Background: The rumen is the hallmark organ of ruminants, playing a vital role in their nutrition and providing products for humans. In newborn suckling ruminants milk bypasses the rumen, while in adults this first chamber of the forestomach has developed to become the principal site of microbial fermentation of plant fibers. With the advent of single-cell transcriptomics, it is now possible to study the underlying cell composition of rumen tissues and investigate how this relates the development of mutualistic symbiosis between the rumen and its epithelium-attached microbes.Results: We constructed a comprehensive cell landscape of the rumen epithelium, based on single-cell RNA sequencing of 49,689 high-quality single cells from newborn and adult rumen tissues. Our single-cell analysis identified six immune cell subtypes and seventeen non-immune cell subtypes of the rumen. On performing cross-species analysis of orthologous genes expressed in epithelial cells of cattle rumen and the human stomach and skin, we observed that the species difference overrides any cross-species cell-type similarity. Comparing adult with newborn cattle samples, we found fewer epithelial cell subtypes and more abundant immune cells, dominated by T helper type 17 cells in the rumen tissue of adult cattle. In newborns, there were more fibroblasts and myofibroblasts, anIGFBP3⁺epithelial cell subtype not seen in adults, while dendritic cells were the most prevalent immune cell subtype. Metabolism-related functions and the oxidation-reduction process were significantly upregulated in adult rumen epithelial cells. Using 16S rDNA sequencing, fluorescence in situ hybridization, and absolute quantitative real-time PCR, we found that epithelialDesulfovibriowas significantly enriched in the adult cattle. Integrating the microbiome and metabolome analysis of rumen tissues revealed a high co-occurrence probability ofDesulfovibriowith pyridoxal in the adult cattle compared with newborn ones while the scRNA-seq data indicated a stronger ability of pyroxidal binding in the adult rumen epithelial cell subtypes. These findings indicate thatDesulfovibrioand pyridoxal likely play important roles in maintaining redox balance in the adult rumen.Conclusions: Our integrated multi-omics analysis provides novel insights into rumen development and function and may facilitate the future precision improvement of rumen function and milk/meat production in cattle.

The aim of this study was to investigate the effects of N-carbamoylglutamate (NCG) supplementation during the transition period on the functions of blood polymorphonuclear neutrophils (PMN), inflammation, and oxidative stress in dairy cows. Thirty multiparous Chinese Holstein dairy cows at wk 4 before parturition were blocked into 2 groups by parity, body weight, and milk yield of previous lactation, and randomly allocated to 2 dietary treatments of basal diet supplemented without (control, n = 15) or with 20 g/d per cow of NCG (NCG, n = 15). The supplementation was carried out from d -21 to 21 relative to calving. Health incidents (mastitis, retained placenta, and lameness) were recorded, and blood samples were collected at d -21, -7, 0 (the calving date), 7, and 21 relative to parturition and analyzed for variables related to inflammation and oxidative stress. In addition, whole blood was collected at d 7 to isolate PMN and used for analysis of the expression of functional genes and from d -21 to 21 for determination of weekly hematological parameters. The number of lymphocytes was greater at d 7 in the blood of NCG cows. The plasma level of malondialdehyde was lower in the NCG group, and blood reactive oxygen species were lower at d 7, whereas total antioxidant capacity tended to be greater in the NCG group and glutathione peroxidase tended to be higher at d 21 in cows fed NCG, suggesting that NCG supplementation improved antioxidation in cows. In addition, the concentration of serum amyloid A was lower in NCG-fed animals during the postpartum stage. Blood concentrations of IL6 and tumor necrosis factor-α were lower and tended to be lower in NCG-fed animals at d 7, respectively. Meanwhile, the concentrations of IL6 tended to be lower in NCG-fed animals at d 21. Furthermore, the expression of S100A9 and MMP9 in the PMN was lower and tended to be lower, respectively, whereas the expression of ITGB2, XBP1 tended to be higher and expression of CLEC6A was higher in NCG-fed cows. Overall, our results indicated that supplementation with NCG during the transition period showed the beneficial effects on animal health, by improving PMN functions and alleviating inflammation status and oxidative stress in dairy cows.

