Influence of rate of inclusion of microalgae on the sensory characteristics and fatty acid composition of cheese and performance of dairy cows.

Modification of milk and cheese fat to contain long-chain n-3 fatty acids (FA) by feeding microalgae (ALG) to dairy cows has the potential to improve human health, but the subsequent effect on the sensory attributes of dairy products is unclear. The objective was to determine the effect of feeding dairy cows different amounts of ALG that was rich in docosahexaenoic acid (DHA) on milk and cheese FA profile, cheese sensory attributes, and cow performance. Twenty Holstein dairy cows were randomly allocated to 1 of 4 dietary treatments in a 4 × 4 row and column design, with 4 periods of 28 d, with cheddar cheese production and animal performance measurements undertaken during the final 7 d of each period. Cows were fed a basal diet that was supplemented with ALG (Schizochytrium limancinum) at 4 rates: 0 (control, C), 50 (LA), 100 (MA), or 150 g (HA) of ALG per cow per day. We found that both milk and cheese fat content of DHA increased linearly with ALG feed rate and was 0.29 g/100 g FA higher in milk and cheese from cows fed HA compared with C. Supplementation with ALG linearly reduced the content of saturated FA and the ratio of n-6:n-3 FA in milk and cheese. Supplementation with ALG altered 20 out of the 32 sensory attributes, with a linear increase in cheese air holes, nutty flavor, and dry mouth aftertaste with ALG inclusion. Creaminess of cheese decreased with ALG inclusion rate and was positively correlated with saturated FA content. We also observed a quadratic effect on fruity odor, which was highest in cheese from cows fed HA and lowest in LA, and firmness and crumbliness texture, being highest in MA and lowest in HA. Supplementation with ALG had no effect on the dry matter intake, milk yield, or live weight change of the cows, with mean values of 23.1, 38.5, and 0.34 kg/d respectively, but milk fat content decreased linearly, and energy-corrected milk yield tended to decrease linearly with rate of ALG inclusion (mean values of 39.6, 38.4, 37.1, and 35.9 g/kg, and 41.3, 41.3, 40.5, and 39.4 kg/d for C, LA, MA, and HA, respectively). We conclude that feeding ALG to high-yielding dairy cows improved milk and cheese content of DHA and altered cheese taste but not cow performance, although milk fat content reduced as inclusion rate increased.

Effect of feeding Yucca schidigera extract and a live yeast on the rumen microbiome and performance of dairy cows fed a diet excess in rumen degradable nitrogen.

Nitrogen (N) loss from livestock agriculture via ammonia and nitrous oxide can reduce feed efficiency, production and negatively affect the environment. One option to reduce N loss is to add dietary supplements such as Yucca schidigera extract which has ammonia-binding properties and contains antimicrobial steroidal saponins, or Saccharomyces cerevisiae yeast, which can stabilise rumen pH and promote fibre degradation, increasing microbial growth and demand for degradable N. To determine the effect of Yucca schidigera extract when fed alone or in combination with a live yeast on the performance, rumen metabolism, microbiome and N balance, six rumen cannulated dairy cows were fed a mixed ration (C), mixed ration with Y. schidigera extract (De-Odorase®, Alltech®; 5 g/cow/day; D), or mixed ration with Y. schidigera extract (5 g/day) and Saccharomyces cerevisiae (Yea-Sacc®, Alltech®, 1 g/cow per day; DY), in a 3 × 3 Latin rectangle design study with three periods of 49-day duration. Digesta samples were collected via the ruminal cannula during the final week of each period and separated into liquid (LPD) and solid (SPD) phases for microbiome analysis using 16S rRNA amplicon sequencing. DM intake was 0.8 kg/d lower (P < 0.05) in cows fed DY than C or D, with milk protein concentration 1.7 g/kg higher in C than D or DY. There was a beta diversity (Bray Curtis) clustering of the LPD in cows fed D or DY compared to C (P < 0.05), driven by an increase in Prevotella ruminicola-related operational taxonomic units (OTUs), and a decrease in P. brevis and P. bryantii OTUs. A methanogen OTU, Methanobrevibacter olleyae, was decreased in cows fed D or DY and an unclassified species of Gammaproteobacteria was increased in DY (LDA > 2.0, P < 0.05) compared to C. Rumen pH, ammonia and total VFA concentration were not affected by treatment (P > 0.05) but the concentration of propionate and iso-butyrate were lower at 1700 and 2000 h in cows fed DY compared to C (P < 0.05). Measurements of N balance were unaffected by supplementation with D or DY, and there was no effect of treatment on slurry pH. In conclusion, supplementing with an extract of Yucca schidigera either alone or in combination with a live yeast had only a small effect on performance, with Yucca schidigera altering species associated with carbohydrate and protein metabolism, and reduced Methanobrevibacter olleyae which is involved in methanogenesis.

