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We evaluated the between-cow (b-cow) variation and repeatability in omasal and milk fatty acids (FA) related to methane (CH4) emission. The dataset was originated from 9 studies with rumen-cannulated dairy cows conducted using either a switch-back or a Latin square design. Production of CH4 per mole of VFA (Y_CH4VFA) was calculated based on VFA stoichiometry. Experiment, diet within experiment, period within experiment, and cow within experiment were considered as random factors. Empirical models were developed between the variables of interest by univariate and bivariate mixed model regression analysis. The variation associated with diet was higher than the b-cow variation with low repeatability (< 0.25) for milk odd- and branch-chain FA (OBCFA). Similarly, for de novo synthesized milk FA, diet variation was ~ 3-fold greater than the b-cow variation; repeatability for these FA was moderate to high (0.34–0.58). Also, for both cis-9 C18:1 and cis-9 cis-12 cis-15 C18:3 diet variation was more than double the b-cow variation, but repeatability was moderate. Among the de novo milk FA, C4:0 was positively related with stoichiometric Y_CH4VFA, while for OBCFA, anteiso C15:0 and C15:0 were negatively related with it. Notably, when analyzing the relationship between omasal FA and milk FA we observed positive intercept estimates for all the OBCFA, which may indicate endogenous post-ruminal synthesis of these FA, most likely in the mammary gland. For milk iso C13:0, iso C15:0, anteiso C15:0, and C15:0 were positively influenced by omasal proportion of their respective FA and by energy balance. In contrast, the concentration of milk C17:0, iso C18:0, C18:0, cis-11 C18:1, and cis-9 cis-12 cis-15 C18:3 were positively influenced by omasal proportion of their respective FA but negatively related to calculated energy balance. Our findings demonstrate that for most milk FA examined, a larger variation is attributed to diet than b-cow differences with low to moderate repeatability. While some milk FA were positively or negatively related with Y_CH4VFA, there was a pronounced effect of calculated energy balance on these estimates. Additionally, even though OBCFA have been indicated as markers of rumen function, our results suggest that endogenous synthesis of these FA may occur, which therefore, may limit the utilization of milk FA as a proxy for CH4 predictions for cows fed the same diet.

