| Summary: | The current study has two objectives: (1) To determine the effect of different lactic acid bacteria (LAB) strains’ inoculant on silage quality of fresh ryegrass (FR) and rain-treated ryegrass (RTR), and (2) to find the optimal way (silage inoculant vs. direct-fed microbial (DFM)) to use LAB strains in order to improve nutrient digestibility and reduce methane emission (CH<sub>4</sub>) in ruminant production. Five LAB strains were tested, <i>Lactiplantibacillus plantarum</i> AGR-1, <i>L. plantarum</i> AGR-2, <i>Lactococcus lactis</i> subsp. <i>lactis</i> biovar <i>diacetylactis</i> AGR-3, <i>L. lactis</i> subsp. <i>lactis</i> AGR-4 and <i>L. lactis</i> subsp. <i>lactis</i> AGR-5. Each LAB strain was inoculated at 10<sup>6</sup> cfu/g fresh weight into the FR and the RTR and ensiled for 60 days. After ensiling, the effect of LAB strains included as a DFM or silage inoculant on rumen digestibility and CH<sub>4</sub> production were measured using an in vitro gas production system with three separate runs. The in vitro experiment consisted of 24 treatments (2 grasses (FR and RTR) × 2 ways (inoculant or DFM) × 6 strains (5 LAB strains + 1 Control)). The results indicated that the LAB strains’ inoculant treatments reduced (<i>p</i> < 0.0001) the dry matter (DM) losses, the NH<sub>3</sub> concentration (<i>p</i> < 0.0001) and the pH (<i>p</i> = 0.0019) upon ensiling in both the FR and the RTR. The lowest values in dry matter (DM) loss and NH<sub>3</sub> concentration were found in the <i>L. plantarum</i> (AGR-2) and <i>L. lactis</i> (AGR-5). The in vitro CH<sub>4</sub> production was lower for silages inoculated with <i>L. plantarum</i> (AGR-1, <i>p</i> = 0.0054), <i>L. lactis</i> (AGR-4, <i>p</i> = 0.026), <i>L. lactis</i> (AGR-5, <i>p</i> = 0.029) and <i>L. plantarum</i> (AGR-2, <i>p</i> = 0.090), compared to the control. Methane production was lower (<i>p</i> = 0.0027) for LABs when used as silage inoculants, compared to being used as DFM. Lactic acid bacteria used as silage inoculants increased (<i>p</i> ≤ 0.0001) the in vitro DM and organic matter (OM) degradability both in the FR and the RTR, whereas LAB strains used as DFM showed no such effect. The DM and OM digestibility were highest in the <i>L. plantarum</i> (AGR-1, <i>p</i> = 0.0175). Among the five LAB strains used in the current study, <i>L. plantarum</i> (AGR-2) was the best candidate to improve silage quality. Our observations suggest that these LAB strains are most promising when used as silage inoculants and to be confirmed in vivo.
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