Effects of dietary fish meal and soybean meal on the ovine innate and acquired immune response during pregnancy and lactation.

In recent years, livestock producers have been supplementing animal diets with fish meal (FM) to produce value-added products for health conscious consumers. As components of FM have unique neuroendocrine-immunomodulatory properties, we hypothesize that livestock producers may be influencing the overall health of their animals by supplementing diets with FM. In this study, 40 pregnant ewes were supplemented with rumen protected (RP) soybean meal (SBM: control diet) or RP FM, commencing gestation day 100 (gd100), in order to evaluate the impact of FM supplementation on the innate and acquired immune response and neuroendocrine response of sheep during pregnancy and lactation. On gd135, half the ewes from each diet (n = 10 FM, n = 10 SBM) were challenged iv with lipopolysaccharide (LPS) to simulate a systemic bacterial infection and the febrile, respiratory and neuroendocrine responses were monitored over time; the other half (n = 10 FM, n = 10 SBM) of the ewes received a saline injection as control. On lactation day 20 (ld20), all ewes (n = 20 FM, n = 20 SBM) were sensitized with hen egg white lysozyme (HEWL) and the serum haptoglobin (Hp) response was measured over time. The cutaneous hypersensitivity response (CHR) to HEWL challenge was measured on ld30 (n = 20 FM, n = 20 SBM), and blood samples were collected over time to measure the primary and secondary immunoglobulin G (IgG) response to HEWL. There was an attenuated trend in the LPS-induced febrile response by the FM treatment when compared with the SBM treatment (P = 0.06), as was also true for the respiratory response (P = 0.07), but significant differences in neuroendocrine function (serum cortisol and plasma ACTH) were not observed between treatments. Basal Hp levels were significantly lower in the FM supplemented ewes when compared with the SBM supplemented ewes (P < 0.01), and the Hp response to HEWL sensitization differed significantly over time between treatments (P < 0.01). The CHR to HEWL was also significantly attenuated in the FM treatment compared with the SBM (P < 0.01); however, treatment differences in the primary and secondary IgG responses to HEWL were not observed. These results indicate that FM supplementation differentially affects the innate and acquired immune responses in pregnant and lactating sheep compared with a typical SBM diet of commercial flocks. The long-term implications of this immunomodulation warrant further investigation.

Fatty acid profile of colostrum and milk of ewes supplemented with fish meal and the subsequent plasma fatty acid status of their lambs.

The objectives of the current study were to 1) determine whether a fish-meal-supplemented diet fed to ewes during late gestation and early lactation would increase the proportion of docosahexaenoic acid (22:6n-3) in colostrum and milk and 2) examine the subsequent effect on the plasma fatty acid profile of nursing lambs. Eight gestating ewes (Rideau-Arcott; 97 +/- 5 kg of initial BW; 100 d of gestation) were used in a completely randomized design. Ewes were individually housed and fed a control diet (supplemented with soybean meal) or a fish-meal-supplemented diet for 6 wk before lambing and throughout 7 wk of lactation. Colostrum at d 0 and milk samples at d 36 and 49 of lactation were collected. Blood samples were collected from lambs throughout the preweaning period (at 0, 36, and 49 d of age). Fatty acids of the samples were analyzed by GLC. The ewes fed the fish-meal-supplemented diet had greater (P C18, 0.70 vs. 0.38), in colostrum and milk compared with the ewes fed the control diet. However, these fatty acids, excluding total n-3-PUFA, did not change over time, nor was there an interaction between diet and time. The percentage of total SFA was increased (P = 0.012) linearly over time without having any diet effect. The ratio of n-6-PUFA to n-3-PUFA in colostrum and milk from the control group was greater (P = 0.003) than that of the fish-meal-supplemented group. This ratio was decreased over time (P = 0.001). At birth (d 0), lambs born to the fish-meal-supplemented ewes had greater (P = 0.001) plasma concentrations (g/100 g of total fatty acids) of eicosapentaenoic acid, docosahexaenoic acid, and total very long chain n-3-PUFA than the lambs born to the control ewes. The concentrations of these fatty acids were further increased over time (P = 0.001) for the lambs nursing ewes fed the fish-meal-supplemented diet. The present findings suggest that the concentrations of docosahexaenoic acid in ewe colostrum and milk can be enhanced through diet supplementation with fish meal. The docosahexaenoic acid status of their suckling lambs can also be further enhanced, and this may contribute to improve neural tissue development and overall performance of suckling lambs.