The objective of this study was to evaluate the performance of existing models predicting enteric methane (CH) emissions, using a large database (3183 individual data from 103 in vivo studies on dairy and beef cattle, sheep and goats fed diets from different countries). The impacts of dietary strategies to reduce CH emissions, and of diet quality (described by organic matter digestibility (dOM) and neutral-detergent fiber digestibility (dNDF)) on model performance were assessed by animal category. The models were first assessed based on the root mean square prediction error (RMSPE) to standard deviation of observed values ratio (RSR) to account for differences in data between models and then on the RMSPE. For dairy cattle, the CH (g/d) predicting model based on feeding level (dry matter intake (DMI)/body weight (BW)), energy digestibility (dGE) and ether extract (EE) had the smallest RSR (0.66) for all diets, as well as for the high-EE diets (RSR = 0.73). For mitigation strategies based on lowering NDF or improving dOM, the same model (RSR = 0.48 to 0.60) and the model using DMI and neutral- and acid-detergent fiber intakes (RSR = 0.53) had the smallest RSR, respectively. For diets with high starch (STA), the model based on nitrogen, ADF and STA intake presented the smallest RSR (0.84). For beef cattle, all evaluated models performed moderately compared with the models of dairy cattle. The smallest RSR (0.83) was obtained using variables of energy intake, BW, forage content and dietary fat, and also for the high-EE and the low-NDF diets (RSR = 0.84 to 0.86). The IPCC Tier 2 models performed better when dietary STA, dOM or dNDF were high. For sheep and goats, the smallest RSR was observed from a model for sheep based on dGE intake (RSR = 0.61). Both IPCC models had low predictive ability when dietary EE, NDF, dOM and dNDF varied (RSR = 0.57 to 1.31 in dairy, and 0.65 to 1.24 in beef cattle). The performance of models depends mostly on explanatory variables and not on the type of data (individual vs. treatment means) used in their development or evaluation. Some empirical models give satisfactory prediction error compared with the error associated with measurement methods. For better prediction, models should include feed intake, digestibility and additional information on dietary concentrations of EE and structural and nonstructural carbohydrates to account for different dietary mitigating strategies., This study is part of the Joint Programming Initiative on Agriculture, Food Security and Climate Change (FACCE-JPI)’s “GLOBAL NETWORK” project and the “Feeding and Nutrition Network” (http://animalscience.psu.edu/fnn) of the Livestock Research Group within the Global Research Alliance for Agricultural Greenhouse Gases (www.globalresearchalliance.org). Authors gratefully acknowledge funding for this project from: USDA National Institute of Food and Agriculture (Grant no. 2014-67003-21979) University of California, Davis Sesnon Endowed Chair Program, USDA, and Austin Eugene Lyons Fellowship (University of California, Davis); Funding from USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN 04539 and Accession number 1000803, DSM Nutritional Products (Basel, Switzerland), Pennsylvania Soybean Board (Harrisburg, PA, USA), Northeast Sustainable Agriculture Research and Education (Burlington, VT, USA), and PMI Nutritional Additives (Shoreview, MN, USA); the Ministry of Economic Affairs (the Netherlands; project BO-20-007-006; Global Research Alliance on Agricultural Greenhouse Gases), the Product Board Animal Feed (Zoetermeer, the Netherlands) and the Dutch Dairy Board (Zoetermeer, the Netherlands); The Cofund for Monitoring & Mitigation of Greenhouse Gases from Agri- and Silvi-culture (FACCE ERA-GAS)’s project Capturing Effects of Diet on Emissions from Ruminant Systems and the Dutch Ministry of Agriculture, Nature and Food Quality (AF-EU-18010 & BO-4400159-01); USDA National Institute of Food and Agriculture (Hatch Multistate NC-1042 Project Number NH00616-R; Project Accession Number 1001855) and the New Hampshire Agricultural Experiment Station (Durham, NH); French National Research Agency through the FACCE-JPI program (ANR-13-JFAC-0003-01); the Department of Agriculture, Food and the Marine, Ireland Agricultural GHG Research Initiative for Ireland (AGRI-I) project; Academy of Finland (No. 281337), Helsinki, Finland; Swiss Federal Office of Agriculture, Berne, Switzerland; the Department for Environment, Food and Rural Affairs (Defra; UK); Defra, the Scottish Government, DARD, and the Welsh Government as part of the UK’s Agricultural GHG Research Platform projects (www.ghgplatform.org.uk); INIA (Spain, project MIT01-GLOBALNET-EEZ); German Federal Ministry of Food and Agriculture (BMBL) through the Federal Office for Agriculture and Food (BLE); Swedish Infrastructure for Ecosystem Science (SITES) at Röbäcksdalen Research Station; Comisión Nacional de Investigación Científica y Tecnológica, Fondo Nacional de Desarrollo Científico y Tecnológico (Grant Nos. 11110410 and 1151355) and Fondo Regional de Tecnología Agropecuaria (FTG/RF-1028-RG); European Commission through SMEthane (FP7-SME-262270).

