Determination of caffeoylquinic acids in feed and related products by focused ultrasound solid-liquid extraction and ultra-high performance liquid chromatography-mass spectrometry.

A method to determine caffeoylquinic acids (CQAs) in three sources (herbal extract, feed additive and finished feed) using for the first time focused ultrasound solid-liquid extraction (FUSLE) followed by ultra-high performance liquid chromatography (UPLC) coupled to quadrupole-time of flight mass spectrometry is presented. Pressurized liquid extraction (PLE) was also tested as extraction technique but it was discarded because cynarin was not stable under temperature values used in PLE. The separation of the CQAs isomers was carried out in only seven minutes. FUSLE variables such as extraction solvent, power and time were optimized by a central composite design. Under optimal conditions, FUSLE extraction was performed with 8mL of an 83:17 methanol-water mixture for 30s at a power of 60%. Only two extraction steps were found necessary to recover analytes quantitatively. Sensitivity, linearity, accuracy and precision were established. Matrix effect was studied for each type of sample. It was not detected for mono-CQAs, whereas the cynarin signal was strongly decreased due to ionization suppression in presence of matrix components; so the quantification by standard addition was mandatory for the determination of di-caffeoylquinic acids. Finally, the method was applied to the analysis of herbal extracts, feed additives and finished feed. In all samples, chlorogenic acid was the predominant CQA, followed by criptochlorogenic acid, neochlorogenic acid and cynarin. The method allows an efficient determination of chlorogenic acid with good recovery rates. Therefore, it may be used for screening of raw material and for process and quality control in feed manufacture.

Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture.

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C(18:1) and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 x 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C(18:0), trans-11 C(18:1) and cis-9, trans-11 CLA, and increased the flow of trans-10 C(18:1), C(18:2n-6), C(18:3n-3), trans-11,cis-15 C(18:2) and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C(18:1) and trans-10 C(18:1), and increased C(18:2n-6), C(18:3n-3) and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C(18:1) and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.

Effects of saponins, quercetin, eugenol, and cinnamaldehyde on fatty acid biohydrogenation of forage polyunsaturated fatty acids in dual-flow continuous culture fermenters.

Four different plant secondary metabolites were screened for their effect on rumen biohydrogenation of forage long-chain fatty acids, using dual-flow continuous culture fermenters. Treatments were as follows: control (no additive), positive control (12 mg/L of monensin), and plant extracts (500 and 1,000 mg/L of triterpene saponin; 250 and 500 mg/L of quercetin; 250 mg/L of eugenol; 500 mg/L of cinnamaldehyde). Monensin increased propionate, decreased acetate and butyrate proportions, and inhibited the complete biohydrogenation of fatty acids resulting in the accumulation of intermediates of the biohydrogenation process (C18:2 trans-11, cis-15 rather than C18:1 trans-11). Cinnamaldehyde decreased total VFA concentration and proportions of odd and branched-chain fatty acids in total fat effluent. Apparent biohydrogenation of C18:2n-6 and C18:3n-3 was also less, and a shift from the major known biohydrogenation pathway to a secondary pathway of C18:2n-6 was observed, as evidenced by an accumulation of C18:1 trans-10 and trans-10, cis-12 CLA. Quercetin (500 mg/L) increased total VFA concentration, but no shifts in the pathways or extent of biohydrogenation were observed. Eugenol resulted in the accumulation of C18:1 trans-15 and C18:1 cis-15, end products of an alternative biohydrogenation pathway of C18:3n-3. Triterpene saponins did not affect the fermentation pattern, the biohydrogenation pathways, or the extent of biohydrogenation. At the doses tested in this study, we could only show a direct relation between changes in the rumen fatty acid metabolism and the presence of cinnamaldehyde but not for eugenol, quercetin, or triterpene saponins.

Changes in rumen microbial fermentation are due to a combined effect of type of diet and pH.

