The analysis of fecal metabolite profiles could provide novel insights into the mechanisms underlying animal responses to environmental stressors and diet. We aimed to evaluate the effects of a 14-day heat stress period and of dietary mineral and vitamin supplementation under heat stress on fecal metabolite profiles and to investigate their associations with physiological markers of heat stress, leaky gut, and inflammation in lactating dairy cows. Twelve multiparous Holstein cows (42.2 ± 5.6 kg milk/d; 83.4 ± 27.1 DIM) were enrolled in an experiment in a split-plot design. The main plot was the level of dietary vitamin E and Se, as follows: (1) low (L-ESe; 20 IU/kg vitamin E, 0.3 ppm Se) or (2) high (H-ESe 200 IU/kg vitamin E, 1.2 ppm Se). Within each plot, six cows were randomly assigned to either (1) heat stress (HS; Total Humidity Index (THI): 82), (2) pair-feeding in thermoneutrality (TNPF; THI = 64), or (3) HS with vitamin D₃ and Ca supplementation (HS+DCa; 1820 IU/kg and 1.5% Ca; THI: 82) in a replicated 3 × 3 Latin square design with 14-day periods and 7-day washouts. The concentrations of 94 metabolites were determined in fecal samples, including amino acids, fatty acids, biogenic amines, and vitamins. Relative to the L-ESe group, the H-ESe group increased α-tocopherol by threefold, whereas δ-tocopherol was decreased by 78% (P_FDR < 0.01). Nevertheless, correlation analysis between α-tocopherol and all the others fecal metabolites or physiological heat stress measures did not show significant associations. No interactions between main plot and treatments were observed. Relative to TNPF, HS increased plasma tumor necrosis factor-alpha (TNF-α), plasma lipopolysaccharide-binding protein (LBP), milk somatic cell counts (SCC), respiratory rates, rectal temperatures, fecal tridecylic and myristic acids, vitamin B₇, and retinol, whereas it decreased fecal amino acids such as histidine, methyl histidine, acetyl ornithine, and arginine (P_FDR < 0.05). In contrast, HS+DCa increased fecal methyl histidine concentrations and reduced milk SCC, plasma TNF-α, and LBP, as well as rectal temperatures. Discriminant analysis revealed fecal histidine, taurine, acetyl ornithine, arginine, β-alanine, ornithine, butyric + iso-butyric acid, plasma non-esterified fatty acids, TNF-α, LBP, C-reactive protein, and milk SCC were predictive of HS. Several metabolites were predictive of HS+DCa, although only tryptophan was discriminant relative to HS. In conclusion, both heat stress and the supplementation of vitamin D₃ and Ca can influence the fecal metabolome of dairy cows experiencing heat stress, independently of dietary levels of vitamin E and Se. Our results suggest that some fecal metabolites are well associated with physiological measures of heat stress and may thus provide insights into the gut-level changes taking place under heat stress in dairy cows.

Our understanding of metabolic alterations triggered by heat stress is incomplete, which limits the designing of nutritional strategies to mitigate negative productive and health effects. Thus, this study aimed to explore the metabolic responses of heat-stressed dairy cows to dietary supplementation with vitamin D₃/Ca and vitamin E/Se. Twelve multiparous Holstein cows were enrolled in a split-plot Latin square design with two distinct vitamin E/Se supplementation levels, either at a low (ESe-, n = 6, 11.1 IU/kg vitamin E and 0.55 mg/kg Se) or a high dose (ESe+, n = 6 223 IU/kg vitamin E and 1.8 mg/kg Se) as the main plot. Treatment subplots, arranged in a replicated 3 × 3 Latin square design, comprised heat challenge (Temperature Humidity Index, THI: 72.0-82.0) supplemented with different levels of vitamin D₃/Ca: either low (HS/DCa-, 1 012 IU/kg and 0.73%, respectively) or high (HS/DCa+, 3 764 IU/kg and 0.97%, respectively), and a pair-fed control group in thermoneutrality (THI = 61.0-64.0) receiving the low dose of vitamin D₃/Ca (TN). The liquid chromatography-mass spectrometry-based metabolome profile was determined in blood plasma and milk sampled at the beginning (day 0) and end (day 14) of each experimental period. The results were analyzed for the effect of (1) TN vs. HS/ESe-/DCa-, and (2) the vitamin E/Se and vitamin D₃/Ca supplementation. No group or group × day effects were detected in the plasma metabolome (false discovery rate, FDR > 0.05), except for triglyceride 52:2 being higher (FDR = 0.03) on day 0 than 14. Taurine, creatinine and butyryl-carnitine showed group × day interactions in the milk metabolome (FDR ≤ 0.05) as creatinine (+22%) and butyryl-carnitine (+190%) were increased (P < 0.01) on day 14, and taurine was decreased (-65%, P < 0.01) on day 14 in the heat stress (HS) cows, compared with day 0. Most compounds were unaffected by vitamin E/Se or vitamin D₃/Ca supplementation level or their interaction (FDR > 0.05) in plasma and milk, except for milk alanine which was lower (-69%, FDR = 0.03) in the E/Se+ groups, compared with E/Se-. Our results indicated that HS triggered more prominent changes in the milk than in the plasma metabolome, with consistent results in milk suggesting increased muscle catabolism, as reflected by increased creatinine, alanine and citrulline levels. Supplementing with high levels of vitamin E/Se or vitamin D₃/Ca or their combination did not appear to affect the metabolic remodeling triggered by HS.

