In this paper, we examined the microbiome of the bovine rumen, feces, and milk and attempted to understand how the bacterial communities at each site affected the production and movement of vitamin B ₁₂ around the animal’s body. It was determined that the composition of the bovine rumen microbiome correlates well with vitamin B ₁₂ concentration, indicating that the rumen microbiota may be a good target for manipulation to improve production of this important vitamin.

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D₃ and Ca (HS+D₃/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D₃/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D₃/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D₃/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D₃/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D₃/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D₃/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D₃/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D₃ and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.

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.

Sub-acute ruminal acidosis (SARA) is sometimes observed along with reduced milk fat synthesis. Inconsistent responses may be explained by dietary fat levels. Twelve ruminally cannulated cows were used in a Latin square design investigating the timing of metabolic and milk fat changes during Induction and Recovery from SARA by altering starch levels in low-fat diets. Treatments were (1) SARA Induction, (2) Recovery and (3) Control. Sub-acute ruminal acidosis was induced by feeding a diet containing 29.4% starch, 24.0% NDF and 2.8% fatty acids (FAs), whereas the Recovery and Control diets contained 19.9% starch, 31.0% NDF and 2.6% FA. Relative to Control, DM intake (DMI) and milk yield were higher in SARA from days 14 to 21 and from days 10 to 21, respectively (P < 0.05). Milk fat content was reduced from days 3 to 14 in SARA (P < 0.05) compared with Control, while greater protein and lactose contents were observed from days 14 to 21 and 3 to 21, respectively (P < 0.05). Milk fat yield was reduced by SARA on day 3 (P < 0.05), whereas both protein and lactose yields were higher on days 14 and 21 (P < 0.05). The ruminal acetate-to-propionate ratio was lower, and the concentrations of propionate and lactate were higher in the SARA treatment compared with Control on day 21 (P < 0.05). Plasma insulin increased during SARA, whereas plasma non-esterified fatty acids and milk β-hydroxybutyrate decreased (P < 0.05). Similarly to fat yield, the yield of milk preformed FA (>16C) was lower on day 3 (P < 0.05) and tended to be lower on day 7 in SARA cows (P < 0.10), whereas yield of de novo FA (<16C) was higher on day 21 (P < 0.01) in the SARA group relative to Control. The t10- to t11-18:1 ratio increased during the SARA Induction period (P < 0.05), but the concentration of t10-18:1 remained below 0.5% of milk fat, and t10,c12 conjugated linoleic acid remained below detection levels. Odd-chain FA increased, whereas branched-chain FA was reduced during SARA Induction from days 3 to 21 (P < 0.05). Sub-acute ruminal acidosis reduced milk fat synthesis transiently. Such reduction was not associated with ruminal biohydrogenation intermediates but rather with a transient reduction in supply of preformed FA. Subsequent rescue of milk fat synthesis may be associated with higher availability of substrates due to increased DMI during SARA.

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.

A wide range of nutritional and non-nutritional factors influence milk fat synthesis and explain the large variation observed in dairy herds. The capacity of the animal to synthesize milk fat will largely depend on the availability of substrates for lipid synthesis, some of which originate directly from the diet, ruminal fermentation or from adipose tissue stores. The mobilization of non-esterified fatty acids from adipose tissues is important to support the energy demands of milk synthesis and will therefore have an impact on the composition of milk lipids, especially during the early lactation period. Such mobilization is tightly controlled by insulin and catecholamines, and in turn, can be affected indirectly by factors that influence these signals, namely diet composition, lactation stage, genetics, endotoxemia, and inflammation. Environmental factors, such as heat stress, also impact adipose tissue mobilization and milk fat synthesis, mainly through endotoxemia and an immune response-related increase in concentrations of plasma insulin. Indeed, as proposed in the present review, the central role of insulin in the control of lipolysis is key to improving our understanding of how nutritional and non-nutritional factors impact milk fat synthesis. This is particularly the case during early lactation, as well as in situations where mammary lipid synthesis is more dependent on adipose-derived fatty acids.