Structural insights into the multiple functions of protein C inhibitor.

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport.

Serpins in thrombosis, hemostasis and fibrinolysis.

Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin-protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure-function relationships is imperative for the development of state-of-the-art pharmaceuticals for the selective fine-tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z-dependent protease inhibitor, alpha(1)-protease inhibitor, protein C inhibitor, alpha(2)-antiplasmin and plasminogen activator inhibitor-1. We focus on the biological function, the important structural elements, their known non-hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

Discovery and characterization of an antibody directed against exosite I of thrombin.

UNLABELLED: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. BACKGROUND: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. OBJECTIVES: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. METHODS: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. RESULTS: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. CONCLUSIONS: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.

Molecular recognition mechanisms of thrombin.

Thrombin is the final protease generated in the blood coagulation cascade, and is the only factor capable of cleaving fibrinogen to create a fibrin clot. Unlike every other coagulation protease, thrombin is composed solely of its serine protease domain, so that once formed it can diffuse freely to encounter a large number of potential substrates. Thus thrombin serves many functions in hemostasis through the specific cleavage of at least a dozen substrates. The solution of the crystal structure of thrombin some 15 years ago revealed a deep active site cleft and two adjacent basic exosites, and it was clear that thrombin must utilize these unique features in recognizing its substrates. Just how this occurs is still being investigated, but recent data from thrombin mutant libraries and crystal structures combine to paint the clearest picture to date of the molecular determinants of substrate recognition by thrombin. In almost all cases, both thrombin exosites are involved, either through direct interaction with the substrate protein or through indirect interaction with a third cofactor molecule. The purpose of this article is to summarize recent biochemical and structural data in order to provide insight into the thrombin molecular recognition events at the heart of hemostasis.

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.

NTF2 monomer-dimer equilibrium.

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 microM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

The influence of grass silage-to-maize silage ratio and concentrate composition on methane emissions, performance and milk composition of dairy cows.

It is well-established that altering the proportion of starch and fibre in ruminant diets can alter ruminal and post-ruminal digestion, although quantitative evidence that this reduces enteric methane (CH4) production in dairy cattle is lacking. The objective of this study was to examine the effect of varying grass-to-maize silage ratio (70 : 30 and 30 : 70 DM basis), offered ad libitum, with either a concentrate that was high in starch or fibre, on CH4 production, intake, performance and milk composition of dairy cows. A total of 20 cows were allocated to one of the four experimental diets in a two-by-two factorial design run as a Latin square with each period lasting 28 days. Measurements were conducted during the final 7 days of each period. Cows offered the high maize silage ration had a higher dry matter intake (DMI), milk yield, milk energy output and lower CH4 emissions when expressed per kg DMI and per unit of ingested gross energy, but there was no difference in total CH4 production. Several of the milk long-chain fatty acids (FA) were affected by forage treatment with the most notable being an increase in 18:0, 18:1 c9, 18:2 c9 c12 and total mono unsaturated FA, observed in cows offered the higher inclusion of maize silage, and an increase in 18:3 c9 c12 c15 when offered the higher grass silage ration. Varying the composition of the concentrate had no effect on DMI or milk production; however, when the high-starch concentrate was fed, milk protein concentration and milk FAs, 10:0, 14:1, 15:0, 16:1, increased and 18:0 decreased. Interactions were observed for milk fat concentration, being lower in cows offered high-grass silage and high-fibre concentrates compared with the high-starch concentrate, and FA 17:0, which was the highest in milk from cows fed the high-grass silage diet supplemented with the high-starch concentrate. In conclusion, increasing the proportion of maize silage in the diets of dairy cows increased intake and performance, and reduced CH4 production, but only when expressed on a DM or energy intake basis, whereas starch-to-fibre ratio in the concentrate had little effect on performance or CH4 production.

