Reproducibility, validity, and reliability of the incremental step test for subjects with moderate to severe asthma.

OBJECTIVE: We investigated the measurement properties of the incremental step test in subjects with moderate to severe asthma. METHODS: Subjects with moderate to severe persistent asthma were recruited from a tertiary university hospital specializing in treating severe asthma. All subjects performed one cardiopulmonary exercise test (CPET) and two incremental step tests (IST) in random sequences. Pulmonary gas exchange was measured during all exercise tests. The measurement properties investigated were reliability by intraclass correlation coefficient (ICC), measurement error by the standard error of measurement and minimum detectable difference, construct validity by Pearson's correlation, and interpretability by the ceiling and floor effects. RESULTS: Fifty subjects (38 females, mean [SD], age 43.7 [11.6] yr, % FEV1 70 [14.3], BMI 28.5 [5.3] kg/m2) completed the study. The peak oxygen uptake (peak VO2) for the CPET was 27.6 [±6.8] ml/kg/min, for the first IST was 22.3 [±5.3] ml/kg/min and for the second IST was 23.3 [±5.3] ml/kg/min. The IST presented excellent reliability (ICC=0.93, CI95% 0.88-0.96), very good measurement error (2.5%), and construct validity for peak VO2 measurement compared to the CPET (r = 0.85; p < 0.001) to assess exercise capacity in subjects with moderate to severe asthma, with appropriate ceiling (10%) and floor (0%) effects. CONCLUSION: The IST presented excellent reliability and very good measurement error and validity to assess exercise capacity in subjects with moderate to severe asthma, without ceiling or floor effects.

Nitrogen isotopic fractionation as a biomarker for nitrogen use efficiency in ruminants: a meta-analysis.

Animal proteins are naturally 15N enriched relative to the diet and the extent of this difference (Δ15Nanimal-diet or N isotopic fractionation) has been correlated to N use efficiency (NUE; N gain or milk N yield/N intake) in some recent ruminant studies. The present study used meta-analysis to investigate whether Δ15Nanimal-diet can be used as a predictor of NUE across a range of dietary conditions, particularly at the level of between-animal variation. An additional objective was to identify variables related to N partitioning explaining the link between NUE and Δ15Nanimal-diet. Individual values from eight publications reporting both NUE and Δ15Nanimal-diet for domestic ruminants were used to create a database comprising 11 experimental studies, 41 treatments and individual animal values for NUE (n=226) and Δ15Nanimal-diet (n=291). Data were analyzed by mixed-effect regression analysis taking into account experimental factors as random effects on both the intercept and slope of the model. Diets were characterized according to the INRA feeding system in terms of N utilization at the rumen, digestive and metabolic levels. These variables were used in a partial least squares regression analysis to predict separately NUE and Δ15Nanimal-diet variation, with the objective of identifying common variables linking NUE and Δ15Nanimal-diet. For individuals reared under similar conditions (within-study) and at the same time (within-period), the variance of NUE and Δ15Nanimal-diet not explained by dietary treatments (i.e. between-animal variation plus experimental error) was 35% and 55%, respectively. Mixed-effect regression analysis conducted with treatment means showed that Δ15Nanimal-diet was significantly and negatively correlated to NUE variation across diets (NUE=0.415 -0.055×Δ15Nanimal-diet). When using individual values and taking into account the random effects of study, period and diet, the relationship was also significant (NUE=0.358 -0.035×Δ15Nanimal-diet). However, there may be a biased prediction for animals close to zero, or in negative, N balance. When using a novel statistical approach, attempting to regress between-animal variation in NUE on between-animal variation in Δ15Nanimal-diet (without the influence of experimental factors), the negative relationship was still significant, highlighting the ability of Δ15Nanimal-diet to capture individual variability. Among the studied variables related to N utilization, those concerning N efficiency use at the metabolic level contributed most to predict both Δ15Nanimal-diet and NUE variation, with rumen fermentation and digestion contributing to a lesser extent. This study confirmed that on average Δ15Nanimal-diet can predict NUE variation across diets and across individuals reared under similar conditions.

HPLC-DAD-ESI/MS(n) profiling of phenolic compounds from Lathyrus cicera L. seeds.

