Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added to the diet.

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 CO2-methane production in four experiments in a herd of 88 to 109 milking Holsteins. Methane and CO2 production increased with twice-daily feeding, but the CO2:CH4 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 CO2:CH4 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 CO2:CH4 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.

Hematological and lipid changes in newborn piglets fed milk-replacer diets containing erucic acid.

Canola oil is not presently permitted in infant formulations in the United States because of lack of information concerning the effects of feeding canola oil to the newborn. We have previously reported a transient decrease in platelet counts and an increase in platelet size in newborn piglets fed canola oil for 4 wk, and have confirmed this in the present study. In canola oil-fed piglets, changes in platelet size and number were overcome by adding either long-chain saturated fatty acids from cocoa butter (16:0 and 18:0), or shorter-chain saturates from coconut oil (12:0 and 14:0). Feeding a high erucic acid rape-seed (HEAR) oil, with 20% 22:1n-9, led to an even greater platelet reduction and increased platelet size throughout the 4-wk trial. Bleeding times were longer in piglets fed canola oil or HEAR oil compared to sow-reared and soybean oil-fed piglets. There were no other diet-related changes. Diet-induced platelet changes were not related to platelet lipid class composition, but there were fatty acid changes. The incorporation of 22:1n-9 into platelet phospholipids of piglets fed canola oil was low (0.2-1.2%), and even for the HEAR oil group ranged from only 0.2% in phosphatidylinositol to 2.4% in phosphatidylserine. A much greater change was observed in the concentration of 24:1n-9 and in the 24:1n-9/24:0 ratio in platelet sphingomyelin (SM). The 24:1n-9 increased to 49% in the HEAR oil group compared to about 12% in animals fed the control diets (sow-reared piglets and soybean oil-fed group), while the 24:1n-9/24:0 ratio increased from about 1 to 12. Even feeding canola oil, prepared to contain 2% 22:1n-9, led to a marked increase in 24:1n-9 to 29% and had a 24:1n-9/24:0 ratio of 5. The canola oil/cocoa butter group, which also contained 2% 22:1n-9, showed a lower level of 24:1n-9 (20%) and the 24:1n-9/24:0 ratio (3) compared to the canola oil group. The results suggest that the diet-related platelet changes in newborn piglets may be related to an increase in 24:1n-9 in platelet SM, resulting from chain elongation of 22:1n-9. The inclusion of canola oil as the sole source of fat in the milk-replacer diets of newborn piglets resulted in significant platelet and lipid changes.

Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids.

Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (18:2) purported to be anticarcinogenic, low levels of essential fatty acids, and trans fatty acids that increase when essential fatty acids are increased in dairy rations. Milk and rumen fatty acid methyl esters (FAME) were prepared using several acid- (HCl, BF3, acetyl chloride, H2SO4) or base-catalysts (NaOCH3, tetramethylguanidine, diazomethane), or combinations thereof. All acid-catalyzed procedures resulted in decreased cis/trans (delta 9c,11t-18:2) and increased trans/trans (delta 9t,11t-18:2) conjugated dienes and the production of allylic methoxy artifacts. The methoxy artifacts were identified by gas-liquid chromatography (Gl.C)-mass spectroscopy. The base-catalyzed procedures gave no isomerization of conjugated dienes and no methoxy artifacts, but they did not transesterify N-acyl lipids such as sphingomyelin, and NaOCH3 did not methylate free fatty acids. In addition, reaction with tetramethylguanidine coextracted material with hexane that interfered with the determination of the short-chain FAME by GLC. Acid-catalyzed methylation resulted in the loss of about 12% total conjugated dienes, 42% recovery of the delta 9c,11t-18:2 isomer, a fourfold increase in delta 9t,11t-18:2, and the formation of methoxy artifacts, compared with the base-catalyzed reactions. Total milk FAME showed significant infrared (IR) absorption due to conjugated dienes at 985 and 948 cm-1. The IR determination of total trans content of milk FAME was not fully satisfactory because the 966 cm-1 trans band overlapped with the conjugated diene bands. IR accuracy was limited by the fact that the absorptivity of methyl elaidate, used as calibration standard, was different from those of the other minor trans fatty acids (e.g., dienes) found in milk. In addition, acid-catalyzed reactions produced interfering material that absorbed extensively in the trans IR region. No single method or combination of methods could adequately prepare FAME from all lipid classes in milk or rumen lipids, and not affect the conjugated dienes. The best compromise for milk fatty acids was obtained with NaOCH3 followed by HCl or BF3, or diazomethane followed by NaOCH3, being aware that sphingomyelins are ignored. For rumen samples, the best method was diazomethane followed by NaOCH3.