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis.

Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin-protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and alpha2-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.

Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis.

BACKGROUND: Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. OBJECTIVES: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. PATIENTS AND METHODS: We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparin-affinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. RESULTS: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. CONCLUSIONS: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.

The conformational basis of thrombosis.

Antithrombin readily undergoes a spontaneous transition from its active five-stranded form to a six-stranded inactive latent form. The recognition of this change in plasma has been obscured by the immediate linkage of newly formed latent antithrombin to a molecule of active antithrombin to give a dimer with an electrophoretic mobility readily confused with that of native active antithrombin. A new micromethod now allows unequivocal identification of latent antithrombin in whole plasma. This shows that at 37 degrees C some 10% of plasma antithrombin is converted to the latent form in 24 h. The rate of conversion is greatly accelerated at increased temperatures, as occurs in the pasteurisation of plasma concentrates that should now be checked for efficacy. But increased transition also occurs in the plasma at the slightly increased temperatures that accompany incidental infections. This is of particular significance if there is a conformationally unstable variant of antithrombin; here fever can provoke a sudden transition with the onset of a characteristically severe episode of thromboembolism. Such variants are not rare and include those previously classified as pleiotropic. The precise structural pathway, now known with antithrombin, provides a model of the changes occurring in other conformational diseases, including Alzheimer's and the prion dementias.

The serpins: nature's molecular mousetraps.

A special family of inhibitors, known as the serpins, has evolved an extraordinary mechanism to enable the control of the proteolytic pathways essential to life. The serpins undergo a profound change in conformation to entrap their target protease in an irreversible complex. The solving of the structure of this complex now completes a video depiction of the changes involved. The serpin, just like a mousetrap, is seen to change with a spring-like movement from an initial metastable state to a final hyperstable form. The structure shows how this conformational shift not only inhibits the protease but also destroys it. A bonus from these structural insights is the realisation that a number of diseases, as diverse as thrombosis, cirrhosis and dementia, all share a common mechanism arising from similar mutations of different serpins.

Thrombin inhibition by the serpins.

Thrombin is the central protease in the blood coagulation network. It has multiple substrates and cofactors, and it appears that four serpins are responsible for inhibiting the thrombin produced in haemostasis and thrombosis. Structural studies conducted over the last 10 years have resolved how thrombin recognises these serpins with the aid of cofactors. Although antithrombin (AT), protein C inhibitor (PCI), heparin cofactor II (HCII) and protease nexin-1 (PN1) all share a common fold and mechanism of protease inhibition, they have evolved radically different mechanisms for cofactor-assisted thrombin recognition. This is likely to be due to the varied environments in which thrombin is found. In this review, I discuss the unusual structural features of thrombin that are involved in substrate and cofactor recognition, the serpin mechanism of protease inhibition and the fate of thrombin in the complex, and how the four thrombin-specific serpins exploit the special features of thrombin to accelerate complex formation.

Type II antithrombin deficiency caused by a large in-frame insertion: structural, functional and pathological relevance.