Low ruminal pH may occur when feeding high-concentrate diets. However, because the reduction in pH occurs at the same time as the amount of concentrate fed increases, the changes observed in rumen fermentation may be attributed to pH or the type of substrate being fermented. Our objective was to determine the contribution of pH and type of substrate being fermented to the changes observed in rumen fermentation after supplying a high-concentrate diet. Eight dual-flow, continuous culture fermenters (1,400 mL) were used in 4 periods to study the effect of pH and type of diet being fermented on rumen microbial fermentation. Temperature (39 degrees C), solid (5%/h), and liquid (10%/h) dilution rates, and feeding schedule were maintained constant. Treatments were the type of diet (FOR = 60% ryegrass and alfalfa hays and 40% concentrate; CON = 10% straw and 90% concentrate) and pH (4.9, 5.2, 5.5, 5.8, 6.1, 6.4, 6.7, and 7.0). Diets were formulated to have similar CP and ruminally undegradable protein levels. Data were analyzed as a mixed-effects model considering the linear, quadratic, and cubic effects of pH, the effects of diet, and their interactions. Semipartial correlations of each independent variable were calculated to estimate the contribution of each factor to the overall relationship. True digestion of OM and NDF were affected by pH, but not by type of diet. Total VFA were reduced by pH and were greater in CON than in FOR. Acetate and butyrate concentrations were reduced by pH but were not affected by diet. Propionate concentration increased as the pH decreased and was greater in CON than in FOR. Ammonia-N concentration decreased with decreasing pH and was lower in CON than in FOR. Microbial N flow was affected by pH, diet, and their interaction. Dietary N flow increased as pH decreased and was greater in CON than in FOR. The degradation of CP followed the opposite pattern, increasing as pH increased, and was less in CON than in FOR. The efficiency of microbial protein synthesis (g of N/kg of OM truly digested) was slightly reduced by pH and was less in CON than in FOR. These results indicate that the effects of feeding a high-concentrate diet on rumen fermentation are due to a combination of pH and substrate. Furthermore, the digestion of OM in high-concentrate diets is likely limited by the pH-induced effects on the microbial population activity.

Invited review: Essential oils as modifiers of rumen microbial fermentation.

Microorganisms in the rumen degrade nutrients to produce volatile fatty acids and synthesize microbial protein as an energy and protein supply for the ruminant, respectively. However, this fermentation process has energy (losses of methane) and protein (losses of ammonia N) inefficiencies that may limit production performance and contribute to the release of pollutants to the environment. Antibiotic ionophores have been very successful in reducing these energy and protein losses in the rumen, but the use of antibiotics in animal feeds is facing reduced social acceptance, and their use has been banned in the European Union since January 2006. For this reason, scientists have become interested in evaluating other alternatives to control specific microbial populations to modulate rumen fermentation. Essential oils can interact with microbial cell membranes and inhibit the growth of some gram-positive and gram-negative bacteria. As a result of such inhibition, the addition of some plant extracts to the rumen results in an inhibition of deamination and methanogenesis, resulting in lower ammonia N, methane, and acetate, and in higher propionate and butyrate concentrations. Results have indicated that garlic oil, cinnamaldehyde (the main active component of cinnamon oil), eugenol (the main active component of the clove bud), capsaicin (the active component of hot peppers), and anise oil, among others, may increase propionate production, reduce acetate or methane production, and modify proteolysis, peptidolysis, or deamination in the rumen. However, the effects of some of these essential oils are pH and diet dependent, and their use may be beneficial only under specific conditions and production systems. For example, capsaicin appears to have small effects in high-forage diets, whereas the changes observed in high-concentrate diets (increases in dry matter intake and total VFA, and reduction in the acetateto-propionate ratio and ammonia N concentration) may be beneficial. Because plant extracts may act at different levels in the carbohydrate and protein degradation pathways, their careful selection and combination may provide a useful tool to manipulate rumen microbial fermentation effectively. However, additional research is required to establish the optimal dose in vivo in units of the active component, to consider the potential adaptation of microbial populations to their activities, to examine the presence of residues in the products (milk or meat), and to demonstrate improvements in animal performance.

Effects of alfalfa extract, anise, capsicum, and a mixture of cinnamaldehyde and eugenol on ruminal fermentation and protein degradation in beef heifers fed a high-concentrate diet.

