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An experiment was conducted to determine the effects of dietary concentrations of Co on vitamin [B.sub.12] production and fermentation of mixed ruminal microbes grown in continuous culture fermentors. Four fermentors were fed 14 g of DM/d. The DM consisted of a corn and cottonseed hull-based diet with Co supplemented as CoC[O.sub.3]. Dietary treatments were 1) control (containing 0.05 mg of Co/kg of DM), 2) 0.05 mg of supplemental Co/kg of DM, 3) 0.10 mg of supplemental Co/kg of DM, and 4) 1.0 mg of supplemental Co/kg of DM. After a 3-d adjustment period, fermentors were sampled over a 3-d sampling period. This process was repeated 2 additional times for a total of 3 runs. Ruminal fluid vitamin [B.sub.12] concentrations were affected by Co supplementation (P < 0.01), and there was a treatment x day interaction (P < 0.01). By sampling d 3, cultures fed the basal diet supplemented with 0.10 mg of Co/kg had greater (P < 0.05) vitamin [B.sub.12] concentrations than those supplemented with 0.05 mg of Co/kg of DM, and increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased (P < 0.01) ruminal fluid vitamin [B.sub.12] con centration. Ruminal fluid succinate also was affected (P < 0.10) by a treatment x day interaction. Cobalt supplementation to the control diet greatly decreased (P < 0.05) succinate in ruminal cultures on sampling d 3 but not on d 1 or 2. Molar proportions of acetate, propionate, and isobutyrate, and acetate:propionate were not affected by the addition of supplemental Co to the basal diet. However, molar proportions of butyrate, valerate, and isovalerate increased (P < 0.05) in response to supplemental Co. The majority of long-chain fatty acids observed in this study were not affected by Co supplementation. However, percentages of C18:0 fatty acids in ruminal cultures tended (P < 0.10) to be greater for Co-supplemented diets relative to the control. Methane, ammonia, and pH were not greatly affected by Co supplementation. The results indicate that a total (diet plus supplemental) Co concentration of 0.10 to 0.15 mg/kg of dietary DM resulted in adequate vitamin [B.sub.12] production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermentors. Key words: cobalt, ruminal culture, succinate, vitamin [B.sub.12]

Five dual-flow fermentors (700 mL) were used to determine the effects of eastern gamagrass (Tripsacum dactyloides [L.] L.) diets on microbial metabolism by mixed rumen cultures. Fermentors were incubated with filtered ruminal contents and allowed to adapt for 4 d to diets followed by 3 d of sample collection. Five dietary treatments were tested: 1) gamagrass hay (GH) + no corn (GHNC), 2) gama grass silage (GS) + no corn (GSNC), 3) GS + low corn (GSLC), 4) GS + medium corn (GSMC); and 5) GS + high corn (GSHC). The experiment was conducted as a randomized complete block design with five treatments and three replications. Total VFA concentrations were not affected by diets. Corn addition linearly decreased (P < 0.001) molar proportion of acetate. In contrast, molar proportion of propionate was reduced in GSLC (cubic effect, P < 0.001) but remained similar across other diets. Corn supplementation linearly increased molar proportion of butyrate (P < 0.001). The acetate + butyrate-to-propionate ratio was highest in cultures offered GSLC (cubic effect, P < 0.001) but similar across other diets. Feeding GSNC resulted in a higher ruminal pH compared with GHNC (P < 0.03). Increasing the level of corn supplementation in GS linearly decreased culture pH (P < 0.001). All diets resulted in similar methane production, with the exception of GSMC, which lowered methane output (quadratic effect, P < 0.004). Total substrate fermented to VFA and gas tended to be greater with GHNC than with GSNC (P < 0.06) and linearly increased with the addition of corn (P < 0.004). Neutral detergent fiber digestibility was similar between GH and GS and was not affected by supplemental corn. Microbial N flow increased in cultures offered GSHC (quadratic effect, P < 0.02). Corn supplementation at the medium and high level linearly decreased [C.sub.18:0] (P < 0.02) and increased trans-[C.sub.18:1] (P < 0.004). Including corn at the high level with GS did not have a detrimental effect on fermentation in dual-flow fermentors. Key Words: Corn Supplementation, Gamagrass, Microbial Nitrogen