The transition period in dairy cows is a critical stage and peripartum oxidative status, negative energy balance (NEB) and inflammation are highly prevalent. Fecal microbial metabolism is closely associated with blood oxidative status and nonesterified fatty acids (NEFA) levels. Here, we investigated dynamic changes in total oxidative status markers and NEFA in blood, fecal microbiome and metabolome of 30 dairy cows during transition (-21d, -7d, +7d, +21d relative to calving). Then the Bayesian network and 9 machine learning algorithms were applied to dismantle their relationship. Our results show that the oxidative status indicator (OSI) of -21d, -7d, +7d was higher than +21d (P < 0.001). The plasma concentration of nonesterified fatty acids (NEFA) peaked on +7d (P < 0.001). For fecal microenvironment, a decline in bacterial α diversity was observed at postpartum (P < 0.001) and in bacterial interactions at +7d (P = 0.014). Conversely, microbial metabolites involved in carbohydrate, lipid and energy metabolism increased on +7d (P < 0.05). A correlation analysis revealed that 11 and 10 microbial metabolites contributed to OSI and NEFA variations, respectively (Arc. strength >0.5). The support vector machine (SVM) radial model showed the highest average predictive accuracy (100% and 88.9% in the test and external data sets) for OSI using 1 metabolite and 3 microbiota. SVM radial model also showed the highest average diagnostic accuracy (100% and 91% in the test and external data sets) for NEFA with 2 metabolites and 3 microbiota. Our results reveal a relationship between variation in the fecal microenvironment and indicators of oxidative status, NEB and inflammation, which provide a theoretical basis for the prevention and precise regulation of peripartum oxidative status and NEB.

This study investigated the effects of ursodeoxycholic acid (UDCA) and bile salt (BAS) supplementation on lactation performance, health, and gut microbiota in periparturient dairy cows. Fifty-one Holstein dairy cows were selected at d -28 before parturition and blocked into 3 dietary treatments, including the control (CON; n = 17) received a basal diet, whereas the UDCA (n = 17) and BAS groups (n = 17) were supplemented with 10 g/d UDCA and 20 g/d BAS from d -21 to +21, with an observation phase until d +35. Milk yield and composition were recorded weekly, whereas the DMI were measured biweekly. Blood samples were collected at d +7 and +21, whereas rumen fluid and fecal samples were collected at d +21. Milk yield was significantly higher in the UDCA group at d +21 compared with the CON group, whereas on d +28, milk yield was significantly higher in both the UDCA and BAS groups compared with the CON group, and the DMI of the UDCA group showed an increased tendency at prepartum. Plasma nonesterified fatty acids were significantly higher in the BAS group, whereas Ala aminotransferase content were significantly lower in the UDCA group compared with the control. Furthermore, the cholesterol, malondialdehyde, oxidative stress index, serum amyloid A, and haptoglobin content were significantly lower in the UDCA and BAS groups. In total, 35, 43, and 45 plasma bile acids (BA) were detected in the control, UDCA, and BAS groups, respectively. Compared with the control, 8 key BA, including UDCA, tauroursodeoxycholic acid, glycoursodeoxycholic acid, and 5 key BA, including tauro-β-muricholic acid and hyocholic acid, were identified in the UDCA and BAS groups, respectively. The concentrations of total VFA and acetate in the UDCA and BAS groups were higher than that in the CON group, and the concentration of propionate tended to be higher. The β-diversity of both rumen and gut microbiota was significantly higher in the CON, UDCA, and BAS groups, whereas no significant changes were observed in α-diversity. Key rumen VFA-production bacteria, including Prevotella_7, Succinivibrionaceae_UCG-001, and Selenomonas, were enriched in the UDCA and BAS groups, along with an increase in beneficial gut microbiota, such as Butyrivibrio, Ruminococcus, and Caproiciproducen, and a reduction in harmful bacteria, such as Stenotrophomonas and Chryseobacterium. These findings suggest that the observed improvements in production performance and health may be mediated by alterations in peripheral BA and rumen and gut microbiota, offering insights for optimizing the nutrition and health of transitional dairy cows.

