Exploratory Characterization of the Rumen DNA Viral Fraction in Hu Sheep with Diet-Induced Contrasting In Vitro Methane Production

Rumen viruses may influence methane-related fermentation indirectly by altering microbial populations that produce or consume hydrogen, formate, and methylated substrates, but evidence in sheep remains limited. The objective of this exploratory study was to determine whether the viral component recovered from bulk rumen metagenomes was associated with diet-induced differences in in vitro methane production through variation in viral community structure, predicted host associations, or metabolic functions. Forty-eight Hu rams were assigned to two dietary treatments with four pen replicates per treatment; one animal per pen was sampled, yielding eight rumen-fluid samples for 12 h in vitro methane determination and metagenomic sequencing. We characterized the viral component recovered bioinformatically from bulk rumen metagenomes. Phage-derived sequences predominated in both the low-methane (LMEG; 80.72%) and high-methane (HMEG; 79.95%) groups, with Uroviricota and Caudoviricetes as the dominant lineages. Global diversity, predicted-host composition, and KEGG and UniProtKB/ViralZone profiles did not show statistically supported group separation; however, two putative genus-level labels, Pakpunavirus and Fromanvirus, were enriched in LMEG after the reported false-discovery-rate correction. More than 99% of CHERRY host assignments were bacterial, and the conservative PHP–CHERRY consensus set was dominated by Bacillota and Bacteroidota. No methanogen-associated viral contig received concordant genus-level support. HMEG showed numerical increases in mtd, cofF, and fdhA, but none remained significant after correction, and canonical methyl-coenzyme M reductase genes were not detected. These findings identify candidate diet-associated viral signals consistent with a possible indirect virus–bacteria–fermentation relationship, but they do not establish a causal role of rumen viruses in methane production or identify phages for direct methane mitigation. Further studies with larger sample sizes, direct virus–host validation, and measurements of rumen fermentation and animal performance are required before potential methane-mitigation applications can be considered.