We measured effects of continuous vs twice-daily feeding, the addition of unsaturated fat to the diet, and monensin on milk production, milk composition, feed intake, and C[O.sub.2]-methane production in four experiments in a herd of 88 to 109 milking Holsteins. Methane and C[O.sub.2] production increased with twice-daily feeding, but the C[O.sub.2]:C[H.sub.4] ratio remained unchanged. Soybean oil did not affect the milkfat percentages, but fatty acid composition was changed. All saturated fatty acids up to and including 16:0 decreased (P < .01), whereas 18:0 and trans 18:1 increased (P < .001). The 18:2 conjugated dienes also increased (P < .01) when the cows were fed soybean oil. Monensin addition to the diet at 24 ppm decreased methane production (P < .01); the C[O.sub.2]:C[H.sub.4] ratios reached 15, milk production increased (P < .01), and milkfat percentage and total milkfat output decreased (P < .01), as did feed consumption, compared with cows fed diets without monensin (P < .05). Milk fatty acid composition showed evidence of depressed ruminal biohydrogenation: saturated fatty acids (P < .05) decreased and 18:1 increased (P < .001); most of the increase was seen in the trans 18:1 isomer. As with soybean oil feeding, addition of monensin also increased (P < .05) the concentration of conjugated dienes. The monensin feeding trial was repeated 161 d later with 88 cows, of which 67 received monensin in the diet in the first trial and 21 cows were newly freshened and had never received monensin. Methane production again decreased (P < .05), but this time the C[O.sub.2]:C[H.sub.4] ratio did not change and all other monensin-related effects were absent. The ruminal microflora in the cows that had previously received monensin seemed to have undergone some adaptive changes and no longer responded as before. Key Words: Methane, Carbon Dioxide, Ionophores, Gas Production, Dairy Cows

High-moisture ear corn (HMEC) was untreated, treated with propionic acid (PA), or inoculated with a mixture of Lactobacillus plantarum and Enterococcus faecium and allowed to ensile in laboratory silos for 0, 7, 21, 42, 138, or 202 d. The silages were evaluated for fermentation quality, microbial populations, and aerobic stability. In all treatments, silage pH declined rapidly within 7 d, but the rate of decline seemed greatest with the inoculum. The lactic acid content of inoculated HMEC was higher (P < .05) than that of control or PA-treated HMEC. Regardless of treatment, the population of lactic acid bacteria (LAB) increased (P < .1) up to 7 to 21 d of fermentation then declined; LAB counts decreased (P < .05) up to 42 d in control and PA-treated silage but continued to decline until 138 d for inoculated silage. Yeast and mold counts tended to decrease up to 42 d of ensiling then increased (P < .05) as fermentation progressed. Between 138 and 202 d of ensiling, the control silage showed a marked increase (P < .10) in pH and yeast and mold populations, providing evidence of secondary fermentation; PA treatment and bacterial inoculation prevented secondary fermentation. Inoculation tended to reduce estimates of sample temperature for silage stored for 138 d and exposed to air, but not for the corresponding silage stored for 202 d. Treatment with PA prevented the loss (P > .05) of acetic acid and the rise (P > .05) in pH during air exposure of the 138-d silage; both control and PA-treated silage showed an increase (P < .05) in yeast and mold populations, but the increments were 38% and 23%, respectively. Compared with PA, the relative efficacy of inoculation in improving aerobic spoilage of HMEC depended on the period of silo storage and the criterion used to assess aerobic stability. Key Words: Inoculation, Aerobic Treatment, Maize Silage, Maize Ears, Propionic Acid

High-moisture ear corn (HMEC) was treated with specific bacterial inoculants and evaluated for its aerobic stability and utilization for growth by beef steers. Immediately after harvest, HMEC (65% DM) was ensiled in tower silos after being either untreated (control) or treated with the following inoculants: 1) Ecosyl(R) (E); 2) Ecosyl plus Serratia rubidaea (E + SR); and 3) Ecosyl plus Streptococcus thermophilus (E + ST). A portion of HMEC was frozen immediately (-20 degrees C) and subsequently treated with eight bacterial inoculants before ensiling in laboratory silos; the fermented material was then exposed to air for 7 d for assessment of aerobic deterioration. The eight inoculants included the three used in the tower silos and four additional ones: Streptococcus thermophilus, Bacillus subtilis (BS), Serratia rubidaea, and a mixture of Ecosyl + B. subtilis (E + BS). The growth trial was conducted for 112 d with 32 crossbred steers (average BW 296 kg). A digestion trial was conducted, according to a 4 x 4 Latin square design, using an additional four steers (average BW 367 kg). In both trials, steers were fed the same four diets containing inoculated (E, E + SR, and E + ST) or control HMEC. Upon exposure to air, Ecosyl-treated ensiled HMEC had the least increase in pH compared with other single inoculants; all inoculant treatments lessened (P < .05) the increase in sample temperature compared with control. During aerobic exposure, treatment of HMEC with BS seemed to reduce the disappearance of water-soluble carbohydrates, whereas Ecosyl seemed to reduce lactic acid. Despite evidence of improved aerobic stability with Ecosyl and BS, inoculation of HMEC did not (P > .10) improve BW gain or feed efficiency; however, all inoculants reduced (P < .05) digestibility of ADF.

