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

Significance of Topic

The Japanese rock ptarmigan is classified as a high-risk species and faces challenges in captive breeding and successful reintroduction. Metabolome analysis of feces offers insights into gut microbiota function and nutritional status, helping to develop diets and breeding protocols that support health and adaptation to the wild.

Study Objectives and Overview

This study compared the fecal metabolite profiles of wild and artificially bred Japanese rock ptarmigans. The goal was to identify key metabolic differences linked to diet and gut flora, informing strategies to reconstruct natural intestinal communities and optimize feeding regimens for captive birds prior to release.

Methodology

• Sample collection: Fresh feces were frozen at –80 °C immediately after excretion.
• Pretreatment: 100 mg of feces was mixed with phosphate buffered saline, stirred, centrifuged, ultrafiltered, and diluted tenfold with ultrapure water.
• Metabolite analysis: An ion-pair–free LC/MS/MS method enabled simultaneous detection of 97 hydrophilic compounds including amino acids, organic acids, nucleosides, and nucleotides.

Used Instrumentation

• Shimadzu Nexera X2 UHPLC system with reversed-phase column and gradient elution (0.1 % formic acid in water and acetonitrile, 0.25 mL/min, 3 µL injection).
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer operating in ESI positive and negative modes with MRM detection.

Main Results and Discussion

• Detection: On average, 56 metabolites were detected in wild bird samples (n=4) and 60 in captive-bred samples (n=6).
• Multivariate analysis: Principal component analysis showed clear clustering separating wild and captive groups.
• Key discriminant metabolites: Captive birds had elevated levels of free amino acids (phenylalanine, leucine, isoleucine, valine), suggesting protein-rich feeding. Wild birds showed higher nucleic acid intermediates (uridine, adenine, cytidine), indicating active microbial conversion supporting amino acid biosynthesis.
• Implications: Excess dietary protein in captive feed may predispose birds to health issues such as nephropathy and developmental disorders, while wild ptarmigans benefit from a microbiota-driven metabolic system akin to grazing species.

Benefits and Practical Applications

• Provides a metabolomic framework to tailor captive diets and reduce metabolic imbalances.
• Supports reconstruction of gut flora essential for oxalate degradation and nutrient synthesis.
• Enhances breeding protocols by linking metabolic markers to health outcomes and reintroduction success.

Future Trends and Opportunities

• Integration of metabolomics with next-generation sequencing for comprehensive gut microbiome profiling.
• Development of bespoke feed formulations that mimic wild dietary components and microbial metabolites.
• Application of this analytical approach to other endangered species for improved conservation breeding.
• Standardization of fecal metabolome screening as a routine tool in wildlife management and ex situ breeding programs.

Conclusion

Fecal metabolome analysis using LC/MS/MS revealed distinct metabolic signatures between wild and captive-bred Japanese rock ptarmigans. These findings highlight the need for optimized feed and gut flora restoration to support health and survival post-release. The approach offers a valuable tool for refining breeding strategies and promoting conservation outcomes.