Lathyrus cicera L. seeds are of interest for food and feed purposes. Despite the recognized antioxidant activity of the seeds, arising from the phenolic fraction, their phenolic compounds have not been studied in depth yet. Therefore, to determine the phenolics profile of these seeds, a target analysis was performed using high-performance liquid chromatography coupled to photodiode-array detection and electrospray ionization/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)). Thirty-seven glycosylated flavonoids were identified for the first time in the seeds of this species and, according to their MS fragmentation, clustered in flavonol-3-O-di-/tri-glycosides-7-O-rhamnosides and other flavonol-glycosides, and flavonol-3-O-(cinnamoyl)glycoside-7-O-rhamnosides, flavonol-3-O-(dihydrophaseoyl, cinnamoyl)glycoside-7-O-rhamnosides and flavonol-3-O-(malonyl)glycoside-7-O-rhamnosides. Glycosides of kaempferol were the main flavonoids found (10 non-acylated and 21 acylated), followed by those of quercetin (3) and those of isorhamnetin, apigenin and luteolin (1). The most abundant flavonols were identified as kaempferol-3-O-(2-hexosyl)hexoside-7-O-rhamnosides. The methodology used allowed to increase the knowledge on a relevant phytochemical class of seeds from L. cicera.

Short communication: Relationship between the efficiency of utilization of feed nitrogen and 15N enrichment in casein from lactating dairy cows.

The objective of the present study was to evaluate the relationship between the efficiency of conversion of feed N into milk N [N-use efficiency (NUE)] and (15)N enrichment of milk casein from lactating cows fed corn silage-based diets. Samples of feeds and milk were obtained from 3 experiments with lactating dairy cows. All diets were based on corn silage and were designed to evaluate the effects of (1) diets with different ratios of effective rumen-degradable protein to fermentable metabolizable energy (experiment 1), (2) different proportions of quickly and slowly rumen-degradable protein (experiment 2), and (3) synchronizing the availability of fermentable metabolizable energy and effective rumen-degradable protein in the rumen (experiment 3). Although no significant effect of diet on casein δ(15)N values was detected, casein was more enriched than the diet in each of the experiments. Nitrogen-use efficiency was negatively related to adjusted Δ(15)N (casein δ(15)N--diet δ(15)N) for experiments 1 and 2 individually and when combining data from all 3 experiments. The relatively low values for Δ(15)N suggest that these productive animals were using dietary N efficiently, with a high proportion of N going to milk protein and less to urea. The weak, although significant, relationship between NUE and adjusted Δ(15)N, is consistent with relatively little variation in hepatic deamination and transamination, with variation in rumen efficiency having the predominant effect on NUE. The present study confirms the lower (15)N enrichment in protein when NUE is high and the potential to use N-isotope fractionation as a marker of NUE.

Effects of protein sources on concentrations of hydrogen sulphide in the rumen headspace gas of dairy cows.

Two Latin square design experiments investigated the relationship between hydrogen sulphide concentration in the rumen headspace gas of dairy cows and the early stages of protein degradation in the rumen. In Expt 1, three protein sources differing in rumen N (nitrogen) degradability (maize gluten feed (MGF); sunflower meal (SFM); and soyabean meal (SBM)) were used, whereas in Expt 2 four different batches of the same feed (MGF) differing in colour (CIE L*, a*, b* (CIELAB) scale) were used. After allowing the concentration of hydrogen sulphide in rumen gas to decline close to zero, a fixed amount of protein sources was offered to cows and the concentrations of hydrogen sulphide were recorded in rumen headspace gas at 30-min intervals. In Expt 1, the concentration of hydrogen sulphide showed considerable variation between protein sources, with MGF having the highest concentration followed by SFM and SBM resulting in very low concentrations. The N wash losses (zero time measurements with nylon bags) ranked the feeds in the same way, from MGF (highest; 61%) to SBM (lowest; 26%). There were marked differences in the degradation of cystine and methionine between protein sources, although the degradation of cystine was always higher than for methionine. MGF (Expt 2) led to increased concentrations of hydrogen sulphide, with peak concentrations achieved between 1 and 2 h after feeding. The concentrations of hydrogen sulphide were higher for MGF1, intermediate for MGF2 and lower for MGF3 and MGF4, agreeing with colour scale. Differences in the early stages of dietary sulphur degradation corresponded with differences in hydrogen sulphide concentrations in rumen gas. The results suggest that hydrogen sulphide concentrations in the rumen headspace gas could be useful to evaluate nutritional parameters not measured by the in sacco technique, contributing to a better understanding of the response of dairy cows to different protein supplements.

