPM-HS than GAA-HS. Total plasma AA concentrations did not differ among treatments (2,056 ± 89 µM), but citrulline concentration was greater in CON-HS than CON-TN (69 vs. 55 µM). Feeding RPM-HS increased plasma cystine concentration compared with CON-HS (24.4 vs. 18.8 µM). At the ruminal level, compared with CON-TN, CON-HS increased the molar proportions of iso-butyrate and isovalerate, and the abundance of Rumicoccus flavefaciens and Succinimonas amylolytica. These variables were not affected by feeding RPM, GAA, or MIX; however, relative to CON-HS, RPM-HS increased cellulase activity and tended to increase xylanase activity. The abundance of Megaspheara elsdenii was greater and Succinimonas amylolytica lower in RPM-HS than CON-HS, whereas GAA-HS increased abundance of Streptococcus bovis approximately 3-fold relative to CON-HS. Ruminal papillae CBS activity more than doubled in CON-HS compared with CON-TN. The HS did not affect mRNA abundance of the 28 target genes evaluated. In contrast, RPM-HS and, to a greater extent, MIX-HS induced coordinated molecular remodeling characterized by upregulation of genes involved in polyamine synthesis, antioxidant responses, and tight-junction signaling. Overall, RPM improved milk protein synthesis and metabolic resilience during HS, whereas MIX primarily enhanced epithelial adaptative responses without additional lactational benefits.