BACKGROUND: The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences. OBJECTIVE: To characterize the first antithrombin deficiency caused by a large in-frame insertion. PATIENTS/METHODS: Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK-EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin. RESULTS: The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in-frame insertion of 24 bp in SERPINC1, affecting strand 3 of ß-sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N-terminal disulphide bonds, and was conformationally sensitive. The variant was non-inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into ß-sheet A as the fourth strand, and maintained a native RCL. CONCLUSIONS: This is the first case of a large in frame-insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non-inhibitory variant, which acquires a hyperstable conformation with a native RCL.

Serpin structure, function and dysfunction.

Serpins have been studied as a distinct protein superfamily since the early 80s. In spite of the poor sequence homology between family members, serpins share a highly conserved core structure that is critical for their functioning as serine protease inhibitors. Therefore, discoveries made about one serpin can be related to the others. In this short review, I introduce the serpin structure and general mechanism of protease inhibition, and illustrate, using recent crystallographic and biochemical data on antithrombin (AT), how serpin activity can be modulated by cofactors. The ability of the serpins to undergo conformational change is critical for their function, but it also renders them uniquely susceptible to mutations that perturb their folding, leading to deficiency and disease. A recent crystal structure of an AT dimer revealed that serpins can participate in large-scale domain-swaps to form stable polymers, and that such a mechanism may explain the accumulation of misfolded serpins within secretory cells. Serpins play important roles in haemostasis and fibrinolysis, and although each will have some elements specifically tailored for its individual function, the mechanisms described here provide a general conceptual framework.

Effect of whole-crop pea (Pisum sativum L.) silages differing in condensed tannin content as a substitute for grass silage and soybean meal on the performance, metabolism, and carcass characteristics of lambs.

Two experiments were conducted to investigate the effect of inclusion of whole-crop pea (WCP) silages, differing in condensed tannin content, as a substitute for grass silage (GS) and soybean meal on lamb metabolism, performance, plasma metabolites, digestibility, and carcass characteristics. In both experiments lambs were offered either solely GS or a 50:50 mix on a DM basis of GS with either low-tannin (LTPS) or high-tannin (HTPS) pea silage ad libitum. Each forage mix was fed with either 400 g/d of low-protein (LP) concentrate or 400 g/d of LP with an additional 200 g/d of pelletized soybean meal (HP), resulting in 6 dietary treatments. Experiment 1 examined the effects of the diets on metabolism, digestibility, and N balance using 6 lambs in 4 periods of 21 d in an incomplete crossover design. Experiment 2 used 48 lambs and examined the effects of the diets on ADG, plasma metabolites, and carcass characteristics over 56 d. Both experiments were analyzed using a 3 × 2 factorial arrangement of treatments. In Exp. 1, lambs offered the LTPS diets had a greater (P < 0.05) digestibility of DM and OM than those offered the GS diets. Lambs offered the WCP silages had an increased (P < 0.05) N intake, N output, and digestibility of GE compared with those offered GS. Mean N digestibility was greatest (P < 0.05) in lambs offered LTPS. Lambs offered HP diets had increased (P < 0.001) digestibility of DM, OM, GE and N, and N- intake, output, retention, and digestibility compared with those offered the LP diets. In Exp. 2, there was no effect (P > 0.05) of forage type on intake, slaughter BW, or feed conversion efficiency (FCE). However, lambs offered the LTPS had a greater (P < 0.05) ADG than those offered the GS diets. Feeding diets containing HP increased (P < 0.001) total DMI, slaughter BW, ADG, and FCE. Lambs offered the WCP had a greater (P < 0.05) plasma ß-hydroxybutyrate and urea concentration compared with those offered the GS diets. Feeding lambs HP diets increased (P < 0.05) plasma urea and total protein. Forage mix had no effect (P > 0.05) on carcass composition except for fat depth, which was greater (P < 0.05) in lambs offered WCP silage. Diets containing the HP increased (P < 0.05) carcass weight, hind leg circumference, chop dimensions, and kidney weight. It was concluded that lambs offered LTPS performed better than those offered GS and that LTPS has a concentrate sparing effect. Additionally, the increased tannin concentration in HTPS did not increase performance over lambs offered either GS or LTPS.