In this study, the effect of 0, 40, and 80 μmol/L of LA on triglyceride (TG) content was evaluated, as well as the mRNA expression of genes involved in milk fat synthesis in bovine mammary epithelial cells (BMECs) which were cultured in two-dimensional (2D) and three-dimensional (3D) models. TG content was measured with a TG determination kit, and the expression of genes was measured with real-time quantitative polymerase chain reactions (RT-qPCR). Adding 80 μmol/L of LA significantly increased intracellular TG content in both 2D and 3D cultured BMECs (p < .05). The mRNA expression of diacylglycerol acyltransferase 2 (DGAT2) and peroxisome proliferator-activated receptor-γ (PPARG) increased with the addition of 80 and 40 μmol/L of LA, respectively (p < .05), whereas the expression of PPARG was downregulated in the 2D model (p < .05). Adding LA significantly reduced the expression of sterol regulatory element-binding transcription factor 1 (SREBF1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), and fatty acid-binding protein 3 (FABP3) compared to the control group (p < .05). Altogether, TG content in BMECs and expression of genes involved in milk fat synthesis were significantly higher with addition of LA under 3D BMECs culture model than those of 2D (p < .05), and 40 and 80 µmol/L of LA inhibited the de novo synthesis of fatty acids and promoted the expression of DGAT2 and accumulation of TG. The mRNA expression of PPARG in 3D model was increased by addition of 40 μmol/L LA, whereas it was downregulated in 2D model.

The purpose of this research was to investigate the impact of chestnut tannins (CHTs) on nutrient digestibility, nitrogen balance, in situ crude protein (CP) digestibility, protease enzymes, and microbial community composition in sheep. Eighteen 1.5-year-old sheep (43.0 ± 2.0 kg initial BW) fitted with permanent ruminal cannula were selected and randomly divided into three groups, which were fed with CHTs added at 0, 2, and 6%/kg DM. The pre-feeding period lasted for 12 days, and the actual trial period was 18 days. Rumen fluid was collected to assess in situ crude protein (CP) degradability, while rumen digesta was analyzed for total and ruminal proteolytic bacterial populations. Using one-way ANOVA in SAS to analyze data, the results indicated that 2% CHT reduced in situ degradability by 26.23%, while 6% reduced it by 58.01% in the rumen of the sheep. The CP apparent digestibility, nitrogen metabolism, and population of proteolytic bacteria of sheep were decreased in the 6% CHT group (p < 0.05), while the above indices of the 2% CHT group were not affected. Furthermore, CHT supplementation significantly altered the ruminal microbial community structure. Particularly in the 2% CHT group, the relative abundances of Bacteroidota and Prevotella increased. LEfSe analysis revealed that Bacteroidale replaced U29-B03 as the dominant microbiota at 2% CHT. Doses of 2% CHT can be incorporated into sheep diets without impairing digestion. These findings support the inclusion of CHT doses of less than 2% for enhancing protein digestion and increasing the types of beneficial bacteria in the rumen, while doses above 6% should be avoided.

: Negative energy balance (NEB) in dairy cows induces excessive lipolysis, leading to elevated levels of β-hydroxybutyric acid (BHBA), which, when accumulated, can cause liver damage. Rutin (RT), a natural flavonoid with antioxidant and anti-inflammatory properties, has demonstrated potential hepatoprotective effects; however, its ability to mitigate BHBA-induced hepatocellular injury in calves remains unclear. This study first assessed the impact of various BHBA concentrations on oxidative stress in calf hepatocytes, then explored the protective effects and underlying mechanisms of RT, and finally employed untargeted metabolomics to further elucidate RT’s mode of action. The results showed that exposure to 1.2 mM BHBA significantly increased malondialdehyde (MDA), nitric oxide (NO) contents, and reactive oxygen species (ROS) levels, while markedly decreasing glutathione (GSH) content and catalase (CAT) activity compared with the blank control. Notably, pretreatment with 100 μg/mL RT resulted in the greatest increase in GSH contents (180%) compared to BHBA treatment alone, while 150 μg/mL RT led to the most pronounced reduction in MDA contents (220%). Furthermore, BHBA treatment significantly upregulated the expression of Kelch-like ECH-associated protein 1 (Keap1) and downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO1), and heme oxygenase-1 (HO-1) at both the mRNA and protein levels. These alterations were effectively reversed by pretreatment with 100 μg/mL RT. Non-targeted metabolomics identified 1525 metabolites in total. Based on OPLS-DA, metabolites with a variable importance in projection (VIP) > 1 and p < 0.05 were considered significantly altered. Compared with the blank control, BHBA treatment upregulated 47 metabolites—including 8-hydroxy-2′-deoxyguanosine, 3-hydroxyisovaleric acid, and N-palmitoyl-sphingosine—and downregulated 58 metabolites, such as betaine, linolenic acid, and arachidonic acid. In contrast, RT pretreatment upregulated 207 metabolites relative to the BHBA treatment, including linolenic acid, taurocholic acid, and 4-hydroxybenzoic acid, and downregulated 126 metabolites, including 3-hydroxyisovaleric acid, 8-hydroxy-2′-deoxyguanosine, and pyruvaldehyde. Pathway enrichment analysis indicated that RT alleviated BHBA-induced hepatocyte injury primarily by modulating the fatty acid degradation pathway. In summary, RT mitigated BHBA-induced oxidative stress in calf hepatocytes by regulating the Keap1/Nrf2 signaling pathway and further exerted protective effects through metabolic reprogramming.