The effect of dietary fiber level on milk fat concentration and fatty acid profile of cows fed diets containing low levels of polyunsaturated fatty acids.

The objective of this study was to investigate the effect of dietary fiber level on milk fat concentration, yield, and fatty acid (FA) profile of cows fed diets low in polyunsaturated fatty acid (PUFA). Six rumen-fistulated Holstein dairy cows (639 +/- 51 kg of body weight) were used in the study. Cows were randomly assigned to 1 of 2 dietary treatments, a high fiber (HF; % of dry matter, 40% corn silage, 27% alfalfa silage, 7% alfalfa hay, 18% protein supplement, 4% ground corn, and 4% wheat bran) or a low fiber (LF; % of dry matter, 31% corn silage, 20% alfalfa silage, 5% alfalfa hay, 15% protein supplement, 19% ground wheat, and 10% ground barley) total mixed ration. The diets contained similar levels of PUFA. The experiment was conducted over a period of 4 wk. Ruminal pH was continuously recorded and milk samples were collected 3 times a week. Milk yield and dry matter intake were recorded daily. The rumen fluid in cows receiving the LF diet was below pH 5.6 for a longer duration than in cows receiving the HF diet (357 vs. 103 min/d). Neither diet nor diet by week interaction had an effect on milk yield (kg/d), milk fat concentration and yield, or milk protein concentration and yield. During wk 4, milk fat concentration and milk fat yield were high and not different between treatments (4.30% and 1.36 kg/d for the HF treatment and 4.31% and 1.33 kg/d for the LF treatment, respectively). Cows receiving the LF diet had greater milk concentrations (g/100 g of FA) of 7:0; 9:0; 10:0; 11:0; 12:0; 12:1; 13:0; 15:0; linoleic acid; FA

Effects of nutritionally induced metabolic acidosis with or without glutamine infusion on acid-base balance, plasma amino acids, and plasma nonesterified fatty acids in sheep.

This study characterized the effects of nutritionally induced metabolic acidosis with or without Gln infusion on acid-base balance, plasma AA, and plasma NEFA in sheep. In a randomized complete block design with a 2 x 2 factorial arrangement of treatments, 24 fully fleeced sheep (Rideau-Arcott, 63.6 +/- 5.9 kg of BW) were fed a control supplement (CS; 300 g/d of canola meal) or an acidosis supplement (AS; 300 g/d of NutriChlor; HCl-treated canola meal), offered twice daily at 0700 and 1100 h. Sheep were infused at 1400 h daily with 0.3 g of L-glutamine per kg of BW or saline via jugular vein catheters for 7 d. The sheep were individually housed and limit-fed a basal diet of dehydrated alfalfa pellets (1.75 kg/d; 90% DM, 22% CP, and 1.2 Mcal of NE(g)/kg on a DM basis) offered twice daily at 1000 and 1300 h. Blood and urine was sampled daily between 1100 and 1130 h, and blood samples were analyzed for hematocrit, plasma pH, gases, strong ions, AA, and NEFA, whereas urine was analyzed for pH. The AS reduced (P < 0.01) DMI, urine and plasma pH, blood urea, partial pressure of CO(2), strong ion difference, and plasma HCO(3)(-), and increased (P < 0.01) plasma K(+), Ca(2+), and Cl(-). The AS with saline infusion increased (P

Effects of monensin and dietary soybean oil on milk fat percentage and milk fatty acid profile in lactating dairy cows.

