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

Importance of the Topic

The Japanese rock ptarmigan is an endangered species experiencing population decline due to nutritional imbalances and loss of native gut microbiota when bred in captivity. Metabolome analysis of fecal samples allows researchers to assess gut microbial activity and dietary impacts, providing critical information for optimizing breeding protocols and ensuring successful reintroduction into wild habitats.

Aims and Study Overview

This study aims to compare the fecal metabolite profiles of wild and artificially bred Japanese rock ptarmigans using LC/MS/MS. By identifying distinct metabolic signatures, the research seeks to guide the development of feed formulations and microbial interventions that restore native gut flora and improve bird health.

Methodology and Used Instrumentation

Sample Preparation

• Fresh feces were collected from wild and captive ptarmigans, flash-frozen, and stored at –80 °C.
• Samples were homogenized in phosphate buffered saline, centrifuged, ultrafiltered, and diluted tenfold for analysis.

LC/MS/MS Analysis

• Chromatography: Shimadzu Nexera X2 system with a reversed-phase column; gradient elution using 0.1 % formic acid in water (A) and acetonitrile (B); flow rate 0.25 mL/min; injection volume 3 µL.
• Mass spectrometry: Shimadzu LCMS-8060 with electrospray ionization in both positive and negative modes; multiple reaction monitoring (MRM) acquisition.

Main Results and Discussion

• An average of 56 hydrophilic metabolites were detected in wild ptarmigan feces and 60 in captive birds, including amino acids, organic acids, nucleosides, and nucleotides.
• Principal component analysis revealed clear separation between wild and captive groups.
• Captive birds showed elevated levels of free amino acids (phenylalanine, leucine, isoleucine, valine), indicating excessive dietary protein.
• Wild birds exhibited higher levels of nucleic acid metabolites (uridine, adenine, cytidine), suggesting active microbial conversion supporting amino acid synthesis in nutrient-poor environments.

These findings imply that current captive diets may predispose birds to metabolic disorders and that wild-type gut microbiota play a vital role in nutrient recycling.

Benefits and Practical Applications

• Provides a basis for adjusting protein content in captive feed to prevent nephropathy and other health issues.
• Identifies metabolic biomarkers for monitoring gut health during breeding and release programs.
• Informs strategies to reintroduce grazing-animal-like microbial communities to captive birds.

Future Trends and Applications

• Combining next-generation sequencing with metabolomics for holistic gut microbiome profiling.
• Developing tailored probiotic formulations to recreate native microbial consortia.
• Applying this integrative approach to other endangered species requiring dietary and microbiome management.

Conclusion

Fecal metabolome analysis effectively distinguishes dietary and microbial differences between wild and captive Japanese rock ptarmigans. The insights gained enable refinement of feeding strategies and microbiome restoration, supporting more successful breeding and conservation efforts.