Identification of C18 intermediates formed during stearidonic acid biohydrogenation by rumen microorganisms in vitro.

In vitro batch incubations were used to study the rumen biohydrogenation of unsaturated fatty acids. An earlier study using increasing supplementation levels of stearidonic acid (18:4n-3), revealed that the rumen microbial population extensively biohydrogenates 18:4n-3 after 72 h of in vitro incubation, though several intermediates formed were not completely characterized. Therefore, in the present study, samples were reanalyzed in order to identify the 18:2, 18:3 and 18:4 biohydrogenation intermediates of 18:4n-3. Gas-liquid chromatography coupled to mass spectrometry was used to characterize these intermediates. The acetonitrile chemical ionization mass spectrometry of the fatty acid methyl esters derivatives enabled the discrimination of fatty acids as non-conjugated or conjugated biohydrogenation intermediates. In addition, the acetonitrile covalent adduct chemical ionization tandem mass spectrometry yielded prominent ions indicative of the double bond position of the major 18:3 isomers, i.e. Δ5,11,15 18:3. Furthermore, the 4,4-dimethyloxazoline derivatives prepared from the fatty acid methyl esters enabled the structure of novel 18:2, 18:3 and 18:4 biohydrogenation intermediates to be elucidated. The intermediates accumulated in the fermentation media after 72 h of incubation of 18:4n-3 suggest that similar to the biohydrogenation pathways of linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, the pathway of the 18:4n-3 also proceeds with the formation of conjugated fatty acids followed by hydrogenation, although no conjugated dienes were found. The formation of the novel biohydrogenation intermediates of 18:4n-3 seems to follow an uncommon isomerization pattern with distinct double bond migrations.

Technical note: stearidonic acid metabolism by mixed ruminal microorganisms in vitro.

Dietary supplementation of stearidonic acid (SDA; 18:4n-3) has been considered a possible strategy to increase n-3 unsaturated fatty acid content in ruminant products; however, little is known about its metabolism in the rumen. In vitro batch incubations were carried out with bovine ruminal digesta to investigate the metabolism of SDA and its biohydrogenation products. Incubation mixtures (4.5 mL) that contained 0 (control), 0.25, 0.50, 0.75, 1.00, 1.25, or 1.50 mg of SDA supplemented to 33 mg (DM basis) of commercial total mixed ration based on corn silage, for dairy cows, were incubated for 72 h at 39°C. The content of most fatty acids in whole freeze-dried cultures was affected by SDA supplementation. Branched-chain fatty acids decreased linearly (P < 0.01), and odd-chain fatty acids decreased quadratically (P < 0.01), particularly from 1.00 mg of SDA and above, whereas most C18 fatty acids increased linearly or quadratically (P ≤ 0.04). Stearidonic acid concentrations at 72 h of incubation were very small (<0.6% of total fatty acids and ≤0.9% of added SDA) in all treatments. The apparent biohydrogenation of SDA was extensive, but it was not affected by SDA concentration (P > 0.05). Biohydrogenation followed a pattern similar to that of other C18 unsaturated fatty acids up to 1.00 mg of SDA. Stearic acid (18:0) and vaccenic acid (18:1 trans-11) were the major fatty acids formed, with the latter increasing 9-fold in the 1.00 mg of SDA treatment. At greater inclusion rates, 18:0 and 18:1 trans isomers decreased (P ≤ 0.03), accompanied by increases in unidentified 18:3 and 18:4 isomers (P = 0.02), suggesting that the biohydrogenation pathway was inhibited. The present results clearly indicate that SDA was metabolized extensively, with numerous 18:4 and 18:3 products formed en route to further conversion to 18:2, 18:1 isomers, and 18:0.

Effects of dietary protein concentration and balance of absorbable amino acids on productive responses of dairy cows fed corn silage-based diets.