Nitrogen is a component of essential nutrients critical for the productivity of ruminants. If excreted in excess, N is also an important environmental pollutant contributing to acid deposition, eutrophication, human respiratory problems, and climate change. The complex microbial metabolic activity in the rumen and the effect on subsequent processes in the intestines and body tissues make the study of N metabolism in ruminants challenging compared with nonruminants. Therefore, using accurate and precise measurement techniques is imperative for obtaining reliable experimental results on N utilization by ruminants and evaluating the environmental impacts of N emission mitigation techniques. Changeover design experiments are as suitable as continuous ones for studying protein metabolism in ruminant animals, except when changes in body weight or carryover effects due to treatment are expected. Adaptation following a dietary change should be allowed for at least 2 (preferably 3) wk, and extended adaptation periods may be required if body pools can temporarily supply the nutrients studied. Dietary protein degradability in the rumen and intestines are feed characteristics determining the primary AA available to the host animal. They can be estimated using in situ, in vitro, or in vivo techniques with each having inherent advantages and disadvantages. Accurate, precise, and inexpensive laboratory assays for feed protein availability are still needed. Techniques used for direct determination of rumen microbial protein synthesis are laborious and expensive, and data variability can be unacceptably large; indirect approaches have not shown the level of accuracy required for widespread adoption. Techniques for studying postruminal digestion and absorption of nitrogenous compounds, urea recycling, and mammary AA metabolism are also laborious, expensive (especially the methods that use isotopes), and results can be variable, especially the methods based on measurements of digesta or blood flow. Volatile loss of N from feces and particularly urine can be substantial during collection, processing, and analysis of excreta, compromising the accuracy of measurements of total-tract N digestion and body N balance. In studying ruminant N metabolism, nutritionists should consider the longer term fate of manure N as well. Various techniques used to determine the effects of animal nutrition on total N, ammonia- or nitrous oxide-emitting potentials, as well as plant fertilizer value, of manure are available. Overall, methods to study ruminant N metabolism have been developed over 150 yr of animal nutrition research, but many of them are laborious and impractical for application on a large number of animals. The increasing environmental concerns associated with livestock production systems necessitate more accurate and reliable methods to determine manure N emissions in the context of feed composition and ruminant N metabolism., The authors acknowledge support for preparation of this manuscript by the USDA National Institute of Food and Agriculture, grant/award number: 2014-67003-21979, NH00616-R, the Department of Animal Science and College of Agricultural Sciences, The Pennsylvania State University; the Swiss Federal Office of Agriculture, INIA (Spain, project MIT01-GLOBALNETEEZ); Natural Resources Institute Finland (Luke); and Ministry of Agriculture, Nature and Food Quality (the Netherlands), GRA project BO-43-003.02.004

In vitro fermentation techniques (IVFT) have been widely used to evaluate the nutritive value of feeds for ruminants and in the last decade to assess the effect of different nutritional strategies on methane (CH4) production. However, many technical factors may influence the results obtained. The present review has been prepared by the 'Global Network' FACCE-JPI international research consortium to provide a critical evaluation of the main factors that need to be considered when designing, conducting and interpreting IVFT experiments that investigate nutritional strategies to mitigate CH emission from ruminants. Given the increasing and wide-scale use of IVFT, there is a need to critically review reports in the literature and establish what criteria are essential to the establishment and implementation of in vitro techniques. Key aspects considered include: i) donor animal species and number of animal used, ii) diet fed to donor animals, iii) collection and processing of rumen fluid as inoculum, iv) choice of substrate and incubation buffer, v) incubation procedures and CH measurements, vi) headspace gas composition and vii) comparability of in vitro and in vivo measurements. Based on an evaluation of experimental evidence, a set of technical recommendations are presented to harmonize IVFT for feed evaluation, assessment of rumen function and CH production., This review is part of the FACCE-JPI ‘Global Network’ project and the ‘Feeding and Nutrition Network’ of Livestock Research Group, in the Global Research Alliance for Agricultural Greenhouse Gases (www.globalresearchalliance.org). Authors acknowledge national funding from INIA (Spain, project MIT01-GLOBALNET-EEZ), the Ministry of Economic Affairs (The Netherlands; project Global Research Alliance on Agricultural Greenhouses Gases, BO-20-007-006), USDA-NIFA (USA), French National Research Agency through the program FACCE-JPI program, the Irish contribution to this JPI-FACCE project was funded by the Agricultural GHG Research Initiative for Ireland (AGRI-I), Department for Environment Food & Rural Affairs (UK), BLW (Switzerland) and Academy of Finland, Helsinki, Finland (Project 281337).

