Combined Metabolomics and Biochemical Analyses of Serum and Milk Revealed Parity-Related Metabolic Differences in Sanhe Dairy Cattle

Abstract

The production performance of dairy cattle is closely related to their metabolic state. This study aims to provide a comprehensive understanding of the production performance and metabolic features of Sanhe dairy cattle across different parities, with a specific focus on evaluating variations in milk traits and metabolites in both milk and serum. Sanhe dairy cattle from parities 1 to 4 (S1, n = 10; S2, n = 9; S3, n = 10; and S4, n = 10) at mid-lactation were maintained under the same feeding and management conditions. The milk traits, hydrolyzed milk amino acid levels, serum biochemical parameters, and serum free amino acid levels of the Sanhe dairy cattle were determined. Multiparous Sanhe dairy cattle (S2, S3, and S4) had a greater milk protein content, lower milk lactose content, and lower solids-not-fat content than primiparous Sanhe dairy cattle (S1). Moreover, S1 had a higher ratio of essential to total amino acids (EAAs/TAAs) in both the serum and milk. The serum biochemical results showed the lower glucose and total protein levels in S1 cattle were associated with milk quality. Furthermore, ultra-high-resolution high-performance liquid chromatography with tandem MS analysis (UPLC-MS/MS) identified 86 and 105 differential metabolites in the serum and milk, respectively, and these were mainly involved in amino acid, carbohydrate, and lipid metabolism. S1 and S2/S3/S4 had significantly different metabolic patterns in the serum and milk, and more vitamin B-related metabolites were significantly higher identified in S1 than in multiparous cattle. Among 36 shared differential metabolites in the serum and milk, 10 and 7 metabolites were significantly and strongly correlated with differential physiological indices, respectively. The differential metabolites identified were enriched in key metabolic pathways, illustrating the metabolic characteristics of the serum and milk from Sanhe dairy cattle of different parities. L-phenylalanine, dehydroepiandrosterone, and linoleic acid in the milk and N-acetylornithine in the serum could be used as potential marker metabolites to distinguish between Sanhe dairy cattle with parities of 1-4. In addition, a metabolic map of the serum and milk from the three aspects of carbohydrates, amino acids, and lipids was created for the further analysis and exploration of their relationships. These results reveal significant variations in milk traits and metabolites across different parities of Sanhe dairy cattle, highlighting the influence of parity on the metabolic profiles and production performance. Tailored nutritional strategies based on parity-specific metabolic profiles are recommended to optimize milk production and quality in Sanhe cattle.

Keywords: cows; lactation; metabolism; multiparous; primiparous.

Figure 1

Multivariate statistical analysis of metabolites identified from Sanhe dairy cattle with 1–4 parities. (A) Principal component analysis (PCA) score plot of serum samples from S1 to S4 based on untargeted metabolomics. (B) PCA score plot of milk samples from S1 to S4 based on untargeted metabolomics. The abscissa PC1 = first principal component and the ordinate PC2 = second principal component.

Figure 2

Identification and classification of differential serum metabolites. (A) Classification of metabolites from serum based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (B) Hierarchical cluster analysis (HCA) of differential metabolites identified from S1-B, S2-B, S3-B, and S4-B groups.

Figure 3

Identification and classification of differential metabolites in milk. (A) Classification of metabolites from serum based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (B) Hierarchical cluster analysis (HCA) of differential metabolites identified from the S1-M, S2-M, S3-M, and S4-M groups.

Figure 4

Functional pathway enrichment analysis of differential metabolites in pairwise comparisons of serum and milk samples. (A–C) Bubble chart analysis of functional pathways from S1-B vs. S2-B, S1-B vs. S3-B, and S1-B vs. S4-B comparisons, respectively. (E–G) Bubble chart analysis of functional pathways from S1-M vs. S2-M, S1-M vs. S3-M, and S1-M vs. S4-M comparisons, respectively. x-axis, pathway impact; y-axis, −log (p). Circles represent metabolic pathways. Darker circles indicate more significant changes in the metabolite levels in the corresponding pathway, whereas the circle size corresponds to the pathway impact score. Significantly enriched functional pathways are marked with colored blocks of the corresponding groups. (D,H) Venn diagrams of functional pathways shared by groups in pairwise comparisons of serum and milk samples. The colors of the Venn circles correspond to the colors of the significantly enriched pathways of the two-by-two comparison groups. The repetitive functional pathways and their corresponding metabolites are marked with rounded rectangles.

Figure 5

Identification and analysis of common differential metabolites in serum and milk. (A) Venn diagrams of identified metabolites in serum and milk samples. Blue and red circles represent milk and serum samples, respectively. (B) Correlation network analysis of shared differential metabolites with indicators of difference in serum and milk. On the left, the associations of 36 shared differential metabolites with significant indicators of difference in S1–S4 serum are shown. On the right, the associations of 36 shared differential metabolites with significant indicators of difference in S1–S4 milk are shown. Correlations between metabolites and differential indicators were determined using Mantel’s tests, with the thickness of the connecting line indicating the correlation coefficient, with a thick line indicating a Mantel’s r ≥ 0.5; The color of the connecting line indicates significance, with orange being a highly significant correlation (p < 0.01) and green being a significant correlation (0.01 < p < 0.05). Pearson’s test tested correlations between shared differential metabolites; box size and color gradient indicate Pearson’s correlation, blue indicates positive correlation, red indicates negative correlation, and white words in the boxes indicate correlation coefficients. Biomarkers in serum are indicated in blue font and biomarkers in milk are indicated in pink font. White text in the boxes represents correlation coefficients, where * denotes 0.01 < p < 0.05, ** denotes 0.001 < p < 0.01, and *** denotes p < 0.001. (C) Predictive analyses of metabolites strongly associated with significantly different physiological indicators in serum (AUC > 0.8). (D) Predictive analyses of metabolites strongly associated with significantly different physiological indicators in milk (AUC > 0.8).

Figure 6

Metabolic networks involving pathways of lipid metabolism, carbohydrate metabolism, and amino acid metabolism consisting of differential metabolites in serum and milk. Metabolites identified in serum and milk samples are marked in red and green, respectively, and metabolites identified in both serum and milk samples are marked in green. In the legend, the four red squares represent serum samples from Sanhe dairy cattle with parities of 1–4, and the four blue squares represent milk samples from Sanhe dairy cattle with parities of 1–4. Blue indicates relatively low levels and red indicates relatively high levels. The color change in the squares is described based on identification intensity (log2) of the metabolite, with blue indicating lower and red indicating higher. The inverted red triangle indicates relatively higher levels of metabolites in serum than in milk, and the blue triangle indicates relatively lower levels of metabolites in milk than in serum. The color change in the triangle is described based on the fold change (log2) of the metabolite between serum and milk, with blue indicating lower and red indicating higher. The lipid metabolism, carbohydrate metabolism, and amino acid metabolism categories are represented by blue, purple, and wheat blocks, respectively. Black solid arrows indicate direct metabolism, and black dashed arrows indicate the presence of more than two intermediate metabolites. The metabolites in the orange boxes are strongly correlated with physiological indicators that are significantly different in serum or milk. S1: first-parity Sanhe dairy cattle; S2: second-parity Sanhe dairy cattle; S3: third-parity Sanhe dairy cattle; S4: fourth-parity Sanhe dairy cattle; S: serum; M: milk.