Hubei Shishou Milu National Nature Reserve is an ideal place to restore the wild population of Père David's deer (Elaphurus davidianus). Understanding foraging ecology and diet composition is essential for assessing population development or establishing long-term effective conservation measures for endangered species. However, little is known about the diet composition of Père David's deer and its diet selection mechanism. In this study, we used stable isotope technology to investigate the diet composition of Père David's deer according to various tissues (i.e., fur, muscle, liver, heart, and feces) and seasons, and evaluated the correlation between the nutrient composition of plants and diet composition. Bayesian isotope analysis showed that the autumn and winter diet estimated by fur and fecal samples indicated a diet dominated by C3 grasses (42.7 to 57.2%, mean), while the summer diet estimated by muscle and liver samples was dominated by C₃ forbs (30.9 to 41.6%, mean). The Pearson correlation test indicated that the contribution of winter diet composition reflected by fur and fecal samples was associated with correlations with crude protein (r = 0.666, P < 0.01) and soluble sugars (r = 0.695, P < 0.01). The results indicated that crude protein and soluble sugars were important factors influencing the winter diet selection of Père David's deer. In the context of the current reintroduction facing many challenges, such as habitat fragmentation, wetland degradation, and human disturbance, comprehensively evaluating the diet selection mechanism of Père David’s deer under different resource specificities and temporal changes should be considered in the future.

Crude fat determination was determined according to the direct extraction method. Freeze-dried plant samples (500 mg) were dissolved in 10 ml of HCl for 50 min. Then, 10 ml of ethanol was added, and the fat was extracted with 25 ml of petroleum ether, mixed and shaken and the supernatant was collected. The supernatant was dried (105 °C for 2 h) and weighed to calculate the crude fat content.

The C/N and N contents were determined using an elemental analyzer (Flash EA 1112HT, Thermo Fisher Scientific, USA) in the laboratory of the Food Inspection and Quarantine Center, Shenzhen Custom, China.

The soluble sugar content was determined according to the anthrone - H₂SO₄ method. A standard curve was established with glucose standards. Freeze-dried plant samples (250 mg) were extracted by adding 10 ml of distilled water in boiling water for 30 min, and the supernatant was mixed with ethyl anthranilate-concentrated H₂SO₄, shaken, and held in a boiling water bath for 1 min. The absorbance was measured at 630 nm by UV spectrophotometry.

The crude protein content was determined by the Coomassie brilliant blue method. A standard curve was established with bovine serum protein standards. Freeze-dried plant samples (100 mg) were weighed and extracted with distilled water for 2 h at room temperature, the supernatant was mixed with Kaumas Brilliant Blue G250, and the absorbance was measured at 595 nm with UV spectrophotometry.

Condensed tannin (proanthocyanidins) was determined by the acid-butanol method. Freeze-dried plant samples (50 mg) were dissolved in 1 ml of methanol and mixed with 6 mL of 95% butan-1-ol and 5% concentrated HCl in 10 mL test tubes. The tubes were sealed and placed in a water bath at 95 °C for 1 h. The color was observed after cooling to room temperature. The alcoholysis under acidic conditions converts the extended units of condensed tannins into colored anthocyanins. The darker the color is, the higher the tannin concentration.

After the pretreatments, the carbon and nitrogen stable isotope ratios of the samples were analyzed using an elemental analyzer (Flash EA 1112HT, Thermo Fisher Scientific, USA) coupled with an isotope ratio mass spectrometer (Delta V Advantage, Thermo Fisher Scientific, USA) in the laboratory of the Food Inspection and Quarantine Center, Shenzhen Custom, China. Approximately 0.200 mg (± 0.001 mg) of sample plant and tissues was weighed and inserted into 4 mm × 6 mm tin cups. The carbon and nitrogen stable isotope ratios of the sample were expressed in delta notation (difference between sample and standard) as parts per thousand (‰):

δ = [(R_sample/R_standard) -1]×1000

where R is the abundance ratio of heavy isotopes to light isotopes in the sample. ¹³C/¹²C and ¹⁵N/¹⁴N. R_sample is the measured isotope ratio; R_standard is the isotope ratio of reference materials. All results are reported relative to atmospheric nitrogen as the standard for δ¹⁵N and to Cretaceous belemnite (Belemnitella Americana) from the Peedee Formation of South Carolina for δ¹³C. Laboratory standards (glycine and urea) were run every 12 samples to correct any instances of instrument drift. The analytical precision was ± 0.1‰ for ¹³C and ± 0.2‰ for ¹⁵N.

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