Metabolome Analysis of Japanese Rock Ptarmigan Feces by LC/MS/MS: Application to the Establishment of Breeding Technology for Endangered Species

Significance of the Topic

Metabolome profiling of fecal samples offers critical insights into gut microbial activity and host nutrition. For endangered species such as the Japanese rock ptarmigan, understanding metabolite patterns is vital to design feeds that reconstruct wild‐type intestinal flora, prevent health issues like oxalate nephropathy, and support successful reintroduction into natural habitats.

Objectives and Study Overview

This study aimed to compare metabolite compositions in feces from wild and captive‐bred Japanese rock ptarmigans. By identifying distinguishing metabolic signatures, researchers sought to inform breeding protocols and feed formulations that mimic the nutritional and microbial conditions of the wild population.

Methodology and Applied Instrumentation

Sample Preparation:

• Feces collected immediately after excretion and stored at –80 °C.
• Extraction with phosphate‐buffered saline, centrifugation, ultrafiltration and 10× dilution with ultrapure water.

Chromatography and Mass Spectrometry:

• UHPLC system: Nexera X2 with a reversed‐phase column.
• Mobile phases: 0.1 % formic acid in water (A) and acetonitrile (B); gradient elution at 0.25 mL/min; 3 µL injection.
• MS instrument: Shimadzu LCMS‐8060, electrospray ionization (both positive and negative modes), multiple reaction monitoring.
• Gas flows: nebulizing 3 L/min, drying 10 L/min, heating 10 L/min; temperatures: DL 250 °C, block heater 400 °C, interface 300 °C.

Main Results and Discussion

Pooled data showed detection of an average of 56 metabolites in wild birds and 60 in captive‐bred birds. Principal component analysis and t‐tests revealed clear separation of groups. Key findings:

• Captive‐bred birds exhibited elevated levels of free amino acids (phenylalanine, leucine, isoleucine, valine), indicating excess dietary protein.
• Wild birds displayed higher concentrations of nucleic acid intermediates (uridine, adenine, cytidine), suggesting active microbial conversion of nucleotides into amino acids, akin to grazing animals.

Excessive protein intake in captive feed may predispose birds to health issues such as infectious diseases, skeletal abnormalities, and oxalate nephropathy due to disrupted gut microbial balance.

Benefits and Practical Applications

Metabolomic profiling of feces enables:

• Optimization of feed formulations to prevent nutritional imbalances and disease.
• Reconstruction of beneficial gut microbiota by identifying key metabolic pathways.
• Noninvasive monitoring of health status and digestive efficiency in wild and captive populations.

Future Trends and Applications

Integrating next‐generation sequencing of gut microbiota with targeted metabolomics will deepen understanding of host–microbe interactions. Future work may focus on:

• Developing synbiotic diets combining specific microbial strains with tailored nutrients.
• Real‐time in situ metabolite monitoring using portable MS platforms.
• Applying machine learning to predict health outcomes and guide conservation strategies.

Conclusion

Fecal metabolome analysis by LC/MS/MS successfully differentiated wild and captive‐bred Japanese rock ptarmigans, highlighting diet‐induced metabolic shifts. These insights support the refinement of breeding practices and feed composition to emulate wild nutritional conditions and improve conservation outcomes.

Reference

Shimadzu Corporation, Application Note No. C201, 2019.