Effect of nigericin, monensin, and tetronasin on biohydrogenation in continuous flow-through ruminal fermenters.

Four ionophores differing in cation selectivity were compared for their effect on microbial fermentation and biohydrogenation by ruminal bacteria in continuous culture. Monensin and nigericin are monovalent antiporters with selective binding affinities for Na+ and K+, respectively. Tetronasin is a divalent antiporter that binds preferentially with Ca2+ or Mg2+. Valinomycin is a monovalent uniporter and does not exchange K+ for H+. Steady-state concentrations of 2 micrograms/ml of monensin, nigericin, tetronasin, or valinomycin were maintained by constant infusion into fermenters. Molar percentages of acetate were lower, and those of propionate were higher, in the presence of monensin, nigericin, and tetronasin; all three ionophores also decreased CH4 production. Concentrations of valinomycin as high as 8 micrograms/ml had no effect on volatile fatty acids or CH4 production. Monensin, nigericin, and tetronasin inhibited the rate of biohydrogenation of linoleic acid. Continuous infusion of C18:2n-6 at a steady-state concentration of 314 micrograms/ml into fermenters receiving monensin, nigericin, or tetronasin resulted in lower amounts of stearic acid and higher amounts of oleic acid. Ionophores increased total C18:2 conjugated acids mainly because of an increase in the cis-9, trans-11-C18:2 isomer. If reflected in milk fat, ionophore-induced changes in ruminal lipids could enhance the nutritional qualities of milk.

Identification of a new sphingolipid 3-O-acyl-D-erythro-sphingomyelin in newborn pig and infant plasma.

A new sphingolipid was found in newborn pig plasma at a level of 2.5 +/- 0.4% of total lipids. The compound decreased to less than half that amount by day one of age and virtually disappeared by the fourth week. On thin-layer chromatography (TLC) the new lipid migrated close to phosphatidylethanolamine. The compound was isolated by TLC from the plasma of newborn piglets and identified as a 3-O-acyl-D-erythro-sphingomyelin by chemical and chromatographic techniques, 1H- and 13C-nuclear magnetic resonance and fast-atom bombardment mass spectrometry. Mild alkaline hydrolysis at room temperature gave mainly C16 and C18 fatty acids and sphingomyelin. Subsequent reaction with Ba(OH)2 released long-chain saturated and monounsaturated fatty acids from C14 to C24, and sphingosine which was identified as the erythro configuration by gas chromatography. Less than 1% of the sphingosine was of the C20 isomer. No hydroxy fatty acids were found. The acylated sphingomyelin was only found in plasma lipids of newborn piglets and not in their red blood cell membranes or platelets of newborn piglets, or in sow plasma. This compound was tentatively identified by chromatography in trace amounts in the serum of cord blood of newborn infants, but not in the plasma lipids of adults.

Methane and carbon dioxide emissions from dairy cows in full lactation monitored over a six-month period.

Methane and CO2 emissions from a herd of 118 lactating cows were measured directly by continuous monitoring with an infrared gas analyzer from 24 gas sampling locations. A total of 112 d of gas output was recorded between June 1993 and November 1993. Recordings were integrated at .5-h intervals, so that there were 48 data points for each 24-h period. The mean 24-h CH4 emission per cow was 587 +/- 61.3 L; the range was 436 to 721 L. The mean 24-h CO2 emission per cow was 6137 +/- 505 L, and the range was 5032 to 7427 L. These values were not corrected for gas emissions from stored manure, which contributed 5.8 and 6.1%, respectively, to CH4 and CO2 output under conditions of this experiment.

Steady-state rates of linoleic acid biohydrogenation by ruminal bacteria in continuous culture.

