The focus on the bioactive effects of plants concerns mainly ruminal microflora for the modulation of rumen fermentation with very little emphasis placed on their consequences on health parameters, including antioxidative and immune stimulating effects. The aim of the present study was to examine the effects of supplementing phytogenic feed additives composed of a mixture of eucalyptus (Eucalyptus citriodora) and poplar (Populus deltoides) leaf-meal (EPLM) on immune response, antioxidant status, metabolic profile, enteric methane production, growth performance, and nutrient utilization in buffalo (Bubalus bubalis) calves. In vitro studies with graded doses of EPLM extract revealed a reduction in total gas and methane production with an increased proportion of propionate without affecting feed degradability. In the in vivo experimentation, eighteen female buffalo calves (10-14 months old, avg. body weight 131.68 ± 7.50 kg) were divided into three groups (CONT, EPLM-1, and EPLM-2) of six each in a completely randomized design. Treatment groups were supplemented with a blend (1:1) of dry grounded eucalyptus (Eucalyptus citriodora) and poplar (Populus deltoids) leaves (50 g, EPLM-1; 150 g, EPLM-2). Feed intake and growth rate of buffalo calves fed on different feeding regimens did not differ (p > 0.05). Haemato-biochemical parameters reveal no variations (p > 0.05) among groups, irrespective of period of collection, except the concentration of blood urea, which was decreased (p < 0.05) in both treatment groups as compared to the control. The levels of reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) increased (p < 0.05) in EPLM supplemented animals as compared to CONT. The extent of lipid peroxidation (LPO) was reduced (p < 0.05) with increased level of EPLM in the diet. The total thiol group (T-SH) was also increased with the supplementation of leaves in the diet. The mean absolute values for skin thickness following the intra-dermal injection of PHA-P were increased (p < 0.05) in all the supplemented animals relative to CONT. However, there was no significant difference among the calves fed graded levels of these feed additives in the diet. The Ab titer against Pasteurella multocida vaccine was higher (p < 0.05) on both day 45 and 90 in both treatments, irrespective of the level of additive supplemented. The enteric methane production was reduced in EPLM supplemented buffaloes; however, digestibility of all the nutrients remained comparable (p > 0.05) among the animals. It may be concluded that blends of eucalyptus (Eucalyptus citriodora) and poplar (Populus deltoides) leaf-meal (50 g/h/d) containing 3.19 g, 2.30 g, and 0.71 g of total phenolics, tannin phenolics, and condensed tannins, respectively, can be used as the phytogenic feed additive for improving antioxidant status and immunity of buffalo calves, and mitigating enteric methane production without affecting performance and nutrient utilization.

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