To develop an alternative high-protein forage resource to alleviate ruminant feed shortages, we investigated the effects of replacing alfalfa (Medicago sativa L.) with different ratios of paper mulberry (Broussonetia papyrifera L., RY) on fermentation quality, protein degradation, and in vitro digestibility of total mixed ration (TMR) silage. The TMR were made with alfalfa and RY mixtures (36.0%), maize meal (35.0%), oat grass (10.0%), soybean meal (7.5%), brewers’ grain (5.0%), wheat bran (5.0%), premix (1.0%), and salt (0.5%) on a dry matter basis, respectively. The alfalfa and RY mixtures were made in the following ratios of dry matter: 36:0 (RY0), 27:9 (RY9), 18:18 (RY18), 9:27 (RY27), and 0:36 (RY36). After ensiling for 7, 14, 28, and 56 days, fermentation quality, protein degradation, and microbial counts were examined, and chemical composition and in vitro digestibility were analyzed after 56 days of ensiling. All TMR silages, irrespective of the substitution level of RY, were well preserved with low pH and ammonia nitrogen content, high lactic acid content, and undetectable butyric acid. After ensiling, the condensed tannin content for RY18 silages was higher than the control, but non-protein nitrogen, peptide nitrogen, and free amino acid nitrogen contents was lower, while the fraction B1 (buffer-soluble protein) was not different among all the silages. Dry matter and crude protein digestibility for RY27 and RY36 silages was lower than the control, but there was no difference between control and RY18 silages. This study suggested that ensiling RY with alfalfa inhibited true protein degradation, but decreased in vitro dry matter and crude protein digestibility of TMR silages, and that 18:18 is the optimal ratio.

Aerobic deterioration of ensiled total mixed ration (ETMR) represents a critical challenge in livestock feed preservation, yet the microbial drivers of this process remain poorly characterized. Although Pichia kudriavzevii has been identified as the predominant spoilage yeast in ETMR, its ecological role in fungal community assembly and succession during aerobic deterioration remains unclear. This study elucidates the impact of P. kudriavzevii on aerobic stability and fungal succession in alfalfa and oat based ETMR, inoculated with or without P. kudriavzevii and analyzed after 42 days ensiling. Chemical analyses and high-throughput sequencing demonstrated that P. kudriavzevii inoculation significantly reduced aerobic stability (P<0.05) without affecting fermentation quality. P. kudriavzevii substantially altered fungal communities during both ensiling and aerobic exposure, displaying increased relative abundancewhile suppressing Saccharomyces cerevisiae and Monascus purpureus. Although interspecific fungal interactions were generally weak, a strong negative correlation was identified between P. kudriavzevii abundance and lactic acid concentration (R=-0.789, P<0.001), coinciding with progressive pH elevation during aerobic exposure. The findings establish that P. kudriavzevii drives aerobic deterioration mainly through lactic acid catabolism, with its effects depending on intraspecific diversity. This study provides fundamental insights into fungal-driven spoilage mechanisms in ETMR, highlighting potential microbiome-based targets for enhancing silage preservation. Specifically, the results suggest that controlling P. kudriavzevii proliferation or its metabolic activity could represent an effective strategy for preventing aerobic spoilage in ensiled feeds.

Ensiling is a common method for preserving total mixed ration (TMR), although exposure to air upon opening often leads to aerobic deterioration associated