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Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; L.) silage increases milk fat 18:3-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis and . The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation on lipolysis. A randomized block design with a 2 × 3 × 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy ( L.) and meadow fescue ( Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3-3 and total fatty acids (TFA) were higher (0.001) for RC than for grass. For both forage species, 18:3-3 and TFA content decreased linearly (0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher (0.001) for RC than grass (11 vs. 0.11 μkatal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease ( = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true ( = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis . For both plant species, total PPO activity was not associated with the extent of lipolysis . However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.

Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.

Two experiments were carried out to determine the effects of feeding grass silages differing in their water-soluble carbohydrate content, with or without red clover silage, on the efficiency of nutrient use. High-sugar grass, control grass, and red clover were ensiled in laboratory silos for use in an in vitro experiment (Exp. 1). For an in vivo experiment (Exp. 2), the same forage types were baled and ensiled. All silages were well preserved; within experiments the grass silages had similar composition, except for greater (P0.05) water-soluble carbohydrate concentrations in the high-sugar than the control grass silage. In Exp. 1, high-sugar grass, control grass, and red clover silages were fed alone or as mixtures (30:70, 50:50, or 70:30 on a DM basis, respectively) of each grass with the red clover silage to a simulated rumen culture system. There were no significant differences in microbial N flow or efficiency of microbial protein synthesis between individual forages. However, the corresponding values for the 70:30 ratio of high-sugar grass:red clover silage were greater (P0.05) than for the red clover silage. The value for the efficiency of N use (g of microbial N/g of feed N) was greater (0.86; P0.05) for high-sugar grass silage than the control grass silage. In addition, the high-sugar grass:red clover silage mixtures all gave greater (P0.05) values for the efficiency of N use than red clover silage alone; this difference was not achieved with the control grass mixture. Experiment 2 was an incomplete Latin square design conducted with 6 Here-ford x Friesian steers (163 +/- 5.9 kg of BW) with rumen and duodenal cannulas fed the following 5 silage diets: high-sugar grass silage; control grass silage; high-sugar grass and red clover silage (50:50 DM basis); control grass and red clover silage (50:50 DM basis); and red clover silage. Rumen NH3-N concentration was lowest (P0.05) with the high-sugar grass silage. Microbial N flows to the duodenum and efficiency of microbial protein synthesis were greater (P0.05) for steers fed the high-sugar grass silage than for control grass and red clover silages, and mixing red clover with grass silages increased (P0.05) these values compared with red clover silage alone. In both experiments, the efficiency of incorporation of silage N into microbial N was more than 20% greater (P0.05) for high-sugar grass than for control grass silage. These data suggest that grass silage with high-sugar content provides a forage-based strategy for balancing N and energy supply and improving the efficiency of use of grass silage N in the rumen.

The experiment investigated the digestion of lipids from different forage silages in beef steers. Six Hereford x Friesian steers prepared with rumen and duodenal cannulas were given ad libitum access to a high-sugar grass silage, control grass silage, red clover silage, or mixtures of the red clover and each of the grass silages (50:50, DM basis). The experiment was conducted as an incomplete 5 x 5 Latin square, with an additional randomly repeated sequence. Total fatty acid and C18:3n-3 concentrations were greater (P0.05) for the high-sugar grass silage than the control grass silage or the red clover silage. Dry matter and total fatty acid intake were less (P0.05) for steers fed the control grass silage than for steers fed the other diets. Duodenal flow of C18:3n-3 was greater (P0.05), and flows of C18:0 and total C18:1 trans were less (P0.05), for the red clover silage compared with the 2 grass silage diets, with the mixtures intermediate. These results were supported by a reduction (P0.05) in biohydrogenation of C18:3n-3 for the red clover silage, with the mixtures again being intermediate. Flows of total branched- and odd-chain fatty acids were greater (P0.05) for the high-sugar grass silage diet, possibly as a result of greater microbial flow, because these fatty acids are associated with bacterial lipid. Duodenal flows of the chlorophyll metabolite, phytanic acid, were greater (P0.05) for animals fed the high-sugar grass silage treatments compared with the other treatments. These results confirm the potential for modifying the fatty acid composition of ruminant products by feeding red clover silage.