A cyclical changeover design experiment (3-wk periods; 12-wk total) was conducted to evaluate whether improving the balance of absorbable AA would allow the feeding of less crude protein (CP) without compromising production, thereby reducing the potential environmental pollution from dairy farms. Sixteen multiparous Holstein cows were assigned to 1 of 8 dietary treatments as total mixed rations (TMR) containing [dry matter (DM) basis] 45% corn silage, 5% coarsely chopped wheat straw, and 50% concentrate mixture. The 8 treatments were formulated to differ in dietary CP (14 and 16%; DM basis) and in the balance of absorbable AA achieved by changing the main protein source (MPS) of the concentrate mixtures [replacing soybean meal (SBM) with corn byproducts (CBP), dried corn distillers grains (DDG), and some corn gluten meal], and by adding a mixture of rumen-protected Lys and Met (RPLM). Feeding lactating dairy cows corn silage-based diets with 16% CP promoted significantly higher DM intakes and milk yields, and lower feed N-use efficiency than feeding diets with 14% CP. Replacing SBM with CBP significantly increased milk yields and decreased milk fat and protein concentrations, but had no effect on the efficiency of conversion of feed N into milk N. With 16% CP diets, the addition of RPLM decreased feed N use efficiency. A significant effect was observed for the MPS × RPLM interaction on milk protein concentrations. Plasma Lys concentration was lower with diets based on CBP, and plasma Met increased with RPLM. We did not find clear benefits of RPLM in facilitating a reduction of dietary protein without loss of production.

Effect of ensiling and silage additives on fatty acid composition of ryegrass and corn experimental silages.

Two experiments were conducted using laboratory mini-silos to study the effect of ensiling and silage additives on fatty acid (FA) composition, including minor or unusual FA, of ryegrass and corn silages. Ryegrass was ensiled for 12 wk with no additives, with the addition of a bacterial inoculant or formic acid. Corn was ensiled for 9 wk without additives, with the addition of a bacterial inoculant or calcium formate. Ensiling affected both total FA content and FA composition of ryegrass silages. Total FA concentration increased (P < 0.001) during ryegrass ensiling. The proportions (g/100 g of total FA) of the major unsaturated FA, 18:3n-3 and 18:2n-6, were not affected (P > 0.05) by ensiling. However, their concentration (mg/g of DM) in silages was greater (P=0.017 and P=0.001, respectively) than in fresh ryegrass. Two 18:2 FA (trans-11,cis-15 and cis-9,cis-15) that were not originally present in the fresh ryegrass were detected in silages. Silage additives affected the FA composition of ryegrass silages, mostly by increasing the proportions of SFA, but not on total FA concentration. Ensiling did not affect (P=0.83) total FA content of corn silages; however, FA composition was affected, mostly by decreasing the proportions of 18:2n-6 and 18:3n-3. Silage additives had no effect on corn silage FA composition. Exposing corn silages to air resulted in no oxidation of FA or reduction in total FA content or composition.

rEnolase maternal immunization confers caries protection on offspring.

Therapeutic vaccination with Streptococcus sobrinus recombinant enolase (rEnolase) protects rats from dental caries. Here, we investigated the effect that maternal rEnolase vaccination before pregnancy had on the offspring's immune response to S. sobrinus oral infection and dental caries progression. Female Wistar rats were immunized by intranasal and subcutaneous routes with rEnolase adsorbed onto aluminum hydroxide as adjuvant or similarly treated with the adjuvant alone (sham-immunized). Ten days after the last administration, the immunized females were paired with a male rat. The oral immune responses to S. sobrinus infection and dental caries in the offspring were evaluated. The results showed that pups born from rEnolase-immunized mothers had higher levels of rEnolase-specific salivary IgA and IgG antibodies (indicating a placental antibody transfer) and lower sulcal and proximal enamel caries scores than rats born from sham-immunized mothers. In conclusion, rEnolase maternal immunization before pregnancy provides offspring with protection against S. sobrinus-induced dental caries.

Rumen biohydrogenation-derived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils.