An experiment was conducted to determine the effects of Cu and soybean oil (SBO) supplementation on ruminal and tissue lipid metabolism and carcass characteristics in finishing steers. Sixty Angus steers (369.0 [+ or -] 10.1 kg) were stratified by weight and randomly assigned to treatments in a 2 x 2 factorial arrangement, with factors being 0 or 20 mg of supplemental Cu/kg DM from Cu sulfate and 0 or 4% SBO. Steers were fed a high-concentrate basal diet that contained 5.3 mg Cu/kg DM. Average daily gain and feed intake were reduced (P [is less than] 0.01) by SBO but were not affected by Cu. Gain:feed ratio was not affected by treatment. Liver Cu concentrations were higher (P [is less than] 0.01) in steers receiving supplemental Cu and lower (P [is less than] 0.04) in SBO-supplemented steers. Copper supplementation tended to reduce (P [is less than] 0.12) and SBO supplementation tended to increase (P [is less than] 0.11) serum cholesterol concentrations. Backfat depth was reduced (P [is less than] 0.10) by Cu and SBO supplementation. Marbling scores and longissimus muscle lipid content were not affected by Cu supplementation; however, SBO supplementation reduced (P [is less than] 0.01) marbling scores. Longissimus muscle polyunsaturated fatty acids tended to be increased (P [is less than] 0.14) in Cu-supplemented steers. Longissimus muscle C 18-conjugated dienes and the 18:1 trans isomer were increased (P [is less than] 0.05) in SBO-supplemented steers. Ruminal fluid 18:3 was increased (P [is less than] 0.05) and the 18:1 trans isomer was decreased (P [is less than] 0.05) in Cu-supplemented steers. These results indicate that as little as 20 mg of supplemental Cu/kg DM can reduce backfat and may alter lipid metabolism in steers fed high-concentrate diets. Key Words: Cholesterol, Copper, Fatty Acids, Steers

An experiment was conducted to determine the effects of dietary concentrations of Co on vitamin B12 production and fermentation of mixed ruminal microbes grown in continuous culture fermentors. Four fermentors were fed 14 g of DM/d. The DM consisted of a corn and cottonseed hull-based diet with Co supplemented as CoCO3. Dietary treatments were 1) control (containing 0.05 mg of Co/kg of DM), 2) 0.05 mg of supplemental Co/kg of DM, 3) 0.10 mg of supplemental Co/kg of DM, and 4) 1.0 mg of supplemental Co/kg of DM. After a 3-d adjustment period, fermentors were sampled over a 3-d sampling period. This process was repeated 2 additional times for a total of 3 runs. Ruminal fluid vitamin B12 concentrations were affected by Co supplementation (P < 0.01), and there was a treatment ×day interaction (P < 0.01). By sampling d 3, cultures fed the basal diet supplemented with 0.10 mg of Co/kg had greater (P < 0.05) vitamin B12 concentrations than those supplemented with 0.05 mg of Co/kg of DM, and increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased (P < 0.01) ruminal fluid vitamin B12 concentration. Ruminal fluid succinate also was affected (P < 0.10) by a treatment × day interaction. Cobalt supplementation to the control diet greatly decreased (P < 0.05) succinate in ruminal cultures on sampling d 3 but not on d 1 or 2. Molar proportions of acetate, propionate, and isobutyrate, and acetate:propionate were not affected by the addition of supplemental Co to the basal diet. However, molar proportions of butyrate, valerate, and isovalerate increased (P < 0.05) in response to supplemental Co. The majority of long-chain fatty acids observed in this study were not affected by Co supplementation. However, percentages of C18:0 fatty acids in ruminal cultures tended (P < 0.10) to be greater for Co-supplemented diets relative to the control. Methane, ammonia, and pH were not greatly affected by Co supplementation. The results indicate that a total (diet plus supplemental) Co concentration of 0.10 to 0.15 mg/kg of dietary DM resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermentors. [ABSTRACT FROM AUTHOR]

