The Effects of L-Citrulline on the Apparent Digestion and Metabolism of Nutrients, Blood Hormone Levels, Amino Acid Metabolism and the Diversity of Faecal Microbiota in Mares in the Later Stage of Pregnancy.

Feeding pregnant mares L-citrulline, especially at 30 g/day, improved protein digestion, nitrogen handling, certain blood amino acids linked to the urea cycle, and fecal microbiome diversity compared with no supplement. Among 0, 15, 30, and 45 g/day, 30 g/day performed best across multiple indicators, suggesting L-citrulline can support metabolic health in late gestation.

What the study asked and why it matters

• Question: Does dietary L-citrulline improve nutrient digestion, metabolic status, blood hormones and amino acids, and gut microbiota in mares late in pregnancy?
• Importance: Late gestation is metabolically demanding; improving protein use, nitrogen balance, and gut health may support mare well-being and fetal development while reducing metabolic strain.
• Rationale: L-citrulline is a precursor to arginine and nitric oxide, central to the urea cycle and vascular function; it may enhance amino acid availability, nitrogen disposal, and gut-microbiome interactions.

Study design and methods

• Animals: 32 healthy pregnant mares in late gestation.
• Groups: Randomly assigned to 4 groups: Control (0 g/day), 15 g/day, 30 g/day, and 45 g/day of L-citrulline.
• Duration: 72 days total (12-day adaptation + 60-day feeding period).
• Feeding: Fixed ration of 11.2 kg/horse/day; complete consumption ensured, keeping dry matter intake equal across groups.
• Design strength: Random allocation and controlled intake minimize confounding from variable feed intake.

What outcomes were measured

• Apparent digestibility of nutrients, with emphasis on crude protein.
• Nitrogen metabolism indices (e.g., nitrogen utilization/retention rate).
• Blood hormone levels (hormones not detailed in the abstract, but assessed).
• Plasma amino acid profile, especially urea-cycle–related amino acids (citrulline, arginine, ornithine, etc.).
• Fecal microbiota: alpha diversity (overall diversity) and taxonomic composition (beneficial bacteria abundance).
• Composite evaluation: A multi-indicator scoring system to identify the optimal L-citrulline dose.

Key findings

• Protein utilization: 30 g/day significantly increased apparent crude protein digestibility versus control (p < 0.05).
• Nitrogen handling: 30 g/day improved nitrogen metabolism rate (p < 0.05), indicating better incorporation or disposal of nitrogen.
• Amino acids: 30 g/day elevated plasma amino acids linked to the urea cycle, consistent with enhanced nitrogen cycling and arginine availability.
• Microbiome: 30 g/day increased fecal microbial diversity and raised the abundance of beneficial taxa (specific genera not listed in the abstract), suggesting a healthier gut ecosystem.
• Hormones: Blood hormone levels were evaluated; the abstract emphasizes metabolic and microbiome improvements, implying no adverse hormonal disruption at 30 g/day.
• Dose response: The multi-indicator score identified 30 g/day as optimal among 0, 15, 30, and 45 g/day.

How L-citrulline could produce these effects (biological plausibility)

• Arginine precursor: L-citrulline is efficiently converted to arginine in the kidneys, often more effectively than giving arginine itself due to lower first-pass metabolism.
• Urea cycle support: More citrulline/arginine can facilitate ammonia detoxification via the urea cycle, improving nitrogen disposal and overall nitrogen balance.
• Protein synthesis: Increased arginine availability can stimulate protein synthesis and reduce protein catabolism, aligning with higher apparent protein digestibility and nitrogen utilization.
• Vascular effects: Arginine-derived nitric oxide may enhance blood flow to the gut and placenta, potentially improving nutrient absorption and metabolic efficiency.
• Microbiome crosstalk: Improved nitrogen handling can alter luminal nitrogen substrates, favoring beneficial microbes and increasing microbial diversity.

Interpretation of the microbiome findings

• Higher diversity: Typically correlates with greater resilience and metabolic flexibility of the gut ecosystem.
• Beneficial taxa: An increase suggests better gut barrier support, fermentation balance, and potentially reduced endotoxin exposure.
• Functional implications: Shifts may improve short-chain fatty acid production and nitrogen salvage, complementing host nitrogen metabolism.

Practical implications for equine management

• Suggested dose: 30 g/day of L-citrulline appears to offer the best balance of benefits in late-gestation mares under the study’s conditions.
• Target outcomes: Improved protein efficiency, better nitrogen handling, and a healthier gut microbiome profile without changing total feed intake.
• Feeding strategy: Maintain consistent forage/concentrate intake and introduce L-citrulline gradually to the full target dose.
• Monitoring: Track body condition, manure consistency, and behavior; coordinate with a veterinarian if integrating into broader peripartum management.

Strengths of the study

• Controlled intake: Equal dry matter intake across groups isolates the supplement’s effect.
• Randomized allocation: Reduces selection bias.
• Multi-domain assessment: Combines digestion, nitrogen metabolism, blood biomarkers, and microbiome profiling.
• Dose-ranging: Enables identification of a practical optimal dose.

Limitations and cautions

• Sample size: 32 mares limits power to detect smaller effects and to analyze subgroup differences.
• Reporting detail: Specific hormone changes and exact microbial taxa were not provided in the abstract.
• Duration and timing: 60-day supplementation in late gestation; effects on foal outcomes, lactation, and postpartum recovery were not assessed.
• Generality: Findings may depend on diet composition, breed, management, and environmental factors not detailed in the abstract.
• Dose ceiling: 45 g/day did not outperform 30 g/day on the composite score, so more is not necessarily better.

What this means for future research

• Mechanistic detail: Map changes in specific urea-cycle intermediates, nitric oxide metabolites, and protein turnover markers.
• Microbiome function: Link taxonomic shifts to fermentation outputs (e.g., short-chain fatty acids) and gut barrier markers.
• Reproductive outcomes: Track foal birthweight, neonatal health, colostrum quality, and postpartum mare recovery.
• Safety and pharmacokinetics: Define upper tolerable intakes, long-term safety, and interactions with typical equine rations.
• Comparative dosing: Explore body-weight–adjusted dosing and timing (e.g., earlier in gestation or peripartum).

Bottom line

• In late-gestation mares, 30 g/day of L-citrulline improved crude protein digestibility, nitrogen metabolism, urea-cycle–related amino acids, and gut microbiome diversity, with this dose outperforming lower and higher tested levels across multiple indicators.
• These results support L-citrulline as a nutritional regulator for metabolic and gut health during the final months of pregnancy, warranting confirmation in larger and longer-term studies that include maternal and foal outcomes.