Ruminant husbandry is a major source of anthropogenic greenhouse gases (GHG). Filling knowledge gaps and providing expert recommendation are important for defining future research priorities, improving methodologies and establishing science-based GHG mitigation solutions to government and non-governmental organisations, advisory/extension networks, and the ruminant livestock sector. The objectives of this review is to summarize published literature to provide a detailed assessment of the methodologies currently in use for measuring enteric methane (CH) emission from individual animals under specific conditions, and give recommendations regarding their application. The methods described include respiration chambers and enclosures, sulphur hexafluoride tracer (SF) technique, and techniques based on short-term measurements of gas concentrations in samples of exhaled air. This includes automated head chambers (e.g. the GreenFeed system), the use of carbon dioxide (CO) as a marker, and (handheld) laser CH detection. Each of the techniques are compared and assessed on their capability and limitations, followed by methodology recommendations. It is concluded that there is no ‘one size fits all’ method for measuring CH emission by individual animals. Ultimately, the decision as to which method to use should be based on the experimental objectives and resources available. However, the need for high throughput methodology e.g. for screening large numbers of animals for genomic studies, does not justify the use of methods that are inaccurate. All CH measurement techniques are subject to experimental variation and random errors. Many sources of variation must be considered when measuring CH concentration in exhaled air samples without a quantitative or at least regular collection rate, or use of a marker to indicate (or adjust) for the proportion of exhaled CH sampled. Consideration of the number and timing of measurements relative to diurnal patterns of CH emission and respiratory exchange are important, as well as consideration of feeding patterns and associated patterns of rumen fermentation rate and other aspects of animal behaviour. Regardless of the method chosen, appropriate calibrations and recovery tests are required for both method establishment and routine operation. Successful and correct use of methods requires careful attention to detail, rigour, and routine self-assessment of the quality of the data they provide., This review is part of the FACCE-JPI ‘Global Network’ project. Authors acknowledge national funding from the Departmentfor Environment, Food and Rural Affairs (UK), INIA (Spain, project MIT01-GLOBALNET-EEZ), the Ministry of Economic Affairs(The Netherlands; project Global Research Alliance on Agricultural Greenhouses Gases, BO-20-007-006), USDA-NIFA (USA),French National Research Agency through the FACCE-JPI program, Agricultural GHG Research Initiative for Ireland (AGRI-I),Academy of Finland, Helsinki, Finland (Project 281337), and BLW (Switzerland).

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.

The potential of milk allantoin as an index ofmicrobial protein supply was evaluated in two experiments conducted with 12 multiparous Holstein-Friesian dairy cows that examined the effects of altering the supply of metabolizable energy (ME) and fermentable metabolizable energy (FME). In the first experiment, late lactation cows received a 14·3 kg dry matter (DM) per day basal diet consisting of (g/kg DM) chopped barley straw (415), soya-bean meal (322), molassed sugar-beet pulp (197), molasses (33), urea (17) and a vitamin and mineral supplement (17) for a 21-day co-variance period. During four 16-day periods, six treatments were allocated randomly to cows, consisting of potato starch (1, 2 or 3 kg DM per day) or fat supplements (0·64, 1·27 or 1·91 kg DM per day). In the second experiment, early lactation cows received 40 kg/day (fresh weight, (FW)) of silage (307 g/kg toluene-corrected dry matter, 149 g/kg DM crude protein and 11·6 MJ/kg ME). During three 21-day periods, four treatments were evaluated consisting of supplements of either 4·1 (LI) or 8·1 (12) kg fresh weight per day of a low fat concentrate (acid hydrolysis ether extract (AHEE) 45 g/kg DM) or 3·8 (HI) or 7·5 (H2) kg fresh weight per day of a high fat concentrate (AHEE 110 g/kg DM). Both experiments showed individual cow milk allantoin concentration or excretion to be poorly correlated with urinary purine derivative excretion or calculated microbial protein supply. Use of treatment mean (TM) values dramatically improved these relationships. For pooled TM (no. = 10) values from both experiments, close relationships existed between milk allantoin excretion and concentration with milk yield (r values 0·991 and 0·883, respectively). Auto-correlation with milk yield appeared to account for milk allantoin excretion and concentration being highly correlated with urinary purine derivative excretion (r values 0·908 and 0·934, respectively) and calculated microbial protein supply (r values 0·938 and 0·945, respectively). Current experimental data indicates that measurement of milk allantoin is not a reliable indicator of microbial protein supply for individual cows.