Eighty lactating Holstein cows from 21 to 91 days in milk were fed a corn silage-based total mixed ration (TMR) formulated to meet National Research Council (2001) recommendations except the Met content was limited (42 g/d) in order to investigate the impact of supplementing rumen-protected (RP) forms of Met, betaine, and choline on performance and metabolism. One of four supplements was blended into the TMR to produce four dietary treatments: 1.) control, 2.) 20 g/d RP-Met, 3.) 45 g/d RP-betaine, and 4.) 40 g/d RP-choline. Calcium salts of fatty acids were used to protect both RP-betaine and RP-choline supplements and were added to both control and RP-Met supplements so that equal amounts of fat were supplied to all treatments. Intake of DM was not different among treatments (P > 0.2). The treatment by parity interaction tended to be different (P = 0.06) for milk yield with 44.3 kg/d produced in MP cows fed RP-choline compared to MP cows fed all other treatments (37.8, 40.0, and 38.7, respectively) while there were no differences among treatments in PP cows. Cows fed RP-Met or RP-choline had higher milk CP yield than cows fed control or RP-betaine (P = 0.02). There were no differences in milk fat yield or milk urea nitrogen (MUN) (P > 0.2). Mean body weight and body condition score (BCS) were not different among treatments (P > 0.2). [ABSTRACT FROM AUTHOR]

Mitigation of enteric methane (CH 4 ) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH 3 ) while the latter targets specific enzymes using structural analogs of CH 4 and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH 4 production by rumen microbes in batch culture. Sodium nitrate (NaNO 3 ), sodium sulfate (Na 2 SO 4 ), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO 3 + Na 2 SO 4 (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH 4 concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH 4 was highest in MF diet followed by HF and LF diets. Culture pH and NH 3 in the control decreased in the order of HF, MF, to LF as expected. NaNO 3 decreased ( p < 0.001) CH 4 and butyrate and increased acetate and propionate ( p < 0.03 and 0.003, respectively). Cultures receiving NaNO 3 had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH 4 at 6h with no further decrease at 24 h ( p < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors ( p < 0.001) while enriching succinate producers and assimilators as well as propionate producers ( p adj < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations ( p < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H 2 concentrations ( p < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio ( p < 0.001). Only the cultures treated with BCM in combination with other additives significantly (p adj < 0.05) decreased the abundance of Methanobrevibacter expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation in-vitro , inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH 3 , and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tanaka, Collins, Polkoff and Fellner.)

Background: Weaning stress reduces growth performance and health of young pigs due in part to an abrupt change in diets from highly digestible milk to fibrous plant-based feedstuffs. This study investigated whether dietary galactooligosaccharide (GOS), supplemented both pre- and post-weaning, could improve growth performance and intestinal health via alterations in the hindgut microbial community., Methods: Using a 3 × 2 factorial design, during farrowing 288 piglets from 24 litters received either no creep feed (FC), creep without GOS (FG-) or creep with 5% GOS (FG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). Pigs were sampled pre- (D22) and post-weaning (D31) to assess intestinal measures., Results: Creep fed pigs grew 19% faster than controls (P < 0.01) prior to weaning, and by the end of the nursery phase (D58), pigs fed GOS pre-farrowing (FG+) were 1.85 kg heavier than controls (P < 0.05). Furthermore, pigs fed GOS in phase 1 of the nursery grew 34% faster (P < 0.04), with greater feed intake and efficiency. Cecal microbial communities clustered distinctly in pre- vs. post-weaned pigs, based on principal coordinate analysis (P < 0.01). No effects of GOS were detected pre-weaning, but gruel creep feeding increased Chao1 α-diversity and altered several genera in the cecal microbiota (P < 0.05). Post-weaning, GOS supplementation increased some genera such as Fusicatenibacter and Collinsella, whereas others decreased such as Campylobacter and Frisingicoccus (P < 0.05). Changes were accompanied by higher molar proportions of butyrate in the cecum of GOS-fed pigs (P < 0.05)., Conclusions: Gruel creep feeding effectively improves suckling pig growth regardless of GOS treatment. When supplemented post-weaning, prebiotic GOS improves piglet growth performance associated with changes in hindgut microbial composition., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Early intestinal development is important to infant vitality, and optimal formula composition can promote gut health., Objectives: The objectives were to evaluate the effects of arachidonate (ARA) and/or prebiotic oligosaccharide (PRE) supplementation in formula on the development of the microbial ecosystem and colonic health parameters., Methods: Newborn piglets were fed 4 formulas containing ARA [0.5 compared with 2.5% of dietary fatty acids (FAs)] and PRE (0 compared with 8 g/L, containing a 1:1 mixture of galactooligosaccharides and polydextrose) in a 2 x 2 factorial design for 22 d. Fecal samples were collected weekly and analyzed for relative microbial abundance. Intestinal samples were collected on day 22 and analyzed for mucosal FAs, pH, and short-chain FAs (SCFAs)., Results: PRE supplementation significantly increased genera within Bacteroidetes and Firmicutes, including Anaerostipes, Mitsuokella, Prevotella, Clostridium IV, and Bulleidia, and resulted in progressive separation from controls as determined by Principal Coordinates Analysis. Concentrations of SCFA increased from 70.98 to 87.37 mM, with an accompanying reduction in colonic pH. ARA supplementation increased the ARA content of the colonic mucosa from 2.35-5.34% of total FAs. PRE supplementation also altered mucosal FA composition, resulting in increased linoleic acid (11.52-16.33% of total FAs) and ARA (2.35-5.16% of total FAs)., Conclusions: Prebiotic supplementation during the first 22 d of life altered the gut microbiota of piglets and increased the abundance of specific bacterial genera. These changes correlated with increased SCFA, which may benefit intestinal development. Although dietary ARA did not alter the microbiota, it increased the ARA content of the colonic mucosa, which may support intestinal development and epithelial repair. Prebiotic supplementation also increased unsaturation of FAs in the colonic mucosa. Although the mechanism requires further investigation, it may be related to altered microbial ecology or biohydrogenation of FA., (Published by Elsevier Inc.)