Few studies have evaluated the impact of a lipid-rich diet with palmitic acid (PA) in ruminal fermentation. The objective was to evaluate the in vitro bath culture of the protected PA on the ruminal fermentative variables. Four diets were used: (a) without protected PA (nPA) and (b) inclusion of protected PA at three levels: PA3 = 3%, PA6 = 6%, and PA9 = 9% dry matter (DM). The maximum gas production occurred without including protected PA and the low gas production given with the inclusion of protected PA9 (p ≤ 0.05). Meanwhile, the inclusion with 3 and 6% of protected PA showed a higher level of gas production kinetics than the other treatments (p ≤ 0.05). DM and organic matter degradation after 72 h of incubation linearly decreased (p ≤ 0.05) with a high level of protected PA. The level of C16:0 in the protected AP increased with the higher level of supplemented AP (p ≤ 0.05). The best efficiency in propionic acid, decrease in methane, and increase in biohydrogenation (79%) occurred with the inclusion of protected PA3 (p ≤ 0.05). In conclusion, supplements with protected PA at 3 and 6% increased PA availability. The results indicate good benefits of protected PA on fermentative variables, and these doses seem ideal for future research in lactating goats.

The aim of this study was to determine plasma biotin (B8), folate (B9), and vitamin B₁₂ (B12) concentrations of multiparous dairy cows under heat stress (HS) fed with adequate or high concentrations of vitamin D₃ (VITD) and E (VITE), Ca, and Se. Twelve multiparous Holstein cows were used in a split-plot design. The main plot was: (1) Adequate VITE and Se; (2) High VITE and Se. Within each main plot, cows were assigned to treatments: (1) HS with adequate concentrations of VITD and Ca; (2) HS with high concentrations of VITD and Ca; (3) pair-feeding (PF) with HS in thermoneutrality with adequate concentrations of VITD and Ca in a duplicated 3 × 3 Latin square. There were no treatment effects on plasma B8 and B12. Plasma B9 was lower by 21% for HS with adequate VITD and Ca than PF cows. Results suggested that HS might modify folate status in dairy cows, regardless of treatments.

Background and Aim: Palmitic acid (PA) (C16: 0) is a rumen-inert long-chain fatty acid (FA) widely used in dairy cattle to increase dietary energy density and milk fat synthesis; however, its effects in dairy goats remain poorly characterized. This study evaluated whether supplementing 3% or 6% PA in the diet of mid-lactation goats could improve milk yield, composition, FA profile, and whole-animal energy balance under semi-arid Mexican production conditions. Materials and Methods: Twenty-one multiparous crossbred goats (45.8 ± 1.2 kg; 21 ± 3 days in milk) were randomly assigned to three treatments for 6 weeks after a 2-week adaptation: (1) Control diet (without PA), (2) diet + 3% PA, and (3) diet + 6% PA on a dry-matter (DM) basis. Diets were isoenergetic and isoproteic before PA addition. Individual DM intake (DMI), milk yield, and composition were measured daily; milk FA profiles and energy balance were determined on days 0, 21, and 42. Data were analyzed using a mixed-model with repeated measures, and means were compared using the Tukey test (p ≤ 0.05). Results: PA inclusion did not affect DMI, body weight, or milk yield. However, milk fat concentration and yield increased significantly (p < 0.01) in both PA treatments, with the highest fat concentration observed at 6% PA. The milk FA profile shifted toward greater C16: 0 and C16: 1 proportion (p < 0.0001) and decreased short-chain (C16) FA fractions. Energy-corrected milk yield rose by ~40% in PA-fed goats, and energy balance improved markedly from week 3 onward, particularly in the 3% group (p < 0.01), indicating superior dietary energy utilization without intake suppression. Conclusion: Moderate PA supplementation (~3% DM) effectively enhances milk fat synthesis and energy efficiency in goats while maintaining stable intake and yield. Increasing PA beyond 3% confers minimal additional benefit and may overly saturate milk fat. These findings provide species-specific evidence that rumen-inert fat inclusion can be an efficient strategy to support metabolic status and product quality in mid-lactation goats under variable forage systems. Keywords: dairy goats, energy balance, fatty acid profile, milk composition, palmitic acid, rumen-inert fat.