Ruminal biohydrogenation of linoleic acid was determined in fermenters with a continuous culture of microorganisms. Rates of biohydrogenation and changes of fatty acids in culture were measured during steady-state concentration of linoleic acid that was achieved by continuous infusion of linoleic acid into the fermenters. A number of trans and cis isomers were identified using a GLC equipped with an infrared detector. The infusion of linoleic acid resulted in a substantial increase in the content of trans-C18:1 and a lesser increase in cis-C18:1. the major trans peak consisted of a mixture of n-9 and n-7 isomers. Biohydrogenation of infused linoleic acid averaged 77%. There was evidence of fatty acid loss, as determined by a decrease in the recovery of linoleic acid after 8 h of infusion. Addition of C18:2n-6 had no major effect on the VFA production by ruminal microorganisms. The results were similar to those measured in vivo, indicating that artificial fermenters were reliable predictors of fatty acid metabolism in vivo.

Hematological and lipid changes in newborn piglets fed milk replacer diets containing vegetable oils with different levels of n-3 fatty acids.

To test if linolenic acid (18:3n-3) from vegetable oils would affect bleeding times and platelet counts in newborns, piglets were used as a model fed milk replacer diets containing 25% (by wt) vegetable oils or oil mixtures for 28 d and compared to sow-reared piglets. The oils tested included soybean, canola, olive, high oleic sunflower (HOAS), a canola/coconut mixture and a mixture of oils mimicking canola in fatty acid composition. All piglets fed the milk replacer diets showed normal growth. Bleeding times increased after birth from 4-6 min to 7-10 min by week 4 (P < 0.001), and were higher in pigs fed diets containing 18:3n-3, as well as in sow-reared piglets receiving n-3 polyunsaturated fatty acids (PUFA) in the milk, as compared to diets low in 18:3n-3. Platelet numbers increased within the first week in newborn piglets from 300 to 550 x 10(9)/L, and remained high thereafter. Milk replacer diets, containing vegetable oils, generally showed a transient delay in the rise of platelet numbers, which was partially associated with an increased platelet volume. The oils showed differences in the length of delay, but by the third week of age, all platelet counts were > 500 x 10(9)/L. The delay in rise in platelet counts appeared to be related to the fatty acid composition of the oil, as the effect was reproduced by a mixture of oils with a certain fatty acid profile, and disappeared upon the addition of saturated fatty acids to the vegetable oil. There were no alterations in the coagulation factors due to the dietary oils. Blood plasma, platelets and red blood cell membranes showed increased levels of 18:3n-3 and long-chain n-3 PUFA in response to dietary 18:3n-3. The level of saturated fatty acids in blood lipids was generally lower in canola and HOAS oil-fed piglets as compared to piglets fed soybean oil or reared with the sow. The results suggest that consumption of milk replacer diets containing vegetable oils rich in 18:3n-3 does not represent a bleeding risk, and that the transient lower platelet count can be counterbalanced by the addition of saturated fatty acids to the vegetable oils.

Ion transport and methane production in Methanobacterium thermoautotrophicum.

In Methanobacterium thermoautotrophicum, the protonmotive force for the H+-translocating ATPase consists mainly of a transmembrane electrical gradient (Deltapsi). These cells do not establish a significant transmembrane pH gradient (inside alkaline) and, in fact, if the suspending medium is of pH >/= 7.0, the pH gradient may be reversed-i.e., inside acid with respect to the extracellular pH. These studies show by both 23Na NMR and 22Na+ distribution that Na+ extrusion with the generation of Deltapsi precedes methanogenesis in Mb. thermoautotrophicum. It is calculated that the newly established Na+ gradients increase Deltapsi by approximately 50 mV (inside negative). There is no detectable H+ extrusion during methane synthesis; instead there is a high rate of H+ consumption for methane synthesis and an increase in internal pH. This was supported by 31P NMR experiments, which showed an internal pH shift from 6.8 to 7.6. With the cells maintained at an external pH of 7.2, the initial transmembrane pH gradient of -0.4 (inside acid) at 60 degrees C is equivalent to Deltapsi of + 27 mV (inside positive); after 20 min of incubation, the transmembrane pH gradient is + 0.4 (inside alkaline), which at 60 degrees C is equivalent to Deltapsi of -27 mV (inside negative). Actively respiring cells generated a protonmotive force of -198 mV. It is proposed that energy for CO2 reduction to the level of formaldehyde (the first step in methane synthesis) in Mb. thermoautotrophicum is derived from the Deltapsi generated by electrogenic Na+ extrusion. The protonmotive force required for ATP synthesis consists primarily of Deltapsi and appears to be the result of both an electrogenic Na+ extrusion and a pH gradient (inside alkaline) which develops during methanogenesis.