The objective of this study was to investigate the effect of monensin (MN) and dietary soybean oil (SBO) on milk fat percentage and milk fatty acid (FA) profile. The study was conducted as a randomized complete block design with a 2 x 3 factorial treatment arrangement using 72 lactating multiparous Holstein dairy cows (138 +/- 24 d in milk). Treatments were [dry matter (DM) basis] as follows: 1) control total mixed ration (TMR, no MN) with no supplemental SBO; 2) MN-treated TMR (22 g of MN/kg of DM) with no supplemental SBO; 3) control TMR including 1.7% SBO; 4) MN-treated TMR including 1.7% SBO; 5) control TMR including 3.4% SBO; and 6) MN-treated TMR including 3.4% SBO. The TMR (% of DM; corn silage, 31.6%; haylage, 21.2%; hay, 4.2%; high-moisture corn, 18.8%; soy hulls, 3.3%; and protein supplement, 20.9%) was offered ad libitum. The experiment consisted of a 2-wk baseline, a 3-wk adaptation, and a 2-wk collection period. Monensin, SBO, and their interaction linearly reduced milk fat percentage. Cows receiving SBO with no added MN (treatments 3 and 5) had 4.5 and 14.2% decreases in milk fat percentage, respectively. Cows receiving SBO with added MN (treatments 4 and 6) had 16.5 and 35.1% decreases in milk fat percentage, respectively. However, the interaction effect of MN and SBO on fat yield was not significant. Monensin reduced milk fat yield by 6.6%. Soybean oil linearly reduced milk fat yield and protein percentage and linearly increased milk yield and milk protein yield. Monensin and SBO reduced 4% fat-corrected milk and had no effect on DM intake. Monensin interacted with SBO to linearly increase milk fat concentration (g/100 g of FA) of total trans-18:1 in milk fat including trans-6 to 8, trans-9, trans-10, trans-11, trans-12 18:1 and the concentration of total conjugated linoleic acid isomers including cis-9, trans-11 18:2; trans-9, cis-11 18:2; and trans-10, cis-12 18:2. Also, the interaction increased milk concentration of polyunsaturated fatty acids. Monensin and SBO linearly reduced, with no significant interaction, milk concentration (g/100 g of FA) of short- and medium-chain fatty acids (

Supplemental algal meal alters the ruminal trans-18:1 fatty acid and conjugated linoleic acid composition in cattle.

The effects of dietary algal supplementation, a source of docosahexaenoic acid, on the fatty acid profile of rumen lipids in cattle were evaluated, with special emphasis on CLA and trans fatty acids produced by rumen microbes. A diet based on corn silage was fed with supplements containing the following: 1) no algal meal and fed at 2.1 kg of DM/d (control), 2) algal meal and fed at 1.1 kg of DM/d (low algal meal), 3) algal meal and fed at 2.1 kg of DM/d (medium algal meal), and 4) algal meal and fed at 4.2 kg of DM/d (high algal meal). A modified lipid extraction procedure was developed to analyze the lipid changes in rumen fluid. The percentage of stearic acid (18:0) in rumen fluid was decreased by algal meal supplementation (P < 0.001) compared with control and was linearly dependent on the level of algal meal supplementation (P = 0.005). Total trans-18:1 in rumen fluid of cattle fed the control diet was 19% of total fatty acids. Addition of algal meal increased (P < 0.001) total trans-18:1 up to 43%, mostly due to 18:1 trans-10 that increased (P = 0.002) to 29.5% of total rumen fatty acids. This increase in 18:1 trans-10 seems to suggest a change in the rumen microbial population. Vaccenic acid (18:1 trans-11) increased quadratically (P = 0.005) with increasing level of algal meal supplementation in the diets. The total CLA content was low in the control (<0.9%) and increased with dietary algal meal addition, although not significantly; the greatest level was 1.5% with the medium algal meal diet. The increase of rumenic acid (cis-9, trans-11 CLA) was quadratic (P = 0.05) with algal meal supplementation, whereas trans-10, cis-12 CLA increased linearly with increased level of algal meal from 0.08 to 0.13% (P = 0.03). The ratio of trans-11 (cis-9, trans-11 CLA + 18:1 trans-11) to trans-10 (trans-10, cis-12 CLA + 18:1 trans-10) decreased from 2.45 to 0.77, 0.87, and 0.21 for the control, low algal meal, medium algal meal, and high algal meal diets, respectively. The content of docosahexaenoic acid in rumen fluid increased (P = 0.002) from 0.3 to 1.4% of total fatty acids with increasing level of algal meal supplementation in the diets. Our results suggest that algal meal inhibits the reduction of trans-18:1 to 18:0, giving rise to the high trans-18:1 content. In conclusion, algal meal could be used to increase the concentration in rumen contents of trans-18:1 isomers that serve as precursors for CLA biosynthesis in the tissues of ruminants.