The effects of supplementation with rapeseed, sunflower, and linseed oils (0.5 kg/d; good sources of oleic, linoleic, and linolenic acids, respectively) on milk responses and milk fat fatty acid (FA) profile, with special emphasis on rumen-derived biohydrogenation intermediates (BI), were evaluated in a replicated 4 x 4 Latin square study using 16 grazing dairy cows. The dietary treatments were 1) control diet: 20-h access to grazing pasture supplemented with 5 kg/d of corn-based concentrate mixture (96% corn; CC); 2) RO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of rapeseed oil; 3) SO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of sunflower oil; and 4) LO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of linseed oil. Milk fatty acids were converted to methyl esters and analyzed by gas-liquid chromatography and silver-ion HPLC. Dietary treatments had no effect on milk production or on milk protein content and milk protein production. Supplementation with rapeseed and sunflower oils lowered milk fat content and milk fat production, but linseed oil had no effect. Inclusion of dietary vegetable oils promoted lower concentrations of short-chain (including 4:0) and medium-chain FA (including odd- and branched-chain FA) and 18:3n-3, and higher concentrations of C(18) FA (including stearic and oleic acids). The BI concentration was higher with the dietary inclusion of vegetable oils, although the magnitude of the concentration and its pattern differed between oils. The RO treatment resulted in moderate increases in BI, including trans 18:1 isomers and 18:2 trans-7,cis-9, but failed to increase 18:1 trans-11 and 18:2 cis-9,trans-11. Sunflower oil supplementation resulted in the highest concentrations of the 18:1 trans-10, 18:1 cis-12, and 18:2 trans-10,trans-12 isomers. Concentrations of 18:1 trans-11 and 18:2 cis-9,trans-11 were higher than with the control and RO treatments but were similar to the LO treatment. Concentration of BI in milk fat was maximal with LO, having the highest concentrations of some 18:1 isomers (i.e., trans-13/14, trans-15, cis-15, cis-16), most of the nonconjugated 18:2 isomers (i.e., trans-11,trans-15, trans-11,cis-15, cis-9,cis-15, and cis-12,cis-15), and conjugated 18:2 isomers (i.e., trans-11,cis-13, cis-12,trans-14, trans-11,trans-13, trans-12,trans-14, and trans-9,trans-11), and all conjugated 18:3 isomers. The LO treatment induced the highest amount and diversity of BI without decreasing milk fat concentration, as the RO and SO treatments had, suggesting that the BI associated with 18:3n-3 intake may not be the major contributors to inhibition of mammary milk fat synthesis.

Effects of grass silage and soybean meal supplementation on milk production and milk fatty acid profiles of grazing dairy cows.

The effects of supplementation with grass silage and replacement of some corn in the concentrate with soybean meal (SBM) on milk production, and milk fatty acid (FA) profiles were evaluated in a replicated 4 x 4 Latin square study using 16 dairy cows grazing pasture composed of ryegrass, Kentucky bluegrass, and white clover. Each experimental period lasted for 3 wk. The 4 dietary treatments were PC, 20 h of access to grazing pasture, supplemented with 6 kg/d of corn-based concentrate mixture (96% corn; C); PCSB, 20 h of access to grazing pasture, supplemented with 6 kg/d of corn- and SBM-based concentrate mixture (78% corn and 18% SBM; CSB); SC, 7 h of access to grazing pasture during the day and 13 h of ad libitum access to grass silage at night, supplemented with 6 kg/d of C concentrate; and SCSB, 7 h of access to grazing pasture during the day and 13 h of ad libitum access to grass silage at night, supplemented with 6 kg/d of CSB concentrate. The concentrate mixtures were offered twice each day in the milking parlor and were consumed completely. Grass silage supplementation reduced dietary crude protein and concentration of total sugars, and dietary SBM inclusion increased dietary crude protein concentration and decreased dietary starch concentration. Milk yield and energy-corrected milk were increased by SBM supplementation of cows with access to grass silage. Milk protein concentration was lower in cows offered grass silage, regardless of whether SBM was fed. Dietary SBM inclusion tended to increase milk fat concentration. Plasma urea N was reduced by silage feeding and increased by SBM supplementation. Supplementation with grass silage overnight could represent a useful strategy for periods of lower pasture availability. Dietary inclusion of SBM in solely grazing cows had no effects on milk production and composition, exacerbated the inefficient capture of dietary N, and increased diet cost. Grass silage supplementation affected milk FA profiles, increasing both the FA derived from de novo synthesis and those derived from rumen microbial biomass, and decreasing the sum of C18 FA (mostly derived from diet or from mobilization of adipose tissue reserves). Milk fat concentrations of conjugated linoleic acid cis-9, trans-11, vaccenic acid (18:1 trans-11), and linolenic acid (18:3n-3) were unaffected by grass silage supplementation, suggesting that partial replacement of pasture by unwilted grass silage does not compromise the dietary quality of milk fat for humans.