Four Holstein heifers (360 +/- 22 and 450 +/- 28 kg of BW in Exp. 1 and 2, respectively) fitted with ruminal trocars were used in 4 x 4 Latin square designs to evaluate the effects on ruminal microbial fermentation of the following: Exp. 1, no additive, alfalfa extract (30 g/d, AEX), a mixture of cinnamaldehyde (0.18 g/d) and eugenol (0.09 g/d; CIE1), and AEX and CIE1 in combination; and Exp. 2, no additive, anise oil (2 g/d), capsicum oil (1 g/d), and a mixture of cinnamaldehyde (0.6 g/d) and eugenol (0.3 g/d). Heifers were fed a 90:10 concentrate:barley straw diet (16% CP; 25% NDF) for ad libitum intake. Each period consisted of 15 d for adaptation and 6 d for sampling. On d 16 to 18, DM and water intakes were measured. On d 19 to 21 ruminal contents were sampled at 0, 3, 6, 9, and 12 h after feeding to determine ruminal pH and the concentrations of VFA, L-lactate, large peptides, small peptides plus AA (SPep+AA), and ammonia N. On d 20 and 21, samples of ruminal fluid were collected at 0 and 3 h after feeding to determine protozoal counts. In Exp. 1, CIE1 and AEX decreased (P < 0.05) total DMI, concentrate DMI, and water intake. The increase (P < 0.05) in SPep+AA and the decrease (P < 0.05) in ammonia N when supplementing CIE1 suggest that deamination was inhibited. Treatment AEX increased (P < 0.05) the acetate to propionate ratio, which is less efficient for beef production. Treatment CIE1 increased (P < 0.05) counts of holotrichs. Effects of AEX and CIE1 were not additive for many of the measured metabolites. In Exp. 2, treatments had no effect on ruminal pH, total VFA concentration, and butyrate proportion. The capsicum oil treatment increased (P < 0.05) DMI, water intake, and SPep+AA N concentration and decreased (P < 0.05) acetate proportion, branched-chain VFA concentration, and large peptide N concentration. The cinnamaldehyde (0.6 g/d) and eugenol (0.3 g/d) treatment decreased (P < 0.05) water intake, acetate proportion, branched-chain VFA, L-lactate, and ammonia N concentrations and increased (P < 0.05) propionate proportion and SPep+AA N concentration. The anise oil treatment decreased (P < 0.05) acetate to propionate ratio, branched-chain VFA and ammonia N concentrations, and protozoal counts. The results indicate that at the doses used a mixture of cinnamaldehyde and eugenol, anise oil, and capsicum oil may be useful as modifiers of rumen fermentation in beef production systems.

Screening for the effects of natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle.

Six natural plant extracts and three secondary plant metabolites were tested at five doses (0, 0.3, 3, 30, and 300 mg/L) and two different pH (7.0 and 5.5) in a duplicate 9 x 5 x 2 factorial arrangement of treatments to determine their effects on in vitro microbial fermentation using ruminal fluid from heifers fed a high-concentrate finishing diet. Treatments were extracts of garlic (GAR), cinnamon (CIN), yucca (YUC), anise (ANI), oregano (ORE), and capsicum (CAP) and pure cinnamaldehyde (CDH), anethole (ATL), and eugenol (EUG). Each treatment was tested in triplicate and in two periods. Fifty milliliters of a 1:1 ruminal fluid-to-buffer solution were introduced into polypropylene tubes supplied with 0.5 g of DM of a 10:90 forage:concentrate diet (15.4% CP, 16.0% NDF; DM basis) and incubated for 24 h at 39 degrees C. Samples were collected for ammonia N and VFA concentrations. The decrease in pH from 7.0 to 5.5 resulted in lower (P < 0.05) total VFA, ammonia N, branched-chain VFA concentration, acetate proportion, and acetate:propionate, and in a higher (P < 0.05) propionate proportion. The interaction between pH and doses was significant for all measurements, except for ATL and CDH for butyrate, ATL and EUG for acetate:propionate ratio, and ORE for ammonia N concentration. The high dose of all plant extracts decreased (P < 0.05) total VFA concentrations. When pH was 7.0, ATL, GAR, CAP, and CDH decreased (P < 0.05) total VFA concentration, and ANI, ORE, CIN, CAP, and CDH increased (P < 0.05) the acetate:propionate. The CIN, GAR, CAP, CDH, ORE, and YUC decreased (P < 0.05), and EUG, ANI, and ATL increased (P < 0.05) ammonia N concentration. The effects of plant extracts on the fermentation profile when pH was 7.0 were not favorable for beef production. In contrast, when pH was 5.5, total VFA concentration did not change (ATL, ANI, ORE, and CIN) or increased (P < 0.05) (EUG, GAR, CAP, CDH, and YUC), and the acetate:propionate (ORE, GAR, CAP, CDH, and YUC) decreased (P < 0.05), which would be favorable for beef production. Ammonia N (ATL, ANI, CIN, GAR, CAP, and CDH) and branched-chain VFA (ATL, EUG, ANI, ORE, CAP, and CDH) concentrations also were decreased (P < 0.05), suggesting that deamination was inhibited. Results indicate that the effects of plant extracts on ruminal fermentation in beef cattle diets may differ depending on ruminal pH. When pH was 5.5, GAR, CAP, YUC, and CDH altered ruminal microbial fermentation in favor of propionate, which is more energetically efficient.

