DISCUSSION

Metabolic signatures associated with milk production performance

This study identified a panel of 15 discriminant metabolites that clearly differentiated HP and LP Sapera goats, suggesting their potential as candidate metabolic biomarkers associated with milk production performance. These metabolites included pyruvate (C00022), N-carbamoyl-L-aspartate (C00233), fumarate (C00417), N-acetylneuraminic acid (C01352), 3-hydroxy-3-methylglutaric acid (C02985), glycerol 3-phosphate (C03981), 2-oxoadipic acid (C04438), L-aminoadipic acid (C05267), β-hydroxybutyric acid (C05983), cystathionine (C05273), sphinganine (C16566), phytosphingosine (C15767), sphingosine (C21963), ceramide-related intermediates (C21937), and cystathionine-related metabolites (C05269). These metabolites displayed clear differences in abundance between HP and LP animals, indicating that milk production performance is associated with systematic metabolic remodeling rather than random biochemical variability. In metabolomics studies, metabolites that consistently discriminate phenotypic groups often represent key metabolic nodes integrating multiple physiological processes, making them strong candidates for biomarker discovery [13, 14].

Central carbon metabolism and energy production

Several metabolites enriched in HP goats were closely associated with central carbon metabolism and energy production, particularly pyruvate and fumarate. Pyruvate is a central intermediate linking glycolysis, gluconeogenesis, and mitochondrial respiration and therefore serves as a key metabolic junction regulating cellular energy metabolism [15]. Higher pyruvate abundance in HP goats may reflect differences in glycolytic activity and carbon substrate availability associated with lactation performance. Similarly, fumarate is an intermediate of the TCA cycle and reflects mitochondrial metabolic activity. Increased fumarate abundance may indicate altered central carbon metabolism between HP and LP goats and is consistent with the high energetic demand of lactation [16, 17]. However, serum metabolite abundance alone cannot confirm mitochondrial flux or determine whether these metabolic differences are a cause or consequence of higher milk production. Because lactation is one of the most energy-demanding physiological states in mammals, efficient mitochondrial metabolism is essential for maintaining sustained milk synthesis [18].

Nucleotide metabolism and glycoprotein biosynthesis

Metabolites such as N-carbamoyl-L-aspartate and N-acetylneuraminic acid highlight the involvement of nucleotide metabolism and glycoprotein biosynthesis in lactation physiology. N-carbamoyl-L-aspartate participates in the de novo pyrimidine biosynthesis pathway and plays an important role in cellular proliferation and biosynthetic activity [19]. Increased levels of this metabolite may reflect enhanced cellular metabolic activity in the mammary gland during high milk production. N-acetylneuraminic acid, commonly referred to as sialic acid, is a key component of glycoproteins and glycolipids involved in cell signaling, immune regulation, and glycosylation processes [20]. Glycosylated molecules are important components of milk proteins and cellular membranes in mammary tissue, suggesting that altered sialic acid metabolism may reflect physiological adaptations that support milk synthesis [21].

Lipid metabolism and membrane remodeling

Several metabolites identified in this study were associated with lipid metabolism and membrane remodeling, including 3-hydroxy-3-methylglutaric acid, glycerol 3-phosphate, 2-oxoadipic acid, L-aminoadipic acid, and β-hydroxybutyric acid. Lipid metabolism is a fundamental component of lactation biology because fatty acids and lipid-derived molecules provide both energy substrates and structural components required for milk fat synthesis [22]. β-hydroxybutyric acid, in particular, is a ketone body produced during fatty acid oxidation and reflects hepatic lipid metabolism and energy balance [23]. Increased levels of lipid-related metabolites may reflect greater lipid mobilization and utilization associated with the energetic requirements of lactation in HP goats [24]. Alternatively, these changes may represent systemic metabolic responses to higher milk output rather than direct mechanisms that drive production.