Physicochemical characterization of nopal pads (Opuntia ficus indica) and dry vacuum nopal powders as a function of the maturation.

This paper presents the physicochemical and nutrimental characterization of fresh nopal (Opuntia ficus indica, Redonda variety) and nopal powder produced at different stages of development. Nopal powder was obtained by dry vacuum technique using 10(2) Torr and low temperature (40 degrees C). The results showed that the nutrimental and mineral composition of nopal changes as a function of the maturation as follow: The ash content increases from 18.41 for nopalitos (60 g of weight) to 23.24% (nopal pads 200 g); calcium content increases from 1.52 to 3.72%, while phosphorous exhibits an opposite trend: 0.43 to 0.27%, respectively. Calcium oxalate was determined by X-ray diffraction and SEM microscopy and quantified by using atomic absorption spectroscopy. Calcium oxalate decreases from 7.95 to 3.47 mg/g and the Ca/P ratio varies from 3.6 to 11. The soluble fibre decreases from 25.22 to 14.91%, while insoluble fibre increases from 29.87 to 41.65%. These results suggest that nopal could be an important source of minerals within the diets of people in Mexico and the rest of Latin America.

Effects of dietary protein and starch on intake, milk production, and milk fatty acid profiles of dairy cows fed corn silage-based diets.

Feed intake, milk production, and milk fatty acid profiles of dairy cows fed corn silage-based diets with different protein and starch concentrations were measured in a 3-period experiment in a changeover design using 12 Holstein cows. Each experimental period lasted for 3 wk. The diet fed as a total mixed ration consisted of 45% corn silage, 5% coarsely chopped wheat straw, and 50% concentrate, on a dry matter (DM) basis. The 4 treatments, formulated to be isoenergetic and to differ in concentrations of dietary crude protein (CP) and starch (DM basis), were as follows: low CP and low starch (LPLS; 14% CP and 15% starch), low CP and high starch (LPHS; 14% CP and 25% starch), high CP and low starch (HPLS; 16% CP and 15% starch), and high CP and high starch (HPHS; 16% CP and 25% starch). The LPLS treatment led to lower DM intake, milk yield, milk protein concentration, and milk lactose yield, probably due to a shortage of both rumen-degradable protein supply to rumen microbes and glucogenic nutrients to the animal. There were no differences between protein-rich diets and LPHS, suggesting that this diet satisfied the rumen-degradable protein requirements of rumen microbes and did not limit feed intake, and the increased supply of glucogenic nutrients spared AA so that the nutrient requirements of mid lactation dairy cows were met. Further increases in CP concentration increased plasma urea concentration and resulted in decreased efficiency of conversion of dietary N into milk N. Milk fatty acid profiles were affected by starch and protein supply, with starch having the largest effect. Additionally, increasing dietary starch concentration decreased the apparent transfer of dietary polyunsaturated fatty acids to milk, suggesting an increased channeling of fatty acids to adipose tissue. The results further suggest that C(15:0) and C(17:0) are synthesized de novo in animal tissues.

Effects of dietary sulphur sources on concentrations of hydrogen sulphide in the rumen head-space gas of dairy cows.

Three change-over design experiments investigated the origin of hydrogen sulphide in the rumen head-space gas of dairy cows, comparing the effects of single iso-S additions of methionine, cysteine and sodium sulphate, as well as the effects of single meals of fresh ryegrass or white clover. The concentration of hydrogen sulphide in rumen gas declined close to zero within 4 h after withdrawal of the previous feed. Sulphur sources were then given to cows and concentrations of hydrogen sulphide recorded in rumen head-space gas at 30-min intervals. Cysteine addition (8 g) led to a rapid (within 30 min) and a large (490 and 957 p.p.m. respectively in two experiments) increase in hydrogen sulphide concentration. Concentrations were significantly less following methionine addition. Increasing levels of cysteine addition led to significant increases in hydrogen sulphide concentrations ( P < 0.001 for the linear effect), although peak hydrogen sulphide was delayed and concentrations remained higher for longer with the highest (12 g) addition of cysteine ( P < 0.01 for the 'cysteine level' × 'time' interaction). The increase in concentration of hydrogen sulphide from sodium sulphate was smaller (230 p.p.m.) and slower (2 h) than for cysteine. Despite the much higher intake of cystine for white clover in comparison with perennial ryegrass ( P < 0.001), there was almost no increase in hydrogen sulphide concentration in rumen head-space gas from cows fed white clover. It seems likely that this is associated with the use of sulphur to produce thiocyanate to detoxify the hydrogen cyanide from cyanogenic white clover.