Long-term effects of feeding monensin on milk fatty acid composition in lactating dairy cows.

The objective of this study was to determine the long-term effects of feeding monensin on milk fatty acid (FA) profile in lactating dairy cows. Twenty-four lactating Holstein dairy cows (1.46 +/- 0.17 parity; 620 +/- 5.9 kg of live weight; 92.5 +/- 2.62 d in milk) housed in a tie-stall facility were used in the study. The study was conducted as paired comparisons in a completely randomized block design with repeated measurements in a color-coded, double blind experiment. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (TMR) with a forage-to-concentrate ratio of 60:40 (control TMR; placebo premix) vs. a medicated TMR [monensin TMR; regular TMR + 24 mg of Rumensin Premix per kg of dry matter (DM)] fed ad libitum. The animals were fed and milked twice daily (feeding at 0830 and 1300 h; milking at 0500 and 1500 h). Milk samples were collected before the introduction of treatments and monthly thereafter for 6 mo and analyzed for FA composition. Monensin reduced the percentage of the short-and medium-chain saturated FA 7:0, 9:0, 15:0, and 16:0 in milk fat by 26, 35, 19, and 6%, respectively, compared with the control group. Monensin increased the percentage of the long-chain saturated FA in milk fat by 9%, total monounsaturated FA by 5%, total n-6 polyunsaturated FA (PUFA) by 19%, total n-3 PUFA by 16%, total cis-18:1 by 7%, and total conjugated linoleic acid (CLA) by 43% compared with the control group. Monensin increased the percentage of docosahexaenoic acid (22:6n-3), docosapentaenoic acid (22:5n-3), and cis-9, trans-11 CLA in milk fat by 19, 13, and 43%, respectively, compared with the control. These results suggest that monensin was at least partly effective in inhibiting the biohydrogenation of unsaturated FA in the rumen and consequently increased the percentage of n-6 and n-3 PUFA and CLA in milk, thus enhancing the nutritional properties of milk with regard to human health.

Long-term effects of feeding monensin on methane production in lactating dairy cows.

