Neonatal calves’ diarrhea, which can be severe enough to cause death, has a significant impact on the global cattle industry. In this study, alfalfa polysaccharides and seaweed polysaccharides were found to significantly improve the diarrhea condition in neonatal calves. To explore the underlying mechanisms, further microbiomic and metabolomic analyses were conducted. This study investigated the impact of alfalfa polysaccharides and seaweed polysaccharides on growth performance, serum metabolites, gut microbiota, and metabolomics in neonatal Holstein calves. A total of 24 newborn calves were randomly assigned to three groups, with 8 calves per treatment group. The control (CON) group was fed a basal diet, the alfalfa polysaccharide (AP) group received a basal diet supplemented with alfalfa polysaccharides (4 g/calf/day), and the seaweed polysaccharide group (SP) received a basal diet supplemented with seaweed polysaccharides (4 g/calf/day). These polysaccharides were plant extracts. Compared to the CON group, the results indicated that SP significantly enhanced the body weight, height, chest circumference, and average daily gain of Holstein calves (p < 0.05), while also reducing the diarrhea rate and improving manure scoring (p < 0.05). Compared to the CON, AP also reduced the diarrhea rate (p < 0.05). In terms of serum biochemistry, supplementation with AP and SP increased serum alkaline phosphatase (ALP) and insulin-like growth factor 1 (IGF-1) levels compared to the CON group (p < 0.05). Both AP and SP elevated serum catalase (CAT) and Total Antioxidant Capacity (T-AOC) levels, indicating enhanced antioxidant status (p < 0.05). Regarding immune responses, supplementation with AP and SP significantly increased serum complement component 3 (C3) and immunoglobulin M (IgM) levels, while significantly reducing pro-inflammatory cytokines interleukin-18 (IL-18), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) compared to the CON group (p < 0.05). Microbiota analysis revealed that AP modulated the abundance of Firmicutes, while SP influenced the abundance of Prevotella and Succiniclasticum. AP and SP differentially influenced intestinal metabolites compared to the CON group, leading to enrichment in pathways related to immunity, antibacterial, and anti-inflammatory functions. These pathways included the biosynthesis of alkaloids from ornithine, lysine, and nicotinic acid, glucocorticoid and mineralocorticoid receptor canothersis/antagonists, secondary metabolite biosynthesis, and alkaloid biosynthesis from histidine and purine, thus alleviating intestinal inflammation. Therefore, by supplementing with AP and SP, the diarrhea rate in calves was reduced, and the immune function of Holstein calves was enhanced, while simultaneously promoting a higher relative abundance of beneficial gut bacteria and suppressing the relative abundance of pathogenic bacteria. Additionally, gut pathways associated with immune response and inflammation were modulated by AP and SP. This study provided valuable insights and theoretical underpinnings for the use of AP and SP in preventing diarrhea in neonatal calves.

Introduction: To elucidate the effects of fluctuating dietary crude protein (CP) on Holstein dairy cows, this study investigated lactation performance, rumen bacterial communities, and metabolome profiles.Methods: In a 60-day trial, twenty-four Holstein cows were randomly assigned to a fluctuating CP diet (FCP), high CP diet (HCP, 18.05% CP), or intermediate CP diet (ICP, 16.05% CP). FCP alternated between high (18.05%, FCP-HCP) and low CP (14.04%, FCP-LCP) every 48 h, forming a 96-h cycle. During the final cycle, blood, milk, and urine samples were collected to measure plasma urea nitrogen (PUN), milk urea nitrogen (MUN), and urine urea nitrogen (UUN). Rumen bacterial diversity and metabolomic profiles were also analyzed.Results: Fluctuating CP did not significantly affect lactation performance (p > 0.05). PUN in FCP was lower than HCP on Day 1 (p < 0.01), while MUN in ICP was lower than HCP and FCP on Day 2 (p < 0.01). On Day 3, PUN and UUN in FCP exceeded HCP and ICP (p < 0.05), and on Day 4, PUN in FCP was again lower than HCP (p < 0.01). FCP had lower Proteobacteria on Day 2 (p < 0.05) and lower Patescibacteria on Days 3–4 (p < 0.05). Metabolomics indicated shifts mainly in amino acid, energy, purine, and pyrimidine metabolism.Discussion: These findings show that short-term CP fluctuations induce dynamic changes in nitrogen excretion, rumen bacterial composition, and key metabolic pathways without affecting milk production. This suggests that dietary CP variation primarily influences nitrogen metabolism and rumen bacterial dynamics rather than lactation performance.

Alpha lipoic acid (α-LA) is a powerful antioxidant, which can reduce oxidative damage and inflammation in the host. In previous research, we found that 600 mg/kg α-LA supplemented in the diet could improve the activity of antioxidant enzymes and regulate the secretion of inflammatory factors in rumen of sheep. However, the mechanism of α-LA driving the antioxidant capacity in rumen of sheep remains unknown. The aim of this current research was to investigate the role of α-LA in antioxidant and inflammatory processes in the rumen of sheep. Transcriptomic and metabolomic analyses were performed to assess the variation of genes and metabolites of rumen epithelial tissue in sheep induced in the LA (600 mg/kg α-LA) group compared with the CTL (0 mg/kg α-LA) group. The results showed that some discovered core genes in the rumen epithelial tissue were negatively correlated with antioxidant activity. G6PD and HK2, the downregulated and upregulated core genes in the comparison of LA/CTL, were enriched in the pentose phosphate pathway (PPP) and the pathways of mannose and oligosaccharide metabolism, respectively. The PPP is a metabolic pathway within cells, primarily functioning to produce nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. The differential metabolites were enriched in the pathway of 2-oxocarboxylic acid metabolism, which improves the antioxidant capacity of the rumen epithelium by increasing enzymatic activities of SOD. In conclusion, the present study demonstrated that α-LA improved antioxidant activity by regulating PPP and 2-oxocarboxylic acid metabolism. This study will provide a theoretical basis for the application of α-LA in the raising of sheep.

κ-casein (κ-CN, CSN3) variants affect milk composition, processing, milk protein content and milk yield. Whether the CSN3 mutation affects the expression of its own genes, which in turn affects the casein content and milk production, is unknown. To address this question, we conducted an experiment on the effects of κ-CN genotypes and transcripts on κ-CN expression and DHI indices. Through PCR sequencing, PCR-RFLP, western blot and PCA analysis, the expression of κ-CN in the κ-CN BB genotype was significantly greater than that in AA genotype and AB genotype (P < 0.05). For the heterozygous κ-CN AB genotype, there was no significant difference in the expression level of κ-CN between transcript A and transcript B (P > 0.05). The κ-CN transcripts were more suitable than the κ-CN genotypes as classification indicators associated with dairy herd improvement (DHI) indices such as milk protein content and milk production. Therefore, κ-CN variants do affect the content of κ-CN in milk, but it cannot be confirmed that the content of the κ-CN B variant is definitely higher than the κ-CN A variant, nor can the difference law in milk protein content be confirmed. This phenomenon may be related to regulatory changes caused by in the non-coding region of κ-CN.