Backfat and carcass composition of piglets fed milk replacers containing vegetable oil compared with sow-reared piglets.

The carcass composition of piglets fed artificial milk was compared to sow-reared piglets. The artificial milk diets contained 25%, by weight, soybean oil or mixtures of canola and high erucic acid rapeseed oil. Both the total lipid and nitrogen (apparent) digestibility of the artificial milk diets was high, even when the dietary oil contained high levels of erucic acid. Sow-reared animals were matched with the piglets receiving the artificial milk by sex and live body weight. On both a relative and an absolute basis, the piglets receiving the artificial milk diets had less carcass fat than sow-reared animals. The per cent nitrogen and ash of the carcasses of sow-reared piglets were significantly reduced compared with piglets eating milk replacer. The fatty-acid patterns of the backfat of the piglets generally resembled the patterns of the dietary lipids. Piglets eating vegetable oil diets had long-chain polyunsaturated fatty acids in their backfat, even though the oils they were consuming did not.

Lipid accumulation in isolated perfused rat hearts has no apparent effect on mechanical function or energy metabolism as measured by 31P NMR.

Male Sprague-Dawley rats were fed diets that contained 20% by weight soybean oil or rapeseed oil (21% and 43% erucic acid) for 7 days. The rapeseed oil diets increased the cardiac triacylglycerol content 5-fold and 25-fold, respectively, above control values. Hearts were removed from the animals and perfused with modified Krebs-Henseleit buffer at 37 degrees C. The calculated rate-pressure product was used as a measure of contractile function. 31P NMR spectra were acquired throughout a protocol that consisted of 12 min control perfusion, followed by 12 min perfusion with 20 microM isoproterenol, 12 min washout, 12 min total global ischemia, and 28 min reperfusion. The steady state levels of creatine phosphate, ATP, intracellular pH, contractile function, and the free energy of ATP hydrolysis (delta GATP) were determined for all three groups of hearts. Isoproterenol more than doubled the rate-pressure product of the hearts on all diets and decreased the concentrations of creatine phosphate and ATP with a concomitant rise in Pi. After global ischemia, creatine phosphate levels recovered fully, ATP levels remained low, and most hearts developed ventricular fibrillation. Changes in intracellular pH were the same for all groups: pH was 7.1 throughout the equilibration and isoproterenol perfusion period, decreased to pH approximately 6.4 during ischemia, and returned to 7.0 during reperfusion. The results indicate that the fat accumulation that occurs in the hearts of rats fed diets rich in high erucic acid rapeseed oil does not interfere with the cardiac high energy phosphate metabolism or contractile function.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary saturated fat on erucic acid induced myocardial lipidosis in rats.

Male Sprague-Dawley rats were fed for one week diets containing 20% by weight fat/oil mixtures with different levels of erucic acid (22:1n-9) (approximately 2.5 or 9%) and total saturated fatty acids (approximately 8 or 35%). Corn oil and high erucic acid rapeseed (HEAR) oil were fed as controls. The same hearts were evaluated histologically using oil red O staining and chemically for cardiac triacylglycerol (TAG) and 22:1n-9 content in cardiac TAG to compare the three methods for assessing lipid accumulation in rat hearts. Rats fed corn oil showed trace myocardial lipidosis by staining, and a cardiac TAG content of 3.6 mg/g wet weight in the absence of dietary 22:1n-9. An increase in dietary 22:1n-9 resulted in significantly increased myocardial lipidosis as assessed histologically and by an accumulation of 22:1n-9 in heart lipids; there was no increase in cardiac TAG except when HEAR oil was fed. An increase in saturated fatty acids showed no changes in myocardial lipid content assessed histologically, the content of cardiac TAG or the 22:1n-9 content of TAG at either 2.5 or 9% dietary 22:1n-9. The histological staining method was more significantly correlated to 22:1n-9 in cardiac TAG (r = 0.49; P less than 0.001) than to total cardiac TAG (r = 0.40; P less than 0.05). The 22:1n-9 content was highest in cardiac TAG and free fatty acids. Among the cardiac phospholipids, the highest incorporation was observed into phosphatidylserine, followed by sphingomyelin. With the addition of saturated fat, the fatty acid composition showed decreased accumulation of 22:1n-9 and increased levels of arachidonic and docosahexaenoic acids in most cardiac phospholipids, despite decreased dietary concentrations of their precursor fatty acids, linoleic and linolenic acids.