Amino acid metabolism

Additional metabolites, including cystathionine, cystathionine-related metabolites, and amino acid-derived intermediates such as L-aminoadipic acid, highlight the involvement of amino acid metabolism in the metabolic differences between HP and LP goats. Amino acids serve as essential substrates for milk protein synthesis and participate in metabolic signaling pathways regulating nutrient utilization and metabolic homeostasis [25]. Alterations in amino acid metabolism may therefore reflect metabolic adjustments that support protein synthesis and nitrogen utilization during lactation [26].

Sphingolipid-related metabolic changes

The identification of sphingolipid-related metabolites, including phytosphingosine, sphinganine, sphingosine, and ceramide-related intermediates, further suggests the involvement of lipid signaling pathways in metabolic adaptation to lactation. Sphingolipids play important roles in membrane structure, cell signaling, and metabolic regulation [27]. In lactating animals, membrane lipid turnover is particularly relevant because mammary epithelial cells undergo active secretory activity and continuous remodeling of cellular and milk fat globule membranes. Sphingolipid intermediates may also be linked to stress- and inflammation-related signaling, which can influence mammary gland function and metabolic adaptation during lactation. These molecules participate in lipid signaling pathways that regulate cellular stress responses, energy metabolism, and membrane remodeling [28]. Thus, the altered abundance of sphingolipid intermediates in this study may reflect differences in membrane lipid turnover, cellular signaling, or inflammation-related metabolic responses associated with lactation performance [29].

Enriched metabolic pathways

The biological significance of these metabolites becomes clearer when examined through metabolite set enrichment analysis. The enrichment analysis revealed that the discriminatory metabolites converged into several interconnected metabolic functions, particularly fatty acid elongation, fatty acid degradation, and central carbon metabolism pathways. Fatty acid metabolism plays a central role in lactation physiology because fatty acids provide both energy substrates and structural components for milk fat synthesis [30]. Efficient lipid mobilization and oxidation enable lactating animals to meet the high energetic demands of continuous milk production [31]. The enrichment of fatty acid metabolic pathways suggests that lipid metabolism was an important systemic feature distinguishing HP and LP goats. This may reflect differences in lipid mobilization, oxidation, or nutrient partitioning associated with lactation performance.

Central carbon metabolism pathways, particularly the TCA cycle, were also identified as important metabolic processes distinguishing HP and LP goats. The TCA cycle integrates carbohydrate, lipid, and amino acid metabolism while generating adenosine triphosphate and biosynthetic precursors required for cellular function [32]. The involvement of the TCA cycle may reflect differences in mitochondrial energy metabolism and substrate oxidation between HP and LP goats [33].

Carbohydrate metabolism pathways, including glycolysis/gluconeogenesis, pyruvate metabolism, and propanoate metabolism, were also detected in the pathway analysis. In ruminants, propanoate produced during ruminal fermentation serves as the primary precursor for hepatic gluconeogenesis, ultimately providing glucose required for lactose synthesis in the mammary gland [34]. Because lactose synthesis largely determines milk volume through osmotic regulation of milk secretion, efficient glucose metabolism is critical for sustaining high milk yield [35].

KEGG pathway topology interpretation

KEGG pathway topology analysis further reinforced the importance of lipid metabolism and central carbon metabolism in the metabolic differentiation between HP and LP goats. Pathways such as fatty acid degradation, fatty acid elongation, and the TCA cycle exhibited both strong statistical significance and notable pathway impact values. Pathway topology analysis evaluates not only the presence of metabolites within pathways but also their connectivity and influence within metabolic networks. Therefore, pathways with higher impact values may represent metabolically relevant hubs associated with differences between HP and LP goats [36].

Comparison with previous studies

The metabolic patterns observed in this study are generally consistent with previous studies in dairy goats and cattle, which reported associations between lactation performance, metabolic efficiency, energy metabolism, lipid mobilization, amino acid metabolism, and ketone body-related pathways [16, 24]. In goats, previous metabolomic and physiological studies have shown that variation in milk yield or lactation status is associated with changes in mammary gland metabolism, mobilization of body energy reserves, and milk metabolite profiles [11, 24]. Similarly, studies in dairy cattle have identified metabolites in milk or blood as indicators of metabolic health and lactation performance, particularly during periods of high metabolic demand [5]. Compared with the previous integrative metabolomics and hormonal profiling study in Sapera goats [11], the present study focuses specifically on serum LC-HRMS-based discrimination between HP and LP groups and provides additional pathway-level interpretation through metabolite enrichment and KEGG pathway topology analyses. Therefore, the present findings extend current knowledge of dairy goat metabolomics by highlighting the combined involvement of fatty acid degradation, fatty acid elongation, central carbon metabolism, and sphingolipid-related metabolites in distinguishing HP and LP Sapera goats under tropical production conditions.