Effects of cinnamaldehyde and garlic oil on rumen microbial fermentation in a dual flow continuous culture.

Eight continuous culture fermentors inoculated with ruminal liquor from heifers fed a 50:50 alfalfa hay:concentrate diet (17.6% crude protein, 28.0% neutral detergent fiber) were used in 3 replicated periods to study the effects of cinnamaldehyde (CIN) and garlic oil (GAR) on rumen microbial fermentation. Treatments were no additive (negative control); 1.25 mg/L (MON) and 12.5 mg/L (MON10) of the ionophore antibiotic monensin (positive control); 31.2 mg/L CIN (CIN) and 312 mg/L (CIN10) of CIN; and 31.2 mg/L GAR (GAR) and 312 mg/L (GAR10) of GAR (Allium sativa). The MON10 caused expected changes in microbial fermentation patterns (a decrease in fiber digestion, ammonia N concentration, and proportions of acetate and butyrate; an increase in the proportion of propionate; and a trend to increase small peptide plus AA N concentration). The CIN decreased the proportion of acetate and branch-chained volatile fatty acids (VFA) and increased the proportion of propionate; CIN10 decreased the proportion of acetate and increased the proportion of butyrate compared with the control. The GAR10 increased the proportion of propionate and butyrate and decreased the proportion of acetate and branch-chained VFA compared with the control. The GAR10 also increased the small peptide plus amino acid N concentration, although no effects were observed on large peptides or ammonia N concentrations. The CIN and GAR10 resulted in similar effects as monensin, with the exception of the effects on the molar proportion of butyrate, which suggests that they might have a different mode of action in affecting in vitro microbial fermentation.

Effects of natural plant extracts on ruminal protein degradation and fermentation profiles in continuous culture.

Eight dual-flow continuous culture fermenters were used in four consecutive periods of 10 d to study the effects of six natural plant extracts on ruminal protein degradation and fermentation profiles. Fermenters were fed a diet with a 52:48 forage:concentrate ratio (DM basis). Treatments were no extract (CTR), 15 mg/kg DM of a mixture of equal proportions of all extracts (MIX), and 7.5 mg/kg DM of extracts of garlic (GAR), cinnamon (CIN), yucca (YUC), anise (ANI), oregano (ORE), or pepper (PEP). During the adaptation period (d 1 through 8), samples for ammonia N and VFA concentrations were taken 2 h after feeding. On d 9 and 10, samples for VFA (2 h after feeding), and peptide, AA, and ammonia N concentrations (0, 2, 4, 6, and 8 h after feeding) were also taken. Differences were declared at P < 0.05. During the adaptation period, total VFA and ammonia N concentrations were not affected by treatments. The acetate proportion was higher from d 2 to 6 in CIN, GAR, ANI, and ORE, and the propionate proportion was lower from d 2 to 4 in CIN and GAR, and from d 2 to 5 in ANI and ORE, compared with CTR. However, the proportion of individual VFA (mol/100 mol) was similar in all treatments after d 6, except for valerate in d 9 and 10, which was lower in PEP (2.8 +/- 0.27) compared with CTR (3.5 +/- 0.27). The average peptide N concentration was 31% higher in MIX, and 26% higher in CIN and YUC compared with CTR (6.5 +/- 1.07 mg/100 mL). The average AA N concentration was 17 and 15% higher in GAR and ANI, respectively, compared with CTR (7.2 +/- 0.77 mg/100 mL). The average ammonia N concentration was 31% higher in ANI and 25.5% lower in GAR compared with CTR (5.5 +/- 0.51 mg/100 mL). The accumulation of AA and ammonia N in ANI suggested that peptidolysis and deamination were stimulated. The accumulation of AA N and the decrease in ammonia N in GAR suggests that deamination was inhibited. The accumulation of peptide N and the numerical decrease in AA N in CIN suggest that peptidolysis was inhibited. Results indicate that plant extracts modified ruminal fermentation, but microbes were adapted to some extracts after 6 d of fermentation. Therefore, data from short-term in vitro fermentation studies may lead to erroneous conclusions, and should be interpreted with caution. Careful selection of these additives may allow the manipulation of protein degradation in the rumen.