Evaluation of palm kernel meal and corn distillers grains in corn silage-based diets for lactating dairy cows.

The effects of increasing levels of solvent-extracted palm kernel meal (SPKM) and corn distillers dried grains (CDG) in corn silage-based diets on feed intake and milk production were examined in 2 experiments. In Experiment 1, 20 Holstein cows averaging 100 d in milk (DIM) (SD = 61.5) at the start of the experiment were utilized in an 11-wk randomized complete block design with 4 treatments in 5 blocks to study effects of increasing levels of SPKM in the diet. During a 3-wk preliminary period, cows were fed a standard diet. At the end of the preliminary period, cows were blocked by 4% fat-corrected milk yield, parity number (primiparous and multiparous), and DIM, and were assigned randomly to 1 of 4 experimental diets. The total mixed ration (TMR) consisted of (dry matter basis) 40% corn silage, 5% coarsely chopped wheat straw, and 55% concentrate. The increasing dietary levels of SPKM were achieved by replacing protein sources and citrus pulp with SPKM and urea (0, 5, 10, and 15% SPKM and 0.06, 0.22, 0.38, and 0.55% urea for SPKM0, SPKM5, SPKM10, and SPKM15, respectively). In Experiment 2, 18 Holstein cows averaging 93 DIM (SD = 49.1) at the start of the experiment were utilized in an 11-wk randomized complete block design with 3 treatments in 6 blocks to study effects of increasing levels of CDG in the diet. The preliminary period lasted for 2 wk. Assignment of cows to treatments was the same as in Experiment 1. The TMR consisted of (dry matter basis) 40% corn silage, 5% coarsely chopped wheat straw, and 55% concentrate. The increasing dietary levels of CDG were achieved by replacing soybean meal and citrus pulp with CDG and urea (0, 7, and 14% CDG and 0, 0.22, and 0.49% urea for CDG0, CDG7, and CDG14, respectively). There were no significant treatment effects on dry matter intake, milk yield, or milk composition in Experiment 1. Inclusion of SPKM tended to increase protein and lactose contents of milk. The SPKM0 diet promoted body weight loss. There were no treatment effects in Experiment 2, except for milk protein content, which was decreased by CDG. Plasma Lys concentration tended to be affected by SPKM and CDG inclusions. Plasma concentrations of 3-methylhistidine and Leu seemed to be related to body protein degradation/synthesis. The feeding of SPKM up to 15% in the diet decreased feed costs without detrimental effects on productive responses and tended to increase milk protein content. The inclusion of CDG in diets based on corn silage and corn byproducts might decrease milk protein content due to an unbalanced supply of AA, particularly Lys.

Therapeutic vaccine against Streptococcus sobrinus-induced caries.

Streptococcus sobrinus produces a virulence-associated immunomodulatory protein (VIP) which suppresses the host-specific immune response and induces the early production of IL-10. In this study, we evaluated the effects of therapeutic immunization with this VIP on the incidence of caries in S. sobrinus-infected rats. Groups of Wistar rats were orally infected with S. sobrinus and fed with sucrose-sweetened drinking water ad libitum. Five days later, rats were immunized intranasally with active or heat-inactivated VIP plus alum as adjuvant or PBS plus adjuvant (sham-immunized). After 3 wks, all rats were re-immunized as above. Evaluation of dental caries showed that VIP-immunized animals had significantly fewer enamel sulcal and proximal caries lesions than did the sham-immunized animals (p < 0.001). The protective effects following therapeutic VIP immunization were attributed to the induced salivary immunoglobulin A specific to the VIP. These results offer a promising and safe strategy for the development of a vaccine against dental caries.

Nitrogen supplementation of corn silages. 1. Effects on feed intake and milk production of dairy cows.

