Anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), sodium citrate (Na-citrate), or heparin are normally used in hematological clinical tests to prevent coagulation. Although anticoagulants are fundamental for the correct application of clinical tests, they produce adverse effects in different fields, such as those involving specific molecular techniques; for instance, quantitative real time polymerase chain reactions (qPCR) and gene expression evaluation. For this reason, the aim of this study was to evaluate the expression of 14 genes in leukocytes that were isolated from the blood of Holstein cows, and which were collected in Li-heparin, K-EDTA, or Na-citrate tubes; then, they were analyzed using qPCR. Only the SDHA gene showed a significant dependence (p ≤ 0.05) on the anticoagulant that was used with the lowest expression; this was observed in Na-Citrate after being compared with Li-heparin and K-EDTA (p < 0.05). Although a variation in transcript abundance with the three anticoagulants was observed in almost all the investigated genes, the relative abundance levels were not statistically significant. In conclusion, the qPCR results were not influenced by the presence of the anticoagulant; thus, we had the opportunity to choose the test tube that was used in the experiment without interfering effects impacting the gene expression levels caused by the anticoagulant.

This study evaluated the effects of supplementing dairy calves with Saccharomyces cerevisiae fermentation-derived postbiotics (SCFP) on growth, metabolism, immune status, and first lactation performance. Eighteen Holstein heifer calves were blocked by birth body weight and serum total protein and randomly assigned to control (CTR; n = 9; no supplementation) or SCFP (n = 9; 1 g/d SmartCare^® in milk replacer until weaning plus 5 g/d NutriTek^® until 70 d; Diamond V™, USA). Calves were weaned at 60 d and monitored until 160 d. Feed intake did not differ between groups. SCFP calves had greater post-weaning average daily gain from 71 to 100 d (0.93 vs. 0.60 kg/d, SCFP and CTR, respectively) and body weight from 100 to 160 d. They tended to have greater plasma β-hydroxybutyrate at 60 (0.32 vs. 0.27 mmol/L, SCFP and CTR, respectively) and 70 d (0.46 vs. 0.42, SCFP and CTR, respectively) and urea at 70 d (4.89 vs. 4.33 mmol/L, SCFP and CTR, respectively) and had greater acetate (515 vs. 384 μmol/L, SCFP and CTR, respectively) and propionate (33.13 vs. 22.4 ± 4.86 μmol/L, SCFP and CTR, respectively) at 60 d. SCFP calves also had lower nonesterified fatty acids at 21 d (0.23 vs. 0.38 mmol/L, SCFP and CTR, respectively), suggesting reduced energy mobilization during the most critical pre-weaning stage. Plasma myeloperoxidase was greater at 70 d (340 vs. 262 U/L, SCFP and CTR, respectively), as was phagocytosis by polymorphonuclear neutrophils at 60 (+10.4%) and 70 d (+8.2%). Feeding SCFP increased rumen activity and plasma volatile fatty acid concentrations, likely due to enhanced nutrient absorption and reduced weaning stress. SCFP calves exhibited a better immune response to lipopolysaccharide stimulation, as indicated by leukocyte gene expression, MPO, and PMN phagocytosis. Metagenomic analyses showed minor but significant changes in early-life microbiota composition at 7, 21, and 42 d. During first lactation, SCFP cows produced 2.1 kg/d more milk in the first 100 days in milk compared with CTR. In conclusion, early supplementation with SCFP supported rumen development, improved metabolic and immune function, and may enhance future productivity in dairy cows.

Early life is a critical phase for dairy calves, influencing their growth, metabolism, and overall health. This study aimed to evaluate the effects of feeding either whole milk (WM) or milk replacer (MR) on growth performance, metabolic markers, and hematological parameters from birth until weaning at 60 d of age in Fleckvieh dairy. Eighteen heifer calves were assigned to one of 2 dietary treatments: WM (n = 9; DM basis, 29.1% protein, 27.3% fat, and 37.3% lactose; 5.25 Mcal/kg of DM) or MR (n = 9; DM basis, 22.9% protein, 19.1% fat, and 51.73% lactose; 4.76 Mcal/kg DM; Elvor, Maen Roch, France). The MR was prepared at 130 g/L to match solids content in WM. Calves were housed in individual hutches, fed 3 L of milk twice daily until 53 d and then 3 L of milk once daily until weaning (60 d). Calves had ad libitum access to starter and intake was recorded daily. Growth performance was assessed through BW and period-specific ADG (gain per day between successive weighings), whereas metabolic and hematological markers were analyzed through plasma biomarkers and blood cell counts, respectively. Data were subjected to ANOVA using the GLM procedure for single measures and the GLIMMIX procedure for repeated measures. Despite similar total DMI, calves fed WM exhibited an overall greater BW and greater period-specific ADG (0.77 vs. 0.85 kg/d in MR and WM, respectively), and from 21 to 60 d, WM calves were 3.8 kg heavier than MR-fed calves. From the metabolism standpoint, feeding WM resulted in overall greater plasma concentrations of glucose, urea, cholesterol, alkaline phosphatase, and Ca, but lower bilirubin. Specifically, WM calves had greater paraoxonase activity at 35, 45, and 60 d (weaning), greater ceruloplasmin levels at 15, 21, and 28 d, and greater Mg at 60 d of age. Among hematological parameters, WM calves had greater circulating total leukocytes at 21 and 28 d of age due to enhanced neutrophils. Although WM calves consumed milk with greater proportions of fat and protein and lower lactose content, the intake of milk and starter DM and total energy was similar compared with MR calves. However, considering the better growth rate of WM calves (especially in the second week of life), the better balance between fat, protein, and lactose suggests that feeding WM can enhance bioavailability of nutrients. Further research is needed to explore the long-term implications of these feeding strategies on calf health and productivity, particularly in relation to sustainability and economic feasibility.