Inhibition of methylcoenzyme M methylreductase by a uridine 5'-diphospho-N-acetylglucosamine derivative.

Uridine-5'-diphospho-N-acetylglucosamine, when oxidized with periodate to the corresponding aldehyde (o-UDP-GlcNAc), was a potent inhibitor of the methylcoenzyme M methylreductase reaction which catalyzes the reductive demethylation of methylcoenzyme M to methane. The oxidation product, o-UDP-GlcNAc, appears to bind to the UDP-GlcNAc site of the enzyme and inhibits the reduction of methylcoenzyme M both by MRF or its active hydrolytic fragment HS-HTP. The kinetic patterns indicate that o-UDP-GlcNAc inhibition is noncompetitive with HS-HTP or MRF, and the Hill coefficient indicated that there was cooperativity between the UDP and HS-HTP binding sites. The methylreductase enzyme was protected from o-UDP-GlcNAc inhibition by prior exposure to low concentrations of MRF. HS-HTP, at the same concentration as MRF, was not effective in protecting the enzyme from inhibition by o-UDP-GlcNAc.

Myocardial changes in newborn piglets fed sow milk or milk replacer diets containing different levels of erucic acid.

This study was undertaken to determine whether the neonate was more susceptible to the effects of dietary erucic acid (22:1n-9) than the adult. Newborn piglets were used to assess the safety of different levels of 22:1n-9 on lipid and histological changes in the heart. Newborn piglets showed no myocardial lipidosis as assessed by oil red 0 staining, but lipidosis appeared with consumption of sow milk and disappeared by seven days of age. Milk replacer diets containing soybean oil, or rapeseed oil mixtures with up to 5% 22:1n-9 in the oil, or 1.25% in the diet, gave trace myocardial lipidosis. Rapeseed oil mixtures with 7 to 42.9% 22:1n-9 showed definite myocardial lipidosis in newborn piglets, which correlated to dietary 22:1n-9, showing a maximum after one week on diet. The severity of the lipidosis was greater than observed previously with weaned pigs. There were no significant differences among diets in cardiac lipid classes except for triacylglycerol (TAG), which increased in piglets fed a rapeseed oil with 42.9% 22:1n-9. TAG showed the highest incorporation of 22:1n-9, the concentration of 22:1n-9 in TAG was similar to that present in the dietary oil. Among the cardiac phospholipids, sphingomyelin and phosphatidylserine had the highest, and diphosphatidylglycerol (DPG) the lowest level of 22:1n-9. The low content of 22:1n-9 in DPG of newborn piglets is not observed in weaned pigs and rats fed high erucic acid rapeseed oil. The relative concentration of saturated fatty acids was lowered in all cardiac phospholipids of piglets fed rapeseed oils, possibly due to the low content of saturated fatty acids in rapeseed oils. The results suggest that piglets fed up to 750 mg 22:1n-9/kg body weight/day showed no adverse nutritional or cardiac effects.

Structure of a novel cofactor containing N-(7-mercaptoheptanoyl)-O-3-phosphothreonine.

The cofactor required in the methylcoenzyme M methylreductase reaction was shown to be a large molecule with an Mr of 1149.21 in the free acid form. The cofactor, named MRF for methyl reducing factor, was identified from analyses by fast atom bombardment mass spectrometry and 1H, 13C, and 31P NMR spectroscopy as uridine 5'-[N-(7-mercaptoheptanoyl)-O-3-phosphothreonine-P-yl(2-acetamido- 2-deoxy- beta-mannopyranuronosyl)(acid anhydride)]-(1----4)-O-2-acetamido-2-deoxy- alpha-glucopyranosyl diphosphate. MRF contains N-(7-mercaptoheptanoyl)threonine O-3-phosphate (HS-HTP) [No11, K. M., Rinehart, K. L., Tanner, R. S., & Wolfe, R. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242] and is linked to C-6 of 2-acetamido-2-deoxymannopyranuronic acid of the UDP-disaccharide through a carboxylic-phosphoric anhydride linkage. It is postulated that this bond is responsible for the instability of the molecule and its hydrolysis during isolation. Analyses of Eadie and Hofstee plots of the methylcoenzyme M methylreductase reaction indicate that MRF has a 6-fold lower Km(app) than HS-HTP and a 50% greater Vmax. This suggests that the UDP-disaccharide moiety may be of importance in the binding of MRF to the enzyme active site.

Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs.

Hearts from 4 week-old weanling pigs were capable of continuous work output when perfused with Krebs-Henseleit buffer containing 11 mM glucose. Perfused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of glucose plus physiological concentrations (0.1 mM) of either palmitate or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and carnitine. When hearts were perfused with 1 mM fatty acids, the work output declined below control values. Erucic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by perfused rat hearts. In addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated perfused pig hearts used in the present study, erucic acid oxidation rates were as high as palmitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during erucic acid perfusion did not change noticeably from those during glucose perfusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to erucic acid are not replicated in the intact pig heart.

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

Antiketogenic effects of monensin in early lactation.

Holstein cows, second lactation or later, were gradually introduced to monensin-containing concentrate 1 wk prepartum and fed complete diets containing 15 and 30 g monensin/ton of DM for 3 wk postpartum. The addition of 30 g monensin/ton of feed decreased the incidence of subclinical ketosis from 6 out of 12 to 1 out of 12. The concentration of beta-hydroxybutyrate in blood from cows in the high monensin group was decreased during the 3-wk postpartum experimental period. Acetate: propionate ratios decreased from 2.32 in the control group to 1.44 in the high monensin group. Feed intake in the low monensin group was less than in the control group, but there were no significant differences in body weight changes or milk production. Monensin, when added to the diet, lowered milk fat in one of the monensin fed groups (low) but not in the other. Milk protein and lactose concentrations were not changed by the addition of monensin to the diet. No other adverse treatment effects were observed. Two cows from each group were culled for causes unrelated to treatment; the remainder completed the normal 305-d lactation and were rebred without problems.

Changes in oxidation reduction potentials and volatile fatty acid production by rumen bacteria when methane synthesis is inhibited.

Rumen inoculum was cultured in specially designed fermenters that allowed simultaneous measurement of pH, oxidation-reduction potentials, and gas production. The cultures were maintained at pH 6.8 by addition of 1 M NaHCO3 and continuous infusion of artificial saliva. Gas flow was maintained at 20.0 ml/min with a stream of O2-free N2. Monensin at 7.0 micrograms/ml inhibited CH4 production 49% below control concentrations. The sodium salt of 2-bromoethanesulfonic acid added at an initial concentration of 5 x 10(-5) M inhibited CH4 production by 86% and increased H2 production from less than .5 mumol/min in the control to 24.5 mumol/min in the inhibited fermenter. The redox potentials in the control fermenter remained above -.20 V and did not change with the addition of monensin. Bromoethanesulfonic acid rapidly decreased the redox potential in the fermenter to -.33 V. Volatile fatty acid production was not significantly altered by the addition of 2-bromoethanesulfonic acid. The addition of monensin gave the expected decrease in acetate:propionate ratios, decreased acetate and butyrate production, and increased valerate (but not propionate) production.

Structure of purified cytoplasmic cofactor from Methanobacterium thermoautotrophicum.

The reduction of methylcoenzyme M to methane is known to require a heat stable and oxygen sensitive cofactor. Recently it has been shown that the active site of this cofactor is 7-mercaptoheptanoylthreonine phosphate. The present study shows that in the complete structure of this cofactor 7-mercaptoheptanoylthreonine phosphate is linked by pyrophosphate to two N-acetyl-glucosamine residues and an unidentified terminal group R with m/z 214. By fast-atom-bombardment mass spectrometry the intact cofactor, isolated as the mixed disulfide with 2-mercaptoethanol, was shown to have a molecular weight of 1084.5. The pyrophosphate bond is quite labile and undergoes hydrolysis or prolonged storage. This lability of the pyrophosphate bond may explain why the intact cofactor has not been isolated until now.