Feed intake and milk production responses to N supplementation of corn silage-based diets were measured in three 3 x 3 Latin square experiments. In each experiment, 9 Holstein cows received total mixed rations (TMR), based on corn silage. In Exp. 1, midlactation cows were used to study effects of diets with different ratios of effective rumen-degradable protein (ERDP; g) to fermentable metabolizable energy (FME; MJ), providing a large deficiency (RL), a slight deficiency (RM), and a slight excess (RH) in relation to the target level of 11 g of ERDP/MJ of FME, respectively, for lactating cows. Diets were formulated to be isoenergetic, and to satisfy the metabolizable protein requirements. In Exp. 2, early-lactation cows were used to evaluate effects of different proportions of quickly and slowly rumen-degradable protein (RDP), achieved by replacing soybean meal with urea in the concentrates (0, 0.5, and 1% urea). Experiment 3 investigated effects of synchronizing the availability of FME and ERDP in the rumen. Midlactation cows received a diet containing, on a dry matter (DM) basis, 45% corn silage, 5% ryegrass hay, 35% energy-rich concentrate, and 15% protein-rich concentrate (crude protein: 38% of DM; urea: 2% of DM). The protein-rich concentrate was fed either once (D1) or twice (D2) per day before the meal, or included in the TMR (DU). Treatment RL led to lower DM intake and milk yield, but higher milk production efficiency; there were no significant differences between treatments RM and RH. There were no significant treatment effects on DM intake, milk yield, or milk composition in Exp. 2. Manipulating rumen synchrony by altering the timing of feeding affected milk yields, with D1 cows producing significantly less than D2 and DU cows, which were similar. The amount of ERDP in the diet should be matched to the amount of fermentable energy available to maximize intake, milk yields, and the conversion of feed N into milk protein. However, this study showed only small benefits to altering the diurnal pattern of supply of RDP and FME, and only with extreme feeding strategies that would not be used in practice. Urine volume increased in response to increased or unbalanced protein supply. Analysis of the allantoin:creatinine ratio in spot samples of urine was not useful in identifying predicted differences in microbial protein yield from the rumen.

Nitrogen supplementation of corn silages. 2. Assessing rumen function using fatty acid profiles of bovine milk.

The effects of N supplementation strategies on milk fatty acid profiles of dairy cows and their use as a noninvasive technique to diagnose rumen function, and to guide protein feeding decisions on-farm were evaluated in three experiments. Each experiment was designed according to three 3 x 3 Latin squares with 9 Holstein cows receiving total mixed rations based on corn silage. Experiment 1 was designed to study effects of diets with different ratios of effective rumen-degradable protein (ERDP; g) to fermentable metabolizable energy (FME; j) providing, respectively, a large deficiency, a slight deficiency, and a slight excess in relation to the target level of 11 g of ERDP/MJ FME for lactating cows. Experiment 2 evaluated effects of different proportions of quickly and slowly rumen-degradable protein achieved by replacing soybean meal with urea in the concentrates (0, 0.5, and 1% urea for U0, U5, and U10, respectively). Experiment 3 investigated effects of synchronizing the availability of FME and ERDP in rumen by offering the protein-rich concentrate once or twice per day before the meal (corn silage, ryegrass hay, and energy-rich concentrate), or included in the total mixed ration. Milk fatty acid profiles were significantly affected by dietary N and carbohydrate supply. Principal component factor analysis provided a reasonable description of the data, clearly discriminating between fatty acids that are synthesized by different metabolic pathways. Several sources/pathways were distinguished: de novo synthesis in the mammary gland (short- and medium-chain fatty acids), delta9-desaturase activity (monoenoic fatty acids), direct absorption from the blood stream (long-chain fatty acids), and de novo synthesis by the rumen microbial populations (odd-chain fatty acids). Discriminant canonical analysis showed that milk odd-chain fatty acids had a higher ability to discriminate between diets than even-chain fatty acids. The anteiso C15:0 increased in line with increasing sugar supply, and C17:0 appears to be a marker of protein deficiency. Additionally, iso C17:0 and anteiso C17:0 were associated with the NDF and CP contents of diets. The results suggests that milk odd-chain fatty acids have the potential to be used as a noninvasive technique to assess rumen function in terms of microbial populations, substrates and interactions.