The potential of the spot urine sampling technique as an alternative to performing a total urine collection was evaluated. Twelve multiparous Holstein-Friesian cows were given two experimental diets in a complete change-over design using two 14-day experimental periods. Experimental diets were either silage offeredad libitumwith 7 kg fresh weight concentrate supplement as a single meal (SF), or a complete diet formulated from the same ingredients with a similar foragexoncentrate ratio (CD). Total urine collections were performed every 2 h on days 11 and 14 of each experimental period. Subsamples of urine were stored at 20°C and subsequently analysed by high-performace liquid chromatography. Daily allantoin and purine derivative (PD) excretion were highly correlated (r = 0·995, no. = 48,P< 0·001). PD and creatinine excretion during each 2-h interval depended on time of collection (PD,P< 0·001 and creatinine,P< 0·05) and on cow (P< 0·01) but were unaffected by sampling day or treatment. Diurnal variations in the molar ratio ofPD or allantoin to creatinine (PD/c and Ale, respectively) followed similar diurnal patterns as observed for PD and allantoin excretion. The data were used to assess the error of prediction of daily mean PD/c or Ale ratios. Three spot sampling regimens (based on the collection of four 4-h samples, three 8-h samples or two 12-h samples) and also on either single or 2-day urine collections were evaluated. Collection of multiple samples within a day was more reliable than collecting fewer samples over several days. Prediction errors were greater for SF compared with CD. Even the most intensive sampling regimen did not allow an acceptable prediction of daily mean PDIc or Ale ratio, minimum r values for PDIc and Ale ratios were 0·098, 0·136 and 0·547, 0·579 for SF and CD, respectively. Furthermore, daily mean PDIc and Ale ratios proved poor predictors of daily PD and allantoin excretion (r values of 0·69 and 0·72, respectively). Total urine collection appears necessary to assess accurately daily PD excretion in dairy cows.

Because of the potential benefits to human health, there is interest in increasing 18:3n-3, 20:5n-3, 22:6n-6, and cis-9,trans-11 CLA in ruminant foods. Four Aberdeen Angus steers (406 ± 8.2 kg of BW) fitted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square experiment with 21-d periods to examine the potential of fish oil (FO) and linseed oil (LO) in the diet to increase ruminal outflow of trans-11 18:1 and total n-3 PUFA in growing cattle. Treatments consisted of a control diet (60:40; forage:concentrate ratio, on a DM basis, respectively) based on maize silage, or the same basal ration containing 30 g/kg of DM of FO, LO, or a mixture (1:1, wt/wt) of FO and LO (LFO). Diets were offered as total mixed rations and fed at a rate of 85 g of DM/(kg of BW(0.75)/d). Oils had no effect (P = 0.52) on DMI. Linseed oil had no effect (P0.05) on ruminal pH or VFA concentrations, whereas FO shifted rumen fermentation toward propionate at the expense of acetate. Compared with the control, LO increased (P0.05) 18:0, cis 18:1 (Δ9, 12-15), trans 18:1 (Δ4-9, 11-16), trans 18:2, geometric isomers of 9,11, 11,13, and 13,15 CLA, trans-8,cis-10 CLA, trans-10,trans-12 CLA, trans-12,trans-14 CLA, and 18:3n-3 flow at the duodenum. Inclusion of FO in the diet resulted in greater (P0.05) flows of cis-9 16:1, trans 16:1 (Δ6-13), cis 18:1 (Δ9, 11, and 13), trans 18:1 (Δ6-15), trans 18:2, 20:5n-3, 22:5n-3, and 22:6n-3, and decreased (P0.001) 18:0 at the duodenum relative to the control. For most fatty acids at the duodenum, responses to LFO were intermediate of FO and LO. However, LFO resulted in greater (P = 0.04) flows of total trans 18:1 than LO and increased (P0.01) trans-6 16:1 and trans-12 18:1 at the duodenum compared with FO or LO. Biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen was independent of treatment, but both FO and LO increased (P0.001) the extent of 18:3n-3 biohydrogenation compared with the control. Ruminal 18:3n-3 biohydrogenation was greater (P0.001) for LO and LFO than FO, whereas biohydrogenation of 20:5n-3 and 22:6n-3 in the rumen was marginally less (P = 0.05) for LFO than FO. In conclusion, LO and FO at 30 g/kg of DM altered the biohydrogenation of unsaturated fatty acids in the rumen, causing an increase in the flow of specific intermediates at the duodenum, but the potential of these oils fed alone or as a mixture to increase n-3 PUFA at the duodenum in cattle appears limited.

Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n = 5) were performed in 100-mL flasks maintained at 39 °C containing 400mg of grass hay, 50 mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0 h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.