Ruminal and gastrointestinal degradability of protein in alfalfa and birdsfoot trefoil estimated by an improved 3-step procedure

Authors: Grabber, John; COBLENTZ, W.K. - US Department Of Agriculture (USDA)

Submitted to: Translational Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2026
Publication Date: 2/23/2026
Citation: Grabber, J.H., Coblentz, W. 2026. Ruminal and gastrointestinal degradability of protein in alfalfa and birdsfoot trefoil estimated by an improved 3-step procedure. Translational Animal Science. https://doi.org/10.1093/tas/txag022.
DOI: https://doi.org/10.1093/tas/txag022

Interpretive Summary: Ruminant livestock such as dairy cattle usually produce more milk and meat when consuming pasture or fresh forage of a legume known as birdsfoot trefoil. This production response is generally attributed to birdsfoot trefoil containing condensed tannins, which can be quite effective for improving protein utilization by increasing the proportion of protein digested in the gastrointestinal tract of livestock. Dairy cattle are, however, often less responsive to birdsfoot trefoil fed as hay or silage, and this might be related to the formation of high proportions of protein-tannin complexes that are completely resistant to digestion. To investigate this, we used a 3-step method that involved digesting forage in the rumen of cows for 12 hours and then placing forage residues in glass flasks for additional digestion by gastric and intestinal enzymes. For this study we evaluated hays and silages prepared from one alfalfa cultivar lacking tannin and five birdsfoot trefoil cultivars that contained 1.3 to 3.0% tannin on a dry matter basis. We found that tannins desirably reduced the solubility and digestion of protein in the rumen. However, only 45% of the protein protected from ruminal digestion by tannin could be subsequently digested by gastrointestinal enzymes. The remaining protein protected from ruminal digestion by tannin was undigestible and would be excreted in feces, providing no benefit to the animal. Our results suggest other approaches, such as formulating diets with a proper balance of legume hay or silage with protein supplements, will likely be more effective than tannins for improving the production of milk by dairy cattle.

Technical Abstract: Unlike many legumes, birdsfoot trefoil (BFT, Lotus corniculatus L.) fed as fresh forage often contains a desirable type and quantity of condensed tannins (CT) that improves the productivity of ruminant livestock. Dairy cattle are, however, often less responsive to BFT fed as hay or silage, and this might be related to the relative partitioning of rumen-undegradable protein (RUP) into gastrointestinal-degraded protein (GIDP) and undegradable protein (UP). To investigate this, we used a 3-step method based on a 12-h in-situ ruminal incubation followed by an in-vitro gastrointestinal digestion to evaluate 12 hay and 12 silage samples prepared from two cuttings of one alfalfa cultivar lacking CT and five BFT cultivars containing 1.3 to 3.0% CT on a dry matter basis. Linear relationships between CP fractions and CT on a crude protein (CP) basis were examined at P = 0.05, using a means model for intercepts and an effects model for slopes. A standard 0-h washing machine extraction of samples sealed within in-situ bags indicated insoluble protein increased from 45.3% to 51.4% in hay and from 18.7% to 32.0% in silage as CT increased from 0% to 16%. Buffer extraction and glass-fiber filtration revealed that 7.2 percentage points of particulate CP was lost from hays and silages during the in-situ washing procedure. The 3-step procedure indicated UP increased from 4.5% in alfalfa to 10.5% in BFT containing 16% CT. Using insoluble CP and 3-step data, a relatively simple 2-timepoint kinetic model corrected for washout of particulate CP was developed to more accurately estimate RUP concentration in forage. The 2-point model indicated RUP increased from 17.9% to 28.8% in hay and from 13.0% to 24.5% in silage while GIDP (calculated from RUP minus UP) increased from 13.5% to 17.5% in hay and from 8.6% to 14.7% in silage as CT concentration increased from 0 to 16%. Hay and silage had similar slopes and intercepts for UP, and similar slopes but differing intercepts for RUP and GIDP. Our results indicate 45% of the RUP formed by CT was available as GIDP to support ruminant livestock production. The larger portion of RUP formed by CT would be excreted in feces. Overall, our study suggests CT are an inefficient means for delivering GIDP from conserved forage. Other approaches, such as formulating diets with a proper balance of conserved legumes, RUP supplements, and other feedstuffs will likely be more effective for improving the productivity of ruminant livestock.