Metabolic Changes in Serum and Milk of Holstein Cows in Their First to Fourth Parity Revealed by Biochemical Analysis and Untargeted Metabolomics

Abstract

The performance of dairy cows is closely tied to the metabolic state, and this performance varies depending on the number of times the cows have given birth. However, there is still a lack of research on the relationship between the metabolic state of Holstein cows and the performance of lactation across multiple parities. In this study, biochemical analyses and metabolomics studies were performed on the serum and milk from Holstein cows of parities 1-4 (H1, N = 10; H2, N = 7; H3, N = 9; H4, N = 9) in mid-lactation (DIM of 141 ± 4 days) to investigate the link between performance and metabolic changes. The results of the milk quality analysis showed that the lactose levels were highest in H1 (p = 0.036). The total protein content in the serum increased with increasing parity (p = 0.013). Additionally, the lipase activity was found to be lowest in H1 (p = 0.022). There was no difference in the composition of the hydrolyzed amino acids in the milk among H1 to H4. However, the free amino acids histidine and glutamate in the serum were lowest in H1 and highest in H3 (p < 0.001), while glycine was higher in H4 (p = 0.031). The metabolomics analysis revealed that 53 and 118 differential metabolites were identified in the milk and serum, respectively. The differential metabolites in the cows' milk were classified into seven categories based on KEGG. Most of the differential metabolites in the cows' milk were found to be more abundant in H1, and these metabolites were enriched in two impact pathways. The differential metabolites in the serum could be classified into nine categories and enriched in six metabolic pathways. A total of six shared metabolites were identified in the serum and milk, among which cholesterol and citric acid were closely related to amino acid metabolism in the serum. These findings indicate a significant influence of blood metabolites on the energy and amino acid metabolism during the milk production process in the Holstein cows across 1-4 lactations, and that an in-depth understanding of the metabolic changes that occur in Holstein cows during different lactations is essential for precision farming, and that it is worthwhile to further investigate these key metabolites that have an impact through controlled experiments.

Keywords: amino acids; dairy cattle; lactation; lactose; metabolites; precision livestock farming.

Figure 1

Orthogonal partial least square discriminant analysis (OPLS-DA) score plot for the serum and milk samples from Holstein dairy cattle of different parities. The vertical pink and blue blocks represent the positive and negative modes, respectively. The horizontal wheat and purple blocks represent serum and milk samples, respectively. Triangles of different colors represent samples from corresponding groups. T score [1] represents the regression coefficient weight of the abscissa, and Orthogonal T score [1] represents the regression coefficient weight of the ordinate.

Figure 2

(A) Hierarchical cluster analysis (HCA) of differential metabolites identified from H1-M, H2-M, H3-M, and H4-M. (B) Classification of metabolites from milk based on KEGG.

Figure 3

(A) Hierarchical cluster analysis (HCA) of differential metabolites identified from H1-B, H2-B, H3-B, and H4-B. (B) Classification of metabolites from serum based on KEGG.

Figure 4

(A) Functional pathway enrichment analysis of differential metabolites from serum. (B) Functional pathway enrichment analysis of differential metabolites from milk. The colour and size of the bubbles indicate the p-value and the pathway impact index, the darker the bubble the higher the p-value and the larger the bubble the higher the pathway impact index.

Figure 5

(A) Venn diagram of the distribution of shared differential metabolites of serum and milk for H1–H4. Green circles indicate milk, red circles indicate serum, and purple circles indicate milk shared with serum. (B) Spearman’s correlation analysis of shared differential metabolites in serum and milk from H1–H4. Horizontal coordinates indicate shared metabolites in milk, vertical coordinates indicate shared metabolites in serum, * represents 0.01 < p < 0.05, ** represents 0.001 < p < 0.01, and *** represents p < 0.001. (C) Correlation network analysis of shared differential metabolites with indicators of difference in serum and milk. On the left, the associations of 6 shared differential metabolites with significant indicators of difference in H1–H4 serum are shown. On the right, the associations of 6 shared differential metabolites with significant indicators of difference in H1–H4 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 indicated 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. (D) Intensity of identification in serum or milk from H1–H4 of important metabolites significantly associated with differential physiological indicators.

Figure 6

Summary diagram showing the main results from the present work. H1 exhibits a higher energy metabolism demand, as evidenced by the higher levels of carbohydrate metabolism in the H1-B, which can stimulate the TCA cycle in milk through the common intermediate product glucose, resulting in higher lactose content in H1-M. On the other hand, H2–H4 show higher levels of total protein and free amino acids in the serum, indicating a more active amino acid metabolism.