The effects of citrulline on horse milk components and fecal metabolism were analyzed based on LC-MS and 16s amplicon.

Overview

• This study investigated the effects of L-citrulline supplementation on milk production, milk quality, foal growth, blood biochemistry, and fecal microbiota and metabolism in lactating mares grazing under natural conditions.
• The research aimed to understand how L-citrulline influences lactation performance and related physiological parameters to optimize nutritional strategies for mares.

Introduction and Research Background

• The lactation period in mares is crucial for the health of foals and future reproductive success, making nutritional optimization vital.
• L-citrulline is a non-protein amino acid that serves as a precursor to arginine; it is known to improve mammary blood flow and reduce oxidative stress in livestock.
• Previous studies in other livestock have suggested benefits for lactation, but there is limited research on its effects in horses (Equus species).
• Prior to this study, little information was available on how L-citrulline affects lactation performance, mare blood biochemistry, and fecal microbiota and metabolism during lactation in mares.
• The study aims to fill this knowledge gap and contribute to improved dietary strategies in the horse industry.

Study Design and Methods

• A total of 32 lactating mares were divided into four groups (8 mares each): a control group (no L-citrulline) and three experimental groups receiving 15 g/day, 30 g/day, or 45 g/day of L-citrulline respectively.
• All groups were grazed under identical environmental and management conditions to isolate the effects of supplementation.
• Measurements taken included milk production volume, milk fat content, foal weight and body size, and blood biochemical markers such as lactate dehydrogenase (LDH) and creatine kinase (CK).
• Fecal samples from the control group and the 30 g/day group (optimal dose based on lactation results) were analyzed using 16S rDNA sequencing to assess microbiota composition and LC-MS/MS metabolomics to detect metabolic changes.

Key Findings

• Milk production and quality: The group receiving 30 g/day L-citrulline showed a significant increase in milk yield (P < 0.01).
• All L-citrulline supplemented groups had increased milk fat percentage compared to control, indicating improved milk quality.
• Foal growth: Foals from all supplemented groups had higher weight and larger body size, suggesting improved growth and development.
• Blood biochemistry: LDH and CK levels were significantly lower in all experimental groups, indicating reduced muscle stress or damage and better physiological status.

Fecal Microbiota and Metabolomic Analysis

• 16S rDNA sequencing identified Firmicutes as the dominant bacterial phylum in feces.
• Significant compositional differences were found in specific bacterial species, including F082 and genus Ruminococcaceae_bacterium_AE2021, between control and 30 g/day groups.
• Functional prediction (Tax4Fun) indicated that genes related to digestive metabolism, genetic information processing, and environmental information processing had the highest abundance.
• LC-MS/MS metabolite profiling revealed differential metabolites mainly linked to biological systems and metabolic pathways, indicating shifts in fecal metabolic processes due to supplementation.

Conclusions and Implications

• L-citrulline supplementation at 30 g/day effectively enhances milk production and milk fat content during lactation in grazing mares.
• Improved milk quality supports better foal growth and development, as evidenced by greater foal weights and body size across supplemented groups.
• The reductions in LDH and CK levels suggest a protective effect on mare muscle status and overall physiological health during lactation.
• Alterations in fecal microbiota and metabolomics profiles indicate that L-citrulline influences gut microbial communities and their metabolic functions, which may be linked to improved nutrient utilization or health.
• These results provide foundational evidence for the inclusion of L-citrulline in nutritional strategies for lactating mares, with positive impacts on both mare and foal health and potential benefits for the horse industry.