To combat the stress of weaning, we utilized novel gruel creep feeders to supplement suckling pigs with divergent soluble (n = 6 litters) versus insoluble (n = 6) diets compared with un-supplemented controls (n = 6). Post-weaning, pigs were fed a common phase 1 diet. Average daily weight gains of pigs fed soluble and insoluble creep diets were 53% and 17% greater than control pigs, respectively (p < 0.01). Creep intake was higher (82%) for pigs fed the soluble diet, and the accompanying weight increase was sustained post-weaning (p < 0.02). Villus measures were prematurely altered in soluble-creep-fed pigs (p < 0.01), with decreases in villi length, crypt depth, and villus area pre-weaning. No effects of treatment were detected for VFA concentrations and pH in the cecum. There was an interaction between treatment and age for several pro- and anti-inflammatory cytokines (p < 0.01), where soluble-creep-fed pigs had increased cytokine levels with age, whereas cytokine levels in the insoluble and control groups decreased over time. We conclude that a soluble creep diet fed in a gruel state during the pre-weaning period has a positive impact on weaning weight that is sustained post-weaning, and is accompanied by alterations in the intestinal health of young pigs.

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolomics of heparinized whole blood drawn from six African savanna elephants ( Loxodonta africana ) maintained on a well characterized diet. Whole blood samples obtained under behavioral restraint, then quickly frozen in liquid nitrogen, were stored at -80 °C until analysis. Frozen samples were thawed under controlled conditions and extracted with methanol and chloroform to separate the polar and non-polar metabolites. We identified 18 polar metabolites and 14 non-polar lipids using one-dimensional (1D) and two-dimensional (2D) NMR spectra. Despite unexpected rouleaux formation in the thawed frozen samples, spectra were consistent among animals and did not vary dramatically with age or the sex of the animal.

Background: African elephants in managed care have presented differences in the balance between omega-3 and omega-6 fatty acids, a situation primarily thought to be due to dietary differences between the managed animals and their free-ranging counterparts. Because of this, circulating fatty acid status is included in routine monitoring of elephant health. A method of blood collection that requires only a few drops of whole blood, dried on filter paper (DBS) and can be used for analyzing full fatty acid profiles offers advantages in clinical application., Methods: This study compared the use of whole blood, and whole blood DBS, serum or plasma for use in evaluating circulating fatty acid composition in African savannah elephants. Samples from six African elephants (two males and four females) were collected during the same week at the NC Zoo, Asheboro, NC., Results: Results found only 2 of 36 individual fatty acids and none of the 10 fatty acid groupings were different when comparing the four blood fraction sample types to each other with Mann-Whitney U-Test pairwise comparisons. Myristic acid (14:0) was lower in the DBS samples than in whole blood, serum, and plasma and pentadecaenoic acid (15:1) was slightly more concentrated in DBS and whole blood., Discussion: Results indicate that fatty acid profile of serum, plasma, whole blood, and DBS are comparable in African elephants. The DBS method offers advantages in acquisition and handling and may be preferable to other methods in both routine health assessment of captive animals and field research on free ranging animals., Competing Interests: Dr. Doug Bibus works for Lipid Technologies LLC., (©2021 Wood et al.)