Spent coffee grounds (SCG), a by-product rich in polyphenols, can form part of enteric CH4 mitigation strategies while promoting the circular economy. This study aimed to evaluate the effect of 3 levels of SCG inclusion in the concentrate on enteric CH4 production, feed intake, apparent digestibility, ruminal fermentation pattern, microbial protein supply and gene expression of immune and antioxidant markers in peripheral blood of dry dairy ewes. In a replicated 4 × 4 Latin square design, 8 non-productive Latxa ewes were assigned to a concentrate that differed in the level of SCG: Control (0g/kg DM), SCG100 (100g/kg DM), SCG150 (150g/kg DM) and SCG200 (200g/kg DM). In each period, 14 days of adaptation were allowed, followed by 7 days in individual metabolic cages, and 3 days in respiratory chambers. To avoid a carry-over effect a minimum of 7 days were allotted between periods in which ewes consumed control concentrate and grass hay. Total organic matter intake (OMI) and CH4 emissions (g/d) presented a quadratic response (P=0.008 and P<0.001, respectively) to increasing levels of SCG in the feed. However, when CH4 emissions were corrected for OMI, a linear decrease was observed with increasing levels of SCG in the concentrate (P=0.009). This reduction in CH4 emissions (g/kg OMI) could be explained by the linear decrease (P=0.034) observed in apparent digestibility of organic matter (OM), particularly in crude protein (CP) and starch (P=0.002 and P=0.003, respectively), with increasing levels of SCG in the concentrate. No significant response was found on CH4 emissions corrected for digestible OM and on ruminal fermentation pattern. Regarding microbial protein supply, a linear increase in microbial protein supply efficiency (P=0.008) was observed with increasing levels of SCG in the concentrate. Moreover, SCG inclusion linearly reduced interleukin 10 (P=0.031), nuclear factor E2-related factor 2 (P=0.007), nuclear factor kappa β (P=0.014), superoxide dismutase 1 (P=0.015) gene expression and tended to linearly reduce those of tumor necrosis factor-α (P=0.074) and glutathione peroxidase 1 (P=0.082). In conclusion, inclusion of SCG up to 200g/kg in the concentrate did not modify ruminal fermentation pattern, but linearly reduced CH4 emissions per kg of OMI, due to a linear decrease in apparent digestibility of CP and starch. Moreover, linearly increased the efficiency of microbial supply and improved sheep's blood antioxidant-immune status.
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