The community structure of colonised bacteria in the gastrointestinal tracts (GITs) of pre-weaned calves is affected by extrinsic factors, such as the genetics and diet of the calves; however, the dietary impact is not fully understood and warrants further research. Our study revealed that a total of 6, 5, 2 and 10 bacterial genera showed biologically significant differences in the GITs of pre-weaned calves fed four waste-milk diets: acidified waste milk, pasteurised waste milk, untreated bulk milk, and untreated waste milk, respectively. Specifically, generic biomarkers were observed in the rumen (e.g.,Bifidobacterium, Parabacteroides, Fibrobacter, Clostridium, etc.), caecum (e.g.,Faecalibacterium, Oxalobacter, Odoribacter, etc.) and colon (e.g.,Megamonas, Comamonas, Stenotrophomonas, etc.) but not in the faeces. In addition, the predicted metabolic pathways showed that the expression of genes related to metabolic diseases was increased in the calves fed untreated waste milk, which indicated that untreated waste milk is not a suitable liquid diet for pre-weaned calves. This is the first study to demonstrate how different types of waste milk fed to pre-weaned calves affect the community structure of colonised bacteria, and the results may provide insights for the intentional adjustment of diets and gastrointestinal bacterial communities.

β-casein is a primary protein in milk, and its variants have been associated with changes in the protein content of bovine milk. However, there has been little research focused on the effects of β-casein variants on milk metabolites. In the present study, dairy cows producing milk with β-casein variant A1/A1 (A1), A2/A2 (A2), and their heterozygote A1/A2 (A12) were screened by a high-resolution melting method. Individual milk samples were then collected from each of the cows, and the milk metabolites were separated and analyzed using nuclear magnetic resonance spectroscopy- and liquid-chromatography mass spectrometry-based metabolomics techniques. Differences in metabolites among the variant groups were evaluated by multivariate statistical analysis. The relative abundances of methionine, proline, and α-lactose were the highest in β-casein variant A2 milk, whereas choline, glycine, citric acid, and cyclic adenosine monophosphate (cAMP) showed the highest abundances in variant A1 milk. Metabolic pathways analysis indicated that the differential metabolites between variants A1 and A2 were involved in pantothenate and coenzyme A biosynthesis, butanoate metabolism, and valine, leucine, and isoleucine biosynthesis. Our results reveal the differences in milk metabolites among the β-casein variants A1, A2, and the heterozygote. These findings, thus, provide novel insights into the effects of β-casein variants on milk metabolites, facilitating further research into the mechanism of the biosynthesis of milk components in the mammary gland and the potential physiological function of milk associated with β-casein variants.

Background: The intestine of young ruminants is in the developmental stage and has weaker resistance to the changes of external environment. Improving intestinal health is vital to promoting growth of young ruminants. This study investigated effects of guanidino acetic acid (GAA) and rumen‐protected betaine (RPB) supplementation on growth, dietary nutrient digestion and GAA metabolism in the small intestine of sheep.Methods: Eighteen healthy Kazakh rams (27.46 ± 0.10 kg of body weight and 3‐month old) were categorized into control, test group I and test group II, which were fed a basal diet, 1500 mg/kg GAA and 1500 mg/kg GAA + 600 mg/kg RPB, respectively.Results: Compared with control group, test group II had increased (p < 0.05) average daily gain, plasma creatine level, ether extract (EE) and phosphorus digestibility on day 30. On day 60, the EE apparent digestibility, jugular venous plasma GAA, GAA content in the duodenal mucosa and GAA content in the jejunal and ileal mucosa of test group II were higher (p < 0.05) than other groups. Transcriptome analysis revealed that the differentially expressed genes (DEGs) involved in the duodenal pathways of oxidative phosphorylation and non‐alcoholic fatty liver disease were significantly altered in test group II versus test group I (p < 0.05). Moreover, in the jejunum, the MAPK signalling pathway, complement and coagulation cascade and B‐cell receptor signalling pathway were significantly enriched, with ATPase, solute carrier transporter protein, DHFR, SI, GCK, ACACA and FASN being the significantly DEGs (p < 0.05).Conclusion: Dietary supplementation of RPB on top of GAA in sheep diets may promote sheep growth and development by improving the body's energy, amino acid, glucose and lipid metabolism capacity.

L-Citrulline (L-Cit), a non-essential amino acid, is characterized by its unique extrahepatic metabolism, which significantly enhances the bioavailability of arginine metabolism in tissues. This study investigated the impact of L-Cit supplementation on ruminal microbiota composition, plasma metabolites, reproductive hormones, and antioxidant capacity in Hu ewes. Sixty non-pregnant Hu ewes, similar in age and parity, with an average body weight of 47 ± 5.05 kg, were randomly assigned to either a Control group or Experimental group. The Control group received a basal diet, while the Experimental group was supplemented with 10 g/d of L-Cit in addition to the basal diet for 65 days. Compared to the Control group, the Experimental group exhibited a significantly higher estrus rate. Plasma estradiol (E₂) levels were significantly reduced (p < 0.01), while luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations showed significant increases (p < 0.05). Testosterone (T) content was also significantly elevated (p < 0.01). Plasma levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly higher in the experimental group, with highly significant differences (p < 0.01). The 16S rRNA sequencing analysis revealed that at the phylum level, the relative abundance of Bacteroidetes was decreased, while that of Firmicutes was increased in the experimental group. At the family level, the relative abundance of norank_o__Clostridia_UCG-014 was significantly increased. At the genus level, the relative abundance of Prevotellaceae_UCG-003 was significantly decreased. The main enriched pathways in the CON group were identified as Lipoic acid metabolism and Nicotinate and nicotinamide metabolism. The main enriched pathways in the experimental group were identified as Prion diseases, Chlorocyclohexane and chlorobenzene degradation, Chloroalkane and chloroalkene degradation, Biofilm formation-Escherichia coli, and Phosphotransferase system (PTS). LC–MS analysis indicated significant upregulation of pathways such as drug metabolism by other enzymes, folate biosynthesis, and valine, leucine, and isoleucine biosynthesis, whereas oxidative phosphorylation and propanoate metabolism were significantly downregulated. These results demonstrate that L-Cit supplementation in the diet modulates the ruminal microbiota of Hu ewes, optimizing volatile fatty acid (VFA) proportions, enhancing carbohydrate metabolism, improving xenobiotic degradation capacity, stimulating the synthesis and release of reproductive hormones. Ultimately, these coordinated effects led to a synergistic increase in estrus and conception rates.