Practical implications for precision nutrition

Beyond their biological significance, the metabolites identified in this study have important implications for precision nutrition strategies in dairy goat production systems. Precision nutrition aims to optimize nutrient supply to meet the metabolic requirements of individual animals, maximizing productivity while maintaining metabolic health [37]. The metabolic biomarkers identified in this study may therefore serve as valuable indicators of metabolic efficiency and nutrient utilization capacity in Sapera goats. Monitoring these metabolites could enable early identification of animals with superior metabolic adaptation to lactation, allowing targeted nutritional interventions to improve production efficiency [38]. Nutritional strategies targeting lipid metabolism, energy balance, and amino acid availability, such as improving dietary fatty acid balance, optimizing gluconeogenic precursor supply, and ensuring adequate amino acid availability, may be explored in future studies to determine whether they can improve lactation performance and metabolic stability in dairy goats [39].

Study limitations and future perspectives

Despite these insights, several limitations should be considered when interpreting the results of this study. The relatively small sample size may limit statistical robustness and influence the predictive performance of the identified metabolites as candidate biomarkers [40]. In addition, the cross-sectional design and single sampling time point limit the ability to evaluate temporal metabolic changes across lactation. Environmental factors under tropical conditions, such as ambient temperature, humidity, and seasonal variation, may also influence feed intake, energy balance, and serum metabolite profiles. Another limitation is that milk composition traits, including milk fat, protein, and lactose contents, were not measured in the present study. Therefore, the metabolic differences observed between HP and LP goats should be interpreted as being associated with milk yield-based classification rather than milk composition or milk quality traits.

Another technical limitation is that pooled quality control samples and technical injection replicates were not included in the LC-HRMS workflow. Therefore, quality control-based coefficient of variation filtering and batch-effect correction could not be applied. Although data quality was assessed using peak quality, retention time alignment, mass accuracy, MS/MS spectral matching, and database-supported annotation, future metabolomics studies should include pooled quality control samples, technical replicates, and quality control-based filtering to improve analytical reproducibility and confidence. In addition, serum metabolomics reflects systemic metabolic responses and does not directly capture tissue-specific metabolic activity within organs directly involved in lactation, particularly the mammary gland and liver. Consequently, the observed metabolite changes represent systemic metabolic signatures rather than direct measurements of mammary gland or hepatic metabolic activity or metabolic flux within specific tissues [7].

Future studies should incorporate larger animal populations, longitudinal sampling across different lactation stages, milk composition analysis, pooled quality control samples, technical replicates, and integration with transcriptomic or proteomic analyses to provide deeper mechanistic insights into the metabolic regulation of milk production. Furthermore, targeted metabolite quantification and validation studies will be necessary to confirm the diagnostic and predictive utility of the candidate biomarkers identified in this study. Future studies integrating serum metabolomics with milk metabolomics, milk composition traits, hormonal profiling, enzyme activity, or metabolic flux analyses would provide stronger mechanistic insight into the relationship between metabolic pathways, milk yield, and milk quality in Sapera goats.

Overall, the findings of this study demonstrate that variation in milk production between HP and LP Sapera goats is associated with coordinated metabolic remodeling across lipid, central carbon, and amino acid metabolism. The 15 candidate metabolites identified in this study represent key intermediates in these metabolic networks and may serve as metabolic biomarkers that reflect lactation efficiency. These results provide new insights into the biochemical mechanisms underlying variation in milk production and highlight the potential of metabolomics for precision livestock nutrition and management strategies to improve productivity in dairy goat production systems.