The objective of this study was to determine the long-term effects of feeding monensin on methane (CH4) production in lactating dairy cows. Twenty-four lactating Holstein dairy cows (1.46 +/- 0.17 parity; 620 +/- 5.9 kg of live weight; 92.5 +/- 2.62 d in milk) housed in a tie-stall facility were used in the study. The study was conducted as paired comparisons in a completely randomized design with repeated measurements in a color-coded, double-blind experiment. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (TMR) with a forage-to-concentrate ratio of 60:40 (control TMR; placebo premix) vs. a medicated TMR (monensin TMR; regular TMR + 24 mg of Rumensin Premix/kg of dry matter) fed ad libitum. The animals were fed and milked twice daily (feeding at 0830 and 1300 h; milking at 0500 and 1500 h) and CH4 production was measured prior to introducing the treatments and monthly thereafter for 6 mo using an open-circuit indirect calorimetry system. Monensin reduced CH4 production by 7% (expressed as grams per day) and by 9% (expressed as grams per kilogram of body weight), which were sustained for 6 mo (mean, 458.7 vs. 428.7 +/- 7.75 g/d and 0.738 vs. 0.675 +/- 0.0141, control vs. monensin, respectively). Monensin reduced milk fat percentage by 9% (3.90 vs. 3.53 +/- 0.098%, control vs. monensin, respectively) and reduced milk protein by 4% (3.37 vs. 3.23 +/- 0.031%, control vs. monensin, respectively). Monensin did not affect the dry matter intake or milk yield of the cows. These results suggest that medicating a 60:40 forage-to-concentrate TMR with 24 mg of Rumensin Premix/kg of dry matter is a viable strategy for reducing CH4 production in lactating Holstein dairy cows.

Effect of supplementing myristic acid in dairy cow rations on ruminal methanogenesis and fatty acid profile in milk.

The objective of this study was to evaluate the effects of supplementing myristic acid in dairy cow rations on ruminal methanogenesis and the fatty acid profile in milk. Twelve multiparous Holstein dairy cows (710 +/- 17.3 kg of live weight; 290 +/- 41.9 d in milk) housed in a tie-stall facility were used in the study. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (control diet), and 2) the regular milking cow total mixed ration supplemented with 5% myristic acid on a dry matter basis (MA diet). The cows were fed and milked twice daily (feeding, 0830 and 1300 h; milking, 0500 and 1500 h). The experiment was conducted as a completely randomized design and consisted of a 7-d pretrial period when cows were fed the control diet to obtain baseline measurements, a 10-d dietary adaptation period, and a 1-d, 8-h measurement period. The MA diet reduced methane (CH4) production by 36% (608.2 vs. 390.6 +/- 56.46 L/d, control vs. MA diet, respectively) and milk fat percentage by 2.4% (4.2 vs. 4.1 +/- 0.006%, control vs. MA diet, respectively). The MA diet increased 14:0 in milk by 139% and cis-9 14:1 by 195%. There was a correlation (r = -0.58) between the 14:0 content in milk and CH4 production and cis-9 14:1 and CH4 production (r = -0.47). Myristic acid had no effect on the contents of CLA or trans-10 18:1 and trans-11 18:1 isomers in milk. These results suggest that MA could be used to inhibit the activities of methanogens in ruminant animals without altering the conjugated linoleic acid and trans-18:1 fatty acid profile in milk.

Fatty acid composition of ruminal bacteria and protozoa, with emphasis on conjugated linoleic acid, vaccenic acid, and odd-chain and branched-chain fatty acids.

Knowledge of the fatty acid profile of microbial lipids is of great nutritional importance to the animals and, subsequently, their products. This study was conducted to examine the fatty acid profiles of mixed rumen bacteria and protozoa. Bacterial and protozoal cells were isolated by differential centrifugation of rumen contents. The main fatty acids were palmitic (16:0) and stearic (18:0) in both the bacterial and protozoal fractions. Palmitic acid was 74% greater in the protozoal fatty acids than in the bacterial fatty acids, whereas bacteria had 2.25-times greater stearic acid (18:0) proportions compared with protozoa. The total odd-chain plus branched-chain fatty acids were 16.5% of bacterial fatty acids and 11.0% of protozoal fatty acids. The anteiso-17:0 proportions in bacterial and protozoal fatty acids were 1.4 and 2.9%, respectively. The most abundant trans-18:1 isomer, vaccenic acid (18:1 trans-11), was 6.6% of total fatty acids in protozoa and 2.0% of total fatty acids in bacteria. The cis-9, trans-11 CLA was 8.6-times greater in the protozoal fraction (1.32% of total fatty acids) than in the bacterial fraction (0.15%). These results suggest that the presence of protozoa in the rumen may increase the supply of CLA and other unsaturated fatty acids for lower gut absorption by ruminants.

Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro.

In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria.

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro.

An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO(2)). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h with or without MSO (1 mM) or MSO(2) (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 micromol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO(2) and 2-aminobutyric acid (2AB) in B and BP. MSO(2) as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO(2); but MSO can be utilized by B and BP for producing Met.

Biosynthesis of methionine from homocysteine, cystathionine and homoserine plus cysteine by mixed rumen microorganisms in vitro.

This study quantitatively investigated the biosynthesis of methionine (Met) and the production of related compounds from homocysteine (Hcys), cystathionine (Cysta), and homoserine (Hser) plus cysteine (Cys) by rumen bacteria (B) or protozoa (P) alone and by a mixture of these bacteria and protozoa (BP). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h. Hcys, Cysta, and Hser plus Cys were catabolized by all rumen microbial fractions to different extents. B, P, and BP converted Hcys to Met with 2-aminobutyric acid (2AB) and methionine sulfoxide. The Met-producing ability of B (83.2 micromol g(-1) microbial nitrogen; MN) from Hcys was about 3.6 times higher than that of P in a 6-h incubation period. The ability of BP, during the same incubation period, was about 30.0% higher than that of B. Hcys, Met, and 2AB were formed when Cysta was incubated with B, P, or BP. Rumen microbial fermentation of Hser plus Cys led to the formation of Cysta, Met (through Hcys), and 2AB. Thus the results indicated that a trans-sulfurylation pathway for Met synthesis was operating in the rumen bacteria and protozoa. The results mentioned above have been demonstrated for the first time in B, P, and BP in the present study.

Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography.

In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150x4.6 mm I.D., 5 microm particle size) equipped with a guard column (4.0x4 mm, 5 microm particle size). Solvent A is composed of acetonitrile-sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile-sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (microM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6-100.6% for an intra-day study and 93.9-99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 microM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 microM) and 5AV (497.6 microM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.

Studies on the possibility of histidine biosynthesis from histodinol, imidazolepyruvic acid, imidazoleacetica acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro.

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.

Biosynthesis of threonine from homoserine by mixed rumen microorganisms: an in vitro study.

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39 degrees C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 micromol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report.

In vitro catabolism of histidine by mixed rumen bacteria and protozoa.

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39 degrees C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 micromol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated.

Catabolism of methionine and threonine in vitro by mixed ruminal bacteria and protozoa.

In vitro studies were conducted to examine the metabolism of methionine (Met) and threonine (Thr) using mixed ruminal bacteria (B), mixed ruminal protozoa (P), and a combination of these two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 1 mM each of the substrates at 39 degrees C for 12h. Met, Thr and their related amino compounds in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. Met was degraded by 58.7, 22.1, and 67.3% as a whole in B, P, and BP suspensions, respectively, during 12h incubation. In the case of Thr, these values were 67.3, 33.4, and 76.2% in B, P, and BP, respectively. Met was catabolized by all of the three microbial suspensions to methionine sulfoxide and 2-aminobutyric acid. Catabolism of Thr by B and BP resulted in the production of glycine and 2-aminobutyric acid, while P produced only 2-aminobutyric acid. From these results, the existence of diverse catabolic routes of Met and Thr in rumen microorganisms was indicated.

Convenient method of threonine, methionine and their related amino compounds by high-performance liquid chromatography and its application to rumen fluid.

A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO2), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150x4.6 mm I.D., 5 microm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (microM) (S/N ratio, 3:1) and quantification (microM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO2, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93-102.3% in the within-day study and 94.52-98.69% on different day (6 days) studies. The average contents (microM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency.

Determination of histidine and related compounds in rumen fluid by liquid chromatography.

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.