Background: Mammary health is important for transition dairy cows and has been well recognized to exert decisive effects on animal welfare. However, the factors influencing mammary health are still unclear. Differential somatic cell count (DSCC) could reflect the mastitis risk since it is the percentage of neutrophils plus lymphocytes in total somatic cells and could be reflective of mammary health of dairy cows. This work aimed to investigate the assessment and prognosis of the health of transition cows based on blood neutrophil extracellular traps (NETs). Results: Eighty-four transition Holstein dairy cows were selected. The serum was sampled in all the animals at week 1 pre- and postpartum, and milk was sampled at week 1 postpartum. Based on the DSCC in milk at week 1, cows with lower (7.4% ± 4.07%, n = 15) and higher (83.3% ± 1.21%, n = 15) DSCCs were selected. High DSCC cows had higher levels of red blood cell counts (P < 0.05), hemoglobin (P = 0.07), and hematocrit (P = 0.05), higher concentrations of serum oxidative variables [(reactive oxygen species (P < 0.05), malondialdehyde (P < 0.05), protein carbonyl (P < 0.05), and 8-hydroxy-2-deoxyguanosine (P = 0.07)], higher levels of serum and milk NETs (P < 0.05) and blood-milk barrier indicators, including serum β-casein (P = 0.05) and milk immunoglobulin G2 (P = 0.09), than those of low DSCC cows. In addition, lower concentrations of serum nutrient metabolites (cholesterol and albumin) (P < 0.05) and a lower level of serum deoxyribonuclease I (P = 0.09) were observed in high DSCC cows than in low DSCC cows. Among the assessments performed using levels of the three prepartum serum parameters (NETs, deoxyribonuclease I and β-casein), the area under the curve (0.973) of NETs was the highest. In addition, the sensitivity (1.00) and specificity (0.93) were observed for the discrimination of these cows using NETs levels with a critical value of 32.2 ng/mL (P < 0.05). Conclusions: The formation of NETs in blood in transition dairy cows may damage the integrity of the blood-milk barrier and thereby increase the risk for mastitis in postpartum cows.

Objective: The aim of this study is to investigate the dynamic changes of plasma bile acids (BA) and their correlations with physiological parameters and fecal microbiome in transitional dairy cows.Methods: Twenty multiparous dairy cows were selected, the blood and fecal samples were collected on d −21, −7, +7, and +21 of calving. The targeted metabolome and 16s rDNA ampicon sequencing were utilized to identify BA profiles and fecal microbial composition, respectively.Results: A total of 32 BAs were found, comprising 9 primary BAs (PBA) and 23 secondary BAs (SBA). Majority of the PBAs (7 out to 9) and SBAs (15 out to 23) exhibited significant increases postpartum compared to prepartum levels. Notably, ursodeoxycholic acid, taurocholic acid and 7-ketodeoxycholic acid showed higher importance. Correlation analysis showed the BAs concentrations positively correlated with the concentrations of aspartate aminotransferase, total antioxidant capacity, and glutathione peroxidase, while exhibiting substantial negative correlation with triglyceride concentrations. A decline in bacterial alpha diversity in postpartum and significantly different β-diversity were observed. Furthermore, 30 distingtive genera were identified over the transition period. Among these, six and eleven biomarkers such as Alistipes and Ruminococcaceae_UCG_014 were identified at +7 d and +21 d, respectively. Furthermore, the abundances of choloylglycine hydrolase and 7-alpha-hydroxysteroid dehydrogenase which are involved in SBA biosynthesis were significantly higher postpartum as determined by PICRUSt2 analysis over the transition period.Conclusion: The BA profile and concentrations underwent significant changes during the transition period in dairy cows and these changes are closely related to the periparturient health of the cows. Ursodeoxycholic acid and Alistipes was identified as the pivotal BA and microbial genus. Our study elucidates these metabolic processes, providing useful insights into strategies for enhancing the nutrition and well-being of perinatal dairy cows.