The transition period is characterized by major metabolic and immunological changes that increase disease susceptibility in dairy cows. Genetic background may influence the regulation of immune responses during this phase. The aim of this study was to compare the expression of immune-related genes in circulating leukocytes of Brown Swiss and Holstein cows across the transition period. Twenty-two pregnant heifers (10 Brown Swiss and 12 Holstein) were managed under identical conditions and sampled at −21, +7, and +28 days relative to calving. Whole blood was collected using PAXgene tubes, and gene expression was analyzed by quantitative PCR. Target genes were associated with immune recognition, leukocyte migration and adhesion, antimicrobial activity, oxidative stress, and inflammatory response. Gene expression profiles differed between breeds over time. Holstein cows showed higher expression of genes involved in pathogen recognition before calving, including CD14 and TLR4. In contrast, Brown Swiss cows showed higher expression of genes related to leukocyte migration (ITGAL, CCR2), antimicrobial activity (MMP9, MPO), inflammatory response (TNFA, IL18), and oxidative stress (ALOX5, ALOX15), particularly after calving. These results indicate breed-related differences in leukocyte gene expression and suggest that genetic background influences immune adaptation during the transition period.

Weaning represents a critical transition in dairy calves, involving simultaneous dietary, social, and environmental changes. This study investigated how the weaning affects immune-related gene expression in circulating leukocytes of Holstein calves and whether post-weaning growth performance is associated with distinct immunometabolic responses. Forty female calves were retrospectively classified into two groups based on average daily gain (ADG) during the 10 days following complete weaning (from 60 to 70 days of age). Calves were fully weaned at 60 days of age using a step-down approach. Animals were categorized as high ADG (HIGH, n = 20) or low ADG (LOW, n = 20) Blood samples collected at 60 and 70 days were analyzed for plasma metabolites and leukocyte transcriptomic profiles targeting 37 genes involved in innate immunity, inflammation, oxidative stress, and metabolic regulation. Weaning induced a marked activation of innate immune pathways in all calves, as evidenced by increased expression of CD14, NFKB1, IL1β, TNFα, and genes related to cell adhesion and trafficking. However, LOW calves exhibited greater transcriptional responses, with higher expression of TLR2, CASP1, IL18, CD16, and markers of oxidative (SOD2, HSPA5) at 70 days, indicating a metabolic and transcriptional profile consistent with a greater physiological burden. Additionally, at 70 days, LOW calves had lower plasma glucose, insulin, triglycerides, and cholesterol, along with higher urea and creatinine, while β-hydroxybutyrate did not differ between groups, suggesting reduced energy availability and increased reliance on amino acid catabolism. These findings suggest that calves with lower post-weaning growth exhibit greater immune activation and altered metabolic status, highlighting variability in resilience to weaning stress. Understanding such differences may support the development of targeted nutritional or management strategies to improve early-life adaptation and long-term productivity in dairy systems

Heat stress (HS) during the transition period disrupts metabolic adaptation and mammary gland (MG) remodeling. Methionine, a rate-limiting amino acid, supports cellular growth and stress responses through one-carbon metabolism pathways. We evaluated whether rumen-protected methionine (RPM) supplementation during the transition period preserves MG structure and function in heat-stressed multiparous Holstein cows. We hypothesized that RPM would mitigate the negative effects of HS on MG microstructure, gene expression, and cellular turnover. A total of 53 multiparous Holstein cows were enrolled at dry-off and assigned to diets with or without RPM (MET, CON). Four weeks prepartum, all MET and half of CON cows were fitted with electric heat blankets to induce HS (METHS, CONHS), while remaining CON cows were thermoneutral (CONTN). MG biopsies (8 and 28 days in milk; DIM) and milk samples were collected to assess microstructure, cellular turnover, gene expression, and mammary epithelial integrity. HS appeared to impair MG remodeling, with fewer but larger alveoli and a thicker epithelial layer, with a slight increase in epithelial cell proliferation, increased occludin fluorescence intensity, and increased milk concentration of K⁺. Meanwhile, RPM under HS increased stromal proliferation, lower occludin fluorescence intensity, and a reduced Na⁺:K⁺ ratio, indicating overall improved mammary epithelial integrity. Additionally, RPM partially promoted greater alveolar organization early postpartum and reduced lumen expansion, with coordinated changes in one-carbon metabolism, amino acid transport, and mTOR-related pathways. Overall, HS disrupts MG cellular dynamics, whereas close-up methionine supplementation partially improves mammary adaptability under chronic transition period HS.